SUMMARY

INDEX

SUMMARY 5

WELCOME SALUTATION 7

COMMISSIONS AND SPONSOR ACKNOWLEDGEMENTS 9

HISTORY 11

THE SYMPOSIUM 12

PRE-SYMPOSIUM “30TH LNNE’S ANNIVERSARY” 13

FINAL PROGRAM 16

INVITED SPEAKERS 24

TO ALL AUTHORS OF ABSTRACTS 40

NEUROSCIENCE AND EPILEPSY ABSTRACTS 41

NEUROSCIENCE-VISUAL ARTS ABSTRACTS 73

MAP 81

TRAVEL 82

WELCOME SALUTATION

RIBEIRÃO PRETO, SEPTEMBER 18TH, 2018

WELCOME SALUTATION

Dear All Participants

International Symposium NEWroscience 2018

Looking for Epilepsy Cure along Centuries and across Cultures

From Demons and Blessings to Brain Complex Networks and DNA Editing

Celebration of the LNNE’s 30th Anniversary (1988-2018)

On behalf of the Organizing, Scientific and Artistic Committees it is with great happiness that I give you a warm welcome to Ribeirão Preto. I am quite positive that the Scientific, Academic and mostly Human Gathering that we have built for this special occasion will be memorable.

This event is organized by the Neurophysiology and Experimental Neuroethology Laboratory (LNNE) at the Ribeirão Preto School of Medicine (FMRP) and the University of São Paulo - (USP).

We are grateful to the Brazilians Research Foundations, especially FAPESP and CAPES (This event was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001), the FMRP and USP, which together with UNIMED RIBEIRÃO PRETO, UNIMED NORDESTE PAULISTA and ADInstruments, allowed us to put together at the NEWroscience 2018, thirty seven internationally recognized specialists from twenty eight Institutions from thirteen countries, from four continents in the fields of Epileptology, Neuropsychiatry, Neurology, Psychology, Behavioral, Cellular, Molecular and Computational Neuroscience, in both Basic and Applied Sciences.

We consider that having near 200 registrants, mostly young Under-Graduate and Graduate Students, as well as Post-Docs, and 58 Abstracts (Epilepsy and Neuroscience), 7 images (Neuroscience and Arts) to be presented as posters in such an integrative field is a big success.

Looking forward to your productive and friendly participation, I welcome all of you to Ribeirão Preto, Brasil and to the NEWroscience 2018.

Norberto Garcia-Cairasco
President of the Organizing Committee

COMMISSIONS AND SPONSOR ACKNOWLEDGEMENTS

ORGANIZING COMMITTEE

Norberto Garcia-Cairasco

Adrieli Oliveira Raminelli

Alexandra Cunha

Ana Luiza Ferreira Donatti

Artur Fernandes

Flavio Del Vecchio

José Antônio Cortes Oliveira

Lívea D. Godoy

Nayara Cobra Barreiro Barroca

Polianna Delfino-Pereira

Samuel Amorin Grisote

Suélen Santos Alves

Rui Milton

Willian Lazarini Lopes

SCIENTIFIC COMMITTEE

Norberto Garcia-Cairasco

Marcio Flavio Dutra Moraes

SCIENTIFIC EVALUATION FOR NEUROSCIENCE ABSTRACTS

Norberto Garcia-Cairasco

Alexandra Cunha

Ana Luiza Ferreira Donatti

Artur Fernandes

Eduardo Henrique Lima Umeoka

Rui Milton

ARTISTIC EVALUATION FOR NEUROSCIENCE-ARTS ABSTRACTS

Maria Luiza Dál-Col

Luciane Strambi

ACKNOWLEDGEMENTS

We would like to extend our appreciation to the Brazilian Research Foundations FAPESP and CAPES (This event was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001), PROEX-CAPES that provided a quite significant financial support to the International Symposium NEWroscience 2018. To the Law School of Ribeirão Preto at the University of São Paulo and its Director for the use of the magnificent Auditorium and nearby areas and structure. To the Institute for Advanced Studies (IEA-USP-RP), Director and staff, for all the logistics for registrations, diffusion and all the support associated with our NEWroscience Symposium. To several foreign invited speakers who kindly payed their flights to attend NEWroscience 2018. To the private sponsors, among them: UNIMED RIBEIRÃO PRETO, UNIMED NORDESTE PAULISTA and ADInstruments, our acknowledgement for the donations that allowed us to offer the welcome cocktail and the coffee-breaks. Indeed, a substantial part of the registration fees will be spent also in the coffee-breaks, therefore we would like to acknowledge also all the registrants for their special contributions to this. Last but not least, I would like to extend my great appreciation to Marcio Moraes from UFMG, Co-Organizer, and to all the Members of the LNNE who, without any restriction and hesitation, have done an excellent, voluntary work, making part of all the Organizing Committees, in the middle of regular Lab activities, in order to help making this event, not only possible, but absolutely special.

HISTORY

The Neurophysiology and Experimental Neuroethology Laboratory (LNNE), under the Direction of Prof. Norberto Garcia-Cairasco, has received during the last 30 years nearly 100 people from High School Students, to Under-Graduate and Graduate Students and Post-Docs. Together with them the LNNE produced during its existence 21 PhD Thesis, 13 Master Dissertations, 4 ongoing PhD Thesis and 4 ongoing Master Dissertations. In addition it has produced near 140 scientific articles, creating a solid network of national and international collaborations in the fields of Behavioral, Cellular, Molecular and Computational Neuroscience, especially in Epilepsy and Neuropsychiatric Comorbidities Research. Furthermore, since the 80´s the LNNE has strong participation in outreach Neuroscience activities, has produced 24 International Journal Covers and in the last 8 years has pioneered, tuned with the Internationally recognized Brain Awareness Week (Dana Foundation for Brain Alliances), the Semana Nacional do Cérebro (Sociedade Braisileira de Neurociências e Comportamento - SBNeC).

We believe that a successful formula for Excellence in Science must contain the right amounts of creative thinking, hard work and sensitivity. That is why the LNNE has stimulated scientific inter and transdisciplinary discussions with a great variety of researchers in their fields over its 30 years of history.

Our first Meeting, celebrating the 10th Aniversary, took place in 1998, and was a quite regional reunion entitled “Neurociências Integrativas” (Integrative Neurosciences) and counted on the participation of local Scientists and Students to talk about “Physiology in the Age of Molecular Biology”, “Genomics and Neuroinformatics”, “Epilepsy and Neuroscience” and “Visual Arts and Neuroscience”.

Prof. Garcia-Cairasco, motivated by the success of the previous meeting decided to organize it again once every five years. Our second meeting, in 2003, was dedicated to discuss the Neuroscience and Arts Connection. Entitled “Neuro-Arte” (Neuro-Art) gathered people from various parts of our country to present their artistic, academic and scientific points of view in Neuroscience. Larger in participants than the earlier event, but yet in a national range, this was a “pilot experiment” for what was about to come.

In 2008 we decided to embrace what we began to call the NEW challenge, inspired by Kuhn´s “paradigm shift” proposal, and for the first time we organized the “International Symposium NEWroscience 2008: Contemporary Neurosciences, Epilepsies and the Arts” with 9 Speakers from abroad and 4 Brazilians. This event culminated in the publication in 2009 of a Supplement of the Journal Epilepsy & Behavior, with 13 Invited Reviews, concerning their areas of expertise and the Abstracts of 48 works, presented as Posters. Without losing the link with Visual Arts we accepted Scientific and Neuro-Art Posters and the best images received Special Awards. In parallel we offered, with the sponsorship of Olympus Latin America, the MicroWorlds Confocal Workshop, with 7 Speakers.

In 2013 the challenge was even bigger. The “International Symposium NEWroscience 2013 - Epilepsies: Complexity and Comorbidities”, counted on the participation of 14 International and 8 Brazilian Speakers who discussed in 6 Thematic Round Tables “Challenges and Perspectives for Epilepsy Research”, “Modeling Epilepsy and Comorbidities: The Complexity of the Mechanisms”, “Complex Systems and the Epilepsies I – From Knowledge Networks to the Connectome”, “Complex Systems and the Epilepsies II – From Complexity in Semiology Analysis to Brain Circuits Modeling”, “Psychiatric Comorbidities and the Epilepsies: From Clinical Insights to Experimental and Molecular Biomarkers” and “Psychiatric Comorbidities and the Epilepsies II: Technological Advances and Future Perspectives”. All the Speakers were invited to write a Review Article and a Special Issue of the Journal Epilepsy & Behavior was published in 2014, with 23 excellent articles, mostly Reviews. The 100 Abstracts presented as Posters for the NEWroscience 2013 were also, after strict criteria of the Editorial Board of Epilepsy & Behavior published in the same Special Issue of Epilepsy & Behavior.

In 2018, the celebration will be even greater because the LNNE celebrates its 30th Anniversary (see details below).

THE SYMPOSIUM

The Neurophysiology and Experimental Neuroethology Laboratory (LNNE) at the Ribeirão Preto School of Medicine - University of São Paulo, promotes the International Symposium NEWroscience 2018. “Looking for Epilepsy Cure along Centuries and across Cultures. From Demons and Blessings to Brain Complex Networks and DNA Editing”

Held in Ribeirão Preto from September 18th to 21st , this event is also part of the Celebration of the LNNE’s 30th Anniversary.

Because in 2018 the LNNE celebrates its 30th Anniversary, we naturally thought that, as a consequence of our social responsibility as Scientists and Educators in a Public University, and because of our commitment with the production of high quality knowledge, necessary to improve the quality of life of people with epilepsy and associated comorbidities, we can give back to the community, indeed, to the society which support us, an academic and scientific gift: The “International Symposium NEWroscience 2018. “Looking for Epilepsy Cure along Centuries and across Cultures. From Demons and Blessings to Brain Complex Networks and DNA Editing”. This is a plural, unique event, which will count on the participation of thirty seven internationally recognized specialists from twenty eight Institutions from thirteen countries, from four continents who will gather to discuss, first in a Keynote Speech “Self-Reflected Brain: Dreams and Consciousness in Neuroscience and the Arts” at the Opening Ceremony on the 18th of September and next from the 19th to the 21st of September, in 6 Thematic Round Tables. The Round Tables themes are: “Controversies and Convergences in the History of Science and the Arts in Neuropsychiatry and the Epilepsies”, “From Connectomes to Multilayered Complex Networks in the Epilepsies and Neuropsychiatric Comorbidities”, “Cell and Molecular Mechanisms of the Epilepsies”, “Epilepsy Hijacking Brain Oscillations: a Gradual Corruption from Normal Activity”, “Precision Medicine and Technological Challenges in Neuroscience and Epileptology”, “Epilepsies and Comorbidities: Challenging and Integrating Multiples Levels of Complexity”. The specific Talks and corresponding Invited Speakers are detailed in the Final Program.

A completely new approach is also included in the NEWroscience 2018 version. At the end of Round Table 1, Round Tables 2 to 4 and Round Tables 5 and 6 we will have 3 of what we called “Brainstorm and Final Statements”, for the first time in any International Epilepsy Meeting. These sections are proposed to be critical reflections associated to the mentioned Round Tables themes, in order to track conflicts, to detect challenges and to propose/trace future creative “paradigm-shift” developments.

Finally, similar to what happened at the Symposia NEWroscience 2008 and NEWroscience 2013, after the NEWroscience 2018, we expect to have almost 40 Review Articles, plus the 3 Brainstorms/Final Statements, as written products of those reflections. Moreover, only Abstracts actually presented at the NEWroscience 2018 and which fulfill the strict criteria of the Editorial Board of Epilepsy & Behavior will be also placed in the corresponding Special Issue of that Journal, which will be published in 2019. This Special Issue of Epilepsy & Behavior will have Dr. Norberto Garcia-Cairasco (USP-Brazil) and Dr. Marcio Flavio Dutra Moraes (UFMG-Brazil) as Guest-Editors and Dr. Steven C. Schachter (Harvard-USA) as Editor in-Chief.

To enrich the event we have strongly encouraged the participation of all Clinical and Basic Neuroscientists, Clinical Neurologists, Neuropsychiatrists, Psychologists, Molecular Biologists, Physical Therapists, Physicists, Biomedical Engineers, Plastic Artists, and anybody interested academically, artistically or socially in this challenging and integrative meeting. We encouraged also the participation of Undergraduate and Graduate Students, Post-Docs and all interested researchers with submission of Abstracts to be presented as Posters. We will have three categories: Neuroscience Abstracts, Epilepsy Abstracts and Neuroscience & Arts Abstracts.

We are confident that this event, which will show the conjunction of different Cultures and Centuries of studies on Epilepsy and Comorbidities, will have a strong impact for the progress of Epilepsy Research by promoting the discussion on how scientists usually look at their object of study. Complex Systems demand Complex ways of analysis and although impossible to be studied by this optic in the past, the growing computational power and the large amount of data generated over the decades, summed with intriguing minds and some sweat will help us solving the Puzzle of the Epilepsies and their associated Comorbidities.

As in previous years this is a great avenue for creative thinking, linking Hard Science and Arts as another way to contribute to the recognized goals of epileptologists around the World and the general population: to remove social stigmas, “taking epilepsy out of the shadows”. In that context, on the 20th of September an open (free) Round Table will be held with the title “Epilepsia no dia-a-dia” (Epilepsy in daily life), offered at the Institute for Advanced Studies (IEA-USP-RP) for all interested people, but particularly for those with epilepsy and their families.

PRE-SYMPOSIUM “30TH LNNE’S ANNIVERSARY”

Pré Simpósio

Evento comemorativo dos 30 ANOS DO LNNE

“LNNE: 30 ANOS DE CIÊNCIA, EDUCAÇÃO E ARTES”

“LNNE: 30 YEARS OF SCIENCE, EDUCATION AND ARTS”

Auditório da Faculdade de Direito da Universidade de São Paulo
Campus de Ribeirão Preto

PROGRAMAÇÃO DO PRÉ-SIMPÓSIO

Comitê Pré-Simpósio

Ana Luiza Ferreira Donatti

Alexandra Olimpio Siqueira Cunha

Guilherme Gozzoli Podolsky Gondim

18  SETEMBRO 2018 | TERÇA-FEIRA

MANHÃ

09:00 – 09:20 | Abertura

Norberto Garcia-Cairasco / Renato M.E. Sabbatini (Video-clip)

FMRP-USP/Instituto EDUMED

09:20 – 09:30

Flávio Del Vecchio e Oswaldo Del Vecchio

FMRP - USP

09:30 – 09:40

Maria Carolina Doretto

EpiBrasil

09:40 – 09:50

José Antonio C. de Oliveira

FMRP-USP

09:50 – 10:00

Jeane Tsutsui (Video-clip)

INCOR-USP

10:00 – 10:10

Maria Cristina Lancia Curi

Hospital das Forças Armadas, Brasília

10:10 – 10:35 | Brainstorm

Márcio Dutra Morais

UFMG

Maria Cristina Lancia Curi 

Hospital das Forças Armadas, Brasília

10:30 – 11:00 | Coffee Break

11:00 – 11:10

Hamilton Wakamatsu

Radiologista, Curitiba, PR

11:10 – 11:20

Glaucia Karime Braga

Farmacopéia Brasileira

11:20 – 11:30

Francisco D’Arruda Montalverne

Neurologista/Neurocirurgião Chefe do Serviço de Neuroradiologia Intervencionista do HGF

11:30 – 11:40

Francisco Santos Leite

Uni-FACEF, Franca, SP

11:40 – 11:50

Marília Gabriela A.G. Pereira

UNIFAL

11:50 – 12:00

Poliana Bertti Dutra

FMRP-USP/Barão de Mauá

12:00 – 12:20 | Brainstorm

Eduardo H. L. Umeoka

FMRP-USP

Victor Rodrigues dos Santos

UFMG

12:20 – 13:20 | Almoço

TARDE

13:30 – 13:40

Marcio Furtado (Video-clip)

Uniformed Services University

13:40 – 13:50

Erica Maria Granjeiro

Universidade Estadual de Feira de Santana

13:50 – 14:00

Alexandra Cunha

FMRP-USP

14:10 – 14:20

Lívea Dornela Godoy

FMRP-USP

14:20 – 14:30

Guilherme Gozzoli Podolsky

FMRP-USP

14:30 – 14:40

Maria Luiza Dál-Col

Prefeitura de Londrina, PR

14:40 – 14:50

Palloma Beatriz Fialho Damas

Iniciação Científica Junior – FMRP-USP

Jonathan Batista Ferreira

Iniciação Científica Junior – FMRP-USP

14:50 – 15:00

Artur Fernandes

FMRP-USP

15:00 – 15:10

Nayara Cobra B. Barroca

FMRP-USP

Suélen Alves

FMRP-USP

15:10 – 15:20

Polianna Delfino

FMRP-USP

Rui Milton Patrício da Silva Jr.

FMRP-USP

15:20 – 15:30 | Brainstorm e Encerramento

Norberto Garcia Cairasco

FMRP-USP

FINAL PROGRAM

International Symposium

NEWroscience 2018

“Looking for Epilepsy Cure along Centuries and across Cultures.

From Demons and Blessings to Brain Complex Networks and DNA Editing”

18th  SEPTEMBER 2018 | TUESDAY

18:00 – 19:20 | Secretariat Opening, Registration and Material Pick-Up

19:20 – 19:30 | Opening Remarks

Margaret de Castro

Diretora da Faculdade de Medicina de Ribeirão Preto – Universidade de São Paulo.

Norberto Garcia-Cairasco

Faculdade de Medicina de Ribeirão Preto – Universidade de São Paulo – Brasil.

19:30 – 20:30 | Opening Lecture

Self-Reflected Brain: Dreams and Consciousness in Neuroscience and the Arts

Greg Dunn

Greg Dunn Design.

20:30 – 23:00 | Opening Cocktail

19th  SEPTEMBER 2018 | WEDNESDAY

Morning – Round Table 1

Controversies and Convergences in the History of Science and the Arts in Neuropsychiatry and the Epilepsies

Chairman: Peter Wolf /Norberto Garcia-Cairasco

Danish Epilepsy Centre – Dianalund, Denmark. / FMRP-USP.

08:00 – 08:25 | The Epilepsies: Dialogues between Traditional Healers and Contemporary Neurologists in Africa

Lamine Gueye

University of Dakar, Dakar, Senegal – África.

08:25 – 08:50 | Contribution of Pre-Columbian and Contemporary Indian Cultures to the Treatment of Epilepsies and Associated Neurological Syndromes

Jaime Fandiño Franky

Fundación Centro Colombiano de Epilepsia y Enfermedades Neurológicas – FIRE, Cartagena, Colombia.

08:50 – 9:15 | Renaissance Philosophy of Nature and Epilepsy

Eduardo Kickhofel

Universidade Federal Paulista – Guarulhos, Brasil.

09:15 – 9:40 | The Epilepsies of Dostoievski (Russia) and Machado de Assis (Brazil): The Working Brains of Bright Writers with Epilepsy

Peter Wolf / Norberto Garcia-Cairasco

Danish Epilepsy Centre – Dianalund, Denmark. / FMRP-USP.

09:40 – 10:10 | Coffee-Break

10:10 – 10:35 | Ayahuasca, an Ancient Substance with Traditional and Contemporary use in Neuropsychiatry and Neuroscience

Rafael Guimarães

Universidade de São Paulo – Ribeirão Preto, Brasil.

10:35 – 11:00 | The Modern Use of Cannabis for Epilepsy: From Bench to Clinical Trials of Cannabidiol

Fabricio Moreira

Universidade Federal de Minas Gerais – Belo Horizonte, Brasil.

11:00 – 11:30 | General Discussion

Moderator: Peter Wolf /Norberto Garcia-Cairasco

Danish Epilepsy Centre – Dianalund, Denmark. / FMRP-USP.

11:30 – 12:00 | Brainstorm and Final Statement 1 – Controversies and Future Challenges (From Round Table 1)

Chairman: Eduardo Kichkofel

Universidade Federal de São Paulo – Guarulhos, Brasil.

Co-Chairman: Gabriel Arisi

Universidade Federal de São Paulo – São Paulo, Brasil.

Afternoon – Round Table 2

From Connectomes to Multilayered Complex Networks in the Epilepsies and Neuropsychiatric Comorbidities

Chairman: Fernando Cendes

Universidade Estadual de Campinas – Campinas, Brasil.

14:00 – 14:25 | Mood, Anxiety Disorders and the Concept of Epilepsy as a Network Disease

Fernando Cendes

Universidade Estadual de Campinas – Campinas, Brasil.

14:25 – 14:50 | Epilepsies, Autistic and Mood Disorders as Complex Networks Diseases

Andres Kanner / Fernando Cendes

University of Miami – Miami, USA. / UNICAMP.

14:50 – 15:15 | Matrix Macromolecules, Gliosis, Mesial Sclerosis and Memory Impairments in Temporal Lobe Epilepsy

João Pereira Leite

Universidade de São Paulo – Ribeirão Preto, Brasil.

15:15 – 15:45 | Coffee Break – Poster

15:45 – 16:10 | Hippocampal-Frontal Networks in Experimental Models of Neuropsychiatric Disorders in Epilepsy Models

Cleiton Lopes Aguiar

Universidade Federal de Minas Gerais – Belo Horizonte, Brasil.

16:10 – 16:35 | Computational Neuroscience and Temporal Lobe Epilepsy: Scaling from Neurons to Networks

Julian Tejada

Universidade Federal de Sergipe – Aracaju, Brasil.

16:35 – 17:00 | Contributions of Basic Science to a Network Analysis of Epilepsy and Comorbidities

Cristiane Q. Tilelli

Universidade Federal de São João Del Rei – Divinópolis, Brasil.

17:00 – 18:00 | General Discussion

Moderators:

Fernando Cendes

Universidade Estadual de Campinas, Campinas, Brasil.

Cleiton Lopes Aguiar

Universidade Federal de Minas Gerais – Belo Horizonte, Brasil.

20th  SEPTEMBER 2018 | THURSDAY

Morning – Round Table 3

Cell and Molecular Mechanisms of the Epilepsies

Chairman: Asla Pitkänen

University of Eastern Finland – Kuopio, Finland.

08:00 – 08:25 | How Might Novel Technologies such as Optogenetics and DREADDS Lead to Better Treatments in Epilepsy?

Esther Krook-Magnuson

University of Minnesota – Minneapolis, USA.

08:25 – 08:50 | Looking for Biomarkers for Epileptogenesis and Functional Outcome after Brain Injury

Asla Pitkänen

University of Eastern Finland – Kuopio, Finland.

08:50 – 09:15 | Interneurons: Role in Maintaining and Restoring Synaptic Plasticity

Maria Elisa Calcagnotto

Universidade Federal do Rio Grande do Sul – Porto Alegre , Brazil.

09:15 – 09:45 | Coffee Break – Poster

09:45 – 10:10 | Characterizing Molecular, Biochemical and Genetic Changes in Neurotransmitters in the Brain of Experimental Models of Drug-Resistant Epilepsy

Luisa Lilia Rocha Arrieta

Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional – D.F., Mexico.

10:10 – 10:35 | New Avenues in Molecular Genetics for Diagnosis and Therapeutics, as Applied to the Epilepsies

Íscia Lopes-Cendes

Universidade Estadual de Campinas – Campinas, Brasil.

10:35 – 11:00 | Molecular Tools to Characterize Seizure Susceptibility in Rodent Genetic Models of Epilepsy

Dolores E. Lopez

Universidad de Salamanca – Salamanca, España.

11:00 – 12:00 | General Discussion

Moderators:

Esther Krook-Magnuson

University of Minnesota – Minneapolis, USA.

Íscia Lopes-Cendes

Universidade Estadual de Campinas – Campinas, Brasil.

Afternoon – Round Table 4

Epilepsy Hijacking Brain Oscillations: a Gradual Corruption from Normal Activity

Chairman: Marcio F. Dutra Moraes

Universidade Federal de Minas Gerais – Belo Horizonte, Brasil.

14:00 – 14:25 | Closed Loop Control of Seizures

Dean Freestone

University of Melbourne – Melbourne, Austrália.

14:25 – 14:50 | Synchronization and Desynchronization in Epilepsy: Controversies and Hypotheses

Premek Jiruska

Czech Academy of Sciences – Prague, Czech Republic.

14:50 – 15:15 | Periodic and Non-Periodic Deep Brain Stimulation in Epilepsy Models: Effects on Network Synchronization

Vinícius Rosa Cota

Universidade Federal de São João del Rei – São João del Rei, Brasil.

15:15 – 15:40 | Coffee Break

15:40 – 16:05 | Deep Brain Stimulation in Epilepsy: Actively Probing Neural Networks

Marcio F. Dutra Moraes

Universidade Federal de Minas Gerais – Belo Horizonte, Brasil.

16:05 – 16:30 | The Genesis of an Epileptic Hippocampus

Claudio Queiroz

Universidade Federal do Rio Grande do Norte – Natal, Brasil.

16:30 – 17:00 | General Discussion

Moderator: Marcio F. Dutra Moraes

Universidade Federal de Minas Gerais – Belo Horizonte, Brasil.

17:00 – 18:00 | Brainstorm and Final Statement – Controversies and Future Challenges (From Round Tables 2, 3 and 4)

Chairman: Dean Freestone

University of Melbourne - Australia.

Co-Chairman: Cristiane Q. Tilelli

Universidade Federal de São João Del Rei – Divinópolis, Brasil.

Moderators: Julian Tejada

Universidade Federal de Sergipe – Aracaju, Brasil.

Claudio Queiroz

Universidade Federal do Rio Grande do Norte – Natal, Brasil.

19:00 – 21:00 | Mesa Redonda aberta ao público e gratuita:

Epilepsia no dia-a-dia

“Conversando sobre Epilepsia: Orientação a pessoas com epilepsia e familiares para uma vida melhor”

Debatedores/Palestrantes:

Dra. Maria Carolina Doretto

Professora aposentada da UFMG. Secretária Executiva da Federação Brasileira de Epilepsia – Epibrasil.

Dra. Vera Cristina Terra

Médica neurologista com experiência no tratamento de epilepsias refratárias na infância e na adolescência. Professora de Neurologia da Universidade Federal do Paraná.

Dr. Artur Fernandes

Pesquisador na área de genética das epilepsias.

OBJETIVO: apoiar as pessoas com epilepsia em suas necessidades, especialmente no âmbito da educação, proporcionar informações corretas sobre a doença, orientar sobre tratamentos e campanhas de combate ao estigma e ao preconceito.

O evento é aberto a todos os interessados e é gratuito. Serão fornecidos certificados a quem solicitar.

21st  SEPTEMBER 2018 | FRIDAY

Morning – Round Table 5

Precision Medicine and Technological Challenges in Neuroscience and Epileptology

Chairman: Ashok Shetty

College Station – Texas, USA.

08:00 – 08:25 | Human Brain Slices for Epilepsy Research: Pitfalls, Solutions and Future Challenges

Mark O. Cunningham

University of Dublin – Dublin, Ireland.

8:25 – 08:50 | Intranasal Mesenquimal Stem Cell-Derived A1-Exosomes ease Inflammation, and Prevent Abnormal Neurogenesis and Memory Dysfunction after Status Epilepticus

Ashok Shetty

College Station – Texas, USA.

08:50 – 09:15 | Complementary MRI/DTI and Chemogenetics/Optogenetics to Pinpoint Epileptogenic Networks in Experimental Models of Epilepsy

Patrick Forcelli

Georgetown University School of Medicine – Washington, DC, USA.

09:15 – 09:45 | Coffee Break – Poster

09:45 – 10:10 | Networks for Tonic Respiratory Drive, Chemoreception and SUDEP in Epilepsies

George Richerson

University of Iowa – Iowa City, USA.

10:10 – 10:35 | Molecular and Neuroanatomical Neuroplasticity in Neurogenetic Niches in Experimental Epilepsy Models

Victor Rodrigues Santos

Universidade Federal de Minas Gerais – Belo Horizonte, Brasil.

10:35 – 11:00 | Experimental and Ethnopharmacological Characterization of Substances with Anti-Epileptogenic and Neuroprotective Actions

Olagide Wagner Castro

Universidade Federal de Alagoas – Maceió, Brasil.

11:00 – 12:00 | General Discussion

Moderators: 

Ashok Shetty

College Station – Texas, USA.

Mark O. Cunningham

Newcastle University – Newcastle upon Tyne, UK.

Afternoon – Round Table 6

Epilepsies and Comorbidities: Challenging and Integrating Multiples Levels of Complexity

Chairman: Norberto Garcia-Cairasco

Universidade de São Paulo – Ribeirão Preto, Brasil.

14:00 – 14:25 | Non-invasive Imaging in Epilepsy using Biomarkers

Edson Amaro Júnior

Hospital Israelita Albert Einstein – São Paulo, Brasil.

14:25 – 14:50 | Computational Neuroscience and the Epilepsies

Antônio Carlos Roque Da Silva Filho

Universidade de São Paulo – Ribeirão Preto, Brasil.

14:50 – 15:15 | Complex Systems Approach for Innovation in AEDs Discovery

Alan Talevi

Universidad Nacional de la Plata – La Plata, Argentina.

15:15 – 15:40 | Role of P-Glycoprotein in Pharmacoresistant Epilepsy and SUDEP

Alberto Lazarowski

Universidad de Buenos Aires – Buenos Aires, Argentina.

15:40 – 16:05 | Coffee Break

16:10 – 16:35 | MicroRNAs, Gene Networks, Gene Therapy: Promises and Challenges for Treating Epilepsies and their Comorbidities

Michele Simonato

University of Ferrara – Ferrara, Italy.

16:35 – 17:00 | Complexity Systems and Emergent Properties Views as Applied to Epilepsy Research: A Paradigm Shift Eagerly Needed

Norberto Garcia-Cairasco

University of São Paulo – Ribeirão Preto, Brasil.

17:00 – 18:00 | Brainstorm and Statement 3 – Controversies and Future Challenges (From Round Tables 5 and 6)

Chairman: Norberto Garcia-Cairasco

University of São Paulo – Ribeirão Preto, Brasil.

Co-Chairman: Alberto Lazarowski

Universidad de Buenos Aires – Buenos Aires, Argentina.

Moderators: Michele Simonato

University of Ferrara – Ferrara, Italy.

Antonio C. Pinheiro de Oliveira

Universidade Federal de Minas Gerais – Belo Horizonte, Brasil.

18:00 – 18:30 | Awards Ceremony

Celebrating Women in Science & Art

Best Scientific Poster in Epilepsy. Cassidy Megan, the Founder of the Purple Day, Award.

Best Scientific Poster in Neuroscience. Rosalind Franklin, the Mother of the DNA, Award.

Best Poster in Neuroscience & Arts. Nise da Silveira, the Rebel Psychiatrist, Award.

The Diploma Leonardo da Vinci Award

Given to LNNE PhD Students Graduated from 2013 to 2018

18:30 – 18:40 | Closing Remarks

Scientific Committee

Norberto Garcia-Cairasco

Marcio F.D. Moraes

19:10 | Farewell Dinner

INVITED SPEAKERS

International Symposium

NEWroscience 2018

“Looking for Epilepsy Cure along Centuries and across Cultures.

From Demons and Blessings to Brain Complex Networks and DNA Editing”

Invited Speakers

Norberto Garcia-Cairasco

University of São Paulo – Ribeirão Preto, Brazil.

Physiology/Neuroscience & Behavioral Sciences Departments. Ribeirão Preto School of Medicine.

Round Table 6 - Epilepsies and Comorbidities: Challenging and Integrating Multiples Levels of Complexity

LECTURE: Complexity Systems and Emergent Properties Views as Applied to Epilepsy Research: A Paradigm Shift Eagerly Needed.

The study of the epilepsies and associated neuropsychiatric comorbidities has evolved over decades into big challenges for complexity system solutions and the consequent emergent properties. Looking first at the semiology descriptions, we have passed from qualitative descriptions to sophisticated digital capturing and graph analysis of behavioral sequences. The complementary EEG parameters needed for epileptogenic zones and lesions detection, have grown in sophistication, from surface EEG to stereo-EEG, allowing the construction of complex electrophysiologically-connected networks, although not necessarily used in its entire magnitude for prognosis modeling, for example, of recurrence. Finally, the evolution of high resolution imaging tools such as MRI, Bold, DTI, have allowed the production of sophisticated connectivity maps (connectomes).

Norberto Garcia-Cairasco will show how little effort, indeed, has been done over the decades to place all these datasets together as a means to reconstruct the ideal, certainly utopic, model of combined electrophysiological, imaging and its final common manifestation, semiology, in a single but complex map. The availability of computational neuroscience tools has increased the analytical power for both diagnosis (etiology and causality), and solutions (a wide variety of selective or network-wise targets for prediction and therapeutic protocols). Looking at the future, he will finally discuss how this totally new trans-disciplinary approach, will help to make the eagerly needed “paradigm shift” in this important field of research.

Greg Dunn

Philadelphia – USA.

Greg Dunn Design.

Keynote Speech

Opening Lecture

LECTURE: Self-Reflected Brain: Dreams and Consciousness in Neuroscience and the Arts.

Dunn is a PhD in Neuroscience at the University of Pennsylvania, and his approach to art mirrors his scientific background.

He uses pipettes to deliver precise amounts of pigment to the canvas, creates wet lab-style workflow protocols so that he can accurately reproduce techniques later, makes digital mock-ups of a project before putting his brush to canvas, and uses his knowledge of chemistry to precisely manipulate the variety of materials he utilizes in his work.

But while his approach to his art is influenced by a knowledge of neuroscience and the scientific method, his approach to life is influenced by the artists and culture he looks to for artistic inspiration.

Greg Dunn’s struggle to emulate and expand upon the work of artists from centuries before his time adds human emotion, conflict, and context to the abstract forms and tendrils of the neurobiological landscape.

Peter Wolf

Danish Epilepsy Centre – Dianalund, Denmark.

Professor and Doctor of Medical Science

Round Table 1 - Controversies and Convergences in the History of Science and the Arts in Neuropsychiatry and the Epilepsies

LECTURE: The epilepsies of Dostoyevsky and Machado de Assis.

It appears like a curious coincidence that, towards the end of the 19th century, two writers who are considered champions of their respective national literatures, Dostoyevsky of Russia and Machado de Assis of Brasil had epilepsy, most probably temporal lobe epilepsy. However, the way both authors dealt with their condition could not have been more different. Dostoyevsky did not hide his diagnosis and used epilepsy amply in his fiction, creating such impressive characters with epilepsy as Prince Myshkin, Kirillov or Smerdyakov, and amply using his seizure experiences for literary purposes.

Machado tried his best to keep the diagnosis a secret (without success) and never talked overtly about seizures and epilepsy in his writings. But his seizure experiences obviously were too fascinating not to describe them, and complex partial seizures can be identified in two of his novels, Brás Cubas and Quincas Borba. This seizure type was so little know that there was no risk his readers would understand that these experiences were epileptic. Quincas Borba (1891) has several features which can be understood as an homage to Dostoyevsky’s Idiot (1869).

Lamine Gueye

University of Dakar, Dakar, Senegal – África.

Full professor Neurophysiology.

Round Table 1 - Controversies and Convergences in the History of Science and the Arts in Neuropsychiatry and the Epilepsies

LECTURE: The Epilepsies: Dialogues between Traditional Healers and Contemporary Neurologists in Africa.

The prevalence of epilepsy in Africa is the highest in the world, 10 to 30‰ in many countries.

The care of patients is still difficult because of the lack of qualified staff and infrastructures, the unavailability of certain drugs, but also the influence of socio-cultural factors.

