UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
TESE DE DOUTORADO
ESTADOS PSICODÉLICOS E SONO NO CÉREBRO DO RATO:
ESTUDOS COMPORTAMENTAIS, ELETROFISIOLÓGICOS E
MOLECULARES
Annie da Costa Souza
Orientador: Prof. Sidarta Ribeiro Co-orientador: Vítor Lopes dos Santos
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UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
TESE DE DOUTORADO
ESTADOS PSICODÉLICOS E SONO NO CÉREBRO DO RATO:
ESTUDOS COMPORTAMENTAIS, ELETROFISIOLÓGICOS E
MOLECULARES
Tese apresentada ao Curso de Pós-Graduação em Neurociências, Instituto do Cérebro, Universidade Federal do Rio Grande do Norte, como requisito parcial para a obtenção do título de Doutora em Neurociências.
Orientador: Prof. Sidarta Ribeiro Co-orientador: Vítor Lopes dos Santos
Annie da Costa Souza
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UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
PhD THESIS
PSYCHEDELIC STATES AND SLEEP IN THE RAT BRAIN:
BEHAVIORAL, ELECTROPHYSIOLOGICAL AND MOLECULAR
STUDIES
A thesis submitted to Neuroscience
Post-Graduation Program, Brain
Institute of the Federal University of Rio Grande do Norte, in partial fulfillment of the requirements for the Ph.D. degree in Neuroscience.
Advisor: Prof. Sidarta Ribeiro Co-advisor: Vítor Lopes dos Santos
Annie da Costa Souza
Souza, Annie da Costa.
Estados psicodélicos e sono no cérebro do rato: estudos comportamentais, eletrofisiológicos e moleculares / Annie da Costa Souza. - Natal, 2020.
277f.: il.
Tese (Doutorado) - Universidade Federal do Rio Grande do Norte, Instituto do Cérebro, 2020.
Orientador: Sidarta Ribeiro.
Coorientador: Vítor Lopes dos Santos.
1. Alucinógenos. 2. Hipocampo. 3. Córtex pré-frontal. 4. Córtex somatossensorial. 5. Sono. I. Ribeiro, Sidarta. II. Santos, Vítor Lopes dos. III. Título.
RN/UF/BSAC CDU 612.8
Universidade Federal do Rio Grande do Norte - UFRN Sistema de Bibliotecas - SISBI
Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial Árvore do Conhecimento - Instituto do Cérebro - ICE
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“Le véritable voyage, ce n'est pas de parcourir le désert ou de franchir de grandes distances sous-marines, c'est de parvenir en un point exceptionnel où la saveur de l'instant baigne tous les contours de la vie intérieure.”
Jean Huguet Saint-Exupéry ou l'Enseignement du désert
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Agradecimentos
Essa tese representa a construção de mais de uma década de formação. No início de 2008 comecei nas neurociências como aluna de iniciação científica júnior e desde então sigo me encantando pela pesquisa nessa área. Essa trajetória não seria possível sem todos que fizeram ou fazem parte dela, portanto agradeço a todos!
Agradeço imensamente à minha família, sem ela nada seria possível. Obrigada aos meus pais Ana Maria e Carlson Pereira, desde minha existência me proporcionaram um lar cheio de amor e carinho. Sem o amor e dedicação deles o caminho até aqui seria impraticável. Agradeço também aos meus irmãos. Bryan (Bê), obrigada por ser esse ser humano incrível que me apresentou um mundo cheio de luz, obrigada pela parceria no trabalho e na vida, obrigada pelo amor fraterno e pela amizade verdadeira. Nicholy (Fia), obrigada por sua energia desde pequena, pelo seu carinho, por me ensinar, por ser minha irmã mais velha sempre que se faz necessário, pela parceria, pelo amor, pela amizade genuína e carinho de sempre. Obrigada Bia e Luca, que por muitos anos não estiveram presentes mas que ultimamente me fizeram reviver uma fraternidade de infância. Obrigada às minhas Vovó Nenzinha (in memoriam) e Vovó Creusa. Obrigada às minhas tias, tios e primas.
Agradeço a todos os meus amigos do ICe que fazem do ambiente de trabalho mais leve e aconchegante. Obrigada especialmente a eterna galera do @container e aos astrócitos, aos atuais e aos que já um dia estiveram lá. Gratidão pelas boas energias!
Agradeço ainda aos amigos Daniel, Felipe, Daiane, Ana Raquel T., Jéssica A., Jéssica W., Brisa, Davi, Joseph, Fernando, Celso, Alan, Belinha, Lívia (Biu), Bessa, Pedrosa, Rafaelzinho, Lívia P, Margareth, Igor, Pri, Ju A., Ju B., Luana, Bruna L., Bruna C., Ana Raquel M, Geissy, Ingrid, Renato, Sara, Annara, Gabi, Jhulimar, Andrea, Lari, Andressa, Caro, Genedy, Markus, Pavão, João, Robson, Conde, Lockman, Sérgio A., Vítor, Greg, Jasiara, Victoria...e tantos outros pela amizade, companheirismo, parceria, troca de conhecimentos e colaboração. Obrigada por estarem sempre disponíveis para ajudar no que for preciso.
Agradeço ao grupo das meninas@neuro, amigas de longa data do ICe que permeiam ao longo dos anos uma fraternidade genuína.
Agradeço ao Sci-Girls, a todas as mulheres que fazem parte dessa rede de apoio e sororidade. Obrigada a esse grupo que faz a vida e a academia mais leve e agradável. Obrigada Natália Mota pela inspiração e criação desse grupo.
Agradeço a todos os integrantes do Sidarta-Lab. Obrigada por fazerem desse laboratório um ambiente colaborativo e cheio de axé! Obrigada principalmente aos integrantes do Sidlab-Rats, com quem tive o prazer de conviver diariamente: Daniel Almeida, o eterno médico iluminado do lab e amigo sempre disposto a ajudar no que for preciso, Daiane Golbert, amiga e parceira para todas as horas juntando arte e ciência com maestria, Felipe Cini, sempre doando o melhor de si e cuidando de todos ao seu redor, Ana
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Raquel, sempre atenciosa e companheira, Priscilla Kelly, sempre doce e gentil, Juliana Ávila e seu astral empolgado e Rebeka Nogueira, com sua dedicação e delicadeza. Obrigada pela amizade e por tornar o lab um ambiente saudável, amoroso, agradável e de colaboração. Obrigada, pelas discussões ricas, pelos devaneios, pelos lanches e cafés, pelas trocas de experiências, pelos ensinamentos e por estarem sempre dispostos a ajudar e compartilhar. Obrigada Ana Elvira, a melhor assistente de laboratório que poderiamos ter, agradeço por sua incrível eficiência, prestatividade, bom humor e amizade.
Agradeço a todos os funcionários, técnicos e terceirizados que estão sempre encontrando soluções e promovendo o bem-estar no instituto.
Agradeço as instituições UFRN/ICe e CAPES pelo suporte estrutural e financeiro a esse trabalho.
Agradeço aos meus amigos externos ao ICe pela amizade incondicional de longa data. Carolina Araújo, Hanoch Griner, Renzo Torrecuso, Natália Boccardi, Arthur França, Vítor Santos, Ana Maria Soares. Agradeço também à todos os meus amigos do Cefet e da Biologia, principalmente à Thalita, Eloysi, Karol, Gurgi, (S)Rafa, Fernando, Paulo Henrique, Maria Louyse, João Paulo, Renato, Diego, Vitão, Pedro, Daíse e Rômulo. Obrigada pela compreensão das ausências e mesmo assim por serem sempre presentes em minha vida.
Gostaria de agradecer a todos que contribuiram indiretamente ou diretamente para a realização desse trabalho.
Agradeço a todos os colaboradores diretos e co-autores dos trabalhos aqui apresentados. Especialmente a Bryan Souza, Vitor Santos, Nicholy Souza, Arthur França, Elena Moradi, Richardson Leão, Adriano Tort, Felipe Cini, Daiane Golbert, Ignacio Gendriz, Daniel Almeida Filho, Sérgio Rushi, Cátia Pereira, Vinícius Cota, Wilfredo Blanco, Jasiara Oliveira, Victoria Andino, Robson Scheffer, Roberto Etchenique, Steven Rehens, Lívia Goto, Marcelo Costa, Rafael Pedrosa, Bruna Koike, Cláudio Queiroz.
