Chronic ethanol exposure produces long-lasting, subregion-specific physiological adaptations in RMTg-projecting mPFC neurons.

Chronic ethanol exposure produces neuroadaptations in the medial prefrontal cortex (mPFC) that are thought to facilitate maladaptive behaviors that interfere with recovery from alcohol use disorder. Despite evidence that different cortico-subcortical projections play distinct roles in behavior, few studies have examined the physiological effects of chronic ethanol at the circuit level. The rostromedial tegmental nucleus (RMTg) is functionally altered by chronic ethanol exposure. Our recent work identified dense input from the mPFC to the RMTg, yet the effects of chronic ethanol exposure on this circuitry is unknown. In the current study, we examined physiological changes after chronic ethanol exposure in prelimbic (PL) and infralimbic (IL) mPFC neurons projecting to the RMTg. Adult male Long-Evans rats were injected with fluorescent retrobeads into the RMTg and rendered dependent using a 14-day chronic intermittent ethanol (CIE) vapor exposure paradigm. Whole-cell patch-clamp electrophysiological recordings were performed in fluorescently-labeled (RMTg-projecting) and -unlabeled (projection-undefined) layer 5 pyramidal neurons 7-10 days following ethanol exposure. CIE exposure significantly increased intrinsic excitability as well as spontaneous excitatory and inhibitory postsynaptic currents (sE/IPSCs) in RMTg-projecting IL neurons. In contrast, no lasting changes in excitability were observed in RMTg-projecting PL neurons, although a CIE-induced reduction in excitability was observed in projection-undefined PL neurons. CIE exposure also increased the frequency of sEPSCs in RMTg-projecting PL neurons. These data uncover novel subregion- and circuit-specific neuroadaptations in the mPFC following chronic ethanol exposure and reveal that the IL mPFC-RMTg projection is uniquely vulnerable to long-lasting effects of chronic ethanol exposure. This article is part of the Special Issue on "PFC circuit function in psychiatric disease and relevant models".

Chronic ethanol exposure produces long-lasting, subregion-specific physiological adaptations in RMTg-projecting mPFC neurons.

Chronic ethanol exposure produces neuroadaptations in the medial prefrontal cortex (mPFC) which facilitate the maladaptive behaviors interfering with recovery from alcohol use disorder. Despite evidence that different cortico-subcortical projections play distinct roles in behavior, few studies have examined the physiological effects of chronic ethanol at the circuit level. The rostromedial tegmental nucleus (RMTg) is a GABAergic midbrain region involved in aversive signaling and is functionally altered by chronic ethanol exposure. Our recent work identified a dense input from the mPFC to the RMTg, yet the effects of chronic ethanol exposure on this circuitry is unknown. In the current study, we examined physiological changes after chronic ethanol exposure in prelimbic (PL) and infralimbic (IL) mPFC neurons projecting to the RMTg. Adult male Long-Evans rats were injected with fluorescent retrobeads into the RMTg and rendered dependent using a 14-day chronic intermittent ethanol (CIE) vapor exposure paradigm. Whole-cell patch-clamp electrophysiological recordings were performed in fluorescently-labeled (RMTg-projecting) and -unlabeled (projection-undefined) layer 5 pyramidal neurons 7-10 days following ethanol exposure. CIE significantly increased intrinsic excitability as well as excitatory and inhibitory synaptic drive in RMTg-projecting IL neurons. In contrast, no lasting changes in excitability were observed in RMTg-projecting PL neurons, although a CIE-induced reduction in excitability was observed in projection-undefined PL neurons. CIE also increased excitatory synaptic drive in RMTg-projecting PL neurons. These data uncover novel subregion- and circuit-specific neuroadaptations in the mPFC following chronic ethanol exposure and reveal that the IL mPFC-RMTg projection is uniquely vulnerable to long-lasting effects of chronic ethanol.

A translational rodent model of individual differences in sensitivity to the aversive properties of ethanol.