Societal beliefs evoke inorganic and mystical causes of the disease, hence the frequent use of traditional methods for the treatment of patients. Some treatments are herbal but also is frequently based on mystical approaches (incantation, wearing amulets, bathing).

Such treatments are not good for patients and for solving this problem, it is necessary to establish a dialogue between neurologists and traditional healers. We will describe the experience made in Senegal.

Jaime Fandiño Franky

Fundación Centro Colombiano de Epilepsia y Enfermedades Neurológicas - FIRE, Cartagena, Colombia.

Fundación Centro Colombiano de Epilepsia y Enfermedades Neurológicas.

Round Table 1 - Controversies and Convergences in the History of Science and the Arts in Neuropsychiatry and the Epilepsies

LECTURE: Contribution of Precolumbian and Contemporary Indian Cultures to the Treatment of Epilepsies and Associated Neurological Syndromes.

Epilepsy was a known disease in pre-Columbian era. Aztecs and Incas stand out and attached it to religion and magic. Many empiric botanic remedies were used, most of them without real results according to reports of Spanish chroniclers such as Garcilaso de la Vega. In the deep Amazon jungles of Colombia, Ecuador and Brazil, the indigenes also had many different botanic remedies and aromatic smokes. Aztecs believed that epilepsy was due to a sin, and gave to the patients a bath with herbs.

In Paraguay, Bolivia and “Misiones” (Argentina), the “paye” or Chaman gives “caaré”, a root that produces hallucinations, and also leafs of “ysaú kaa” were given to the patients. “Sonco nanay”, as Incas named the epilepsy, has been described confusedly. The “Sircay” (surgeon) performed decompressive craniectomies for epilepsy. Doubts exist about what the results were. “Momentary Interruption” means the whole seizure. It is nowadays very difficult to know how the native people prepared the mixtures.

Perhaps it was a fiddle or flimflam. But it is true that some alkaloid herbs are still used in Modern Medicine. In contrast, in 1494, 200.000 epileptic women were killed in Europe because they were considered “witches”, which showed the big cultural difference and also ignorance, when compared the two continents.

Eduardo Kickhofel

Universidade Federal Paulista – Guarulhos, Brasil.

Universidade Federal Paulista.

Round Table 1 - Controversies and Convergences in the History of Science and the Arts in Neuropsychiatry and the Epilepsies

LECTURE: Renaissance Philosophy of Nature and Epilepsy.

In his presentation Eduardo Kickhofel will reflect on the relationship between “art” and “science” so far as it concerns a symposium on neurosciences. He undertakes a historical overview of that relationship, paying particular attention to the sui generis case of Leonardo da Vinci, who very often is regarded as the man who worked on art and science with equal ease.

He then explains why Leonardo´s idea of merging these two forms of knowledge failed, considering the clear-cut distinction between art and science in his time. With this clarification, he explore the matter today.

Eduardo looks, for example, at Raphael’s The Transfiguration, in which the representation of the possessed boy is seen by neuroscientists as indicative of an epileptic seizure. He also looks at the ideas of neuroscientists such as Semir Zeki and Vilayanur Ramachandran, who study particular aspects of brain function and suggest a new merging of art and science.

Rafael Guimarães

Universidade de São Paulo – Ribeirão Preto, Brasil.

Departamento de Neurociências e Ciências do Comportamento.

Round Table 1 - Controversies and Convergences in the History of Science and the Arts in Neuropsychiatry and the Epilepsies

LECTURE: Ayahuasca, an Ancient Substance with Traditional and Contemporary use in Neuropsyquiatry and Neuroscience.

Ayahuasca is a botanical hallucinogenic preparation traditionally used by indigenous populations of Northwestern Amazonian countries for ritual and therapeutic purposes. It is rich in β-carboline alkaloids and N,N-dimethyltryptamine (DMT). Preclinical, observational, and experimental studies suggest that Ayahuasca and its alkaloids have anxiolytic and antidepressive effects.

Rafael Guimarães will show, among other studies, a recently report in an open-label trial that Ayahuasca administration was associated with significant decreases in depression symptoms for 2-3 weeks after the experimental session in 17 patients with treatment-resistant major depressive disorder.

Fabricio Moreira

Universidade Federal de Minas Gerais – Belo Horizonte, Brasil.

Lecturer/Assistant Professor (Pharmacology).

Round Table 1 - Controversies and Convergences in the History of Science and the Arts in Neuropsychiatry and the Epilepsies

LECTURE: The Modern Use of Cannabis for Epilepsy: From Bench to Clinical Trials of Cannabidiol.

The use of Cannabis sativa in the treatment of diseases has been highly controversial. In fact, the prejudice people have about marijuana fuels a debate that contrasts conservative arguments with the already proven benefits of the medicinal use of the plant.

The researcher Fabrício Moreira will discuss cannabinoids and their effects, seeking to develop substances of medical interest that will aid in the treatment of various diseases and disorders, such as epilepsy.

Gabriel Maisonnave Arisi

Universidade Federal de São Paulo – São Paulo, Brasil.

Assistant Professor of Physiology at the Federal University of São Paulo.

Round Table 1 - Controversies and Convergences in the History of Science and the Arts in Neuropsychiatry and the Epilepsies

Co-Chairman: Brainstorm and Final Statement 1 – Controversies and Future Challenges.

Gabriel Arisi is MSc and PhD from the Ribeirão Preto School of Medicine, University of São Paulo, with Post-Doctoral training at the University of Texas A&M. He has research experience with neurogenesis, experimental models of epilepsy, dendritic morphology and immune-nervous systems interface. Since last year Gabriel has started his teaching career, being currently an Assistant Professor of Physiology at the Federal University of São Paulo.

Fernando Cendes

Universidade Estadual de Campinas – Campinas, Brasil.

Departamento de Neurologia.

Round Table 2 - From Connectomes to Multilayered Complex Networks in the Epilepsies and Neuropsychiatric Comorbidities

LECTURE: Mood, Anxiety Disorders and the Concept of Epilepsy as a Network Disease.

After decades of focused research into complex disorders such as dementia, schizophrenia, and epilepsy, it has become clear that there are overlapping components. Often, this interrelationship is simplified by stating that seizures are a “comorbidity” of what is considered to be “another” disorder like Alzheimer’s disease, or epilepsy is a “risk factor” for Alzheimer’s disease.

The implication is that “neurology” and “psychiatry” are less divided than one might think, and our approach to research and treatment should be adapted accordingly. In this presentation Fernando Cendes will discuss the a new assess of the relationships between neurological and psychiatric conditions in general, and specifically autism, depression, Alzheimer’s disease, schizophrenia, and epilepsy.

Andres Kanner

University of Miami – Miami, USA.

Department of Neurology – Epilepsy Division.

Round Table 2 - From Connectomes to Multilayered Complex Networks in the Epilepsies and Neuropsychiatric Comorbidities

LECTURE: Epilepsies, Autistic and Mood Disorders as Complex Networks Diseases.

Epilepsy is a neurologic condition which often occurs with other neurologic and psychiatric disorders. The relationship between epilepsy and these conditions is complex. Some population-based studies have identified a bidirectional relationship, whereby not only patients with epilepsy are at increased risk of suffering from some of these neurologic and psychiatric disorders (migraine, stroke, dementia, autism, depression, anxiety disorders, Attention deficit hyperactivity disorder (ADHD), and psychosis), but also patients with these conditions are at increased risk of suffering from epilepsy.

The existence of common pathogenic mechanisms has been postulated as a potential explanation of this phenomenon. Andrés Kanner , will discuss data from several studies, as well as different hypotheses proposing that psychiatric disorders worsen the course and/or severity of an epileptic disorder and also he will suggest research strategies to test these hipotheses.

João Pereira Leite

Universidade de São Paulo – Ribeirão Preto, Brasil.

Ribeirão Preto School of Medicine.

Round Table 2 - From Connectomes to Multilayered Complex Networks in the Epilepsies and Neuropsychiatric Comorbidities

LECTURE: Matrix Macromolecules, Gliosis, Mesial Sclerosis and Memory Impairments in Temporal Lobe Epilepsy.

Temporal lobe epilepsy (TLE) caused by hippocampal sclerosis (HS) is the most common cause of drug-resistant epilepsy in adults leading to surgical treatment.

Histologic evaluation of the sclerotic hippocampus reveals neuron loss, gliosis, and changes in molecules associated with water homeostasis, such as aquaporin (AQP4) and extracellular matrix (ECM) proteoglycans.

In his lecture, João Pereira Leite will discuss the molecular, histologial and behavioral alterations, such as the presence of episodic memory impairment in adults and children with TLE.

Cleiton Lopes Aguiar

Universidade Federal de Minas Gerais – Belo Horizonte, Brasil.

Núcleo de Neurociências e Comportamento, Instituto de Ciências Biológicas.

Round Table 2 - From Connectomes to Multilayered Complex Networks in the Epilepsies and Neuropsychiatric Comorbidities

LECTURE: Hippocampal-Frontal Networks in Experimental Models of Neuropsychiatric Disorders in Epilepsy Models.

Mounting evidence suggests that the limbic system is pathologically involved in cases of psychiatric comorbidities in temporal lobe epilepsy (TLE) patients.

The objective of the presentation of Cleiton Lopes Aguiar, mostly based in experimental models data, will be to discuss a conceptual framework describing how neuropathological and connectivity changes might contribute to the development of psychosis and to the potential neurobiological mechanisms that cause schizophrenia-like psychosis in TLE patients.

Julian Tejada

Universidade Federal de Sergipe – Aracaju, Brasil.

Centro de Educação de Ciências Humanas, Departamento de Psicologia.

Round Table 2 - From Connectomes to Multilayered Complex Networks in the Epilepsies and Neuropsychiatric Comorbidities

LECTURE: Computational Neuroscience and Temporal Lobe Epilepsy: Scaling from Neurons to Networks.

Epilepsy research has been strongly involved in the main neuroscience discoveries, and the Computational Neuroscience area is not an exception. One of the most studied models is the simulation of abrupt changes in neuronal responses or networks induced by continuous seizures or Status Epilepticus. Due to the fact that a number of different models have been developed, from lumped up to detailed deterministic approaches, the development of additional models depends on the availability of experimental data which, usually are not valued, several times not recorded or even published.

In his talk Julian Tejada will show the process to build realistic computational models of dentate gyrus and will try to make evident the necessary information and the sequential steps to do it. Finally, he will show how capturing data and building the models are important tasks, not only for Theoretical and Computational Neuroscience, but to generate hypotheses to be tested at the bench or to be applied for Translational Neuroscience challenges.

Cristiane Q. Tilelli

Universidade Federal de São João Del Rei – Divinópolis, Brasil.

Professora Associada da Universidade Federal de São João del-Rei (UFSJ).

Round Table 2 - From Connectomes to Multilayered Complex Networks in the Epilepsies and Neuropsychiatric Comorbidities

LECTURE: Contributions of Basic Science to a Network Analysis of Epilepsy and Comorbidities.

Epilepsy is a complex disease, with epileptic seizures having diverse etiologies. One of the most common and well characterized epilepsy types, mesial temporal lobe epilepsy (MTLE), is specially related to one brain region, the hippocampal formation. MTLE can be modeled in animals, using several approaches, such as electrical stimulation and local or systemic drug injections.

Of importance, great morphological and electrographical alterations are observed in the hippocampal formation both in patients and animals affected by MTLE. On the other hand, a great amount of studies recognizes that other areas, besides the hippocampal formation, are involved in the expression of MTLE.

Immediate early genes, such as c-Fos, inflammation-related biomarkers, neurodegeneration, axonal and dendritic reorganization, and MRI/DTI alterations, have been detected in extra-hippocampal sites, such as amygdaloid complex, cortical non-temporal regions, thalamus, hypothalamus, basal ganglia, brainstem and cerebellum.

However, extra-hippocampal networks are commonly neglected in the evaluation of epilepsies, since they are usually morphologically, functionally and, based upon connectivity, more complex than hippocampus. Cristiane Q. Tilelli will, therefore, present here a critical review, discussing extra-hippocampal circuits as part of MTLE disease, suggesting future directions for experimental epilepsy research.

Asla Pitkänen

University of Eastern Finland – Kuopio, Finland.

Department of Neurobiology, A.I. Virtanen Institute for Molecular Sciences.

Round Table 3 - Cell and Molecular Mechanisms of the Epilepsies

LECTURE: Looking for Biomarkers for Epileptogenesis and Functional Outcome after Brain Injury.

Traumatic brain Injury (TBI) is defined as an alteration in brain function, or other evidence of brain pathology, caused by an external force. Clinically the post-TBI clinical phenotype can include post-traumatic epilepsy.

Asla Pitkanen presentation will discuss the hypothesis that early identification of injury severity with quantitative magnetic resonance imaging (MRI) provides biomarkers for predicting increased seizure susceptibility and epileptogenesis after traumatic brain injury TBI.

Esther Krook-Magnuson

University of Minnesota – Minneapolis, USA.

Neuroscience Department.

Round Table 3 - Cell and Molecular Mechanisms of the Epilepsies

LECTURE: How Might Novel Technologies such as Optogenetics and DREADDS Lead to Better Treatments in Epilepsy?

Tools which allow cell-type selective neuromodulation, such as optogenetics, allow circuit dissection in epilepsy and, importantly, the potential to better understand the disorder and develop novel treatment strategies. Our work has shown that targeting specific populations of neurons in the hippocampal formation using on-demand optogenetics can be an effective strategy for inhibiting temporal lobe seizures. However, care must be applied, because optogenetic methods can also be used to induce, rather than inhibit, seizures. Exciting data also points to the ability of selective neuromodulation targeting areas remote from the seizure focus to powerfully inhibit seizures. Together, our work has led to new insights not only into epilepsy, but neuronal circuits in healthy animals as well, and may lead to the development of novel therapeutic approaches.

Maria Elisa Calcagnotto

Universidade Federal do Rio Grande do Sul – Porto Alegre , Brasil.

Biochemistry Department.

Round Table 3 - Cell and Molecular Mechanisms of the Epilepsies

LECTURE: Interneurons: Role in Maintaining and Restoring Synaptic Plasticity.

Inhibitory circuits play an important role in synaptic plasticity during development and adulthood. Changes in interneuronal activity induce structural and synaptic rearrangements of inhibitory interneurons, network oscillations, and homeostatic plasticity.

In addition to epileptic seizures, deficits in the inhibitory system lead to aberrant information processing and cognitive impairment in various neurological disorders.

In her talk Maria Elisa Calcagnotto will discuss the role of inhibitory circuits in synaptic plasticity and how cellular replacement strategies can remodel changes in circuit function and homeostasis in the context of brain repair.

Luisa Lilia Rocha Arrieta

Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional – D.F., Mexico.

Pharmacobiology Department.

Round Table 3 - Cell and Molecular Mechanisms of the Epilepsies

LECTURE: Characterizing Molecular, Biochemical and Genetic Changes in Neurotransmitters in the Brain of Experimental Models of Drug-Resistant Epilepsy.

Luisa Rocha´s talk will be based on her studies focused on the characterization of multiple receptor binding, as well as subsequent transductional mechanisms in tissue obtained from patients with pharmacoresistent temporal lobe epilepsy (TLE).

Her results suggest significant alterations in the GABAergic neurotransmission and altered signal transduction mechanisms downstream opioid receptor activation in neocortex of patients with TLE, suggesting important influences of seizure frequency and duration of illness on these alterations.

Íscia Lopes-Cendes

Universidade Estadual de Campinas – Campinas, Brasil.

Departamento de Genética.

Round Table 3 - Cell and Molecular Mechanisms of the Epilepsies

LECTURE: New Avenues in Molecular Genetics for Diagnosis and Therapeutics, as Applied to the Epilepsies.

The development of the cerebral cortex involves complex steps, requiring tightly regulated molecular mechanisms for the efficient and precise control of gene expression. Some of the molecular pathways that control gene expression are mediated by microRNAs, a class of noncoding RNAs that regulate gene expression at the posttranscriptional level.

Gene regulations mediated by microRNAs have emerged as potential regulators of numerous biological processes within the brain, especially in neurodevelopment. In her lecture Iscia Lopes-Cendes will discuss the use of molecular genetics as a possible target for diagnosis and therapy in neurological diseases such as epilepsy.

Dolores E. Lopez

Universidad de Salamanca – Salamanca, España.

Instituto de Neurociencias.

Round Table 3 - Cell and Molecular Mechanisms of the Epilepsies

LECTURE: Molecular Tools to Characterize Seizure Susceptibility in Rodent Genetic Models of Epilepsy.

The complexity of epilepsy requires intense interdisciplinary research for better understanding of its etiology. Thus, a variety of models are available for exploring different aspects of epilepsy such as in silico, in vivo, and in vitro. Some in vivo models represent the natural association between genetic predisposition and external events that trigger seizures, like genetic models of epilepsy audiogenic, which seizures are triggered by high intensity acoustic stimulation.

Dolores E. López presentation will discuss the basic mechanisms associating genetic predisposition to epilepsy, microarrays-detected early gene expression after seizures, and the recognition of new targets that could be consequently tested in the development of antiepileptic drugs.

Marcio Flavio Dutra Moraes

Universidade Federal de Minas Gerais – Belo Horizonte, Brasil.

Professor Titular da Universidade Federal de Minas Gerais.

Round Table 4 - Epilepsy Hijacking Brain Oscillations: a Gradual Corruption from Normal Activity

LECTURE: Deep Brain Stimulation in Epilepsy: Actively Probing Neural Networks.

The desire to find alternative treatments for epilepsy has led to a resurgence of interest in the use of deep brain stimulation (DBS), which has been used quite successfully in movement disorders. Because of the diversity of regions involved with seizure initiation and propagation, a variety of targets for stimulation has been examined.

In this context, Marcio F.D. Moraes will discuss about local electrical and neurochemical alterations produced by specific protocols of DBS, as applied to current stimulation models in epilepsy. If successful they will enable future technologies with translational value, even nanotechnologies to flourish in the developing field of therapeutic neuromodulation.

Dean Freestone

University of Melbourne – Melbourne, Austrália.

Department of Electrical and Electronic Engineering.

Round Table 4 - Epilepsy Hijacking Brain Oscillations: a Gradual Corruption from Normal Activity

LECTURE: Closed loop control of seizures.

Amongst the novel therapeutic strategies for treatment for epilepsy is being investigated the delivery of electrical stimuli to neural targets thought to be responsible for the generation, propagation or termination of epileptic seizures in order to prevent or more rapidly terminate seizure events upon their automatic detection. Therefore, Dean Freestone lecture will explore the utilization of a closed-loop system for the automated detection and control of epileptic seizures.

Premek Jiruska

Czech Academy of Sciences – Prague, Czech Republic.

Department of Developmental Epileptology.

Round Table 4 - Epilepsy Hijacking Brain Oscillations: a Gradual Corruption from Normal Activity

LECTURE: Synchronization and Desynchronization in Epilepsy: Controversies and Hypotheses.

Epilepsy has been historically seen as a functional brain disorder associated with excessive synchronization of large neuronal populations leading to a hypersynchronous state. Recent evidence showed that epileptiform phenomena, particularly seizures, result from complex interactions between neuronal networks characterized by heterogeneity of neuronal firing and dynamical evolution of synchronization.

Desynchronization is often observed preceding seizures or during their early stages; in contrast, high levels of synchronization observed towards the end of seizures may facilitate termination. In his talk, Premek Jiruska will discuss cellular and network mechanisms responsible for such complex changes in synchronization.

Vinícius Rosa Cota

Universidade Federal de Minas Gerais – Belo Horizonte, Brasil.

Professor associado I no Departamento de Engenharia Elétrica da Universidade Federal de São João Del-Rei na área de Neuroengenharia.

Round Table 4 - Epilepsy Hijacking Brain Oscillations: a Gradual Corruption from Normal Activity

LECTURE: Periodic and Non-Periodic Deep Brain Stimulation in Epilepsy Models: Effects on Network Synchronization.

Regular high-frequency stimulation (HFS) commonly has anticonvulsant effects and sparse antiepileptogenic properties. On the other hand, HFS is related to acute and long-term increases in excitability related to direct neuronal activation, long-term potentiation, and kindling, raising concerns regarding its safety and jeopardizing in-depth understanding of its mechanisms. In turn, the safer regular low-frequency stimulation (LFS) has a robust antiepileptogenic effect, but its pro- or anticonvulsant effect seems to vary at random among studies.

In his presentation Vinicius Rosa Cota will discuss the development of a protocol of temporally unstructured electrical stimulation applied to the amygdala. In that scenario, his group has shown that nonperiodic stimulation (NPS), a nonstandard form of low frequency stimulation, is capable of suppressing both acute and chronic spontaneous seizures.

Claudio Queiroz

Universidade Federal do Rio Grande do Norte – Natal, Brasil.

Instituto do Cérebro.

Round Table 4 - Epilepsy Hijacking Brain Oscillations: a Gradual Corruption from Normal Activity

LECTURE: The Genesis of an Epileptic Hippocampus.

In rodents, systemic administration of kainic acid (KA) or pilocarpine (PILO) acutely produces long-lasting epileptiform discharges or Status Epilepticus (SE) leading to cell death, synaptic reorganization and spontaneous seizures. In both cases, it has been suggested that granule cells in the dentate gyrus are spared, and reorganization of their synaptic projections are responsible for generating a recurrent excitatory network at the gate of the hippocampus.

Claudio Queiroz will show in his talk the identification of the SE effects on the reorganization of mature and immature granule cells, their morphology and localization after intrahippocampal administration of KA or PILO in mice, using granule cells labeled with conditioned doublecortin-dependent expression of green fluorescent protein (GFP) at the chronic phase.

Local administration of both neurotoxins induced bilateral paroxysmal activity and hilar and CA1 cell death, among other alterations. It will be discussed how KA and PILO models of TLE will lead to different cellular alterations in the hippocampus and how these data need to be interpreted as complementary approaches to the study of epilepsy.

Ashok Shetty

College Station – Texas, USA.

Department of Molecular and Cellular Medicine.

Round Table 5 - Precision Medicine and Technological Challenges in Neuroscience and Epileptology

LECTURE: Intranasal Mesenquimal Stem Cell-Derived A1-Exosomes ease Inflammation, and Prevent Abnormal Neurogenesis and Memory Dysfunction after Status Epilepticus.

In his lecture, Ashok Shetty will demonstrate that intranasal (IN) administration of A1-exosomes alleviates multiple adverse changes that typically emerge after status epilepticus (SE), a medical crisis that presents a high propensity to evolve into chronic hippocampus dysfunction. Specifically, A1-exosome treatment after SE reduced neuron loss and inflammation, maintains normal neurogenesis and preserve cognitive and memory functions.

He will show also significant those results are for clinical application of A1-exosomes for curbing the evolution of SE-induced injury into chronic hippocampus dysfunction. The results also imply that IN administration of A1-exosomes is therapeutic for other neurological conditions that present with significant neuroinflammation.

Mark O. Cunningham

University of Dublin – Dublin, Ireland.

Senior Lecturer in Neuronal Dynamics.

Round Table 5 - Precision Medicine and Technological Challenges in Neuroscience and Epileptology

LECTURE: Human Brain Slices for Epilepsy Research: Pitfalls, Solutions and Future Challenges.

Mark Cunningham’s presentation will highlight the use of human neocortical tissue, the in vitro brain slice characterized by electrophysiological studies.

The approach permits experimentalists to determine the ability of neocortical microcircuits to generate pathological electrical activity as the human neocortical tissue is, in the majority of cases, removed during elective neurosurgery for the treatment of drug-refractory epilepsy.

This presentation will highlight recent work concerning the mechanistic features of electrophysiological signatures only observed previously with in vivo recordings in epileptic human patients.

Patrick Forcelli

Georgetown University School of Medicine – Washington, DC, USA.

Department of Pharmacology & Physiology.

Round Table 5 - Precision Medicine and Technological Challenges in Neuroscience and Epileptology

LECTURE: Complementary MRI/DTI and Chemogenetics/Optogenetics to Pinpoint Epileptogenic Networks in Experimental Models of Epilepsy.

Epilepsy is the quintessential circuit disorder, with seizure activity propagating through anatomically constrained pathways. These pathways, necessary for normal sensory, motor, and cognitive function, are hijacked during seizures. Understanding the network architecture at the level of both local microcircuits and distributed macrocircuits may provide new therapeutic avenues for the treatment of epilepsy.

In his lecture, Patrick Forcelli will discuss examples of the application of optogenetics/chemogenetics and MRI/DTI to epilepsy research and a critical discussion will be raised on the comparative strengths and weaknesses of these approaches, the importance of microcircuit–macrocircuit interactions in seizure mapping.

Moreover, he will address how these approaches allow testing causality within specific seizure pathways for both preclinical and translational applications, and in other areas of neuroscience.

George Richerson

University of Iowa – Iowa City, USA.

Department of Neurology.

Round Table 5 - Precision Medicine and Technological Challenges in Neuroscience and Epileptology

LECTURE: Networks for Tonic Respiratory Drive, Chemoreception and SUDEP in Epilepsies.

Patients with refractory epilepsy have a high risk of sudden unexpected death (SUDEP). In recent years it has been discovered that many cases of SUDEP are due to respiratory arrest induced by convulsive seizures, and that the amygdala plays an important role as a pathway for forebrain activity to invade the lower brainstem.

Serotonergic mechanisms also contribute to the process, either by influencing the seizure severity or respiratory drive. It is also now clear that some cases of sudden infant death syndrome (SIDS) share pathophysiological mechanisms with SUDEP.

In this talk, data from animal models and human studies will be described that have helped explain the mechanisms and pathways involved in SUDEP and SIDS, how this knowledge is leading to identification of biomarkers of those at highest risk, and the potential for treatments and strategies to prevent these deaths.

Victor Rodrigues Santos

Universidade Federal de Minas Gerais – Belo Horizonte, Brasil.

Universidade Federal de Minas Gerais.

Round Table 5 - Precision Medicine and Technological Challenges in Neuroscience and Epileptology

LECTURE: Molecular and Neuroanatomical Neuroplasticity in Neurogenetic Niches in Experimental Epilepsy Models.

Hippocampal neurogenesis continues throughout life in mammals – including humans. During the development of temporal lobe epilepsy (TLE), newly-generated hippocampal granule cells integrate abnormally into the brain. Abnormalities include abnormal proliferation, differentiation, ectopic localization of newborn cells, formation of abnormal basal dendrites, disruptions of the apical dendritic tree and mossy fiber sprouting.

Changes in granule cell position, dendritic and axonal structure alter the types of inputs and the circuitry of dentate gyrus. These abnormalities are hypothesized to contribute to the development of epilepsy, and may underlie cognitive disorders associated with the disease as well. One pathway candidate to modulate these abnormalities is the PTEN/AKT/mTOR pathway. Hyperactivation of the mTOR pathway is associated with numerous neurological diseases, including autism and epilepsy.

In his talk Victor Rodrigues Santos willdemonstrate that deletion of the mTOR negative regulator, PTEN, from a subset of hippocampal dentate granule impairs dendritic patterning, increases excitatory input and decreases inhibitory input.

Olagide Wagner Castro

Universidade Federal de Alagoas – Maceió, Brasil.

Laboratório de Farmacologia e Fisiologia Integrativa.

Round Table 5 - Precision Medicine and Technological Challenges in Neuroscience and Epileptology

LECTURE: Experimental and Ethnopharmacological Characterization of Substances with Anti-Epileptogenic and Neuroprotective Actions.

The hippocampus is one of the most susceptible regions in the brain to be distraught with Status Epilepticus (SE) induced injury. SE can occur from multiple causes and is more frequent in children and the elderly population. Administration of a combination of antiepileptic drugs (AED) can abolish acute seizures in most instances of SE but cannot prevent the morbidity typically seen after SE such as cognitive and memory impairments and spontaneous recurrent seizures (SRS).

This is primarily due to the inefficiency of AED to modify the evolution of SE-induced initial precipitating injury into epileptogenic changes followed by SRS, cognitive and memory impairments, associated with chronic inflammation, significantly waned neurogenesis and abnormal synaptic reorganization. Thus, alternative approaches that are efficient not only for curtailing SE-induced initial brain injury and its complex neuroplastic phenomena, but also for restraining the progression of SE into a chronic epileptic state are needed.

Olagide W. Castro will introduce three emerging therapies for preventing or easing SE-induced neurodegeneration, neuroinflammation, cognitive and memory impairments. Specifically, therapeutic efficacy of the natural products Resveratroland Citrus limon essential oil, and the effect of reduction in glucose metabolism restricted to the hippocampus, by inhibition of Na+/glucosecotransporters( SGLTs) using intrahippocampal administration of phlorizin PZN before pilocarpine-induced SE will be discussed.

Edson Amaro Júnior

Hospital Israelita Albert Einstein – São Paulo, Brasil.

IIEPAE · Instituto do Cérebro

Round Table 6 - Epilepsies and Comorbidities: Challenging and Integrating Multiples Levels of Complexity

LECTURE: Non-invasive Imaging in Epilepsy using Biomarkers.

Several approaches for the use of contrast agents in magnetic resonance imaging (MRI) to label cells have been developed for experimental and clinical applications. The development of methods for in vivo cell detection and the differentiation of cells labeled with a contrast agent that is composed of magnetic nanoparticles (MNPs) that are based on iron oxide have made MRI an essential technique in the monitoring of cells labeled with implanted MNPs.

The MRI technique is advantageous because it is non-invasive and does not cause ionizing radiation damage, making it ideal for studies of cell labeling by analyzing the characteristic relaxation times, i.e., the longitudinal relaxation time (T1) and the transverse relaxation time (T2). Edson Amaro Júnior’s presentation will highlight the use of biomarkers in MRI for epilepsy.

Antônio Carlos Roque da Silva Filho

Universidade de São Paulo – Ribeirão Preto, Brasil.

Departamento de Física Médica, FFCLRP.

Round Table 6 - Epilepsies and Comorbidities: Challenging and Integrating Multiples Levels of Complexity

LECTURE: Computational Neuroscience and the Epilepsies.

Temporal lobe epilepsy (TLE) provokes a number of different morphological alterations in granule cells of the hippocampus dentate gyrus. These alterations may be associated with the hyperactivity and hypersynchrony found in the epileptic dentate gyrus, and its study requires the use of different kinds of approaches including computational modeling.

Conductance-based models of both normal and epilepsy-induced morphologically altered granule cells have been used in the construction of network models of dentate gyrus to study the effects of these alterations on epilepsy.

In his presentation, Antonio Carlos Roque will review these models and discuss their contributions to the understanding of the association between alterations in neuronal morphology and epilepsy.

Alan Talevi

Universidad Nacional de la Plata – La Plata, Argentina.

Professor (Associate)

Round Table 6 - Epilepsies and Comorbidities: Challenging and Integrating Multiples Levels of Complexity

LECTURE: Complex Systems Approach for Innovation in AEDs Discovery.

The term drug design describes the search of novel compounds with biological activity, on a systematic basis. In its most common form, it involves modification of a known active scaffold or linking known active scaffolds, although de novo drug design (i.e., from scratch) is also possible.

In his presentation, Alan Talevi will discuss the recent applications of modern drug discovery technologies in the field of epilepsy, including high-throughput screening, in silico screening and computer-aided drug design. The suitability and perspectives of each approach will be discussed, depending on the context, along with the technology and translational gaps influencing them.

Alberto Lazarowski

Universidad de Buenos Aires – Buenos Aires, Argentina.

Physiology/Neuroscience & Behavioral Sciences Departments. Ribeirão Preto School of Medicine.

Round Table 6 - Epilepsies and Comorbidities: Challenging and Integrating Multiples Levels of Complexity

LECTURE: Role of P-Glycoprotein in Pharmacoresistant Epilepsy and SUDEP.

Brain overexpression of P-glycoprotein (P-gp) is considered one of the most relevant factors related with refractory epilepsy (RE), the epileptic condition with higher risk of SUDEP. Neurons and cardiomyocytes, normally do not express P-gp, however, those cells can express it by a wide spectrum of damage signals including stress, inflammation, hypoxia or glutamate-dependent neuronal excitotoxicity (convulsive stress).

Dr. Alberto Lazarowski will show that because P-gp activity can modify the resting membrane potential producing depolarization with values from −70 to −10 mV, P-gp can also alter the normal physiological function of neurons and/or cardiomyocytes. Under repetitive severe seizures, as classically observed in RE, an increased neuronal P-gp expression could reduce the convulsive threshold, facilitating the recurrence of new and more severe seizures or Status Epilepticus.

Similarly, quite severe convulsive stress will induce a hypoxic-ischemic heart, with a HIF-1a-dependent P-gp ovexpression in cardiomyocytes, with a concomitant depolarization, evidenced by low heart retention of 99mTc-SESTAMIBI, malignant bradycardia,which ends in fatal acute heart deficiency.

All these mechanisms, could explain the potential role of P-gp overexpression in the development of SUDEP in RE.

Michele Simonato

University of Ferrara – Ferrara, Italy.

Department of Medical Sciences.

Round Table 6 - Epilepsies and Comorbidities: Challenging and Integrating Multiples Levels of Complexity

LECTURE: MicroRNAs, Gene Networks, Gene Therapy: Promises and Challenges for Treating Epilepsies and their Comorbidities.

Gene therapy has been employed to pursue anti-epileptogenic and anti-seizure effects. Michele Simonato will show how anti-epileptogenic effects have been obtained supplementing the neurotrophic factors FGF-2 and BDNF during l, the period between the Status Epilepticus (insult) and the beginning of spontaneous recurrent seizures in experimental models of epilepsy (latency), by means of a Herpes-based vector.

Anti-seizure effects may be obtained increasing the strength of inhibitory signals or reducing it on excitatory signals. The most promising results have been obtained with the neuropeptides galanin and neuropeptide Y (NPY). In particular, NPY expressing AAV vectors produced robust anti-seizure effects but not overt side effects, supporting their application in human epilepsy.

TO ALL AUTHORS OF ABSTRACTS

TO ALL AUTHORS OF ABSTRACTS

International Symposium NEWroscience 2018

Looking for Epilepsy Cure along Centuries and across Cultures

From Demons and Blessings to Brain Complex Networks and DNA Editing

It is with great pleasure and satisfaction that we confirm 58 Scientific Neuroscience and Epilepsy Abstracts and 7 Neuroscience & Arts Abstracts submitted for evaluation by three referees each, to be presented as Posters at our International Symposium NEWroscience 2018. “Looking for Epilepsy Cure along Centuries and across Cultures. From Demons and Blessings to Brain Complex Networks and DNA Editing”

Although you have received already the acceptance, most of the Abstracts were sent back to the authors for format adaptation, language corrections or improvement of their contents. We have accepted the Abstracts for presentation as Posters at the NEWroscience 2018 and that gives you the bonus of having them published adequately numbered and identified, in our electronic NEWroscience 2018 Annals. However, because of strict Editorial regulations coupled to the high standards of Epilepsy & Behavior, all the Abstracts need to pass throughout additional editorial monitoring. Those Abstracts that in the opinion of the Editors of Epilepsy & Behavior do not fill or attend the quality of the Journal will not be published automatically on the Special Issue of the Journal. Please, remember that the Abstracts will be companions of the Reviews written by the Invited Speakers of our event and every text included in the Special Issue will be reviewed by the Guest-Editor and the Editor-in-Chief of Epilepsy & Behavior. That decision will be final, which means that there will be not additional appeal.

With kind appreciation for your understanding I look forward to see you all in our International Symposium NEWroscience 2018.

