Desire or Dread from Nucleus Accumbens Inhibitions: Reversed by Same-Site Optogenetic Excitations.

Microinjections of a glutamate AMPA antagonist (DNQX) in medial shell of nucleus accumbens (NAc) can cause either intense appetitive motivation (i.e., 'desire') or intense defensive motivation (i.e., 'dread'), depending on site along a flexible rostrocaudal gradient and on environmental ambience. DNQX, by blocking excitatory AMPA glutamate inputs, is hypothesized to produce relative inhibitions of NAc neurons. However, given potential alternative explanations, it is not known whether neuronal inhibition is in fact necessary for NAc DNQX microinjections to generate motivations. Here we provide a direct test of whether local neuronal inhibition in NAc is necessary for DNQX microinjections to produce either desire or dread. We used optogenetic channelrhodopsin (ChR2) excitations at the same local sites in NAc as DNQX microinjections to oppose relative neuronal inhibitions induced by DNQX in female and male rats. We found that same-site ChR2 excitation effectively reversed the ability of NAc DNQX microinjections to generate appetitive motivation, and similarly reversed ability of DNQX microinjections to generate defensive motivation. Same-site NAc optogenetic excitations also attenuated recruitment of Fos expression in other limbic structures throughout the brain, which was otherwise elevated by NAc DNQX microinjections that generated motivation. However, to successfully reverse motivation generation, an optic fiber tip for ChR2 illumination needed to be located within <1 mm of the corresponding DNQX microinjector tip; that is, both truly at the same NAc site. Thus, we confirm that localized NAc neuronal inhibition is required for AMPA-blocking microinjections in medial shell to induce either positively-valenced 'desire' or negatively-valenced 'dread'.SIGNIFICANCE STATEMENT A major hypothesis posits neuronal inhibitions in nucleus accumbens generate intense motivation. Microinjections in nucleus accumbens of glutamate antagonist, DNQX, which might suppress local neuronal firing, generate either appetitive or defensive motivation, depending on site and environmental factors. Is neuronal inhibition in nucleus accumbens required for such pharmacologically-induced motivations? Here we demonstrate that neuronal inhibition is necessary to generate appetitive or defensive motivations, using local optogenetic excitations to oppose putative DNQX-induced inhibitions. We show that excitation at the same site prevents DNQX microinjections from recruiting downstream limbic structures into neurobiological activation, and simultaneously prevents generation of either appetitive or defensive motivated behaviors. These results may be relevant to roles of nucleus accumbens mechanisms in pathological motivations, including addiction and paranoia.

The Role of Orexin Signaling in the Ventral Tegmental Area and Central Amygdala in Modulating Binge-Like Ethanol Drinking Behavior.

BACKGROUND: Recent reports have demonstrated that binge-like ethanol (EtOH) drinking leads to an increase in hypothalamic orexin (OX) signaling and that suppressing this signaling via systemic administration of an orexin receptor (OXR) antagonist blocks this behavior; however, the specific OX pathways that modulate this behavior remain unknown. The goal of this study was to further elucidate the role of the OX system in binge-like EtOH drinking using behavioral, molecular, and pharmacological techniques. METHODS: The drinking-in-the-dark (DID) paradigm was used to model binge-like drinking behavior in male C57BL/6J mice. Experiment 1 examined changes in the OX precursor, prepro-orexin, within the hypothalamus following multiple cycle EtOH or sucrose DID using polymerase chain reaction (PCR) analysis. In experiments 2a and 2b, we used site-directed infusion of an OXR antagonist to examine the individual contribution of each OXR subtype within the ventral tegmental area (VTA) and central nucleus of the amygdala (CeA), respectively, in binge-like EtOH or sucrose drinking. RESULTS: Findings from our PCR study revealed that multiple cycles of binge-like EtOH drinking did not lead to changes in prepro-orexin mRNA as a function of binge-like EtOH drinking. However, data from site-directed pharmacology studies indicate that the orexin-1 receptor (OX1R) is the predominate receptor subtype within the VTA and CeA that regulates binge-like EtOH drinking. Interestingly, inhibition of OX1Rs did not affect binge-like sucrose intake, which suggests that these OX circuits are specific for EtOH consumption. CONCLUSIONS: As a whole, these data suggest that the VTA and CeA are important regions in which OX regulates binge-like EtOH drinking behavior. Moreover, these findings identify OXR antagonists as a potential treatment option that may be used to ameliorate problematic drinking behavior while leaving responding to natural rewards relatively intact.

