SNAP25 differentially contributes to Gi/o-coupled receptor function at glutamatergic synapses in the nucleus accumbens.

The nucleus accumbens (NAc) guides reward-related motivated behavior implicated in pathological behavioral states, including addiction and depression. These behaviors depend on the precise neuromodulatory actions of Gi/o-coupled G-protein-coupled receptors (GPCRs) at glutamatergic synapses onto medium spiny projection neurons (MSNs). Previous work has shown that discrete classes of Gi/o-coupled GPCR mobilize Gßγ to inhibit vesicular neurotransmitter release via t-SNARE protein, SNAP25. However, it remains unknown which Gαi/o systems in the NAc utilize Gßγ-SNARE signaling to dampen glutamatergic transmission. Utilizing patch-clamp electrophysiology and pharmacology in a transgenic mouse line with a C-terminal three-residue deletion of SNAP25 (SNAP25Δ3) weaking the Gßγ-SNARE interaction, we surveyed a broad cohort of Gi/o-coupled GPCRs with robust inhibitory actions at glutamatergic synapses in the NAc. We find that basal presynaptic glutamate release probability is reduced in SNAP25Δ3 mice. While κ opioid, CB1, adenosine A1, group II metabotropic glutamate receptors, and histamine H3 receptors inhibit glutamatergic transmission onto MSNs independent of SNAP25, we report that SNAP25 contributes significantly to the actions of GABAB, 5-HT1B/D, and µ opioid receptors. These findings demonstrate that presynaptic Gi/o-coupled GPCRs recruit heterogenous effector mechanisms at glutamatergic synapses in the NAc, with a subset requiring SNA25-dependent Gßγ signaling.

Accumbal Histamine Signaling Engages Discrete Interneuron Microcircuits.

BACKGROUND: Central histamine (HA) signaling modulates diverse cortical and subcortical circuits throughout the brain, including the nucleus accumbens (NAc). The NAc, a key striatal subregion directing reward-related behavior, expresses diverse HA receptor subtypes that elicit cellular and synaptic plasticity. However, the neuromodulatory capacity of HA within interneuron microcircuits in the NAc remains unknown. METHODS: We combined electrophysiology, pharmacology, voltammetry, and optogenetics in male transgenic reporter mice to determine how HA influences microcircuit motifs controlled by parvalbumin-expressing fast-spiking interneurons (PV-INs) and tonically active cholinergic interneurons (CINs) in the NAc shell. RESULTS: HA enhanced CIN output through an H2 receptor (H2R)-dependent effector pathway requiring Ca2+-activated small-conductance K+ channels, with a small but discernible contribution from H1Rs and synaptic H3Rs. While PV-IN excitability was unaffected by HA, presynaptic H3Rs decreased feedforward drive onto PV-INs via AC-cAMP-PKA (adenylyl cyclase-cyclic adenosine monophosphate-protein kinase A) signaling. H3R-dependent plasticity was differentially expressed at mediodorsal thalamus and prefrontal cortex synapses onto PV-INs, with mediodorsal thalamus synapses undergoing HA-induced long-term depression. These effects triggered downstream shifts in PV-IN- and CIN-controlled microcircuits, including near-complete collapse of mediodorsal thalamus-evoked feedforward inhibition and increased mesoaccumbens dopamine release. CONCLUSIONS: HA targets H1R, H2R, and H3Rs in the NAc shell to engage synapse- and cell type-specific mechanisms that bidirectionally regulate PV-IN and CIN microcircuit activity. These findings extend the current conceptual framework of HA signaling and offer critical insight into the modulatory potential of HA in the brain.

TAAR1 regulates drug-induced reinstatement of cocaine-seeking via negatively modulating CaMKIIα activity in the NAc.

