Functional Alterations in the Dorsal Raphe Nucleus Following Acute and Chronic Ethanol Exposure

Alcoholism is a pervasive disorder perpetuated in part to relieve negative mood states like anxiety experienced during alcohol withdrawal. Emerging evidence demonstrates a role for the serotonin-rich dorsal raphe (DR) in anxiety following ethanol withdrawal. The current study examined the effects of chronic ethanol vapor exposure on the DR using slice electrophysiology in male DBA2/J mice. We found that chronic ethanol exposure resulted in deficits in social approach indicative of increased anxiety-like behavior at both 24 h and 7 days post-ethanol exposure. At 24 h post-ethanol exposure, we observed increased excitability and decreased spontaneous inhibitory transmission (inhibitory postsynaptic currents, IPSCs) in the DR. At 7 days post-ethanol exposure, we observed increased spontaneous and miniature excitatory transmission (excitatory postsynaptic currents, EPSCs). Because acute ethanol alters GABA transmission in other brain regions, we assessed the effects of ex vivo ethanol (50 mM) on miniature IPSCs (mIPSCs) in the DR 24-h post-ethanol exposure. Bath application of ethanol enhanced the amplitude of mIPSCs in cells from ethanol-naive and chronic intermittent ethanol-exposed (CIE) mice, but significantly enhanced the frequency of mIPSCs only in cells from CIE mice, suggesting that DR neurons are more sensitive to the inhibitory effects of acute ethanol following CIE. On the basis of these findings, we hypothesize that net excitation of DR neurons following chronic ethanol exposure contributes to enhanced anxiety during ethanol withdrawal, and that increased sensitivity of DR neurons to subsequent ethanol exposure may mediate acute ethanol's ability to relieve anxiety during ethanol withdrawal

Potent and Selective Peptide-based Inhibition of the G Protein Gαq

In contrast to G protein-coupled receptors, for which chemical and peptidic inhibitors have been extensively explored, few compounds are available that directly modulate heterotrimeric G proteins. Active Gα q binds its two major classes of effectors, the phospholipase C (PLC)-β isozymes and Rho guanine nucleotide exchange factors (RhoGEFs) related to Trio, in a strikingly similar fashion: a continuous helix-turn-helix of the effectors engages Gα q within its canonical binding site consisting of a groove formed between switch II and helix α3. This information was exploited to synthesize peptides that bound active Gα q in vitro with affinities similar to full-length effectors and directly competed with effectors for engagement of Gα q A representative peptide was specific for active Gα q because it did not bind inactive Gα q or other classes of active Gα subunits and did not inhibit the activation of PLC-β3 by Gβ 1 γ 2 In contrast, the peptide robustly prevented activation of PLC-β3 or p63RhoGEF by Gα q ; it also prevented G protein-coupled receptor-promoted neuronal depolarization downstream of Gα q in the mouse prefrontal cortex. Moreover, a genetically encoded form of this peptide flanked by fluorescent proteins inhibited Gα q -dependent activation of PLC-β3 at least as effectively as a dominant-negative form of full-length PLC-β3. These attributes suggest that related, cell-penetrating peptides should effectively inhibit active Gα q in cells and that these and genetically encoded sequences may find application as molecular probes, drug leads, and biosensors to monitor the spatiotemporal activation of Gα q in cells

Lateral Hypothalamus GABAergic Neurons Modulate Consummatory Behaviors Regardless of the Caloric Content or Biological Relevance of the Consumed Stimuli

It was recently reported that activation of a subset of lateral hypothalamus (LH) GABAergic neurons induced both appetitive (food-seeking) and consummatory (eating) behaviors in vGat-ires-cre mice, while inhibition or deletion of GABAergic neurons blunted these behaviors. As food and caloric-dense liquid solutions were used, the data reported suggest that these LH GABAergic neurons may modulate behaviors that function to maintain homeostatic caloric balance. Here we report that chemogenetic activation of this GABAergic population in vGat-ires-cre mice increased consummatory behavior directed at any available stimulus, including those entailing calories (food, sucrose, and ethanol), those that do not (saccharin and water), and those lacking biological relevance (wood). Chemogenetic inhibition of these neurons attenuated consummatory behaviors. These data indicate that LH GABAergic neurons modulate consummatory behaviors regardless of the caloric content or biological relevance of the consumed stimuli

Mu Opioid Receptor Modulation of Dopamine Neurons in the Periaqueductal Gray/Dorsal Raphe: A Role in Regulation of Pain