Norberto Garcia-Cairasco
President of the Organizing Committee

NEUROSCIENCE AND EPILEPSY ABSTRACTS

International Symposium

NEWroscience 2018

“Looking for Epilepsy Cure along Centuries and across Cultures.

From Demons and Blessings to Brain Complex Networks and DNA Editing”

Neuroscience Abstracts And Epilepsy Abstracts

001 - Genetic basis of neurosurgically resected hemimegalencephaly and epilepsy.

Garcia, C.A.B.1; Silva Jr, W.A.3; Gleeson, J.G.2; Machado, H.R.1

1 - Department of Surgery and Anatomy, School of Medicine of Ribeirão Preto, University of São Paulo, Brazil

2 - Laboratory for Pediatric Brain Disease, Howard Hughes Medical Institute, Department of Neurosciences, University of California, San Diego, La Jolla, California, USA.

3 - Center for Medical Genomics at University Hospital of the Ribeirão Preto Medical School-USP, SP, Brazil

Rationale: Hemimegalencephaly (HME) is a clinically devastating malformation of cortical development in children and is considered the most common cause of refractory epilepsy. Some patients with HME do not achieve a satisfactory index in seizures control, even after all types of treatment. In this context, genetic and molecular analysis is an important tool to aid in the understanding of molecular and cellular pathogenesis and future viability therapeutic alternatives. Thus, the aim of this study is to identify the specific HME genetic alterations in the mTOR pathway. Methods: Ten patients diagnosed with HME were selected, with an indication for hemispherectomy surgery in the Neuropediatrics outpatient clinic of the Clinical Hospital of the Medical School of Ribeirão Preto - USP. All those whose legal guardian and/or child did not agree to sign the informed consent form and those whose histopathological diagnosis was different from HME were excluded from the study. The samples were obtained from the biopsy of affected brain tissue, previously identified by preoperative image, during the proposed surgical procedure, and with the aid of a neuronavigator. The biopsied tissue was then fractionated into two parts. In the first, brain portions were coronally cut into 5-μm-thick sections, mounted on histological slides and were sent for anatomopathological analysis. They were prepared on microscopic slides and stained with hematoxylin-eosin (H&E), in order to evaluate general cellular architecture patterns, distribution of structures, and cellular density. The second part of the brain sample was sent for further analysis by mean of exome sequencing. The whole exome capture was performed with the Sure Select All Exon Target Enrichment System (Agilent Human All Exon 50 Mb kit). Libraries underwent paired-end sequencing (2 × 100 bp) on an Illumina HiSeq 2000 instrument according to manufacturer’s protocol. For each case sample, >90% of the exome was covered at >30×. The selection of 50 most expressed genes, with several types of mutations, and their impact on the phenotype were also performed in these patients. The protocol for this study received prior approval from the Research Ethics Committee of the HCFMRP / USP and all patients signed the informed consent form. As samples were sent to the Gleeson Laboratory for Pediatric Brain Disease, University of California at San Diego, CA, the study was also approved by CONEP (# 6978/2015).

Results: The qualitative data analysis of histological sections of 2 patients show extensive areas of neuronal loss and clusters resulting in architectural distortion, mainly of the laminar distribution of the neocortex compared with histological analysis of healthy brain tissue. Additionally, in 2 cases there were rare foci of giant neurons, with large nuclei and wide cytoplasm. In some areas, the cortex presents reduction of the number of layers (four layers), with fusion of the gyri. This study identified somatic mutations in the PIK3CA gene in two cases (details in methods section), which is considered one of the genetic causes for patients diagnosed with HME. For case 1, with a rate of 22%, c.1258T> C (p.C420R) and case 2, with a rate of 11%, c.1624G> A (p.E542K).

Discussion/Conclusions: This study suggests mutations in the PIK3CA gene are associated with increased mTOR signaling in affected brain regions. These results may contribute to the identification of the genes associated with HME and thus to understand the mechanisms involved with the evolution of the pathology and to the discovery of new therapies for epileptic seizures caused by these developmental lesions, with modulators for the mTOR pathway.

Acknowledgements/Financial Support: Howard Hughes Medical Institute at San Diego, Center for Medical Genomics at University Hospital of the Ribeirão Preto Medical School.

002 - A probing approach to assess pre-ictal excitability and synchrony in a computational model of the Hippocampus

Carvalho, V.R.1,3; Moraes, M.F.D.1,2,3; Mendes, E.M.A.M.2,3

1 - Núcleo de Neurociências (NNC), Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas – Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brasil.

2 - Centro de Tecnologia e Pesquisa em Magneto Ressonância, Programa de Pós-Graduação em Engenharia Elétrica - Universidade Federal de Minas Gerais, Belo Horizonte, Brasil

3 - Programa de Pós-Graduação em Engenharia Elétrica - Escola de Engenharia - Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brasil.

Rationale: We believe the study of transitions from inter-ictal to ictal states would benefit from the use of probing approaches. Neuronal systems could be stimulated to elicit responses that would highlight intrinsic features involved in state transitions, providing information which would be difficult to obtain with the passive analysis of electroencephalographic data. This study aims to evaluate the probing approach in an established computational neuronal model. It is hypothesized that, since the model parameters are shifted towards seizure states, stimuli would reveal hyperexcitability and synchrony dynamics which would be otherwise unobtainable.

Methods: Stimuli were applied as inputs to Wendling’s lumped-parameter model of the hippocampal CA1 circuit. This model consists of three interacting sub-populations, each corresponding to excitatory (pyramidal) and inhibitory (dendritic and soma-projecting, representing oriens-lacunosum moleculare and basket cells, respectively) neurons. Stimuli consisted of monophasic pulses to the excitatory population, applied every 2 seconds, with 10 ms duration. In order to assess synchrony, two model subsets were coupled through fast-somatic inhibition (FSI, reflecting basket cells) populations, with coupling strength K. Using Python 2.7, simulations were done with the model in “normal” state, but shifting parameters towards the ictal state. These were synaptic gains (increasing A = excitatory, decreasing B = slow dendritic inhibition) and coupling factor K, which can elicit seizures in specific parameter configurations. Features were extracted from post-stimulus epochs (400 ms) of the mean simulated local field potential (LFP); statistical moments (Variance, Skewness, Kurtosis) and Hjorth Parameters. Phase locking value (PLV), a synchrony measure, was also calculated between the LFPs of the two coupled population subsets. Evaluation of probing efficiency in highlighting feature changes (or trends) before seizure states was done by fitting a 2nd order model to each normalized (z-score) feature series and comparing the resulting residuals and coefficients.

Results: When decreasing B (slow dendric inhibition synaptic gain), increases in signal variance and Hjorth Complexity, as well as decreases in skewness, kurtosis and Hjorth mobility were observed, only when probing stimuli were employed. Respective model fit residuals were (17.1, 12.3, 18.8, 18.8, 14.4) for control (without probing) and (6.0, 1.2, 1.2, 0.33, 1.0). Changes were also visible in synchrony between model subsets with fixed coupling; when decreasing B, PLV increased, with model fit residuals equal to 15.8 for control and 1.9 with stimuli. Similar results were found when increasing A; as a seizure approaches, higher values of variance and lower values of Hjorth mobility, skewness and kurtosis are observed with probing, with respective residuals (0.29, 16.0, 18.2, 18.0) for control and (0.57, 2.82, 6.70, 5.53) for probing. In this case however, the increase in variance can be also observed without stimuli, albeit more subtle. Synchrony was also increased, only when probing was used (residuals 15.7 and 2.26). Increasing the coupling factor K yielded similar changes in univariate features, but not in synchrony, which presented an irregular, slight increasing trend. Control and probing fit residuals were, respectively: variance (14.0, 2.57), Hjorth Mobility (1.26, 0.83), skewness (13.27, 0.80), kurtosis (19.9, 0.92) and PLV (19.1, 15.1).

Discussion/Conclusions: The results show marked increases in excitability and synchrony measures, traditional hallmarks related to ictogenesis. Except on the case of increasing A, these changes were visible only when stimuli were used, supporting the predictive value of probing. Furthermore, the decrease in Skewness, Kurtosis and Hjorth mobility can be interpreted as an increase in the order of the simulated electroencephalographic signal, which is in accordance with findings in the literature. These results motivate further studies with probing approaches in conjunction with seizure prediction methods.

Acknowledgements/Financial Support: This work was carried out with the financial support of CAPES - Brazil. We also thank CNPq and FAPEMIG for developing and supporting research.

003 - Differential effects of Angiotensin II and Angiotensin (1-7) on GFAP and GLAST expression in hippocampal astrocytes.

Pereira, M.G.A.G.1; Donato, L.F.1; Souza, M.M.2; Ruginsk, S.G.2

1 - Department of Biochemistry; Biomedical Sciences Institute, Federal University of Alfenas, Alfenas, Minas Gerais, Brazil.

2 - Department of Physiological Sciences; Biomedical Sciences Institute, Federal University of Alfenas, Alfenas, Minas Gerais, Brazil.

Rationale: Called “cement” by Virchow in the middle of the 19th century, astrocytes are non-neuronal cells that have a crucial role in the central nervous system, including structural and metabolic support, transmitter reuptake and release, modulation of synaptic transmission, etc. The Renin Angiotensin System (RAS) described initially as a hormonal system that regulates the arterial pressure, nowadays it is related to regulation of local physiological process and pathogenesis of numerous diseases. Thus, the central focus of the present study was to clarify by which mechanisms the peptides of the RAS would act to modulate astrocytic function in the hippocampus, an important region of the limbic system that participates in the formation of memories and emotions, drastically affected by several neurological disorders.

Methods: We performed a primary astrocyte culture with cells isolated from the hippocampus of neonatal male Wistar rats (P02). Tissue fragments were then dissociated and cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin for a period of 7-9 days under controlled atmosphere (37ºC, 95% of O2 and 5% of CO2). After this period, astrocytes were separated by mechanical agitation (200 rpm, 2 h at 37°C) and cultured for an additional period of 2 days in 12-well plates. Cells were incubated with Angiotensin II (1 nM) or Angiotensin-(1-7) (1 nM), in the presence of an AT1 (Losartan, 10 μM) or a Mas (A779, 10 μM) receptor antagonist, respectively. Next, cells were fixed and processed for glial fibrillary acidic protein (GFAP) or Glutamate transporter (GLAST) fluorescent staining. Protocol approved by Local Ethics Committee on the Use of Animals (n° 585/2014). The results were expressed as means ± mean standard errors (MSE) and analyzed by One-Way Analysis of Variance, followed by Bonferroni’s post-test (Graph-Pad Prism®).

Results: Ang-(1-7) promoted a highly significant increase in GFAP expression (3 times, p<0.001), which was completely inhibited by the incubation with A779. Conversely, AngII reduced GFAP expression (4 times, p<0.01), being this effect partially reversed by Losartan. Regarding glutamate balance, Ang-(1-7) increased GLAST expression (2 times, p<0.001), an effect partially reversed by A779 and not observed in response to Ang II incubation.

Discussion/Conclusions: We conclude that Ang-(1-7) promotes an increase in both GFAP expression in a mas receptor-dependent pathway, and GLAST expression. On the other hand, AngII decreased GFAP expression in an AT1 receptor dependent pathway. Further studies could evaluate the association of the current data with the known neuroprotective activity of Ang (1-7).

Acknowledgements/Financial Support: UNIFAL-MG, CNPq, FAPEMIG, FAPESP.

004 - The role of the Dorsal Raphe Nucleus’ serotoninergic system on behavioural consequences induced by ethanol withdrawal

Tarrega, J.P.M.¹; Cardoso, R.C.2; Padovan, C.M.1

1 - Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto - Universidade de São Paulo

2 - Faculdade de Medicina de Ribeirão Preto - Universidade de São Paulo

Rationale: Even with its wide consumption and influence on socio-economic aspects of most modern western societies, ethanol’s neurobiological mechanism of action is not fully known. Data from the literature and our laboratory suggests that serotonin (5-HT) is involved in the modulation of the behavioural consequences of ethanol withdrawal. However, the brain sites involved in such effects remain to be elucidated. The dorsal raphe nucleus (DRN) is activated during ethanol withdrawal and several data from the literature point to its role on anxiety. Therefore, the aim of this study was to investigate the role of 5-HT mediated-neurotransmission within the DRN in the anxiety and depression-like behavioral responses induced by ethanol withdrawal.

Methods: The study consisted of two experiments: (1) Male Wistar rats (7 weeks old; Ethical Committee Protocol 15.1.1351.59.8) with cannulas aimed to the DRN received three different doses (0.1, 1 and 10 ηmol/0.2 μL) of the selective 5-HT reuptake inhibitor Fluoxetine (FLX) five minutes before testing and were submitted to the Elevated Plus Maze (EPM) test in order to map the dose-response relationship of the drug. (2) Another group of male Wistar rats underwent a semi-voluntary ethanol consumption protocol, with free access to an ethanol solution (EtOH; 6% v/v) during three weeks. Then, they were submitted to 48 h of abstinence and tested in the EPM. Intra-DRN treatment with FLX (0.1 and 1 ηmol/0.2 μL) or Saline (SAL; 0.2 μL) occurred five minutes before test in the EPM. Frequency of entrances and time spent in the EPM arms were recorded and analyzed (ANOVA test, significance value: 0.05).

Results: None of the of FLX doses used significantly changed the behaviour of rats regarding the frequency of entrances in the closed arm (FC; F3,47 = 1.336; p >0.05; One-Way ANOVA) and percentage of entries in the open arms (PFO; F3,47 = 2.043; p >0.05). The percentage of time spent in the open arms (PTO; F3,47 = 2.938; p <0.05) had significant difference between treatments. In the second experiment, intra-DRN treatment did not alter significantly the test parameters (FC: F6,89 = 1.421; p > 0.05; PFO: F6,89 = 1,582, p > 0.05; PTO: F6,89 = 0.905, p > 0.05; Two-Way ANOVA).

Discussion/Conclusions: Our data show that intra-DRN FLX did not attenuate the abstinence-induced decrease in EPM exploration, thus suggesting that the serotonergic system of the DRN is not involved in the anxiety-like responses induced by ethanol withdrawal.

Acknowledgements/Financial Support: Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq.

005 - Effects of Verapamil on GFAP expression and aspartate uptake in hypothalamic astrocytes challenged with an osmotic stimulus

SOUZA, M. M.1; RUGINSK, S. G2.

1 - Master’s student of the Graduate Program of Biosciences Applied to Health - UNIFAL-MG

2 - Assistant Professor, Department of Physiological Sciences, Institute of Biomedical Sciences - UNIFAL-MG

Rationale: Recent evidence shows that astrocytes are essential elements for the adaptive responses induced by osmotic challenges, actively participating in neurotransmission. However, the precise mechanisms involved in the transduction of the osmotic signal in astrocytes are still uncertain. In neurons, this transduction is mediated by the opening or closing of ion channels, including the L-type Ca2+ channels (LTCC), also expressed by astrocytes. Therefore, the present study investigated the participation of LTCCs in the transduction of the osmotic signal in cultured hypothalamic astrocytes, performing the indirect assessment of cell morphology through the expression of glial fibrillary acid protein (GFAP), as well as the evaluation of the uptake of tritiated aspartate (3H-aspartate).

Methods: For this purpose, male Wistar rats (2 days-old) were euthanized for removal of the medial basal hypothalamus, which was dissociated with 0.05% trypsin. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM containing fetal bovine serum (10%) and 1% penicillin-streptomycin for a period of approximately 10-15 days. The astrocytes were then separated and cultured in 12-well and 6-well plates (1 x 104 and 1 x 105 cells/well, respectively) containing poly-D-lysine coverslips for approximately 4 days. On the day of the immunofluorescence experiment, each well was incubated with DMEM (109 mM NaCl) containing or not verapamil (10 μM, LTCC selective inhibitor) for 30 minutes. After preincubation, DMEM containing or not verapamil was maintained in some wells (designated controls) and in the others was replaced by DMEM containing high sodium (132 mM NaCl), containing or not the specific inhibitor, for an additional period of 2 hours. Subsequently, the cells were fixed and subjected to the immunofluorescence protocol for GFAP labeling. On the day of the 3H- aspartate uptake assay, the experimental design was the same as described above. However, after 15 minutes of stimulation, a volume of 10 μL (0.1 μCi) of 3H-aspartate was added to each well for another 15 minutes. A volume of 500 μL of medium was recovered into vials containing scintillation liquid. Adhered cells were lysed with deionized water and 500 μL were collected into flasks containing scintillation liquid. Radiation was estimated on a β-counter. All procedures were previously approved by the Animal Research Ethics Committee (Protocol 585/2014).

Results: Incubation with hypertonic DMEM significantly reduced GFAP expression in hypothalamic astrocytes (9308.35 ± 1608.23 versus 5737.35 ± 527.71 µm2, p < 0.05). The similar effect of reducing GFAP expression was observed at baseline conditions in response to incubation with verapamil (9308.35 ± 1608.23 versus 5715.49 ± 629.66 µm2, p < 0.01). The uptake of 3H-aspartate reduced significantly in astrocytes incubated with hypertonic DMEM (p <0.01). Preincubation with verapamil did not change the 3H-aspartate uptake response, neither under basal nor hypertonic conditions.

Discussion/Conclusions: LTCCs play an important role in astrocyte activity, since incubation with verapamil alone was able to alter the expression of GFAP, a protein of the astrocytic cytoskeleton associated with morphological changes of these cells. Similarly, hypertonic conditions lead to an altered glutamate balance in these cells, apparently not by LTCCs.

Acknowledgements/Financial Support: Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Federal University of Alfenas (UNIFAL-MG).

006 - Evaluation of Nitrous Oxide acute treatment in the learned helplessness model.

Dias, I. C. S.1*, Rossignoli, M.T.1, Abrao, J.2, Hallak, J. E. C1.

1 - Dept. Neuroscience and Behavioral Neuroscience, Ribeirão Preto School of Medicine, University of São Paulo, SP-Brazil.

2 - Dept. of Biomechanics, Medicine and Rehabilitation of Locomotor System.

Rationale: NMDA antagonists have demonstrated a rapid and long-lasting antidepressant effect. Considering the search for new and more effective antidepressants, we propose to study the effect of nitrous oxide (N2O), an NMDA antagonist, in the learned helplessness model of depression.

Methods: Adult male Wistar rats (250-350 g) were subjected to a four-days protocol of learned helplessness. On the first day were applied 60 cycles of 10 seconds randomized shocks (0.8 mA), preceded by light as the conditioned stimulus, in a gray vanilla aroma shuttle box (inescapable shock group-IS). On days 2, 3 and 7, all rats were submitted to the test itself (test 1, 2 and 3 respectively). Then, 30 cycles of shock (0.6 mA) were applied, being each cycle preceded by sound (75 dB) as conditioned stimulus, in the shuttle box but with striped walls and lemon aroma. The IS group was divided between animals that inhaled oxygen (IS-O2) and those who inhaled N2O (IS-N2O), during half an hour both of them. The treatments occurred on the third day to evaluate acute effects (test 2) and late effects on day 7 (test 3). During the tests, we measured the number of failures, when animals did not escape the shock, as well the latency time to realize the flight, when they escaped. Statistical analyses were performed using two-way ANOVA-repeated measures (Two-way ANOVA RM), followed by Bonferroni post-hoc tests (Bonferroni test). Statistical significance defined as p<0.05. The protocol was approved by the Ethical Committee on Animal Research (126/2017).

Results: Preliminary data show that in the test 2 (acute effect) and test 3 (late effect) there was no difference between latency time in IS-N2O (n=4) and IS-O2 (n=10) group (Two-way ANOVA RM, F(2,16)=26.9, IS vs IS-N2O, Bonferroni test, p>0.05). Similarly, the failures amounts between these groups were also not different (Two-way ANOVA RM, F(2,16)=39.8, IS vs IS-N2O, Bonferroni test, p>0.05). There are ongoing experiments to increase animals number in IS-N2O group, as well to test subchronic treatment of the drug in another group of subjects.

Discussion/Conclusions: Acute nitrous oxide treatment did not reverse acute and late behavioral deficits occurred in learned helplessness.

Acknowledgements/Financial Support: CAPES, CNPq and FAPESP.

007 - Cancer survivorship and neuroinflammation: effects of tumor experience on sickness behavior after an immune challenge

Santos, J.C. 1, Jordan, K.2,3, Bever, S.R.3,4, Pereira-da-Silva, G.1,5, Pyter, L.M.2,4,6

1 - Department of Basic and Applied Immunology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil

2 - Department of Neuroscience, Ohio State University, Columbus, OH, USA

3 - Institute for Behavioral Medicine Research, Ohio State University Wexner Medical Center, Columbus, OH, USA

4 - Departments of Psychiatry and Behavioral Health, Ohio State University, Columbus, OH, USA

5 - Postgraduate Program in Public Health Nursing, Ribeirão Preto College of Nursing, University of São Paulo, Ribeirão Preto, SP, Brazil

6 - Behavioral Neuroendocrinology Group, Ohio State University, Columbus, OH, USA

Rationale: Cancer survivors report several physical and mental health issues, even after a successful antitumor treatment, which increase the psychosocial burden and reduce quality of life. Inflammatory responses are also altered in cancer survivors and have been linked to behavioral comorbidities (e.g., fatigue). Indeed, rodent tumor models indicate that tumors by themselves are sufficient to alter neuroinflammatory responses necessary for sickness behaviors, after an immune challenge, but these immune and behavioral changes are poorly understood for survivors after tumor resection. In this study, we tested the extent to which tumors and tumor resection alters neuroinflammation and resulting sickness behaviors (lethargy, fever, anorexia) following an acute immune challenge (i.p. lipopolysaccharide [LPS] injection).

Methods: Tumors were induced in female 8- to 9-weeks old Balb/c mice, using 5 x 106 murine non-metastatic mammary cancer 67NR cells injected into the 4th mammary fat pad. Some tumors were resected after two and a half weeks (“tumor-resected”) and some tumors remained intact (“tumor”); surgical controls received sham surgery. Two and a half weeks after tumor treatments, some surgical controls (n=10) received an i.p. injection of vehicle, whereas the rest of the mice (control, tumor and tumor-resected mice; n=7-20/ group) received an LPS injection (E. coli, 026:B6, 250 μg/kg, i.p). Food intake, body mass, body temperature and locomotion were measured in all groups 24 h before LPS and either 4 or 24 h after injection. Telemetry transmitters implanted intraperitoneally were used to assess locomotion (i.e., lethargy) and body temperature (i.e., fever) responses.

Results: The mammary tumors decreased Tnfα and Il-6 gene expression levels in the frontal cortex, hippocampus, and hypothalamus, while tumor resection mostly reversed the neuroinflammatory responses (p<0.05 by 2-way ANOVA). Tumors decreased the LPS-induced lethargy (p<0.05 by 2-way ANOVA and RM-ANOVA) and hypothermia/fever over time during the 24 h after LPS injection compared to LPS treated surgical control (by 2-way ANOVA and RM-ANOVA; p<0.05 in all cases). Tumor-resected mice exhibited an intermediate response for body temperature, showing an incomplete recovery after LPS injection, since the fever response observed in the surgical control mice between 2 and 8 h after LPS injection was absent in tumor-resected mice (p<0.05 by 2-way ANOVA in all cases). LPS treatment significantly reduced body mass and food intake (anorexia) in all groups compared to control mice treated with vehicle 24 h after the immune challenge (p<0.0001, by 2-way ANOVA in all cases).

Discussion/Conclusions: In contrast to other chronic inflammatory states, our data indicate that some mammary cancers may decrease instead of exacerbating neuroinflammatory responses to subsequent challenges. Decreased neuroinflammatory responses may be maladaptive by influencing the bacterial clearance in live infectious challenges, as well as other types of behavior (e.g. cognition, affect). The tumor-induced changes in neuroinflammatory responses contributed to impaired sickness behavior response (lethargy and the biphasic hypothermic/fever response to the LPS challenge) and persisted even after tumor resection (fever response to the LPS challenge). Taken together, our results indicate that functional changes to neuroinflammatory pathways persist after cancer experience.

Acknowledgements/Financial Support: The authors thank Hsin-Yun Lin, Lindsay Strehle, Jasskiran Kaur, and Shireen Desai for technical assistance, Jen Staten for animal care. CAPES (J.S. - process number 88881.133418/2016-01), Ohio State University Medical Center, NIH grant CA201681 and a CCTS core grant UL1TR001070 (L.P.).

008 - Anxiety-like behavior induced by ethanol withdrawal evolves to depressive-like behavior in Wistar rats: role of serotoninergic system in the dorsal hippocampus and corticosterone signaling

Campos-Cardoso, R1; Carolino, ROG2; Anselmo-Franci, JA2and Padovan, CM1,3

1 - Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, 14040-900, Ribeirão Preto, SP, Brasil;

2 - Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, 14040-902, Ribeirão Preto, SP, Brasil;

3 - Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, 14040-901, Ribeirão Preto, SP, Brasil.

Rationale: Chronic ethanol consumption (CEC) leads to the development of cardiovascular, psychiatric and neurodegenerative diseases. Nevertheless, the abrupt interruption on ethanol consumption leads to symptoms of anxiety. Once abstinence is sustained, anxiety symptoms can evolve to depressive syndromes, which, in turn, can be responsible for relapse and back to ethanol consumption. Data using animal models point to the involvement of serotoninergic system in the consequences of abstinence. Furthermore, corticosterone signalling has a great importance modulating rodent’s response to behavioral tests and ethanol withdrawal. Therefore, we investigated whether Imipramine (IMI) would attenuate the effects induced by abstinence to CEC on behavioral, neurochemical and neuroendocrine parameters.

Methods: Male adult Wistar rats (n=123) had free access to water (WATER) or an ethanol solution (EtOH; 6% v/v) during three weeks. Then, they were submitted to 48 h of abstinence. After that, they were tested in the Elevated Plus Maze (EPM). IMI (15mg/kg) or saline (SAL;1 ml/kg) was given 24, 19 and 1h before EPM test. The number of enclosed arm entries (EAE) and percentage of entries (OAE) and time spent (TOA) in the open arms were recorded. Numbers of rearing and stretched-attempt posture (EAP) were counted to EPM, as a risk-assessment measure. Immediately after tests, half of each group of rats was euthanized, had their blood collected to measure corticosterone levels and dorsal hippocampus was dissected to measure 5-HT levels by HPLC. The other half returned to their home cage and remained undisturbed for three weeks, with free access to water and food, after which they were submitted to the forced swim test (FST). A two-way ANOVA was used to analyse data, with DRINK (EtOH or WATER) and DRUG (IMI or SAL) being the factors considered. Significance was set at p<0.05. All procedures were previously approved by the local Ethical Committee on Animal Use on Research (#15.1.1351.59.8).

Results: IMI decreased EAE in WATER-treated rats (DRUG:F=12.5;p<0.05). No effects of DRINK (F=0.05;p>0.05) nor an interaction DRINKxDRUG (F=3.69;p>0.05) were found on EAE. IMI attenuated the EtOH-induced decrease in OAE (effects (p<0.05) of DRINK (F=9.04), DRUG (F=14.38), and an interaction DRINKxDRUG (F=6.23)). DRUG significantly increased TOA (F=16.3; p<0.05), while DRINK did not (F=0.72; p>0.05). On the other hand, we only found a significant interaction DRINKXDRUG on TOA (F=14.8; p<0.05). IMI also attenuated the abstinence-induced effects on rearing (DRUG:F=11.8;p<0.05; DRINK:F=2.1;p>0.05; DRINKxDRUG:F=0.01;p>0.05) and EAP (DRINK:F=14.6;p<0.05; DRUG:F=56.04;p<0.05 and DRINKxDRUG:F=30,9;p<0,05), thus decreasing these behaviors. We did not observe significant differences on corticosterone levels (DRUG:F=0.81;p>0,05; DRINKxDRUG:F=0.04;p>0,05), however EtOH-animals tended to have a higher corticosterone concentration (F=3.4;p=0,08) when compared to WATER. IMI (DRUG:F=0.087;p>0.05) and EtOH (DRINK:F=1.13;p>0.05) or interaction DRINKXDRUG (F=0.31;p>0.05) did not change 5-HT levels in the dorsal hippocampus of rats. On the FST, IMI attenuated the ethanol-induced increased immobility time (DRINK:F=14.79;p<0.05; DRUG:F=28.89;p<0.05; and interaction DRINKxDRUG:F=5.18;p<0.05), without changing the latency for immobility behavior (EtOH:F=0.06; DRUG:F=0.13; DRINKxDRUG:F=0.03;p>0.05).

Discussion/Conclusions: Our results show that EtOH abstinence induced a decrease on exploratory activity and an increase in risk-assessment behaviors in the EPM. Also, protracted abstinence induced an increased immobility time in FST. These effects were attenuated by subchronic treatment with IMI, thus suggesting the involvement of the serotoninergic and noradrenergic systems. Although significant changes were not found on corticosterone levels, it may influence these behaviours, since there was a tendency of significance.

Acknowledgements/Financial Support: CAPES and CNPq (471917/2014-1).

009 - Role of TRP family receptors in seizures and their consequences

Batista, C.R.A.; Gomes, G.F.; Rosa, W.M.B.; Botelho, G.C.; Ferrari, C.Z.; Vieira, L.B.; Oliveira, A.C.P.

Department of Pharmacology, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.

Rationale: The family of transient potential receptors (TRP) are a group of cation channels involved in different physiological processes, such as sensations related to different stimuli or ion homeostasis. Epilepsy is a neurological disorder, defined by a state of recurrent, spontaneous seizures, affecting millions of people around the world. Epileptogenesis is associated with neuronal loss, mainly in the hippocampus happening in temporal lobe epilepsy (TLE). The latter is the most common type of adult epilepsy and is characterized by seizures that initiate locally and may spread throughout the entire brain. TRPV1 channels are expressed in the dentate gyrus of hippocampus which is critical in epileptogenesis. Furthermore, the increase of calcium entry in hippocampal neurons has been associated to epilepsy’s etiology, motivating the study of the TRP family, whose channels are highly permeable to calcium. Ca2+ influx, via TRPV1 channel, was increased in the hippocampal neurons in pentylenetetrazol-induced epileptic rats. There is still no conclusive evidence showing the involvement of the TRPA1 and the TRPV1 channels in seizures. In this context, we aimed to evaluate the role of these channels in the modulation of seizures and associated events in mice.

Methods: C57Bl/6 animals (10-12 weeks, n= 7-10) received intrahippocampal pilocarpine injection (40 μg), 30 minutes after vehicle (DMSO 1%, tween 80 5%, saline 94%) or TRP receptor modulators. AP18, the TRPA1 blocker, was administered at doses of 1, 3 and 10 mg/Kg, intraperitoneally, and TRPV1 blocker, SB366791 0.1; 1; 10 and 30 μM, for in vitro protocol. The seizures were evaluated for 90 minutes after pilocarpine injection, according to the Racine’s scale of seizures. Neuronal death and synaptosomes preparations were evaluated. This work was approved by the Ethics Committee on Animal Use of UFMG: CEUA 197/2017.

Results: Chi-square statistical analysis revealed a strong association between AP18 and the development of Status Epilepticus (SE) as well as AP18 and tonic-clonic seizures (P<0.0001). Sixty percent of pilocarpine group developed SE. AP18, 1, 3 and 10 mg/Kg, increased the SE to 85%, 75% and 70%, respectively. Twenty percent of the pilocarpine group developed tonic-clonic seizures, while 1, 3 and 10 mg/Kg of AP18 increased the frequency of tonic-clonic seizures to 71%, 63% and 70%, respectively. AP18 (10 mg/Kg) increased the latency for the animals to develop the SE (23 minutes) as compared with pilocarpine group (12 minutes; p<0.05). AP18 (1 mg/Kg) also increased the fluorojadeC staining in comparison with pilocarpine group in CA1 region of hippocampus, indicating an increasing in neurodegeneration. Finally, SB366791 (30 μM) increased more than 15 times the glutamate release as compared to the control group (P<0.0001).

Discussion/Conclusions: To date, our results suggest that AP18 is pro-convulsant and the TRPA1 receptor has an important role in neuroprotection, once TRPA1 blockade increased the susceptibility of mice to seizures and neurodegeneration induced by pilocarpine. Although TRPV1 blockade enhanced the glutamate release induced by KCl, it is not possible to speculate whether this drug would increase seizures and neurodegeneration induced by pilocarpine. Therefore, further studies are necessary to clarify this issue.

Acknowledgements/Financial Support: CNPq, FAPEMIG.

010 - Phase-amplitude coupling between neural oscillations is decreased by non-periodic electrical stimulation in epileptic seizures

Oliveira, J.C.1; Rodrigues,S.M.A.F.1; Maciel,R.M.1; Réboli, L.A.1; Cota, V.R.1,2

1 - Laboratório Interdisciplinar de Neuroengenharia e Neurociências (LINNce) / UFSJ

2 - Departamento de Engenharia Elétrica (DEPEL), Universidade Federal de São João del-Rei (UFSJ)

Rationale: Epilepsy is characterized by a state of permanent neural hyperexcitability. On the other hand, epileptic seizures - behavioral and neurobiological expression of this hyperexcitability - also depend on the hypersynchronous coupling of neural oscillators. With a special focus in the hypersynchronization aspect of the disease, our group has shown that temporally-unstructured electrical stimulation, with random intervals between pulses at a fixed mean frequency of 4 stimuli per second (non-periodic stimulation – NPS) applied to the basolateral amygdala is able to suppress seizures in animal models of epilepsy. One way to measure neural synchronism would be through the interaction of neural oscillations in different frequency bands. In this sense, the aim of this study is to investigate the hypothesis that NPS suppresses seizures by desynchronizing neural oscillators responsible for ictogenesis, and to characterize this phenomenon through modulation index (MI), a technique capable of measuring the coupling between the phase of a low-frequency component and the amplitude of a high-frequency component of electrical brain activity.

Methods: Electrophysiological recordings (band-pass filtered from 0.3 to 300 Hz, and sampling rate at 1000 Hz) from cortex (CX), hippocampus (HP), and thalamus (TH) were performed and NPS (pulses were square waves of 100 μs total duration and current amplitudes varied from 100 to 400 μA) was applied to both amygdala (AMY) in male Wistar rats, weighing 250–320 g, subjected to the pentylenetetrazole (PTZ) continuous infusion model (10 mg/ml/min). Two animal groups were evaluated according to the use or not of NPS until the onset of seizure behaviors: control (CTRL; n= 29) and stimulated (NPS; n = 62) (all methods were previously approved by the Ethics Committee on Animal Research of our university - protocol 31/2014 / CEUA-UFSJ). Threshold of PTZ required to trigger forelimb clonus (FC) or generalized tonic-clonic seizures (GTCS) behaviors were calculated. The MI was calculated from a sliding time windows with duration of 30 s, displaced every 10 s, from the period spanning from the beginning of PTZ infusion until the appearance of the first epileptiform spike in the electrographic recording (period preceding the myoclonic phase).

Results: The PTZ threshold to GTCS was significantly increased in NPS group when compared to control group (p < 0.05, Student t test). The CTRL group had a low MI initially, but as the convulsant insult was delivered and the ictogenic process progressed, the MI increased considerably in the last time windows near the first spike (preceding the onset of epileptiform activity) in CX and HP, mainly for pairs of phase frequency 0-2 Hz and amplitude frequency 8-14 Hz, 14-30 Hz, and 30-60 Hz (p < 0.05, Student t test). In contrast, the NPS group showed a not significant decreasing trend in the MI in the last windows preceding the first spike.

