Kristen E. Pleil

Distinct extended amygdala circuits for divergent motivational states

The co-morbidity of anxiety and dysfunctional reward processing in illnesses such as addiction and depression suggests that common neural circuitry contributes to these disparate neuropsychiatric symptoms. The extended amygdala, including the bed nucleus of the stria terminalis (BNST), modulates fear and anxiety, but also projects to the ventral tegmental area (VTA), a region implicated in reward and aversion, thus providing a candidate neural substrate for integrating diverse emotional states. However, the precise functional connectivity between distinct BNST projection neurons and their postsynaptic targets in the VTA, as well as the role of this circuit in controlling motivational states, have not been described. Here we record and manipulate the activity of genetically and neurochemically identified VTA-projecting BNST neurons in freely behaving mice. Collectively, aversive stimuli exposure produced heterogeneous firing patterns in VTA-projecting BNST neurons. By contrast, in vivo optically identified glutamatergic projection neurons displayed a net enhancement of activity to aversive stimuli, whereas the firing rate of identified GABAergic (γ-aminobutyric acid-containing) projection neurons was suppressed. Channelrhodopsin-2-assisted circuit mapping revealed that both BNST glutamatergic and GABAergic projections preferentially innervate postsynaptic non-dopaminergic VTA neurons, thus providing a mechanistic framework for in vivo circuit perturbations. In vivo photostimulation of BNST glutamatergic projections resulted in aversive and anxiogenic behavioural phenotypes. Conversely, activation of BNST GABAergic projections produced rewarding and anxiolytic phenotypes, which were also recapitulated by direct inhibition of VTA GABAergic neurons. These data demonstrate that functionally opposing BNST to VTA circuits regulate rewarding and aversive motivational states, and may serve as a crucial circuit node for bidirectionally normalizing maladaptive behaviours.

Chronic alcohol remodels prefrontal neurons and disrupts NMDAR-mediated fear extinction encoding

Alcoholism is frequently co-morbid with post-traumatic stress disorder, but it is unclear how alcohol affects the neural circuits mediating recovery from trauma. We found that chronic intermittent ethanol (CIE) impaired fear extinction and remodeled the dendritic arbor of medial prefrontal cortical (mPFC) neurons in mice. CIE impaired extinction encoding by infralimbic mPFC neurons in vivo and functionally downregulated burst-mediating NMDA GluN1 receptors. These findings suggest that alcohol may increase risk for trauma-related anxiety disorders by disrupting mPFC-mediated extinction of fear.

A New DREADD Facilitates the Multiplexed Chemogenetic Interrogation of Behavior

DREADDs are chemogenetic tools widely used to remotely control cellular signaling, neuronal activity, and behavior. Here we used a structure-based approach to develop a new Gi-coupled DREADD using the kappa-opioid receptor as a template (KORD) that is activated by the pharmacologically inert ligand salvinorin B (SALB). Activation of virally expressed KORD in several neuronal contexts robustly attenuated neuronal activity and modified behaviors. Additionally, co-expression of the KORD and the Gq-coupled M3-DREADD within the same neuronal population facilitated the sequential and bidirectional remote control of behavior. The availability of DREADDs activated by different ligands provides enhanced opportunities for investigating diverse physiological systems using multiplexed chemogenetic actuators.

Corticotropin Releasing Factor Signaling in the Central Amygdala is Recruited during Binge-Like Ethanol Consumption in C57BL/6J Mice

A well established body of work indicates a crucial role for corticotropin-releasing factor (CRF) in neurobiological responses associated with excessive dependence-like ethanol drinking in ethanol-vapor-exposed rodents. Recent evidence demonstrates a role for CRF in the modulation of binge-like ethanol consumption by nondependent mice, a behavior that can precede ethanol dependence. The CRF circuitry that is engaged by binge-like ethanol exposure, however, is unknown. Using converging approaches, we provide evidence that, similar to ethanol-vapor-induced increases in ethanol intake, CRF signaling in the central nucleus of the amygdala (CeA) is engaged during binge-like ethanol consumption by C57BL/6J mice. Specifically, we found that binge-like consumption of an ethanol solution (20% ethanol v/v) was attenuated by pretreatment with the CRF1R antagonists antalarmin, 4-ethyl-[2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]amino-1-butanol, and NBI-27914 at doses (30 mg/kg, i.p.) that did not alter nonbinge-like ethanol consumption. Binge-like ethanol consumption resulted in significant increases of CRF immunoreactivity in the CeA immediately following ethanol drinking and 18–24 h following ethanol removal and also blocked the ability of CRF to enhance GABAergic transmission in the CeA 18–24 h following ethanol removal. Pretreatment with bilateral injections of antalarmin (1 μg/0.5 μl per side) into the CeA, but not the adjacent basolateral amygdala, significantly attenuated binge-like ethanol consumption. These findings suggest that CRF signaling in the CeA is recruited during excessive ethanol intake, before the development of dependence. We hypothesize that plastic changes in CRF signaling develop with repeated binge-like drinking episodes, contributing to the transition to dependence.