Binge-like consumption of ethanol and other salient reinforcers is blocked by orexin-1 receptor inhibition and leads to a reduction of hypothalamic orexin immunoreactivity.

BACKGROUND: Orexin (OX) neurons originating in the lateral hypothalamus (LH) are ideally positioned to modulate reward processing as they form connections with several key brain regions known to be involved in the reward pathway. Consistent with these findings, a growing number of studies have implicated the OX system in modulating the rewarding properties of several drugs of abuse, including ethanol (EtOH). However, the role of the OX system in excessive binge-like EtOH intake remains relatively unexplored. Here, we assessed changes in OX immunoreactivity (IR) in the hypothalamus following repeated cycles of binge-like EtOH drinking and assessed the participation of the OX-1 receptor (OX1R) in binge-like EtOH consumption. METHODS: The drinking-in-the-dark (DID) paradigm was used to model binge-like EtOH drinking in male C57BL/6J mice. In the first experiment, mice experienced 1 or 3 cycles of binge-like EtOH or sucrose drinking with DID procedures to assess changes in OX IR in distinct subregions of the hypothalamus. Subsequent experiments examined binge-like EtOH and saccharin drinking following peripheral injections of 0.0, 5.0, or 10.0 mg/kg SB-334867 (SB), a selective OX1R antagonist. Finally, mice were given peripheral injections of SB and open-field locomotor activity was measured. RESULTS: Relative to water drinking controls, binge-like consumption of EtOH and sucrose resulted in a marked reduction in OX IR in the LH. Inhibition of the OX1R via SB blunted EtOH and saccharin drinking, but did not alter open-field locomotor activity. CONCLUSIONS: Our observed reduction in OX IR in the LH indicates that the OX system in engaged during binge-like consumption of EtOH and sucrose. The observation that inhibition of the OX1R signaling blunted binge-like EtOH, and saccharin drinking suggests that reward-related OX circuits originating in the LH participate in the consumption of salient reinforcers regardless of calories.

Repeated cycles of binge-like ethanol (EtOH)-drinking in male C57BL/6J mice augments subsequent voluntary EtOH intake but not other dependence-like phenotypes.

BACKGROUND: 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 [EtOH] vapor exposure). Similar neuropeptide systems modulate excessive EtOH 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 this present project was to study the effects of a history of binge-like EtOH drinking, using DID procedures, on phenotypes that have previously been described with procedures to model dependence-like drinking. METHODS: Male C57BL/6J mice first experienced 0 to 10 four-day binge-like drinking episodes (3 days of rest between episodes). Beginning 24 hours after the final binge-like drinking session, mice were tested for anxiety-like behaviors (with elevated plus maze [EPM] and open-field locomotor activity tests), ataxia with the rotarod test, and sensitivity to handling-induced convulsions (HICs). One week later, mice began a 40-day 2-bottle (water vs. EtOH) voluntary consumption test with concentration ranging from 10 to 20% (v/v) EtOH. RESULTS: A prior history of binge-like EtOH drinking significantly increased subsequent voluntary EtOH consumption and preference, effects most robust in groups that initially experienced 6 or 10 binge-like drinking episodes and completely absent in mice that experienced 1 binge-like drinking episode. Conversely, a history of binge-like EtOH drinking did not influence anxiety-like behaviors, ataxia, or HICs. CONCLUSIONS: Excessive EtOH drinking stemming from DID procedures does not initially induce phenotypes consistent with a dependence-like state. However, the subsequent increases in voluntary EtOH consumption and preference that become more robust following repeated episodes of binge-like EtOH drinking may reflect the early stages of EtOH dependence, suggesting that DID procedures may be ideal for studying the transition to EtOH dependence.

Chemosensory responsiveness to ethanol and its individual sensory components in alcohol-preferring, alcohol-nonpreferring and genetically heterogeneous rats.

