Understanding sex differences and the translational value of models of persistent substance use despite negative consequences.

Persistent substance use despite negative consequences is a key facet of substance use disorder. The last decade has seen the preclinical field adopt the use of punishment to model adverse consequences associated with substance use. This has largely involved the pairing of drug use with either electric foot shock or quinine, a bitter tastant. Whilst at face value, these punishers may model aspects of the physical and psychological consequences of substance use, such models are yet to assist the development of approved medications for treatment. This review discusses progress made with animal models of punishment to understand the behavioral consequences of persistent substance use despite negative consequences. We highlight the importance of examining sex differences, especially when the behavioral response to punishment changes following drug exposure. Finally, we critique the translational value these models provide for the substance use disorder field.

A balancing act: the role of pro- and anti-stress peptides within the central amygdala in anxiety and alcohol use disorders.

The central nucleus of the amygdala (CeA) is widely implicated as a structure that integrates both appetitive and aversive stimuli. While intrinsic CeA microcircuits primarily consist of GABAergic neurons that regulate amygdala output, a notable feature of the CeA is the heterogeneity of neuropeptides and neuropeptide/neuromodulator receptors that it expresses. There is growing interest in the role of the CeA in mediating psychopathologies, including stress and anxiety states and their interactions with alcohol use disorders. Within the CeA, neuropeptides and neuromodulators often exert pro- or anti- stress actions, which can influence anxiety and alcohol associated behaviours. In turn, alcohol use can cause adaptions within the CeA, which may render an individual more vulnerable to stress which is a major trigger of relapse to alcohol seeking. This review examines the neurocircuitry, neurochemical phenotypes and how pro- and anti-stress peptide systems act within the CeA to regulate anxiety and alcohol seeking, focusing on preclinical observations from animal models. Furthermore, literature exploring the targeting of genetically defined populations or neuronal ensembles and the role of the CeA in mediating sex differences in stress x alcohol interactions are explored.

Anterior Insular Cortex is Critical for the Propensity to Relapse Following Punishment-Imposed Abstinence of Alcohol Seeking.

Humans with alcohol use disorder typically abstain because of the negative consequences associated with excessive drinking, and exposure to contexts previously associated with alcohol use can trigger relapse. We used a rat model that captures a characteristic of this human condition: namely voluntary abstinence from alcohol use because of contingent punishment. There is substantial variability in the propensity to relapse following extended periods of abstinence, and this is a critical feature preventing the successful treatment of alcohol use disorder. Here we examined relapse following acute or prolonged abstinence. In male alcohol preferring P rats, we found an increased propensity to relapse in Context B, the punishment context after prolonged abstinence. Next, we found that neither alcohol intake history nor the motivational strength of alcohol predicted the propensity to relapse. We next examined the putative circuitry of context-induced relapse to alcohol seeking following prolonged abstinence using Fos as a marker of neuronal activation. The anterior insular cortex (AI) was the only brain region examined where Fos expression correlated with alcohol seeking behavior in Context B after prolonged abstinence. Finally, we used local infusion of GABAA and GABAB receptor agonists (muscimol + baclofen) to show a causal role of the AI in context-induced relapse in Context B, the punishment context after prolonged abstinence. Our results show that there is substantial individual variability in the propensity to relapse in the punishment-associated context after prolonged abstinence, and this is mediated by activity in the AI.SIGNIFICANCE STATEMENT A key feature of alcohol use disorder is that sufferers show an enduring propensity to relapse throughout their lifetime. Relapse typically occurs despite the knowledge of adverse consequences including health complications or relationship breakdowns. Here we use a recently developed rodent model that recapitulates this behavior. After an extended period of abstinence, relapse propensity is markedly increased in the "adverse consequence" environment, akin to humans with alcohol use disorder relapsing in the face of adversity. From a circuitry perspective, we demonstrate a causal role of the anterior insular cortex in relapse to alcohol seeking after extended abstinence following punishment imposed voluntary cessation of alcohol use.

Pattern of neural activation following yohimbine-induced reinstatement of alcohol seeking in rats.