Agradeço aos laboratórios parceiros e aos seus respectivos Professores Richardson Leão, Katarina Leão, Adriano Tort, Diego Laplagne, Dráulio Araújo, Rodrigo Pereira, Cláudio Queiroz e Tarciso Velho por sempre estarem de portas abertas e dispostos à ajudar das diversas formas possíveis, seja trazendo equipamentos/reagentes necessários à pesquisa, trocando ideias, discutindo resultados ou emprestando alguma ferramenta. Obrigada pela disponibilidade.
Agradeço aos membros da banca por aceitarem participar da construção desse trabalho, Prof Cleiton Aguiar, Prof Richardson Leão, Prof Adriano Tort, esses últimos que acompanham ativamente esse e outros trabalhos de longa data, e Prof Vinícius Cota, que fomentou meus primeiros passos na neurociência juntamente com Cátia e Sidarta, e com quem até hoje colaboro.
Agradeço ao meu co-orientador e amigo Vítor Santos, que desde o início me acompanhou e esteve presente na minha vida acadêmica e pessoal. Obrigada pelas parcerias, fofuras disfarçadas de bullyings e amizade.
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Agradeço ao meu orientador Prof Sidarta Ribeiro pela orientação de longa data e pela amizade. Obrigada por me apresentar desde cedo o brilho nos olhos de quem faz o que gosta, e por manter vivo o otimismo dentro de mim, mesmo nos momentos mais difíceis. Obrigada pelo apoio de sempre, serei eternamente grata pela confiança e parceria que construímos durante esses anos.
Gratidão a vocês todos e ao universo por me proporcionar uma história repleta de boa gente, repleta de Ubuntu!
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SUMMARY
Resumo... 12
Abstract ... 14
List of abbreviations... 16
1 CHAPTER 1: INVESTIGATION OF THE NEUROPHYSIOLOGICAL AND BEHAVIORAL EFFECTS OF SEROTONERGIC AGONISTS IN THE RAT ... 19
1.1 On herein presented work – Chapter 1... 19
1.2 INTRODUCTION ... 19
1.2.1 Serotonin and the serotonergic system ... 20
1.2.1.1 Receptor distribution ... 21
1.2.2 Prefrontal cortex circuitry ... 26
1.2.3 Prefrontal cortex and serotonergic system ... 28
1.2.4 Hippocampal-cortical circuitry ... 32
1.2.5 The hippocampal formation ... 32
1.2.6 Memory and spatial navigation ... 34
1.2.7 Hippocampus and serotonergic system ... 35
1.2.8 Serotonergic agonist/hallucinogens/psychedelics ... 37
1.2.8.1 Therapeutic use of psychedelics: on depression and anxiety ... 39
1.2.8.2 5-MeO-DMT ... 40
1.2.8.3 d-LSD ... 41
1.2.8.4 d-LSD: receptors binding, neurophysiology and behavior alterations ... 44
1.2.8.5 Behavioral and Electrophysiological correlates of Psychedelics in rodents ... 45
1.2.8.6 Neural correlates of hallucinogens in humans ... 48
1.2.9 On the present study‘s motivation ... 50
1.3 AIMS ... 52
1.4 METHODS ... 52
1.4.1 Electrodes manufacturing (Append Chapter) ... 52
1.4.2 Surgery and animal care... 53
1.4.3 Experimental design ... 54 1.4.3.1 5-MeO-DMT ... 54 1.4.3.2 d-LSD ... 55 1.4.4 Experimental setup ... 56 1.4.5 LFP analysis ... 58 1.5 RESULTS ... 61
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1.5.1 Behavioral effects of 5-MeO-DMT and d-LSD... 61
1.5.2 Electrophysiological effects of 5-MeO-DMT and d-LSD ... 65
1.5.3 Supplementary figures ... 81
1.6 DISCUSSION ... 87
1.6.1 Animal behavior ... 87
1.6.2 Electrophysiological findings: LFP power ... 90
1.6.3 Electrophysiological findings: LFP Coherence ... 91
1.6.4 Differences of i.p. versus i.c.v. experiments ... 92
1.6.5 Mechanistic hypothesis based on cortical organization, 5-HT receptors location and literature /present results ... 94
1.6.6 Comparing results to human psychedelic literature ... 96
1.6.7 Sleep-waking cycle alterations ... 98
1.7 FINAL CONSIDERATIONS ... 102
2 CHAPTER 2: ON SLEEP, SYNAPTIC PLASTICITY AND MEMORY – Investigation of the sleep proteomic in the rat hippocampus and primary somatosensory cortex ... 103
2.1 On herein presented work – Chapter 2 ... 103
2.2 INTRODUCTION ... 103
2.2.1 Sleep, Synaptic Plasticity, and Memory ... 103
2.2.2 Synaptic homeostasis and restructuring across the sleep-wake cycle ... 130
2.3 PROTEOMIC ANALYSIS ACROSS THE SLEEP-WAKE CYCLE ... 136
2.3.1 ABSTRACT ... 136
2.3.2 AIM ... 138
2.3.3 METHODS ... 138
2.3.4 RESULTS and DISCUSSION ... 145
2.4 FINAL CONSIDERATIONS ... 178
3 REFERENCES ... 179
Appendix A – On sleep and memory ... 197
1 Sleep deprivation and gene expression ... 197
2 Recording Day and Night: Advice for New Investigators in the Sleep and Memory Field 225 3 Investigation of the sleep-dependent role played by the hippocampus during declarative memory consolidation in rats ... 247
Appendix B – In vitro electrophysiology in brain organoids – a pilot study ... 251
1 Introduction ... 251
2 Methods ... 251
3 Preliminary results and discussion ... 252
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Resumo
Psicodélicos clássicos são substâncias conhecidas por induzir estados alterados de consciência. Esses estados dependem da atividade de receptores serotonérgicos 5-HT2A e 5-HT1A.
Porém, devido à longa proibição dessas drogas, pouco é conhecido quanto aos correlatos eletrofisiológicos no cérebro. No primeiro capítulo do presente trabalho, investigamos os efeitos da 5-MeO-DMT(DMT) e do d-LSD, dois potentes agonistas serotonérgicos, na atividade eletrofisiológica do hipocampo (HP) e do córtex pré-frontal (PF) de ratos. Encontramos alterações típicas de comportamento nos 15 minutos subsequentes à injeção das drogas, tais como aumento da locomoção, ocupância da arena, e ocorrência de comportamentos estereotipados (wetdog shake, andar descoordenado etc). Em acordo com resultados prévios, encontramos alterações nos potenciais de campo local (‗local field potential‘, LFP) no PF, bem como no HP. Enquanto a potência na faixa de frequência de theta (5-10Hz) gama (30-100Hz) diminuiu em ambas as áreas nos primeiros 30 minutos após a injeção de DMT, a potência na faixa de delta (0.5-4.5Hz) não apresentou variação significativa. De modo semelhante, porém tardio, o HP apresentou diminuição da potência na faixa de gama após ~4h30min da injeção de d-LSD. Além disso, encontramos um aumento da coerência entre PF e HP na faixa de delta e gama para os experimentos DMT na condição experimental de injeção intraperitoneal (i.p.). Considerando que os achados acima mencionados sugerem que DMT e d-LSD levam a estados cerebrais alterados em termos de eletrofisiologia, decidimos compará-los ao ciclo sono-vigília. Análises de mapa de estados definidos por razões espectrais demonstraram que ambas as substâncias promovem uma mudança no ciclo sono-vigília. Os animais apresentaram maior velocidade durante estados classificados eletrofisiologicamente como sono de movimento rápido dos olhos (‗rapid eye movement‘, REM) e sono de ondas lentas (‗slow wave sleep‘, SWS). Em alguns casos foi possível observar também transições pouco usuais entre estados (WK para REM). Em outras palavras, ainda que o animal estivesse comportamentalmente acordado, essa vigília é similar a estados de sono, pelo menos em termos eletrofisiológicos. Em suma, esses resultados corroboram parcialmente achados anteriores e trazem alguns pontos ao debate. Adicionalmente, os novos resultados encontrados aqui contribuem para um melhor entendimento das mudanças eletrofisiológicas causadas por alucinógenos no cérebro.