Relapse remains a major challenge to the treatment of cocaine addiction. Recent studies suggested that the trace amine-associated receptor 1 (TAAR1) could be a promising target to treat cocaine addiction and relapse; however, the underlying mechanism remains unclear. Here, we aimed to investigate the neural mechanism underlying the role of TAAR1 in the drug priming-induced reinstatement of cocaine-seeking behavior in rats, an animal model of cocaine relapse. We focused on the shell subregion of nucleus accumbens (NAc), a key brain region of the brain reward system. We found that activation of TAAR1 by systemic and intra-NAc shell administration of the selective TAAR1 agonist RO5166017 attenuated drug-induced reinstatement of cocaine-seeking and prevented drug priming-induced CaMKIIα activity in the NAc shell. Activation of TAAR1 dampened the CaMKIIα/GluR1 signaling pathway in the NAc shell and reduced AMPAR-EPSCs on the NAc slice. Microinjection of the selective TAAR1 antagonist EPPTB into the NAc shell enhanced drug-induced reinstatement as well as potentiated CaMKIIα activity in the NAc shell. Furthermore, viral-mediated expression of CaMKIIα in the NAc shell prevented the behavioral effects of TAAR1 activation. Taken together, our findings indicate that TAAR1 regulates drug-induced reinstatement of cocaine-seeking by negatively regulating CaMKIIα activity in the NAc. Our findings elucidate a novel mechanism of TAAR1 in regulating drug-induced reinstatement of cocaine-seeking and further suggests that TAAR1 is a promising target for the treatment of cocaine relapse.

Cocaine restricts nucleus accumbens feedforward drive through a monoamine-independent mechanism.

Parvalbumin-expressing fast-spiking interneurons (PV-INs) within feedforward microcircuits in the nucleus accumbens (NAc) coordinate goal-directed motivational behavior. Feedforward inhibition of medium spiny projection neurons (MSNs) is initiated by glutamatergic input from corticolimbic brain structures. While corticolimbic synapses onto MSNs are targeted by the psychostimulant, cocaine, it remains unknown whether cocaine also exerts acute neuromodulatory actions at collateralizing synapses onto PV-INs. Using whole-cell patch-clamp electrophysiology, optogenetics, and pharmacological tools in transgenic reporter mice, we found that cocaine decreases thalamocortical glutamatergic drive onto PV-INs by engaging a monoamine-independent mechanism. This mechanism relies on postsynaptic sigma-1 (σ1) activity, leading to the mobilization of intracellular Ca2+ stores that trigger retrograde endocannabinoid signaling at presynaptic type-1 cannabinoid receptors (CB1R). Cocaine-evoked CB1R activity occludes the expression of CB1R-dependent long-term depression (LTD) at this synaptic locus. These findings provide evidence that acute cocaine exposure targets feedforward microcircuits in the NAc and extend existing models of cocaine action on mesolimbic reward circuits.

Kappa opioid receptor modulation of excitatory drive onto nucleus accumbens fast-spiking interneurons.

The dynorphin/kappa opioid receptor (KOR) system within the nucleus accumbens (NAc) contributes to affective states. Parvalbumin fast-spiking interneurons (PV-FSIs), a key component of feedforward inhibition, participate in integration of excitatory inputs to the NAc by robustly inhibiting select populations of medium spiny output neurons, therefore greatly influencing NAc dependent behavior. How the dynorphin/KOR system regulates feedforward inhibition in the NAc remains unknown. Here, we elucidate the molecular mechanisms of KOR inhibition of excitatory transmission onto NAc PV-FSIs using a combination of whole-cell patch-clamp electrophysiology, optogenetics, pharmacology, and a parvalbumin reporter mouse. We find that postsynaptic KOR stimulation induces long-term depression (LTD) of excitatory synapses onto PV-FSI by stimulating the endocytosis of AMPARs via a PKA and calcineurin-dependent mechanism. Furthermore, KOR regulation of PV-FSI synapses are input specific, inhibiting thalamic but not cortical inputs. Finally, following acute stress, a protocol known to elevate dynorphin/KOR signaling in the NAc, KOR agonists no longer inhibit excitatory transmission onto PV-FSI. In conclusion, we delineate pathway-specific mechanisms mediating KOR control of feedforward inhibitory circuits in the NAc and provide evidence for the recruitment of this system in response to stress.

Patch-clamp and multi-electrode array electrophysiological analysis in acute mouse brain slices.

Patch-clamp and multi-electrode array electrophysiology techniques are used to measure dynamic functional properties of neurons. Whole-cell and cell-attached patch-clamp recordings in brain slices can be performed in voltage-clamp and current-clamp configuration to reveal cell-type-specific synaptic and cellular parameters governing neurotransmission. Multi-electrode array electrophysiology can provide spike activity recordings from multiple neurons, enabling larger sample sizes, and long-term recordings. We provide our guide to preparing acute rodent brain slices with example experiments and analyses intended for novice and expert electrophysiologists. For complete details on the use and execution of this protocol, please refer to Manz et al. (2020b).

Noradrenergic Signaling Disengages Feedforward Transmission in the Nucleus Accumbens Shell.

The nucleus accumbens shell (NAcSh) receives extensive monoaminergic input from multiple midbrain structures. However, little is known how norepinephrine (NE) modulates NAc circuit dynamics. Using a dynamic electrophysiological approach with optogenetics, pharmacology, and drugs acutely restricted by tethering (DART), we explored microcircuit-specific neuromodulatory mechanisms recruited by NE signaling in the NAcSh of parvalbumin (PV)-specific reporter mice. Surprisingly, NE had little direct effect on modulation of synaptic input at medium spiny projection neurons (MSNs). In contrast, we report that NE transmission selectively modulates glutamatergic synapses onto PV-expressing fast-spiking interneurons (PV-INs) by recruiting postsynaptically-localized α2-adrenergic receptors (ARs). The synaptic effects of α2-AR activity decrease PV-IN-dependent feedforward inhibition onto MSNs evoked via optogenetic stimulation of cortical afferents to the NAcSh. These findings provide insight into a new circuit motif in which NE has a privileged line of communication to tune feedforward inhibition in the NAcSh.SIGNIFICANCE STATEMENT The nucleus accumbens (NAc) directs reward-related motivational output by integrating glutamatergic input with diverse neuromodulatory input from monoamine centers. The present study reveals a synapse-specific regulatory mechanism recruited by norepinephrine (NE) signaling within parvalbumin-expressing interneuron (PV-IN) feedforward inhibitory microcircuits. PV-IN-mediated feedforward inhibition in the NAc is instrumental in coordinating NAc output by synchronizing the activity of medium spiny projection neurons (MSNs). By negatively regulating glutamatergic transmission onto PV-INs via α2-adrenergic receptors (ARs), NE diminishes feedforward inhibition onto MSNs to promote NAc output. These findings elucidate previously unknown microcircuit mechanisms recruited by the historically overlooked NE system in the NAc.

Cannabinoid type 1 receptors in A2a neurons contribute to cocaine-environment association.

RATIONALE: Cannabinoid type 1 receptors (CB1Rs) are widely expressed within the brain's reward circuits and are implicated in regulating drug induced behavioral adaptations. Understanding how CB1R signaling in discrete circuits and cell types contributes to drug-related behavior provides further insight into the pathology of substance use disorders. OBJECTIVE AND METHODS: We sought to determine how cell type-specific expression of CB1Rs within striatal circuits contributes to cocaine-induced behavioral plasticity, hypothesizing that CB1R function in distinct striatal neuron populations would differentially impact behavioral outcomes. We crossed conditional Cnr1fl/fl mice and striatal output pathway cre lines (Drd1a -cre; D1, Adora2a -cre; A2a) to generate cell type-specific CB1R knockout mice and assessed their performance in cocaine locomotor and associative behavioral assays. RESULTS: Both knockout lines retained typical locomotor activity at baseline. D1-Cre x Cnr1fl/fl mice did not display hyperlocomotion in response to acute cocaine dosing, and both knockout lines exhibited blunted locomotor activity across repeated cocaine doses. A2a-cre Cnr1fl/fl, mice did not express a preference for cocaine paired environments in a two-choice place preference task. CONCLUSIONS: This study aids in mapping CB1R-dependent cocaine-induced behavioral adaptations onto distinct striatal neuron subtypes. A reduction of cocaine-induced locomotor activation in the D1- and A2a-Cnr1 knockout mice supports a role for CB1R function in the motor circuit. Furthermore, a lack of preference for cocaine-associated context in A2a-Cnr1 mice suggests that CB1Rs on A2a-neuron inhibitory terminals are necessary for either reward perception, memory consolidation, or recall. These results direct future investigations into CB1R-dependent adaptations underlying the development and persistence of substance use disorders.

