Recent Perspectives on Sex Differences in Compulsion-Like and Binge Alcohol Drinking.

Alcohol use disorder remains a substantial social, health, and economic problem and problem drinking levels in women have been increasing in recent years. Understanding whether and how the underlying mechanisms that drive drinking vary by sex is critical and could provide novel, more targeted therapeutic treatments. Here, we examine recent results from our laboratories and others which we believe provide useful insights into similarities and differences in alcohol drinking patterns across the sexes. Findings for binge intake and aversion-resistant, compulsion-like alcohol drinking are considered, since both are likely significant contributors to alcohol problems in humans. We also describe studies regarding mechanisms that may underlie sex differences in maladaptive alcohol drinking, with some focus on the importance of nucleus accumbens (NAcb) core and shell regions, several receptor types (dopamine, orexin, AMPA-type glutamate), and possible contributions of sex hormones. Finally, we discuss how stressors such as early life stress and anxiety-like states may interact with sex differences to contribute to alcohol drinking. Together, these findings underscore the importance and critical relevance of studying female and male mechanisms for alcohol and co-morbid conditions to gain a true and clinically useful understanding of addiction and neuropsychiatric mechanisms and treatment.

Neuronal modulation of hepatic lipid accumulation induced by bingelike drinking.

Excessive alcohol consumption, including binge drinking, is a common cause of fatty liver disease. Binge drinking rapidly induces hepatic steatosis, an early step in the pathogenesis of chronic liver injury. Despite its prevalence, the process by which excessive alcohol consumption promotes hepatic lipid accumulation remains unclear. Alcohol exerts potent effects on the brain, including hypothalamic neurons crucial for metabolic regulation. However, whether or not the brain plays a role in alcohol-induced hepatic steatosis is unknown. In the brain, alcohol increases extracellular levels of adenosine, a potent neuromodulator, and previous work implicates adenosine signaling as being important for the development of alcoholic fatty liver disease. Acute alcohol exposure also increases both the activity of agouti-related protein (AgRP)-expressing neurons and AgRP immunoreactivity. Here, we show that adenosine receptor A2B signaling in the brain modulates the extent of alcohol-induced fatty liver in mice and that both the AgRP neuropeptide and the sympathetic nervous system are indispensable for hepatic steatosis induced by bingelike alcohol consumption. Together, these results indicate that the brain plays an integral role in alcohol-induced hepatic lipid accumulation and that central adenosine signaling, hypothalamic AgRP, and the sympathetic nervous system are crucial mediators of this process.

Drinking despite adversity: behavioral evidence for a head down and push strategy of conflict-resistant alcohol drinking in rats.

Compulsive alcohol drinking, where intake persists regardless of adverse consequences, plays a major role in the substantial costs of alcohol use disorder. However, the processes that promote aversion-resistant drinking remain poorly understood. Compulsion-like responding has been considered automatic and reflexive and also to involve higher motivation, since drinking persists despite adversity. Thus, we used lickometry, where microstructural behavioral changes can reflect altered motivation, to test whether conflict-resistant intake [quinine-alcohol (QuiA)] reflected greater automaticity or motivation relative to alcohol-only drinking (Alc). Front-loading during QuiA and Alc suggested incentive to drink in both. However, the relationship between total licking and intake was less variable during QuiA, as was lick volume, without changes in average responding. QuiA bout organization was also less variable, with fewer licks outside of bouts (stray licks) and fewer gaps within bouts. Interestingly, QuiA avoidance of stray licking continued into short bouts, with fewer short and more medium-length bouts, which was striking given their minor impact on intake. Instead, more effort at bout onset could allow short bouts to persist longer. Indeed, while QuiA licking was overall faster, QuiA bouts were especially fast at bout initiation. However, few QuiA changes individually predicted greater intake, perhaps suggesting an overarching strategy during aversion-resistant responding. Thus, our results indicate that aversion-resistant intake exhibited less variability, where increased automaticity could decrease need for awareness, and stronger bout initiation, which might prolong responding despite adversity. This may reflect a collective strategy, which we call Head Down and Push responding that facilitates conflict-resistant, compulsion-like intake.