O segundo capítulo dessa tese é dedicado à investigação do papel cognitivo das fases do sono em termos moleculares (e eletrofisiológicos). O sono desempenha um papel importante na consolidação de memórias, entretanto, pouco se sabe sobre os aspectos moleculares que estão por trás desse papel e sobre qual dinâmica tal perfil apresentaria durante as fases de SWS e sono REM. Em estudo prévio, demonstramos que a CaMKIIα fosforilada, uma proteína quinase relacionada à
13 plasticidade sináptica, diminui durante o SWS e encontra-se com níveis aumentados durante o sono REM no HP de ratos. Tal efeito ocorre somente em animais que foram previamente expostos a objetos novos na vigília precedente, assim como na indução transcricional de genes imediatos (IEG) dependentes de CaMKII durante o sono REM, conforme estudos anteriores de nosso grupo. Tal indução de IEG se dá inicialmente no HP e depois, gradualmente, se transfere ao córtex somestésico primário (S1). Hipotetizamos que o SWS e o sono REM possuem papéis e perfis de fosforilação proteica distintos no processamento de memórias durante o sono. Mais especificamente, fizemos um ‗screening‘ das proteínas fosforiladas no S1 e no HP durante ambas fases do sono em animais expostos (+) ou não expostos (-) à novidade na vigília anterior. Identificamos 535 fosfoproteínas no total para as regiões HP (198) e S1 (337), dentre as quais 90 proteínas foram significativamente moduladas (S1=69; HP=21). Através de análises ontogenéticas encontramos que as proteínas moduladas pertencem a diversas classes, por exemplo, relacionadas à organização de citoesqueleto, processamento de RNA, vias de sinalização por cálcio etc. Os resultados apontam para uma maior abundância de proteínas significativamente moduladas no S1 de animais expostos a novidade durante o SWS (S1=23 HP=9, SWS+ x SWS-). Possivelmente essa modulação (diminuição/aumento) da abundância de proteínas fosforiladas está relacionada aos estados de ativação e desativação (‗up and down‘ states) que ocorrem no SWS. Já durante o sono REM, há menor número de proteínas moduladas (S1=3; HP=3, REM+ x REM-). É possível que a novidade/estímulo gere uma fosforilação mais seletiva de proteínas relacionadas ao processamento sensorial, principalmente durante o sono REM. Encontramos que proteínas relacionadas à plasticidade sináptica estão moduladas e que algumas delas se correlacionam com fusos corticais durante o SWS e sono intermediário (IS). Por exemplo, a Relina, proteína que regula positivamente a morfogênese sináptica, está aumentada durante o sono REM+ comparado ao SWS+, e se correlaciona positivamente com o número de fusos durante o SWS e IS. Comparando os mesmos grupos encontramos que a CaCNA, um canal de cálcio dependente de voltagem, encontra-se com níveis diminuídos de fosforilação. A CaCNA desfosforilada forma um complexo (com Ca+2\Calcineurina) que atua na regulação transcricional de genes relacionados a plasticidade sináptica. As análises funcionais das proteínas que servem de marcadores dos grupos experimentais e análises de correlação com fusos indicam que há enriquecimento concomitante de vias relacionadas a cascatas envolvidas na regulação positiva e negativa da ‗força sináptica‘ (por exemplo, proteínas quinases e fosfatases). Tal ideia é corroborada por achados prévios do nosso grupo e também por evidências recentes de outros grupos (e.x. poda e ‗reforço‘ de sinapses durante o REM após aprendizado motor). Nosso estudo indica que o SWS e o sono REM possuem diferentes perfis de fosforilação de proteínas relacionadas à modulação sináptica, sugerindo que possuem papéis distintos e complementares na consolidação de memórias. Nossos achados corroboram a teoria do entalhamento de memórias durante o sono, no qual algumas sinapses são ‗fortalecidas‘ enquanto outras são ‗enfraquecidas‘.
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Abstract
Classic psychedelics are substances known for altering consciousness. Its psychoactive effect is dependent on 5-HT2A and 5-HT1A serotonergic receptors, but due to the long prohibition of
these drugs, little is known about their electrophysiological effects on the brain. In the current work I set out to investigate the effects of 5-MeO-DMT (DMT) and d-LSD, two potent serotonergic agonists, on the local field potentials (LFP) recorded from the hippocampus and prefrontal cortex of rats. Typical behavioral alterations ~15 min after drug injection were observed, such as increased locomotion, space occupancy, and the occurrence of stereotyped behaviors (wet-dog shake, uncoordinated gaiting etc). Similar to previous results, LFP alterations were detected in prefrontal cortical areas (PFC), as well as in the hippocampus (HP). The power in the theta (5-12 Hz) and gamma band (30-100 Hz) decreased in the two areas within the first 30 min after (i.p. and i.c.v.) DMT injection for all experiments, except for the highest dose of DMT (i.c.v.) in the PFC. Likewise, we found a similar result for d-LSD in the long-term analysis, there was a decrease in the gamma power after ~4h30min after d-LSD (i.p.) injection. Moreover, coherence analysis revealed that DMT (i.p.) increased the coherence between HP and PFC in the delta and gamma range. Next, we assessed how similar the changes caused by classic psychedelics are to the changes observed across the sleep-wake cycle. State map analysis revealed that both substances promoted a shift in the spectral profile typical of waking (WK) towards that of slow-wave sleep (SWS) or intermediated sleep (IS)/REM. Although animals remain awake after being treated with psychedelics, it is not a normal WK in terms of the LFP spectral profile. While some of the results obtained corroborate previous studies (e.g., the decrease in gamma power in the PFC), we also found divergent results, such as the decrease in PFC theta power. Altogether, the results are novel and promote a better understanding of the neurophysiological alterations caused by classic hallucinogens.
The second chapter of this thesis is dedicated to the investigation of the cognitive role of the distinct sleep stages in terms of the molecular (and electrophysiological) correlates. Sleep plays an important role in memory consolidation and cognition, however little is known on the molecular mechanism that underlies this function, and which dynamic it would have across the different sleep stages (SWS and REM sleep). In previous works, we have demonstrated that phosphorylated CaMKIIα, a kinase protein related to synaptic plasticity, and CaMKII-dependent immediate-early gene (IEG), zif-268, are down-regulated during SWS and up-regulated during REM sleep in the HP of rats exposed to novelty in the previous waking. That IEG induction is initiated in the HP and is gradually transferred to the somatosensory cortex (S1). We hypothesized that the SWS and REM sleep play distinct roles in memory processing during sleep and that the phosphoproteomic profiles are as well distinct. More specifically, we screened the phosphorylated proteins of the S1 and the HP during both sleep stages of animals that were exposed (+) or not exposed (-) to novelty in the previous waking. We identified a total of 535 phosphoproteins in both HP (198) and S1 (337), and 90 were significantly modulated across the sleep cycle (S1=69; HP=21). The ontogenetic analysis
15 revealed that the modulated proteins belong to several classes, for instance, cytoskeleton organization, RNA processing, calcium signalling pathways etc. Overall the results point that novelty-induced changes in protein phosphorylation levels are more pronounced in the S1 mainly during SWS (S1=23, HP=9, SWS+ x SWS-). Possibly, this upward/downward modulation could be related to the characteristics of the ‗up and down‘ states of the slow oscillations of SWS. A variety of functions were identified and a great part of that refers to the general functioning of the neurons, especially during SWS. During REM sleep there are fewer modulated proteins (S1=3, HP=3, REM+ x REM-). It is possible that novelty/stimuli would narrow the phosphorylation to more specific pathways related to sensory processing, mainly during REM sleep. We found that synaptic plasticity-related proteins are significantly modulated and some are correlated to SWS and intermediate sleep (IS) cortical spindle occurrence. For instance, Reelin, a protein that positively regulates synaptic morphogenesis, is upregulated during REM sleep compared to SWS of novelty exposed animals, and it is positively correlated to SWS and IS spindle count. Comparing the same groups (REM+ x SWS+) we found that CaCNA, a calcium voltage-dependent channel, is downregulated. The dephosphorylated CaCNA forms a complex (with Ca+2\Calcineurin) that regulates synaptic plasticity-related gene transcription. The functional analysis of proteins that are markers of experimental groups and spindles correlation analysis indicate that there is a concomitant enrichment of pathways related to synaptic reinforcement and weakening (e.g. activation of proteins kinases and phosphatases). Such an idea is corroborated by previous findings of our group and also by other recent evidence by other groups (e.g. REM sleep synaptic pruning and strengthening after motor learning). Our study indicates that SWS and REM sleep have different phosphorylation profiles, suggesting they have distinct and complementary roles in memory consolidation. Finally, our findings corroborate the theory of synaptic embossing during sleep, in which synapses are ‗reinforced‘ or ‗weakened‘.
