An Adolescent Porcine Model of Voluntary Alcohol Consumption Exhibits Binge Drinking and Motor Deficits in a Two Bottle Choice Test.

AIMS: Alcohol is the most commonly abused substance leading to significant economic and medical burdens. Pigs are an attractive model for studying alcohol abuse disorder due to the comparable alcohol metabolism and consumption behavior, which are in stark contrast to rodent models. This study investigates the usage of a porcine model for voluntary binge drinking (BD) and a detailed analysis of gait changes due to motor function deficits during alcohol intoxication. METHODS: Adolescent pigs were trained to drink increasing concentration (0-8%) of alcohol mixed in a 0.2% saccharin solution for 1 h in a two bottle choice test for 2 weeks. The training period was followed by a 3-week alcohol testing period, where animals were given free access to 8% alcohol in 0.2% saccharin solution and 0.2% saccharin water solution. Blood alcohol levels were tested and gait analysis was performed pre-alcohol consumption, last day of training, and Day 5 of each testing period. RESULTS: Pigs voluntarily consumed alcohol to intoxication at all timepoints with blood alcohol concentration (BAL) ≥80 mg/dl. Spatiotemporal gait parameters including velocity, cadence, cycle time, swing time, stance time, step time, and stride length were perturbed as a result of intoxication. The stratification of the gait data based on BAL revealed that the gait parameters were affected in a dose-dependent manner. CONCLUSION: This novel adolescent BD porcine model with inherent anatomical and physiological similarities to humans display similar consumption and intoxication behavior that is likely to yield results that are translatable to human patients.

Intermittent Ethanol Access Increases Sensitivity to Social Defeat Stress.

BACKGROUND: Comorbidity between alcoholism and depression is extremely common. Recent evidence supports a relationship between alcohol exposure and stress sensitivity, an underlying factor in the development of depression. Our laboratory has recently shown that chronic alcohol gavage increases sensitivity to social defeat stress (SDS). However, the effects of voluntary alcohol consumption, resulting from protocols such as intermittent ethanol access (IEA), on defeat stress sensitivity have yet to be elucidated. METHODS: We first assessed the effects of 4 weeks of IEA to 20% alcohol on sensitivity to subthreshold SDS exposure. Next, to examine neuroinflammatory mechanisms, we analyzed gene expression of inhibitor of NFkB (IkB) following IEA or chronic alcohol exposure (10 days of 3.0 g/kg alcohol via intragastric gavage). Then, we quantified NFkB activation via ß-galactosidase immunohistochemistry following IEA or chronic alcohol gavage in NFkB-LacZ mice. RESULTS: IEA-exposed mice displayed an increase in sensitivity to subthreshold SDS compared to water-drinking controls. We also found that IkB gene expression was decreased in the nucleus accumbens (NAC) and amygdala (AMY) following IEA but was not altered following chronic alcohol gavage. Finally, we observed increased NFkB activity in the central amygdala (CEA), basolateral amygdala (BLA), and medial amygdala (MEA) after IEA, and increased NFkB activity solely in the CEA following chronic alcohol gavage. CONCLUSIONS: These findings further corroborate that prior alcohol exposure, in this case intermittent voluntary consumption, can impact development of depressive-like behavior by altering stress sensitivity. Furthermore, our results suggest the CEA as a potential mediator of alcohol's effects on stress sensitivity, as NFkB was activated in this region following both IEA and chronic alcohol gavage. Thus, this study provides novel insight on alterations in the NFkB pathway and identifies specific regions to target in future experiments assessing the functional role of NFkB in these processes.

Sex differences in oral oxycodone self-administration and stress-primed reinstatement in rats.

The opioid epidemic has become a severe public health problem, with approximately 130 opioid-induced deaths occurring each day in the United States. Prescription opioids are responsible for approximately 40% of these deaths. Oxycodone is one of the most commonly abused prescription opioids, but despite its prevalent misuse, the number of preclinical studies investigating oxycodone-seeking behaviors is relatively limited. Furthermore, preclinical oxycodone studies that include female subjects are even more scarce, and it is critical that future work includes both sexes. Additionally, the oral route of administration is one of the most common routes for recreational users, especially in the early stages of drug experimentation. However, currently, only two studies have been published investigating operant oral oxycodone self-administration in rodents. Therefore, the primary goal of the present study was to establish an oral oxycodone operant self-administration model in adult male and female rats, as well as to examine a potential mechanism of stress-primed reinstatement. We found that females consumed significantly more oral oxycodone than males in operant self-administration sessions. We also found that active oxycodone self-administration was reduced by mu opioid receptor antagonism and by substitution of water for oxycodone solution. Lastly, we induced stress-primed reinstatement and found that this behavior was significantly attenuated by antagonism of the neurokinin-1 receptor, consistent with our prior work examining stress-induced reinstatement of alcohol- and cocaine-seeking.