Limited Excessive Voluntary Alcohol Drinking Leads to Liver Dysfunction in Mice.

BACKGROUND: Liver damage is a serious and sometimes fatal consequence of long-term alcohol intake, which progresses from early-stage fatty liver (steatosis) to later-stage steatohepatitis with inflammation and fibrosis/necrosis. However, very little is known about earlier stages of liver disruption that may occur in problem drinkers, those who drink excessively but are not dependent on alcohol. METHODS: We examined how repeated binge-like alcohol drinking in C57BL/6 mice altered liver function, as compared with a single binge-intake session and with repeated moderate alcohol consumption. We measured a number of markers associated with early- and later-stage liver disruption, including liver steatosis, measures of liver cytochrome P4502E1 (CYP2E1) and alcohol dehydrogenase (ADH), alcohol metabolism, expression of cytokine mRNA, accumulation of 4-hydroxynonenal (4-HNE) as an indicator of oxidative stress, and alanine transaminase/aspartate transaminase as a measure of hepatocyte injury. RESULTS: Importantly, repeated binge-like alcohol drinking increased triglyceride levels in the liver and plasma, and increased lipid droplets in the liver, indicators of steatosis. In contrast, a single binge-intake session or repeated moderate alcohol consumption did not alter triglyceride levels. In addition, alcohol exposure can increase rates of alcohol metabolism through CYP2E1 and ADH, which can potentially increase oxidative stress and liver dysfunction. Intermittent, excessive alcohol intake increased liver CYP2E1 mRNA, protein, and activity, as well as ADH mRNA and activity. Furthermore, repeated, binge-like drinking, but not a single binge or moderate drinking, increased alcohol metabolism. Finally, repeated, excessive intake transiently elevated mRNA for the proinflammatory cytokine IL-1B and 4-HNE levels, but did not alter markers of later-stage liver hepatocyte injury. CONCLUSIONS: Together, we provide data suggesting that even relatively limited binge-like alcohol drinking can lead to disruptions in liver function, which might facilitate the transition to more severe forms of liver damage.

Ethanol drives aversive conditioning through dopamine 1 receptor and glutamate receptor-mediated activation of lateral habenula neurons.

There has been increasing interest in the lateral habenula (LHb) given its potent regulatory role in many aversion-related behaviors. Interestingly, ethanol can be rewarding as well as aversive; we therefore investigated whether ethanol exposure alters pacemaker firing or glutamate receptor signaling in LHb neurons in vitro and also whether LHb activity in vivo might contribute to the acquisition of conditioned place aversion to ethanol. Surprisingly, in epithalamic slices, low doses of ethanol (1.4 mM) strongly accelerated LHb neuron firing (by ~60%), and ethanol's effects were much reduced by blocking glutamate receptors. Ethanol increased presynaptic glutamate release, and about half of this effect was mediated by dopamine subtype 1 receptors (D1Rs) and cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. In agreement with these findings, c-Fos immunoreactivity in LHb regions was enhanced after a single administration of a low dose of ethanol (0.25 g/kg i.p.). Importantly, the same dose of ethanol in vivo also produced strong conditioned place aversion, and this was prevented by inhibiting D1Rs or neuronal activity within the LHb. By contrast, a higher dose (2 g/kg) led to ethanol conditioned place preference, which was enhanced by inhibiting neuronal activity or D1Rs within the LHb and suppressed by infusing aminomethylphosphonic acid or the D1R agonist SKF38393 within the LHb. Our in vitro and in vivo observations show, for the first time, that ethanol increases LHb excitation, mediated by D1R and glutamate receptors, and may underlie a LHb aversive signal that contributes to ethanol-related aversion.

