Neurobiologia da memória temporal e contextual aversiva: engramas, redes neurais e conexões funcionais e neuroanatômicas do córtex pré-límbico
Data
2022-02-24
Autores
Santos, Thays Brenner dos 
Orientadores
Oliveira, Maria Gabriela Menezes de
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Em associações separadas no tempo, uma representação de um estímulo condicionado (CS) deve ser mantida para ser associada com um estímulo aversivo (US) posterior. Entretanto, o efeito deste intervalo de tempo no circuito neural que subjaz memórias aversivas, que pode requerer, diferencialmente, grupos neuronais seletivamente ativados (neuronal ensembles) que contribuem para a memória transiente do CS, permanece desconhecido. Visto que o córtex pré-límbico (PL) é engajado em tarefas que requerem a persistência de estímulos ao longo do tempo, neuronal ensembles no PL podem participar da neurobiologia de associações temporais. O objetivo deste estudo foi determinar o circuito neural que contribui para associações separadas no tempo. Para isto, nós utilizamos uma tarefa na qual um intervalo de 5s separa um CS contextual do US, chamada de condicionamento de medo ao contexto em descontinuidade temporal (CFC-5s), um modelo que também permite a investigação de fatores contextuais no circuito neural de associações temporais e a formação e manutenção de representações neurais conjuntivas ao longo do tempo. Nós examinamos a atividade relacionada com a aquisição e a expressão de um fator de transcrição relacionada a consolidação em seguida ao treino do CFC-5s. Nós também avaliamos células ativadas pelo aprendizado e reativadas pela evocação do CFC-5s para inferir o engrama de associações temporais e contextuais. Nós então usamos a co-ativação das regiões cerebrais para gerar redes funcionais subjacentes a aquisição do CFC-5s, e a analisamos usando teoria de grafos. Em seguida, nós investigamos se a reativação de neuronal ensembles no PL, ativados pela aprendizagem do CFC-5s, era suficiente para induzir a expressão de respostas condicionada, e se sua inibição prejudicava a evocação da memória. Além disso, nós investigamos a contribuição de aferentes do PL e da amígdala basolateral (BLA), uma região relacionada a convergência do CS e US para sua associação, na aquisição do CFC-5s, e a contribuição de conexões funcionais do PL para a consolidação do CFC-5s. Por último, nós também investigamos se a conexão funcional entre o PL e a região CA1 do hipocampo ventral (vCA1) relacionada ao aprendizado contextual, era necessária para a aquisição do CFC-5s. Para isto, nós utilizamos reações imuno-histoquímica (c-Fos e para pCREB), traçador retrógrado (retrobead), inativação temporária (muscimol), optogenética e duas estratégias genéticas para acessar neurônios ativados permanentemente (TRAP e RAM). Para determinar o circuito neural do processo de formar, manter e associar representações neurais, nós comparado ratos ou camundongos treinados no CFC-5s com grupos para o controle de associações sobrepostas no tempo (condicionamento de medo ao contexto), aprendizado contextual, aprendizado não associativo e condições basais. Os resultados mostraram que a aquisição de associações de medo separadas no tempo recruta especificadamente 11 regiões cerebrais do córtex prefrontal medial, da amígdala, do hipocampo ventral, do córtex entorrinal e do córtex parahippocampal. O CFC-5s também engaja a ativação de projeções do córtex perirrinal e do vCA1 ao PL, e de projeções do PL para a BLA. A expressão de pCREB induzida pelo treino do CFC-5s requereu interação funcional entre o PL e a amígdala, e entre o PL e o hipocampo dorsal. O CFC-5s também induziu neuronal ensembles no PL relacionados ao engrama, que são necessários para a evocação do CFC-5s e que reativados induzem à resposta condicionada. Redes funcionais do CFC-5s tiveram como hubs os núcleos da amígdala, incluindo uma maior conectividade interna e externa com o tálamo e o hipocampo. Os núcleos da amígdala e o hipocampo ventral também foram regiões cerebrais altamente centrais (hubs) na rede funcional. Em conjunto, a conectividade entre estas regiões cerebrais poderia fornecer uma memória transiente para associações separadas no tempo, o que pode ser relevante para entender associações que se apoiam em processos similares, como o condicionamento de traço e a memória de trabalho. Nós propomos que este circuito neural mantenha de forma incidental representações neurais para sua associação aversiva.