BACKGROUND: A strong relationship exists between individual sensitivity to the aversive properties of ethanol and risk for alcohol use disorder (AUD). Despite this, our understanding of the neurobiological mechanisms underlying the subjective response to ethanol is limited. A major contributor to this lack of knowledge is the absence of preclinical models that enable exploration of this individual variability such as is possible in studies of humans. METHODS: Adult male and female Long-Evans rats were trained to associate a novel tastant (saccharin) with acute exposure to either saline or ethanol (1.5 g/kg or 2.0 g/kg i.p.) over three conditioning days using a standard conditioned taste aversion (CTA) procedure. Variability in sensitivity to ethanol-induced CTA was phenotypically characterized using a median split across the populations studied. RESULTS: When examining group averages, both male and female rats exposed to saccharin paired with either dose of ethanol exhibited lower saccharin intake relative to saline controls indicative of ethanol-induced CTA. Examination of individual data revealed a bimodal distribution of responses uncovering two distinct phenotypes present in both sexes. CTA-sensitive rats exhibited a rapid and progressive reduction in saccharin intake with each successive ethanol pairing. In contrast, saccharin intake was unchanged or maintained after an initial decrease from baseline levels in CTA-resistant rats. While CTA magnitude was similar between male and female CTA-sensitive rats, among CTA-resistant animals females were more resistant to the development of ethanol-induced CTA than males. Phenotypic differences were not driven by differences in baseline saccharin intake. CONCLUSIONS: These data parallel work in humans by revealing individual differences in sensitivity to the aversive properties of ethanol that emerge immediately after initial exposure to ethanol in both sexes. This model can be used in future studies to investigate the neurobiological mechanisms that confer risk for AUD.

Attenuated incubation of ethanol-induced conditioned taste aversion in a model of dependence.

RATIONALE: Preclinical studies report attenuated ethanol-induced conditioned taste aversion (CTA) following chronic ethanol exposure, suggesting that tolerance develops to the aversive properties of ethanol. However, these studies are confounded by pre-exposure to the unconditioned stimulus (US; ethanol), which is well known to hinder conditioning. OBJECTIVES: This study was designed to determine whether chronic ethanol exposure produces tolerance to the aversive properties of ethanol in the absence of a US pre-exposure confound. METHODS: CTA was performed in adult male and female Long-Evans rats by pairing 0.1% ingested saccharin with an intraperitoneal injection of ethanol (1.5 or 2.0 g/kg) or saline. Rats were then rendered ethanol dependent using chronic intermittent ethanol (CIE) vapor exposure. Controls were exposed to room air (AIR). The effect of chronic ethanol on CTA expression and reconditioning were examined following vapor exposure. RESULTS: Prior to vapor exposure, both sexes developed CTA to a comparable degree with 2.0 g/kg producing greater CTA than 1.5 g/kg ethanol. Following vapor exposure, AIR controls exhibited an increase in CTA magnitude compared to pre-vapor levels. This effect was largely absent in CIE-exposed rats. Re-conditioning after vapor exposure facilitated increased CTA magnitude to a similar degree in AIR- and CIE-exposed males. In contrast, CTA magnitude was unchanged by re-conditioning in females. CONCLUSIONS: These data suggest that chronic ethanol does not facilitate tolerance to the aversive properties of ethanol but rather attenuates incubation of ethanol-induced CTA. Loss of CTA incubation suggests that CIE exposure disrupts circuits encoding aversion.

Attenuated incubation of ethanol-induced conditioned taste aversion in a model of dependence.

Rationale: Preclinical studies report attenuated ethanol-induced conditioned taste aversion (CTA) following chronic ethanol exposure, suggesting that tolerance develops to the aversive properties of ethanol. However, these studies are confounded by pre-exposure to the unconditioned stimulus (US; ethanol), which is well known to hinder conditioning. Objectives: This study was designed to determine whether chronic ethanol exposure produces tolerance to the aversive properties of ethanol in the absence of a US pre-exposure confound. Methods: CTA was performed in adult male and female Long-Evans rats by pairing 0.1% ingested saccharin with an intraperitoneal injection of ethanol (1.5 or 2.0 g/kg) or saline. Rats were then rendered ethanol dependent using chronic intermittent ethanol (CIE) vapor exposure. Controls were exposed to room air (AIR). The effect of chronic ethanol on CTA expression and reconditioning were examined following vapor exposure. Results: Prior to vapor exposure, both sexes developed CTA to a comparable degree with 2.0 g/kg producing greater CTA than 1.5 g/kg ethanol. Following vapor exposure, AIR controls exhibited an increase in CTA magnitude compared to pre-vapor levels. This effect was absent in CIE-exposed rats. These group differences were eliminated upon re-conditioning after vapor exposure. