Ventral hippocampal projections to infralimbic cortex and basolateral amygdala are differentially activated by contextual fear and extinction recall.

Fear and extinction learning are thought to generate distinct and competing memory representations in the hippocampus. How these memory representations modulate the expression of appropriate behavioral responses remains unclear. To investigate this question, we used cholera toxin B subunit to retrolabel ventral hippocampal (vHPC) neurons projecting to the infralimbic cortex (IL) and basolateral amygdala (BLA) and then quantified c-Fos immediate early gene activity within these populations following expression of either contextual fear recall or contextual fear extinction recall. Fear recall was associated with increased c-Fos expression in vHPC projections to the BLA, whereas extinction recall was associated with increased activity in vHPC projections to IL. A control experiment was performed to confirm that the apparent shift in projection neuron activity was associated with extinction learning rather than mere context exposure. Overall, results indicate that hippocampal contextual fear and extinction memory representations differentially activate vHPC projections to IL and BLA. These findings suggest that hippocampal memory representations orchestrate appropriate behavioral responses through selective activation of projection pathways.

A cortico-amygdala neural substrate for endocannabinoid modulation of fear extinction.

Preclinical and clinical studies implicate endocannabinoids (eCBs) in fear extinction, but the underlying neural circuit basis of these actions is unclear. Here, we employed in vivo optogenetics, eCB biosensor imaging, ex vivo electrophysiology, and CRISPR-Cas9 gene editing in mice to examine whether basolateral amygdala (BLA)-projecting medial prefrontal cortex (mPFC) neurons represent a neural substrate for the effects of eCBs on extinction. We found that photoexcitation of mPFC axons in BLA during extinction mobilizes BLA eCBs. eCB biosensor imaging showed that eCBs exhibit a dynamic stimulus-specific pattern of activity at mPFC→BLA neurons that tracks extinction learning. Furthermore, using CRISPR-Cas9-mediated gene editing, we demonstrated that extinction memory formation involves eCB activity at cannabinoid CB1 receptors expressed at vmPFC→BLA synapses. Our findings reveal the temporal characteristics and a neural circuit basis of eCBs' effects on fear extinction and inform efforts to target the eCB system as a therapeutic approach in extinction-deficient neuropsychiatric disorders.

Selective sub-nucleus effects of intra-amygdala oxytocin on fear extinction.

There is growing evidence that the neuropeptide oxytocin (OT) modulates fear and extinction in humans and rodents through actions in corticolimbic circuits including the central amygdala (CeA). Prior studies have, however, been limited to subjects that exhibit intact basal extinction, rather than extinction-impaired populations that could potentially therapeutically benefit from viable OT-targeting treatments. Here, we assessed the effects of pre-extinction training infusion of OT into the CeA, or basolateral amygdala (BLA), on extinction in an inbred mouse strain (S1) model of impaired extinction. We found that intra-CeA OT, at a dose of 0.01 µg, enabled extinction memory formation, as evidenced by lesser freezing as compared to vehicle-infused controls on a drug-free retrieval test. Conversely, infusion of a higher, 1.0 µg OT dose, markedly reduced freezing and increased grooming during extinction training and produced elevated freezing on drug-free retrieval. Infusion of the 0.01 µg dose into the BLA was without behavioral effects. Together, our data show that OT acts in a dose-dependent manner within the CeA to promote extinction in otherwise extinction-deficient mice. These findings provide further support for the potential utility of OT as an adjunctive treatment to extinction-based therapies for trauma and anxiety disorders.

Distinct hippocampal engrams control extinction and relapse of fear memory.

Learned fear often relapses after extinction, suggesting that extinction training generates a new memory that coexists with the original fear memory; however, the mechanisms governing the expression of competing fear and extinction memories remain unclear. We used activity-dependent neural tagging to investigate representations of fear and extinction memories in the dentate gyrus. We demonstrate that extinction training suppresses reactivation of contextual fear engram cells while activating a second ensemble, a putative extinction engram. Optogenetic inhibition of neurons that were active during extinction training increased fear after extinction training, whereas silencing neurons that were active during fear training reduced spontaneous recovery of fear. Optogenetic stimulation of fear acquisition neurons increased fear, while stimulation of extinction neurons suppressed fear and prevented spontaneous recovery. Our results indicate that the hippocampus generates a fear extinction representation and that interactions between hippocampal fear and extinction representations govern the suppression and relapse of fear after extinction.

Dorsolateral Striatum Engagement Interferes with Early Discrimination Learning.

In current models, learning the relationship between environmental stimuli and the outcomes of actions involves both stimulus-driven and goal-directed systems, mediated in part by the DLS and DMS, respectively. However, though these models emphasize the importance of the DLS in governing actions after extensive experience has accumulated, there is growing evidence of DLS engagement from the onset of training. Here, we used in vivo photosilencing to reveal that DLS recruitment interferes with early touchscreen discrimination learning. We also show that the direct output pathway of the DLS is preferentially recruited and causally involved in early learning and find that silencing the normal contribution of the DLS produces plasticity-related alterations in a PL-DMS circuit. These data provide further evidence suggesting that the DLS is recruited in the construction of stimulus-elicited actions that ultimately automate behavior and liberate cognitive resources for other demands, but with a cost to performance at the outset of learning.

Impact of [d-Lys(3)]-GHRP-6 and feeding status on hypothalamic ghrelin-induced stress activation.

Ghrelin administration directly into hypothalamic nuclei, including the arcuate nucleus (ArcN) and the paraventricular nucleus (PVN), alters the expression of stress-related behaviors. In the present study we investigated the effect of feeding status on the ability of ghrelin to induce stress and anxiogenesis. Adult male Sprague Dawley rats were implanted with guide cannula targeting either the ArcN or PVN. In the first experiment we confirmed that ArcN and PVN ghrelin treatment produced anxiety-like behavior as measured using the elevated plus maze (EPM) paradigm. Ghrelin was administered during the early dark cycle. Immediately after microinjections rats were placed in the EPM for 5min. Both ArcN and PVN treatment reduced open arm exploration. The effect was attenuated by pretreatment with the ghrelin 1a receptor antagonist [d-Lys(3)]-GHRP-6. In a separate group of animals ghrelin was injected into either nucleus and rats were returned to their home cages for 60min with free access to food. An additional group of rats was returned to home cages with no food access. After 60min with or without food access all rats were tested in the EPM. Results indicated that food consumption just prior to EPM testing reversed the avoidance of the open arms of the EPM. In contrast, rats injected with ghrelin, placed in their home cage for 60min without food, and subsequently tested in the EPM, exhibited an increased avoidance of the open arms, consistent with stress activation. Overall, our findings demonstrate that ghrelin 1a receptor blockade and feeding status appear to impact the ability of ArcN and PVN ghrelin to elicit stress and anxiety-like behaviors.