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List of abbreviations
DMT or 5-MeO-DMT: 5-METHOXY-N,N-DIMETHYLTRYPTAMINE NN-DMT: N,N-DIMETHYLTRYPTAMINE
d-LSD or DLS or LSD or LSD-25: N, N-DIETHYLLYSERGAMIDE, aka Lysergic acid diethylamide
I.P.: Intra-peritoneal
I.C.V.: Intra-cerebro-ventricular LFP: Local field potential AP: Antero-posterior DV: Dorso-ventral ML: Medio-lateral
HP: Hippocampus (dorsal)
CA: HP subfield, Cornu Ammonis area 1-3 EC: Entorhinal cortex
PFC or PF: Prefrontal cortex mPFC: medial Prefrontal cortex ACC: Anterior cingulate cortex NR: Nucleus reuniens
LG: Low gamma SWS: Slow wave sleep
REM: Rapid eye movement (sleep) WK: Waking
IS: Intermediate sleep
DOI: 2,5-Dimethoxy-4-iodoamphetamine DOM: 4-methyl-2,5-dimethoxyamphetamine
DMT15: Refers to the first 15min after DMT injection DMT30: Refers to 15 to 30min after DMT injection DLS15: Refers to the first 15min after DMT injection
17 DLS30: Refers to 15 to 30min after DMT injection
SWS -: Animals killed during SWS, not previously exposed to novel objects SWS +: Animals killed during SWS, previously exposed to novel objects
REM -: Animals killed during REM sleep, not previously exposed to novel objects REM +: Animals killed during REM sleep, previously exposed to novel objects PNS: Peripheral nervous system
CNS: Central nervous system
fMRI: functional Magnetic resonance imaging BOLD: Blood-oxygen-level-dependent EEG: Electroencephalogram
DMN: Default mode network RSN: Resting state networks LFP: Local field potentials
TPOH2: enzyme tryptophan hydroxylase AADC: L-amino acid decarboxylase MAO: monoamine oxidase enzyme AD: Alzheimer disease
IP3: Inositol trisphosphate
CREB: cAMP response element-binding protein EGR-1: Early growth response protein 1, aka Zif-268 BDNF: Brain-derived neurotrophic factor
DR: Dorsal raphe MDT: Medial Thalamus
RSFC: Resting state functional connectivity PH: Parahippocampal
V1: Primary visual cortex
S1: Primary somatosensory cortex
APT: alphamethyl-p-tyrosine, a catecholamine synthesis inhibitor HTR: head-twitch response, aka wet-dog shake
18 CTC: Communication through coherence
TCB-2: high-affinity 5-HT2A agonist (4-Bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine
hydrobromide
NMDAr: NMDA receptor AMPAr: AMPA receptor
CaMKII: Ca2+/calmodulin-dependent protein kinase II MAPK: Mitogen Activated Protein Kinase
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1 CHAPTER 1: INVESTIGATION OF THE NEUROPHYSIOLOGICAL
AND BEHAVIORAL EFFECTS OF SEROTONERGIC AGONISTS IN THE RAT
1.1
On herein presented work – Chapter 1In this first chapter, I present the main research developed during my PhD. I investigated the neurophysiological effects of 5-MeO-DMT and d-LSD, two potent serotonergic psychedelics. More specifically, I report behavioral and electrophysiological results obtained with these substances in the rat brain. I also compare the altered state induced by serotonergic psychedelics with those observed across the normal sleep-wake cycle.
1.2
INTRODUCTIONThe notion that medicine, religion and culture correspond to completely separate fields is quite modern. In the prehistoric past, our ancestors likely learned to use plants and fungi for medicinal purposes and to transmit the acquired knowledge by oral communication through countless generations. In this context, as well as in the context of extant hunter-gatherer societies, treating disease could involve both the administration of actual medicine, as well as engagement in a religious ritual (e.g., intake of psychedelics followed by praying, chanting or dancing). Although this relation changed with the advance of science, some of these traditions are still present in our modern society. For instance, many classic hallucinogens, or psychedelics, are used in a religious context, for example, the Ayahuasca tea used in several Brazilian religions (e.g. Santo Daime, União do Vegetal, Barquinha)(Labate, B. C. 2006), or the Peyote used in Native American Religion (Albert Hofmann 1979).
A considerable variety of plants are known for their hallucinogens effects (Smet e Rivier 1985; D. E. Nichols 2004; Albert Hofmann 1979). Despite having been used for a long time, little is known about the neural mechanisms that underlie the action of psychedelics in the alteration of perception and thought. Such scarcity of knowledge reflects the wide drug prohibition implemented in the 1960s. Recently, however, interest in psychedelic research has been revived because of the major therapeutic potential of these
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substances (Palhano-Fontes et al. 2019; F. S. da Silva et al. 2018; Grob et al. 2011; Ross et al. 2016; Reiche et al. 2018; Cameron et al. 2019). Beyond their therapeutic use, psychedelics provide a unique tool to investigate how perception and thought occur in the brain. In this thesis, I present the results of behavioral and neurophysiological studies of the effects of classic psychedelics in rats. Because these substances act on the brain mainly as serotonergic agonists (Richard A. Glennon, Titeler, e McKenney 1984), in the following sections I will review the serotonergic system and the related brain areas that underlie altered perception.
1.2.1 Serotonin and the serotonergic system
Serotonin, or 5-hydroxytryptamine (5-HT), is a monoamine synthesized from tryptophan by a two-step reaction involving the enzyme tryptophan hydroxylase (TPOH2) and L-amino acid decarboxylase (AADC), and degraded by monoamine oxidase enzyme (MAO). Serotonin is present in a wide variety of organisms, from very early invertebrates to quite recent vertebrates (C. D. Nichols e Sanders-Bush 2001). It is one of the most important neurotransmitters of the peripheral and central nervous system (CNS), and takes part in a variety of essential functions. While serotonin is a major controller of gastrointestinal and blood vessel motility in the peripheral system, it also impacts the CNS in a widespread manner and modulates a variety of brain areas and functions. This modulation includes eating, reward, thermoregulation, cardiovascular regulation, locomotion, pain, reproduction, sleep-wake cycle, memory, cognition, aggressiveness, responses to stressors, emotion, and mood (Charnay e Léger 2010).
In the CNS, the major producers of serotonin are the raphe nuclei, which are located in the brainstem and that project to many forebrain regions (Figure 1.2.1). These forebrain regions include cortical and subcortical areas such as the thalamus, limbic system and frontal cortex. Because these projections vary in density between and within brain regions (Hornung 2003), cortical areas receive more serotonergic afferents on more superficial layers, while projections to the amygdala are more concentrated in the basal nucleus. Serotonergic afferents can differ substantially regarding their morphology: 5-HT axons innervating the hippocampus are more spaced and form small and elongated varicosities, whereas in the cerebral cortex they are tightly packed, large and round (Kosofsky e Molliver 1987). The hippocampal formation and the prefrontal cortex display a highly
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dense distribution of serotonergic receptors and are relevant areas for a variety of cognitive functions (Charnay e Léger 2010).
Figure 1.2.1: The raphe nuclei are the main producers of serotonin in the brain and project to almost every structure of the brain (C. D. Nichols e Sanders-Bush 2001). Adapted by D. Drieskens from Lesch e Waider 2012.
