The parasubthalamic nucleus refeeding ensemble delays feeding initiation and hastens water drinking.

The parasubthalamic nucleus (PSTN) is activated by refeeding after food deprivation and several PSTN subpopulations have been shown to suppress feeding. However, no study to date directly addressed the role of PSTN neurons activated upon food access in the control of ensuing food consumption. Here we identify consumption latency as a sensitive behavioral indicator of PSTN activity, and show that, in hungry mice, the ensemble of refeeding-activated PSTN neurons drastically increases the latency to initiate refeeding with both familiar and a novel, familiar food, but does not control the amount of food consumed. In thirsty mice, this ensemble also delays sucrose consumption but accelerates water consumption, possibly reflecting anticipatory prandial thirst, with again no influence on the amount of fluid consumed. We next sought to identify which subpopulations of PSTN neurons might be driving these latency effects, using cell-type and pathway-specific chemogenetic manipulations. Our results suggest a prominent role of PSTN Tac1 neurons projecting to the central amygdala in the hindrance of feeding initiation. While PSTN Crh neurons also delay the latency of hungry mice to ingest familiar foods, they surprisingly promote the consumption of novel, palatable substances. Furthermore, PSTN Crh neurons projecting to the bed nucleus of the stria terminalis accelerate rehydration in thirsty mice. Our results demonstrate the key role of endogenous PSTN activity in the control of feeding and drinking initiation and delineate specific circuits mediating these effects, which may have relevance for eating disorders.

Ethanol's interaction with BK channel α subunit residue K361 does not mediate behavioral responses to alcohol in mice.

Large conductance potassium (BK) channels are among the most sensitive molecular targets of ethanol and genetic variations in the channel-forming α subunit have been nominally associated with alcohol use disorders. However, whether the action of ethanol at BK α influences the motivation to drink alcohol remains to be determined. To address this question, we first tested the effect of systemically administered BK channel modulators on voluntary alcohol consumption in C57BL/6J males. Penitrem A (blocker) exerted dose-dependent effects on moderate alcohol intake, while paxilline (blocker) and BMS-204352 (opener) were ineffective. Because pharmacological manipulations are inherently limited by non-specific effects, we then sought to investigate the behavioral relevance of ethanol's direct interaction with BK α by introducing in the mouse genome a point mutation known to render BK channels insensitive to ethanol while preserving their physiological function. The BK α K361N substitution prevented ethanol from reducing spike threshold in medial habenula neurons. However, it did not alter acute responses to ethanol in vivo, including ataxia, sedation, hypothermia, analgesia, and conditioned place preference. Furthermore, the mutation did not have reproducible effects on alcohol consumption in limited, continuous, or intermittent access home cage two-bottle choice paradigms conducted in both males and females. Notably, in contrast to previous observations made in mice missing BK channel auxiliary ß subunits, the BK α K361N substitution had no significant impact on ethanol intake escalation induced by chronic intermittent alcohol vapor inhalation. It also did not affect the metabolic and locomotor consequences of chronic alcohol exposure. Altogether, these data suggest that the direct interaction of ethanol with BK α does not mediate the alcohol-related phenotypes examined here in mice.

Central amygdala corticotropin-releasing factor neurons promote hyponeophagia but do not control alcohol drinking in mice.