NPY signaling inhibits extended amygdala CRF neurons to suppress binge alcohol drinking.

Binge alcohol drinking is a tremendous public health problem because it leads to the development of numerous pathologies, including alcohol abuse and anxiety. It is thought to do so by hijacking brain systems that regulate stress and reward, including neuropeptide Y (NPY) and corticotropin-releasing factor (CRF). The central actions of NPY and CRF have opposing functions in the regulation of emotional and reward-seeking behaviors; thus, dysfunctional interactions between these peptidergic systems could be involved in the development of these pathologies. We used converging physiological, pharmacological and chemogenetic approaches to identify a precise neural mechanism in the bed nucleus of the stria terminalis (BNST), a limbic brain region involved in pathological reward and anxiety behaviors, underlying the interactions between NPY and CRF in the regulation of binge alcohol drinking in both mice and monkeys. We found that NPY Y1 receptor (Y1R) activation in the BNST suppressed binge alcohol drinking by enhancing inhibitory synaptic transmission specifically in CRF neurons via a previously unknown Gi-mediated, PKA-dependent postsynaptic mechanism. Furthermore, chronic alcohol drinking led to persistent alterations in Y1R function in the BNST of both mice and monkeys, highlighting the enduring, conserved nature of this effect across mammalian species. Together, these data provide both a cellular locus and signaling framework for the development of new therapeutics for treatment of neuropsychiatric diseases, including alcohol use disorders.

Chemogenetic inactivation of ventral hippocampal glutamatergic neurons disrupts consolidation of contextual fear memory.

Synaptic consolidation is a process thought to consolidate memory in the brain. Although lesion studies have mainly implicated the hippocampus (HPC) in this process, it is unknown which cell type(s) or regions of the HPC might be essential for synaptic consolidation. To selectively and reversibly suppress hippocampal neuronal activity during this process, we developed a new Gi-DREADD (hM4Di) transgenic mouse for in vivo manipulation of neuronal activity in freely moving animals. We found that CA1 pyramidal neurons could be dose-dependently inactivated by clozapine-n-oxide (CNO). Inactivation of hippocampal neurons within 6 h immediately after conditioned fear training successfully impaired the consolidation of contextual memory, without disturbing cued memory. To anatomically define the brain subregion critical for the behavioral effects, hM4Di viral vectors were transduced and selectively expressed in the glutamatergic neurons in either the dorsal or ventral HPC. Significantly, we found that selective inactivation of ventral but not dorsal glutamatergic hippocampal neurons suppressed the synaptic consolidation of contextual memory.

Central neuropeptide y modulates binge-like ethanol drinking in C57BL/6J mice via Y1 and Y2 receptors

Frequent binge drinking has been linked to heart disease, high blood pressure, type 2 diabetes, and the development of ethanol dependence. Thus, identifying pharmaceutical targets to treat binge drinking is of paramount importance. Here we employed a mouse model of binge-like ethanol drinking to study the role of neuropeptide Y (NPY). To this end, the present set of studies utilized pharmacological manipulation of NPY signaling, immunoreactivity (IR) mapping of NPY and NPY receptors, and electrophysiological recordings from slice preparations of the amygdala. The results indicated that central infusion of NPY, a NPY Y1 receptor (Y1R) agonist, and a Y2R antagonist significantly blunted binge-like ethanol drinking in C57BL/6J mice (that achieved blood ethanol levels >80 mg/dl in control conditions). Binge-like ethanol drinking reduced NPY and Y1R IR in the central nucleus of the amygdala (CeA), and 24 h of ethanol abstinence after a history of binge-like drinking promoted increases of Y1R and Y2R IR. Electrophysiological recordings of slice preparations from the CeA showed that binge-like ethanol drinking augmented the ability of NPY to inhibit GABAergic transmission. Thus, binge-like ethanol drinking in C57BL/6J mice promoted alterations of NPY signaling in the CeA, and administration of exogenous NPY compounds protected against binge-like drinking. The current data suggest that Y1R agonists and Y2R antagonists may be useful for curbing and/or preventing binge drinking, protecting vulnerable individuals from progressing to the point of ethanol dependence.

