Chronic Intermittent Ethanol Exposure Dysregulates Nucleus Basalis Magnocellularis Afferents in the Basolateral Amygdala.

Nucleus basalis magnocellularis (NBM) cholinergic projections to the basolateral amygdala (BLA) regulate the acquisition and consolidation of fear-like and anxiety-like behaviors. However, it is unclear whether the alterations in the NBM-BLA circuit promote negative affect during ethanol withdrawal (WD). Therefore, we performed ex vivo whole-cell patch-clamp electrophysiology in both the NBM and the BLA of male Sprague Dawley rats following 10 d of chronic intermittent ethanol (CIE) exposure and 24 h of WD. We found that CIE exposure and withdrawal enhanced the neuronal excitability of NBM putative "cholinergic" neurons. We subsequently used optogenetics to directly manipulate NBM terminal activity within the BLA and measure cholinergic modulation of glutamatergic afferents and BLA pyramidal neurons. Our findings indicate that CIE and withdrawal upregulate NBM cholinergic facilitation of glutamate release via activation of presynaptic nicotinic acetylcholine receptors (AChRs). Ethanol withdrawal-induced increases in NBM terminal activity also enhance BLA pyramidal neuron firing. Collectively, our results provide a novel characterization of the NBM-BLA circuit and suggest that CIE-dependent modifications to NBM afferents enhance BLA pyramidal neuron activity during ethanol withdrawal.

Chronic Alcohol Dysregulates Glutamatergic Function in the Basolateral Amygdala in a Projection-and Sex-Specific Manner.

Chronic intermittent ethanol and withdrawal (CIE/WD) produces alcohol dependence, facilitates anxiety-like behavior, and increases post-CIE alcohol intake. The basolateral amygdala (BLA) is one of several brain regions that regulates anxiety-like behavior and alcohol intake through downstream projections to the nucleus accumbens (NAC) and bed nucleus of the stria terminalis (BNST), respectively. Previous studies revealed that CIE/WD induces input- and sex-specific adaptations to glutamatergic function in the BLA. The BLA receives information from two distinct input pathways. Glutamatergic afferents from medial structures like the thalamus and prefrontal cortex enter the BLA through the stria terminalis whereas lateral cortical structures like the anterior insula cortex enter the BLA through the external capsule. CIE/WD increases presynaptic glutamatergic function at stria terminalis synapses and postsynaptic function at external capsule synapses. Previous studies sampled neurons throughout the BLA, but did not distinguish between projection-specific populations. The current study investigated BLA neurons that project to the NAC (BLA-NAC neurons) or the BNST (BLA-BNST neurons) as representative "reward" and "aversion" BLA neurons, and showed that CIE/WD alters glutamatergic function and excitability in a projection- and sex-specific manner. CIE/WD increases glutamate release from stria terminalis inputs only onto BLA-BNST neurons. At external capsule synapses, CIE/WD increases postsynaptic glutamatergic function in male BLA-NAC neurons and female BLA-BNST neurons. Subsequent experiments demonstrated that CIE/WD enhanced the excitability of male BLA-NAC neurons and BLA-BNST neurons in both sexes when glutamatergic but not GABAergic function was intact. Thus, CIE/WD-mediated increased glutamatergic function facilitates hyperexcitability in male BLA-NAC neurons and BLA-BNST neurons of both sexes.

Structural, functional, and behavioral significance of sex and gonadal hormones in the basolateral amygdala: A review of preclinical literature.

The basolateral amygdala (BLA) is intimately involved in the development of neuropsychiatric disorders such as anxiety and alcohol use disorder (AUD). These disorders have clear sex biases, with women more likely to develop an anxiety disorder and men more likely to develop AUD. Preclinical models have largely confirmed these sex-specific vulnerabilities and emphasize the effects of sex hormones on behaviors influenced by the BLA. This review will discuss sex differences in BLA-related behaviors and highlight potential mechanisms mediated by altered BLA structure and function, including the composition of GABAergic interneuron subpopulations, glutamatergic pyramidal neuron morphology, glutamate/GABA neurotransmission, and neuromodulators. Further, sex hormones differentially organize dimorphic circuits during sensitive developmental periods (organizational effects) and initiate more transient effects throughout adulthood (activational effects). Current literature indicates that estradiol and allopregnanolone, a neuroactive progestogen, generally reduce BLA-related behaviors through a variety of mechanisms, including activation of estrogen receptors or facilitation of GABAA-mediated inhibition, respectively. This enhanced GABAergic inhibition may protect BLA pyramidal neurons from the excitability associated with anxiety and alcohol withdrawal. Understanding sex differences and the effects of sex hormones on BLA structure and function may help explain sex-specific vulnerabilities in BLA-related behaviors and ultimately improve treatments for anxiety and AUD.

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.