There is substantial evidence implicating the serotonergic system in cognition (Švob Štrac, Pivac, and Mück-Šeler 2016; Harvey 2003). The serotonergic circuitry interacts with other neurotransmitter networks in a complex manner. Serotonergic circuits are primarily composed by serotonin-producing neurons that can have 5-HT receptors (autoreceptors) as well as other neurotransmitters‘/hormones‘ receptors (heteroreceptors) involved in neuronal firing or serotonin release (Yoshida et al. 2009; Di Giovanni, Esposito, e Di Matteo 2010; Di Matteo et al. 2008; Fink e Göthert 2007). Other neurotransmitters influence the serotonergic neurotransmission in return (Adell et al. 2010). For example, glutamatergic, GABAergic, cholinergic and catecholaminergic systems do modulate serotonergic activity in the raphe nuclei (Haj-Dahmane e Shen 2009; Monti 2010b; Michelsen, Prickaerts, e Steinbusch 2008; Monti 2010a).
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Serotonergic receptors can be separated into 7 families: 5-HT1 to 7; and divided into several subtypes: 5-HT1A, B, D, E and F; 5-HT2A-C; 5-HT3, 5-HT4, 5-HT5, 5-HT6 and 5-HT7
(D. Hoyer et al. 1994; Barnes e Sharp 1999). While 5-HT1 and 5-HT5 families increase inhibitory potential (neuronal hyperpolarization mediated by GIRK channels), the remaining ones are excitatory. The heterogeneous receptor subtypes, as well as their different distribution in the brain, are in agreement with the widespread serotonin function.
Receptor family 5-HT1A is a Gi-coupled protein receptor; its activation inhibits the adenylate cyclase pathway and leads to K+ channel opening (Figure 1.2.2). It is mainly located on the axon hillock, thus its activation increases inhibitory potentials (neuron hyperpolarization by the opening of GIRK (G-protein-gated inwardly rectifying K+) channels) (Barnes e Sharp 1999; Czyrak et al. 2003; Efrain C. Azmitia et al. 1996; Rojas e Fiedler 2016). Also, this receptor is highly abundant in the prefrontal cortex and hippocampus and is considered a central receptor type in the regulation of the 5-HTergic system as a whole, and possibly that is behind its role in cognition. As an auto-receptor, the 5-HT1A receptor occurs at the soma of neurons that produce serotonin, providing negative feedback of serotonergic activation and regulating the release of serotonin (Hjorth et al. 2000). As heteroreceptors, occurring at the postsynaptic serotonergic and non-serotonergic neurons (Daniel Hoyer, Hannon, e Martin 2002), they are mainly distributed at the limbic system and regulate cognition, mood and emotions (Popova e Naumenko 2013). Depending on the 5-HT1A receptor location, its activation leads to opposing outcomes. For instance, its postsynaptic activation impairs emotional memory, while its presynaptic activation leads to improved retention in the passive avoidance task (Stiedl et al. 2015).
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Figure 1.2.2: 5-HT1A receptor is a Gi/o protein-coupled. The 5-HT1A – Gi/o complex activation leads to inhibition of adenylate cyclase and opening of potassium channels, while active Go leads to calcium channels closing (Adapted by D. Drieskens from C. D. Nichols e Sanders-Bush 2001).
Regarding other neurotransmitters, 5-HT1A activation leads to dopamine increased levels in the prefrontal cortex, striatum and hippocampus. Ogren et al. also showed that by altering glutamatergic, cholinergic, GABAergic neurotransmission in the hippocampus, cortex and medial septum projections, 5-HT1A receptors activity can affect declarative and non-declarative memory (Ogren et al. 2008). Besides neurotransmission regulation, 5-HT1A receptors can also regulate immediate-early genes expression important to memory consolidation (Ogren et al. 2008). These interactions may help to explain the relation of 5-HT1A receptor to memory formation.
As well as 5-HT1A receptor type, 5-HT1B receptors are inhibitors and occur in the
presynaptic terminals inhibiting the release of 5-HT and other neurotransmitters (Pauwels 1997). They also occur in non-serotonergic neurons at the postsynaptic terminals and are present in the hippocampus, cortex as well as basal ganglia and striatum (Daniel Hoyer, Hannon, e Martin 2002). Consistent evidence has implicated 5-HT1B receptor alteration in severe pathologies as Alzheimer disease (AD) and schizophrenia. For instance, in postmortem tissue analysis of AD patients, the density of 5-HT1B receptors is altered in frontal and hippocampal areas which is accompanied by a cognitive decline (Garcia-Alloza
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et al. 2004). Also, in schizophrenic patients evidence showed that 5-HT1B is increased in the hippocampal formation (López-Figueroa et al. 2004).
Unlike 5-HT1A-B receptors, receptor family 5-HT2A is a major excitatory receptor and its activation is necessary to induce the hallucinogenic effects of the classic hallucinogens (González-Maeso et al. 2007; R. A. Glennon, Rosecrans, e Young 1983; Winter et al. 1999; Vollenweider et al. 1998), including 5-MeO-DMT and d-LSD studied here. Those receptors are Gq-coupled proteins and among other targets, their activation leads to an increase in Ca+2 intracellular levels in the postsynaptic terminal and triggers calcium-dependent cascades (Figure 1.2.3) (Barnes e Sharp 1999). For instance, as a downstream target, it leads to CREB phosphorylation and increased mRNA levels of erg-1 (Masson et al. 2012). 5-HT2A receptors are mainly located in the apical dendrites of pyramidal neurons (Puig e Gulledge 2011) and likely their activation act amplifying excitatory inputs (George K. Aghajanian e Marek 1999). The 5-HT2A receptor is well distributed in the neocortex, including prefrontal, parietal and somatosensory cortex, as well in the basal ganglia and hippocampus, with a slightly less extent (Daniel Hoyer, Hannon, e Martin 2002; Beliveau et al. 2017). Concerning the cortical laminar location, it is mainly found in the cortical layers 2/3 (Puig e Gulledge 2011; Santana e Artigas 2017; Amargós-Bosch et al. 2004). In the PFC 5-HT2A is present in both pyramidal and GABAergic neurons (Santana et al. 2004), and are also present in feed-forward inhibitory neurons that target the pyramidal neurons (Puig et al. 2010). *See further details of 5-HT2A receptor activity in the prefrontal cortex in the 1.2.3 section.
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Figure 1.2.3: 5-HT2A receptor is a Gq protein-coupled. The Gq protein activation leads
to the hydrolysis of PIP2 to IP3 and DAG by the phospholipase C. IP3 leads to the release of Ca+2 from intracellular stores and DAG leads to activation of PKC and the formation of arachidonic acid. The increase in intracellular calcium levels activated calmodulin leading to potassium channels closing. In return, Ca+2 and calmodulin induce downstream immediate-early gene expression. (Adapted by D. Drieskens from C. D. Nichols e Sanders-Bush 2001).
Evidence suggests that the 5-HT2A receptor family is implicated in cognition and memory. In patients with Alzheimer‘s disease, lower levels of 5-HT2A receptors correlate with severity of symptoms (Marner et al. 2012; Versijpt et al. 2003; Lai et al. 2005). Concerning experiments using serotonergic agonists and antagonists, results diverge regarding memory. For instance, working memory in rats is enhanced by the serotonergic agonist TCB-2 (L.-B. Li et al. 2015), while ritanserin, an antagonist, leads to better spatial learning and memory (Naghdi e Harooni 2005). Also, a study of postmortem tissue found decreased levels of 5-HT2A receptors in the prefrontal cortex of schizophrenic subjects, but this could be due to treatment with 5-HT2A antagonists (Selvaraj et al. 2014).
The 5-HT1A and 5-HT2A receptors occur in more than half of the cortical pyramidal neurons, and in the majority of them in the same neuron (Santana et al. 2004; Amargós-Bosch et al. 2004). However, while 5-HT2A receptors are concentrated on the apical dendrites near the soma, 5-HT1A receptors are located closer to the axon hillock (Puig e Gulledge 2011).
