Contextual fear conditioning with a time interval induces CREB phosphorylation in the dorsal hippocampus and amygdala nuclei that depend on prelimbic cortex activity.

In temporal associations, a conditioned stimulus (CS) is separated by a time interval from the unconditioned stimulus (US), which activates the prelimbic cortex (PL) to maintain a CS representation over time. However, it is unknown whether the PL participates, besides the encoding, in the memory consolidation, and thus directly, with activity-dependent changes or indirectly, by modulation of activity-dependent changes in other brain regions. We investigated brain regions supporting the consolidation of associations with intervals and the influence of PL activity in this consolidation process. For this, we observed in Wistar rats the effect of pre-training PL inactivation by muscimol in CREB (cAMP response element-binding protein) phosphorylation, which is essential for memory consolidation, in subdivisions of the medial prefrontal cortex (mPFC), hippocampus, and amygdala 3 h after the training in the contextual fear conditioning (CFC) or CFC with 5-s interval (CFC-5s), fear associations without or with an interval between the CS and US, respectively. Both the CFC-5s and CFC training increased phosphorylation of CREB in the PL and infralimbic cortex (IL); lateral (LA) and basolateral (BLA) amygdala; dorsal CA1 (dCA1); dorsal (dDG), and ventral dentate gyrus, and the CFC-5s training in the central amygdala (CEA). PL activity was necessary for the CREB phosphorylation in the PL, BLA, CEA, dCA1, and dDG only in animals trained in the CFC-5s. The cingulate cortex, ventral CA1, and ventral subiculum did not have learning-induced phosphorylation of CREB. These results suggest that the mPFC, hippocampus, and amygdala support the consolidation of associations with or without intervals and that PL activity influences consolidation in the dorsal hippocampus and amygdala in temporal associations. Thereby, the PL contributes directly and indirectly by modulation to memory consolidation. The time interval engaged the PL early in recent memory consolidation. Results expanded PL's role beyond the time interval and remote memory consolidation.

Functional interaction of ventral hippocampal CA1 region and prelimbic cortex contributes to the encoding of contextual fear association of stimuli separated in time.

Although stimuli that are associated often overlap in time, previous events can also predict the occurrence of a later aversive stimulus and be associated with it to better guide future behavior. Associations of stimuli separated in time have been studied using discrete stimulus as the conditioned stimulus (CS) in trace conditioning or, more recently in our lab, using the context as the CS in contextual fear conditioning with temporal discontinuity (CFC-5s), a task that simultaneously includes the processing of time and space components. It is thought that fear memories are encoded by the strengthening of synaptic connections in a distributed neural network. However, it is unclear how this temporal factor, which may differentially require the maintenance of the stimulus over time, affects the interactivity between brain regions to form the association. Because the prelimbic cortex (PL) and the hippocampus have been individually engaged in trace conditioning, they may functionally interact to encode associations separated in time. This is anatomically supported by direct ipsilateral projections from the ventral hippocampal CA1 region (vCA1) to PL. The aim of the present study was to investigate the effect of the functional disconnection of vCA1 and PL on CFC-5s using pre-training asymmetric reversible inactivation with muscimol. For comparison, we also observed its effect on contextual fear conditioning (CFC). Results showed that the functional disconnection impaired the encoding of the CFC-5s, an association of stimuli separated in time, while did not affect the CFC, an association of stimuli overlapped in time. In addition, the preserved connection in one hemisphere was sufficient to support the encoding of CFC-5s. The time interval by itself did not increase freezing responses and both CFC and CFC tasks had similar generalization and higher freezing responses than unconditioned groups. These findings suggest that the time factor alters the requirement of the interactivity of the brain regions underlying fear conditioning and extend the relevance of hippocampal-prefrontal interactions in memory.

Involvement of the prelimbic cortex in contextual fear conditioning with temporal and spatial discontinuity.

Time plays an important role in conditioning, it is not only possible to associate stimuli with events that overlap, as in delay fear conditioning, but it is also possible to associate stimuli that are discontinuous in time, as shown in trace conditioning for a discrete stimuli. The environment itself can be a powerful conditioned stimulus (CS) and be associated to unconditioned stimulus (US). Thus, the aim of the present study was to determine the parameters in which contextual fear conditioning occurs by the maintenance of a contextual representation over short and long time intervals. The results showed that a contextual representation can be maintained and associated after 5s, even in the absence of a 15s re-exposure to the training context before US delivery. The same effect was not observed with a 24h interval of discontinuity. Furthermore, optimal conditioned response with a 5s interval is produced only when the contexts (of pre-exposure and shock) match. As the pre-limbic cortex (PL) is necessary for the maintenance of a continuous representation of a stimulus, the involvement of the PL in this temporal and contextual processing was investigated. The reversible inactivation of the PL by muscimol infusion impaired the acquisition of contextual fear conditioning with a 5s interval, but not with a 24h interval, and did not impair delay fear conditioning. The data provided evidence that short and long intervals of discontinuity have different mechanisms, thus contributing to a better understanding of PL involvement in contextual fear conditioning and providing a model that considers both temporal and contextual factors in fear conditioning.

