Evaluation of Alcohol Preference and Drinking in msP Rats Bearing a Crhr1 Promoter Polymorphism

Alcoholism is a pervasive societal problem, yet available pharmacotherapies fail to treat most sufferers. The type 1 corticotropin-releasing factor (CRF1) receptor has received much attention for its putative role in the progression to alcohol dependence, although at present its success in clinical trials has been limited. Two single-nucleotide polymorphisms in the rat Crhr1 promoter have been identified in the Marchigian substrain of Sardinian alcohol-preferring (msP) rats. Unlike other Wistar-derived alcohol-preferring lines, nondependent msP rats reduce their alcohol self-administration in response to CRF1 antagonists and show increased brain CRF1 expression. The current study tested the hypotheses that the A alleles in the Crhr1 promoter polymorphisms are: (1) unique to msP (vs. CRF1 antagonist-insensitive) alcohol-preferring lines and (2) associate with greater alcohol preference or intake. Two related polymorphisms were observed in which both loci on a given chromosome were either mutant variant (A) or wild-type (G) alleles within the distal Crhr1 promoter of 17/25 msP rats (68%), as compared to 0/23 Indiana P rats, 0/20 Sardinian alcohol-preferring rats bred at Scripps (Scr:sP) and 0/21 outbred Wistar rats. Alcohol consumption in msP rats did not differ according to the presence of Crhr1 A alleles, but greater alcohol preference (98%) was observed in A allele homozygous msP rats (AA) compared to msP rats with wild-type (GG, 91%) or heterozygous (GA, 91%) genotypes. The greater alcohol preference reflected decreased water intake, accompanied by reduced total calories consumed by AA rats. The data show that msP rats differentially possess mutant A variant alleles in the polymorphic promoter region of the Crhr1 gene that may differentially regulate consumption

Molecular tools to elucidate factors regulating alcohol use

Alcohol use disorders (AUD) are pervasive societal problems, marked by high levels of alcohol intake and recidivism. Despite these common disease traits, individuals diagnosed with AUD display a range of disordered drinking and alcohol-related behaviors. The diversity in disease presentation, as well as the established polygenic nature of the disorder and complex neurocircuitry, speak to the variety in neurochemical changes resulting from alcohol intake that may differentially regulate alcohol-related behaviors. Investigations into the molecular adaptations responsible for maladaptive alcohol-related behavioral outcomes require an ever-evolving set of molecular tools to elucidate with increasing precision how alcohol alters behavior through neurochemical changes. This review highlights recent advances in molecular methodology, addressing how incorporation of these cutting-edge techniques not only may enhance current knowledge of the molecular bases of AUD, but also may facilitate identification of improved treatment targets that may be therapeutic in specific subpopulations of AUD individuals

Sex differences in responses of the basolateral-central amygdala circuit to alcohol, corticosterone and their interaction

Alcohol use disorders are chronically relapsing conditions that pose significant health challenges for our society. Stress is a prevalent trigger of relapse, particularly for women, yet the mechanisms by which alcohol and stress interact, and how this differs between males and females, remain poorly understood. The glutamatergic circuit connecting the basolateral (BLA) and central (CeA) nuclei of the amygdala is a likely locus for such adaptations, yet the impact of alcohol, corticosterone and their interaction on this circuit has been understudied. In particular, no studies have addressed sex differences in these effects or potential differential responses between the lateral and medial subdivisions of the central nucleus. Thus, we assessed the effects of alcohol and corticosterone treatments on BLA-evoked compound glutamatergic responses in medial and lateral CeA neurons from male and female rats. We observed minimal differences between medial and lateral CeA responses to alcohol and corticosterone in male rats, which were primarily sensitive to alcohol-induced inhibition of glutamatergic postsynaptic potentials. Unlike male neurons, cells from female rats displayed reduced sensitivity to alcohol’s inhibitory effects. In addition, female neurons diverged in their sensitivity to corticosterone, with lateral CeA neuronal responses significantly blunted following corticosterone treatment and medial CeA neurons largely unchanged by corticosterone or subsequent co-application of alchol. Together these data highlight striking differences in how male and female amygdala respond to alcohol and the stress hormone corticosterone, factors which may impact differential susceptibility of the sexes to alcohol- and stress-related disorders

Sexual dimorphism in the neural impact of stress and alcohol

Alcohol use disorder is a widespread mental illness characterized by periods of abstinence followed by recidivism, and stress is the primary trigger of relapse. Despite the higher prevalence of alcohol use disorder in males, the relationship between stress and behavioral features of relapse, such as craving, is stronger in females. Given the greater susceptibility of females to stress-related psychiatric disorders, understanding sexual dimorphism in the relationship between stress and alcohol use is essential to identifying better treatments for both male and female alcoholics. This review addresses sex differences in the impact of stressors on alcohol drinking and seeking in rodents and humans. As these behavioral differences in alcohol use and relapse originate from sexual dimorphism in neuronal function, the impact of stressors and alcohol, and their interaction, on molecular adaptations and neural activity in males and females will also be discussed. Together the data reviewed herein, arising from a symposium entitled “Sex matters in stress-alcohol interactions” presented at the Fourth Volterra Conference on Stress and Alcohol, will highlight the importance of identifying sex differences to improve treatments for comorbid stress and alcohol use disorder in both populations

Sex differences in the long-term effects of past stress on alcohol self-administration, glucocorticoid sensitivity and phosphodiesterase 10A expression

Stress responses differ by sex, and females are more susceptible to developing mental illnesses because of past stress, including alcohol use disorder. Investigation of neuroadaptations governing the interaction between past stress and future alcohol intake remains understudied in females. A history of footshock stress previously was shown to increase alcohol self-administration under relapse-like conditions in male rats, associated with elevated phosphodiesterase 10A (PDE10A) mRNA expression in the dorsomedial prefrontal cortex and basolateral amygdala. To identify sex differences in long-term stress effects, male and female Wistar rats were exposed to light-cued footshock stress prior to alcohol self-administration training. While past stress did not alter acquisition or extinction, reacquisition self-administration was oppositely impacted by past stress. Stress history slightly increased reacquisition self-administration in males, but reduced alcohol self-administration in females, relative to same-sex controls. Control females self-administered less alcohol following glucocorticoid receptor inhibition by mifepristone, which did not significantly alter alcohol consumption in the other groups. PDE10A expression in synaptically enriched fractions also differed by sex and stress history in a brain region-specific manner. Females expressed more synaptic PDE10A than males in basolateral amygdala and dorsolateral striatum, regardless of stress history, whereas dorsomedial prefrontal cortex PDE10A protein levels matched group differences in reacquisition drinking, but also were expressed at much lower levels than all other regions examined. Together, these data show stress history differentially impacts alcohol self-administration and PDE10A expression by sex, with control females consuming alcohol in a glucocorticoid receptor-sensitive fashion that may relate to sex differences in PDE10A expression

Emerging targets for addiction neuropharmacology: From mechanisms to therapeutics

Drug abuse represents a considerable burden of disease and has enormous economic impacts on societies. Over the years, few medications have been developed for clinical use. Their utilization is endowed with several limitations, including partial efficacy or significant side effects. On the other hand, the successful advancement of these compounds provides an important proof of concept for the feasibility of drug development programs in addiction. In recent years, a wealth of information has been generated on the psychological mechanisms, genetic or epigenetic predisposing factors, and neurobiological adaptations induced by drug consumption that interact with each other to contribute to disease progression. It is now clear that addiction develops through phases, from initial recreational use to excessive consumption and compulsive drug seeking, with a shift from positive to negative reinforcement driving motivated behaviors. A greater understanding of these mechanisms has opened new vistas in drug development programs. Researchers' attention has been shifted from investigation of classical targets associated with reward to biological substrates responsible for negative reinforcement, impulse loss of control, and maladaptive mechanisms resulting from protracted drug use. From this research, several new biological targets for the development of innovative therapies have started to emerge. This chapter offers an overview of targets currently under scrutiny for the development of new medications for addiction. This work is not exhaustive but rather it provides a few examples of how this research has advanced in recent years by virtue of studies carried out in our laboratory

Preclinical evidence implicating corticotropin-releasing factor signaling in ethanol consumption and neuroadaptation

The results of many studies support the influence of the corticotropin-releasing factor (CRF) system on ethanol (EtOH) consumption and EtOH-induced neuroadaptations that are critical in the addiction process. This review summarizes the preclinical data in this area after first providing an overview of the components of the CRF system. This complex system involves hypothalamic and extra-hypothalamic mechanisms that play a role in the central and peripheral consequences of stressors, including EtOH and other drugs of abuse. In addition, several endogenous ligands and targets make up this system and show differences in their involvement in EtOH drinking and in the effects of chronic or repeated EtOH treatment. In general, genetic and pharmacological approaches paint a consistent picture of the importance of CRF signaling via type 1 CRF receptors (CRF1) in EtOH-induced neuroadaptations that result in higher levels of intake, encourage alcohol seeking during abstinence and alter EtOH sensitivity. Furthermore, genetic findings in rodents, non-human primates and humans have provided some evidence of associations of genetic polymorphisms in CRF-related genes with EtOH drinking, although additional data are needed. These results suggest that CRF1 antagonists have potential as pharmacotherapeutics for alcohol use disorders. However, given the broad and important role of these receptors in adaptation to environmental and other challenges, full antagonist effects may be too profound and consideration should be given to treatments with modulatory effects.The authors were supported by the Department of Veterans Affairs; NIH NIAAA grants P60AA010760, R24AA020245 and U01AA013519 and NIH NIDA grant P50DA018165, during the writing of this manuscript. The authors have no financial conflict of interest to disclose