Greater inhibition of female rat binge alcohol intake by adrenergic receptor blockers using a novel Two-Shot rat binge drinking model.

Binge drinking (BD) contributes strongly to the harms of alcohol use disorder. Most rodent models do not result in binge-level blood alcohol concentrations (BACs), and to better understand individual and sex differences in neurobiological mechanisms related to BD, the use of outbred rat strains would be valuable. Here, we developed a novel BD model where after 3+ months of intermittent access to 20% alcohol Wistar rats drank, twice a week, with two 5-minute intake (what we called Two-shot) separated by a 10-minute break. Our findings showed during Two-Shot that most animals reached ≥ 80mg% BAC levels (when briefly food-restricted). However, when increasing alcohol concentrations from 20% to 30%, 40%, or 50%, rats titrated to similar intake levels, suggesting rapid sensing of alcohol effects even when front-loading. Two-Shot drinking was reduced in both sexes by naltrexone (1mg/kg), validating intake suppression by a clinical therapeutic agent. Further, both propranolol (ß adrenergic receptor antagonist) and prazosin (α1 adrenergic receptor antagonist) reduced female but not male BD at the lower dose. Thus, our results provide a novel model for BD in outbred rats and suggest that female binging is more sensitive to adrenergic modulation than males, perhaps providing a novel sex-related therapy.

Greater inhibition of female rat binge alcohol intake by adrenergic receptor blockers using a novel Two-Shot rat binge drinking model.

Binge drinking (BD) contributes strongly to the harms of alcohol use disorder. Most rodent models do not result in binge-level blood alcohol concentrations (BACs), and to better understand individual and sex differences in neurobiological mechanisms related to BD, the use of outbred rat strains would be valuable. Here, we developed a novel BD model where after 3+ months of intermittent access to 20% alcohol Wistar rats drank, twice a week, with two 5-min intake (what we called Two-shot) separated by a 10-min break. Our findings showed during Two-Shot that most animals reached ≥ 80 mg% BAC levels (when briefly food-restricted). However, when increasing alcohol concentrations from 20 to 30%, 40%, or 50%, rats titrated to similar intake levels, suggesting rapid sensing of alcohol effects even when front-loading. Two-Shot drinking was reduced in both sexes by naltrexone (1 mg/kg), validating intake suppression by a clinical therapeutic agent for human problem drinking. Further, both propranolol (ß-adrenergic receptor antagonist) and prazosin (α1-adrenergic receptor antagonist) reduced female but not male BD at the lower dose. Thus, our results provide a novel model for BD in outbred rats and suggest that female binging is more sensitive to adrenergic modulation than males, perhaps providing a novel sex-related therapy.

Sex- and estrous-related response patterns for alcohol depend critically on the level of compulsion-like challenge.

Alcohol use disorder is a substantial social and economic burden. During the last years, the number of women with drinking problems has been increasing, and one main concern is that they are particularly more vulnerable to negative consequences of alcohol. However, little is known about female-specific response patterns for alcohol, and potential underlying differences in brain mechanisms, including for compulsion-like alcohol drinking (when intake persists despite adverse consequences). We used lickometry to assess behavioral microstructure in adult Wistar male and female rats (n = 28-30) during alcohol-only drinking or moderate- or higher-challenge alcohol compulsion (10 or 60 mg/l quinine in alcohol, respectively). Estrous stages were determined and related to drinking levels and patterns of responding to alcohol, as was ovariectomy. Our findings showed that females (where we didn't determine estrus stage) had similar total licks in a session as males, but significantly longer licking bouts under alcohol-only and moderate-challenge, suggesting greater persistence. Further, greater intake under alcohol-only and moderate-challenge was related to faster licking in males, while female consumption was not related to licking speed. Thus, females could have increased persistence without greater vigor, unlike males. However, under higher-challenge, faster licking did predict higher intake in females, similar to males. To better understand female higher-challenge responding, we examined drinking in relation to phases of the estrous cycle. Higher-challenge had longer bouts only in late diestrus. In addition, ovariectomy led to longer bouts only under higher-challenge, suggesting that conditions with reduced hormone levels could increase female persistence for alcohol under higher-challenge. However, ovariectomy also reduced alcohol-only and moderate-challenge drinking but did not reduce bout length. Thus, intake level and response strategy could be regulated somewhat differently by ovarian hormones. Finally, moderate-challenge licking speed was less variable during early diestrus, and we previously showed more stereotyped responding specifically under moderate-challenge in males. By combining behavioral microstructure and sex- and estrus-related changes in drinking patterns, our results suggest that females have greater persistence for alcohol under lower-challenge drinking, while late diestrus and ovariectomy unmasked greater persistence under higher-challenge. Together, our novel insights could help develop more effective and personalized treatments for problematic alcohol use.