In associations separated in time, a neural representation of a conditioned stimulus (CS) must be sustained over time to be associated with a later unconditioned stimulus (US). However, the effect of this time factor remains unknown in the neural circuit subserving fear associations, which may differentially include an ensemble of distributed neurons that cooperate for a transient memory of the CS. Given that the prelimbic cortex (PL) has been engaged in tasks requiring persistence of stimuli over time, neuronal ensembles in PL and brain regions that connect functionally or neuroanatomically with the PL may subserve temporal associations. The present study aimed to determine the neural circuit supporting fear associations separated in time. For this, we used a task in which a 5 second interval separates the context from the US, called contextual fear conditioning (CFC) with temporal discontinuity, the CFC-5s, a model that also allows the investigation of contextual factors in the neural circuit of fear associations and the formation and incidental maintenance of conjunctive neural representations over time. We brain-wide examined the activity associated with the encoding and transcription factors associated with the consolidation following the CFC-5s training. We also evaluated the overlap of cells activated by the CFC-5s learning and reactivated by the CFC-5s retrieval to infer the memory engram of temporal and contextual associations. We then used the co-activation of brain regions and co-activation of engram cells to generate functional networks and analyzed them by graph theory. After that, we interrogated encoding ensembles identified in the PL, observing if their reactivation was sufficient to induce fear expression and their inhibition to disrupt the memory retrieval. In addition, we investigated the contributions of direct afferents to the PL and the basolateral amygdala nucleus (BLA), a brain region related to CS-US convergence and fear association, in the encoding, and the contribution of functional interaction with the PL for the consolidation. Finally, we evaluated whether the functional connection of the vCA1, a brain region related to contextual learning, with the PL, was necessary to encode the CFC-5s. For this, we used a combined approach of immunohistochemistry, retrograde tracing (retrobeads), temporary inactivation (muscimol), optogenetics, and two genetic strategies to access activated neurons permanently, the targeted recombination in active populations (TRAP) transgenic mice and the virally mediated robust activity marking (RAM) system. We quantified the c-Fos expression, the c-Fos expression in PL-projecting or BLA-projecting neurons 90 minutes following the CFC-5s training, and the phosphorylation of cAMP response element-binding protein (pCREB) 3 hours following the CFC-5s training, preceded or not by pre-training temporary inactivation of the PL by muscimol. We also observed the overlapping of TRAPed neurons following the training and c-Fos-positive cells following the test session. We tagged PL neurons activated during the CFC-5s training with ChR2 or NpACY using RAM viruses to opto-stimulate or -inhibit them in the test session. We functionally disconnected the vCA1-PL using pre-training asymmetrical reversible inactivation by muscimol. To specifically determine the neural circuit supporting the processes of forming, maintaining, and associating neural representations over time, we compared rats or mice trained in the CFC-5s with control groups for associations overlapped in time (standard CFC), contextual and non-associative learning, and basal activation. Results showed that the encoding of contextual fear associations separated in time is specifically supported by activity in 11 brain regions from the medial prefrontal cortex, amygdala, ventral hippocampus, lateral entorhinal cortex, and parahippocampal cortex. The CFC-5s also engaged the activation of the perirhinal cortex and vCA1 projections to the PL and PL projections to the BLA. pCREB expression induced by CFC-5s required functional interaction between the PL and the amygdala, and PL and the dorsal hippocampus. Functional disconnection of the vCA1-PL impaired the encoding of the CFC-5s and spared the CFC. The CFC-5s, but not the CFC, has engram cells in the PL necessary for memory retrieval, and that reactivation induced fear expression. Functional networks of the CFC-5s had increased importance of the amygdala nuclei, including higher intrinsic connectivity and external connectivity with the thalamus and the hippocampus. Amygdala nuclei were also hub regions, and thalamic nuclei were connector hubs. Connectivity among these brain regions could provide the source of transient memory for fear associations separated in time to occur, which may be relevant to understanding associations that rely on similar processes, such as trace conditioning and working memory. We proposed a role of this neural circuit in the incidental transient memory of conjunctive neural representations for fear associations.