Discussion/Conclusions: The present findings of electrographic recording contributed to confirm the hypothesis that NPS works by desynchronizing neural networks while showing that this temporally unstructured pattern of electrical stimulation is able to induce a substantial decrease of phase-amplitude couplings between particular neural oscillations of frequencies bands of structures involved in ictogenic phenomena.

Acknowledgements/Financial Support: CAPES (post-doctoral fellowship), FAPEMIG and CNPq.

011 - Glucose injection reduces seizure severity and neuronal death after Status Epilepticus

Melo, I.S.1, Santos, Y.M.O.1, Pacheco, A.L.D.1, Silva, S. R.1, Silva, V.O.1, Costa, M.A.1, Cavalcante, C.M.B.1, Freitas-Santos, J1, Oliveira, K. O.1, Duzzioni, M.1, Sabino-Silva, R.1,2, Borbely, A.U.1, Castro, O.W1.

1 - Department of Physiology and Pharmacology, Institute of Biological Sciences and Health, Federal University of Alagoas (UFAL), Maceió/AL.

2 - Institute of Biomedical Science (ICBIM), Federal University of Uberlandia (UFU). Uberlândia/MG.

Rationale: Glucose is the main source of energy for the brain and its absence can cause neuronal dysfunction. During Status Epilepticus (SE), neurons become overexcited, increasing energy consumption. Glucose uptake is increased via the sodium/glucose cotransporter 1 (SGLT1) in the hippocampus under epileptic conditions. In addition, a supply of glucose can prevent neuronal damage and memory consolidation impairments caused by SE. We evaluated the effect of increased glucose availability in memory consolidation alterations, behavior of limbic seizures, neurodegeneration process and SGLT1 expression.

Methods: Experimental procedures were approved by the Ethical Committee for Animal Research of UFAL (04/2016). Male Wistar rats (n=35 [240-340g]) were submitted to stereotaxic surgery for cannula implantation in the hilus of dentate gyrus of hippocampus. Before intrahippocampal microinjections, some animals were trained in an inhibitory avoidance test and were evaluated one day later. Animals received microinjections of vehicle (VEH, saline 0.9%, 1μL) or glucose (GLU, 3mM [diluted in saline]) in hippocampus followed 30 minutes later by pilocarpine (PILO) (1.2 mg/μL), VEH+PILO and GLU+PILO, respectively. In addition, another group (PILO+GLU) received GLU (3mM) after 5 minutes of PILO. Behavioral analysis of seizures was performed for 90 minutes during SE, according to Racine scale (1972). Animals were perfused after 24 hours of SE and neurodegeneration was evaluated by histochemistry of Fluoro-Jade (FJ). FJ positive neurons (FJ+) were counted (ImageJ–NIH) in hippocampus and thalamic nuclei. SGLT1 expression was evaluated by immunofluorescence and quantified by densitometry (ImageJ–NIH). In inhibitory avoidance test, results were expressed as median with interquartile range, compared by the Kruskal-Wallis test; while for limbic seizures, FJ+ neurons and SGLT1 expression, as mean±SEM, compared with unpaired t-test.

Results: The administration of glucose (3 mM) prior to PILO did not prevent the impairment of memory consolidation caused by PILO-induced SE (H27 (2) = 13.21, p<0.05) Furthermore, the same concentration of glucose after PILO reduced the severity of seizures (VEH+PILO, 0.69±0.02; PILO+GLU, 0.57±0.03), as well as the number of limbic seizures classes 3 (VEH+PILO, 1.12±0.10; PILO+GLU, 0.55±0.17), 4 (VEH+PILO, 1.15±0.08; PILO+GLU, 0.58±0.14), and 5 (VEH+PILO, 1.46±0.23; PILO+GLU, 0.57±0.24). Similarly, glucose after SE reduced the number of FJ+ neurons in hippocampus [CA1 (VEH+PILO, 335.7±36.96; PILO+GLU, 51.67±25.18), CA3 (VEH+PILO, 300.9±39.37; PILO+GLU, 82.17±49.04) and hilus (VEH+PILO, 161.3±31.87; PILO+GLU, 25.0±9.41)] and thalamic nuclei [lateral posterior (VEH+PILO, 175.1±68.19; PILO+GLU, 5.33±1.14), centrolateral (VEH+PILO, 134.1±47.57; PILO+GLU, 5.33±1.54) and posterior paraventricular (VEH+PILO, 197.9±44.94; PILO+GLU, 10.00±6.01)]. Finally, SGLT1 expression was elevated in hippocampus [CA1 (VEH+PILO, 1.45±0.24; PILO+GLU, 2.33±0.24), CA3 (VEH+PILO, 1.46±0.27; PILO+GLU, 3.00±0.28) and hilus (VEH+PILO, 1.29±0.24; PILO+GLU, 2.33±0.22)] after increasing glucose levels.

Discussion/Conclusions:These preliminary data suggest that the administration of intrahippocampal glucose protects brain in the earlier stages of epileptogenic processes and increase the expression of SGLT1.

Acknowledgements/Financial Support: Members of the Laboratory of Neuropharmacology and Integrative Physiology of the Department of Physiology and Pharmacology, Institute of Biological Sciences and Health, Federal University of Alagoas (UFAL). FAPEAL, CNPQ, CAPES.

012 - Migration potential and β-Catenin expression analysis in fibroblasts, brain tissue and induced pluripotent stem cells (iPSC) neurodifferentiation in focal cortical dysplasia (FCD) development

Rodrigues, F.V.F.1,3; Marinowic, D.1,2; Zanirati, G.1,2; Azevedo, P.N.1,3; Souza, E.1; Palmini, A.1; Paglioli, E.1; Machado, D.C.1,2; Costa, J.C.1,4

1 - Brain Institute of Rio Grande do Sul (BraIns);

2 - Graduate Program in Medicine and Health Sciences, Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Brazil;

3 - Graduate Program in Medicine, Pediatrics and Child Health, Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Brazil;

4 - School of Medicine, Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Brazil.

Rationale: Focal cortical dysplasia (FCD) is the most frequent malformation in cortical development and is associated with drug refractory epilepsy. The mechanisms involved in the genesis of FCD are poorly described and here we propose that it can be related to clonal somatic mutations that affect signaling pathways in FCD patients. Β-Catenin adhesion protein acts in the intracellular signal WNT pathway. This pathway regulates the formation of complex and organized tissues acting from adhesion to cell migration. Changes in cell migration potential may be related to cortex malformation in FCD genesis.

Methods: In this study we analyzed the migration potential and the expression of β-catenin in iPSC cells during different time points in neurodifferentiation and fibroblasts of patients with FCD. Fibroblasts from two FCD patients (a 45-year-old male and a 12-year-old female) and two healthy individuals (a 41year-old and a 50-year-old females) were obtained from skin biopsy and cultured until the 5th passage. The iPSC generation was performed by exposing fibroblasts to viral and neurodifferentiated vectors (viral vectors containing the genes OCT4, SOX2, KLF4, and c-MYC using the CytoTune®-iPS 2.0 Sendai Reprogramming kit -Thermo Fisher Scientific Massachusetts, EUA) for 35 days. For migration analysis, cells were cultured in inserts with porosity of 8 μm by 24, 48 and 72 hours (fibroblasts) and 3 and 7 days (iPSC). The relative expression levels of β-catenin were analyzed by qRT-PCR. The present study was reviewed and approved by the Committee of Research Ethics of the Pontifical Catholic University of Rio Grande do Sul (approval no. 17943213.9.0000.5336) through the system Platafoma Brasil. Written informed consent was obtained from patient 1 and from the parents of patient 2 (a minor) enrolled in the present study, according to Brazil Resolution no. 466/12.

Results: iPSCs from FCD patients and controls demonstrated no difference in migration. In contrast, fibroblast cell migration assay showed a higher cell migration capacity in patients with FCD at 24 and 48 h compared to control. After 24 hours of cell migration induction, the marked area for the migrating fibroblasts was 33.2 mm2 (SEM 13.6) for the control patients and 154.2 mm2 (SEM 18.2) for FDC patients (p<0.0001). At 48 hours from cell migration induction, the area of the cell migration in the control group was 65.5 mm2 (SEM 21.4) and 160.2 mm2 (SEM 41.7) in the FCD patients (p<0.001). iPSCs and fibroblasts from FCD patients showed an increase in β-Catenin expression when compared to healthy individuals (4.3 times and 3.1 times, respectively) During control neurodifferentiation an increase of 5 times in β-catenin gene expression was observed in the 14, 22 and 35-day compared to undifferentiated iPSC. In FCD samples, after 35 days the expression was 2.5 times when compared to undifferentiated iPSC. The expression levels are preliminary data and more robust statistics will be done with more patients

Discussion and Conclusions: Our results indicate that the migratory stimulus is not fully interpreted or does not act as a transcription factor for β-catenin in patients with FCD. Furthermore, it may influence the malformation of the cortex due to deficiency of this important molecule. In fibroblasts the migratory stimulus seems to be more pronounced in patients with FCD, indicating a possible compensation in the differentiated cells by lack of β-catenin during neurodevelopment. New patients and different research methodologies are underway to better understand this mechanism.

Acknowledgements/Financial Support: PUCRS, CAPES, CNPq, FAPERGS and Panduratta.

013 - Maternal inhalation of crack cocaine promotes deficit in memory consolidation, anxiogenic-like behavior and increased susceptibility to epileptic seizures in F1 generation

Pacheco, A.L.D., Nicácio, D.C.S.P., Silva, S.R., Serejo, V.F.M., Melo, I.S., Freitas-Santos, J., Costa, M.A., Cavalcante, C.M.B., Souza, F.M.A., Neto, J.G.S, Silva, B.R.M., Oliveira, K.L.S., Duzzioni, M., Castro, O.W.

Department of Physiology and Pharmacology, Institute of Biological Sciences and Health, Federal University of Alagoas (UFAL), Maceió / AL.

Rationale: Crack cocaine, cocaine in its smoked form, is currently one of the major public health problems, reflecting increased crime. Another alarming factor is the increase in the number of children intoxicated by crack cocaine during the gestational period, mother’s children recognized in literature as “crack babies”. Cocaine, crack cocaine and their metabolites cross the placenta and promote effects on the fetus. Studies have indicated the incidence of prematurity, irritability, sweating and seizures. In children, exposure to crack cocaine leads to cognitive deficits, depression and altered susceptibility to epileptic seizures in adulthood. We evaluated the effects of maternal inhalation of crack cocaine on susceptibility and patterns of Status Epilepticus (SE), as well cognitive performance and anxiety-like behavior.

Methods: Experimental procedures were approved by the Ethical Committee for Animal Research of the Federal University of Alagoas (UFAL) (protocol # 54/2017). Pregnant Wistar rats from the 5th to the 21st gestational day were exposed to the products of crack cocaine pyrolysis. Pups were kept with their mothers until the 21st day of postnatal life. After 30 days, animals were subjected to behavioral anxiety [elevated plus maze (EPM)] and memory [step down (SD)] tests. At 60 days, the male and female adult animals (F1 offspring) were submitted to stereotaxic surgery for cannula implantation in the hilus of dentate gyrus of the hippocampus. Animals received microinjection of intrahippocampal pilocarpine (H-PILO) in subconvulsant doses (sH-PILO, 0.6 mg/μL) to analyze the susceptibility and severity of epileptic seizures. Results were expressed as mean ± SEM with the unpaired t-test.

Results: Exposure to crack cocaine during the embryonic stage leads to impairment of consolidation of long-term memory of young female rats (F1) (control 171.5 ± 38.30; exposed 67.25 ± 40.26; p= 0.03). In addition, maternal exposure to crack cocaine also leads to anxiogenic-like behaviors; reduced the time spent (control 39.48 ± 3.753; exposed 20.23 ± 5.400; p= 0.01) and entries into the open arms (control 38.50 ± 1.924; exposed 26.53 ± 4.292; p= 0.02), and a lower frequency of risk assessment measured by head-dipping (control 4.500 ± 0.6368; exposed 2.200 ± 0.5925; p= 0.02) in young male rats. Animals exposed to sH-PILO showed increased susceptibility reducing latency to SE (sH-PILO 4844 ± 380.3 seconds ; CRACK+sH-PILO 3333 ± 602.0 seconds; p= 0.05), increased frequency of seizures (sH-PILO 11.33 ± 6.952 seconds; CRACK+sH-PILO 36.11 ± 9.286 seconds; p= 0.05) and total time of seizures (sH-PILO 117,3 ± 89,62 seconds ; CRACK+sH-PILO 600,3 ± 183,1 seconds; p= 0.03).

Discussion/Conclusions: These preliminary data suggest that exposure, to crack cocaine pyrolysis products during the gestational period leads to pup’s impairment of long-term memory formation, increased propensity for anxiety, and reduced threshold for epileptic seizures.

Acknowledgements/Financial Support: Members of the Laboratory of Neuropharmacology and Integrative Physiology of the Department of Physiology and Pharmacology, Institute of Biological Sciences and Health, Federal University of Alagoas (UFAL). FAPEAL, CAPES, CNPQ.

014 - Crack cocaine inhalation potentiates the effect of pilocarpine by reduction of acetylcholinesterase activity

Bastos-Cavalcante C. M.¹, Santos J. F. ¹, Pacheco A. L. D. ¹, Melo, I. S. ¹, Costa, M. A. ¹, Silva, J. M. ², Machado S. S. ², Borbely A. U. ¹, Castro O. W. ¹

1 - Institute of Biological Sciences and Health – Federal University of Alagoas, Maceió, Brazil

2 - Institute of Chemistry and Biotechnology, Federal University of Alagoas Maceió, Brazil

Rationale: Crack cocaine is a powerful addictive stimulant drug. Animal studies have shown that cholinergic system may play a role in neurotoxicity induced by cocaine or the inhalation of its active metabolites. Here we evaluate the acetylcholinesterase (AChE) activity, behavior and the threshold for epileptic seizures of rats that received a low dose of pilocarpine (PILO) followed by the exposure to the crack cocaine smoke.

Methods: Experimental procedures were approved by the Ethical Committee for Animal Research of UFAL (protocol #27/2016). Male Wistar rats (n=48; 240-340g) were submitted to stereotaxic surgery for implantation of a cannula into hilus of dentate gyrus of hippocampus. Animals received microinjections of a low dose of PILO (0.75 mg/μL) or vehicle (VEH [saline 0.9%]). Immediately after PILO administration animals were individually exposed to air (PILO) or 400 mg (VEH+CRACK and PILO + CRACK) for 10 minutes. We analyzed latency, number of wet dog shakes (WDS), frequency, duration and severity of limbic seizures according to Racine Scale (1972), during 120 min after crack cocaine exposure. Acetylcholinesterase activity (n=24) was performed after 10 (NAÏVE, SE, PILO, CRACK and PILO+CRACK) and 60 min (NAÏVE, SE, PILO, CRACK and PILO+CRACK) of crack cocaine exposure in cell-free extracts of hippocampus as described by Ellman et al. (1961) and total contents of the extracts were determined according to the Bradford’s method (1976). Results were expressed as mean±SEM, compared by one-way ANOVA followed by post-hoc test Student-Newman-Keuls.

Results: Animals subjected to PILO + CRACK demonstrated increased severity of limbic seizures (stages 0-3) in comparison to PILO and CRACK (stages 0-2). The latency to seizures decreased in PILO+CRACK (0.66 ± 0.33 minutes) when compared CRACK (7.85 ± 1.28 minutes) e a PILO (6.33 ± 2.01 minutes) (p<0.01). PILO + CRACK displayed higher seizure frequency (107.7 ± 24.41 seizures) than PILO (24.83 ± 6.44 seizures) and CRACK (11.43 ± 2.44 seizures) (p< 0.01). Similarly, PILO + CRACK revealed increase in the duration of seizures (2138 ± 723.9 seconds) when compared to PILO (442.0 ± 198.5 seconds) e CRACK (124.9 ± 42.49 seconds) (p< 0.01). The number of WDS increased in PILO + CRACK (162.79 ± 45.40 WDS) and PILO (127.3 ± 52.73 WDS) compared to CRACK (8.00± 2.646 WDS) (p<0.02). We observed an increase in the AChE activity, after 10 min in the CRACK (0.534±0.055 mmol.min-1.ml-1) group when compared to NAIVE (0.438±0.019 mmol.min-1.ml-1) (p<0.01) and after 60 min there was a reduction of AChE activity in CRACK (0.277±0.027 mmol.min-1.ml-1) group and PILO + CRACK (0.294±0.030 mmol.min-1.ml-1) in relation to NAIVE (0.438±0.019 mmol.min-1.ml-1) (p<0.01) indicating a decrease in the activity of AChE.

Discussion/Conclusions: These data suggest that crack cocaine inhalation potentiates the effects of a sub dose of PILO. We also showed that AChE activity has a biphasic response that could be associated with the observed effects of crack exposure.

Acknowledgements/Financial Support: To all those involved in the UFAL. FAPEAL/PPSUS and CAPES.

015 - High-frequency stimulation of the CA1 hippocampal subfield prevents ketamine-induced aberrant oscillations and synaptic plasticity in the prefrontal cortex in vivo

Lopes-Aguiar, C.1; Ruggiero, R.N. 2; Rossignoli, M.T.2; Esteves, I.M. 2; Peixoto-Santos, J.E.2; Romcy-Pereira, R.N.3 ; Leite, J.P.2

1 - Núcleo de Neurociências, Dept. Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais.

2 - Dept. Neuroscience and Behavioural Neuroscience, Ribeirão Preto School of Medicine, University of São Paulo, Brazil.

3 - Brain Institute, Federal University of Rio Grande do Norte, Natal, Brazil.

Rationale: Psychotic disorders affect around 20% of the patients with temporal lobe epilepsy (TLE) and its mechanisms are still poorly understood. In animal models, it has been shown that the excitatory/inhibitory unbalance in cortico-limbic circuits produces secondary dysfunctions in the dopaminergic system, leading to psychotic-like behavioral abnormalities. In the long-term, cortical synaptic plasticity dysfunction seems to underlie the negative symptoms and cognitive deficits observed in schizophrenia or psychosis in TLE. Interestingly, enhancement of glutamatergic receptors activity by treatment with positive allosteric modulators seems to attenuate cognitive impairments in some cases of psychosis. Ketamine (KET), an N-methyl-D-aspartate (NMDA) antagonist, has been regarded as a useful pharmacological tool to test certain principles of the glutamatergic hypothesis of schizophrenia. In rodents, KET produces several alterations, such as hyperlocomotion, working memory deficits, glutamatergic hyperactivation, and aberrant oscillatory cross-frequency coupling. To further explore the relationship between glutamatergic synaptic efficacy and oscillatory alterations induced by KET, we sought to test whether the induction of cortical long-term potentiation (LTP) by electric high-frequency stimulation (HFS) in hippocampal CA1 cells is able to prevent the oscillatory changes induced by KET.

Methods: Electrodes were positioned into CA1 and mPFC in adult urethane-anesthetized male rats. Synaptic efficacy in the CA1-mPFC pathway were assessed by electrical stimulation at CA1 (monophasic pulses, duration = 0.2 ms, frequency = 0.05 Hz, intensity = 150 to 300 µA) with concomitant recordings of field post-synaptic potentials (fPSPs) in the mPFC. Electrophysiological signals were amplified (gain 100x), band-pass filtered (0.3 Hz – 3 kHz) and digitized at 10 kHz before being visualized and recorded in a dedicated computer. Short-term plasticity was evaluated by measuring paired-pulse facilitation (PPF). After 90 min of baseline recordings, two independent groups of animals received either KET-S+ (12.5mg/kg, i.p.) or vehicle, followed by 120 min electrophysiological monitoring. In a different set of animals, two applications of high-frequency stimuli (HFS) were performed before KET-S+ treatment. Oscillatory patterns were continuously monitored by local field potentials (LFPs) recordings in the mPFC and CA1 during the whole experiment. For each rat, the amplitude of fPSPs was divided into 10 min epochs and normalized against the baseline. Statistical analysis was performed by two-way ANOVA for repeated measures followed by Tukey’s post hoc test. All procedures were performed according to the Ethical Committee for Animal Experimentation of the University of São Paulo-FMRP (193/2009).

Results: Our results showed that KET-S+ enhanced (+10%, p<0.05; two-way ANOVA RM) CA1-mPFC synaptic efficacy. In addition, KET-S+ impaired PPF (-15%, p<0.05; two-way ANOVA RM). Prior induction of LTP prevented the short-term plasticity disruption induced by KET-S+. Besides, analysis of prefrontal oscillations revealed that KET-S+ promoted an aberrant enhancement of the phase-amplitude coupling between delta and high-gamma in the mPFC (+200%, p<0.001; two-way ANOVA RM). Interestingly, prior induction of LTP was also sufficient to prevent the enhancement of mPFC phase-amplitude coupling induced by KET-S+.

Discussion and Conclusions: Altogether, our findings extend our understanding on the relationship between support recent discoveries that positive allosteric modulators of glutamatergic receptors attenuate cognitive impairments in animal models of psychosis and in patients with schizophrenia.

Acknowledgements/Financial Support: FAPESP (09/54410-0), CAPES, CNPq.

016 - Effects of litter reduction on metabolic parameters in male and female young offspring

Mangerona, B. A.; Ruginsk, S.G.

Universidade Federal de Alfenas, Instituto de Ciências Biomédicas, Alfenas – MG

Rationale: Obesity is a serious clinical condition that constitutes one of the major public health problems nowadays. Its origin is multifactorial and involves genetic, metabolic and behavioral factors that result in an imbalance between caloric intake and expenditure, with consequent positive energy balance and weight gain.

Methods: All experiments were performed under protocols approved by Ethical Committee for Animal Experimentation (protocol n. 11/2018). Using the experimental model of reduced litter that mimics the current human demographic and nutritional transition scenarios, characterized by a reduction in the number of children with a proportional greater access to food, we aimed to analyze obesity through a translational approach. In order to investigate and characterize this model, this study employed 21-day-old Wistar male and female rats obtained from control mothers (10 pups/dam; normal litter: NL) or submitted to the reduced offspring protocol (3 pups/dam; small litter: SL), evaluating the effects of sexual dimorphism on the weight gain, the relative masses of tissues involved in metabolic functional (adrenal, pituitary, brown adipose tissue - BAT and perigonadal and retroperitoneal stores of white adipose tissue - WAT) and plasma lipid profile (total cholesterol and triglycerides). All data were submitted to two-way ANOVA analysis followed by Tukey’s or Sidak’s multiple comparisons test using the software Graphpad Prism 7.0.

Results: A significant increase in body mass was observed in the offspring originated from SL dams (p < 0.0001), as well as in the relative mass of BAT (p < 0.05) and in both perigonadal (p < 0.0001) and retroperitoneal WAT stores (p < 0.05). No significant differences were found in the relative adrenal and pituitary masses among groups. Regarding the lipid profile, both females and males originated from SL dams exhibited increased plasma levels of total cholesterol (p < 0.01) and a reduction in plasma triglycerides concentrations (p < 0.05).

Discussion/Conclusions: In conclusion, the present study shows that postnatal overfeeding is effective in inducing obesity in young animals of both sexes, influencing metabolic parameters. Therefore, these results demonstrate the importance of nutrition as a factor for neonatal programming, determining the development of obesity in the adult offspring.

Acknowledgements/Financial Support: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

017 - Complexity of different spatial and temporal patterns of non-periodic electrical stimulation, as assessed by Shannon and spectral entropies, correlates with their anticonvulsant power

Silva, W.S.1; Oliveira, J.C.1; Cota, V.R.1

1 - Laboratório Interdisciplinar de Neuroengenharia e Neurociências (LINNce)/ Departamento de Engenharia Elétrica (DEPEL-UFSJ)/Universidade Federal de São João del-Rei (UFSJ).

Rationale: Epilepsy is a brain disorder characterized by hypersynchronous neural activity caused by hypersensitivity of the tissue. In the last years, our research group has shown satisfactory results of the use of non-periodic electrical stimulation (NPS) in breaking the hypersynchronized activity by imposing a non-rhythmic pattern. Recent results have shown that biphasic, bilateral, and asynchronous stimulation has greater anticonvulsant effect and putatively greater desynchronizing power. In this sense, the aim of this study was to investigate the relationship between the complexity of different patterns of NPS with their anticonvulsant power against pentylenetetrazole (PTZ)-induced seizures in animals.

Methods: A total of 74 male Wistar rats were divided in one control (without stimulation) and six stimulated groups according to distinct temporal and spatial variations of NPS (i.e. mono versus biphasic, uni versus bilateral, and synchronous versus asynchronous stimulation). All animals were submitted to a surgical procedure for the bilateral implantation of stimulation electrodes in the amygdalae. Pulses were square waves with 50 μs width for each phase and varied between 100 – 400 μA according to animal’s susceptibility. Animals were subjected to the PTZ continuous infusion model (10 mg/ml/min). Latency to generalized tonic clonic seizure (GTCS) was measured to calculate the amount of drug necessary to elicit convulsive behavior and these values were normalized by body weight to obtain threshold data (ethical protocol 01/2011). The complexity of each NPS pattern was assessed by two different approaches, considering different aspects of the neurobiology underlying neural response to electrical stimulation (ES): Shannon entropy and spectral entropy. The former calculates how non-periodic a stimulation pattern is, considering the two channels simultaneously in matrix form. The latter uses the power spectrum density (PSD) of each channel. After adding the two PSD, the number of frequencies with power above 10-16 is computed. Spectral and Shannon entropies values obtained for each NPS pattern were then correlated to PTZ threshold to elicit GTCS, using Pearson correlation.

Results: The stimulation disorder calculated through entropy showed a strong correlation (r > 0.97) with the PTZ threshold needed to trigger GTCS in each group. The Shannon entropy obtained a correlation coefficient of 0.9703 (p < 0.01) and spectral entropy obtained a correlation of 0.9808 (p < 0.001). The spectral entropy obtained better results when comparing unilateral monophasic to bilateral biphasic pulses, and synchronous to asynchronous stimulation.

Discussion/Conclusions: Results from both approaches indicate a high correlation between the complexity of the NPS stimulation pattern and its anticonvulsant effect. Increasing the number of phases, stimulating both hemispheres, and desynchronizing stimuli between amygdalae gradually resulted in higher entropy values of the NPS pattern and consequently its anticonvulsant power, showing that these ES parameters have additive effects. These results corroborate the notion of neural desynchronization as a mechanism underlying the therapeutic effect of NPS.

Acknowledgements/Financial Support: This work was supported by FAPEMIG APQ 02485-15.

018 - Puzzles benefits for Education: TECGrupo.com experience

Paiva, D.C.

TECGrupo.com, Northwest Fluminense Institute, Federal Fluminense University (INFES - UFF) Santo Antônio de Pádua, Rio de Janeiro, Brazil

Rationale: This abstract presents a project in which puzzles assembly were used by considering its benefits for cognitive aspects related to attention, concentration, detailed vision. Focus on a group relationship, the students under 10 and 14 years were separated in groups of four. Observing the activities important aspects to show is that the students were concentrated on the puzzle dynamics. TECGrupo.com is a research group hold in Santo Antônio de Pádua, RJ, at a campus of Federal Fluminense University. The main activities of the group are concerning on Technology, Education, and Cognition (TEC) aspects. The group leader is a cognition interested since the master degree where he developed a simulator of crowd behavior. During the doctorate, he had studied at the University of Sao Paulo where had been part of Cognitio group with professor Henrique Schützer Del Nero. Because of this, since 2016 TECGrupo.com have been organized the Brain Awareness Week named Semana Nacional do Cérebro, Professor Henrique Schützer Del Nero and other events concerning the TEC areas every year.

Methods: The activity reported was based on gamification concept, a competition was held as part of TEC-Encontro event. Students from public and private schools participate at two editions. The first edition was held in 2016 supported by Coordination for the Improvement of Higher Education Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES, in Portuguese) through Event in the Country Support Program (Programa de Apoio a Eventos no País, PAEP, in Portuguese). Eight schools from the cities of Santo Antônio de Pádua - RJ, Miracema - RJ, Pirapetinga - MG and Ubá - MG, were selected to take part in the event promoted by the Technology, Education, and Cognition research group (TECGrupo.com). Children up to 14 years old participated on selective or local phase. This one was conducted by eleven undergraduates in Computing of the Northwest Fluminense Institute of Higher Education of the Fluminense Federal University (INFES-UFF). The students were separated into groups of 4 members, where those under 10 years old set up puzzles of 250 pieces and those under 14 years old assembled of 500 pieces. A period of up to 4 hours was provided for completion. After selectives in all schools, the classification was performed for the shortest time and quantity of assembled pieces and the best teams were classified for the “Final”. The 2017 edition was supported by Pró-Reitoria de Pesquisa e Pós-Graduação of Federal Fluminense University (PROPPI - UFF). The competition occurred at public and cultural places due to the partnership with the director of culture at Santo Antônio de Pádua, RJ, and in two public schools. The editions reached almost 600 students from four cities.

Discussion/Conclusions: As predicted was observed the great potential of the puzzles in the aid to basic education. Children were mostly attentive and entertained, talking only to members of their groups, while exercising their persistence and logical reasoning. Those belonging to private colleges were accustomed to teamwork, while a few students from the public school had participated in such activities. However, despite the difference, the best times were obtained by public school students, which shows great unexplored capacity in such children. In 2016 final, students, parents and school professionals were gathered, the latter being engaged to the point of requesting puzzles to assemble. The awards were given by the director of publications of the Brazilian Computer Society (SBC). After the events and the competitions, the puzzle collection acquired in partnership with Grow® remains available for free use by schools with only a simple application form. Due to the success of the activities, TECGrupo.com was awarded the best work at NeuroEdu 2017, invited and held the TEC Day at the Casa das Irmãs in Tanguá, RJ and was also invited by the management of a private school to organize a free activity of puzzles for his students. The 2018 edition is confirmed to be performed in November.

Acknowledgements/Financial Support: Federal Fluminense University (UFF) and the Coordination for the Improvement of Higher Education Personnel (CAPES) for the support considering the edital 03/2016 (PAEP - CAPES) and 2017 (PAEC-UFF).

019 - Morphofunctional and molecular defects of hearing sensitivity in the genetic audiogenic seizure-prone hamster GASH/Sal

Damasceno, S.1, Álvarez-Morujo, D.2, Sánchez-Benito, D.2, Díaz-Casado, M.E2,3, Rosa Mª García-Aparicio4, Medeiros, D.C.1, Álvarez-Morujo, A.J.2,4, López, D.E.2,3, Godard, A.L.B.1, Gómez-Nieto, R.2,3.

1 - Institute of Biological Sciences, Federal University of Minas Gerais, Brazil

2 - Institute of Neuroscience of Castilla y León / Institute of Biomedical Research of Salamanca, University of Salamanca, Spain.

3 - Department of Cell Biology and Pathology, University of Salamanca, Spain

4 - Department of Anatomy and Histology, University of Salamanca, Spain

Rationale: Audiogenic reflex seizures are precipitated by an abnormal response of the brain to environmental auditory stimuli. The genetic audiogenic seizure hamster from Salamanca (GASH/Sal) is an experimental model of epilepsy that exhibits generalized tonic–clonic seizures in response to loud sound. Here, we used the GASH/Sal model for studying the morphofunctional and molecular defects of its cochlear receptor that might be associated with seizure susceptibility.

Methods: We used 9 GASH/Sal and compared them with 9 control animals, all males 80 days old, in a multi-technical approach that includes functional hearing test, histology and gene expression studies. Auditory brainstem responses (ABR) elicited by click stimuli were used to assess the functionality of the cochlear and auditory neural function. The organ of Corti and spiral ganglion cells were studied using light microscopy and morphometric analyses with ImageJ software. To determine the transcriptional changes in cochlear gene expression, quantitative analyses of mRNA expression through qRT-PCR was performed for the genes Gpr98, Cdh23, Pcdh15 and Prestin in naïve and seizure-induced animals. The transcript levels were normalized using Gapdh as the housekeeping gene and analyzed according the comparative Ct method (2-ΔΔCT). The significance of the qPCR analysis results was determined using a one-tailed t-test for each gene, considering as significant p <0.05. All the experiments with animals were carried out in compliance with European legal regulations and approved by the Bioethics Committee of the University of Salamanca.

Results: ABR thresholds of GASH/Sal hamsters were significantly higher than ABR thresholds of control animals (p < 0.05). The amplitude of ABR wave I that corresponds to the auditory nerve fibers was drastically reduced (p < 0.01) in the GASH/Sal. This correlates with morphological alterations in the cochlear hair cells and spiral ganglion neurons that were reduced in number. In naïve animals, the relative expression of the genes Gpr98, Cdh23, Pcdh15 and Prestin was lower in the cochlea of the GASH/Sal as compared to controls (p < 0.05). In control animals, the auditory stimulus caused an increase in the expression of the Gpr98 and Prestin genes (p < 0.001 and p < 0.05 respectively), as well as a decrease in the expression of the Cdh23 and Pcdh15 genes (p < 0.05), in comparison with naïve controls. In the GASH/Sal hamsters, the sound-induced seizure caused a significant increase (p < 0.01) in the expression of all genes, in relation to both the naïve state and the auditory stimulated control group.

Discussion/Conclusions: The GASH/Sal showed alterations in auditory hearing sensitivity, including elevated auditory thresholds and alteration of the amplitude of the ABR components. The GASH/Sal exhibits morphological defects in the organ of Corti and spiral ganglion neurons that matched those functional alterations. Expression of key genes involved in functionality, structure, and development of cochlear hair cells are altered in the GASH/Sal. This study provides insights into morphofunctional and molecular defects of the cochlear receptor that might contribute to the seizure susceptibility of the GASH/Sal model.

Acknowledgements/Financial Support: This study has been sponsored by CNPq and CAPES for Damasceno S, the University of Salamanca Support Grant for the GIRs 2017 (IP-DE López) and Junta de Castilla y León #SA070P17 (IP- DE López).

020 - Influence of experimental epileptic seizures in the function and expression of p-glycoprotein

Hummel, M.T.R.1; Placido, R.V.1; Kawano, T.1; Rodrigues, R.F.1; Martinez, G.B.1; Bernardes, J.S.1; Silva-Júnior, R.M.P.2; Costa, L.H.3; Garcia-Cairasco, N.2; Pereira, M.G.A.G.4; Boralli, V.B.1

1 - Department of Clinical and Toxicological Analysis, Federal University of Alfenas, Alfenas, Minas Gerais, Brazil.

2 - Neurophysiology and Experimental Neuroethology Laboratory, Physiology Department, University of São Paulo, Ribeirão Preto, Brazil.

3 - Department of Medicine, Federal University of Piauí, Brazil.

4 - Department of Biochemistry, Federal University of Alfenas, Alfenas, Minas Gerais, Brazil.

Rationale: Epilepsy is a worldwide spread pathology prevalent in developing countries; about fifty million people in the world are affected by this pathology. The disease is characterized by recurrent seizures, which are brief episodes and involuntary, and occur due to excessive electrical discharges that affect the brain cells. Patients who have epilepsy, in some cases do not respond successfully to the treatment. Some molecular carriers, such as the P-glycoprotein (Pg-P), present in the blood-brain barrier may be involved in resistance mechanisms to antiepileptic drugs, which are substrates of Pg-P. Therefore, the evaluation of Pg-P function and its expression may help to understand epilepsy’s pathological mechanisms.