Alcohol activates orosensory circuits that project to motivationally relevant limbic forebrain areas that control appetite, feeding and drinking. To date, limited data exists regarding the contribution of chemosensory-derived ethanol reinforcement to ethanol preference and consumption. Measures of taste reactivity to intra-orally infused ethanol have not found differences in initial orofacial responses to alcohol between alcohol-preferring (P) and alcohol-non-preferring (NP) genetically selected rat lines. Yet, in voluntary intake tests, P rats prefer highly concentrated ethanol upon initial exposure, suggesting an early sensory-mediated attraction. Here, we directly compared self-initiated chemosensory responding for alcohol and prototypic sweet, bitter and oral trigeminal stimuli among selectively bred P, NP and non-selected Wistar (WI) outbred lines to determine whether differential sensory responsiveness to ethanol and its putative sensory components are phenotypically associated with genetically influenced alcohol preference. Rats were tested for immediate short-term lick responses to alcohol (3-40%), sucrose (0.01-1 M), quinine (0.01-3 mM) and capsaicin (0.003-1 mM) in a brief-access assay designed to index orosensory-guided behavior. P rats exhibited elevated short-term lick responses to both alcohol and sucrose relative to NP and WI lines across a broad range of concentrations of each stimulus and in the absence of blood alcohol levels that would produce significant post-absorptive effects. There was no consistent relationship between genetically mediated alcohol preference and orosensory avoidance of quinine or capsaicin. These data indicate that enhanced initial chemosensory attraction to ethanol and sweet stimuli are phenotypes associated with genetic alcohol preference and are considered within the framework of downstream activation of oral appetitive reward circuits.

Dissecting Mechanisms of Motivation within the Nucleus Accumbens Using Optogenetics.

Studies mapping psychological functions to discrete brain regions often require manipulations that yield changes in a particular area and observing a subsequent shift in behavior. As investigators tap into neural underpinnings of behavior, it is useful to utilize technologies that permit temporally and spatially discrete shifts in neural signaling and neurobiological processes. This chapter contains protocols for creating "Fos plumes," a means of mapping alterations in neural activity induced by neural manipulations. By localizing increases or decreases in c-Fos in targeted brain regions, the relative spread of each manipulation can be mapped, and the functional roles of individual mechanisms within particular brain areas can be defined. The chapter also provides examples of behavioral testing protocols using optogenetics to localize psychological functions in the nucleus accumbens (NAc), a brain region involved in the production of motivated behaviors. Together, these methods provide avenues for researchers to localize and causally demonstrate the impact of neural manipulations in the brain.

Assessment of depression-like behavior and anhedonia after repeated cycles of binge-like ethanol drinking in male C57BL/6J mice.

Psychological depression is frequently linked to alcohol abuse and even serves as key indicators of an alcohol use disorder (AUD). This relationship is supported by preclinical findings in which depression-like phenotypes develop in animals exposed to chronic intermittent ethanol vapor, a common preclinical model of alcohol dependence. However, the emergence of these maladaptive phenotypes following repeated binge-like ethanol drinking remains relatively unexplored. The purpose of this study was to evaluate depression-like behaviors associated with binge-like consumption in mice. Using the drinking-in-the-dark (DID) paradigm, we examined the impact of multiple binge-like cycles (1, 3, or 6) on depression-like behaviors in the forced swim test (FST) and sucrose preference as a test for anhedonia. We also assessed the effect of repeated binge cycles on the consumption of bitter and sweet tastants over a range of concentrations. Results indicated that binge-like ethanol drinking did not lead to depression-like behavior as repeated cycles of DID did not alter sucrose consumption or preference nor did it impact time spent immobile during the FST. Animals that experienced six cycles of DID showed increased quinine consumption and increased quinine preference, which may be indicative of an escalated preference for tastants that resemble the gustatory aspects of ethanol. Interestingly, an unexpected ~20% increase in hypermobility was observed after three cycles of binge-like ethanol drinking. Although the FST is most frequently used to model depression-like behavior, emerging evidence suggests that increased hypermobility during the FST could be indicative of an inability to cope in a stressful situation, suggesting that repeated ethanol exposure in the present experiment transiently enhances stress reactivity.

Current perspectives on incentive salience and applications to clinical disorders.