Corticotropin-releasing factor (CRF) signaling in the central nucleus of the amygdala (CeA) plays a critical role in rodent models of excessive alcohol drinking. However, the source of CRF acting in the CeA during alcohol withdrawal remains to be identified. In the present study, we hypothesized that CeA CRF interneurons may represent a behaviorally relevant source of CRF to the CeA increasing motivation for alcohol via negative reinforcement. We first observed that Crh mRNA expression in the anterior part of the mouse CeA correlates positively with alcohol intake in C57BL/6J males with a history of chronic binge drinking followed by abstinence and increases upon exposure to chronic intermittent ethanol (CIE) vapor inhalation. We then found that chemogenetic activation of CeA CRF neurons in Crh-IRES-Cre mouse brain slices increases gamma-aminobutyric acid (GABA) release in the medial CeA, in part via CRF1 receptor activation. While chemogenetic stimulation exacerbated novelty-induced feeding suppression (NSF) in alcohol-naïve mice, thereby mimicking the effect of withdrawal from CIE, it had no effect on voluntary alcohol consumption, following either acute or chronic manipulation. Furthermore, chemogenetic inhibition of CeA CRF neurons did not affect alcohol consumption or NSF in chronic alcohol drinkers exposed to air or CIE. Altogether, these findings indicate that CeA CRF neurons produce local release of GABA and CRF and promote hyponeophagia in naïve mice, but do not drive alcohol intake escalation or negative affect in CIE-withdrawn mice. The latter result contrasts with previous findings in rats and demonstrates species specificity of CRF circuit engagement in alcohol dependence.

Brain-wide functional architecture remodeling by alcohol dependence and abstinence.

Alcohol abuse and alcohol dependence are key factors in the development of alcohol use disorder, which is a pervasive societal problem with substantial economic, medical, and psychiatric consequences. Although our understanding of the neurocircuitry that underlies alcohol use has improved, novel brain regions that are involved in alcohol use and novel biomarkers of alcohol use need to be identified. The present study used a single-cell whole-brain imaging approach to 1) assess whether abstinence from alcohol in an animal model of alcohol dependence alters the functional architecture of brain activity and modularity, 2) validate our current knowledge of the neurocircuitry of alcohol abstinence, and 3) discover brain regions that may be involved in alcohol use. Alcohol abstinence resulted in the whole-brain reorganization of functional architecture in mice and a pronounced decrease in modularity that was not observed in nondependent moderate drinkers. Structuring of the alcohol abstinence network revealed three major brain modules: 1) extended amygdala module, 2) midbrain striatal module, and 3) cortico-hippocampo-thalamic module, reminiscent of the three-stage theory. Many hub brain regions that control this network were identified, including several that have been previously overlooked in alcohol research. These results identify brain targets for future research and demonstrate that alcohol use and dependence remodel brain-wide functional architecture to decrease modularity. Further studies are needed to determine whether the changes in coactivation and modularity that are associated with alcohol abstinence are causal features of alcohol dependence or a consequence of excessive drinking and alcohol exposure.

Affective Disturbances During Withdrawal from Chronic Intermittent Ethanol Inhalation in C57BL/6J and DBA/2J Male Mice.

BACKGROUND: Alcohol use disorders are characterized by a complex behavioral symptomatology, which includes the loss of control over alcohol consumption and the emergence of a negative affective state when alcohol is not consumed. Some of these symptoms can be recapitulated in rodent models, for instance following chronic intermittent ethanol (EtOH; CIE) vapor inhalation. However, the detection of negative affect in mice withdrawn from CIE has proven challenging and variable between strains. This study aimed to detect reliable indices of negative emotionality in CIE-exposed C57BL/6J (C57) and DBA/2J (DBA) mice. Males were used because they are known to escalate their voluntary EtOH consumption upon CIE exposure, which is hypothesized to be driven by negative reinforcement (relief from negative affect). METHODS: Adult male mice were exposed to 4 to 6 weeks of CIE and were evaluated 3 to 10 days into withdrawal in the social approach, novelty-suppressed feeding, digging, marble burying, and bottle brush tests. RESULTS: Withdrawal from CIE decreased sociability in DBA mice but not in C57 mice. Conversely, hyponeophagia was exacerbated by CIE in C57 mice but not in DBA mice. Withdrawal from CIE robustly increased digging activity in both strains, even in the absence of marbles. Aggressive responses to bottle brush attacks were elevated in both C57 and DBA mice following CIE exposure, but CIE had an opposite effect on defensive responses in the 2 strains (increase in C57 vs. decrease in DBA). CONCLUSIONS: Our results indicate that withdrawal from CIE elicits negative emotionality in both C57 and DBA mice, but different tests need to be used to measure the anxiogenic-like effects of withdrawal in each strain. Increased digging activity and irritability-like behavior represent novel indices of affective dysfunction associated with withdrawal from CIE in both mouse strains. Our findings enrich the characterization of the affective symptomatology of protracted withdrawal from CIE in mice.