Binge ethanol-drinking potentiates corticotropin releasing factor R1 receptor activity in the ventral tegmental area.

BACKGROUND: Corticotropin releasing factor (CRF) and urocortin play an important role in many stress responses and also can regulate ethanol (EtOH) intake. Adaptations in CRF signaling in the central amygdala promote EtOH consumption after long-term EtOH intake in dependent animals and also after brief periods of binge EtOH intake. Thus, even brief episodes of EtOH consumption can alter the function of the CRF system, allowing CRF to regulate EtOH intake. Here, we examined whether brief binge EtOH consumption leads to CRF receptor adaptations within the ventral tegmental area (VTA), a structure involved in signaling rewarding and aversive events and important in the development and expression of drug and alcohol addiction. METHODS: We utilized a mouse model of binge drinking known as drinking in the dark (DID), where C57BL/6J mice drink approximately 6 g/kg in 4 hours and achieve blood EtOH concentrations of approximately 100 mg/dl, which is equivalent to binge drinking in humans. We used ex vivo whole-cell recordings from putative VTA dopamine (DA) neurons to examine CRF regulation of NMDA receptor (NMDAR) currents. We also examined the impact of CRF receptor antagonist injection in the VTA on binge EtOH intake. RESULTS: Ex vivo whole-cell recordings from putative VTA DA neurons showed enhanced CRF-mediated potentiation of NMDAR currents in juvenile mice that consumed EtOH in the DID procedure. CRF-induced potentiation of NMDAR currents in EtOH-drinking mice was blocked by administration of CP-154,526 (3 µM), a selective CRF1 receptor antagonist. Furthermore, intra-VTA infusion of CP-154,526 (1 µg) significantly reduced binge EtOH consumption in adult mice. These results were not due to alterations of VTA NMDAR number or function, suggesting that binge drinking may enhance signaling through VTA CRF1 receptors onto NMDARs. CONCLUSIONS: Altered CRF1 receptor-mediated signaling in the VTA promotes binge-like EtOH consumption in mice, which supports the idea that CRF1 receptors may therefore be a promising pharmacological target for reducing binge drinking in humans.

Engagement for Alcohol Escalates in the 5-Choice Serial Reaction Time Task After Intermittent Access.