Methods: The evaluation was performed comparing pharmacokinetics parameters in Wistar Audiogenic Rats (WAR) with their control (Wistar) submitted to the protocol of chronic audiogenic seizures (audiogenic kindling), which mimics temporal lobe epilepsy. The WAR group was sub-divided into: (1) NORMAL WAR which presented limbic recruitment during audiogenic kindling, and (2) RESISTANT WAR which presented no seizures during kindling. All animals received fexofenadine (dose of 10 mg/kg dissolved in water, oral gavage), a Pg-P substrate. The blood was collected in 0.25, 0.5, 1, 1.25, 2, 3, 4, 6, 8 and 12 hours after administration (Protocol Ethics Committee nº 658/2015 CEUA-UNIFAL/MG). The brain was collected for immunohistochemical analysis of Pg-P. The blood concentrations of fexofenadine were determined by ultra-high performance liquid chromatography coupled to mass spectrometry. Chromatographic conditions: column C18, with mobile phase of methanol: acetonitrile: ammonium acetate 10 mM (45:45:10, v:v), with flow of 0.4 mL/min, and monitoring of the following transitions to fexofenadine: ion precursor m/z 502 and ions products 484, 466 and 171.

Results: The pharmacokinetic analysis results showed an increase in fexofenadine bioavailability (AUC) and half-life due to a clearance reduction in RESISTANT WAR (group 2) when compared to the control group (p<0.05). Immunohistochemical brain tissue analysis showed an increase in Pg-P expression on the WAR groups (NORMAL AND RESISTANT WARS) in the blood-brain barrier when compared to control group (P<0.01).

Discussion/Conclusions: This increase in the expression of Pg-P demonstrated that the access to substrates of this transporter to the central nervous system is diminished, causing an increase in the plasma availability, evidenced by the parameter AUC, then antiepileptic drugs cannot play their pharmacological effect. Most of the drugs used in the treatment to epilepsy are substrates of Pg-P, then they can be affected with this increase in expression of the efflux transporter and therefore cannot perform the pharmacological action, explaining those cases of patients refractory to drug therapy.

Acknowledgements/Financial Support: UNIFAL-MG, CAPES, CNPq, FAPEMIG, FAPESP.

021 - Early-life Status Epilepticus induces psychotic-like behavior and affects hippocampal-prefrontal connectivity without neuronal loss

Ruggiero, R.N.1; Marques, D.B.1; Beraldo, I.K.S.2; Pereira, V.F.2; Bueno-Junior, L.S.1; Kandratavicius, L.1; Rossignolli, M.T.1; De Ross, J.B.1; Lopes-Aguiar, C.2; Leite J.P.1

1 - Dept. Neuroscience and Behavioral Neuroscience, Ribeirão Preto School of Medicine, University of São Paulo, SP-Brazil.

2 - Dept. Physiology and Biophysics Federal University of Minas Gerais, MG-Brazil.

Rationale: Early-life seizures are associated with cognitive and psychiatric impairments later in life. Since animals subjected to early-life Status Epilepticus (SE) do not show the neuropathological features classically associated with temporal lobe epilepsy, the neural mechanisms for these deficits are not known. Our hypothesis is that these behavioral dysfunctions could emerge from mild connectivity alterations between hippocampal CA1 and prefrontal cortex (PFC) since they are pathologically involved in psychiatric comorbidities of adult limbic epilepsy. Our aim was to investigate how early-life SE affects synaptic plasticity and the oscillatory activity of the CA1-PFC pathway, and how these effects relate to behavioral abnormalities.

Methods: We induced a 2 h SE by means of lithium-pilocarpine in P12 male Wistar rats. Once adults, they were tested for spatial working memory (radial arm maze), exploratory behavior (open field), and sensorimotor gating (pre-pulse inhibition of the acoustic startle, PPI). They were submitted to an acute electrophysiological session, with PFC field responses being recorded upon electrical pulses (200 μs, 0.05 Hz, 150–300 μA) into CA1, both before (30 min) and after (240 min) CA1 high-frequency stimulation (HFS, 2 series of 10 trains, each train with 50 pulses at 250 Hz). In a second experiment, SE and control (Ctrl) animals were chronically implanted with microwire bundles in the PFC and CA1 for extracellular recordings (1000x gain, 0.03–3 kHz band pass and digitized at 10 kHz) during free behavior for 48 h. Stages of wake/sleep cycle were classified as slow wave sleep (SWS), rapid eye movement (REM) sleep, active wake and quiet wake using a machine learning algorithm. The protocol was approved by the Ethical Committee on Animal Research (159/2014).

Results: SE rats showed impaired radial arm maze performance (Student t-test, t(23)=-2141, p<0.05, n=11/14), hyperlocomotion (Student t-test, t(23)=-2.886, p<0.05, n=11/14), PPI deficit (Student t-test, t(23)=3.693, p<0.05, n=11/14), and a stronger long-term potentiation (two-way repeated measures ANOVA, F(1,12)=19.687; p<0.001, n=7/7). In the chronic recording session SE animals do not present differences in time spent in each stage of sleep cycle. However, SE rats showed a reduction in delta (1-4 Hz, Student t-test, t(14)=3.59, p<0.05, n=7/9) and an increase in theta frequencies power (5-10 Hz) during SWS (Student t-test, t(14)=-2.43, p<0.05, n=7/9) and a disruption in hippocampus cross-frequency coupling during SWS (Student t-test, t(14)=1.72, p<0.05, n=7/9) and REM (Student t-test, t(14)=1.66, p<0.05, n=7/9), between slow frequencies and high-gamma frequencies (65-100 Hz). No changes in neuronal density were detected with NeuN immunohistochemistry.

Discussion/Conclusions: Our results indicate that typical behavioral abnormalities of animal models of psychosis after early-life SE can occur independently of neuronal loss and are related to long-lasting CA1-PFC alterations. These data suggest possible functional impairments that can underlie psychiatry comorbidities in limbic epilepsy.

Acknowledgements/Financial Support: CNPq, CAPES and FAPESP.

022 - Electrical stimulation in hippocampal CA1 evokes delta waves and inhibits unitary neuronal activity in the medial prefrontal cortex of rats in vivo

Carmo, F.A.C.1; Castro, G.Z.2; Mendes, E.M.A.M.2; Moraes, M.F.D.1; Leite, J.P.3; Diba, K.4; Lopes-Aguiar, C.1

1 - Núcleo de Neurociências, Depto. Fisiologia e Biofísica, Instituto de Ciências Biológicas, UFMG.

2 - Escola de Engenharia, Universidade Federal de Minas Gerais (UFMG).

3 - Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo.

4 - Department of Anesthesiology, University of Michigan Medical School, USA.

Rationale: Recent studies point the coupling between hippocampal sharp wave ripples (SWR) and delta waves followed by spindles in the medial prefrontal cortex (mPFC) to be critical for consolidations of certain types of episodic memory. Despite the abundance of evidence reporting the hippocampal influence on mPFC, it remains unknown whether hippocampal high-frequency activity during SWR events is sufficient to trigger long-term changes in the connectivity of mPFC circuits. Our first objective was to evaluate changes in local field potentials, as well as in neuronal activity in the mPFC, caused by time-specific activation in CA1 in vivo. Afterward, we used a high-frequency stimulus (HFS) protocol, within the ripple natural frequency band, to stimulate CA1 and investigate whether it was sufficient for modulation of long-term cortical responses.

Methods: Three male adult long-evans rats (weighting 300-400 g), under urethane anesthesia (1.2 g/kg, i.p.), were implanted with stimulation electrodes in the CA1 region of the hippocampus and a 64-channel silicon probe in the mPFC. The electrophysiological signals were digitized, filtered, amplified and recorded using the head-stage Intan Technologies and the Open Ephys acquisition board. During the baseline, evoked responses in mPFC were registered after electrical pulses in CA1 (200 ⲙA; 0,05 Hz). Then two series of HFS were applied and had their effects recorded for 45 minutes. Windows of 300 ms were separated immediately after each electrical pulse for analysis of response. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparisons test. All procedures were performed according to the Department of University of Wisconsin Milwaukee Safety and Assurances (13-14 #28).

Results: Preliminary results suggest that electrical stimulation of CA1 was capable of consistently inducing the occurrence of delta waves in mPFC and that local field potential (LFP) changes were associated with transient inhibition of unitary neuronal activity in the same region (50 units analyzed). The HFS caused a change in parameters of the evoked delta waves when compared to the baseline, such as duration [F (2, 97) = 32.03; p<0.0001; one-way ANOVA], amplitude [F (2, 97) = 6.08; p<0.01; one-way ANOVA], and slope [F (2, 97) = 4.98; p<0.01; one-way ANOVA] for a minimum of 30 subsequent minutes. Lastly, following the second HFS, a sole stimulus in CA1 was able to induce multiple delta waves in mPFC.

Discussion/Conclusions: Our results showed that CA1 stimulation leads to changes in LFP in mPFC and also suppresses neuronal activity in this region. These LFP changes were shown to be modified by Long-Term Potentiation (LTP) induction. HFS in the hippocampus, within the natural ripple band, induced LTP between the two regions and suggests hippocampal ripple events may be sufficient to provoke this modified plasticity. Furthermore, the changes observed in the characteristics of mPFC delta waves after HFS suggest that changes in the synaptic plasticity can modulate the coupling of hippocampal SWR and mPFC delta and thalamo-cortical spindles.

Acknowledgements/Financial Support: São Paulo Research Foundation (FAPESP), CAPES, CNPq, USP, UFMG.

023 - Non-periodic stimulation decreases ictal coincidence between neural substrates in pentylenetetrazole animal models

Rodrigues, S.M.A.F.1,2; Oliveira, J.C.1,2; Maciel, R.M.2; Réboli, L.A.2; Cota, V.R.1,2

1 - Programa de Pós-Graduação em Engenharia Elétrica – Associação ampla UFSJ e CEFET-MG (PPGEL)/UFSJ.

2 - Laboratório Interdisciplinar de Neuroengenharia e Neurociências (LINNce)/UFSJ.

Rationale: Electrical stimulation of the nervous system is a promising alternative to treat epilepsy patients who do not respond well or are not eligible for first-choice treatments. In this sense, our group developed a non-standard form of electrical stimulation termed non-periodic stimulation (NPS) with randomized inter-pulse intervals, successfully tested to suppress ictogenesis and epileptogenesis. Our hypothesis is that NPS would impair reverberation of ictal activity and thus promote desynchronization of neural oscillators. In order to further investigate the effect of NPS, we assessed ictal activity by quantifying spike coincidence rate between different neural substrates of animals submitted to pentylenetetrazole (PTZ) model of seizures.

Methods: A total of 57 male Wistar rats were divided in two control (CTRL-MYO; n=14; CTRL-GTCS; n=9) and four NPS stimulated groups (NPS-MYO; n=9; NPS-GTCS; n=12; NPS-GTCSt; n = 8; NPScont-GTCSt; n = 5) (animal experimentation ethical committee protocol 31/2014). Controlled intravenous infusion of PTZ provided a gradual transition of seizure states at varying levels of neural excitability/synchronism. This infusion was carried out until the onset of forelimb myoclonus (MYO-groups) or generalized tonic-clonic seizure (GTCS-groups). The threshold for PTZ-induced seizures was calculated. In NPS-GTCSt and NPScont-GTCSt groups, PTZ was injected only at the average threshold (t) to elicit GTCS in CTRL-GTCS group. For NPScont-GTCSt, NPS remained after cessation of PTZ infusion. Local field potentials (LFP) were recorded from cortex (CX), hippocampus (HP), and thalamus (TH), band-pass filtered (0.3 to 300 Hz), digitized (sampling rate at 1000 Hz), and digitally band-pass filtered (10 to 100 Hz) again to highlight spikes. The ictal coincidence between two substrates was computed whenever there was a co-occurrence of spikes on both channels within a given time window of variable sizes (0.001 to 0.05 s). This coincidence, expressed as a percentage of total spike counting, was calculated during MYO, tonic, and clonic ictal phases, for all three possible pairs of channels. NPS was applied asynchronously to both amygdalae; pulses were biphasic square waves during 100 µs with amplitudes varying from 100 to 400 µA.

Results: Behavior results showed that PTZ threshold to GTCS was significantly increased in NPS-GTCS when compared to CTRL-GTCS (p < 0.05, Student t test). Moreover, NPS-GTCSt and NPScont-GTCSt groups had a lower number of convulsive behavior occurrence (p < 0.01, Fisher’s test) and deaths (p < 0.01, Fisher’s test). Electrographically, NPS caused a decrease in the coincidence rate of spikes. Significant differences (p < 0.05, one-way ANOVA) during MYO phases were found mainly in smaller time windows of comparison between CX and HP in NPS-GTCSt when compared to CTRL-MYO group. During the clonic phase, significant differences were found between HP and TH, CX and TH of NPS-GTCS when compared with CTRL-GTCS group (p < 0.05, Student t test). There was a not significant trend in the tonic phase.

Discussion/Conclusions: Behavior results corroborate the anticonvulsant effect of NPS previously reported by our research group. The coincidence of spikes between channels is related to the propagation of epileptiform activity in ictogenic circuits. Thus, electrophysiological results support the notion that NPS promotes desynchronization of neural circuits involved in ictogenesis, an effect that may underlie its therapeutic effect.

Acknowledgements/Financial Support: CAPES and FAPEMIG (APQ 02485-15).

024 - Effects of calcineurin inhibition on excitotoxicity consequences in a NMDA-induced model.

Gomes, G.F.1; Batista, C.R.A.1; Rosa, W.M.B.1; Botelho, G.C.1; Oliveira, A.C.P.1

1 - Laboratory of Neurofarmacology, Departament of Farmacology, Biological Science Institute, Federal University of Minas Gerais, MG, Brazil.

Rationale: Neurodegenerative disorders represent an important public health issue around the world, mainly because aging is a common risk factor of this group of diseases. One component of the neurodegenerative diseases is the excitotoxicity, a pathological condition in which there is neuron damage, primarily induced by the entry of high levels of calcium ions into cell, caused by the overactivation of receptors for the excitatory neurotransmitter glutamate, such as the NMDA receptor. In the context of calcium modulation, calcineurin (CN) - a Ca2+/calmodulin-dependent serine/threonine phosphatase protein that is expressed in almost all cell types, including brain tissue, can be an important target to better understand processes involved in excitotoxicity. The aim of the present study was to evaluate the effect of calcineurin inhibition on NMDA-induced excitotoxicity on the hippocampus and its impact on memory impairment induced by NMDA toxicity.

Methods: To this end, we used the NMDA-induced excitotoxicity model by injecting 40 pmol of NMDA or vehicle (in 0.5 µL of volume) in the hippocampus of 10-weeks-old male C57BL/6 mice, by the insertion of a guide cannula on the following coordinates: AP: -1.9; ML: -1.5; and DV: +1.8. Eighty minutes before NMDA intra-hippocampal injection, we administrated 2.5, 5 or 10 mg/kg (i.p) of FK506 (tacrolimus) or vehicle (DMSO 1% and Tween 80 in saline) (groups = Vehicle+saline; Vehicle+NMDA; FK2.5+NMDA; FK5+NMDA; and FK10+NMDA). To evaluate the impact of FK506 pretreatment on NMDA-induced memory impairment, the object recognition test (ORT) was applied 24 h after NMDA microinjection. In addition to that, we evaluated the number and presence of seizures, as well as the animals mortality. Finally, we investigated neurodegeneration by FluoroJade C staining (FJC), measured by fluorescence total area with the support of ImageJ software (NIH). For ORT statistical analysis, we used one-sample t-test (theoretical mean = 0.5), and for FJC fluorescence total area statistical analysis, we used on-way ANOVA, considering significant differences always that p < 0.05 (95% confidence interval). Experimental procedures were performed in accordance with the guide for the use of laboratory animals of the Federal University of Minas Gerais under protocol number 200/2017.

Results: Our results showed that NMDA (40 pmol) microinjection, in non-pretreated group, induced deficit in the ORT (one-sample t-test, t=1.336, p=0.252), seizures in 30% of animals and 30% of death, in addition to the neurodegeneration evidenced by FJC. Pretreatment with FK506 (5 mg/kg; i.p) retrieved memory impairment in the ORT (one-sample t-test, t=2.710, p=0.042) and abolished death, despite the maintenance of seizure. On the other hand, FK506 (2.5 and 10 mg/kg) pretreatment increased the number of seizures and deaths induced by NMDA hippocampal microinjection. Pretreatment with FK506 (10 mg/kg) also increased neurodegeneration, as compared to 5mg/kg and non-pretreated groups (fluorescence total area; one-way ANOVA, F=5.560, p=0.018).

Discussion/Conclusions: These preliminary results demonstrate an important role of calcineurin in the NMDA-dependent excitotoxicity process and evidence this molecule as a potential target to better understand basic aspects of neurodegenerative disease.

Acknowledgements/Financial Support: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG).

025 - Hippocampal atrophy and language skills analysis of patients with drug-resistant mesial Temporal Lobe Epilepsy

Assis, A.J.1; Silva, T.F.1; Pereira, P.P.N.2; Fonseca, A.G.A.R.2; Siqueira, J.M.2; Drumond, L.E.1

1 - Universidade Federal de São João del Rei, campus Centro-Oeste, Divinópolis, MG, Brazil.

2 - Núcleo Avançado de Tratamento das Epilepsias (NATE) do Hospital Felício Rocho, Belo Horizonte, MG, Brazil.

Rationale: Temporal lobe epilepsy (TLE) is the most common focal form of epilepsy in adults, representing 40% of all cases, being often observed alongside with hippocampal sclerosis (HS) in 50-70% of cases. Because language is a domain that relies on pre-frontal and temporal lobes, language skills difficulties are commonly found in patients with drug-resistant mesial TLE. This study was designed to assess whether language skills are correlated to the laterality of HS in patients with drug-resistant TLE and to evaluate if the diagnosis of laterality of epilepsy, given by the electroencephalogram (EEG) is corroborated by the structural changes observed in the magnetic resonance imaging (MRI), through the hippocampus segmentation analysis.

Methods: This is a cross-sectional quantitative study. The sample was composed of patients over 18 years old, submitted to surgical treatment for mesial TLE between 2014-2018, in Núcleo Avançado de Tratamento das Epilepsias (NATE), Hospital Felício Rocho, Belo Horizonte/Brazil. An informed consent form approved by the Ethics Committee of Universidade Federal de São João Del Rei - Campus Centro Oeste (CAAE:54549816.0.0000.5545) and by Hospital Felício Rocho (CAAE:54549816.0.3001.5125), was signed by all patients. For the morphological evaluation of the hippocampus (HC), the MRI was used for manual segmentation to calculate its volume, based on the EADC-ADNI protocol, using OsirixTM software. The laterality of TLE was correlated with the patients’ language skills, through neuropsychological tests (Verbal Fluency - FAS – and Boston Naming Test - BNT). Data are presented as means ± S.E.M. Statistical comparisons were made using Student’s t-test.

Results: 57 patients, 19 men, and 38 women, between 19 and 67 years old, composed the sample that was divided into subgroups, according to the side of the EEG ictal onset zone. The patients with right TLE (n=24) showed performance below the average in 41.7% of the FAS tests and in 29.2% of the BNT’s. The patients with left TLE (n=30) had results below the average in 60% and 53.3%, respectively, FAS and BNT. Bitemporal (n=3) patients showed results below the average in 100% of the cases in both tests. Of all the patients, 15 had their hippocampal volumes (HV) measured, 53.34% of them showed atrophy of the hippocampus ipsilateral to the TLE. For patients with left TLE (n=8) the average right HV was 2.3 ±0.12 cm³ and the average Left HV was 1.7±0.17 cm³ (p<0.05). For patients with right TLE (n= 4), the average right HV was 1.9±0.25 cm³ and the left HV was 2.3 ±0.23cm³ (p<0.05). For patients with bilateral TLE (n= 3), right and left HV’s were, respectively, 2.0 ±0.22cm³ and 2.1±0.14 cm³ (p>0.05).

Discussion/Conclusions: The patients with left-sided TLE had a worse performance in language skills tests than right-sided TLE patients. In addition, hippocampal atrophy was observed when the ictal onset zone was unilateral and there was no asymmetry for bitemporal patients, corroborating the laterality detected in the EEG.

Acknowledgements/Financial Support: FAPEMIG and UFSJ.

026 - Effects of hypothyroidism on endocrine parameters and on depressive behavior in adult Wistar rats

Aquino Jr, M.K.1; Ruginsk, S.G.2; Marques, V.B.B.1

1 - Federal University of Alfenas, Faculty of Phrmaceutical Sciences, Minas Gerais, 37130-001

2 - Federal University of Alfenas, Institute of Biomedical Sciences, Minas Gerais, 37130-001

Rationale: Hypothyroidism is a high global incidence disorder which presents a gradual development and is characterized by low or no thyroid hormone (TH) production. Its most common cause is dietary iodine deficiency, followed by autoimmune factors, infiltrative diseases or abnormal thyroid gland development. Individuals with hypothyroidism usually develop comorbidities related to the lack of action of TH in several tissues, such as depression. Therefore, the present work sought to correlate hypothyroidism with the existence of suggestive behaviors of depressive symptoms in adult male Wistar rats.

Methods: The present work was approved by the Ethical Commission on the use of animals (CEUA-UNIFAL) with protocol number 67/2017. Two experimental models of hypothyroidism [thyroidectomy (surgical removal of the gland, Tx) and methimazole treatment (MMI, 0.04% w/v)] were used in animals with or without thyroxine replacement (T4, 20 μg/kg). Control animals of the surgical procedure were those submitted to sham surgery (Sham). In order to characterize the hypothyroidism, the free T4 (FT4) and thyroid stimulating hormone (TSH) dosages were measured, as well as the measurement of the thyroid axis function related tissue masses [adenohypophysis, brown adipose tissue (BAT), perigonadal white adipose tissue (WAT) and thyroid)]. For the depressive behavior evaluation, forced swimming and sucrose ingestion tests were performed. All evaluations were performed 21 days after the surgical procedure and/or days of treatment.

Results: According to the results, the Tx and MMI groups without hormone replacement showed significantly lower FT4 plasma concentrations (p<0.0001), compared to the Sham group and the hormone replacement groups. These results were corroborated by the adenohypophysis mass analysis, which was shown to be increased in the non-hormone-replacement Tx and MMI groups (p<0.005). On the other hand, the thyroid mass was increased in those animals treated solely with MMI, an effect that was brought to the basal levels (Sham group) when the T4 replacement was done (p<0.0001). Regarding BAT and WAT, no significant changes were found in these tissues masses yet. Concerning the depressive behavior evaluation, no differences between the groups in the immobility time through the forced swimming test were identified until now. On the other hand, the animals in the Tx and MMI groups without T4 replacement had a significantly lower sucrose intake (p<0.05) than in the groups receiving hormone replacement. All data were submitted to two-way ANOVA analysis followed by Tukey’s test using Graphpad Prism version 6.0. Values of p <0.05 were considered significant.

Discussion/Conclusions: Therefore, it can be concluded that both hypothyroidism experimental models were able to induce depressive-like behaviors, showing the important effects of the absence of TH on several tissues, particularly the central nervous system.

Acknowledgements/Financial Support: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo e Pesquisa do estado de Minas Gerais (FAPEMIG).

027 - Effects of the association between anticonvulsant and antihypertensive drugs in an experimental model of epilepsy

De Battisti, L.F.F.1; Costa, L.H.2; Guerra, I.O.F.1, Boralli, V.B.3; Pereira, M.G.A.G.1

1 - Department of Biochemistry, Federal University of Alfenas, Alfenas, Minas Gerais, Brazil.

2 - Department of Medicine, Federal University of Piauí, Parnaíba, Piauí, Brazil.

3 - Department of Clinical and Toxicological Analysis, Federal University of Alfenas, Alfenas, Minas Gerais, Brazil.

Rationale: Epilepsy is a brain disorder characterized by the occurrence of recurrent epileptic seizures. Epilepsy is a complex disease by itself, but there are comorbidities associated with this disease that makes it more complicated to be studied and properly treated, one of these associated comorbidities is hypertension and cardiovascular diseases in general. One of the most prescribed antiepileptic drugs, the carbamazepine has a low therapeutic index, small changes in plasma concentration may result in loss of efficacy or signs of intoxication. Enalapril is the drug most commonly used to treat hypertension, decreasing the peripheral vasoconstriction by angiotensin-converting enzyme inhibition. The aim of this study was to investigate the effect of the association between carbamazepine and enalapril on the severity of seizures induced in rats by pilocarpine, an experimental model of epilepsy.

Methods The study was previously approved by the Committee on Ethics in the Use of Animals (CEUA - protocol number 686/2015). The animals (Wistar, male, 2 months) were divided into six experimental groups (n = 8 to 13): G1, carbamazepine (25 mg/kg) in trough concentration; G2, carbamazepine in peak concentration; G3, carbamazepine in trough concentration associated with enalapril (30 mg/kg); G4, carbamazepine in peak concentration associated with enalapril; G5, enalapril and G6, vehicle (control group). The animals received a single shot of the each drug or vehicle before pilocarpine administration. Peak or trough concentration means the highest and the lowest drug blood concentration in the moment of pilocarpine administration. For carbamazepine the peak and the trough concentration is achieved in 2 or 6 hours, respectively. For enalapril, only peak concentration was used for this analysis (2 hours). All pilo-treated animals had status epilepticus (SE) for 90 minutes and the behavior was recorded for drug effect analysis. For this, we analyzed the latency time until the SE and the highest severity behavior into 18 windows of 5 min each. For statistical analysis we performed the one-way ANOVA Kruskal–Wallis test, followed by Bonferroni’s post-hoc test. P ≤ 0.05 was considered statistically significant.

Results: The seizure severity in the G2 group was increased when compared to G6 group (6.05±3.07 and 2.3±0.94, respectively; p<0.05). The animals receiving carbamazepine in peak associated with enalapril (G4 group) presented an increase of seizure severity when compared to the control group (G6 group) (4.1±2.9 and 2.3±0.94, respectively; p<0.05).

Discussion/Conclusions: According to the results, the bioavailability of this antiepileptic drug is directly related to the severity of seizures in the pilocarpine-induced epilepsy model. In addition, the use of carbamazepine associated with enalapril is able to increase the severity of seizures in this model, demonstrating a negative effect when these two drugs are used in combination.

Acknowledgements/Financial support: UNIFAL-MG, CAPES, CNPq, FAPEMIG, FAPESP.

028 - Role of wake-sleep cycle in cardiovascular responses to seizures induced by electric amygdala kindling model

Ghazale, P.P.1; Gomes, K.P.2; Junior, C.Q. de Lima2; Cravo, S.L.D.4; Biancardi, M.F.3; dos Santos, F.C.A3; Mendes, E.P.2; de Castro, C.H.2; Schoorlemmer, G.4; Colugnati, D.B.2; Scorza, F.A.1; Pansani, A.P.2

1 - Departamento de Neurologia e Neurociência, Universidade Federal de São Paulo, São Paulo, SP

2 - Departamento de Ciências Fisiológicas, Universidade Federal de Goiás, Goiânia, GO

3 - Departamento de Histologia, Embriologia e Biologia Celular, Universidade Federal de Goiás, GO

4 - Departamento de Fisiologia, Universidade Federal de São Paulo, São Paulo, SP

Rationale: Cardiovascular alterations have been identified as the main cause of sudden unexpected death in epilepsy (SUDEP). There is an intimate relationship between sleep and epilepsy. Most cases of SUDEP occur during sleep.

Methods: We evaluated cardiovascular changes in rats submitted to electric kindling model of epilepsy at different stages of sleep-wake cycle. Male Wistar rats (250-350g) were fitted with electrodes in the right amygdala, cortex, trapezius and close to the heart for electrical stimulation, EEG, EMG and ECG records, respectively (CEUA/UFG 035/2015). The stimulus (2s, 1ms biphasic wave, 60Hz) was applied once a day. The seizure development was classified according to Racine’s scale (R1-R5). Rats were either not stimulated (sham, n=5-10), stimulated while awake (n=5-13), during rapid eye movements (REM) sleep (n=4-7) or non-REM sleep (n=4-11). Sleep-wake detection was made according to Louis et al., Journal of Neuroscience Methods 2003;33:71. Rats were stimulated until fully kindled (three consecutives R5). We evaluated the cardiovascular parameters in vivo and ex vivo. Heart and aortic rings with (E+) and without (E-) endothelium were submitted to Langendorff and organ bath techniques, respectively. Picrosirius red, hematoxylin and eosin staining were used to cardiac histology. One-way ANOVA or Kruskal-Wallis were performed.

Results: No difference was found in kindling’s latency, seizure duration and heart rate variability. During generalized seizures, there was bradycardia followed by tachycardia. The tachycardia in NREM and REM groups was lesser intense than Awake group (Awake:542.2±12.6bpm; NREM:474.0±11.9bpm; REM:465.9±19.1bpm). After full kindling, the QTc interval was longer in REM group than in Sham (Sham:0.14±0.004s; REM:0.19±0.010s). Intraventricular systolic pressure (Sham:110.2±7.7mmHg; Awake:74.5±7.8mmHg; REM:72.5±4.8mmHg) and developed intraventricular pressure (Sham:98.0±7.5mmHg; Awake:64.4±8.1mmHg; REM:65.5±5.2mmHg) were decreased in Awake and REM groups compared to Sham. The log EC50 of Phenylephrine-induced aortic contraction in E+ (Sham:6.8±0.06; REM:6.4±0.06) and E- (Sham:6.9±0.08; REM:6.5±0.06) was increased in REM group compared to Sham. There was no difference in log EC50 of relaxation induced by acetylcholine and sodium nitroprusside. The cardiomyocytes diameter was reduced in kindled rats compared to Sham; REM group had the highest reduction (Sham:13.5±0.1µm; Awake:11.9±0.1µm; NREM:12.0±0.1µm; REM:10.5±0.1µm). Interstitial fibrosis was decreased in NREM group compared to Awake and increased in REM group compared to all the groups (Sham:9.5±0.7%; Awake:10.5±0.5%; NREM:7.8±0.7%; REM:12.5±0.8%). Perivascular fibrosis index was increased in Awake and NREM group compared to Sham (Sham:2.9±0.12µm2; Awake:2.4±0.10µm2; NREM:2.5±0.08µm2).

Discussion/Conclusions: The preliminary results conclude that sleep, mainly REM phase, is an important factor to cardiovascular alterations, as long QTc, vascular reactivity and heart morphology, but not to ictal heart rate and cardiac function ex vivo in rats with epilepsy.

Acknowledgements/Financial Support: CNPq, CAPES and FAPEG.

029 - Persistent cocaine effects on hippocampal-prefrontal oscillations in vivo

Gusmão, A.P.S.1; Polanczyk, R.S.1; Fernandes, L.da.S.A.1; Mendes, R.A.V.1; Carmo, F.de A.C.1; Beraldo, I.J.da.S.1; Santana, J.S.1; Leite, J.P. 2; Mendes, E.M.A.M. 1; Moraes, M.F.D.1; Moreira, F.de.A.1; Lopes-Aguiar, C. 1

1 - Núcleo de Neurociências, UFMG, Brazil

2 - Faculdade de Medicina de Ribeirao Preto, USP, Brazil

Rationale: Conversion between rapid eye movement (REM) and non-REM sleep states is consequence of precise interactions between neurotransmitter systems, such as cholinergic and serotonergic. Although it is less investigated, dopamine also seems to participate in the control of brain states along the sleep-wake cycle. In rodents, the deletion of dopamine active transporter gene reduced NREM sleep and produces a specific oscillatory state that resembles intrusion of REM into wakefulness. Disturbance of sleep is a frequently reported effect caused by cocaine intake, which induces longer sleep latency, reduced total sleep time and suppression or increase of REM sleep duration, depending on the dose. However, little is known about the cocaine effects on hippocampal-prefrontal cortex oscillations. The aim of this study was to test whether acute cocaine is sufficient to disrupt hippocampal-cortical coordination during urethane-induced sleep-like oscillations.

Methods: Experiment 1: We first video-monitored 14 adult male Wistar rats, approved by CEP-UFMG nº 47/2017, in an open field and injected them with either cocaine (15 mg/kg, i.p.), or vehicle (NaCl, 0.9%). Experiment 2: Eight adult male Wistar rats were anesthetized with urethane (1.25 g/kg, i.p.) and implanted with tungsten electrodes in the medial prefrontal cortex (mPFC) and hippocampal CA1. Local field potentials (LFPs) were filtered, amplified (1 Hz – 1 KHz, 100x), digitized at 4 kHz, visualized and recorded in a computer. Data were exported and analyzed using Matlab 2016b. Cocaine or vehicle was injected systemically after 60 min of baseline recordings. LFPs in CA1 and prefrontal cortex were recorded continuously during 3 hours after cocaine to assess coordination between these two areas.

Results: We first observed that systemic cocaine produces hyperlocomotion during 35 min when applied at the 15 mg/Kg dose (Ctr group: 15.79 ± 2.87 m vs Cocaine group: 96.34 ± 16.04 m, p < 0.001, Student t test) and not in control group. Then, as described before in the literature, we observed that, at baseline conditions, urethane-anesthetized animals displayed spontaneous fluctuations between NREM-like (high amplitude and slow oscillations) and REM-like (low amplitude and faster oscillations) brain states. However, after cocaine injection, we observed persistent REM-like oscillations for at least 3 hours, with no spontaneous transitions to NREM-like oscillations. Next, we compared spontaneous with cocaine-induced REM-like oscillations to examine whether cocaine was either blocking spontaneous transitions or producing an aberrant oscillatory state. Cocaine-induced REM-like oscillations showed higher prefrontal gamma (p < 0.001, Student t test) and hippocampal theta power (p < 0.001, Student t test) when compared with spontaneous REM-like oscillations.

Discussion/Conclusions: Thus, hyperdopaminergic state induced by systemic cocaine seems to produce aberrant oscillatory patterns that resemble spontaneous REM-like state, but with higher cortical gamma and hippocampal theta power. Our findings may further our understanding of the dopaminergic control of brain states along the sleep-wake cycle in normal and pathological conditions, such as Parkinson´s disease. In the following steps, chronic recordings in freely moving animals will be performed to assess how cocaine affects oscillatory patterns during sleep.

Acknowledgements/Financial Support: FAPEMIG, PRPq-UFMG, CAPES e CNPq.

030 - Longitudinal in vivo imaging of cerebral glucose metabolism in the neonatal hypoxia-ischemia model

Azevedo, P.N.1; Zanirati, G.1; Odorcyk, F.K.2; Carabali, L.H.D.2; Ghilardi, I.M.1; Venturin, G.T.1; Zimmer, E.R.1,2; Netto, C.A.2; Greggio, S.1; Costa, J.C.1

1 - Brain Institute of Rio Grande do Sul (BraIns), Pontifical Catholic University of Rio Grande do Sul (PUCRS)

2 - Department of Biochemistry, Federal University of Rio Grande do Sul (UFRGS)

Rationale: Perinatal hypoxia-ischemia (HI) is an important cause of childhood morbidity and mortality. It is estimated that approximately 25% of HI survivors will exhibit permanent neuropsychological disabilities, as well cerebral palsy, autism, hyperactivity, cognitive deficits and epilepsy. HI has been associated to the refractory epilepsy development in the childhood. Despite the scientific and technological advances in the care of neonates at risk, the clinical management of asphyxiated children has been limited only to supportive measures. Due to the clinical significance and the socioeconomic impact generated by this pathology, new strategies have been studied. Noninvasive imaging systems could elucidate the mechanisms of HI injury and contribute to the research for effective therapy strategies. This study proposes to verify in vivo, longitudinally, alterations in the metabolism of cerebral glucose in rats previously submitted to the neonatal model of HI by microPET scanning, correlating the results of the functional imaging with the anatomical, motor and cognitive parameters.