Affective neuroscience research has revealed that reward contains separable components of 'liking', 'wanting', and learning. Here we focus on current 'liking' and 'wanting' findings and applications to clinical disorders. 'Liking' is the hedonic impact derived from a pleasant experience, and is amplified by opioid and related signals in discrete sites located in limbic-related brain areas. 'Wanting' refers to incentive salience, a motivation process for reward, and is mediated by larger systems involving mesocorticolimbic dopamine. Deficits in incentive salience may contribute to avolitional features of depression and related disorders, whereas deficits in hedonic impact may produce true anhedonia. Excesses in incentive salience, on the other hand, can lead to addiction, especially when narrowly focused on a particular target. Finally, a fearful form of motivational salience may even contribute to some paranoia symptoms of schizophrenia and related disorders.

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.

Chronic Voluntary Ethanol Consumption Induces Favorable Ceramide Profiles in Selectively Bred Alcohol-Preferring (P) Rats.

Heavy alcohol consumption has detrimental neurologic effects, inducing widespread neuronal loss in both fetuses and adults. One proposed mechanism of ethanol-induced cell loss with sufficient exposure is an elevation in concentrations of bioactive lipids that mediate apoptosis, including the membrane sphingolipid metabolites ceramide and sphingosine. While these naturally-occurring lipids serve as important modulators of normal neuronal development, elevated levels resulting from various extracellular insults have been implicated in pathological apoptosis of neurons and oligodendrocytes in several neuroinflammatory and neurodegenerative disorders. Prior work has shown that acute administration of ethanol to developing mice increases levels of ceramide in multiple brain regions, hypothesized to be a mediator of fetal alcohol-induced neuronal loss. Elevated ceramide levels have also been implicated in ethanol-mediated neurodegeneration in adult animals and humans. Here, we determined the effect of chronic voluntary ethanol consumption on lipid profiles in brain and peripheral tissues from adult alcohol-preferring (P) rats to further examine alterations in lipid composition as a potential contributor to ethanol-induced cellular damage. P rats were exposed for 13 weeks to a 20% ethanol intermittent-access drinking paradigm (45 ethanol sessions total) or were given access only to water (control). Following the final session, tissues were collected for subsequent chromatographic analysis of lipid content and enzymatic gene expression. Contrary to expectations, ethanol-exposed rats displayed substantial reductions in concentrations of ceramides in forebrain and heart relative to non-exposed controls, and modest but significant decreases in liver cholesterol. qRT-PCR analysis showed a reduction in the expression of sphingolipid delta(4)-desaturase (Degs2), an enzyme involved in de novo ceramide synthesis. These findings indicate that ethanol intake levels achieved by alcohol-preferring P rats as a result of chronic voluntary exposure may have favorable vs. detrimental effects on lipid profiles in this genetic line, consistent with data supporting beneficial cardioprotective and neuroprotective effects of moderate ethanol consumption.

Targeting central melanocortin receptors: a promising novel approach for treating alcohol abuse disorders.

The melanocortin (MC) peptides are produced centrally by propiomelanocortin (POMC) neurons within the arcuate nucleus of the hypothalamus and act through five seven-transmembrane G-protein coupled melanocortin receptor (MCR) subtypes. The MC3R and MC4R subtypes, the most abundant central MCRs, are widely expressed in brain regions known to modulate neurobiological responses to ethanol, including regions of the hypothalamus and extended amygdala. Agouti-related protein (AgRP), also produced in the arcuate nucleus, is secreted in terminals expressing MCRs and functions as an endogenous MCR antagonist. This review highlights recent genetic and pharmacological findings that have implicated roles for the MC and AgRP systems in modulating ethanol consumption. Ethanol consumption is associated with significant alterations in the expression levels of various MC peptides/protein, which suggests that ethanol-induced perturbations of MC/AgRP signaling may modulate excessive ethanol intake. Consistently, MCR agonists decrease, and AgRP increases, ethanol consumption in mice. MCR agonists fail to blunt ethanol intake in mutant mice lacking the MC4R, suggesting that the protective effects of MCR agonists are modulated by the MC4R. Interestingly, recent evidence reveals that MCR agonists are more effective at blunting binge-like ethanol intake in mutant mice lacking the MC3R, suggesting that the MC3R has opposing effects on the MC4R. Finally, mutant mice lacking AgRP exhibit blunted voluntary and binge-like ethanol drinking, consistent with pharmacological studies. Collectively, these preclinical observations provide compelling evidence that compounds that target the MC system may provide therapeutic value for treating alcohol abuse disorders and that the utilization of currently available MC-targeting compounds- such as those being used to treat eating disorders- may be used as effective treatments to this end.