GIRK3 gates activation of the mesolimbic dopaminergic pathway by ethanol.

G protein-gated inwardly rectifying potassium (GIRK) channels are critical regulators of neuronal excitability and can be directly activated by ethanol. Constitutive deletion of the GIRK3 subunit has minimal phenotypic consequences, except in response to drugs of abuse. Here we investigated how the GIRK3 subunit contributes to the cellular and behavioral effects of ethanol, as well as to voluntary ethanol consumption. We found that constitutive deletion of GIRK3 in knockout (KO) mice selectively increased ethanol binge-like drinking, without affecting ethanol metabolism, sensitivity to ethanol intoxication, or continuous-access drinking. Virally mediated expression of GIRK3 in the ventral tegmental area (VTA) reversed the phenotype of GIRK3 KO mice and further decreased the intake of their wild-type counterparts. In addition, GIRK3 KO mice showed a blunted response of the mesolimbic dopaminergic (DA) pathway to ethanol, as assessed by ethanol-induced excitation of VTA neurons and DA release in the nucleus accumbens. These findings support the notion that the subunit composition of VTA GIRK channels is a critical determinant of DA neuron sensitivity to drugs of abuse. Furthermore, our study reveals the behavioral impact of this cellular effect, whereby the level of GIRK3 expression in the VTA tunes ethanol intake under binge-type conditions: the more GIRK3, the less ethanol drinking.

BK Channel ß1 Subunit Contributes to Behavioral Adaptations Elicited by Chronic Intermittent Ethanol Exposure.

BACKGROUND: Large conductance, calcium- and voltage-activated potassium (BK) channels regulate neuronal excitability and neurotransmission. They can be directly activated by ethanol (EtOH) and they may be implicated in EtOH dependence. In this study, we sought to determine the influence of the auxiliary ß1 and ß4 subunits on EtOH metabolism, acute sensitivity to EtOH intoxication, acute functional tolerance, chronic tolerance, and handling-induced convulsions during withdrawal. METHODS: Motor coordination, righting reflex, and body temperature were evaluated in BK ß1 and ß4 knockout, heterozygous, and wild-type mice following acute EtOH administration. Chronic tolerance and physical dependence were induced by chronic intermittent inhalation of EtOH vapor. RESULTS: Constitutive deficiency in BK ß1 or ß4 subunits did not alter the clearance rate of EtOH, acute sensitivity to EtOH-induced ataxia, sedation, and hypothermia, nor acute functional tolerance to ataxia. BK ß1 deletion reduced chronic tolerance to sedation and abolished chronic tolerance to hypothermia, while BK ß4 deletion did not affect these adaptations to chronic EtOH exposure. Finally, the absence of BK ß1 accelerated the appearance, while the absence of BK ß4 delayed the resolution, of the hyperexcitable state associated with EtOH withdrawal. CONCLUSIONS: Altogether, the present findings reveal the critical role of BK ß1 in behavioral adaptations to prolonged, repeated EtOH intoxication.

MeCP2 regulates ethanol sensitivity and intake.