Uncontrollable binge drinking is becoming an increasingly prevalent issue in our society. This is a factor that plays a role in the development of alcohol use disorder (AUD). AUD impacts 15 million Americans annually, with approximately 88,000 dying from alcohol related deaths. There are several aspects of AUD that encourage a strong dependence on alcohol. Impulsivity, motivation, and attention are the primary behavioral facets we contribute to AUD. Many past studies have used the 5-Choice Serial Reaction Time Task (5-Choice) to analyze these types of behaviors using sugar as the reward. We have recently published a study where alcohol was used as a reward in the 5-Choice. 48 mice were trained to respond for alcohol in the 5-Choice, and the analyses for these animals were originally categorized by their alcohol preference and consumption. Upon looking at the data, we became more interested in a new way to classify these mice into groups. High engaged (HE) and low engaged (LE) mice were classified based on their number of correct responses in the last five late-stage sessions. During early-stage training, mice began to separate themselves into two groups based on their interaction with the task. The high-engaged (HE) mice were much more engaged with the task by having a high number of trials and correct responses, as well as a much lower percentage of omissions. The low engaged (LE) mice were not as engaged, this was apparent because of their lower number of trials and correct responses. They also had a much higher percentage of omissions in comparison to HE mice. LE mice presented no significant changes in late-stage training, while HE mice began responding and engaging more. These mice went through a period of intermittent access (IA), where they were allowed to drink alcohol in their cage for 3 weeks. After intermittent access, LE mice increased their responding which suggests an increase in motivation for alcohol as a reward. Engagement analysis presents two clearly different groups, one being motivated to work for alcohol and the other not wanting to work for this reward. These two distinct phenotypes in the 5-Choice could be used to model alcohol motivated behavior, which could help us further understand AUD.

Animal models of compulsion alcohol drinking: Why we love quinine-resistant intake and what we learned from it.

Alcohol Use Disorder (AUD) ranks among the most prevalent mental disorders, extracting ~$250 billion/year in the US alone and producing myriad medical and social harms. Also, the number of deaths related to problem drinking has been increasing dramatically. Compulsive alcohol drinking, characterized by intake that persists despite negative consequences, can be particularly important and a major obstacle to treatment. With the number of people suffering from AUD increasing during the past years, there is a critical need to understand the neurobiology related to compulsive drives for alcohol, as well as the development of novel AUD pharmacological therapies. Here we discuss rodent compulsion-like alcohol drinking (CLAD) models, focusing on the two most widely used adverse stimuli to model rodent compulsion-like responding, quinine adulteration of alcohol and footshook-resistant alcohol intake. For both cases, the goal is to uncover behavior patterns and brain circuits that underlie drive for alcohol even in the face of negative consequences. We discuss caveats, benefits, and potential brain mechanisms, of models for consequence-resistant responding for alcohol more generally, and especially highlight some advantages of quinine-resistance over footshook-resistance. Further, since this review contributes to a Special issue focused on Molecular Aspects of Compulsive Drug Use, we discuss our new findings showing how the noradrenergic system is related to CLAD responding. In particular, we comment on the importance of α1 and ß adrenergic receptors (ARs) as potential targets for treating AUD.

The role of beta- and alpha-adrenergic receptors on alcohol drinking.

Alcohol Use Disorders (AUD) is characterized by compulsion-like alcohol drinking (CLAD), where intake despite negative consequences can be a major clinical obstacle. With few treatment options available for AUD, there is a significant need for novel therapies. The noradrenergic system is an important hub for regulating stress responses and maladaptive drives for alcohol. Studies have shown that drugs targeting α1 adrenenergic receptors (ARs) may represent a pharmacological treatment for pathological drinking. However, the involvement of ß ARs for treating human drinking has received scant investigation, and thus we sought to provide pre-clinical validation for possible AR utility for CLAD by analyzing whether ß AR antagonists propranolol (ß1/2), betaxolol (ß1), and ICI, 118,551 (ß2) impacted CLAD and alcohol-only drinking (AOD) in male Wistar rats. We found that the highest dose of propranolol tested systemically (10 mg/kg) reduced alcohol drinking, while 5 mg/kg propranolol reduced drinking with a trend to impact CLAD more than AOD, and with no effects of 2.5 mg/kg. Betaxolol (2.5 mg/kg) also decreased drinking, while ICI 118.551 had no effects. Also, while AR compounds might have utility for AUD, they can also lead to undesirable side effects. Here, a combination of ineffective doses of propranolol and prazosin reduced both CLAD and AOD. Finally, we investigated the effect of propranolol and betaxolol in two brain areas related to pathological drinking, the anterior insula (aINS) and medial prefrontal cortex (mPFC). Surprisingly, propranolol (1-10 µg) in aINS or mPFC did not affect CLAD or AOD. Together, our findings provide new pharmacological insights into noradrenergic regulation of alcohol consumption, which may inform AUD therapy.