Sex Differences in Aversion-Resistant Ethanol Intake in Mice.

AIMS: Compulsive ethanol intake, characterized by persistent consumption despite negative consequences, is an addictive behavior identified by the DSM-5 as a central criterion in diagnosing alcohol use disorders (AUD). Epidemiological data suggest that females transition from recreational alcohol use to AUD more rapidly than males. Because of this potential sex difference in the etiology of AUD, it is critical to assess addictive behaviors such as compulsive intake in both males and females in preclinical studies. METHODS: We used the model of aversion-resistant ethanol consumption to assess compulsive-like ethanol intake. In these experiments, C57BL6/J mice were first provided with continuous access two-bottle choice between water and ethanol to establish baseline intake. Ethanol solution was then adulterated with increasing concentrations of the bitter tastant quinine hydrochloride. Animals that consume ethanol solution despite its pairing with this negative stimulus are thought to be exhibiting compulsive-like behavior. RESULTS: We found that higher concentrations of quinine were required to suppress ethanol consumption in female mice relative to males. We found no effect of estrous cycle phase on baseline ethanol intake or on quinine-adulterated ethanol intake in females. CONCLUSIONS: Collectively, these data suggest that females exhibit a higher degree of aversion-resistance than male mice. Because we observed no effect of estrous cycle phase, it is likely that the presence of threshold levels of estradiol or progesterone, as opposed to their natural fluctuation across the estrous cycle, mediates increased aversion-resistance in females. Alternatively, or in combination, developmental effects of sex hormones could contribute to aversion-resistant ethanol intake.

Cellular and behavioral effects of lipopolysaccharide treatment are dependent upon neurokinin-1 receptor activation.

BACKGROUND: Several psychiatric conditions are affected by neuroinflammation and neuroimmune activation. The transcription factor nuclear factor kappa light-chain-enhancer of activated B cells (NFkB) plays a major role in inflammation and innate immunity. The neurokinin-1 receptor (NK1R) is the primary endogenous target of the neuroactive peptide substance P, and some data suggests that NK1R stimulation may influence NFkB activity. Both NK1R and NFkB have been shown to play a functional role in complex behaviors including stress responsivity, depression, and addiction. In this study, we test whether NFkB activity in the brain (stimulated by lipopolysaccharide administration) is dependent upon the NK1R. METHODS: Adult male Wistar rats were treated systemically with the NK1R antagonist L822429 followed by administration of systemic lipopolysaccharide (LPS, a strong activator of NFkB). Hippocampal extracts were used to assess expression of proinflammatory cytokines and NFkB-DNA-binding potential. For behavioral studies, rats were trained to consume 1% (w/v) sucrose solution in a continuous access two-bottle choice model. After establishment of baseline, animals were treated with L822429 and LPS and sucrose preference was measured 12 h post-treatment. RESULTS: Systemic LPS treatment causes a significant increase in proinflammatory cytokine expression and NFkB-DNA-binding activity within the hippocampus. These increases are attenuated by systemic pretreatment with the NK1R antagonist L822429. Systemic LPS treatment also led to the development of anhedonic-like behavior, evidenced by decreased sucrose intake in the sucrose preference test. This behavior was significantly attenuated by systemic pretreatment with the NK1R antagonist L822429. CONCLUSIONS: Systemic LPS treatment induced significant increases in NFkB activity, evidenced by increased NFkB-DNA binding and by increased proinflammatory cytokine expression in the hippocampus. LPS also induced anhedonic-like behavior. Both the molecular and behavioral effects of LPS treatment were significantly attenuated by systemic NK1R antagonism, suggesting that NK1R stimulation lies upstream of NFkB activation following systemic LPS administration and is at least in part responsible for NFkB activation.

Developmental exposure to the organochlorine insecticide endosulfan alters expression of proteins associated with neurotransmission in the frontal cortex.

Exposure to environmental contaminants, such as organochlorine insecticides during critical periods of neurodevelopment has been shown to be a major contributor to several neuropsychological deficits seen in children, adolescence, and adults. Although the neurobehavioral outcomes resulting from exposure to these compounds are known the neurotransmitter circuitry and molecular targets that mediate these endpoints have not been identified. Given the importance of the frontal cortex in facilitating numerous neuropsychological processes, our current study sought to investigate the effects of developmental exposure to the organochlorine insecticide, endosulfan, on the expression of specific proteins associated with neurotransmission in the frontal cortex. Utilizing in vitro models we were able to show endosulfan reduces cell viability in IMR-32 neuroblastoma cells in addition to reducing synaptic puncta and neurite outgrowth in primary cultured neurons isolated from the frontal cortex of mice. Elaborating these findings to an in vivo model we found that developmental exposure of female mice to endosulfan during gestation and lactation elicited significant alterations to the GABAergic (GAT1, vGAT, GABAA receptor), glutamatergic (vGlut and GluN2B receptor), and dopaminergic (DAT, TH, VMAT2, and D2 receptor) neurotransmitter systems in the frontal cortex of male offspring. These findings identify damage to critical neurotransmitter circuits and proteins in the frontal cortex, which may underlie the neurobehavioral deficits observed following developmental exposure to endosulfan and other organochlorine insecticides.