Histamine H3 Receptor Function Biases Excitatory Gain in the Nucleus Accumbens.

BACKGROUND: Histamine (HA), a wake-promoting monoamine implicated in stress-related arousal states, is synthesized in histidine decarboxylase-expressing hypothalamic neurons of the tuberomammillary nucleus. Histidine decarboxylase-containing varicosities diffusely innervate striatal and mesolimbic networks, including the nucleus accumbens (NAc). The NAc integrates diverse monoaminergic inputs to coordinate motivated behavior. While the NAc expresses various HA receptor subtypes, mechanisms by which HA modulates NAc circuit dynamics are undefined. METHODS: Using male D1tdTomato transgenic reporter mice, whole-cell patch-clamp electrophysiology, and input-specific optogenetics, we employed a targeted pharmacological approach to interrogate synaptic mechanisms recruited by HA signaling at glutamatergic synapses in the NAc. We incorporated an immobilization stress protocol to assess whether acute stress engages these mechanisms at glutamatergic synapses onto D1 receptor-expressing [D1(+)] medium spiny neurons (MSNs) in the NAc core. RESULTS: HA negatively regulates excitatory gain onto D1(+)-MSNs via presynaptic H3 receptor-dependent long-term depression that requires Gßγ-directed Akt-GSK3ß signaling. Furthermore, HA asymmetrically regulates glutamatergic transmission from the prefrontal cortex and mediodorsal thalamus, with inputs from the prefrontal cortex undergoing robust HA-induced long-term depression. Finally, we report that acute immobilization stress attenuates this long-term depression by recruiting endogenous H3 receptor signaling in the NAc at glutamatergic synapses onto D1(+)-MSNs. CONCLUSIONS: Stress-evoked HA signaling in the NAc recruits H3 heteroreceptor signaling to shift thalamocortical input onto D1(+)-MSNs in the NAc. Our findings provide novel insight into an understudied neuromodulatory system within the NAc and implicate HA in stress-associated physiological states.

Calcium-Permeable AMPA Receptors Promote Endocannabinoid Signaling at Parvalbumin Interneuron Synapses in the Nucleus Accumbens Core.

Synaptic plasticity is a key mechanism of learning and memory. Synaptic plasticity mechanisms within the nucleus accumbens (NAc) mediate differential behavioral adaptations. Feedforward inhibition in the NAc occurs when glutamatergic afferents onto medium spiny neurons (MSNs) collateralize onto fast-spiking parvalbumin (PV)-expressing interneurons (PV-INs), which exert GABAergic control over MSN action potential generation. Here, we find that feedforward glutamatergic synapses onto PV-INs in the NAc core selectively express Ca2+-permeable AMPA receptors (CP-AMPARs). Ca2+ influx by CP-AMPARs on PV-INs triggers long-term depression (LTD) mediated by endocannabinoid (eCB) signaling at presynaptic cannabinoid type-1 (CB1) receptors (CB1Rs). Moreover, CP-AMPARs authorize tonic eCB signaling to negatively regulate glutamate release probability. Blockade of CP-AMPARs in the NAc core in vivo is sufficient to disinhibit locomotor output. These findings elucidate mechanisms by which PV-IN-embedded microcircuits in the NAc undergo activity-dependent shifts in synaptic strength.

Heterosynaptic GABAB Receptor Function within Feedforward Microcircuits Gates Glutamatergic Transmission in the Nucleus Accumbens Core.

