Tatiana Lima Ferreira

Contextual fear memories become less specific as they age, modeling fear overgeneralization seen in post-traumatic stress disorder. Glucocorticoid receptor (GR) signaling in the dorsomedial prefrontal cortex (dmPFC) during the immediate post-learning period may govern both endocrine recovery from an aversive experience and the eventual specificity of the resulting memory, but this link remains untested. We infused vehicle or the GR antagonist mifepristone into the dmPFC of rats immediately after contextual fear conditioning, then measured corticosterone dynamics, fear expression at recent and remote time points, and c-Fos coactivation networks. Mifepristone accelerated corticosterone recovery without changing total hormone release, spared recent memory, and produced stronger, less context-specific freezing at the remote time point. This behavioral shift coincided with reorganization of the retrieval network from a salience-network-like to a default-mode-network-like configuration. These findings identify dmPFC glucocorticoid signaling as a mechanism constraining fear memory generalization as memories transition to a remote, cortically dependent state.

The ability to retrieve contextual fear memories depends on the coordinated activation of a brain-wide circuitry. Transition from recent to remote memories seems to involve the reorganization of this circuitry, a process called systems consolidation that has been associated with time-dependent fear generalization. However, it is unknown whether emotional memories acquired under different stress levels can undergo different systems consolidation processes. Here, we explored the activation pattern and functional connectivity of key brain regions associated with contextual fear conditioning (CFC) retrieval after recent (2 days) or remote (28 days) memory tests performed in rats submitted to strong (1.0mA footshock) or mild (0.3mA footshock) training. We used brain tissue from Wistar rats from a previous study, where we observed that increasing training intensity promotes fear memory generalization over time, possibly due to an increase in corticosterone (CORT) levels during memory consolidation. Analysis of Fos expression across 8 regions of interest (ROIs) allowed us to identify coactivation between them at both timepoints following memory recall. Our results showed that strong CFC elicits higher Fos activation in the anterior insular and prelimbic cortices during remote retrieval, which was positively correlated with freezing along with the basolateral amygdala. Rats trained either with mild or strong CFC showed broad functional connectivity at the recent timepoint whereas only animals submitted to the strong CFC showed a widespread loss of coactivation during remote retrieval. Post-training plasma CORT levels are positively correlated with FOS expression during recent retrieval in strong CFC, but negatively correlated with FOS expression during remote retrieval in mild CFC. Our findings suggest that increasing training intensity results in differential processes of systems consolidation, possibly associated with increased post-training CORT release, and that strong CFC engages activity from the aIC, BLA and PrL – areas associated with the Salience Network in rats – during remote retrieval.

Introdução. O envolvimento do sistema estriatal na aprendizagem S-R é geralmente baseado na plasticidade neural relacionada aos genes de expressão imediata (IEGs). Estudos anteriores também mostraram que o estriado dorsal desempenha um papel no condicionamento de medo do som (TFC). Objetivos. Dado que a expressão de IEGs no estriado dorsal está relacionada com a aprendizagem S-R, analisamos os eventos moleculares iniciais de consolidação no estriado medindo os níveis protéicos de EGR1, C-Fos e Arc, 30 e 90 minutos após o treino de TFC. Além disso, para minimizar uma possível interferência do hipocampo dorsal, a transmissão glutamatérgica foi interrompida durante o treino de condicionamento de medo usando a injeção de antagonista do receptor NMDA AP5 no hipocampo. Método. Ratos Wistar receberam AP5 ou injeção de solução salina no hipocampo cinco minutos antes de serem submetidos ao condicionamento de medo de som (pareamento som e choque) ou apenas o som. Resultados. Os animais que receberam pareamento som e choque nas patas apresentaram diminuição da proteína ARC 30 minutos após o treino quando comparados aos grupos controles. O grupo tratado com AP5, exposto à condição apenas de som, apresentou uma diminuição na proteína EGR 90 minutos após o treino quando comparada à solução salina e som. Não foram observadas diferenças nos níveis de proteína FOS. Conclusões. Nossos resultados sugerem que é possível que alguma interação entre estriado e hipocampo na experiência de processamento do som e que níveis reduzidos de ARC possam estar relacionados às características associativas desta tarefa pavloviana.

Stressful and emotionally arousing experiences create strong memories that seem to lose specificity over time. It is uncertain, however, how the stress system contributes to the phenomenon of time-dependent fear generalization. Here, we investigated whether the glucocorticoid hormonal system affects the specificity of contextual fear memories at several timepoints. We trained male Wistar rats on the contextual fear conditioning (CFC) task using two footshock intensities (mild CFC, 3 footshocks of 0.3 mA, or moderate CFC, 3x 0.6 mA) and immediately after the training session we administered corticosterone (CORT-HBC) systemically. We first tested the animals in a novel context and then in the training context at different intervals following training (2, 14, 28 or 42 days). By measuring freezing in the novel context and then contrasting freezing times shown in both contexts, we inferred contextual fear generalization for each rat, classifying them into Generalizers or Discriminators. Following mild CFC training, the glucocorticoid induced an increased discriminative contextual memory that lasted from two up to 28 days. In contrast, after moderate CFC training, CORT-HBC facilitated contextual generalization at 14 days, compared to the control group that maintained contextual discrimination at this timepoint. For this training intensity, however, CORT-HBC did not have any effect on recent memory specificity. These findings indicate that manipulating glucocorticoid levels after mild or moderately arousing experiences may differentially modulate memory consolidation and time-dependent fear generalization. HIGHLIGHTS - Rats were trained in a contextual fear task and tested for fear generalization. - Corticosterone improved accuracy in mild fear conditioning (FC) up to 28 days. - Same treatment elicited time-dependent fear generalization in moderate FC training.

The insular cortex (IC) is notably implicated in emotional and cognitive processing; however, little is known regarding to what extent its two main subregions play functionally distinct roles on memory consolidation of conditioned fear tasks. Here we verified the effects of temporary functional inactivation of the anterior (aIC) and posterior IC (pIC) on contextual and tone fear memory. Rats received post-training bilateral infusions of the GABA _A receptor agonist muscimol into either the aIC or pIC and were tested 48 and 72 hours after the conditioning session to assess contextual (CFC) and tone (TFC) fear conditioning, respectively. Inactivation of the aIC during memory consolidation did not affect fear memory for CFC or TFC. On the other hand, post-training inactivation of the pIC impaired TFC but not CFC. Our findings indicate that the pIC is a necessary part of the neural circuitry related to the consolidation of cued-fear memories. Highlights: We studied the role of the anterior (aIC) and posterior (pIC) insula in fear memory Post-training inactivation of aIC and pIC did not impact contextual fear conditioning The pIC but not aIC is necessary for the consolidation of tone fear conditioning

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