A Genetic Animal Model of Alcoholism for Screening Medications to Treat Addiction

The purpose of this review is to present up-to-date pharmacological, genetic, and behavioral findings from the alcohol-preferring P rat and summarize similar past work. Behaviorally, the focus will be on how the P rat meets criteria put forth for a valid animal model of alcoholism with a highlight on its use as an animal model of polysubstance abuse, including alcohol, nicotine, and psychostimulants. Pharmacologically and genetically, the focus will be on the neurotransmitter and neuropeptide systems that have received the most attention: cholinergic, dopaminergic, GABAergic, glutamatergic, serotonergic, noradrenergic, corticotrophin releasing hormone, opioid, and neuropeptide Y. Herein, we sought to place the P rat's behavioral and neurochemical phenotypes, and to some extent its genotype, in the context of the clinical literature. After reviewing the findings thus far, this chapter discusses future directions for expanding the use of this genetic animal model of alcoholism to identify molecular targets for treating drug addiction in general

Glucocorticoid receptor antagonism decreases alcohol seeking in alcohol-dependent individuals

Alcoholism, or alcohol use disorder, is a major public health concern that is a considerable risk factor for morbidity and disability; therefore, effective treatments are urgently needed. Here, we demonstrated that the glucocorticoid receptor (GR) antagonist mifepristone reduces alcohol intake in alcohol-dependent rats but not in nondependent animals. Both systemic delivery and direct administration into the central nucleus of the amygdala, a critical stress-related brain region, were sufficient to reduce alcohol consumption in dependent animals. We also tested the use of mifepristone in 56 alcohol-dependent human subjects as part of a double-blind clinical and laboratory-based study. Relative to placebo, individuals who received mifepristone (600 mg daily taken orally for 1 week) exhibited a substantial reduction in alcohol-cued craving in the laboratory, and naturalistic measures revealed reduced alcohol consumption during the 1-week treatment phase and 1-week post-treatment phase in mifepristone-treated individuals. Mifepristone was well tolerated and improved liver-function markers. Together, these results support further exploration of GR antagonism via mifepristone as a therapeutic strategy for alcoholism

Genetic and Pharmacologic Manipulation of TLR4 Has Minimal Impact on Ethanol Consumption in Rodents

Toll-like receptor 4 (TLR4) is a critical component of innate immune signaling and has been implicated in alcohol responses in preclinical and clinical models. Members of the Integrative Neuroscience Initiative on Alcoholism (INIA-Neuroimmune) consortium tested the hypothesis that TLR4 mediates excessive ethanol drinking using the following models: (1) Tlr4 knock-out (KO) rats, (2) selective knockdown of Tlr4 mRNA in mouse nucleus accumbens (NAc), and (3) injection of the TLR4 antagonist (+)-naloxone in mice. Lipopolysaccharide (LPS) decreased food/water intake and body weight in ethanol-naive and ethanol-trained wild-type (WT), but not Tlr4 KO rats. There were no consistent genotypic differences in two-bottle choice chronic ethanol intake or operant self-administration in rats before or after dependence. In mice, (+)-naloxone did not decrease drinking-in-the-dark and only modestly inhibited dependence-driven consumption at the highest dose. Tlr4 knockdown in mouse NAc did not decrease drinking in the two-bottle choice continuous or intermittent access tests. However, the latency to ethanol-induced loss of righting reflex increased and the duration decreased in KO versus WT rats. In rat central amygdala neurons, deletion of Tlr4 altered GABAA receptor function, but not GABA release. Although there were no genotype differences in acute ethanol effects before or after chronic intermittent ethanol exposure, genotype differences were observed after LPS exposure. Using different species and sexes, different methods to inhibit TLR4 signaling, and different ethanol consumption tests, our comprehensive studies indicate that TLR4 may play a role in ethanol-induced sedation and GABAA receptor function, but does not regulate excessive drinking directly and would not be an effective therapeutic target., SIGNIFICANCE STATEMENT Toll-like receptor 4 (TLR4) is a key mediator of innate immune signaling and has been implicated in alcohol responses in animal models and human alcoholics. Members of the Integrative Neuroscience Initiative on Alcoholism (INIA-Neuroimmune) consortium participated in the first comprehensive study across multiple laboratories to test the hypothesis that TLR4 regulates excessive alcohol consumption in different species and different models of chronic, dependence-driven, and binge-like drinking. Although TLR4 was not a critical determinant of excessive drinking, it was important in the acute sedative effects of alcohol. Current research efforts are directed at determining which neuroimmune pathways mediate excessive alcohol drinking and these findings will help to prioritize relevant pathways and potential therapeutic targets