Methods: Male Wistar rats at postnatal day 7 (P7) were subjected to an HI model by permanent occlusion of right common carotid artery and systemic hypoxia for 1 h (n = 22). Additionally, Sham operated rats received the same procedural surgical. However, they were not subimitted neither to carotid occlusion nor to hypoxia environment (n = 13). MicroPET associated with fluorodeoxyglucose (18F-FDG) was used to investigate the cerebral metabolism in vivo of rats at 8, 10, 15, 36 and 60 days of age. Considering the mean of right hippocampus metabolism of Sham animals (18F-FDG-SUVR = 1.2) at P60, HI animal were divided into two groups: HI animals that not show alterations in the right hippocampus metabolism (HI non-hypometabolic, 18F-FDG-SUVR > or equal 1,2; n = 9) and HI animals that show hypometabolism in the right hippocampus (HI hypometabolic, 18F-FDG-SUVR < 1.2; n = 13). All the experimental procedures were performed with the approval of the Animal Care and Ethics Committee of UFRGS, Brazil (protocol number 31939).

Results: We detected so far that there is a transitory hypometabolism in the contralateral hemisphere at P15, and a hypometabolism restricted to the ipsilateral cerebral hemisphere at P36 and P60 for the HI hypometabolic group compared to Sham group. Histological analysis showed that brain volume loss analyzed by Cavalieri method was also restricted to the ipsilateral hemisphere of hypometabolic HI animals group. Besides, we found a negative correlation between the glucose cerebral metabolism and cognitive performance in the Morris water maze task.

Discussion/Conclusions: This is the first study that proposes a longitudinal in vivo analysis of the cerebral metabolism in neonatal HI model. The understanding of the pathophysiology in the first 24 hours after the HI insult, as well as the monitoring of the progression of the cerebral damage until the adult age, is of great clinical interest and the challenge of this study. We hope that this study provides an important tool to search for biomarkers and, a posteriori, adequate interventions for different degrees of severity of neonatal brain injury.

Financial support: CAPES, CNPq, FAPERGS, PUCRS and PANDURATA.

031 - Depression comorbidity in epileptic rats is related to alterations in both cerebral glucose metabolism and functional metabolic network

Zanirati, G.1; Azevedo, P.N.1; Venturin, G.T.1; Greggio, S.1; Alcará, A.M.1; Zimmer, E.R.1,2; Feltes, P.K.1; Marinowic, D.1; da Costa, J.C.1

1 - Brain Institute of Rio Grande do Sul (BraIns), Pontifical Catholic University of Rio Grande do Sul (PUCRS), Porto Alegre, Brazil

2 - Department of Biochemistry and Department of Pharmacology, Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, Brazil

Rationale: Depression is the most frequent psychiatric comorbidity in patients with epilepsy. Although the psychosocial aspects of these patients may contribute to a depressive condition associated with epilepsy, current investigations have increasingly suggested that the relationship between these two pathologies is associated with common pathogenic neurobiological mechanisms. However, these mechanisms involved in the manifestation of depression in epilepsy are not fully understood. Here, we investigated the alterations in brain glucose metabolism and the metabolic network in an experimental model of temporal lobe epilepsy (TLE) and correlated it with depressive behavior during the chronic phase of epilepsy.

Methods: Fifty male Wistar rats (40-45 days of age) were used for the control and epilepsy groups. Experimental model of epilepsy was induced via intraperitoneal injection of pilocarpine (320 mg/kg). The animals received diazepam (10 mg/kg) after 90 minutes of status epilepticus (SE) induction, to control the behavioral seizures. Fluorodeoxyglucose (18F-FDG) was used to investigate the brain metabolism, and a cross-correlation matrix was used to examine the metabolic network using micro–positron emission tomography (microPET) imaging. Forced swim test (FST), sucrose preference test (SPT), and eating-related depression test (ERDT) were used to evaluate depressive-like behavior. Fifteen days after SE induction the animals were video-monitored 12 h/d until the last scan day for the observation of spontaneous recurrent seizures and only animals that showed grade 5 seizures of Racine scale were included in the study as epileptic animals. Epileptic animals were divided in two groups: Pilo depressed and Pilo non-depressed. Control animals received saline injection instead of pilocarpine. Brain glucose metabolism was performed 23, 29, 36, and 46 days post-SE. Depression-like behavioral tests were performed from 47 days to 60 days post-SE. All experimental procedures were approved by the Animal Care and Use Committee of PUCRS (#7293).

Results: Our results showed an association between epilepsy and depression based on changes in both cerebral glucose metabolism and functional metabolic network. More specifically, we observed that epilepsy caused a brain glucose hypometabolism when compared to the control group in the rats, however depression in epileptic animals was associated with an even greater reduction in brain glucose metabolism in the whole brain in comparison to the epileptic non-depressed and control groups. Furthermore, we found that the epileptic depressed group presents a more compromised hypersynchronous brain metabolic network in relation to the epileptic non-depressed group, with aberrant connections in brain regions related to the pathophysiology of both epilepsy and depression. In addition, Pearson’s linear correlation was assessed, and we have identified a significant correlation between brain glucose hypometabolism and depressive-like behavior in chronically epileptic animals. Statistical analysis of data generated from microPET imaging was conducted using IBM SPSS Statistics. Generalized Estimation Equation was used for the longitudinal design. For behavioral data, ANOVA followed by Tukey’s post hoc or unpaired Student’s t-test were conducted using PrismGraph.

Discussion/Conclusions: Thus, our study demonstrated that depression plays an important role in the epileptic brain by revealing relevant and complex alterations in brain glucose metabolism and the metabolic network architecture. Clinically, the emergence of depression as a comorbidity associated with epilepsy may cause a significant worsening of quality of life in patients. Within this scenario, our findings provide novel evidence for a better understanding of the mechanisms of this condition and can be fundamental in order to be able to search for new effective and safe strategies for the treatment and prevention of this comorbidity in epilepsy.

Acknowledgements/Financial Support: CAPES, CNPq, FAPERGS, PUCRS and PANDURATA.

032 - Histological Correlates of Magnetization Transfer changes in the Hippocampus of Drug-resistant Temporal Lobe Epilepsy Patients

Peixoto-Santos, J.E.1; Velasco, T.R.1; Assirati, J.A.2; Carlotti, C.G.2; Matias, C.C.M.S.2; Scandiuzzi, R.C.1; Salmon, C.E.G.3; dos Santos, A.C.4; Leite, J.P.1

1 - Department of Neurosciences and Behavioral Sciences, Ribeirao Preto Medical School, University of Sao Paulo – Ribeirao Preto-SP, Brazil

2 - Department of Surgery and Anatomy, Ribeirao Preto Medical School, University of Sao Paulo – Ribeirao Preto-SP, Brazil

3 - Department of Physics and Mathematics, Faculty of Philosophy, Science and Languages of Ribeirao Preto, University of Sao Paulo – Ribeirao Preto-SP, Brazil

4 - Department of Internal Medicine, Ribeirao Preto Medical School, University of Sao Paulo – Ribeirao Preto-SP, Brazil

Rationale: In temporal lobe epilepsy (TLE) patients, the histological correlates of the reduced hippocampal magnetization transfer (MTR) are not known. Since MTR is dependent on the tissue’s macromolecules, our aim was to evaluate the correlations between extracellular matrix molecules, as well as the pathological changes in cellular populations, and the MTR in drug-resistant TLE patients.

Methods: Twenty-seven TLE patients went through quantitative MRI in a clinical 3 Tesla MRI scanner. Magnetization transfer was evaluated with two 3D sequences with minimal T1 and T2 weighting (TFE = 3; TE = 3 ms; TR = 3.6 ms; flip angle = 8o; magnetization transfer saturation pulse on resonance; voxel = 1x1x3 mm; FOV = 240x180, acquisition time = 5 minutes). Age-matched controls were obtained from voluntaries (RC, n = 20) for the MRI measurements, and hippocampal sections were obtained from autopsy cases (HC, n = 10) for the pathological measurements. Immunohistochemistry was performed in formalin fixed, paraffin embedded hippocampal sections from patients that underwent surgery, as well as autopsy cases, for the evaluation of neurons, (NeuN), reactive astrocytes (GFAP), and extracellular chondroitin sulfate (CS) with specific antibodies. Neurons were evaluated as neuron density, and immunopositive area fraction was used for the evaluation of reactive astrocytes and chondroitin sulfate. Results were considered significant at p<0.05.

Results: Compared to the respective controls, TLE patients had reduced hippocampal MTR (TLE = 47.8, RC = 50.4, p < 0.001). TLE patients had increased immunopositive GFAP area (CA3: TLE = 26 %, HC = 13 %, p = 0.039; CA1: TLE = 51 %, HC = 4 %, p < 0.001; subiculum: TLE = 10 %, HC = 3 %, p = 0.002), increased CS-56 immunopositive area (CA3: TLE = 29 %, HC = 4 %, p < 0.001; CA1: TLE = 27 %, HC = 2 %, p < 0.001; subiculum: TLE = 38 %, HC = 4 %, p < 0.001), and reduced neuron density (CA3: TLE = 10x103 neurons/mm3, HC = 24x103 neurons/mm3, p < 0.001; CA1: TLE = 2x103 neurons/mm3, HC = 24x103 neurons/mm3, p < 0.001). In TLE cases, MTR correlated with neuron density in CA3 (r = 0.581, p = 0.005) and with CS immunopositive areas in CA3 (r = 0.660, p = 0.004), CA1 (r = 0.544, p = 0.01), and in the subiculum (r = 0.578, p = 0.02).

Discussion/Conclusions: Our data indicate that extracellular matrix molecules are the most significant histological correlates of magnetization transfer ratio in the hippocampus of TLE patients.

Acknowledgements/Financial Support: FAPESP (grants #2015/20840-9, #2016/17882-4, and #2017/03739-8).

033 - Hippocampal-prefrontal coordination during sleep in physiological and pathological conditions

L. R. Zacharias1; R. N. Ruggiero1; L.B. Peres1; C. Lopes-Aguiar2; J. P. Leite1

1 - Laboratório de Investigação em Epilepsia, FMRP, USP

2 - Núcleo de Neurociências, UFMG

Rationale: Hippocampal-prefrontal coordination is critical for memory formation and cognition. In slow wave sleep (SWS), sharp-wave ripples (SPW-Rs, 100-250 Hz) are coupled with cortical delta waves (2-5 Hz) and spindles (9-16 Hz) in the prefrontal cortex (PFC). During REM sleep, hippocampal theta (6-12 Hz) oscillations coordinate gamma activity (30-100 Hz). Animal models of temporal lobe epilepsy (TLE) can present rhythmopathies along with cognitive deficits. In electrical kindling model, SPW-Rs are gradually substituted by interictal epileptiform discharges (IEDs), pathological high-frequency oscillations that are also coupled to Delta and spindle events in PFC. Furthermore, theta and slow-gamma coupling can be disrupted during awake states in the kainate model. Here, we aimed to examine potential oscillatory and coupling changes in PFC and CA1 circuits during the epileptogenic process, performing a fast-kindling protocol (FK) in the basolateral amygdala (BLA) in freely moving animals and accessing correlation between substitution of SPW-Rs by IEDs, slow oscillations in PFC and cross-frequency coupling between these areas.

Methods: In adult male Wistar rats (n = 6), single tungsten wires were stereotaxically implanted in PFC and CA1 for local field potential (LFP) recordings, and stimulation electrodes were implanted in BLA for FK protocols. Sleep recordings were performed before, during and after a 3 days FK protocol (biphasic square pulses, 10 trains of 10 s duration per day, 20 min inter-train interval, 50 pps, 1 ms pulse length; n=3 rats for FK group and n = 3 for the control group). Brain states were semi-automatically classified as awake, SWS and REM states based on LFP oscillations, electromyogram and video recordings. During REM sleep we calculate the phase-amplitude modulation index (MI) of theta-gamma oscillations in CA1. During SWS, we analyze the incidence and substitution of SPW-Rs by IEDs in CA1 during epileptogenesis and their coupling with delta-waves and spindles in PFC. All procedures were approved by the Ethical Committee on Animal Research (protocol 107/2016).

Results: Coupling between theta and low-gamma oscillations in CA1 during REM sleep was increased along kindling application (+44%, 33% and 108% compared with baseline conditions), in the first, second and third day of FK application, respectively (MI vs days, ANOVA (F5,708 = 19.69, p < 0.001)). In control group, MI didn’t differ between days of experiment (MI vs days, ANOVA (F5,664 = 1.91, p = 0.091)). In SWS, preliminary data analysis suggested a gradual replacement of SPW-Rs (from 0.6 to 0.1 events/s) by increasing IEDs (from 0 to 0.3 events/s) along kindling application, and IEDs coupling with delta and spindle oscillations in PFC.

Discussion/Conclusions: In this study, we present electrophysiological evidence that alterations in theta-low-gamma coupling in REM sleep can occur during epileptogenesis process, which could alter memory consolidation processes. In SWS, preliminary data analysis suggests that FK can reproduce electrophysiological effects of classical kindling protocols, inducing substitution of SPW-Rs by IEDs. Ongoing experiments will test whether the aforementioned findings are associated with cognitive impairments after FK.

Acknowledgements/Financial Support: The authors would like to thank CAPES, CNPq and FAPESP for the financial support.

034 - Positive allosteric modulation of L-Glutamate transporter EAAT2 as strategy for neuroprotection in an in vivo model of Temporal Lobe Epilepsy induced by pilocarpine.

Prizon, T.1; Liberato, J.L.1,2; Fontana, A.C.K.3; Santos, W.F.1,2

1 - Laboratory of Neurobiology and Venom, Postgraduate in Psychobiology, Faculty of Philosophy, Sciences and Literature of Ribeirão Preto (FFCLRP), University of São Paulo.

2 - Institute of Neuroscience and Behavior ‒ INeC, Ribeirão Preto, São Paulo.

3 - Department of Pharmacology and Physiology, Drexel University College of Medicine Philadelphia, Pennsylvania.

Rationale: Temporal Lobe Epilepsy (TLE) is the most frequent type of epilepsy characterized by the presence of spontaneous and recurrent seizures and hippocampal sclerosis. The basic premise in epileptic syndromes is an unbalance between excitatory (L-Glu) and inhibitory (GABA) neurotransmissions, resulting in hyperexcitability, due to an increase of extracellular L-Glu concentration in the hippocampus and reduced expression of L-Glu transporters subtype EAAT2. These processes trigger various alterations as mitochondrial damage and neuronal inflammation leading to cell death, aspects that were reportedin an initial injury by Status Epilepticus (SE). Emerging perspectives into modulation of glutamatergictransporters have risen. In this work, we have investigated a novel compound that was characterized as a positive allosteric modulator (PAM) of EAAT2, with the goal of developing therapies for the treatment of TLE.

Methods: This experiment was performed in accordance with the guidelines of the Ethics Committee in Animal Use of FFCLRP (protocol number 2017.5. 290.59.4). Three groups of male Wistar rats (280-350g/ 60 days old) were submitted to a model of TLE, by administration of methylscopolamine (1mg/kg; i.p.) followed 30 minutes later by pilocarpine-induced (380 mg/Kg, i.p.) SE. After a 90 min period of SE, rats were anesthetized with thiopental sodium (30 mg/Kg, i.p.) and just two groups (PILO-Saline and PILO-PAM) were undergone for surgical implantation of an osmotic pump (Alzet, 2001D model, s.c.). The pumps were used for continuous administration of either (a) 0.9% saline (n=8) or (b) PAM compound (6.35 µg/µl) with a final concentration after 25h of infusion 1.27 mg/ml, equivalent to 4.06 µg/g(n=7). One group of rats (n=7) was submitted to the all procedures before SE except osmotic pump implantation (group PILO) and, consequently, received no treatment. 72h later, the rats were euthanized, and the hippocampus was removed and processed for histological analysis of neurodegeneration with Fluoro-Jade C (FJC) and glial immunoreactivity with GFAP. The results were expressed as the means +-S.E.M and one-way analysis of variance (ANOVA) with Student-Newman-Keuls post-test considering p<0.05 was used as statistical analyses.

Results: Significant differences were observed at the level of neuronal death, as observed by lower levels number of positive cells of FJC staining in the hilus region of the PILO-PAM group compared to PILO-Saline and PILO groups (F(2.22)=4.780;p=0.0189). No FJC-labeled cells were observed in the dentate gyrus (DG), on the animals that were treated with the PAM compound. No significant differences were observed in the levels of neuronal inflammation with GFAP, among the three groups evaluated.

Discussion/Conclusions: It is known that the initial injury triggered by the SE in the pilocarpine model promotes changes in neuronal circuits, modifying protein function through post-transcriptional regulation andkinetics of ion channels due to membrane depolarization, and activation of an inflammatory process, resulting in increased extracellular concentration of L-Glu and neuronal death. The hippocampal excitation evidenced in this TLE model results in cellular loss in the hilus, CA1 and CA3 regions of the hippocampus, with the DG region being more resistant to cellular death. In addition, the treatment with the PAM compound resulted in areduction of FJC+ cells in the hilus, the most susceptible region, and complete absence of FJC+ staining in the DG, the first layer that receives inputs from the entorhinal cortex. Despite nodifferences in the inflammatory process observed among groups, we hypothesize that administration of the PAM compound could be developed into a treatment for TLE since neuroprotection was observed in the hilus and DG regions of the hippocampus.

Acknowledgements/ Financial Support: The authors would like to thank Joseph Salvino (Department of Pharmacology and Physiology, Drexel University College of Medicine) for the synthesis of the compound and the team of the Laboratory of Epilepsy Research (Department of Neuroscience and Behavioral Sciences, Ribeirão Preto Medical School, University of São Paulo) for the materials to immunohistochemistry process.

035 - Epilepsy comorbidities in the Wistar Audiogenic Rat (WAR) strain: physiological and behavioral consequences of inbreeding selection for seizure susceptibility.

Godoy, L.D.1; Bacchiega, I.B.1; Guzzi, J.P.M.1; Umeoka, E.H.L.2; de Oliveira, J.A.C.1; Garcia-Cairasco, N.1,2.

1 - USP - Departamento de Fisiologia, FMRP, Universidade de São Paulo (Av. Bandeirantes, 3900).

2 - USP - Departamento de Neurociências e Ciências do Comportamento – Programa de Saúde Mental, Universidade de São Paulo (R. Ten. Catão Roxo, 2650).

Rationale: Depression and stress-associated disorders are very common psychiatric comorbidities in epilepsy, often followed by complex neurobiological changes that may lead to worse outcomes such as increased pharmacoresistence. Early life adverse events constitute an important environmental aspect for both epilepsy and depression susceptibility. Wistar Audiogenic Rat (WAR) is a strain selected from the inbreeding of Wistar rats susceptible to audiogenic seizures. Accordingly, physiological and behavioral abnormalities make WAR a valuable tool to investigate comorbidities in epilepsy.

Methods: The current study aims to evaluate: (1). Maternal care in WAR as compared to Wistar rats under undisturbed and stressful conditions. Dams (n=5-9) were daily monitored during the lactating period for 1 h in undisturbed condition (30 events/block) and were subjected to the pup retrieval test on lactating day 2 and maternal defense test on lactating day 7. (2). Depressive-like behaviors basally and after audiogenic kindling (Auk; 20 acoustic stimuli 2x/day). Male rats (n=8-13) were submitted to AuK (Wistar-AuK;WAR-Auk) either post-natal (PN) at PN60-70 or PN70-80, while respective control groups were not acoustically stimulated (Wistar; WAR). All groups were subjected to sucrose preference (PN82-85) and forced swim tests (PN88-89). Animal experimentation was approved by CETEA under number 080/2014.

Results: (1). Maternal behavior: There was no difference for arched back nursing posture, licking/grooming or time of dam in the nest in undisturbed conditions and aggressive behaviors on the maternal defense test. However, WAR had a higher latency (p=0.06; t=2.1) to retrieve the first pup, half of the litter (p=0.03; t=2.6) and last pup (p=0.04; t=2.4) in the pup retrieve test. Also, all Wistar dams grouped the entire litter, whereas in WAR this happened only in 40% of the dams (p=0.07; x2=3.11). (2). Depressive-like behaviors: WAR-AuK group significantly increased sucrose consumption compared to control groups (p<0.001; F(1, 46)=10.13), both when tested 3 or 13 days after AuK. To test if the increased sucrose consumption was not related to metabolic function, Wistar and WARs were tested in individual metabolic cages. WAR present a significant decrease in body weight, water and food intake, compared to Wistar rats (p<0.001; t-test). Although there is no difference in urine volume between the two strains, WAR showed a significant lower urine osmolarity (p<0.05) than Wistar. However, since WAR did not differ from Wistar in sucrose preference test under basal conditions, these metabolic changes do not seem to be related to reward-seeking behavior. In the neuroethological analysis (behavioral sequences) of the forced swim test, WARs presented behavioral repertoire poorly connected in comparison to Wistar, in both pretest and test sessions. Furthermore, WAR and WAR-AuK presented significant reduction (p=0.0162; Two-way RM-ANOVA) of climbing in the test session, when compared to Wistar.

Discussion/Conclusions: Based on these findings we suggest that inbreeding selection had a significant impact on stress-coping behavioral strategies during lactation, adulthood and on metabolic function. Both this early- and late-life changes related to stress might have implications on seizure susceptibility. In a bidirectional perspective it is noteworthy that chronic seizures (Auk) also change depressive-like behaviors and may alter the reward system. To conclude, strains such as the WAR strain provide interesting contributions for underpinning aspects on epilepsy comorbidities.

Acknowledgements/Financial Support: FAPESP, CAPES-PROEX, FAEPA, CNPQ.

036 - Behavioral consequences of intrahippocampal pilocarpine-induced Status Epilepticus in the mouse

Sales, I.R.P.*¹; Araújo, C.P.*¹; Santos, A.N.¹; Queiroz, C.M.¹

1 - Brain Institute, Federal University of Rio Grande do Norte.

*Equal contribution

Rationale: Temporal lobe epilepsy (TLE), the most common form of focal epilepsy, is usually associated with a high prevalence of psychiatric comorbidities. Systemic administration of pilocarpine (PL) or kainic acid (KA) in rodents are used to model the human pathology by inducing long-lasting Status Epilepticus (SE) and chronic cognitive impairment. However, systemic administration of chemoconvulsants results in widespread cell death and synaptic reorganization throughout the brain, raising the question of which brain region is responsible for each behavioral deficit. Unilateral intrahippocampal administration of KA has long been used to constrain abnormal reorganization to the hippocampus and limbic system, although severe behavioral impairment is also observed in the chronic period. To our knowledge, few studies have used local injection of PL, instead of KA, as a model of TLE and none have evaluated the acute and chronic effects of its unilateral intrahippocampal administration in mice.

Methods: Male adult C57Bl/6 mice (10-14 weeks old) received 1 μL either PL in 4 doses: 70 (n = 21), 245 (n = 5), 400 (n = 7) and 700 μg (n = 30), or saline (n = 21) in the dorsal hippocampus through a glass micropipette during isoflurane anesthesia (1-1.5%). Positive controls included animals treated with intrahippocampal administration of KA (20 mM, 50 nL, n = 30) or hyperosmotic saline solution (NaCl 30%, n = 5) and intraperitoneal administration of PL (280 mg/kg, n = 11). Abnormal behavior and SE were video-monitored, and seizure occurrence and severity were scored for 2 hours after substance administration, some animals (PL700 and KA group) underwent electrode implantation to record local field potential in the hippocampus and cortex during SE. One month after treatment, animals were behaviorally evaluated for their cognitive performance using open field arena, object recognition task, Barnes maze and saccharin consumption (approval by the ethical committee # 070/2015).

Results: Unilateral intrahippocampal administration of PL resulted in a long-lasting SE without tonic seizures and a very low mortality rate (4/62 and 10/11 for intrahippocampal and systemic routes, respectively). SE severity, and the associated weight loss in the following days, positively correlated (R2 = 0.96; p = 0.024) with the dose of PL given. Despite locally applied, epileptiform were recorded in both the ipsilateral and the contralateral hippocampi after 700 μg PL and KA. After 30 days, PL-treated animals showed increased ambulation in the open-field (PL70 and PL700, p < 0.05) and impaired performance in the object recognition task, like KA-treated animals (p < 0.01). However, only the latter performed worse than controls on the Barnes maze and saccharin consumption tests (p < 0.05 in both). No acute seizures were observed in animals receiving hyperosmotic saline.

Discussion/Conclusions: The present data validate intrahippocampal administration of PL in mice as an alternative model of TLE in which the cognitive impairments are subtle in comparison to KA-induced SE. Of note, intrahippocampal injection showed lower mortality rate in comparison to systemic administration. Although spontaneous behavioral seizures were observed after the high dose of PL, future studies will determine whether these events differ in frequency, severity, and propagation from the ones presented in the KA and systemic PL models.

Acknowledgements/Financial Support: CAPES, CNPq, FINEP.

037 - Oscillatory disturbances of hippocampal-prefrontal circuits during sleep in an experimental model of Parkinson’s disease

Mendes, R.A.V¹; Padovan-Neto, F.E.²; Ribeiro, D.²; Fisone G.³; Carmo, F. A. C¹; Beraldo, I.J.S.¹; Mendes, E.M.A.M.¹; Moraes, M.F.D.¹; Lopes-Aguiar, C.¹

1 - Núcleo de Neurociências, Depto Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais

2 - Departamento de Psicologia, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo

3 - Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.

Rationale: Nonmotor symptoms are an increasingly urgent problem for the therapy of Parkinson’s disease (PD), a common neurodegenerative disorder typically characterized by tremor, rigidity and bradykinesia. Sleep disturbances, such as sleep fragmentation and rapid eye movement (REM) sleep behavioral disorder (RBD), as well as circadian rhythm dysfunction affect the vast majority of parkinsonian patients. Psychiatric conditions, including affective and cognitive disorders, are also commonly associated with PD and still not well characterized. In this work, our aim was to study the sleep fragmentation, the RBD and associated abnormal oscillatory activity in the 6-hydroxydopamine (6-OHDA) rat model of PD. Interestingly, this model also shows impaired cognitive performance, which is another frequent non-motor symptoms observed in PD patients.

Methods: Studies were performed on 19 male Wistar rats weighing 250 g at the beginning of the experiments. Rats were deeply anesthetized with a mixture of ketamine and xylazine and received a microinjection of either vehicle (0.1% ascorbic acid in 0.9% saline solution; Sham group, n=10) or 6-OHDA (8 μg/4 μL of vehicle, n=9) into the right medial forebrain bundle (MFB). We evaluated the effectiveness of the 6-OHDA lesion 4 weeks post-lesion by assessing potential deficits in forelimb movements using a stepping test. Five weeks after the injection, all rats were implanted with tungsten electrodes in CA1 stratum pyramidale of hippocampus and in medial prefrontal cortex, ipsilateral to the lesion. In a different set of animals, we performed LFP recordings of hippocampal CA1 and prefrontal cortex, which has been shown to play a role in memory consolidation during sleep. We also implanted an electrode in the neck muscles for EMG recordings. All protocols were approved by CEP-UFMG nº 41/2017.

Results: To select rats with presumably severe dopaminergic cell depletion in the substantia nigra compacta, forelimb akinesia was assessed using the stepping test. Animals were tested before surgery and 4 weeks after unilateral vehicle or 6-OHDA administrations into the right MFB. Microinjection of 6-OHDA into the right MFB induced a significant effect of lesion on contralateral stepping test performance (F (1, 17) = 192.4, P < 0.0001). The effect of time was significant (F (1, 17) = 221.5, P < 0.0001) with a significant interaction (F (1, 17) = 149.5, P < 0.0001; two-way RM-ANOVA and Bonferroni post hoc test). Post-hoc comparisons revealed a significant decrease in the number of contralateral adjusting steps 4 weeks after 6-OHDA microinjection into the right MFB (P < 0.0001 vs Sham). Postmortem assessment of striatal dopamine depletion in the striatum was assessed by means of tyrosine hydroxylase immunohistochemistry. Statistical analysis revealed that all 6-OHDA-microinjected animals included in this study presented 90% dopaminergic fiber loss in the ipsilateral striatum (t(17)=12.05; P < 0.0001 vs. Sham; t test). To test whether this animal model of PD displays oscillatory disturbances we recorded LFPs in hippocampal-prefrontal circuits during sleep (3 controls and 3 lesioned). Data analysis is ongoing.

Discussion/Conclusions: First, we successfully standardized the total lesion animal model of PD. Now, we are analyzing the recordings we performed to access phase and amplitude coherence between the hippocampus-prefrontal pathway, as well as triple coupling of hippocampal Sharp Wave Ripple (SWR), thalamic spindles and prefrontal cortex delta waves. We hypothesize that this oscillatory activity is decoupled, and it may play a role in the cognitive disorder seen in PD patients.

Acknowledgements/Financial Support: FAPESP, CNPq, CAPES, PRPq-UFMG.

038 - Computational study of the alterations induced by Temporal Lobe Epilepsy in neuronal models of granule cell neurons

Figueiredo, A.1; Santos, A.P.S.2; Tejada, J.1,2

1 - Programa de Pós-Graduação em Ciências Fisiológicas - Universidade Federal de Sergipe

2 - Departamento de Psicologia - Universidade Federal de Sergipe

Rationale: Animal models to induce temporal lobe epilepsy, such as pilocarpine injection, provoke structural and morphological changes in the dentate gyrus such as granule cells mossy fiber (granule cell axon) sprouting, hilar cell loss, alterations in the dendritic tree of newborn granule cells, and also electrophysiological alterations. Previously, computational studies have investigated the role of morphological alterations, and in this study, we used the same methodology to investigate the possible effects of ion channel alterations on the electrophysiology of the cells.

Methods: Computational models of dentate gyrus granule cells were built using almost all of the rat granule cells three-dimensional reconstructions available on NeuroMorpho.org, generating three families of models: mature, newborn (30 days old) and altered-newborn. The latter corresponds to reconstructions from a sample of cells from animals with pilocarpine-induced Status Epilepticus. The biophysical properties of our models are based on the nine ion channels of a dentate gyrus granule cell model (‎Aradi et al. J COMPUT NEUROSCI, v.6, p.215, 1999). The granule cells were modeling following the conductance-based Hodgkin and Huxley formalism, implemented using NEURON v7.4, and changes in the density of each one of ion channels were evaluated. The effects of these changes were measured using different stimulation protocols, which consist in sets of electrodes connected to the dendritic branch in different distances from the soma. We estimated eight different measures of the neuron activity: Rheobase, Chronaxine, input resistance, membrane time constants, action potential amplitude, half-wide and threshold, firing rate and interspike interval. Using two factor ANOVA, with the respectively Bonferroni post-hoc tests, to identify significant differences.

Results: Granule cells from newborn and altered-newborn family models showed significant higher firing rates and significant lower values of Rheobase current than the mature family models (p-value < 0.05). However, the newborn family models showed values even higher than altered-newborn. A similar pattern, in which the models based on altered-newborn were more excitable than the mature ones, but less than the newborn models, was found for all of the other measures.

Discussion/Conclusions: The electrophysiological response of our models suggests that the morphological alteration presented on the altered-newborn family models contributes to reduce the neuron excitability when no other alteration, besides the reduction of ion channel density, was insert. This hypothesis is restricted, and we are aware that it is also possible, that along with the morphological alterations, the neurons present, not only alteration in the density but also in the dynamic of the ion channels, for instance, changes in some of the families of the sodium ion channels; other possibility is while the newborn neurons get less excitable the mature ones become more active. All these possibilities constitute our roadmap, and further simulation including alteration on the dynamic of the ion channels, as well as, network interactions are going preparing.

Acknowledgements/Financial Support: PROCFIS.

039 - Computational study of the alterations induced by Alzheimer disease in a neuronal model of a CA3 pyramidal neuron

Nascimento, N.M.1, Andrade, B.S.2; Tejada, J.1,2

1 - Programa de Pós-Graduação em Ciências Fisiológicas - Universidade Federal de Sergipe

2 - Departamento de Psicologia - Universidade Federal de Sergipe

Rationale: Alzheimer disease (AD) is a chronic neurodegenerative disease which, generally, affects people of 65 years and older, and it is considered the most common cause of dementia, affecting memory and cognitive functions. AD provokes alterations in the hippocampus and its primary neurons: the pyramidal cells. Different studies have tried to understand the cause of the AD, however it is still poorly understood. There are several competing hypotheses, one of them is the beta-amyloid theory (Aβ), which states that AD begins with an abnormal processing of the Aβ trans-membrane precursor protein, which accumulates in the cell membrane from the soma growing towards the dendrites, forming the senile plaques, and causing alterations in both the structure and function of the pyramidal cells. Our study is an attempt to computationally reproduce the effects of Aβ accumulation, such as alterations in ion channel density, and reduction of dendritic diameter and spine size.

Methods: Our models are based on a hippocampal pyramidal CA3 cell with realistic morphology (‎Migliore et al. J. Neurophysiol, v. 73, n.3, p.1157, 1995) available on ModelDB. The model includes nine ion channels modeling following the conductance-based Hodgkin and Huxley formalism, implemented using NEURON v7.4. The Aβ accumulation out of the cell membrane was simulated as progressive reduction not only of ion channel density, but also the diameter thickness as well as the spine number and the alterations starting on the soma and spreading along the dendritic tree. We consider three different stages of progression: accumulation restricted to the soma, in a radius of 100 μm and 200 μm from the soma, and evaluating an accumulation range from 0 up to 100%. The effects of the alteration were evaluated using different stimulation protocols, which consist in sets of electrodes connected in different regions of the dendritic branch. We estimated eight different measures of subthreshold and suprathreshold neuron activity: Rheobase, Chronaxine, input resistance, membrane time constants, action potential amplitude, half-wide and threshold, firing rate and interspike interval.

Results: There were found significant differences (p-value<0.05) in the subthreshold and suprathreshold measures for the Na currents and for diameter reduction starting from 50% of Aβ accumulation. When evaluated separately, the Na current reduction reduces the neuron excitability, in opposition to the diameter reduction which increases that. However, when placed together the reduction of excitability predominates. But, in all of the cases, the neuronal electrophysiological behavior was robust, resisting to the alterations until an important percent of Aβ accumulation.

Discussion/Conclusions: As expected, some of the alterations induced by the Aβ accumulation reduce and some others increase the excitability of the cells, showing that the interactions between these effects are not trivial, and that even in simulated environment, the identification of Aβ accumulation effects are a challenge, in special, in the beginning of these alterations. On the other hand, the fact that in our models, the reduction of calcium and potassium currents did not show significant effects, could be explained by the relation of these currents with intracellular processes, which are not reproduced by the conductance-based model used in our simulations, due to that, more complex models are necessaries to understand the effects of the AB accumulation.

Acknowledgements/Financial Support: FAPITEC/SE.

040 - Effect of heparin trisulfated disaccharide (TD) in neuronal dead triggered by calcium overload

Chiarantin, G.M.D.1,3; Delgado-Garcia, L.M.1; Lima, M.A.2; Nader, H.B.2; Porcionatto, M.1; Tersariol, I.L.S.2

1 - Department of Biochemistry, Laboratory of Molecular Neurobiology, Federal University of Sao Paulo, Sao Paulo, Brazil.

2 - Department of Biochemistry, Federal University of Sao Paulo, Sao Paulo, Brazil.