The periaqueductal gray (PAG) is a brain region involved in nociception modulation, and an important relay center for the descending nociceptive pathway through the rostral ventral lateral medulla. Given the dense expression of mu opioid receptors and the role of dopamine in pain, the recently characterized dopamine neurons in the ventral PAG (vPAG)/dorsal raphe (DR) region are a potentially critical site for the antinociceptive actions of opioids. The objectives of this study were to (1) evaluate synaptic modulation of the vPAG/DR dopamine neurons by mu opioid receptors and to (2) dissect the anatomy and neurochemistry of these neurons, in order to assess the downstream loci and functions of their activation. Using a mouse line that expresses eGFP under control of the tyrosine hydroxylase (TH) promoter, we found that mu opioid receptor activation led to a decrease in inhibitory inputs onto the vPAG/DR dopamine neurons. Furthermore, combining immunohistochemistry, optogenetics, electrophysiology, and fast-scan cyclic voltammetry in a TH-cre mouse line, we demonstrated that these neurons also express the vesicular glutamate type 2 transporter and co-release dopamine and glutamate in a major downstream projection structure—the bed nucleus of the stria terminalis. Finally, activation of TH-positive neurons in the vPAG/DR using Gq designer receptors exclusively activated by designer drugs displayed a supraspinal, but not spinal, antinociceptive effect. These results indicate that vPAG/DR dopamine neurons likely play a key role in opiate antinociception, potentially via the activation of downstream structures through dopamine and glutamate release

Nociceptin receptor antagonist SB 612111 decreases high fat diet binge eating

Binge eating is a dysregulated form of feeding behavior that occurs in multiple eating disorders including binge-eating disorder, the most common eating disorder. Feeding is a complex behavioral program supported through the function of multiple brain regions and influenced by a diverse array of receptor signaling pathways. Previous studies have shown the overexpression of the opioid neuropeptide nociceptin (orphanin FQ, N/OFQ) can induce hyperphagia, but the role of endogenous nociceptin receptor (NOP) in naturally occurring palatability-induced hyperphagia is unknown. In this study we adapted a simple, replicable form of binge eating of high fat food (HFD). We found that male and female C57BL/6J mice provided with daily one-hour access sessions to HFD eat significantly more during this period than those provided with continuous 24 hour access. This form of feeding is rapid and entrained. Chronic intermittent HFD binge eating produced hyperactivity and increased light zone exploration in the open field and light-dark assays respectively. Treatment with the potent and selective NOP antagonist SB 612111 resulted in a significant dose-dependent reduction in binge intake in both male and female mice, and, unlike treatment with the serotonin selective reuptake inhibitor fluoxetine, produced no change in total 24-hour food intake. SB 612111 treatment also significantly decreased non-binge-like acute HFD consumption in male mice. These data are consistent with the hypothesis that high fat binge eating is modulated by NOP signaling and that the NOP system may represent a promising novel receptor to explore for the treatment of binge eating

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

Repeated Cycles of Binge-Like Ethanol Drinking in Male C57BL/6J Mice Augments Subsequent Voluntary Ethanol Intake But Not Other Dependence-Like Phenotypes

Recently, procedures have been developed to model specific facets of human alcohol abuse disorders, including those that model excessive binge-like drinking (i.e., “drinking in the dark”, or DID procedures) and excessive dependence-like drinking (i.e., intermittent ethanol vapor exposure). Similar neuropeptide systems modulate excessive ethanol drinking stemming from both procedures, raising the possibility that both paradigms are actually modeling the same phenotypes and triggering the same central neuroplasticity. Therefore, the goal of the present project was to study the effects of a history of binge-like ethanol drinking, using DID procedures, on phenotypes that have previously been described with procedures to model dependence-like drinking

Urocortins: CRF's siblings and their potential role in anxiety, depression and alcohol drinking behavior

It is widely accepted that stress, anxiety, depression and alcohol abuse-related disorders are in large part controlled by corticotropin-releasing factor (CRF) receptors. However, evidence is accumulating that some of the actions on these receptors are mediated not by CRF, but by a family of related Urocortin (Ucn) peptides Ucn1, Ucn2 and Ucn3. The initial narrow focus on CRF as the potential main player acting on CRF receptors appears outdated. Instead it is suggested that CRF and the individual Ucns act in a complementary and brain region-specific fashion to regulate anxiety-related behaviors and alcohol consumption. This review, based on a symposium held in 2011 at the research meeting on “Alcoholism and Stress” in Volterra, Italy, highlights recent evidence for regulation of these behaviors by Ucns. In studies on stress and anxiety, the roles of Ucns, and in particular Ucn1, appear more visible in experiments analyzing adaptation to stressors rather than testing basal anxiety states. Based on these studies, we propose that the contribution of Ucn1 to regulating mood follows a U-like pattern with both high and low activity of Ucn1 contributing to high anxiety states. In studies on alcohol use disorders, the CRF system appears to regulate not only dependence-induced drinking, but also binge drinking and even basal consumption of alcohol. While dependence-induced and binge drinking rely on the actions of CRF on CRFR1 receptors, alcohol consumption in models of these behaviors is inhibited by actions of Ucns on CRFR2. In contrast, alcohol preference is positively influenced by actions of Ucn1, which is capable of acting on both CRFR1 and CRFR2. Because of complex distribution of Ucns in the nervous system, advances in this field will critically depend on development of new tools allowing site-specific analyses of the roles of Ucns and CRF

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

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