Complex circuit interactions within the nucleus accumbens (NAc) facilitate goal-directed behavior. Medium spiny neurons (MSNs) mediate NAc output by projecting to functionally divergent brain regions, a property conferred, in part, by the differential projection patterns of D1- and D2 dopamine receptor-expressing MSNs. Glutamatergic afferents to the NAc direct MSN output by recruiting feedforward inhibitory microcircuits comprised of parvalbumin (PV)-expressing interneurons (INs). Furthermore, the GABAB heteroreceptor (GABABR), a Gi/o-coupled G-protein-coupled receptor, is expressed at glutamatergic synapses throughout the mesolimbic network, yet its physiological context and synaptic mechanism within the NAc remains unknown. Here, we explored GABABR function at glutamatergic synapses within PV-IN-embedded microcircuits in the NAc core of male mice. We found that GABABR is expressed presynaptically and recruits a noncanonical signaling mechanism to reduce glutamatergic synaptic efficacy at D1(+) and D1(-) (putative D2) MSN subtypes. Furthermore, PV-INs, a robust source of neuronal GABA in the NAc, heterosynaptically target GABABR to selectively modulate glutamatergic transmission onto D1(+) MSNs. These findings elucidate a new mechanism of feedforward inhibition and refine mechanisms by which GABAB heteroreceptors modulate mesolimbic circuit function.SIGNIFICANCE STATEMENT Glutamatergic transmission in the nucleus accumbens (NAc) critically contributes to goal-directed behaviors. However, intrinsic microcircuit mechanisms governing the integration of these synapses remain largely unknown. Here, we show that parvalbumin-expressing interneurons within feedforward microcircuits heterosynaptically target GABAB heteroreceptors (GABABR) on glutamate terminals. Activation of presynaptically-expressed GABABR decreases glutamatergic synaptic strength by engaging a non-canonical signaling pathway that interferes with vesicular exocytotic release machinery. These findings offer mechanistic insight into the role of GABAB heteroreceptors within reward circuitry, elucidate a novel arm to feedforward inhibitory networks, and inform the growing use of GABABR-selective pharmacotherapy for various motivational disorders, including addiction, major depressive disorder, and autism (Cousins et al., 2002; Kahn et al., 2009; Jacobson et al., 2018; Stoppel et al., 2018; Pisansky et al., 2019).

A novel adolescent chronic social defeat model: reverse-Resident-Intruder Paradigm (rRIP) in male rats.

Psychosocial stress is linked to the etiology of several neuropsychiatric disorders, including Major Depressive Disorder and Post-Traumatic-Stress-Disorder. Adolescence is a critical neurobehavioral developmental period wherein the maturing nervous system is sensitive to stress-related psychosocial events. The effects of social defeat stress, an animal model of psychosocial stress, on adolescent neurobehavioral phenomena are not well explored. Using the standard Resident-Intruder-Paradigm (RIP), adolescent Long-Evans (LE, residents, n = 100) and Sprague-Dawley (SD, intruders, n = 100) rats interacted for five days to invoke chronic social stress. Tests of depressive behavior (forced-swim-test (FST)), fear conditioning, and long-term synaptic plasticity are affected in various adult rodent chronic stress models, thus we hypothesized that these phenomena would be similarly affected in adolescent rats. Serendipitously, we observed the Intruders became the dominant rats and the Residents were the defeated/submissive rats. This robust and reliable role-reversal resulted in defeated LE-Residents showing a depressive-like state (increased time spent immobile in the FST), enhanced fear conditioning in both hippocampal-dependent and hippocampal-independent fear paradigms and altered hippocampal long-term synaptic plasticity, measured electrophysiologically in vitro in hippocampal slices. Importantly, SD-Intruders, SD and LE controls did not significantly differ from each other in any of these assessments. This reverse-Resident-Intruder-Paradigm (rRIP) represents a novel animal model to study the effects of stress on adolescent neurobehavioral phenomenon.

Synaptic Plasticity in the Nucleus Accumbens: Lessons Learned from Experience.

Synaptic plasticity contributes to behavioral adaptations. As a key node in the reward pathway, the nucleus accumbens (NAc) is important for determining motivation-to-action outcomes. Across animal models of motivation including addiction, depression, anxiety, and hedonic feeding, selective recruitment of neuromodulatory signals and plasticity mechanisms have been a focus of physiologists and behaviorists alike. Experience-dependent plasticity mechanisms within the NAc vary depending on the distinct afferents and cell-types over time. A greater understanding of molecular mechanisms determining how these changes in synaptic strength track with behavioral adaptations will provide insight into the process of learning and memory along with identifying maladaptations underlying pathological behavior. Here, we summarize recent findings detailing how changes in NAc synaptic strength and mechanisms of plasticity manifest in various models of motivational disorders.