Alcohol use disorders represent an extensive socioeconomic burden, yet effective treatment options are suboptimal. A major hurdle in treating alcohol use disorders is the high rate of relapse. Stress is a major factor that promotes relapse in abstinent drug users; therefore, understanding neural mechanisms that underpin the effects of stress on alcohol seeking is critical. In rodent models of stress-induced relapse, the α2 -adrenoceptor antagonist, yohimbine, is a widely used chemical stressor to elicit reinstatement of drug/alcohol seeking. However, the exact mechanism how yohimbine precipitates reinstatement of alcohol seeking and the pattern of neural activation associated with yohimbine-induced reinstatement is poorly understood. Therefore, we counted Fos-protein positive nuclei across 42 brain regions in alcohol-experienced alcohol preferring rats that received either yohimbine in the home-cage (1 mg/kg i.p.) or following yohimbine-induced reinstatement of alcohol seeking. The number of Fos-protein positive nuclei was increased in the prefrontal cortex and extended amygdala after home-cage yohimbine compared to naïve- and vehicle-treated rats. Yohimbine-induced reinstatement increased the number of Fos-protein expressing nuclei in multiple other regions including the thalamus, hypothalamus and hippocampus. We then examined inter-regional correlations in Fos-protein expression for all 42 brain regions, which showed Fos expression was more strongly positively correlated following yohimbine-induced reinstatement of alcohol seeking, compared to home-cage yohimbine. These data suggest low-dose yohimbine in a non-drug-associated context activates stress/impulsivity centres within the brain, whereas yohimbine in the drug-associated context recruits additional brain regions to drive alcohol seeking.

Nucleus incertus corticotrophin-releasing factor 1 receptor signalling regulates alcohol seeking in rats.

Alcoholism is a chronic relapsing disorder, and stress is a key precipitant of relapse. The nucleus incertus (NI) is highly responsive to corticotrophin-releasing factor (CRF) and psychological stressors, receives a CRF innervation and expresses CRF1 and CRF2 receptor mRNA. Furthermore, the ascending NI relaxin-3 system is implicated in alcohol seeking in rats. Therefore, in alcohol-preferring rats, we examined the effect of bilateral injections into the NI of the CRF1 receptor antagonist, CP376395 or the CRF2 receptor antagonist, astressin-2B on yohimbine-induced reinstatement of alcohol seeking. Using quantitative PCR analysis of NI micropunches, we assessed the effects of chronic alcohol consumption on gene expression profiles for components of the relaxin-3 and CRF systems. Bilateral intra-NI injections of CP376395 (500 ng/0.25 µl) attenuated yohimbine-induced reinstatement of alcohol seeking. In contrast, intra-NI injections of astressin-2B (200 ng/0.25 µl) had no significant effect. In line with these data, CRF1 , but not CRF2 , receptor mRNA was upregulated in the NI following chronic ethanol intake. Relaxin family peptide 3 receptor mRNA was also increased in the NI following chronic ethanol. Our quantitative PCR analysis also identified CRF mRNA within the rat NI, and the existence of a newly identified population of CRF-containing neurons was subsequently confirmed by detection of CRF immunoreactivity in rat and mouse NI. These data suggest that NI neurons contribute to reinstatement of alcohol seeking, via an involvement of CRF1 signalling. Furthermore, chronic ethanol intake leads to neuroadaptive changes in CRF and relaxin-3 systems within rat NI.

Orphan peptide and G protein-coupled receptor signalling in alcohol use disorder.

Neuropeptides and G protein-coupled receptors (GPCRs) have long been, and continue to be, one of the most popular target classes for drug discovery in CNS disorders, including alcohol use disorder (AUD). Yet, orphaned neuropeptide systems and receptors (oGPCR), which have no known cognate receptor or ligand, remain understudied in drug discovery and development. Orphan neuropeptides and oGPCRs are abundantly expressed within the brain and represent an unprecedented opportunity to address brain function and may hold potential as novel treatments for disease. Here, we describe the current literature regarding orphaned neuropeptides and oGPCRs implicated in AUD. Specifically, in this review, we focus on the orphaned neuropeptide cocaine- and amphetamine-regulated transcript (CART), and several oGPCRs that have been directly implicated in AUD (GPR6, GPR26, GPR88, GPR139, GPR158) and discuss their potential and pitfalls as novel treatments, and progress in identifying their cognate receptors or ligands.

Emerging GPCR targets for AUD: Insights from preclinical studies.

