Projection-Targeted Photopharmacology Reveals Distinct Anxiolytic Roles for Presynaptic mGluR2 in Prefrontal- and Insula-Amygdala Synapses.

Dissecting how membrane receptors regulate neural circuit function is critical for deciphering basic principles of neuromodulation and mechanisms of therapeutic drug action. Classical pharmacological and genetic approaches are not well-equipped to untangle the roles of specific receptor populations, especially in long-range projections which coordinate communication between brain regions. Here we use viral tracing, electrophysiological, optogenetic, and photopharmacological approaches to determine how presynaptic metabotropic glutamate receptor 2 (mGluR2) activation in the basolateral amygdala (BLA) alters anxiety-related behavior. We find that mGluR2-expressing neurons from the ventromedial prefrontal cortex (vmPFC) and posterior insular cortex (pIC) preferentially target distinct cell types and subregions of the BLA to regulate different forms of avoidant behavior. Using projection-specific photopharmacological activation, we find that mGluR2-mediated presynaptic inhibition of vmPFC-BLA, but not pIC-BLA, connections can produce long-lasting decreases in spatial avoidance. In contrast, presynaptic inhibition of pIC-BLA connections decreased social avoidance, novelty-induced hypophagia, and increased exploratory behavior without impairing working memory, establishing this projection as a novel target for the treatment of anxiety disorders. Overall, this work reveals new aspects of BLA neuromodulation with therapeutic implications while establishing a powerful approach for optical mapping of drug action via photopharmacology.

CB1R blockade unmasks TRPV1-mediated contextual fear generalization in female, but not male rats.

Increasing evidence suggests that the neurobiological processes that govern learning and memory can be different in males and females, but many of the specific mechanisms underlying these sex differences have not been fully defined. Here we investigated potential sex differences in endocannabinoid (eCB) modulation of Pavlovian fear conditioning and extinction, examining multiple defensive behaviors, including shock responsivity, conditioned freezing, and conditioned darting. We found that while systemic administration of drugs acting on eCB receptors did not influence the occurrence of darting, females that were classified as Darters responded differently to the drug administration than those classified as Non-darters. Most notably, CB1R antagonist AM251 produced an increase in cue-elicited freezing and context generalization selectively in female Non-darters that persisted across extinction and extinction retrieval tests but was prevented by co-administration of TRPV1R antagonist Capsazepine. To identify a potential synaptic mechanism for these sex differences, we next employed biochemical and neuroanatomical tracing techniques to quantify anandamide (AEA), TRPV1R, and perisomatic CB1R expression, focusing on the ventral hippocampus (vHip) given its known role in mediating contextual fear generalization. These assays identified sex-specific effects of both fear conditioning-elicited AEA release and vHip-BLA circuit structure. Together, our data support a model in which sexual dimorphism in vHip-BLA circuitry promotes a female-specific dependence on CB1Rs for context processing that is sensitive to TRPV1-mediated disruption when CB1Rs are blocked.

CB1R blockade unmasks TRPV1-mediated contextual fear generalization in female, but not male rats.

Increasing evidence suggests that the neurobiological processes that govern learning and memory can be different in males and females, and here we asked specifically whether the endocannabinoid (eCB) system could modulate Pavlovian fear conditioning in a sex-dependent manner. Systemic (i.p.) injection of CB1R antagonist AM251 in adult male and female Sprague Dawley rats prior to auditory cued fear conditioning produced a female-specific increase in freezing that persisted across extinction and extinction retrieval tests but was prevented by co-administration of TRPV1R antagonist Capsazepine. Notably, AM251 also produced robust freezing in a novel context prior to auditory cue presentation the day following drug administration, but not the day of, suggesting that CB1R blockade elicited contextual fear generalization in females. To identify a potential synaptic mechanism for these sex differences, we next used liquid chromatography/tandem mass spectrometry, Western Blot, and confocal-assisted immunofluorescence techniques to quantify anandamide (AEA), TRPV1R, and perisomatic CB1R expression, respectively, focusing on the ventral hippocampus (vHip). Fear conditioning elicited increased vHip AEA levels in females only, and in both sexes, CB1R expression around vHip efferents targeting the basolateral amygdala (BLA) was twice that at neighboring vHip neurons. Finally, quantification of the vHip-BLA projections themselves revealed that females have over twice the number of neurons in this pathway that males do. Together, our data support a model in which sexual dimorphism in vHip-BLA circuitry promotes a female-specific dependence on CB1Rs for context processing that is sensitive to TRPV1-mediated disruption when CB1Rs are blocked.