We have investigated the expression of chromatin-regulating genes in the prefrontal cortex and in the shell subdivision of the nucleus accumbens during protracted withdrawal in mice with increased ethanol drinking after chronic intermittent ethanol (CIE) vapor exposure and in mice with a history of non-dependent drinking. We observed that the methyl-CpG binding protein 2 (MeCP2) was one of the few chromatin-regulating genes to be differentially regulated by a history of dependence. As MeCP2 has the potential of acting as a broad gene regulator, we investigated sensitivity to ethanol and ethanol drinking in MeCP2(308/) (Y) mice, which harbor a truncated MeCP2 allele but have a milder phenotype than MeCP2 null mice. We observed that MeCP2(308/) (Y) mice were more sensitive to ethanol's stimulatory and sedative effects than wild-type (WT) mice, drank less ethanol in a limited access 2 bottle choice paradigm and did not show increased drinking after induction of dependence with exposure to CIE vapors. Alcohol metabolism did not differ in MeCP2(308/) (Y) and WT mice. Additionally, MeCP2(308/) (Y) mice did not differ from WT mice in ethanol preference in a 24-hour paradigm nor in their intake of graded solutions of saccharin or quinine, suggesting that the MeCP2(308/) (Y) mutation did not alter taste function. Lastly, using the Gene Set Enrichment Analysis algorithm, we found a significant overlap in the genes regulated by alcohol and by MeCP2. Together, these results suggest that MeCP2 contributes to the regulation of ethanol sensitivity and drinking.

Mouse parasubthalamic Crh neurons drive alcohol drinking escalation and behavioral disinhibition.

Corticotropin-releasing factor (CRF, encoded by Crh) signaling is thought to play a critical role in the development of excessive alcohol drinking and the emotional and physical pain associated with alcohol withdrawal. Here, we investigated the parasubthalamic nucleus (PSTN) as a potential source of CRF relevant to the control of alcohol consumption, affect, and nociception in mice. We identified PSTN Crh neurons as a neuronal subpopulation that exerts a potent and unique influence on behavior by promoting not only alcohol but also saccharin drinking, while PSTN neurons are otherwise known to suppress consummatory behaviors. Furthermore, PSTN Crh neurons are causally implicated in the escalation of alcohol and saccharin intake produced by chronic intermittent ethanol (CIE) vapor inhalation, a mouse model of alcohol use disorder. In contrast to our predictions, the ability of PSTN Crh neurons to increase alcohol drinking is not mediated by CRF1 signaling. Moreover, the pattern of behavioral disinhibition and reduced nociception driven by their activation does not support a role of negative reinforcement as a motivational basis for the concomitant increase in alcohol drinking. Finally, silencing Crh expression in the PSTN slowed down the escalation of alcohol intake in mice exposed to CIE and accelerated their recovery from withdrawal-induced mechanical hyperalgesia. Altogether, our results suggest that PSTN Crh neurons may represent an important node in the brain circuitry linking alcohol use disorder with sweet liking and novelty seeking.

Chronic MAP4343 reverses escalated alcohol drinking in a mouse model of alcohol use disorder.

Alcohol use disorders can be driven by negative reinforcement. Alterations of the microtubule cytoskeleton have been associated with mood regulation in the context of depression. Notably, MAP4343, a pregnenolone derivative known to promote tubulin assembly, has antidepressant properties. In the present study, we tested the hypothesis that MAP4343 may reduce excessive alcohol drinking in a mouse model of alcohol dependence by normalizing affect during withdrawal. Adult male C57BL/6J mice were given limited access to voluntary alcohol drinking and ethanol intake escalation was induced by chronic intermittent ethanol (CIE) vapor inhalation. Chronic, but not acute, administration of MAP4343 reduced ethanol intake and this effect was more pronounced in CIE-exposed mice. There was a complex interaction between the effects of MAP4343 and alcohol on affective behaviors. In the elevated plus maze, chronic MAP4343 tended to increase open-arm exploration in alcohol-naive mice but reduced it in alcohol-withdrawn mice. In the tail suspension test, chronic MAP4343 reduced immobility selectively in Air-exposed alcohol-drinking mice. Finally, chronic MAP4343 countered the plasma corticosterone reduction induced by CIE. Parallel analysis of tubulin post-translational modifications revealed lower α-tubulin acetylation in the medial prefrontal cortex of CIE-withdrawn mice. Altogether, these data support the relevance of microtubules as a therapeutic target for the treatment of AUD.

Prefrontal cortex glutamatergic adaptations in a mouse model of alcohol use disorder.