G protein-coupled receptors (GPCRs) are the largest group of membrane receptors in the central nervous system and one of the key proteins for signal transduction between cells. Currently, many drugs available on the market act via GPCRs and these receptors remain attractive targets for the treatment of brain disorders, including alcohol use disorder (AUD). Here, we describe the most recent literature, with a primary focus on the past 5 years, on GPCR targets with the potential for reducing behaviours associated with excessive alcohol intake. Specifically, we focus on preclinical evidence of compounds with attractive pharmacological profiles and potential for future clinical investigation for the treatment of AUD.

Cocaine and amphetamine regulated transcript (CART) mediates sex differences in binge drinking through central taste circuits.

The neuropeptide cocaine- and amphetamine-regulated transcript (CART) has been implicated in alcohol consumption and reward behaviours, yet mechanisms mediating these effects have yet to be identified. Using a transgenic CART knockout (KO) mouse line we uncovered a sexually dimorphic effect of CART in binge drinking, with male CART KO mice increasing intake, whilst female CART KO mice decreased their alcohol intake compared to controls. Female CART KO mice show greater sensitivity to bitter solutions that can be overshadowed through addition of a sweetener, implicating taste as a factor. Further we identify that this is not driven through peripherally circulating sex hormones, but the central nucleus of the amygdala (CeA) is a locus where CART contributes to the regulation of alcohol consumption, with CeA CART neutralisation specifically reducing plain alcohol, but not sweetened alcohol consumption in female mice. These findings may have implications for the development of sex-specific treatment options for alcohol use disorders through targeting the CART system.

Sex Differences in Alcohol Use: Is It All About Hormones?

Risky alcohol use and alcohol use disorders (AUD) are a rising problem in women, yet a major disparity in our understanding of what drives alcohol consumption in women remains. Historically biomedical research has focused on male subjects; however, recent increases in reporting of females, have highlighted major differences between the sexes. Here we review the current literature of the effect of gonadal steroid hormones (estrogens, androgens, and progestins), neurosteriods, and neurobiological factors on alcohol use in clinical and preclinical studies of both sexes. Further, we briefly discuss how fundamental sex differences in genetics, metabolism, neuroimmune, and stress responses may influence sex differences in alcohol intake. Comparing the sexes could aid in the discovery of novel therapeutics to treat AUD, and implementation of current treatment options in women.

Neuroanatomical distribution of fluorophores within adult RXFP3 Cre-tdTomato/YFP mouse brain.

Relaxin-family peptide 3 receptor (RXFP3) is activated by relaxin-3 in the brain to influence arousal and related functions, such as feeding and stress responses. Two transgenic mouse lines have recently been developed that co-express different fluorophores within RXFP3-expressing neurons: either yellow fluorescent protein (YFP; RXFP3-Cre/YFP mice) or tdTomato (RXFP3-Cre/tdTomato mice). To date, the characteristics of neurons that express RXFP3-associated fluorophores in these mice have only been investigated in the bed nucleus of the stria terminalis and the hypothalamic arcuate nucleus. To better determine the utility of these fluorophore-expressing mice for further research, we characterised the neuroanatomical distribution of fluorophores throughout the brain of these mice and compared this to the published distribution of Rxfp3 mRNA (detected by in situ hybridisation) in wildtype mice. Coronal sections of RXFP3-Cre/YFP (n = 8) and RXFP3-Cre/tdTomato (n = 8) mouse brains were imaged, and the density of fluorophore-expressing cells within various brain regions/nuclei was qualitatively assessed. Comparisons with our previously reported RXFP3 mRNA distribution revealed that of 212 brain regions that contained either fluorophore or RXFP3 mRNA, approximately half recorded densities that were within two qualitative measurements of each other (on a 9-point scale), including hippocampal dentate gyrus and amygdala subregions. However, many brain areas with likely non-authentic, false-positive, or false-negative fluorophore expression were also detected, including the cerebellum. Therefore, this study provides a guide to which brain regions should be prioritized for future study of RXFP3 in these mice, to better understand the neuroanatomy and function of this intriguing, neuronal peptide receptor.

Targeting muscarinic receptors for the treatment of alcohol use disorders: Opportunities and hurdles for clinical development.