Extended Amygdala to Ventral Tegmental Area Corticotropin-Releasing Factor Circuit Controls Binge Ethanol Intake

BACKGROUND Corticotropin-releasing factor (CRF) signaling at the CRF1 receptor (CRF1R) in the ventral tegmental area (VTA) can modulate ethanol consumption in rodents. However, the effects of binge-like ethanol drinking on this system have not been thoroughly characterized, and little is known about the role of CRF2R or the CRF neurocircuitry involved. METHODS The effects of binge-like ethanol consumption on the VTA CRF system were assessed following drinking-in-the-dark procedures. Intra-VTA infusions of selective CRF1R and/or CRF2R compounds were employed to assess the contributions of these receptors in modulating binge-like ethanol consumption (n = 89). To determine the potential role of CRF projections from the bed nucleus of the stria terminalis (BNST) to the VTA, CRF neurons in this circuit were chemogenetically inhibited (n = 32). Binge-induced changes in VTA CRF system protein and messenger RNA were also assessed (n = 58). RESULTS Intra-VTA antagonism of CRF1R and activation of CRF2R resulted in decreased ethanol intake, which was eliminated by simultaneous blockade of both receptors. Chemogenetic inhibition of local CRF neurons in the VTA did not alter binge-like ethanol drinking, but inhibition of VTA-projecting CRF neurons from the BNST significantly reduced intake. CONCLUSIONS We provide novel evidence that 1) blunted binge-like ethanol consumption stemming from CRF1R blockade requires intact CRF2R signaling, and CRF2R activation reduces binge-like drinking; 2) inhibiting VTA-projecting BNST CRF neurons attenuates binge-like drinking; and 3) binge-like ethanol drinking alters protein and messenger RNA associated with the VTA-CRF system. These data suggest that ethanol-induced activation of BNST-to-VTA CRF projections is critical in driving binge-like ethanol intake.

Effects of chronic ethanol exposure on neuronal function in the prefrontal cortex and extended amygdala.

Chronic alcohol consumption and withdrawal leads to anxiety, escalated alcohol drinking behavior, and alcohol dependence. Alterations in the function of key structures within the cortico-limbic neural circuit have been implicated in underlying the negative behavioral consequences of chronic alcohol exposure in both humans and rodents. Here, we used chronic intermittent ethanol vapor exposure (CIE) in male C57BL/6J mice to evaluate the effects of chronic alcohol exposure and withdrawal on anxiety-like behavior and basal synaptic function and neuronal excitability in prefrontal cortical and extended amygdala brain regions. Forty-eight hours after four cycles of CIE, mice were either assayed in the marble burying test (MBT) or their brains were harvested and whole-cell electrophysiological recordings were performed in the prelimbic and infralimbic medial prefrontal cortex (PLC and ILC), the lateral and medial central nucleus of the amygdala (lCeA and mCeA), and the dorsal and ventral bed nucleus of the stria terminalis (dBNST and vBNST). Ethanol-exposed mice displayed increased anxiety in the MBT compared to air-exposed controls, and alterations in neuronal function were observed in all brain structures examined, including several distinct differences between subregions within each structure. Chronic ethanol exposure induced hyperexcitability of the ILC, as well as a shift toward excitation in synaptic drive and hyperexcitability of vBNST neurons; in contrast, there was a net inhibition of the CeA. This study reveals extensive effects of chronic ethanol exposure on the basal function of cortico-limbic brain regions, suggests that there may be complex interactions between these regions in the regulation of ethanol-dependent alterations in anxiety state, and highlights the need for future examination of projection-specific effects of ethanol in cortico-limbic circuitry.

Neuropeptide regulation of signaling and behavior in the BNST

Recent technical developments have transformed how neuroscientists can probe brain function. What was once thought to be difficult and perhaps impossible, stimulating a single set of long range inputs among many, is now relatively straight-forward using optogenetic approaches. This has provided an avalanche of data demonstrating causal roles for circuits in a variety of behaviors. However, despite the critical role that neuropeptide signaling plays in the regulation of behavior and physiology of the brain, there have been remarkably few studies demonstrating how peptide release is causally linked to behaviors. This is likely due to both the different time scale by which peptides act on and the modulatory nature of their actions. For example, while glutamate release can effectively transmit information between synapses in milliseconds, peptide release is potentially slower [See the excellent review by Van Den Pol on the time scales and mechanisms of release (van den Pol, 2012)] and it can only tune the existing signals via modulation. And while there have been some studies exploring mechanisms of release, it is still not as clearly known what is required for efficient peptide release. Furthermore, this analysis could be complicated by the fact that there are multiple peptides released, some of which may act in contrast. Despite these limitations, there are a number of groups making progress in this area. The goal of this review is to explore the role of peptide signaling in one specific structure, the bed nucleus of the stria terminalis, that has proven to be a fertile ground for peptide action.