Alcohol use disorder (AUD) produces cognitive deficits, indicating a shift in prefrontal cortex (PFC) function. PFC glutamate neurotransmission is mostly mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type ionotropic receptors (AMPARs); however preclinical studies have mostly focused on other receptor subtypes. Here we examined the impact of early withdrawal from chronic ethanol on AMPAR function in the mouse medial PFC (mPFC). Dependent male C57BL/6J mice were generated using the chronic intermittent ethanol vapor-two bottle choice (CIE-2BC) paradigm. Non-dependent mice had access to water and ethanol bottles but did not receive ethanol vapor. Naïve mice had no ethanol exposure. We used patch-clamp electrophysiology to measure glutamate neurotransmission in layer 2/3 prelimbic mPFC pyramidal neurons. Since AMPAR function can be impacted by subunit composition or plasticity-related proteins, we probed their mPFC expression levels. Dependent mice had higher spontaneous excitatory postsynaptic current (sEPSC) amplitude and kinetics compared to the Naïve/Non-dependent mice. These effects were seen during intoxication and after 3-8 days withdrawal, and were action potential-independent, suggesting direct enhancement of AMPAR function. Surprisingly, 3 days withdrawal decreased expression of genes encoding AMPAR subunits (Gria1/2) and synaptic plasticity proteins (Dlg4 and Grip1) in Dependent mice. Further analysis within the Dependent group revealed a negative correlation between Gria1 mRNA levels and ethanol intake. Collectively, these data establish a role for mPFC AMPAR adaptations in the glutamatergic dysfunction associated with ethanol dependence. Future studies on the underlying AMPAR plasticity mechanisms that promote alcohol reinforcement, seeking, drinking and relapse behavior may help identify new targets for AUD treatment.

Blockade of IL-17 signaling reverses alcohol-induced liver injury and excessive alcohol drinking in mice.

Chronic alcohol abuse has a detrimental effect on the brain and liver. There is no effective treatment for these patients, and the mechanism underlying alcohol addiction and consequent alcohol-induced damage of the liver/brain axis remains unresolved. We compared experimental models of alcoholic liver disease (ALD) and alcohol dependence in mice and demonstrated that genetic ablation of IL-17 receptor A (IL-17ra-/-) or pharmacological blockade of IL-17 signaling effectively suppressed the increased voluntary alcohol drinking in alcohol-dependent mice and blocked alcohol-induced hepatocellular and neurological damage. The level of circulating IL-17A positively correlated with the alcohol use in excessive drinkers and was further increased in patients with ALD as compared with healthy individuals. Our data suggest that IL-17A is a common mediator of excessive alcohol consumption and alcohol-induced liver/brain injury, and targeting IL-17A may provide a novel strategy for treatment of alcohol-induced pathology.

Morphological and functional evidence of increased excitatory signaling in the prelimbic cortex during ethanol withdrawal.

Excessive alcohol consumption in humans induces deficits in decision making and emotional processing, which indicates a dysfunction of the prefrontal cortex (PFC). The present study aimed to determine the impact of chronic intermittent ethanol (CIE) inhalation on mouse medial PFC pyramidal neurons. Data were collected 6-8 days into withdrawal from 7 weeks of CIE exposure, a time point when mice exhibit behavioral symptoms of withdrawal. We found that spine maturity in prelimbic (PL) layer 2/3 neurons was increased, while dendritic spines in PL layer 5 neurons or infralimbic (IL) neurons were not affected. Corroborating these morphological observations, CIE enhanced glutamatergic transmission in PL layer 2/3 pyramidal neurons, but not IL layer 2/3 neurons. Contrary to our predictions, these cellular alterations were associated with improved, rather than impaired, performance in reversal learning and strategy switching tasks in the Barnes maze at an earlier stage of chronic ethanol exposure (5-7 days withdrawal from 3 to 4 weeks of CIE), which could result from the anxiety-like behavior associated with ethanol withdrawal. Altogether, this study adds to a growing body of literature indicating that glutamatergic activity in the PFC is upregulated following chronic ethanol exposure, and identifies PL layer 2/3 pyramidal neurons as a sensitive target of synaptic remodeling. It also indicates that the Barnes maze is not suitable to detect deficits in cognitive flexibility in CIE-withdrawn mice.