3 - Center of Mathematics, Computation and Cognition, Laboratory of Neurogenetics, Federal University of ABC, Sao Bernardo do Campo, Brazil.

Rationale: Neurons submitted to hypoxia undergo intense cytoplasmic Ca2+ overload. High concentrations of intracellular Ca2+ can trigger processes of cell dead along the neural tissue, a pathological hallmark of stroke. Ca2+ homeostasis in neurons involves the regulation of the intracellular concentration of Ca2+ ([Ca2+]i) by the Na+/Ca2+ exchanger (NCX), which plays an important role in the efflux of this ion. Previous data published by our group showed that the Heparin Trisulfated Disaccharide (TD), ΔU, 2S-GlcNS, 6S, a product of the enzymatic degradation of the heparin, is able to accelerate Na+/Ca2+ exchange via NCX in hepatocytes, cardiomyocytes and in smooth muscle cells of aorta. Thus, the objective of this work was to verify if TD is a neuroprotective agent able to prevent the neuronal dead by the reduction of [Ca2+]i induced by the cytoplasmic Ca2+ overload.

Methods: 1. in vitro induction of neuronal dead by cytoplasmic Ca2+ overload in the murine neuroblastoma cell line (Neuro-2a). Neuronal dead was induced by elevation of cytoplasmic Ca2+ through: ATP (via purinergic receptor activation), Thapsgargin (via Ca-ATPase inhibition/ SERCA) of the endoplasmic reticulum), Aspartate and Glutamate (via NMDAR activation) in the presence or absence of TD. (2) Murine model of stroke. Was used a traditional model of stroke submitted to somatocortical ischemia by photocoagulation (Rose Bengal Dye; 0.15mg/g, intraperitoneal, single dose) - Ethics Committee number 4949190916, Federal University of Sao Paulo.

Results: (1) in vitro TD pretreatment (30 µM) was able to reduce cell dead and increase the neuronal viability triggered by cytoplasmic Ca2+ overload. Cytoplasmic transient analysis of Ca2+ showed that TD had a dose-dependent effect in the reduction of [Ca2+]i, and this effect was blocked by the NCX inhibitor, KBR7943. (2) The data indicated that the in vivo pretreatment with TD (30 µM, intraventricular, single dose) was able to decrease the estimated area of the ischemic lesion, in around 60%, effect blocked by the NCX antagonist, as well as to decrease in about 20% the apoptotic dead as showed in the TUNEL staining.

Discussion/Conclusions: Taken together, the results indicate that the protective effect of TD in the cell viability is linked to the intracellular calcium homeostasis. Also, was discussed the influence of the TD in the neuroprotection at the pre-ischemic moment, when the NCX would be acting in the forward mode. To date, TD is the only compound described that is capable of activating the forward mode of the NCX exchanger. Thus, we propose DT-NCX as a new therapeutic axis in the prevention of dead triggered by cytoplasmic calcium overload.

Acknowledgements/Financial Support: Coordination of Superior Level Staff Improvement (CAPES), National Council for Scientific and Technological Development (CNPq) and São Paulo Research Foundation (FAPESP).

041 - A behavioral and neurochemical view of the treatment of cocaine addiction, using Ayahuasca as a therapeutic agent

Bruno, V.1; Neugebauer, V.R.1; Koiama, J.R.1; Pacheco, L.H.L.T.2; Garcia, R.C.T.3; Camarini, R.1; Marcourakis, T.1

1 - Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of São Paulo;

2 - Department of Food and Medicine; School of Pharmaceutical Sciences; Federal University of Alfenas;

3 - Federal University of São Paulo, Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences;

4 - Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo.

Rationale: Considered the second most used illegal drug in Brazil, cocaine is a substance with psychostimulant properties that induces robust neuroplastic changes that may lead to addiction. Currently, there are some harm reduction measures aimed at preventing relapse and reducing comorbidities. In this context, research with psychedelics resurfaces with a new focus: treatment of drug addiction. Used in religious movements such as Santo Daime, ayahuasca (AYA) is a psychedelic brew, which contains β-carbolines and dimetiltriptamina (DMT) - the active principle. There’re some evidences that it might be applicable in the treatment of alcohol and nicotine addiction. However, for cocaine, there is a lack of studies. Thus, this work aims to evaluate the possible therapeutic potential of AYA in the model of behavioral sensitization (BS) to cocaine.

Methods: Six-week-old C57Bl/6 mice were habituated in the open field apparatus (OF) for three consecutive days after administration of saline 0.9% (SAL) i.p. On the following day, the behavioral sensitization induced by cocaine (COC) protocol started. Thus, mice received, for 5 alternate days, COC 10 mg/kg or SAL 0.9% i.p. and were immediately submitted to the evaluation of locomotor activity in the OF. At the end of BS protocol, the period of withdrawal or treatment with AYA began, in which they received water or AYA (DMT 3 mg/kg v.o.) for 8 consecutive days. After 30 min of administration, the mice had their locomotor activity evaluated in the OF. Twenty-four hours later, they were challenged with SAL and the next day, a COC challenge was performed. After evaluation in OF, the animals were euthanized and had their prefrontal cortex, striatum and hippocampus dissected and prepared for analysis of D1 and D2 dopaminergic receptors by immunoblotting. (CEUA/FCF 33.2016-P518)

Results: On the second COC administration day, the acquisition of BS was confirmed, since the locomotor activity of the animals was significantly higher on this day (n=24; p<0.05). When performing a COC challenge, cocaine-sensitized mice treated with AYA presented similar locomotor activity to the control group (n=12; p>0.05). Also, a significant decrease of dopaminergic D1 receptors in the striatum on Sal-Aya-COC group was observed, when comparing to Sal-Sal- COC group (n=12; p>0.05).

Discussion/Conclusions: DMT is known to be a potent agonist and β-carbolines are partial agonists of 5-HT2A receptors and has affinity for D1R, but not for D2R. The stimulation of 5-HT2AR reduces the release of DA in the mesolimbic, nigroestrial and mesocortical pathways, via GABAergic interneurons. Therefore, AYA is expected to act in the dopaminergic system reducing the hypersensitivity triggered by COC use. Treatment with AYA at the dose of 3.0 mg DMT/kg was not able to reverse the behavioral sensitization process triggered by the acquisition of COC sensitization.

Acknowledgements/Financial Support: SENAD, FAPESP, CNPq.

042 - Popularization of neuroscience and health education focusing on the brain and in the five senses of elderly people

Granjeiro, E.M.1; Novaes, A.L.2; Souza, L.M.2; Santana, E.A.A.2; Santos, B.P.A.1; Araújo, R.H.O.1; Kalile, M.O.1, Barboni, S.A.V.1

1 - Biological Sciences, Department State University of Feira de Santana, Transnordestina Avenue, Novo Horizonte, Feira de Santana - BA, 44036-900

2 - Heath Department, State University of Feira de Santana, Transnordestina Avenue, Novo Horizonte, Feira de Santana - BA, 44036-900

Rationale: Of great impact in the regional community, the State University of Feira de Santana (UEFS) is a public institution of higher education in Brazil, based in the city of Feira de Santana, the second-most populous city in the state Bahia, with a population of 612,000 according to IBGE’s estimate. Until the 1990s, UEFS was also the only University in this city. Currently, the UEFS has 28 graduate courses, offering 1.926 places by year for the third degree, with important graduate programs (e.g., Biological Sciences and Health Department), as well as, important programs to attend the community, including the University Open to the Third Age (UATI). UATI UEFS has as main objectives to stimulate the promotion of health, the socialization of knowledge, sports practice, conscious exercise of citizenship, social relations, leisure, art, culture, inclusion digital integration, reintegration into the productive sector, socio-community reintegration, strengthening of family ties, psychosomatic balance, preparation for the Third Age and lifelong education. Recently, in order to contribute to the development of studies related to the dissemination and popularization of Neuroscience in the Third Age, the Nucleus of Research in Teaching and Extension in Sciences and Biology (NUPEECBio) and the Inter-transdisciplinary Nucleus of Education, Research and Extension of Health Education (NIEPEXES), Biological Sciences and Heath Department, respectively, organized the workshop “The Brain and the five senses in the Third Age”.

Methods: The workshop included a cluster of activities of Health Education involving students from several courses in Education and Health at UEFS, including Medicine, Nursing, Licentiate courses in Physical Education and Biological Sciences. Educational actions were carried out emphasizing the knowledge of the relation between brain, body, consciousness and emotion, as well as, the five senses and aging. Several activities were directed to prevention, stimulating activities, and daily exercise needed to keep their five senses (taste, touch, sight, hearing, and smell) at the peak of their capabilities for as long as possible, since with aging, these five senses undergo changes that can cause to decrease their effectiveness, which can have an impact on health and mental mindset. For this, there were dialogues, debates, informative murals, workshops and application of educational games as goals to rescue, pass on and discuss basic information on health with the elderly group. After the activities, we used the Likert Scale to measure the level of satisfaction of individuals.

Results: The actions related to the workshop had great acceptance by elderly people. In this context, 95% of the individuals related enthusiasm to participate of the Educational activities and said that they wish to return in new editions of the event.

Discussion/Conclusions: The workshop provided interdisciplinary work, promoting health education, contributing to the dissemination of the benefits of scientific research in the Neuroscience area. Considering that, it is important to live a lifestyle that promotes healthy living, more efforts are going to be made to that new editions may be executed in a greater number of elderly people, narrowing the gap and increasing the dialogue between university and community.

Acknowledgements/Financial Support: The Pro-Rector of Extension (PROEX), Biological Sciences Department (DCBIO), Health Department (DSAU), Open University of the Third Age (UATI) of State University of Feira de Santana by the technical support. New Talents Program CAPES UEFS (055/2012), PIBID CAPES UEFS (061/2013), PROEX UEFS.

043 - Analyses of gene expression and DNA methylation pattern of the β-catenin gene in iPSCs of patients with focal cortical dysplasia

Marinowic, D.R.1,2; Zanirati, G.1,3; Zanatta, A.1,2; de Souza, E.V.1,4; Ghilardi, I.1,4; Santos, P.G.1,4; Majolo, F.1,2; Paglioli, E.5; Palmini, A.5; Machado, D.C.1; Costa, J.C.1

1 - Brain Institute of Rio Grande do Sul (BraIns), Porto Alegre, Brazil;

2 - Graduate Program in Medicine and Health Sciences, Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Brazil;

3 - Graduate Program in Medicine, Pediatrics and Child Health, Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Brazil;

4 - School of Science, Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Brazil;

5 - Epilepsy Surgery Program, Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Brazil.

Rationale: Focal cortical dysplasia (FCD) is a brain disorder closely related to refractory epilepsy. It is characterized by brain cortex malformation and may present some cytological alterations and abnormalities in the cortical structure. The adhesion protein β-catenin plays a key role in cell migration and cell adhesion mainly during the embryonic development. Cytosine methylation is an epigenetic mechanism of gene regulation which is often related to down-regulation of gene expression. The technique of induced pluripotent stem cells (iPSCs) generation is an attractive alternative for disease modeling because the easiness to have access of different cell types, such as skin fibroblasts and blood cells, from several patients. The objective of this study is to analyze the expression and the specific methylation pattern of CTNNB1 gene, which codifies the β-Catenin protein, in different tissues and iPSCs of patients with FCD.

Methods: Fibroblasts were isolated from residual skin fragments obtained from 7 patients submitted to surgical treatment, and brain samples were obtained during surgical resection (Pct#1: 45-year-old, male; Pct#2: 12-year-old, female; Pct#3: 37-year-old, male; Pct#4: 17 year- old, 17 female, Pct#5: 13 year-old, male, for FCD patients and Ctr#1 35-years-old, male; Ctr#2 29 year-old, female) iPSCs were generated through exposure of fibroblasts to viral vectors containing OCT4, SOX2, KLF4, and c-MYC genes. The iPSCs were neurodifferentiated in neurogenic medium with BDNF for 35 days. The RNA and DNA were extracted in 14, 22 and 35 days of iPSC neurodifferentiation. The CTNNB1 gene expression was performed by qRT-PCR and DNA was bisulfite converted. The present study was approved by the Committee of Research Ethics of the Pontifical Catholic University of Rio Grande do Sul (no. 17943213.9.0000.5336) through the system Platafoma Brasil.

Results: The β-Catenin expression was very similar in comparison of tissues (fibroblast: 1.3 times, brain: 1.2 times, and iPSC: 2 times) from FCD patients and control. During iPSC neurodifferentiation, the control group showed an increase of β-Catenin expression (6 times) comparing with non-differentiation iPSCs whereas the FCD group did not show this increase (1.48 times in 14 days, 3.3 times in 22 days and 2.5 times in 35 days).

Discussion/Conclusions: Our results suggest that the lower expression of β-Catenin during the neurodifferentiation of patients with FCD may not be performing migration through an appropriately radial framework due to the difficulty of adhesion of the migrating neurons to the radial glia, altering the migratory course or making it difficult to reach the cortical plate or generating retardant neurons that do not reach its destination and consequently forming the aberrant cortex with presence of giant or balloon cells in the white. These samples which are bisulfite converted need to be amplified by PCR and their methylated sequences determined after performing the Methylation-sensitive high-resolution melting, providing useful results which may be correlated to the lower expression of the β-Catenin gene in FCD patients. Sequencing of this gene will also be performed to check for possible mutations associated with cortex malformation of these patients.

Acknowledgements/Financial Support: CNPq, CAPES and Pandurata.

044 - Lack of association between COMT Val158met polymorphism and Juvenile Myoclonic Epilepsy

Santos Júnior, E.D.1; Silva, I.l.1; Santos, Y.D.R.1; Araújo, M.A.1; Santos, B.P.1; Gitai, D.l.G.1.

1 - Department of Cellular and Molecular Biology, Institute of Biological Sciences and Health, Federal University of Alagoas, Maceió, Alagoas, Brazil.

Rationale: Juvenile myoclonic epilepsy (JME) is the most common form of generalized epilepsy. The most frequent clinical manifestations are myoclonus, predominantly when awakening, generalized tonic-clonic seizures and absence seizures. The genetic component is an important factor in the etiology of JME, which may show Mendelian or genetically complex inheritance. Therefore, the identification of susceptibility genes for JME has aroused the interest of researchers. In this context, the gene encoding for the enzyme Catecholamine O-methyl Transferase (COMT), whose function is the deactivation of catecholamines in the synaptic cleft, is a strong candidate. The synaptic activities performed by these neurotransmitters may contribute to the generation and modulation of epileptic seizures. Thus, the objective of this study is to investigate whether the COMT val158met polymorphism is associated with JME.

Methods: Genotyping of patients diagnosed with EMJ and controls from state of Alagoas was performed by PCR-RFLP and the statistical analyzes were done through the SNPStats platform. In this investigation, we used samples from the DNA bank of patients with JME (n = 96, 63 females, mean age of 25 years) and control subjects (n = 200, 133 females, mean age of 30.5 years) established by our research group and maintained at the Laboratory of Molecular and Cellular Biology - UFAL. To verify if the populations studied were in Hardy-Weinberg equilibrium the chi-square statistical test was performed, using different inheritance models. The study was approved by the Ethics Committee of the Federal University of Alagoas, Brazil and all subjects gave written informed consent before blood collection was performed (CAEE: 17265313.6.0000.5013).

Results: No significant differences were observed in the genotype and allele frequencies of this polymorphism between cases and controls. The mean age at onset of the JME probands’ was 13.4 (range 4-27) years, and 65.6% of them were females. The triad of myoclonus, absences, and generalized tonic-clonic seizures was observed in 69.8% of the patients, the combination of myoclonus and generalized tonic-clonic seizures in 25%, the combination of myoclonus and absence was observed in 2% and the myoclonus alone in 2% of patients. Of the 96 patients, 66.7% of patients were on monotherapy and the remaining patients were on polytherapy. The genotype distribution did not deviate significantly from that expected by Hardy–Weinberg equilibrium (p = 0.28), as estimated by chi-square test. The Proportions of G homozygotes, G/A heterozygotes and A/A were 34%, 47% and 19% in EMJ patients, whereas in control group were 32%, 55% and 14%, respectively. The allele frequencies of G and A were, respectively, 58% and 42% in EMJ patients and 41% and 59% in control group. Genotype proportions and allele frequencies for the COMT val158met polymorphism in both groups were not significantly different (p=0.22).

Discussion/Conclusions: The results present no evidence for an association of COMT val158met with JME. Further studies including other functional polymorphisms are required to investigate the involvement of COMT gene in the genetic susceptibility to JME.

Acknowledgements/Financial Support: The authors thank all the individuals who participated in the study and the Neurology care unit team of Professor Alberto Antunes Hospital/Federal University of Alagoas.

045 - A new model of experimental hemispherotomy in young adult Rattus norvegicus: a neuro tract-tracing and SPECT in vivo study

Matias Jr., I.1, Elias-Filho, D. H.1, Garcia, C. A. B., Silva, G. H.1, Mejia, J.2, Cabral, F. R.2, Miranda, A. C. C.2, da Silva, S. G.2, Lopes, L. da S.1, Coimbra, N. C.2, Machado, H. R.1

1 - Ribeirão Preto Medical School of the University of São Paulo.

2 - Hospital Israelita Albert Einstein

Rationale: The experimental areas of neurosurgery and rehabilitation do not address the technique of cortical resection and disconnection, hemispherotomy, which reproduces the clinical conditions after surgical disconnection for control of seizures in epilepsy syndromes children. The use of a new experimental surgical model is relevant to investigate the mechanisms and effects of the recovery in the developing brain.

Methods: Twenty - six young male Wistar rats (weighing 260 ± 10g) were distributed in two groups (experimental surgery - ES and control - CT). The CT group was composed of five animals divided into two subgroups: neuro-trace (n = 4) and SPECT (n = 1). The ES group consisted of 21 animals. Regarding this group: we used 13 rats (eight animals died) and were used with the following distribution: neuro-trace (n = 10) and SPECT (n = 3). Stages of surgery: (a) frontal disconnection; (b) cortical resection; (c) callosotomy; (d) hypocampectomy; (e) temporary disconnection; (f) posterior disconnection. To identify disconnected fibers and ways after ES, the non-fluorescent anterograde neutron-biotinylated amine dextran (BDA 10000 MW) was microinjected on the right side of motor cortex (M1), according to   atlas  stereotaxic   Paxinos and Watson   (2007) as follows: anteroposterior = 0.12 mm; mid-lateral = 2.0 mm and dorso-ventral = 5.8 mm. Two weeks after surgery, the rats of both groups: (a) CT (n = 1) and ES (n = 3) were anesthetized and obtained ( SPECT images), to verify perfusion in functional areas of the brains of animals. Evaluation of locomotor activity (Rotarod Test), we used 30 young adult male Wistar rats, equally and randomly distributed in three groups: CT, Sham ( craniotomy ) and ES.

Results: In CT, BDA-labeled corticostriatal pathways were found in the microinjection sites of BDA, with visualization of the neurotracer reaching the contralateral cerebral cortex. In the ES group, microinjections of the neurotracer in M1 showed fibers marked ipsilaterally with BDA, rarefaction of interhemispheric projection and absence of BDA marking in the contralateral hemisphere. In the CT group in the SPECT images, we can observe that the cerebral perfusion was uniformly distributed in the cerebral hemispheres, but in the ES group, there was hypoperfusion ipsilateral to surgery. With regard to behavioral activity, there was a tendency to recover the functional deficit over time. From the analysis of two-way variance ( ANOVA), we observed a significant treatment effect [F (34,24) = 2; P <0.0001], time [F (2.19) = 27; P <0.007], and treatment versus time interaction [F (4) = 21.26; P <0.0001]. Initially, there was an increase in the number of attempts to maintain the balance in the rotating rod of the Rota-Rod Test in 10 days [post hoc Bonferroni test ; P <0.001 and 30 days [post hoc Bonferroni test ; P <0.001] after ES, compared to the CT and Sham group.

Discussion/Conclusions: The proposed experimental model (hemisferotomy) was technically feasible and comparable to the surgical procedures currently used in patients with refractory epilepsy it allows investigating the mechanisms and effects of recovery in the developing brain.

Acknowledgemens/ Financial support: National Counsel of Technological and Scientific Development – CNPq; Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo – FAEPA; The São Paulo Research Foundation – FAPESP; Coordination for the Improvement of Higher Education Personnel- CAPES.

046 - N-formyl-methionyl-leucyl-phenylalanine (fMLP) and annexin A1 N-terminal derived peptide (Ac2-26) decreased neurodegeneration process in hippocampi in the pilocarpine-induced Status Epilepticus

Freitas-Santos, J.1; Melo, I.S.1; Pacheco, A.L.D.1; Santos, Y.M.O.1; Costa, M.A.1; Cavalcante, C.M.B.1; Oliveira, K.B.1; Duzzioni, M.1; Sabino-Silva, R. 1,2; Borbely, A.U.1; Castro, O.W1.

1 - Department of Physiology, Institute of Biological Sciences and Health, Federal University of Alagoas (UFAL), Maceió/AL.

2 - Institute of Biomedical Science (ICBIM), Federal University of Uberlandia (UFU). Uberlândia/MG.

Rationale: Status Epilepticus (SE) is defined as continuous and self-sustaining seizures, which trigger hippocampal neurodegeneration, inflammation and gliosis. From the first seizure, astrocytes become reactive, secreting cytokines and chemokines such as IL-1B, tumor necrosis factor (TNF-α), interleukins (IL-1, IL-6, IL10) and interferons (IFN- IFN-β). Annexin A1 (AnxA1) protein is an important endogenous inhibitory regulator of inflammation that is activated in response to tissue injury. Evidence indicates that AnxA1 mediates most of biological functions via interaction with N-formyl peptide receptor (FPR). In addition, it has been reported that N-formyl-methionyl-leucyl-phenylalanine (fMLP), a N-formylated peptide and bioactive peptide derived from AnxA1 N-terminus (Ac2-26) exert an anti-inflammatory role, mediated by activation of G-protein-coupled formylpeptide receptor (FPR). Reduction of inflammation process may contribute to attenuate neurodegeneration caused by epileptic seizures. However, the effects of the administration of fMLP or Ac2-26 during SE are unknown. In this study, we evaluated the influence of these peptides in hippocampal neurodegeneration.

Methods: Experimental procedures were approved by the Ethical Committee for Animal Research of UFAL (04/2016). Male Wistar rats (n=15; 240-340g) were submitted to stereotaxic surgery for cannula implantation in the hilus of the dentate gyrus of left hippocampus. Animals received unilateral microinjections of pilocarpine (PILO) in hippocampus (H-PILO, 1.2mg/μL, 1 μL). After 5 minutes of H-PILO, groups received microinjections of peptides fMLP (1 mg/mL, 1 μL), or Ac2-26 (3 mM, 1 μL) or vehicle (VEH, saline 0.9%, 1 μL) [in the same site]. Animals were perfused after 24 hours of SE and neurodegeneration was evaluated by histochemistry of Fluoro-Jade (FJ). FJ positive neurons (FJ+) were counted (ImageJ–NIH) in hippocampus. Results were expressed as mean±SEM, compared by unpaired t test and one-way ANOVA followed by post-hoc test Student-Newman-Keuls.

Results: The administration of peptides (fMLP or Ac2-26) after H-PILO resulted in a different pattern of neurodeneration in both hippocampi. In ipsilateral hippocampus, Ac2-26 reduced the total number of FJ+ neurons [t(6) = 2.94, p < 0.05] only in the hilus of the dentate gyrus, while fMLP promoted the same effect in CA1 [t(9) = 2.35, p < 0.05]. On the other hand, both peptides were able to decrease neurodegeneration process in all areas of contralateral hippocampus [Hilus (F2, 10 = 9.46; p < 0.005), CA3 (F2, 11 = 23.51; p < 0.0001) and CA1 (F2, 11 = 22.79; p < 0.0001)].

Discussion/Conclusions: Our preliminary findings suggest a new insight into the functional role of fMLP and Ac2-26 peptides, with important implications for their potential use as therapeutic agents for the treatment of brain disorders, such as epilepsy.

Acknowledgements/Financial Support: FAPEAL, CAPES, CNPQ. Members of the Laboratory of Neuropharmacology and Integrative Physiology of the Department of Physiology and Pharmacology, Institute of Biological Sciences and Health, Federal University of Alagoas (UFAL).

047 - Investigation of the influence of typical sleep oscillations states in the synaptic plasticity in hippocampus-prefrontal cortex pathway of rats in vivo

Peres, L.B.¹; Zacharias, L. R.¹; Leite, J. P.¹; Lopes-Aguiar, C.²

1 - Laboratório de Investigação em Epilepsia, FMRP, USP;

2 - Núcleo de Neurociências, UFMG.

Rationale: Hippocampal-cortical communication seems to be crucial in task learning and declarative memory consolidation. However, the mechanisms by which hippocampal activity modulate the synaptic efficacy in the CA1-mPFC pathway remain poorly understood. In vivo studies propose that different oscillatory sleep states could trigger synaptic homeostatic processes. Our hypothesis considers that oscillatory states would be critical in synaptic weight adjustment of hippocampal-cortical connections. Based on that, this study aims to evaluate if different oscillation states (rapid eye movement (REM)/activated or n-REM/deactivated) and cortical delta waves incidence are sufficient to modulate synaptic efficacy in the CA1-mPFC pathway.

Methods: (CEUA protocol n° 208/2017) Wistar rats (300-350 g, n = 3, males) were anesthetized with Urethane (1.25 g/kg), and tripolar electrodes were stereotaxically implanted in both CA1 and medial prefrontal cortex (mPFC) for electrophysiological recordings and electrical stimulation. Two stainless steel screws were used as electrical references, implanted over the occipital and parietal bone. The experiment consisted in one hour of local field potential (LFP) recording as baseline and field post-synaptic potential stimulation in the CA1-mPFC pathway (fPSPs, biphasic square-pulses, 0.05 Hz, 0.2 ms pulse length, applied in CA1), with mPFC and CA1 recording. Later, high- frequency stimulation (HFS, biphasic square-pulse, 2 trains, 0.2 s train length, 250 Hz, 0.2 ms pulse length) was applied to induce long-term potentiation (LTP) and followed by two more hours of mPFC recording along with fPSPs stimulation. LFP oscillations were classified as REM-like (or active) and n-REM-like (or deactivated) states for comparison of fPSPs amplitude and slope in both states, and analysis were performed before and after LTP inducing. Occipital reference was used for state classification, and parietal reference for fPSPs analysis. All the mathematical analyses were done using MatLab 2017a, SigmaStat 3.1 and Minitab 17 softwares.

Results: The amplitude of fPSPs incidents in activated states was higher than in deactivated states, with a mean difference of 8.5% (p=0.000, t-test) during baseline and a mean difference of 36.6% (p < 0.002, t-test) post-LTP. The slope of fPSPs also presented higher values in activated states, with a mean difference of 16.7% (p=0.000, t- test) in baseline and 9.5% post-LTP (p<0.001, t-test).

Discussion/Conclusions: We observed that synaptic efficacy in the CA1-mPFC pathway can be modulated by oscillatory states (activated/deactivated). This modulation appears to be higher after induction of LTP, reinforcing the idea of the synaptic downscaling and homeostasis role of slow wave (n-REM/deactivated) oscillatory state.

Acknowledgements/Financial Support: We would like to acknowledge FMRP-USP and NNC-UFMG for the structure that allowed the accomplishment of this work, and funding agencies Capes, CNPq e Fapesp for the financial support.

048 - Evaluation of the antiepileptic potential of the bioinspired peptide Occidentalina-1202

Quintanilha, M.V.T.1; Mortari, M.R.1

1 - Laboratório de Neurofarmacologia, Departamento de Ciências Fisiológicas, Universidade de Brasília.

Rationale: Epilepsy refers to a set of neurological diseases characterized by excessive activity of neurons which generate hypersynchronized electrical discharges. It is one of the most prevalent neurological diseases in the world, generating a need for investments in treatments that guarantee patients to control epileptic seizures. Antiepileptic drugs (AED) are commonly associated with side effects and are ineffective in seizure control in about 70% of patients with Temporal Lobe Epilepsy (TLE). Research has shown that venom from Polybia occidentalis was able to protect Wistar rats from chemically induced seizures. Thus, the characterization of a new antiepileptic peptide denominated Occidentalina-1202 was made. Peptides designed from isolated venom compounds have emerged as candidates for drug development, and therefore the purpose of this study was to evaluate the antiepileptic activity of the new bioinspired peptide Occidentalina-1202 in an acute model of epileptic seizures and thus to define EC50.

Methods: Male Swiss mice (Mus Musculus), weighing between 18 and 30 g, at 6 to 8 weeks of age (CEUA / IB, protocol 17/2017) underwent a procedure for implantation of a guide cannula in the lateral ventricle 5-6 before the Pilocarpine (250 mg.Kg-1) intraperitoneal (i.p.) injection. Prior to the injection of pilocarpine, the unilateral intraventricle administration of the peptide (6; 3 or 0.3 μg/animal) or 0.9% physiological solution was performed. The positive control animals received a prior treatment with Diazepam (4 mg/kg i.p.), n = 8 for all groups. The influence of the peptide on the occurrence and latency were recorded to detect the beginning of generalized seizures and/or death. Seizures were evaluated using a score for seizure severity and generalized seizure was considered when the animal repeatedly jumped with intense myoclonus followed by death.

Results: The Kruskal-Wallis test, followed by Dunn’s post-test, considered p <0.05 [K = 20.57] showed that the group treated with the peptide at its highest dose and the diazepam group obtained significant differences in latency for the first generalized seizure in comparison to the negative control. They also revealed that the peptide significantly increased the latency for animal death p <0.05 [K = 19.99]. The calculated ED50 was 0.8722 μg / animal, with a confidence interval of 0.3066 - 2.481 μg / animal (R2 = 0.9978).

Discussion/Conclusions: Increased the latency for generalized seizures is a parameter chosen in the work for the investigation of a possible new AED because the drugs available in the market today are ineffective in the treatment of drug-resistant epilepsies they only increase the time between the occurrence of seizures. The data described above demonstrate the potential that the peptide Occidentalina-1202 presents as a possible new antiepileptic substance.

Financial Support: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior- CAPES.

049 - Mg-Al-LDH-based nanosystem for functional interference of microRNAs as a potential new therapeutic method in epilepsy

Araújo, M.A; Santos, B.P; Santos, Y.D.R; Júnior, E.D; Paulino, P.A.T, de Melo, T.S; Bassi, E.J; Dornelas, C.B; Gitaí, D.L.G.

Department of Cellular and Molecular Biology, Institute of Biological Sciences and Health, Federal University of Alagoas, Maceió, Alagoas, Brazil.

Rationale: Differential gene expression studies have shown that microRNAs (miRs) are critical in epileptogenesis, which makes them highly attractive targets for developing therapeutic drugs. Recently, by using microarray coupled RT-qPCR approach, we identified that miR-196b and miR-352 are up-regulated in post Status Epilepticus rat hippocampus. Functional studies based on the depleting of these miRs through inhibitor molecules are crucial for a better understanding of their role in epileptogenesis. However, there are still some challenges in the use of miRs inhibitors (AMOs) in vitro assay, since they suffer from problems such as biodegradability and poor cell uptake. In the current study, we addressed this shortfall by development of an inorganic nanoparticle Mg-Al-Layered Double Hydroxide (Mg-Al-LDH) loaded with AMOs against miR-196b-5p.

Methods: LDHs were synthesized by a co-precipitation method, and they were characterized as hydrodynamic size and zeta potential by DLS (Nanosizer Nano ZS instrument). The LDH morphology was evaluated by electronic transmission microscopy (TEM). The chemical group’s composition was assessed by Fourier transform infrared spectroscopy (FTIR) SSU-8000 (Shimadzu). X-ray diffraction (XRD) patterns were recorded by XRD-7000 (Shimadzu). Quantification of LDHs was measured by inductively coupled plasma atomic emission spectroscopy (ICP-OES) and energy dispersive X-ray (EDX). Nanosystem was formed by vigorous agitation of Mg-Al-LDH and oligonucleotides (AMO or Alexa-fluor 555 labeled dsDNAs) and characterized as described for Mg-Al-LDH alone. For the in vitro assays, the thymic endothelioma cells (tEnd.1) were chosen due its high levels of miR-196b by RT-qPCR. After the treatment with the nanosystem, we assessed the cell viability by MTT and trypan blue exclusion assays, transfection efficiency by flow cytometry, and the miR-196b-5p depletion by RT-qPCR.

Results: LDH nanoparticles are stable in aqueous solutions, presenting zeta potential of +45mV, 0.15 of PDI, size mean of 90nm and a physical-chemical pattern of the Mg-Al-LDH group. TEM images revealed LDHs with a regular hexagonal shape. No discernible cytotoxic effect on tEnd.1cells was observed after LDH administration at concentrations up to 100 μg/ml. HDL loaded with Alexa-fluor 555 labeled dsDNA at concentration of 310 nM resulted in 93% of the tEnd.1cells as Alexa-fluor positive. AMOs carried by LDH resulted in a significant depletion of miR-196b-5p transcripts at 48 h after the transfection as measured by RT-qPCR (p=0.0079).

Discussion/Conclusions: We developed stable Mg-Al-LDH nanoparticles and nanosystem suitable for functional interference of microRNAs studies. As a perspective, we will investigate the role of miR-196b-5p in temporal lobe epilepsy models using treatment with the Mg-Al-LDH nanosystem.

Acknowledgements/Financial Support: CNPq, CAPES, FAPEAL, UFAL.

050 - Auditory Brainstem Response to tones are altered in Wistar Audiogenic Rats (WARs): implications to mesencephalic and limbic seizures during audiogenic kindling.

Cunha, A. O. S.1, de Deus J. L.1, Moradi, M.1, Barcellos Filho, P. C., G.1, Oliveira, J. A. C.1, Garcia-Cairasco, N.1, Leão, R. M.1

1 - Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo. Ribeirão Preto-SP. Brazil.

Rationale: Audiogenic seizures are reflex seizures in response to a high intensity acoustic stimulus due to hyperactivation of brainstem auditory areas. The repetition of acoustic stimulus may lead to the spread of epileptic activity to limbic structures possibly by the enhancement of colliculus-limbic pathways. The Wistar Audiogenic Rat (WAR) strain was selected at the Neurophysiology and Experimental Neuroethology Laboratory during the late 80’s and has provided a variety of experimental data. Here, we investigate Auditory Brainstem Response (ABR) in WARs and checked for correlations with seizure severity.

Methods: Wistar and WARs females (n=7, 70-80 days, both groups) were anesthetized (ketamine, 65 mg/kg and xylazine 10 mg/kg, i.p.) and laid in thermic pad inside a sound-proof chamber (Ethical Committee for Animal Use protocol # 013/2018). Three subcutaneous electrodes were placed in the vertex (recording), ipsilateral ear (reference) and contralateral ear (ground). A 10-cm tube was placed inside each ear and connected to speaker. Clicks or tones (4, 8, 16, 24 and 32 kHz), were presented in decreasing intensity, from 90 to 20 dB, in a rate of 21/s and 512 responses were averaged and recorded. We measured the peak and the latencies of recorded waves (I, II, III, IV and V) for each intensity. The smallest intensity able to evoke a response was considered the hearing threshold. One week later, the audiogenic kindling (AK) protocol started. It consisted of two daily 1 min 120-dB sound stimuli for 10 days, after which the animals were euthanized. Sound stimuli was interrupted when tonic-clonic seizures were observed. All sessions were filmed, and the analysis of seizures was performed after experiments based on limbic and audiogenic seizure scores. Estrous cycle was daily monitored until the first sound stimulation.