Emerging evidence suggests muscarinic acetylcholine receptors represent novel targets to treat alcohol use disorder. In this review, we draw from literature across medicinal chemistry, molecular biology, addiction and learning/cognition fields to interrogate the proposition for muscarinic receptor ligands in treating various aspects of alcohol use disorder, including cognitive dysfunction, motivation to consume alcohol and relapse. In support of this proposition, we describe cholinergic dysfunction in the pathophysiology of alcohol use disorder at a network level, including alcohol-induced adaptations present in both human post-mortem brains and reverse-translated rodent models. Preclinical behavioural pharmacology implicates specific muscarinic receptors, in particular, M4 and M5 receptors, as potential therapeutic targets worthy of further interrogation. We detail how these receptors can be selectively targeted in vivo by the use of subtype-selective allosteric modulators, a strategy that overcomes the issue of targeting a highly conserved orthosteric site bound by acetylcholine. Finally, we highlight the intense pharma interest in allosteric modulators of muscarinic receptors for other indications that provide an opportunity for repurposing into the alcohol use disorder space and provide some currently unanswered questions as a roadmap for future investigation.

A paraventricular thalamus to insular cortex glutamatergic projection gates "emotional" stress-induced binge eating in females.

It is well-established that stress and negative affect trigger eating disorder symptoms and that the brains of men and women respond to stress in different ways. Indeed, women suffer disproportionately from emotional or stress-related eating, as well as associated eating disorders such as binge eating disorder. Nevertheless, our understanding of the precise neural circuits driving this maladaptive eating behavior, particularly in women, remains limited. We recently established a clinically relevant model of 'emotional' stress-induced binge eating whereby only female mice display binge eating in response to an acute "emotional" stressor. Here, we combined neuroanatomic, transgenic, immunohistochemical and pathway-specific chemogenetic approaches to investigate whole brain functional architecture associated with stress-induced binge eating in females, focusing on the role of Vglut2 projections from the paraventricular thalamus (PVTVglut2+) to the medial insular cortex in this behavior. Whole brain activation mapping and hierarchical clustering of Euclidean distances revealed distinct patterns of coactivation unique to stress-induced binge eating. At a pathway-specific level, PVTVglut2+ cells projecting to the medial insular cortex were specifically activated in response to stress-induced binge eating. Subsequent chemogenetic inhibition of this pathway suppressed stress-induced binge eating. We have identified a distinct PVTVglut2+ to insular cortex projection as a key driver of "emotional" stress-induced binge eating in female mice, highlighting a novel circuit underpinning this sex-specific behavior.

How the brain regulates alcohol intake.

A neural pathway involved in goal-oriented behaviours becomes dysregulated during binge drinking and alcohol use disorder.

Clinical Effectiveness of Muscarinic Receptor-Targeted Interventions in Neuropsychiatric Disorders: A Systematic Review.

BACKGROUND: For decades, treatment of mood disorders, psychoses, anxiety and dementia have been confounded by limited efficacy and high rates of treatment resistance. Preclinical and clinical evidence have highlighted disruption of cholinergic signalling in several neuropsychiatric conditions and examined intervention strategies including acetylcholinesterase inhibitors and nicotinic receptor-targeted intervention. However, the effectiveness of these approaches is often curtailed by on-target side effects. Post mortem studies implicate muscarinic receptor dysregulation in neuropsychiatric pathophysiology; therefore, we conducted a systematic review and meta-analysis to investigate the therapeutic efficacy and safety of muscarinic receptor-targeted interventions in adults with neuropsychiatric disorders. METHODS: PubMed, EMBASE, PsycINFO, EBSCO and Web of Science were searched using relevant keywords from database inception to 7 August 2022. Randomised, double-blind, placebo-controlled studies were included if they investigated the effect of muscarinic receptor-targeted intervention in adults with a diagnosis of a neuropsychiatric disorder and were published in English. A narrative synthesis approach was adopted to describe the findings. Wherever three or more studies with a similar intervention were available, effect sizes were calculated, and a meta-analysis was performed. Cochrane risk-of-bias-2 tool was utilised to assess the risk of bias, and sensitivity analyses were performed to identify publication bias. Certainty analysis (high, moderate, low and/or very low) was conducted using GRADE criteria. RESULTS: Overall, 33 studies met the inclusion criteria and 5 were included in the meta-analysis. Despite a limited pool with several different interventions, we found therapeutic efficacy of xanomeline (M1/M4 agonist) in primary psychotic disorders plus behavioural and psychological symptoms of dementia. Scopolamine showed a significant antidepressant effect in a combined cohort of major depressive and bipolar disorders in the short-term outcome measure, but no effect following cessation of treatment. Results from bias assessments suggest "very low" certainty in the antidepressant effect of scopolamine. Critical limitations of the current literature included low power, high heterogeneity in the patient population and a lack of active comparators. CONCLUSION: While the results are not definitive, findings on muscarinic receptor-targeted interventions in several mental disorders are promising in terms of efficacy and safety, specifically in treating schizophrenia, mood disorders, and behavioural and psychiatric symptoms of Alzheimer's disease. However, orthosteric muscarinic receptor-targeted interventions are associated with a range of peripheral adverse effects that are thought to be mediated via M2/M3 receptors. The orthosteric binding site of muscarinic acetylcholine receptors is remarkably conserved, posing a challenge for subtype-selective interventions; nonetheless allosteric ligands with biased signalling pathways are now in development. We conclude that adequately powered prospective studies with subtype-selective interventions are required to determine the clinical effectiveness of muscarinic-receptor targeted interventions for the treatment of neuropsychiatric disorders.