In associations separated in time, a neural representation of a conditioned stimulus (CS) must be sustained over time to be associated with a later unconditioned stimulus (US). However, the effect of this time factor remains unknown in the neural circuit subserving fear associations, which may differentially include an ensemble of distributed neurons that cooperate for a transient memory of the CS. Given that the prelimbic cortex (PL) has been engaged in tasks requiring persistence of stimuli over time, neuronal ensembles in PL and brain regions that connect functionally or neuroanatomically with the PL may subserve temporal associations. The present study aimed to determine the neural circuit supporting fear associations separated in time. For this, we used a task in which a 5 second interval separates the context from the US, called contextual fear conditioning (CFC) with temporal discontinuity, the CFC-5s, a model that also allows the investigation of contextual factors in the neural circuit of fear associations and the formation and incidental maintenance of conjunctive neural representations over time. We brain-wide examined the activity associated with the encoding and transcription factors associated with the consolidation following the CFC-5s training. We also evaluated the overlap of cells activated by the CFC-5s learning and reactivated by the CFC-5s retrieval to infer the memory engram of temporal and contextual associations. We then used the co-activation of brain regions and co-activation of engram cells to generate functional networks and analyzed them by graph theory. After that, we interrogated encoding ensembles identified in the PL, observing if their reactivation was sufficient to induce fear expression and their inhibition to disrupt the memory retrieval. In addition, we investigated the contributions of direct afferents to the PL and the basolateral amygdala nucleus (BLA), a brain region related to CS-US convergence and fear association, in the encoding, and the contribution of functional interaction with the PL for the consolidation. Finally, we evaluated whether the functional connection of the vCA1, a brain region related to contextual learning, with the PL, was necessary to encode the CFC-5s. For this, we used a combined approach of immunohistochemistry, retrograde tracing (retrobeads), temporary inactivation (muscimol), optogenetics, and two genetic strategies to access activated neurons permanently, the targeted recombination in active populations (TRAP) transgenic mice and the virally mediated robust activity marking (RAM) system. We quantified the c-Fos expression, the c-Fos expression in PL-projecting or BLA-projecting neurons 90 minutes following the CFC-5s training, and the phosphorylation of cAMP response element-binding protein (pCREB) 3 hours following the CFC-5s training, preceded or not by pre-training temporary inactivation of the PL by muscimol. We also observed the overlapping of TRAPed neurons following the training and c-Fos-positive cells following the test session. We tagged PL neurons activated during the CFC-5s training with ChR2 or NpACY using RAM viruses to opto-stimulate or -inhibit them in the test session. We functionally disconnected the vCA1-PL using pre-training asymmetrical reversible inactivation by muscimol. To specifically determine the neural circuit supporting the processes of forming, maintaining, and associating neural representations over time, we compared rats or mice trained in the CFC-5s with control groups for associations overlapped in time (standard CFC), contextual and non-associative learning, and basal activation. Results showed that the encoding of contextual fear associations separated in time is specifically supported by activity in 11 brain regions from the medial prefrontal cortex, amygdala, ventral hippocampus, lateral entorhinal cortex, and parahippocampal cortex. The CFC-5s also engaged the activation of the perirhinal cortex and vCA1 projections to the PL and PL projections to the BLA. pCREB expression induced by CFC-5s required functional interaction between the PL and the amygdala, and PL and the dorsal hippocampus. Functional disconnection of the vCA1-PL impaired the encoding of the CFC-5s and spared the CFC. The CFC-5s, but not the CFC, has engram cells in the PL necessary for memory retrieval, and that reactivation induced fear expression. Functional networks of the CFC-5s had increased importance of the amygdala nuclei, including higher intrinsic connectivity and external connectivity with the thalamus and the hippocampus. Amygdala nuclei were also hub regions, and thalamic nuclei were connector hubs. Connectivity among these brain regions could provide the source of transient memory for fear associations separated in time to occur, which may be relevant to understanding associations that rely on similar processes, such as trace conditioning and working memory. We proposed a role of this neural circuit in the incidental transient memory of conjunctive neural representations for fear associations.
Descrição
Citação
SANTOS, Thays Brenner dos. Neurobiology of temporal and contextual aversive memory: engrams, neural networks and functional and neuroanatomical connections of the prelimbic cortex. São Paulo, 2022. 417 f. Tese (Doutorado em Psicobiologia) – Escola paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2022.