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Another relevant receptor type is the orphan receptor Sigma-1. This receptor is present to a great extent in the central nervous system and also in the periphery (Teruo Hayashi e Su 2004), including nervous (Fontanilla et al. 2009) and immune systems (Szabo et al. 2014). It is a transmembrane chaperone that exhibits affinity to several ligands, and among them are neurosteroids, antipsychotics, antidepressants, cocaine, also a variety of synthetic molecules (Teruo Hayashi e Su 2004). Interestingly, they also have an affinity to NN-DMT (Fontanilla et al. 2009) and 5-MeO-DMT (Szabo et al. 2014). They act by regulating Ca+2 signalling via IP3 at the endoplasmic reticulum (T. Hayashi, Maurice, e Su 2000), and also, at the plasma membrane, they regulate K+ ion channels, NMDA receptors and Ca+2 voltage-dependent channels (T. Hayashi, Maurice, e Su 2000; Aydar et al. 2002; Karasawa et al. 2002; Teruo Hayashi et al. 1999; T. Hayashi et al. 1995; Yamamoto et al. 1995; Church, Lodge, e Berry 1985; Klette et al. 1997; H. Zhang e Cuevas 2002). Possibly they also regulate the K+ current by coupling to the channel as a modulatory subunit inhibiting it (Aydar et al. 2002). In the central nervous system, they are mostly present in the prefrontal cortex, hippocampus and striatum (Teruo Hayashi e Su 2004). They are implicated in synaptic plasticity and learning and memory (Maurice e Goguadze 2017; Tsai et al. 2009, 1) and also in pathologies such as schizophrenia, depression, anxiety and dementia (Ishikawa e Hashimoto 2009).
1.2.2 Prefrontal cortex circuitry
The prefrontal cortex is the most anterior portion of the frontal lobe and is one of the latest regions to develop in the ontogeny. It is known to play a role in working memory, attention, response initiation, behavioral flexibility and autonomic controlling and emotion. Since it connects to both other cortical and subcortical regions, the prefrontal cortex can be considered an association area in the brain. The main regions projecting to prefrontal are: the thalamus, the basal ganglia, the amygdala, the hippocampus and the temporal and parietal association cortices. In addition, some peculiar extrinsic connections are present in there such as: reciprocal connections to 1) the anterior and dorsal thalamic nuclei (especially to nucleus dorsalis medialis); 2) the posterior sensory processing areas (receives afferents input from auditory, somatic, visual, olfactory areas); 3) to the limbic system, mainly to the hypothalamus, the amygdala and the hippocampus; And efferent connections to subcortical motor control areas, for instance to the basal ganglia and indirectly to the
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cerebellum and thalamus. Additionally, it has reciprocal connections to diencephalic and mesencephalic nuclei and reticular formations (Fuster 2009, 2001).
The prefrontal cortex can be distinguished from the other cortices by its cytoarchitecture, anatomical features/neural connectivity. In rats, it can be essentially divided into three main parts: 1) a major medially located region along the wall of the hemisphere, the medial prefrontal cortex; 2) A more ventral part, located in the dorso-ventral portion close to the olfactory bulb, the anterior orbital frontal cortex; and 3) the more lateral part, the lateral or sulcal prefrontal cortex, also known as agranular insular cortex. In this thesis we will focus on the medial prefrontal cortex, which can be divided into four subareas: dorsal anterior cingulate, prelimbic, infralimbic and medial orbital cortices (Heidbreder and Groenewegen 2003; Krettek and Price 1977; Van Eden and Uylings 1985).
About cortico-cortical connections, the infralimbic prefrontal cortex sends efferent projections to the prelimbic and medial orbital cortex, and less significant to the anterior cingulate cortex (ACC). The prelimbic area tends to project more to dorsal cortical areas compared to infralimbic (Room et al. 1985; Sesack et al. 1989). Also, it sends fibers to infralimbic and anterior cingulate cortex (ACC), and with a less extent, to the premotor area FR2 and the caudal portion of the cingulate region (Datiche e Cattarelli 1996). In turn, the anterior cingulate projections target mainly the sensory-motor and visual-related areas, and also the caudal cingulate and the retrosplenial cortex (Sesack et al. 1989; Reep, Goodwin, e Corwin 1990). Regarding the afferent connections, the ventromedial prefrontal cortex (the infralimbic and ventral prelimbic areas) receives cortical inputs from perirhinal, ventral agranular insular and piriform areas (Datiche e Cattarelli 1996; Reep, Goodwin, e Corwin 1990; Van Eden, Lamme, e Uylings 1992). While the rostral part of ACC and the motor frontal area FR2 receive more projections from the frontal and parietal motor and somatosensory areas, and temporal association and agranular insular regions (Condé et al. 1995; Reep et al. 1987; Reep, Goodwin, e Corwin 1990; Van Eden, Lamme, e Uylings 1992). In general lines, the dorsal prefrontal cortex efferents and afferents are primarily connected to the somatosensory association cortex, while the more ventral part of the prefrontal is mainly related to cortical interactions, limbic and association areas (Condé et al. 1995; Sesack et al. 1989; Van Eden, Lamme, e Uylings 1992).
Besides cortico-cortical relations, the medial prefrontal cortex (mPFC) projects to the basal forebrain, to the olfactory and limbic structures. Since the subject of study of this
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thesis is the medial prefrontal cortex and the limbic structure of the hippocampus, I will focus mainly on its relationship to mPFC.
The ventral mPFC is predominantly connected to the hippocampus and the amygdala, both main limbic structures. The mPFC connects to the hippocampal formation almost exclusively in a unidirectional way since it receives direct hippocampal projections and only a few projections seem to occur in the opposite direction (Carr e Sesack 1996; Gabbott, Headlam, e Busby 2002; Jay e Witter 1991; Hurley et al. 1991; Sesack et al. 1989). Hippocampal projections originating in the subiculum and CA1 reach mainly the infralimbic and ventral prelimbic regions (Jay e Witter 1991; Swanson 1981). Nevertheless, the mPFC indirectly communicates with the hippocampus through the entorhinal or subcortical structures. The prefrontal cortex communicates bi-directionally with the parahippocampal region. For instance, while the entorhinal cortex receives projections mainly from the infralimbic region, the perirhinal cortex receives inputs from the dorsal mPFC (Hurley et al. 1991; Sesack et al. 1989). Also, there an indirect pathway from the mPFC to the hippocampus through the thalamic nucleus reuniens (NR) (Vertes et al. 2007). The dense projection of mPFC to nucleus reuniens and further to the hippocampus suggests that the NR plays a major role in the information transference from the mPFC to the hippocampus (Vertes 2002). The HP - mPFC interaction is essential to cognitive processing especially for learning and memory, and evidence suggest that both bidirectional pathways are involved (Laroche, Davis, e Jay 2000; Takita et al. 1999).
Although several neurotransmitters are present in the prefrontal cortex, it is mainly mediated by dopamine and acetylcholine. Monoaminergic afferents from the brain stem and limbic system extensively innervate the prefrontal cortex, and in the opposite direction, PFC sends efferent projections to the thalamus, subthalamus, hypothalamus and lower brain stem. The prefrontal cortex produces a great amount of norepinephrine and dopamine compared to the posterior sensory and association cortex. Although PFC is not a major serotonin producer, it receives a dense amount of serotonergic axons terminals from dorsal and medial raphe nuclei (Fuster 2009; 2001; E. C. Azmitia e Segal 1978). Bearing in mind this interaction, we will review the relationship between the serotonergic system and the PFC in the following section.
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As previously mentioned, the main producers of serotonin in the brain are the raphe nuclei. The dorsal and median raphe nuclei send projections broadly to the forebrain including all cortices, predominantly the frontal lobe (Pau Celada, Puig and Artigas 2013). There are bidirectional projections between the medial prefrontal cortex and the dorsal and medial raphe (Hajos et al. 1998). By using a retrograde tracer, Pickel et al found that serotonergic neurons from the midline portion of the dorsal raphe project to the mPFC (Van Bockstaele, Biswas, e Pickel 1993). In the other direction, among other frontal subregions, the ventral mPFC (the infralimbic and dorsal peduncular cortices) projects to the dorsal raphe nucleus (Hajos et al. 1998; Peyron et al. 1998). The high density of axon terminals and serotonergic receptors, especially 5-HT1A, 5-HT2A and 5-HT2C family, in the prefrontal cortex suggests that the serotonergic system is implicated in the prefrontal cortex modulation and its role in cognitive and emotional function (Pau Celada, Puig and Artigas 2013).