Organisation of enkephalin inputs and outputs of the central nucleus of the amygdala in mice.

The central nucleus of the amygdala (CeA) is a key hub integrating sensory inputs and modulating behavioural outputs. The CeA is a complex structure with discrete subdivisions, high peptidergic heterogeneity and broad CNS afferent and efferent projections. While several neuropeptide systems within the CeA have been examined in detail, less is known about CeA preproenkephalin (ppENK) cells. Here, we used a recently developed transgenic Penk-Cre mouse line to advance our understanding of the efferent and afferent connectivity of ppENK in the CeA. First, to determine the fidelity of Cre expression in Penk-Cre transgenic mice, we conducted RNAscope in the CeA of Penk-Cre mice. Our analysis revealed that 96.6 % of CeA Cre+ neurons co-expressed pENK mRNA, and 99.7 % of CeA pENK+ neurons co-expressed Cre mRNA, indicating faithful recapitulation of Cre expression in CeA ppENK-expressing cells, supporting the fidelity of the Penk-Cre reporter mouse. Anterograde tracing of CeAPenk cells showed strong efferent projections to the extended amygdala, midbrain and hindbrain PBN and NTS. Retrograde tracing of Penk afferents to the CeA were more restricted, with primary innervation originating within the amygdala complex and bed nucleus of the stria terminalis, and minor innervation from the parabrachial nucleus and nucleus of the solitary tract. Together, our data provide a comprehensive map of ENKergic efferent and afferent connectivity of the CeA in Penk-Cre mice. Further, we highlight both the utility and limitations of the Penk-Cre mice to study the function of CeA, PBN and NTS ppENK cells.

M1 muscarinic receptor activation decreases alcohol consumption via a reduction in consummatory behavior.

Muscarinic acetylcholine receptors (mAChRs) have been shown to mediate alcohol consumption and seeking. Both M4 and M5 mAChRs have been highlighted as potential novel treatment targets for alcohol use disorders (AUD). Similarly, M1 mAChRs are expressed throughout reward circuitry, and their signaling has been implicated in cocaine consumption. However, whether the same effects are seen for alcohol consumption, or whether natural reward intake is inadvertently impacted is still unknown. To determine the role of M1 mAChRs in alcohol consumption, we tested operant self-administration of alcohol under both fixed ratio (FR3) and progressive ratio (PR3-4) schedules. Enhancing M1 mAChR signaling (via the M1 PAM-Agonist PF-06767832, 1 mg/kg, i.p.) reduced operant alcohol consumption on a fixed schedule but had no effect on motivation to acquire alcohol. To determine whether these actions were specific to alcohol, we examined the effects of M1 enhancement on natural reward (sucrose) self-administration. Systemic administration of PF-06767832 (1 mg/kg, i.p.) also reduced operant sucrose self-administration, suggesting the actions of the M1 receptor may be non-selective across drug and natural rewards. Finally, to understand whether this reduction extended to natural consummatory behaviors, we assessed home cage standard chow and water consumption. M1 enhancement via systemic PF-06767832 administration reduced food and water consumption. Together our results suggest the M1 PAM-agonist, PF-06767832, non-specifically reduces consummatory behaviors that are not associated with motivational strength for the reward. These data highlight the need to further characterize M1 agonists, PAMs, and PAM-agonists, which may have varying degrees of utility in the treatment of neuropsychiatric disorders including AUD.

