Short-term memory reactivation of a weak CS-US association promotes long-term memory persistence in conditioned odor aversion.

In conditioned odor aversion (COA), the association of a tasteless odorized solution (the conditioned stimulus [CS]) with an intraperitoneal injection of LiCl (the unconditioned stimulus [US[), which produces visceral malaise, results in its future avoidance. The strength of this associative memory is mainly dependent on two parameters, that is, the strength of the US and the interstimuli interval (ISI). In rats, COA has been observed only with ISIs of ≤15 min and LiCl (0.15 M) doses of 2.0% of bodyweight, when tested 48 h after acquisition (long-term memory [LTM]). However, we previously reported a robust aversion in rats trained with ISIs up to 60 min when tested 4 h after acquisition (short-term memory [STM]). Since memories get reactivated during retrieval, in the current study we hypothesized that testing for STM would reactivate this COA trace, strengthening its LTM. For this, we compared the LTM of rats trained with long ISIs or low doses of LiCl initially tested for STM with that of rats tested for LTM only. Interestingly, rats conditioned under parameters sufficient to produce STM, but not LTM, showed a reliable LTM when first tested for STM. These observations suggest that under suboptimal training conditions, such as long ISIs or low US intensities, a CS-US association is established but requires reactivation in the short-term in order to persist in the long-term.

A Corticotropin Releasing Factor Network in the Extended Amygdala for Anxiety.

The central amygdala (CeA) is important for fear responses to discrete cues. Recent findings indicate that the CeA also contributes to states of sustained apprehension that characterize anxiety, although little is known about the neural circuitry involved. The stress neuropeptide corticotropin releasing factor (CRF) is anxiogenic and is produced by subpopulations of neurons in the lateral CeA and the dorsolateral bed nucleus of the stria terminalis (dlBST). Here we investigated the function of these CRF neurons in stress-induced anxiety using chemogenetics in male rats that express Cre recombinase from a Crh promoter. Anxiety-like behavior was mediated by CRF projections from the CeA to the dlBST and depended on activation of CRF1 receptors and CRF neurons within the dlBST. Our findings identify a CRFCeA→CRFdlBST circuit for generating anxiety-like behavior and provide mechanistic support for recent human and primate data suggesting that the CeA and BST act together to generate states of anxiety.SIGNIFICANCE STATEMENT Anxiety is a negative emotional state critical to survival, but persistent, exaggerated apprehension causes substantial morbidity. Identifying brain regions and neurotransmitter systems that drive anxiety can help in developing effective treatment. Much evidence in rodents indicates that neurons in the bed nucleus of the stria terminalis (BST) generate anxiety-like behaviors, but more recent findings also implicate neurons of the CeA. The neuronal subpopulations and circuitry that generate anxiety are currently subjects of intense investigation. Here we show that CeA neurons that release the stress neuropeptide corticotropin-releasing factor (CRF) drive anxiety-like behaviors in rats via a pathway to dorsal BST that activates local BST CRF neurons. Thus, our findings identify a CeA→BST CRF neuropeptide circuit that generates anxiety-like behavior.

Muscarinic receptor signaling in the amygdala is required for conditioned taste aversion.

The sense of taste provides information regarding the nutrient content, safety or potential toxicity of an edible. This is accomplished via a combination of innate and learned taste preferences. In conditioned taste aversion (CTA), rats learn to avoid ingesting a taste that has previously been paired with gastric malaise. Recent evidence points to a role of cholinergic muscarinic signaling in the amygdala for the learning and storage of emotional memories. The present study tested the participation of muscarinic receptors in the amygdala during the formation of CTA by infusing the non-specific antagonist scopolamine into the basolateral or central subnuclei before or after conditioning, as well as before retrieval. Our data show that regardless of the site of infusion, pre-conditioning administration of scopolamine impaired CTA acquisition whereas post-conditioning infusion did not affect its storage. Also, infusions into the basolateral but not in the central amygdala before retrieval test partially reduced the expression of CTA. Our results indicate that muscarinic receptors activity is required for acquisition but not consolidation of CTA. In addition, our data add to recent evidence pointing to a role of cholinergic signaling in peri-hippocampal structures in the process of memory retrieval.