Reactivation of encoding ensembles in the prelimbic cortex supports temporal associations.

Fear conditioning is encoded by strengthening synaptic connections between the neurons activated by a conditioned stimulus (CS) and those activated by an unconditioned stimulus (US), forming a memory engram, which is reactivated during memory retrieval. In temporal associations, activity within the prelimbic cortex (PL) plays a role in sustaining a short-term, transient memory of the CS, which is associated with the US after a temporal gap. However, it is unknown whether the PL has only a temporary role, transiently representing the CS, or is part of the neuronal ensembles that support the retrieval, i.e., whether PL neurons support both transient, short-term memories and stable, long-term memories. We investigated neuronal ensembles underlying temporal associations using fear conditioning with a 5-s interval between the CS and US (CFC-5s). Controls were trained in contextual fear conditioning (CFC), in which the CS-US overlaps. We used Robust Activity Marking (RAM) to selectively manipulate PL neurons activated by CFC-5s learning and Targeted Recombination in Active Populations (TRAP2) mice to label neurons activated by CFC-5s learning and reactivated by memory retrieval in the amygdala, medial prefrontal cortex, hippocampus, perirhinal cortices (PER) and subiculum. We also computed their co-reactivation to generate correlation-based networks. The optogenetic reactivation or silencing of PL encoding ensembles either promoted or impaired the retrieval of CFC-5s but not CFC. CFC-5s retrieval reactivated encoding ensembles in the PL, PER, and basolateral amygdala. The engram network of CFC-5s had higher amygdala and PER centralities and interconnectivity. The same PL neurons support learning and stable associative memories.

Functional network of contextual and temporal memory has increased amygdala centrality and connectivity with the retrosplenial cortex, thalamus, and hippocampus.

In fear conditioning with time intervals between the conditioned (CS) and unconditioned (US) stimuli, a neural representation of the CS must be maintained over time to be associated with the later US. Usually, temporal associations are studied by investigating individual brain regions. It remains unknown, however, the effect of the interval at the network level, uncovering functional connections cooperating for the CS transient memory and its fear association. We investigated the functional network supporting temporal associations using a task in which a 5-s interval separates the contextual CS from the US (CFC-5s). We quantified c-Fos expression in forty-nine brain regions of male rats following the CFC-5s training, used c-Fos correlations to generate functional networks, and analyzed them by graph theory. Control groups were trained in contextual fear conditioning, in which CS and US overlap. The CFC-5s training additionally activated subdivisions of the basolateral, lateral, and medial amygdala; prelimbic, infralimbic, perirhinal, postrhinal, and intermediate entorhinal cortices; ventral CA1 and subiculum. The CFC-5s network had increased amygdala centrality and higher amygdala internal and external connectivity with the retrosplenial cortex, thalamus, and hippocampus. Amygdala and thalamic nuclei were network hubs. Functional connectivity among these brain regions could support CS transient memories and their association.

Chronic corticosterone increases ΔFOSB and CRFR1 immunoreactivity in brain regions that modulate aversive conditioning.

Previous studies showed that chronic treatment with corticosterone facilitates elevated T-maze (ETM) inhibitory avoidance and a step-down avoidance task, responses that have been used to investigate aversive conditioning and memory processes. On the other hand, chronic corticosterone does not alter ETM escape from the open arms. The purpose of the present study was to further investigate the effects of chronic corticosterone treatment (200 mg pellets, 21-day release) in an animal model of anxiety that does not involve aversive conditioning: the light/dark transition model. We also investigated the pattern of ΔFosB immunoreactivity (ΔFosB-ir) in different brain regions. To examine how treatment with chronic corticosterone interferes with CRFR1 expression we measured CRFR1 in the same brain structures that exhibited increased ΔFosB-ir. Results showed that chronic treatment with corticosterone did not alter behavioral measurements performed in the light/dark transition model. On the other hand, ΔFosB-ir was increased in several structures that modulate aversive conditioning: the cingulate cortex, the ventro and dorsolateral septum, the amygdala, the paraventricular, dorsomedial and ventromedial hypothalamus, the periaqueductal grey matter, the dorsal raphe, and the median raphe nucleus. Chronic treatment with corticosterone also increased CRFR1-immunoreactivity in the ventrolateral septum, central amygdala, dorsomedial hypothalamus, ventral region of the dorsal raphe and median raphe. These results contribute to a better understanding of the behavioral and neurobiological alterations induced by chronic exposure to glucocorticoids.