Electrical stimulation of the ventral mPFC leads to broad inhibition of the serotonergic neurons in the dorsal and medial raphe, while it does not occur for non-serotonergic neurons or stimulation of the dorsal PFC (Hajos et al. 1998; P. Celada et al. 2001). Possibly this effect is mediated by the 5-HT binding to the 5-HT1A (P. Celada et al. 2001), which occur almost exclusively as an autoreceptor in the raphe serotonergic neurons (Riad et al. 2000). Additionally to the inhibitory effect of 5-HT application, Avesar et al found mixed responses in in vitro experiments: the local application of 5-HT leads to hyperpolarization, depolarization and a third mixed biphasic response of layer V pyramidal neurons in the mPFC. The hyperpolarization effect was mediated by the activation of 5-HT1B receptors, while the depolarization occurred by 5-HT2A receptors activation. The biphasic response profile exhibited a hyperpolarization followed by a depolarization (Avesar e Gulledge 2012), an effect that can be explained by the activation of 5-HT1A followed by 5-HT2A receptors, respectively (Araneda e Andrade 1991). Those findings are compatible with the overlapped expression of 5-HT1A and 5-HT2A receptor family in the same neurons in the PFC (80%) (Santana et al. 2004; Amargós-Bosch et al. 2004). Although, this overlap does not occur in the inhibitory parvalbumin PFC interneurons, which either express 5-HT1A or 5-HT2A receptor type, and are inhibited and excited via 5-HT1A and 5-HT2A receptors, respectively (Puig et al. 2010). The excitatory effect of 5-HT in pyramidal neurons occurs mainly through the inhibition of the after-hyperpolarizing current that usually occurs after burst activity induced by 5-HT2A receptor activation
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(Araneda e Andrade 1991; Andrade 1998). The serotonergic agonist DOI led to an increase, decrease or no change in firing rate of PFC neurons. Such an inhibitory effect is dependent on GABAa, while the excitatory effect of 5-HT2A receptor seems to be dependent on glutamatergic transmission since DOI increases the glutamate effect. Also, blockade of GluR II and AMPA receptor decreases the EPSC mediated by 5-HT2A receptor (George K. Aghajanian e Marek 1999; G. K. Aghajanian e Marek 1997).
Those literature findings are in agreement with their expression in different parts of the neurons: 5-HT1A more concentrated in the axon hillock while 5-HT2A in the apical dendrites of pyramidal cells (Puig e Gulledge 2011). In the same sense, the electrical stimulation of the medial forebrain bundle leads to inhibition of hippocampal pyramidal neurons mediated by 5-HT1A receptors (blocking its activity reversed the inhibition)(Chaput e de Montigny 1988). Also, in vivo 1Hz stimulation of the dorsal raphe regulated the frequency and the amplitude of slow-wave activity through 5-HT2A receptor activation in the PFC of anesthetized rats (Puig et al. 2010). Amagos-Bosch et al found that electrical stimulation of dorsal and medial raphe elicited inhibitory potentials mediated by 5-HT1A receptors and excitatory potentials mediated by 5-HT2A receptors in the mPFC pyramidal neurons. In addition, they observed an opposite response at the same recorded neuron whenever placed the stimulation electrode in a slightly different coordinate. Yet in the same work, by performing microdialysis experiments they found that 5-HT2A/C receptor agonist injection increased serotonin levels and that this effect is reversed by 5-HT1A receptor agonist. Overall, those results indicate the existence of a complex and highly specific network arrangement between both areas (Amargós-Bosch et al. 2004).
It is worth noting that mPFC pyramidal neurons inhibition resulting from raphe stimulation has also a GABA component since this effect is blocked by GABAa antagonist (Puig, Artigas, e Celada 2005). This activation of GABAergic neurons may be mediated by 5-HT3A receptor activation, a possible mechanism that could explain the resultant predominant cortical inhibition. Latency and cytoarchitectonic pieces of evidence indicate the existence of a direct monosynaptic pathway from raphe GABAergic neurons into mPFC, favoring hyperpolarization of mPFC pyramidal neurons as a result of 5-HT release (Pau Celada, Puig, e Artigas 2013).
Besides individual neuronal activity, cortical oscillations can as well be affected by pharmacological or electrophysiological interference on the serotonergic network. For
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instance, Puig et al observed an alteration of frequency and amplitude of slow wave-like oscillations in anesthetized rats upon DR stimulation. They found an increase in frequency, following the stimulation frequency; and an increase in up states potentials and a decrease of the down states. Additionally, high-frequency stimulation in the DR eliminated the slow waves and promoted long-lasting depolarization, eliminating the down-states of slow-like oscillation. Yet in the same work, authors reported an increase in gamma oscillation amplitude after blocking 5-HT1A receptors, as well as an augmented firing rate of fast-spiking interneurons and its synchronization to gamma cycles. The opposite effect was observed after blocking 5-HT2A receptors (Puig et al. 2010).
A summary review of receptor location and interaction between the prefrontal cortex and dorsal raphe is proposed by Celada et al. (2013) and reproduced in Figure 1.2.4.
Figure 1.2.4: Schematic representation of the cellular location of 5-HT1A and 5-HT2A
receptors in the prefrontal cortex and dorsal raphe nucleus, and connections between both regions. Adapted by D.Drieskens from Pau Celada, Puig, e Artigas 2013.
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1.2.4 Hippocampal-cortical circuitry
The hippocampus (named in Latin after its shape similar to a seahorse) is a well-known brain area that is closely related to memory function and spatial navigation. It is part of the forebrain and located in the temporal medial lobes. In this section, I will review its circuitry, and how it communicates with the cortex. I will also present the main relevant findings regarding hippocampal functional communication with other cortical areas, and how it could be affected by the serotonergic agonists used in this work.
1.2.5 The hippocampal formation
The hippocampal formation is part of the limbic system and it comprehends the entorhinal cortex (EC), the pre/parasubiculum, the subiculum and the hippocampus (HP) itself. The hippocampus is a unique structure and in some parts is anatomical organization resembles the cortex. However, differently from cortical organizations, the hippocampal information flow is unidirectional, a remarkable feature, as well as the fact that it is one of the few areas of the brain that process and integrate multimodal sensory information from a variety of cortical regions.
The hippocampus can be divided into the main areas of: CA1, CA2, CA3 and dentate gyrus (DG). The CAs are composed by the layers stratum oriens, stratum pyramidale, stratum lucidum (only in CA3), stratum radiatum, stratum lacunosum/moleculare. The DG layers are the polymorphic layer, stratum granulosum, stratum moleculare (inner and external). The str oriens is the most superficial layer right below the alveus and contains the inhibitory basket cells, OLM and horizontal trilaminal cells. This layer also concentrates the dendrites of the pyramidal cells, which receive inputs from the entorhinal cortex, septum, commissure and contralateral hippocampus. The str pyramidale, as it says, has the pyramidal cell bodies, in CA3 it also contains the mossy fibers and interneurons bodies, as axo-axonic and bistratified cells.
The afferent sensory input arrives at the EC and then at the hippocampus. The EC layer II neurons project to the hippocampal subregion CA3 and DG, through the perforant pathway. While the layer III neurons make synapses to the CA1 and subiculum, through the perforant and alveolar pathways. From DG the granular cells send projections to the CA3
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through the mossy fibers. The CA3 pyramidal cells axons then project to CA1 cells, through the Schaffer collaterals. Finally, CA1 then send axons projections to the subiculum and the deeper layers of the EC. The subiculum also projects to deeper layers of the EC, as well to the pre and parasubiculum (See Figure 1.2.5, reproduced from The Hippocampus Book (Andersen et al. 2006)).
Besides EC connection, the hippocampus also receives input from the perirhinal cortex, that projects to CA1 at the str lacunosum-moleculare. In the other direction, CA1 cells also project to the perirhinal cortex. The amygdala, another structure from the limbic system, also has a bidirectional connection to the hippocampal CA1 region (Yang e Wang 2017). Moreover, the CA1 septal region projects to the retrosplenial and the more medial CA1 region presents considerable projections to the medial frontal lobe.