Can we enhance the clinical efficacy of cognitive and psychological approaches to treat substance use disorders through understanding their neurobiological mechanisms?

Despite decades of research in the field of addiction, relapse rates for substance use disorders remain high. Consequently, there has been growing focus on providing evidence-based treatments for substance use disorders, resulting in the increased development and use of cognitive and psychological interventions. Such treatment approaches, including contingency management, community-reinforcement approach, and cognitive bias modification, have shown promising clinical efficacy in reducing substance use and promoting abstinence during treatment. However, these interventions are still somewhat limited in achieving sustained periods of abstinence post-treatment. The neurobiological mechanisms underpinning these treatment approaches remain largely unknown and under-studied, in part, due to a lack of translational animal models. The adoption of a reverse translational approach may assist in development of more representative models that can facilitate elucidation of the mechanisms behind these clinically relevant interventions. This review examines our current understanding of addiction neurobiology from clinical, preclinical research and existing animal models, and considers how the efficacy of such behavioral-oriented interventions alone, or in combination with pharmacotherapy, may be enhanced to improve treatment outcomes.

Gray areas: Neuropeptide circuits linking the Edinger-Westphal and Dorsal Raphe nuclei in addiction.

The circuitry of addiction comprises several neural networks including the midbrain - an expansive region critically involved in the control of motivated behaviors. Midbrain nuclei like the Edinger-Westphal (EW) and dorsal raphe (DR) contain unique populations of neurons that synthesize many understudied neuroactive molecules and are encircled by the periaqueductal gray (PAG). Despite the proximity of these special neuron classes to the ventral midbrain complex and surrounding PAG, functions of the EW and DR remain substantially underinvestigated by comparison. Spanning approximately -3.0 to -5.2 mm posterior from bregma in the mouse, these various cell groups form a continuum of neurons that we refer to collectively as the subaqueductal paramedian zone. Defining how these pathways modulate affective behavioral states presents a difficult, yet conquerable challenge for today's technological advances in neuroscience. In this review, we cover the known contributions of different neuronal subtypes of the subaqueductal paramedian zone. We catalogue these cell types based on their spatial, molecular, connectivity, and functional properties and integrate this information with the existing data on the EW and DR in addiction. We next discuss evidence that links the EW and DR anatomically and functionally, highlighting the potential contributions of an EW-DR circuit to addiction-related behaviors. Overall, we aim to derive an integrated framework that emphasizes the contributions of EW and DR nuclei to addictive states and describes how these cell groups function in individuals suffering from substance use disorders. This article is part of the special Issue on 'Neurocircuitry Modulating Drug and Alcohol Abuse'.

Cocaine and amphetamine regulated transcript (CART) signalling in the central nucleus of the amygdala modulates stress-induced alcohol seeking.

The central nucleus of the amygdala (CeA) is a key hub of the neural circuitry regulating alcohol and stress interactions. However, the exact neuronal populations that govern this interaction are not well defined. Here we examined the role of the neuropeptide cocaine and amphetamine regulated transcript (CART) within the CeA in stress-induced alcohol seeking. We found that CART-containing neurons are predominantly expressed in the capsular/lateral division of the CeA and are a subpopulation of protein kinase Cδ (PKCδ) cells, distinct from corticotrophin releasing factor (CRF)-expressing cells. Both stress (yohimbine) and stress-induced alcohol seeking activated CART cells within the CeA, while neutralisation of endogenous CeA CART signalling (via antibody administration) attenuated stress-induced alcohol, but not sucrose seeking. Further, blocking CART signalling within the CeA did not alter the motivation to obtain and consume alcohol but did attenuate stressor-induced anxiety-like behaviour during abstinence from alcohol. Together, these data identify CeA CART cells as a subpopulation of PKCδ cells that influence stress × alcohol interactions and mediate stress-induced alcohol seeking behaviours.