Regulation of social interaction in mice by a frontostriatal circuit modulated by established hierarchical relationships.

Social hierarchies exert a powerful influence on behavior, but the neurobiological mechanisms that detect and regulate hierarchical interactions are not well understood, especially at the level of neural circuits. Here, we use fiber photometry and chemogenetic tools to record and manipulate the activity of nucleus accumbens-projecting cells in the ventromedial prefrontal cortex (vmPFC-NAcSh) during tube test social competitions. We show that vmPFC-NAcSh projections signal learned hierarchical relationships, and are selectively recruited by subordinate mice when they initiate effortful social dominance behavior during encounters with a dominant competitor from an established hierarchy. After repeated bouts of social defeat stress, this circuit is preferentially activated during social interactions initiated by stress resilient individuals, and plays a necessary role in supporting social approach behavior in subordinated mice. These results define a necessary role for vmPFC-NAcSh cells in the adaptive regulation of social interaction behavior based on prior hierarchical interactions.

An automated platform for Assessing Working Memory and prefrontal circuit function.

Working memory is a process for actively maintaining and updating task-relevant information, despite interference from competing inputs, and is supported in part by sustained activity in prefrontal cortical pyramidal neurons and coordinated interactions with inhibitory interneurons, which may serve to regulate interference. Chronic stress has potent effects on working memory performance, possibly by interfering with these interactions or by disrupting long-range inputs from key upstream brain regions. Still, the mechanisms by which chronic stress disrupts working memory are not well understood, due in part to a need for scalable, easy-to-implement behavioral assays that are compatible with two-photon calcium imaging and other tools for recording from large populations of neurons. Here, we describe the development and validation of a platform that was designed specifically for automated, high-throughput assessments of working memory and simultaneous two-photon imaging in chronic stress studies. This platform is relatively inexpensive and easy to build; fully automated and scalable such that one investigator can test relatively large cohorts of animals concurrently; fully compatible with two-photon imaging, yet also designed to mitigate head-fixation stress; and can be easily adapted for other behavioral paradigms. Our validation data confirm that mice could be trained to perform a delayed response working memory task with relatively high-fidelity over the course of ∼15 days. Two-photon imaging data validate the feasibility of recording from large populations of cells during working memory tasks performance and characterizing their functional properties. Activity patterns in >70% of medial prefrontal cortical neurons were modulated by at least one task feature, and a majority of cells were engaged by multiple task features. We conclude with a brief literature review of the circuit mechanisms supporting working memory and their disruption in chronic stress states-highlighting directions for future research enabled by this platform.

Prelimbic cortex drives discrimination of non-aversion via amygdala somatostatin interneurons.

The amygdala and prelimbic cortex (PL) communicate during fear discrimination retrieval, but how they coordinate discrimination of a non-threatening stimulus is unknown. Here, we show that somatostatin (SOM) interneurons in the basolateral amygdala (BLA) become active specifically during learned non-threatening cues and desynchronize cell firing by blocking phase reset of theta oscillations during the safe cue. Furthermore, we show that SOM activation and desynchronization of the BLA is PL-dependent and promotes discrimination of non-threat. Thus, fear discrimination engages PL-dependent coordination of BLA SOM responses to non-threatening stimuli.