Using lickometry to infer differential contributions of salience network regions during compulsion-like alcohol drinking.

Alcohol use disorder extracts substantial personal, social and clinical costs, and continued intake despite negative consequences (compulsion-like consumption) can contribute strongly. Here we discuss lickometry, a simple method where lick times are determined across a session, while analysis across many aspects of licking can offer important insights into underlying psychological and action strategies, including their brain mechanisms. We first describe studies implicating anterior insula (AIC) and dorsal medial prefrontal cortex (dMPF) in compulsion-like responding for alcohol, then review work suggesting that AIC/ventral frontal cortex versus dMPF regulate different aspects of behavior (oral control and overall response strategy, versus moment-to-moment action organization). We then detail our lickometer work comparing alcohol-only drinking (AOD) and compulsion-like drinking under moderate- or higher-challenge (ModChD or HiChD, using quinine-alcohol). Many studies have suggested utilization of one of two main strategies, with higher motivation indicated by more bouts, and greater palatability suggested by longer, faster bouts. Instead, ModChD shows decreased variability in many lick measures, which is unexpected but consistent with the suggested importance of automaticity for addiction. Also surprising is that HiChD retains several behavior changes seen with ModChD, reduced tongue variability and earlier bout start, even though intake is otherwise disrupted. Since AIC-related measures are retained under both moderate- and higher-challenge, we propose a novel hypothesis that AIC sustains overall commitment regardless of challenge level, while disordered licking during HiChD mirrors the effects of dMPF inhibition. Thus, while AIC provides overall drive despite challenge, the ability to act is ultimately determined within the dMPF.

Adaptation of the 5-choice serial reaction time task to measure engagement and motivation for alcohol in mice.

Alcohol use disorder (AUD) is related to excessive binge alcohol consumption, and there is considerable interest in associated factors that promote intake. AUD has many behavioral facets that enhance inflexibility toward alcohol consumption, including impulsivity, motivation, and attention. Thus, it is important to understand how these factors might promote responding for alcohol and can change after protracted alcohol intake. Previous studies have explored such behavioral factors using responding for sugar in the 5-Choice Serial Reaction Time Task (5-CSRTT), which allows careful separation of impulsivity, attention, and motivation. Importantly, our studies uniquely focus on using alcohol as the reward throughout training and testing sessions, which is critical for beginning to answer central questions relating to behavioral engagement for alcohol. Alcohol preference and consumption in male C57BL/6 mice were determined from the first 9 sessions of 2-h alcohol drinking which were interspersed among 5-CSRTT training. Interestingly, alcohol preference but not consumption level significantly predicted 5-CSRTT responding for alcohol. In contrast, responding for strawberry milk was not related to alcohol preference. Moreover, high-preference (HP) mice made more correct alcohol-directed responses than low-preference (LP) during the first half of each session and had more longer reward latencies in the second half, with no differences when performing for strawberry milk, suggesting that HP motivation for alcohol may reflect "front-loading." Mice were then exposed to an Intermittent Access to alcohol paradigm and retested in 5-CSRTT. While both HP and LP mice increased 5-CSRTT responding for alcohol, but not strawberry milk, LP performance rose to HP levels, with a greater change in correct and premature responding in LP versus HP. Overall, this study provides three significant findings: (1) alcohol was a suitable reward in the 5-CSRTT, allowing dissection of impulsivity, attention, and motivation in relation to alcohol drinking, (2) alcohol preference was a more sensitive indicator of mouse 5-CSRTT performance than consumption, and (3) intermittent alcohol drinking promoted behavioral engagement with alcohol, especially for individuals with less initial engagement.

Modeling Aversion Resistant Alcohol Intake in Indiana Alcohol-Preferring (P) Rats.