NMDA receptor and nitric oxide synthase activity in the central amygdala is involved in the acquisition and consolidation of conditioned odor aversion.

Rats readily learn to avoid a tasteless odorized solution if they experience visceral malaise after consuming it. This phenomenon is referred to as conditioned odor aversion (COA). Several studies have shown that COA depends on the functional integrity of the amygdala, with most studies focusing on the basolateral nucleus. On the other hand, the role of the central amygdala (CeA) which is known to be involved in the consolidation of conditioned taste aversion (CTA) remains to be established. To address this issue, we evaluated the effect of inhibiting NMDA receptor activity in this structure on COA memory formation. Intra-CeA infusions of non-competitive NMDA receptor inhibitor MK-801 prevented memory formation both when administered before and up to 15 min after COA conditioning, while no effect of this drug was observed when given before long-term memory test. We next evaluated the role of one of the main downstream effectors of brain NMDA receptor signaling, nitric oxide synthase (NOS), known to play a key role in a wide variety learning tasks including some types of olfactory conditioning. Similar results were obtained with inhibition of either NOS or neuron-specific NOS; which proved to be required both during and after COA training, though for a shorter time span than NMDA receptors. Also, neither isoform showed to be required to memory retrieval. These results suggest that the US signaling during acquisition and the initial consolidation step of COA depends on glutamate-NO system activation in the CeA.

Cooperative CRF and α1 Adrenergic Signaling in the VTA Promotes NMDA Plasticity and Drives Social Stress Enhancement of Cocaine Conditioning.

Stressful events rapidly trigger activity-dependent synaptic plasticity, driving the formation of aversive memories. However, it remains unclear how stressful experience affects plasticity mechanisms to regulate appetitive learning, such as intake of addictive drugs. Using rats, we show that corticotropin-releasing factor (CRF) and α1 adrenergic receptor (α1AR) signaling enhance the plasticity of NMDA-receptor-mediated glutamatergic transmission in ventral tegmental area (VTA) dopamine (DA) neurons through distinct effects on inositol 1,4,5-triphosphate (IP3)-dependent Ca2+ signaling. We find that CRF amplifies IP3-Ca2+ signaling induced by stimulation of α1ARs, revealing a cooperative mechanism that promotes glutamatergic plasticity. In line with this, acute social defeat stress engages similar cooperative CRF and α1AR signaling in the VTA to enhance learning of cocaine-paired cues. These data provide evidence that CRF and α1ARs act in concert to regulate IP3-Ca2+ signaling in the VTA and promote learning of drug-associated cues.

Association of stimuli at long intervals in conditioned odor aversion.

Rats learn to avoid a tasteless odorized solution if they experience visceral malaise after consuming it. This phenomenon is referred as Conditioned Odor Aversion (COA). It is widely accepted that an odor can only be associated with illness if the inter-stimulus interval (ISI) is shorter than 15 min. However, this conclusion is based on long-term memory tests usually made 48 h after conditioning, thus precluding the possibility to discriminate between a specific failure to make the odor-malaise association rather than the failure to consolidate the short-term association into long-term memory. In the present study, we compared the short-term and long-term memories for COA in rats trained with long ISIs. Independent groups of male rats were conditioned using 5, 15, 30, 60 or 90 min ISIs and tested either 4 or 48 h after conditioning. We found a reliable odor aversion at 5, 15, 30 and 60 min, but not at 90 min ISIs, when tested 4h after conditioning. In contrast, odor aversion was only found at 5 and 15 min ISIs in the groups tested 48 h after training. Our results show that COA can be acquired when malaise follows the odor CS by at least 60 min. This finding indicates that the lack of aversion at long ISIs is not due to an association failure, but rather to a limitation in consolidating short-term memory into long-term memory of COA.