Virus-mediated shRNA knockdown of prodynorphin in the rat nucleus accumbens attenuates depression-like behavior and cocaine locomotor sensitization.

Dynorphins, endogenous opioid peptides that arise from the precursor protein prodynorphin (Pdyn), are hypothesized to be involved in the regulation of mood states and the neuroplasticity associated with addiction. The current study tested the hypothesis that dynorphin in the nucleus accumbens (NAcc) mediates such effects. More specifically, we examined whether knockdown of Pdyn within the NAcc in rats would alter the expression of depressive-like and anxiety-like behavior, as well as cocaine locomotor sensitization. Wistar rats were injected with adeno-associated viral (AAV) vectors encoding either a Pdyn-specific short hairpin RNA (AAV-shPdyn) or a scrambled shRNA (AAV-shScr) as control. Four weeks later, rats were tested for anxiety-like behavior in the elevated plus maze test and depressive-like behavior in the forced swim test (FST). Finally, rats received one daily injection of saline or cocaine (20 mg/kg, i.p.), followed by assessment of locomotion for 4 consecutive days. Following 3 days of abstinence, the rats completed 2 additional daily cocaine/saline locomotor trials. Pdyn knockdown in the NAcc led to a significant reduction in depressive-like behavior in the FST, but had no effect on anxiety-like behavior in the elevated plus maze. Pdyn knockdown did not alter baseline locomotor behavior, the locomotor response to acute cocaine, or the initial sensitization of the locomotor response to cocaine over the first 4 cocaine treatment days. However, following 3 days abstinence the locomotor response to the cocaine challenge returned to their original levels in the AAV-shPdyn rats while remaining heightened in the AAV-shScr rats. These results suggest that dynorphin in a very specific area of the nucleus accumbens contributes to depressive-like states and may be involved in neuroadaptations in the NAcc that contribute to the development of cocaine addiction as a persistent and lasting condition.

BK channel ß1 and ß4 auxiliary subunits exert opposite influences on escalated ethanol drinking in dependent mice.

Large conductance calcium-activated potassium (BK) channels play a key role in the control of neuronal activity. Ethanol is a potent activator of BK channel gating, but how this action may impact ethanol drinking still remains poorly understood. Auxiliary ß subunits are known to modulate ethanol-induced potentiation of BK currents. In the present study, we investigated whether BK ß1 and ß4 subunits influence voluntary ethanol consumption using knockout (KO) mice. In a first experiment, mice were first subjected to continuous two-bottle choice (2BC) and were then switched to intermittent 2BC, which progressively increased ethanol intake as previously described in wildtype mice. BK ß1 or ß4 subunit deficiency did not affect ethanol self-administration under either schedule of access. In a second experiment, mice were first trained to drink ethanol in a limited-access 2BC paradigm. BK ß1 or ß4 deletion did not affect baseline consumption. Weeks of 2BC were then alternated with weeks of chronic intermittent ethanol (CIE) or air inhalation. As expected, a gradual escalation of ethanol drinking was observed in dependent wildtype mice, while intake remained stable in non-dependent wildtype mice. However, CIE exposure only produced a mild augmentation of ethanol consumption in BK ß4 KO mice. Conversely, ethanol drinking increased after fewer CIE cycles in BK ß1 KO mice than in wildtype mice. In conclusion, BK ß1 or ß4 did not influence voluntary ethanol drinking in non-dependent mice, regardless of the pattern of access to ethanol. However, deletion of BK ß4 attenuated, while deletion of BK ß1 accelerated, the escalation of ethanol drinking during withdrawal from CIE. Our data suggest that BK ß1 and ß4 subunits have an opposite influence on the negative reinforcing properties of ethanol withdrawal. Modulating the expression, distribution or interactions of BK channel auxiliary subunits may therefore represent a novel avenue for the treatment of alcoholism.