Results: We did not observe any differences between amplitudes or latencies of waves I – V in response to clicks in our ABR recordings from WARs compared to Wistars in any of the used intensities. We also analyzed the ratio between waves I/V and asymmetry of right and left ears and compared between groups, and we did not observe differences. Interestingly, when we analyzed the responses to tones, we observed differences in the amplitude of wave II in response to 4 kHz (p = 0.0001), 8 kHz (p = 0.015) and 16 kHz (p = 0.0023), as well as asymmetry between right and left ears in 4 kHz (p = 0.0021). The amplitude of wave III is also bigger for WARs upon 8 kHz tones (p = 0.002). In 16 kHz, we found that wave V is bigger in recordings from WARs (p = 0.04). The ratio between waves I/V reflect the relationship between the evoked potentials of auditory nerve and inferior colliculus and is often referred to as the gain control of auditory information. Our data show asymmetry in the I/V for 4 kHz and 8 kHz pure tones (p = 0.03, for both cases). Finally, we did not find correlations between the amplitude of wave II and seizures severity (r = 0.6; 8kHz; r = 0.18; 4 kHz), ratio I/V (r = -0.3; 8 kHz) or asymmetry of ears (r = 0.1; wave II, 8 kHz). Our data also did not detect differences in the phases of estrous cycle.

Discussion/Conclusions: The present data show that WARs have alterations in primary auditory processing. We do not know how these alterations would impact the sensory motor integration in these animals. Therefore, further research would clarify these and other aspects of the audiogenic treat in these animals.

Acknowledgements/Financial Support: FAPESP, CAPES and CNPq, CNPq/FAPESP INCT Translational Medicine.

051 - Dual Biological Effects of the Immune Regulatory Molecule Galectin-1 in experimental epilepsy

Costa, M.A.1; Pacheco, A.L.D.1; Melo, I.S.1; Cavalcante, C.M.B.1; Rodrigues, L.C.2; Riul, T.B.2,3; Freitas-Santos, J.1; Amaral, M.M.C.1; Garcia-Cairasco, N.2; Dias-Baruff M.2; Castro, O.W1.

1 - Institute of Biological Sciences and Health, Federal University of Alagoas (UFAL), Maceió-AL-Brazil.

2 - University of São Paulo (USP- Ribeirão-Preto), Brazil.

3 - Federal University of Mato Grosso do Sul, Brazil.

Rationale: Inflammation plays a critical role in the pathophysiology of epilepsy. Curiously, the anti-epileptic drug valproic acid is able to induce the gene expression of an endogenous anti-inflammatory molecule, Galectin-1 (Gal-1), both on in vivo and in vitro experimental models. In addition, several lines of scientific evidence point out the potential neuroprotective effect of Gal-1. However, the impact of this immune regulatory molecule on epileptic seizures is not fully understood. Here, we analyzed the efficacy of Gal-1 in brain neuroprotection using a temporal lobe epilepsy model.

Methods: Experimental procedures were approved by the Ethical Committee for Animal Research of Federal University of Alagoas-Brazil (protocols numbers: 22/2013 and 19/2014). Male Wistar rats (n=45; 240-340g) were submitted to stereotaxic surgery for cannula implantation in the hilus of the dentate gyrus of hippocampus. Initially, the animals were intrahippocampally (IH) injected with a single dose of recombinant Gal-1 (5 µg in 1.0 mL PBS) or PBS (1µL; vehicle control). After 30 minutes, Gal-1-pretreated or vehicle-pretreated animals were submitted to induction of Status Epilepticus (SE) by microinjection (IH) of pilocarpine (1.2 mg/µL). Seizures severity and the incidence of spontaneous recurrent seizures (SRS) were evaluated using behavioral activities analysis according to Racine’s scale (1972). Neurodegeneration was evaluated histologically by localization of neuronal damage using Fluoro-Jade (FJ) staining and by quantification of Glial Fibrillary Acidic Protein (GFAP) positive (GFAP+) cells. FJ and GFAP labelling were done in subfields of hippocampus (Hilus, CA3 and CA1) and in Stratum Oriens (SO), Stratum Pyramidale (SP) and Stratum Radiatum (SR). Results were expressed as mean ± SEM and analyzed using unpaired t-test.

Results: On day 1 after SE induction, subfields of hippocampus of Gal-1-pretreated rats exhibited lower number of FJ positive (FJ+) neurons [CA1 (10.5±5.27) and CA3 (21.25±10.36)] when compared to vehicle-pretreated rats [CA1 (67.25±22.56) and CA3 (60.25±7.00)]. Also, 15 days post-SE, the Gal-1 pre-treatment promoted a significative reduction on the number of FJ+ neurons in CA1 (40.25±8.10) and hilus (23.67±12.00) when compared with vehicle-pretreated rats CA1 (119.80±17.92) and hilus (87.67±14.05). However, on day 30 after SE induction, Gal-1-pretreated animals showed high levels of FJ+ cells in the CA1 (219.9 ± 81.8) region, when compared to vehicle-pretreated rats (22.1 ± 22.1). After 24 hours of SE induction, the number of GFAP+ cells were lower in the subfields of hippocampus from Gal-1-pretreated rats [SO (190±27.7), SP (65±6.2) and SR (179.3±26.6)] than in vehicle-pretreated animals [(SO, 382.3±48.8; SP, 170.3±22.3; SR, 442.7±62.4)]. In addition, the quantity of GFAP+ cells were similar in the hilus of the DG in both animal groups.

Discussion/Conclusions: Overall, our data suggest that the Gal-1 has neuroprotective effects in the early, not in the late stage of epileptogenesis and provides new insights into the roles of this endogenous immune regulatory agent in experimental temporal lobe epilepsy.

Acknowledgements/Financial Support: FAPEAL, FAPESP, CNPQ, CAPES. Members of the Laboratory of Neuropharmacology and Integrative Physiology of the Department of Physiology and Pharmacology, Institute of Biological Sciences and Health, Federal University of Alagoas (UFAL).

052 - Prepulse inhibition of the acoustic startle response is enhanced in Wistar Audiogenic Rats (WARs)

Moradi, M.1,2; Cunha, A.O.S.1; de Deus, J. L.1; Barcellos Filho, P. C. G. 1; Oliveira, J. A. C.1; Garcia-Cairasco, N.1; Leite, J.P.2; Leão, R.M.1

1 - Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo. Ribeirão Preto-SP. Brazil

2 - Department of Neuroscience and Behavioral Sciences, Ribeirao Preto School of Medicine, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil

Rationale: Deficits in prepulse inhibition (PPI) are observed in several neurological and psychiatric disorders suggesting the relevance of its underlying neural mechanisms. PPI, a reliable test in humans and rodents, is the measurement of the sensorimotor gating which represents the normal reduction of the startle response as a result of a brief, low intensity non-startling stimulus (prepulse) preceding a startle-evoking stimulus (pulse). The Wistar Audiogenic Rats (WARs) which were derived from audiogenic susceptible Wistar progenitors is a well-studied model of audiogenic seizures, triggered by high-intensity acoustic stimuli. Here we compared the response of PPI in Wistar and WAR animals to look for a potential association between the circuits that process WARs’ innate susceptibility to sound-triggered seizures and the PPI.

Methods: Wistar and WARs females (n=7, 70-80 days) were tested for the acoustic startle (AS)/PPI paradigm, which consisted of a one-week protocol. First day, we placed the animals in the silent chamber for 20 min habituation (Ethical Committee for Animal Use protocol # 013/2018). The following day, animals were placed in the chamber and after 5 min acclimation period, the trials of the AS and PPI were presented randomly. The startle stimulus consisted of a white noise burst with duration of 50 ms at 120 dB in the silent background. In PPI trials, the prepulse stimulus consisted of a broad band noise centered at 4, 8, 16, 24 kHz or white noise at 70 dB which were presented 100 ms before the startle stimulus, with the duration of 50 ms and silent background. We repeated this protocol twice after 2 days of rest. We measured the peak and the latencies of the startle amplitude in both AS and PPI Trials. Results were calculated as the amplitude of the startle response in PPI sessions divided by the amplitude of the startle response in AS sessions multiplied by 100. We also checked for differences in the latencies to the startle reflex. The estrous cycle was continuously monitored. And no differences were observed.

Results: We did not observe any differences between the latencies of ASR in WARs compared to Wistars in any of the tested frequencies. However, percentage of startle reflex inhibited by the pre-pulse for WARs was significantly bigger than for Wistars in 4 kHz (Wistar = 13% ± 5.5 and WAR = 45% ± 6.5; p = 0.025, unpaired t-test) and 8 kHz (Wistar = 18% ± 4.7 and WAR = 52% ± 5.4; p = 0.0005, unpaired t-test), but not in other frequencies or white noise. Interestingly, the mean amplitude of startle reflex was smaller for WARs in all tested frequencies and white noise (4 kHz, p = 0.0274; 8 kHz, p = 0.02; 16 kHz, p = 0.0079; 24 kHz, p = 0.02; WN, p = 0.008; unpaired t-test).

Discussion and Conclusions: Our results show that PPI is enhanced in WARs compared to Wistar controls. Our results contrast with previous data observed in WAG/Rij rats where PPI is impaired. These differences could be associated to intrinsic alterations in PPI circuitry for these models.

Acknowledgements/Financial Support: FAPESP, PROEX-CAPES, CNPq, FAEPA.

053 - Wistar Audiogenic Rat (WAR): a possible model for the study of Alzheimer’s disease related to insulin resistance

Alves, S.S.1; Pereira, P.D.1; Silva Junior, R.M. P.1; Donatti, A.L.F.1; Espreafico, E.M.1; Almeida, S.S.2; Sebolella, A.S.1; Garcia-Cairasco, N.1

1 - Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (FMRP-USP).

2 - Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo (FFCLRP-USP).

Rationale: The Wistar Audiogenic Rat (WAR), a genetic model of epilepsy, presents many comorbidities. Previous data suggested elevated brain levels of phosphorylated tau (pTau) and beta-amyloid protein (Aβ), the main hallmarks of Alzheimer’s Disease (AD). Recent studies have hypothesized AD as the third type of diabetes. The aim of this study was to validate the WAR strain as a model to study AD and correlate with alterations in insulin signaling pathways.

Methods: We have tested 14 Wistar and 14 WAR (4 months old, adult group) and 14 Wistar and 7 WAR (17 months old, aged group). Animals were subjected to the Morris Water Maze (MWM), in order to evaluate spatial learning and memory performance. The MWM consisted of 6 trials per day, for 4 days (learning period), a probe trial and three retests (immediately, 24 hours, and one week after probe). Then, rats were euthanized and their dorsal and ventral hippocampi were dissected for Western Blot (WB) analysis to measure the expression levels of Aβ precursor, pTau (S396), insulin receptor (IR) and phosphorylated insulin receptor substrate 1 (pIRS-1, S312). CEUA protocol 167/2016.

Results: In adult group, the MWM task was learned by both strains (p<0.001), but WARs showed higher latency to reach the platform during the learning period (p<0.01), and in the first (p<0.05) and second retests (p<0.001). We observed longer distances reached by adult WARs in the second retest (p<0.01) and lower velocity during the learning period (p<0.01), first (p<0.001), second (p<0.001) and third retests (p<0.001). In aged group, both strains learned the MWM task (p<0.001). However, it took longer to aged WARs to reach the platform during the learning period (p<0.01), the first (p<0.05) and the third retests (p<0.05). The distances reached by aged WAR were also longer in the first retest (p<0.05) and velocity was also lower during the learning period (p<0.01), first (p<0.01), second (p<0.05) and third retests (p<0.01). In the WB analysis, we observed a reduction of Aβ precursor and IR expression in the ventral and dorsal hippocampus of adult WARs (p<0.05, for both proteins). Similar results were observed for aged WARs which showed a reduction of Aβ precursor and pIRS-1 (p<0.05) and increase in pTau expression in both ventral and dorsal hippocampi (p<0.01).

Discussion/Conclusions: Animals of the WAR strain showed worse performance than the control Wistar in the MWM at both ages, suggestive of spatial learning and memory (short and/or long-term) deficits. The reduction of Aβ precursor might be suggestive of an increase in Aβ cleavage, corroborating the increase in pTau. The alterations in the insulin pathway (IR and pIRS-1 reduction) may be indicative of insulin resistance in the hippocampus of WARs. Because the WAR strain was primarily developed as a model of epilepsy, other molecular and histological studies are being executed to better understand how the current data might validate the WAR strain as a model to study AD related to insulin resistance.

Acknowledgements/Financial Support: FAPESP, CAPES, INCT (FAPESP/CNPQ).

054 - Epilepsy-related comorbidities in the Wistar Audiogenic Rats (WAR) strain: Anxiety-like behaviors associated with seizure susceptibility

Lopes, W.L.1,2; Do Val-da Silva, R.A.¹; Leite, J.P.¹; Garcia-Cairasco, N. 1,2

1 - Neuroscience and Behavioral Sciences Department, Ribeirão Preto School of Medicine, University of São Paulo.

2 - Neurophysiology and Experimental Neuroethology Laboratory, Physiology Department, Ribeirão Preto School of Medicine, University of São Paulo.

Rationale: Epilepsies are neurological disorders characterized not only by the presence of epileptic seizures but also by neuropsychiatric comorbidities. Anxiety is one of the most frequent psychopathologies in people with epilepsies, and more severe seizures are associated with high anxiety levels. Epilepsies-related comorbidities, such as anxiety, are present even in animal models of epileptic seizures, like rodent strains genetically susceptible to seizures. Specifically, the Wistar Audiogenic Rat (WAR) strain has been selected in the early 1990’s and those animals develop epileptic seizures in response to intense sound (110 dB) stimulation (called audiogenic seizures). Seizure profile in WARs is well described and along the chronic protocol of audiogenic seizures (audiogenic kindling, AuK), the initially midbrain-dependent seizures, give rise to forebrain-dependent seizures through a process called limbic recruitment. However, recently, studies demonstrated that the genetic selection in response to seizure phenotype has also selected endogenous alterations reflecting epilepsies-related comorbidities in WARs. In this context, this study aims to characterize the anxiety-like behaviors presented by WARs and the effects of the AuK in these behaviors.

Methods: Adult (2-5 months) male rats were divided into four experimental groups: WAR+AuK, WAR+Sham, Wistar+AuK, and Wistar+Sham (n=6-11/group; CEUA: 057/2017). In the AuK protocol, were applied 20 acoustic stimulations (or Sham expositions), twice a day. In the morning after the last stimulus, rats were placed in the center of the open field (OF) (50 lux), and the free exploratory activity was recorded for 5 minutes. Immediately after, animals were placed on the lit side of the light/dark box (LDB) (65 lux), and the exploratory activity was registered for 5 minutes.

Results: In the OF, WARs presented less locomotor activity (p<0.01) and elevation than Wistars (p<0.05). Additionally, grooming frequency was increased in WARs. AuK effects on anxiety-like behaviors were not observed in this apparatus. In the LDB, WARs explored the lit side less than Wistars (p<0.01) and both, frequency (p<0.01) and time of elevation (p<0.01), were reduced in WARs. The latency to the first return to the lit side was increased in WARs (p<0.05). There are statistical tendencies in respect to AuK in the LDB. The time spent in the lit side was reduced in WAR+AuK (p<0.1) if compared to WAR+Sham and risk assessment behaviors were increased in WAR+AuK if compared to WAR+Sham (p=0.06). All WARs presented seizures in the AuK protocol, whereas seizures were not observed in Wistars, confirming the specificity of this protocol.

Discussion/Conclusion: These results indicate that locomotor activity was reduced in naïve WARs, confirming previous data from our laboratory. In the same way, WARs are more anxious than Wistars, in both apparatuses, in the OF and in the LDB. The increase in grooming behavior indicates the presence of compulsive disorder in WAR, confirming also previous data from our Lab. Additionally, for the first time, it was shown that AuK is capable of increasing the anxiety-like behaviors endogenously presented by naïve WARs.

Acknowledgements/Financial Support: INCT-Translational Medicine, FAPESP, CAPES, CNPq, FAEPA.

055 - Ketogenic diet does not exhibit neuroprotective and anticonvulsant effects on lithium-pilocarpine-induced Status Epilepticus in rats

Pereira, W.S.1; Barros, C.R.A.1; Santos, G.F.C.1; Silva, C.L.A.1; Ataíde, T.R.1; Lima, S.O.1, Gitaí, D.L.G.1

1 - Universidade Federal de Alagoas, Maceió, Brasil.

Rationale: Temporal lobe epilepsy (TLE) is a neurological disorder characterized by complex partial seizures and a high index of pharmacorresitence. Ketogenic diet (KD) is a high-fat, low-carbohydrate diet used as an alternative treatment for refractory epilepsy which simulates biochemical changes similar to fasting. However, whether KD has a preventive effect on Status Epilepticus (SE) induced epileptogenesis is unclear. In the current study, we investigated the effects of KD on astrogliosis, inflammation, cell death and seizures severity on animals submitted to SE.

Methods: Young male (weight/age?) Wistar rats were divided into 2 groups and fed a control diet (7 % lipids, group PILO, n=15) or a KD containing coconut oil (69.8 % lipids, group KD+PILO, n=16). The KD and control diets had a lipid: carbohydrate+protein ratio of 1:11.8 and 3.5:1, respectively. All rats were subjected to lithium chloride (127 mg/kg; i.p); 16-18 h later, rats received an i.p. injection of pilocarpine hydrochloride (30mg/kg) for the induction of SE. In KD+PILO group, the SE induction was performed 30 days after the dietary treatment onset. The latency, number, and severity of limbic seizures before and during SE were analyzed by video-recording. Neuronal death in hippocampus was evaluated 24 h after SE by the count of Fluoro-Jade positive neurons (FJ+). RT-qPCR was used to analyze the relative expression of GFAP and TNF-α mRNAs. Unpaired Student’s t-test was used for comparative analysis and mean differences were considered statistically significant when p≤0.05.

Results: KD group showed lesser weight gain (p<0.05) but a higher feed efficiency compared to the PILO group (p<0.0001). The concentration of b-hydroxybutyrate measured from a small amount of tail blood (Precision Xtra system; Abbott Laboratories) was significantly higher in the rats of KD group (0.70 ± 0.1mol/L and 1.28±0.3mol/L in PILO and KD rats, respectively), p=0.005. There was no difference in latency for SE or seizure severity between control and KD diet. The number of FJ+ neurons in Hilus, CA1 and CA3 hippocampal subfields did not differ between groups. The expression levels of both GFAP and TNF-α were similar in the hippocampus of rats submitted to SE comparing the control and KD groups, but GFAP expression was higher in the naïve group under KD, when compared to the naïve group under the control diet.

Discussion/Conclusions: The use of KD did not exhibit neither anticonvulsant nor neuroprotective effects during the acute phase of the epileptogenic process. It was found that KD, per se, modulates positively the GFAP expression. However, additional studies should be performed to better clarify the role of KD on epileptogenesis.

Acknowledgements/Financial Support: Fapeal/CNPq/Capes.

056 - Effects of HUF-101 in different experimental models of epilepsy

Delfino-Pereira, P.1; Santos, V.R.2; Umeoka, E.H.L.1; Oliveira, J.A.C.2; Del Vecchio, F.2; da Silva Jr., R. M. P.2; Garcia-Cairasco, N.1,2

1 - Neurosciences and Behavioral Sciences Department; Ribeirão Preto School of Medicine; Universiy of São Paulo; Brazil. 

2 - Physiology Department; Ribeirão Preto School of Medicine; University of São Paulo; Brazil.

Rationale: The search for new therapeutic options is extremely necessary, given the dynamic and complex scenario of the epilepsies. Our objective was to evaluate the effect of a single-dose of HUF-101, a cannabidiol analogue, on behavioral, electroencephalographic (EEGraphic) and histochemical parameters in experimental models of the epilepsies.

Methods: (Protocol 1) Adult male WARs (n=11) were exposed to high intensity (120 dB) acoustic stimulation and developed tonic-clonic (brainstem) seizures. After 20 repeated stimuli (2 per day; audiogenic kindling; AuK), all animals developed limbic seizures. Based upon the behavioral profile after the 20 acoustic stimuli, animals were assigned into two groups: those that presented severe brainstem seizures and those that presented moderate brainstem seizures. Thus, 30 minutes before the 21st stimulus (test) animals with severe seizures were treated with HUF-101 (10 mg/kg; i.p) whereas rats from the moderate group were injected with vehicle (Tween 80 in 0.9% NaCl). (Protocol 2) Animals were treated with either vehicle (n= 6) or HUF-101 (n= 6) 30 min before intra-hippocampal microinjection of pilocarpine (PILO) (1.2 mg/1μL) to induce Status Epilepticus (SE). SE was abolished 90 min after its EEGraphic onset by means of a dose of diazepam (5 mg/kg; i.p.). Animals were sacrificed 24 h after the SE and brains were processed for morphological analysis of necrotic neurodegeneration, using Fluorojade C (FJ) histochemistry. While for Protocol 3, naïve male Wistar rats (n=14) were exposed to amygdala rapid kindling (ARK), which consisted in ten daily electrical stimuli, for two consecutive days. In the third day half of the rats were injected with HUF-101 (10 mg/kg; i.p) and the other half was injected with vehicle (Tween 80 in 0.9% NaCl; i.p). Thirty minutes after, the animals were exposed to the 21st stimulus (test). Behavioral responses were classified according to the Racine scale for limbic seizures (1972) modified by Pinel & Rovner (1978). EEGraphic data were acquired from piriform cortex, hippocampal formation and amygdaloid complex. All protocols were approved by the Ethical Committee for Animal Experimentation of the Ribeirão Preto School of Medicine at the University of São Paulo (CEUA, protocol 133/2015).

Results: Results with a single dose of HUF-101 in the AuK with WAR were negative for the following parameters: latencies for wild running/tonic-clonic seizures, as well as seizures severity. However, we observed neuroprotection monitored by the reduction in FJ+ cells (CA3 and CA1 hippocampal neurons) in animals treated with HUF-101, in the PILO SE model. On the other hand, ARK animals (1st-20th stimuli, p<0.0001, repeated measures ANOVA, seizures progression) treated with HUF-101 had a reduction of the severity of limbic seizures after the 21st stimulus (p<0.05, Mann Whitney test). Spectral analysis confirms the HUF-101 effect in limbic seizures, with reduction of epileptogenic activity, demonstrated by comparisons of amplitudes of EEG signal, signal duration, over the distribution of the frequencies (with presence of lower frequencies) at the 20th stimulus compared to the same period at the 21st stimulus.

Discussion/Conclusions: Although data with Auk was negative, combined behavioral and EEG analysis with the ARK model and neuroprotection data with the PILO model indicate quite promising anticonvulsant and neuroprotection effects of HUF-101. Further experiments will investigate a possible neuroprotection in animals treated with HUF-101 (10 mg/kg i.p.) with potential reduction specifically of apoptotic neurons after ARK.

Acknowledgements/Financial Support: We would like to thank all members of the Neurophysiology and Experimental Neuroethology Laboratory (LNNE) at the FMRP-USP. This study was supported by FAPESP (2014/50891-1), CNPq/FAPESP INCT Translational Medicine, CAPES: PROEX-Physiology and PROEX-Neurology, FAEPA.

057 - Expression of the estrogen receptor alpha and beta in Proechimys females submitted to the lithium-pilocarpine epilepsy model

Sanabria, V.1; Perosa, S.1; Bittencourt, S.1; de la Rosa, T.1; Scorza, C.1; Naffah-Mazzacoratti, M.G.2; Cavalheiro, E.1; Amado, D.1*

1 - Department of Neurology and Neurosurgery –Discipline of Experimental Neurology, Universidade Federal de São Paulo, UNIFESP, Rua Pedro de Toledo, 669, 2° Andar, São Paulo, SP, Brazil.

2 - Department of Biochemistry, Universidade Federal de São Paulo, UNIFESP, Rua 3 de Maio, 100, São Paulo, SP, Brazil.

Rationale: Estrogen plays a critical role in brain activities, but the interaction between estrogen and epilepsy is complex, so far not completely understood. It is known that estrogen influence brain excitability, thus mediates proconvulsant effects. Even though, estrogen also has anticonvulsant properties, making its mechanism of action even more complex. Proechimys guyannensis, an Amazonian rodent commonly described as an animal model of resistance to epileptogenesis, still intrigue us with this natural endogenous mechanism. The aim of this study was to determine the expression of estrogen receptors (ER) alpha and beta in the hippocampus of Proechimys females submitted to the lithium-pilocarpine epilepsy model.

Methods: The study was based on 36 adult Proechimys guyannensis females (one-year-old; CEUA N°5594280316): twelve ovariectomized (ovx) and twelve non-ovariectomized (no-ovx) treated with repeated administration of pilocarpine (Pilo) (250 mg/kg, i.p. every 30 min) and six no-ovx and six ovx control animals. All the no-ovx females were at the estrous phase. Seizures were scored in each rat by Racine’s scale. After 30 and 90 min post Status Epilepticus (SE), animals were decapitated, and hippocampi were removed immediately frozen in liquid nitrogen and stored at -80°C.The samples were sonicated, and protein concentration was determined. A standard curve was drawn to determine the linear range. The blots were incubated with ER-beta and ER-alpha antibodies. Peroxidase-conjugated goat anti-mouse IgG was used, and the incubation with anti-β-actin immunoglobulins was performed as control. Blots were revealed using a chemiluminescence detection system and densitometry analysis was used. Data was transformed with Z-score, then latency of the seizures and levels of ER-alpha and ER-beta was analyzed with analysis of variance test (ANOVA) followed by Tukey post-hoc.

Results: All animals presented SE and reached stage 4 of Racine. Six animals (two ovx and four no-ovx) presented SE with the first dose of pilocarpine (250 mg/kg, i.p.), and 18 animals (ten ovx and eight no-ovx) presented SE with the second dose of pilocarpine (500 mg/kg, i.p.). However, no significant difference between ovx and no-ovx in SE latency was observed. Catalepsy like state, and rare chronic movements of the head and neck were observed in Proechimys after SE. Western blotting analysis of Proechimys hippocampi showed higher levels of ER-alpha protein expression in all groups (Control, Control-ovx, Pilo-30, Pilo-30 ovx, Pilo-90 and Pilo-90 ovx) and lower levels of ER-beta (F(1,24)11.300, p=0.003). The expression of ER-alpha for control no-ovx animals was 3.18± 1.21, however, Pilo-90 no-ovx group showed a significant reduction in the expression of ER-alpha,1.50 ± 1.73 (p<0.001).

Discussion/Conclusions: This work confirms the resistance of this rodent to epileptogenesis. Surprisingly, Proechimys showed higher levels of ER-alpha protein expression in all groups than ER-beta. This finding may suggest some protective effect since ER-alpha has shown a critical role in estrogen-induced protection against brain injury. Nevertheless, it is difficult to predict whether the expression of beta and alpha ER lead to seizure resistance or if seizure events lead to beta and alfa expression changes. To understand the role of estrogens in the development of seizures, other studies are being performed in these rodents.

Acknowledgment/Financial Support: This research was supported by grants from FAPESP, CAPES, CNPq and PAEC OEA-GCUB.

058 - Breathing activity in a Temporal Lobe Epilepsy model induced by pilocarpine injection into the hippocampus

Maia, O.A.C.1; Oliveira, M.A.2; Castro, C.L.1; Moriya, H.T.3; Lima, W.T.2; Takakura, A.C.2; Moreira, T.S.1

1 - Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo/SP;

2 - Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo, São Paulo/SP;

3 - Biomedical Engineering Laboratory, University of São Paulo, São Paulo/SP.

Rationale: One of the possible causes of death in epilepsy is breathing disorders, especially apneas, which cause an increase in CO2 levels (hypercapnia) and/or a decrease in O2 levels (hypoxemia) in arterial blood. The respiratory neurons located in the ventral respiratory column (VRC) are one of the main groups responsible for controlling respiratory activity. Recent data from our group has demonstrated respiratory changes in two experimental models of epilepsy: audiogenic epilepsy (WAR) and rapid kindling of the amygdala. Here, we aimed to evaluate respiratory changes in the model of temporal lobe epilepsy (TLE) with intra-hippocampal injection of pilocarpine.

Methods: Adult Wistar rats (250-430 g); N = 16; CEUA ICB/USP: 108/2014) with a unilateral stainless-steel cannula implanted in the ventral hippocampus (VH) region were used. After 5-7 days, the animals were submitted to unilateral injection of pilocapine (PILO: n = 8) (2.4 mg/μL) or saline (CON: n = 8) into the VH. PILO group received methyl scopolamine (1 mg/kg) 30 minutes before pilocarpine administration and CON group received saline 0.9%. Three hours after the onset of the status epilepticus (SE) both groups received diazepam (7.5 mg/kg). The respiratory parameters analyzed by whole-body plethysmography were respiratory rate (fR), tidal volume (VT) and ventilation (VE). In these same animals, we also evaluated the respiratory mechanics such as Newtonian airway resistance (Rn), viscance of the pulmonary parenchyma (G) and the elastance of the pulmonary parenchyma (H). At the end of the experiments, rats were deeply anesthetized with pentobarbital and a 2% solution of Evans blue was injected into the VH for histological confirmation.

Results: From a total of 48 animals, 8 developed SE, and from those, only 3 had spontaneous seizure. Only animals that developed the score 5 of the Racine´s scale were included in the present study. Thirty days after the injection of PILO, no significant changes were observed in basal VE, neither during hypoxic (8% O2) or hypercapnic (7% CO2) stimuli compared to CON group. We also did not observe changes in airway resistance, viscance and elastance between PILO and CON groups.

Discussion/Conclusions: Thus, in the TLE model with the injection of PILO, we did not observe changes in the respiratory variables and in the respiratory mechanics, suggesting that, at least in the period of 30 days, the respiratory pattern and rhythm are intact.

Acknowledgements/Financial Support: FAPESP, CNPq and CAPES/PROEX.

NEUROSCIENCE-VISUAL ARTS ABSTRACTS

International Symposium

NEWroscience 2018

“Looking for Epilepsy Cure along Centuries and across Cultures.

From Demons and Blessings to Brain Complex Networks and DNA Editing”

Neuroscience-Visual Arts Abstracts

001 - CYTOMEGALIC NEURONS AND BALLOON CELLS GENERATING EPILEPSY IN PEDIATRIC CORTICAL DYSPLASIA - A REIMAGINING OF A GRAFFITI

CHODRAUI, F.I

Ribeirão Preto Medical School – USP

Legend: The art consists of a reimagining of a famous Banksy´s graffiti. The histological structures are represented as the balloons lifting the child and getting struck by a lightning bolt that symbolizes the erratic neural activation in cortical dysplasia.

002 - AUTUMN OF THE FAKE NEWRONS
SILVA JR, R. M. P. and ESPREAFICO, E. M..

Department of Cell and Molecular Biology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil

Legend: Dynamic instability in microtubules of murine fibroblast NIH/3T3 after dynein light chain 2 (DYNLL2) knock-down and short time of exposure to nocodazole, an antineoplastic agent which exerts its effect in cells by interfering with the polymerization of microtubules. The microtubule network was double-labeled, with an antibody against beta-tubulin (green) and against alpha-tubulin-tyrosine (red), evidencing dynamic microtubules. The nuclei were stained with DAPI (light red). After exposure to nocodazole, we observed a restructuring of the cytoskeleton, which may explain this modification in cellular morphology, resembling neurons network. Artificial colors, processing was performed by Icy Bioimage software.

Acknowledgements/Financial Support: FAPESP, CNPq and CAPES

003 - ETERNAL SUNSHINE OF THE SPOTLESS MIND

ALVES, S. S. SILVA JUNIOR, R. P. M.1, BARROCA, N. C. B. and

GARCIA-CAIRASCO N.

Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (FMRP-USP).

Legend: Wistar Audiogenic Rats (WAR) hippocampus staining with anti-beta-amyloid (red), one of the main hallmarks of Alzheimer’s Disease. The nuclei were stained with DAPI (blue). The image was acquired with a confocal microscopy ZEISS LSM 780. Artificial colors, processing was performed by Icy Bioimage software.

Acknowledgements/Financial Support: FAPESP 2017/21155-3

004 - IMMUNOFLUORESCENCE CO-LOCALIZATION OF PROGESTERONE RECEPTOR WITH GLUTAMATERGIC INTERNEURONS IN THE CA3 HIPPOCAMPAL REGION OF AN OVARIECTOMIZED FEMALE PROECHIMYS GUYANNENSIS
Viviam Sanabria1, Sandra Perosa1, Simone Bittencourt1, Tomás de la Rosa1, Carla Scorza 1, Maria G. Naffah-Mazzacoratti2, Esper Cavalheiro1, Débora Amado1

1 Department of Neurology and Neurosurgery, Universidade Federal de São Paulo, UNIFESP, São Paulo, SP, Brazil.

2 Department of Biochemistry, Universidade Federal de São Paulo, UNIFESP, São Paulo, SP, Brazil

Legend: Fluorescent imaging of a coronal section of the CA3 hippocampal region of the Amazon rodent P. guyannensis, shows the co-localization of monoclonal anti-progesterone receptor antibody (ab2765, Abcam) followed by Alexa-fluor 594 conjugated secondary antibody (red) with the second primary antibody mGluR 2/3 (NB300-124, Novus) followed by Alexa-fluor 488 conjugated secondary antibody (green). Co-localization is observed in orange and nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (blue). Image was collected by confocal microscopy at 20x and imageJ Software was used.

005 - NEURONS SYNAPKISS

Cunha, A.1; SILVA JUNIOR, R. M. P.2

1 Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil

2 Department of Cell and Molecular Biology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil

Legend: Electron micrograph in the hippocampus (CA1) of Wistar Audiogenic Rats (WAR) evidencing a synapse.

Acknowledgements/Financial Support: FAPESP 2015/22237-7

006 - TWIN STARS

Souza, M. M.1; Moda, L. M.2; Ruginsk, S. G.3

1 Federal University of Alfenas (UNIFAL-MG)

2 Department of Developmental and Cellular Biology, Institute of Biomedical Sciences - Federal University of Alfenas (UNIFAL-MG)

3 Department of Physiological Sciences, Institute of Biomedical Sciences - Federal University of Alfenas (UNIFAL-MG)

Legend: Imunohistochemical staining for Glial Fibrillary Acidic Protein (GFAP, red, AlexaFluor-594) and DAPI (nuclei, blue) in cultured hypothalamic astrocytes. Cells were incubated in DMEM (Dulbecco’s Modified Eagle’s Medium) containing fetal bovine serum and penicillin-streptomycin for a period of approximately 10-15 days. In order to investigate the participation of L-type Ca2+ channels (LTCCs) in the transduction of the osmotic signal, these cells underwent treatment with verapamil, a LTCC blocker, which consistently decreased GFAP expression in comparison to the basal condition. The image was captured using a Nikon C2 microscope, under 63X magnification.

Financial Support: FAPEMIG; CAPES and UNIFAL-MG

007 - TAKE A DEEP LOOK INSIDE EPILEPSY PHYSIOLOGY

GODOY, L. D.

USP - Departamento de Fisiologia, FMRP, Universidade de São Paulo

Legend: Understanding the physiology of epilepsies in the current world requires a systemic and integrated perspective from Science. Embodying all different and complex systems into the scientific discoveries has the potential to translate into real and significant advances to patients’ lives. Therefore, this striking pencil drawing encompass the challenges and interesting connections in epilepsy research.

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