With the substantial social and medical burden of addiction, there is considerable interest in understanding risk factors that increase the development of addiction. A key feature of alcohol use disorder (AUD) is compulsive alcohol (EtOH) drinking, where EtOH drinking becomes "inflexible" after chronic intake, and animals, such as humans with AUD, continue drinking despite aversive consequences. Further, since there is a heritable component to AUD risk, some work has focused on genetically-selected, EtOH-preferring rodents, which could help uncover critical mechanisms driving pathological intake. In this regard, aversion-resistant drinking (ARD) takes >1 month to develop in outbred Wistar rats (and perhaps Sardinian-P EtOH-preferring rats). However, ARD has received limited study in Indiana P-rats, which were selected for high EtOH preference and exhibit factors that could parallel human AUD (including front-loading and impulsivity). Here, we show that P-rats rapidly developed compulsion-like responses for EtOH; 0.4 g/L quinine in EtOH significantly reduced female and male intake on the first day of exposure but had no effect after one week of EtOH drinking (15% EtOH, 24 h free-choice paradigm). Further, after 4−5 weeks of EtOH drinking, males but not females showed resistance to even higher quinine (0.5 g/L). Thus, P-rats rapidly developed ARD for EtOH, but only males developed even stronger ARD with further intake. Finally, rats strongly reduced intake of quinine-adulterated water after 1 or 5 weeks of EtOH drinking, suggesting no changes in basic quinine sensitivity. Thus, modeling ARD in P-rats may provide insight into mechanisms underlying genetic predispositions for compulsive drinking and lead to new treatments for AUDs.

Better living through understanding the insula: Why subregions can make all the difference.

Insula function is considered critical for many motivated behaviors, with proposed functions ranging from attention, behavioral control, emotional regulation, goal-directed and aversion-resistant responding. Further, the insula is implicated in many neuropsychiatric conditions including substance abuse. More recently, multiple insula subregions have been distinguished based on anatomy, connectivity, and functional contributions. Generally, posterior insula is thought to encode more somatosensory inputs, which integrate with limbic/emotional information in middle insula, that in turn integrate with cognitive processes in anterior insula. Together, these regions provide rapid interoceptive information about the current or predicted situation, facilitating autonomic recruitment and quick, flexible action. Here, we seek to create a robust foundation from which to understand potential subregion differences, and provide direction for future studies. We address subregion differences across humans and rodents, so that the latter's mechanistic interventions can best mesh with clinical relevance of human conditions. We first consider the insula's suggested roles in humans, then compare subregional studies, and finally describe rodent work. One primary goal is to encourage precision in describing insula subregions, since imprecision (e.g. including both posterior and anterior studies when describing insula work) does a disservice to a larger understanding of insula contributions. Additionally, we note that specific task details can greatly impact recruitment of various subregions, requiring care and nuance in design and interpretation of studies. Nonetheless, the central ethological importance of the insula makes continued research to uncover mechanistic, mood, and behavioral contributions of paramount importance and interest. This article is part of the special Issue on 'Neurocircuitry Modulating Drug and Alcohol Abuse'.

Sex differences in specific aspects of two animal tests of anxiety-like behavior.

RATIONALE: Anxiety, a negative state of high arousal and vigilance, is especially prevalent in women, making identification of underlying mechanisms critical for developing effective therapies. With the challenge of disentangling biological and social factors in humans, animal tests can provide valuable insights, although such tests, developed in males, have unclear validity for females. OBJECTIVE: To better understand patterns of sex differences across multiple measures within two classical rodent anxiety tests. METHODS: We examined female and male adult Wistar rats (n = 15-18/group) that were single-housed in the novelty suppression of feeding test (NSFT) that involves food under a bright light in food-restricted animals, and light-dark test (LDT), which reflects innate aversion to bright light. To further validate these tests in females, we also examined the impact of 1 mg/kg diazepam. RESULTS: NSFT measures of the most direct interaction with food, latency to grab food and food consumed, indicated increased anxiety-like behavior in females versus males, with diazepam altering these behaviors in females but not males. Most other measures showed more similar effects of diazepam across the sexes, with some evidence of reduced anxiety-like behavior in LDT for females. Principal component analyses indicated limited relationships across behavioral factors, underscoring previous suggestions of the importance of assessing multiple measures to maximize information and ethological relevance. CONCLUSIONS: Combining our findings and previous studies, we speculate that increased anxiety-like behavior in females manifests especially when there is a specific, life-relevant condition (e.g., food in the NSFT). Our findings also validate NSFT and LDT use in females.