Increased Alcohol Consumption in Mice Lacking Sodium Bicarbonate Transporter NBCn1.

The previous reports on an addiction vulnerability marker in the human SLC4A7 gene encoding the Na/HCO3 transporter NBCn1 suggest that this pH-regulating protein may affect alcohol-related behavior and response. Here, we examined alcohol consumption and sensitivity to the sedative effects of alcohol in male NBCn1 knockout mice. These mice displayed lower pH in neurons than wildtype controls, determined by intracellular pH in hippocampal neuronal cultures. Neurons from knockout mice had a higher action potential threshold and a more depolarized membrane potential, thus reducing membrane excitability. In a two-bottle free choice procedure, knockout mice consumed more alcohol than controls and consistently increased alcohol consumption after repeated alcohol deprivation periods. Quinine and sucrose preference was similar between genotypes. Knockout mice showed increased propensity for alcohol-induced conditioned place preference. In loss of righting reflex assessment, knockout mice revealed increased sensitivity to alcohol-induced sedation and developed tolerance to the sedation after repeated alcohol administrations. Furthermore, chronic alcohol consumption caused NBCn1 downregulation in the hippocampus and striatum of mice and humans. These results demonstrate an important role of NBCn1 in regulation of alcohol consumption and sensitivity to alcohol-induced sedation.

Escalated Alcohol Self-Administration and Sensitivity to Yohimbine-Induced Reinstatement in Alcohol Preferring Rats: Potential Role of Neurokinin-1 Receptors in the Amygdala.

Genetic factors significantly contribute to the risk for developing alcoholism. To study these factors and other associated phenotypes, rodent lines have been developed using selective breeding for high alcohol preference. One of these models, the alcohol preferring (P) rat, has been used in hundreds of preclinical studies over the last few decades. However, very few studies have examined relapse-like behavior in this rat strain. In this study, we used operant self-administration and yohimbine-induced reinstatement models to examine relapse-like behavior in P rats. Our previous work has demonstrated that P rats show increased expression of the neurokinin-1 receptor (NK1R) in the central nucleus of the amygdala (CeA), and this functionally contributes to escalated alcohol consumption in this strain. We hypothesized that P rats would show increased sensitivity to yohimbine-induced reinstatement that is also mediated by NK1R in the CeA. Using Fos staining, site-specific infusion of NK1R antagonist, and viral vector overexpression, we examined the influence of NK1R on the sensitivity to yohimbine-induced reinstatement of alcohol seeking. We found that P rats displayed increased sensitivity to yohimbine-induced reinstatement as well as increased neuronal activation in the CeA after yohimbine injection compared to the control Wistar strain. Intra-CeA infusion of NK1R antagonist attenuates yohimbine-induced reinstatement in P rats. Conversely, upregulation of NK1R within the CeA of Wistar rats increases alcohol consumption and sensitivity to yohimbine-induced reinstatement. These findings suggest that NK1R upregulation in the CeA contributes to multiple alcohol-related phenotypes in the P rat, including alcohol consumption and sensitivity to relapse.

The neurokinin-1 receptor mediates escalated alcohol intake induced by multiple drinking models.

We have previously demonstrated that the neurokinin-1 receptor (NK1R) is upregulated in the central nucleus of the amygdala of alcohol preferring (P) rats and that this receptor mediates escalated alcohol consumption in this strain. However, it is unclear if non-genetic models of escalated consumption are also mediated by NK1R signaling, and if so, what brain regions govern this effect. In the experiments presented here, we use two methods of inducing escalated alcohol intake in outbred Wistar rats: yohimbine pretreatment and intermittent alcohol access (Monday, Wednesday, and Friday availability; 20% alcohol). We found that escalated alcohol consumption induced by both yohimbine injection and intermittent access is attenuated by systemic administration of the NK1R antagonist L822429. Also, when compared to continuous alcohol access or access to water alone, NK1R expression was increased in the nucleus accumbens (NAC) and dorsal striatum, but not the amygdala. Escalated consumption induced by intermittent access was attenuated when the NK1R antagonist L822429 was infused directly into the dorsal striatum, but not when infused into the NAC. Taken together, these results suggest that NK1R upregulation contributes to escalated alcohol consumption that is induced by genetic selection, yohimbine injection, and intermittent access. However there is a dissociation between the regions involved in these behaviors with amygdalar upregulation contributing to genetic predisposition to escalated consumption and striatal upregulation driving escalation that is induced by environmental exposures.