The hippocampus also presents some subcortical connections. Those include a significant input of medial septum in the polymorphic layer of DG, str oriens and lacunosum-moleculare of the CA3 region and str oriens in the CA1. In the opposite direction CA3 projects mainly to the lateral septum. There are also hippocampal bidirectional connections to the hypothalamic and thalamic regions, as well as to the brain stem nuclei of noradrenaline and serotonin, locus coeruleus and raphe nuclei, respectively. For instance, the DG receives projections from locus coeruleus, ventral tegmental area (dopamine) and medial/dorsal raphe nuclei mainly in the polymorphic layer. Also, a great portion of the Gabaergic interneurons of DG receives serotonergic terminals from raphe (e.g. pyramidal basket cells). Regarding CA1 and CA3, the serotonergic afferents are diffuse and display different characteristics. On the other hand, the noradrenergic terminals arrive at the str lacunosum-moleculare and str ludicum.
The raphe nuclei axons that arrive at the hippocampus can be grouped into ‗thin‘ or ‗thick‘. While the varicosities from the thin axons are spaced and likely deliver the neurotransmitter in the extracellular space in a non-standard synaptic configuration, the thick axons form asymmetric synapses predominantly to the GABAergic interneurons projecting to the hippocampal pyramidal dendrites.
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Figure 1.2.5. Schematic representation of the hippocampal formation. The information flow in the hippocampus occurs in the following sequence: The input signal arrives in the hippocampus (HP) through the entorhinal cortex (EC). Neurons from EC layer II project to Dentate gyrus (DG) and CA3 through the perforant pathway, while neurons from layer III make synapses with CA1 and subiculum through the perforant and alvelar pathways. Reproduced from Andersen et al. 2006.
1.2.6 Memory and spatial navigation
The hippocampus is a conserved structure among mammals and has been implicated in declarative memory consolidation and spatial navigation. A well-known case in the field is the one of Henry Molaison, aka HM patient, who had both hippocampi removed to treat epilepsy and became amnesiac. He was not able to consolidate newly acquired declarative memories, as well as he did not recall events from ~2 years before surgery. Although he presented severe anterograde and lesser retrograde amnesia, he was able to acquire and consolidate new procedural memories (Scoville and Milner 1957; Dossani, Missios, and Nanda 2015; Milner and Klein 2016).
Since then, many studies have linked this structure with learning and memory giving rise to the hypothesis of a hippocampal-cortex memory system (Zola-Morgan e Squire 1990; Eichenbaum 2000). According to this hypothesis, memories would be initially represented in the hippocampus and then gradually propagating to other parahippocampal areas and then to the cortex. In that way, the hippocampus could work integrating the ‗what‘, ‗where‘ and ‗when‘ components of a memory (Eichenbaum 2000). In fact, the
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hippocampus and other parahippocampal regions are also important for spatial navigation (e.g., the where component of a memory). In 1971, O‘Keefe and Dostrovsky showed that the firing of some hippocampal cells correlated with the animal position, naming those cells as place cells (O‘Keefe e Dostrovsky 1971). Since then, it has been shown a lot of other spatial correlates with this region: speed cells, head direction cells, grid cells, border cells; suggesting its role in processing spatial information (Moser, Kropff, e Moser 2008; Taube, Muller, e Ranck 1990; Lever et al. 2009; Kropff et al. 2015).
In terms of electrophysiological activity, the rat hippocampus has a prominent oscillation in the theta frequency band (usually 6-12Hz). The theta rhythm is present during various rat behaviors, including active waking, animal‘s locomotion and REM sleep (Sławińska e Kasicki 1998; Jonathan Winson 1974; Vanderwolf 1969). In addition, several studies have demonstrated the cognitive function of theta rhythm, especially for memory consolidation and spatial navigation (J. Winson 1978; Easton et al. 2011). For example, the abolishment of hippocampal theta rhythm by medial septum lesion, one of the ‗theta oscillators‘, can lead to impaired memory consolidation and spatial navigation (J. Winson 1978). Theta is also related to place cell activity, that coordinates their spikes within the theta cycle according to the animal position (Skaggs et al. 1996).
In addition to theta oscillations, the hippocampus also presents other faster oscillations in the gamma range (30-100 Hz) and high-frequency oscillation (120-160 Hz). Those faster rhythms can also be coupled to theta, showing a form of cross-frequency coupling. Interestingly, a low gamma rhythm (30-50 Hz) has been associated with CA3 while a faster one (high-gamma, 60-100 Hz) has been associated with EC (Colgin et al. 2009). Those two oscillators are believed to coordinate the inputs reaching the hippocampus, either highlighting the sensory information or memory traces.
1.2.7 Hippocampus and serotonergic system
In this section, I will briefly review the interaction between the hippocampus and serotonergic system, highlighting the occurrence of serotonergic receptors, especially those activated by the serotonergic agonists investigated in this thesis.
36 All serotonergic receptors are distributed in the hippocampus and therefore do somehow modulate its function, which is compatible with the presence of serotonergic afferents previously mentioned. The serotonergic receptor 5-HT1 is abundant in the hippocampus, and most of them are of 1A subtype (Mestikawy et al. 1989; D. Hoyer et al. 1994). They are present in the postsynaptic CA1 pyramidal and DG granular cells and also in extrasynaptic structures (Hannon e Hoyer 2008; Lanfumey e Hamon 2004). In addition, 5-HT1A is present as an autoreceptor in the presynaptic neuron: the somatodendritic autoreceptor‘s activation led to a decrease in 5-HT levels (Muchimapura, Mason, e Marsden 2003). Besides present in neurons, 5-HT1A receptors are also found in other cell types, e.g. astrocytes, radial glia cells (Efrain C. Azmitia et al. 1996).
Another particularly relevant serotonin receptor, especially for this thesis, is the 5-HT2A, which as the 5-HT1A is activated by both serotonergic agonists here investigated. Although, it is reported to be ten times less expressed in the HP compared to cortical levels, the 5-HT2A is also found in the hippocampus (Julius et al. 1990). More specifically, it is found in the presynaptic terminals of CA1 and CA3 pyramidal cells as well as in the DG granular cells (Q.-H. Li et al. 2004); and it is also reported in astrocytes (Xu e Pandey 2000). Considering the other receptors family, they are as well widely distributed in the hippocampus. As already mentioned, the hippocampus plays an important cognitive role, especially in memory consolidation. Mechanisms as long term potentiation and long term depression, crucial for those cognitive processes and long term memory, can be modulated by serotonergic transmission. In the majority, serotonergic receptors activation alters the cAMP levels in the cells and can trigger, for instance, immediate-early gene expression (e.g. BDNF) (Jiang et al. 2016). It has also been implicated in hippocampal neurogenesis regulation in the dentate gyrus (Brezun e Daszuta 1999).
Overall, as summarized and illustrated in Figure 1.2.6, the hippocampal function seems to be highly modulated by serotonergic transmission.
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Figure 1.2.6: Schematic representation of serotonergic receptors distribution on the hippocampus. Reproduced from The Hippocampus Book (Andersen et al. 2006).
1.2.8 Serotonergic agonist/hallucinogens/psychedelics
The term psychedelic came from ancient Greek psukhḗ, ―mind/soul‖ and dêlos, ―manifest, visible‖; and also it is referred to as a classic hallucinogen or serotonergic hallucinogen. Those molecules have very similar chemical structures (Figure 1.2.7. Adapted from Nichols (2004)). Considering that the literature is not completely consistent regarding the use of terms in this field, in order to avoid misleading interpretations, it is important to delimitate the use of the term hallucinogen as a substance that differs from hallucinatory
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state described in diseases as schizophrenia. As previously delimited by Glennon (1999) and adopted by Lopes-dos-Santos (2015), here I use the same criteria for a serotonergic hallucinogen:
It is a substance that produces thought, mood and perceptual changes, without causing addiction, craving, nor major physical disturbances, cognitive impairments, amnesia or delirium (Hollister 1972).
It is dependent on the activation of the 5-HT2A receptor; It is recognized as DOM by rats in a discriminative task;
Figure 1.2.7: Chemical structure of serotonin and serotonergic hallucinogens. Note their common chemical structure highlighted in blue. Adapted by S. Ruschi from Nichols 2004.
The serotonergic agonists can be divided into two main classes: Tryptamines and Phenylethylamines. Tryptamines are monoamine alkaloids that include serotonin and a series of analogues, including the dimethyltryptamines (DMTs), 5-MeO-DMT, NN-DMT, and psilocybin, the prodrug of psilocin, which have relative conformational flexibility and