The role of anterior insula-brainstem projections and alpha-1 noradrenergic receptors for compulsion-like and alcohol-only drinking.

Compulsion-like alcohol drinking (CLAD), where consumption continues despite negative consequences, is a major obstacle to treating alcohol use disorder. The locus coeruleus area in the brainstem and norepinephrine receptor (NER) signaling in forebrain cortical regions have been implicated in adaptive responding under stress, which is conceptually similar to compulsion-like responding (adaptive responding despite the presence of stress or conflict). Thus, we examined whether anterior insula (aINS)-to-brainstem connections and alpha-1 NERs regulated compulsion-like intake and alcohol-only drinking (AOD). Halorhodopsin inhibition of aINS-brainstem significantly reduced CLAD, with no effect on alcohol-only or saccharin intake, suggesting a specific aINS-brainstem role in aversion-resistant drinking. In contrast, prazosin inhibition of alpha-1 NERs systemically reduced both CLAD and AOD. Similar to systemic inhibition, intra-aINS alpha-1-NER antagonism reduced both CLAD and AOD. Global aINS inhibition with GABAR agonists also strongly reduced both CLAD and AOD, without impacting saccharin intake or locomotion, while aINS inhibition of calcium-permeable AMPARs (with NASPM) reduced CLAD without impacting AOD. Finally, prazosin inhibition of CLAD and AOD was not correlated with each other, systemically or within aINS, suggesting the possibility that different aINS pathways regulate CLAD versus AOD, which will require further study to definitively address. Together, our results provide important new information showing that some aINS pathways (aINS-brainstem and NASPM-sensitive) specifically regulate compulsion-like alcohol consumption, while aINS more generally may contain parallel pathways promoting CLAD versus AOD. These findings also support the importance of the adaptive stress response system for multiple forms of alcohol drinking.

Differential importance of nucleus accumbens Ox1Rs and AMPARs for female and male mouse binge alcohol drinking.

Alcohol use disorder exhausts substantial social and economic costs, with recent dramatic increases in female problem drinking. Thus, it is critically important to understand signaling differences underlying alcohol consumption across the sexes. Orexin-1 receptors (Ox1Rs) can strongly promote motivated behavior, and we previously identified Ox1Rs within nucleus accumbens shell (shell) as crucial for driving binge intake in higher-drinking male mice. Here, shell Ox1R inhibition did not alter female mouse alcohol drinking, unlike in males. Also, lower dose systemic Ox1R inhibition reduced compulsion-like alcohol intake in both sexes, indicating that female Ox1Rs can drive some aspects of pathological consumption, and higher doses of systemic Ox1R inhibition (which might have more off-target effects) reduced binge drinking in both sexes. In contrast to shell Ox1Rs, inhibiting shell calcium-permeable AMPA receptors (CP-AMPARs) strongly reduced alcohol drinking in both sexes, which was specific to alcohol since this did not reduce saccharin intake in either sex. Our results together suggest that the shell critically regulates binge drinking in both sexes, with shell CP-AMPARs supporting intake in both sexes, while shell Ox1Rs drove drinking only in males. Our findings provide important new information about sex-specific and -general mechanisms that promote binge alcohol intake and possible targeted therapeutic interventions.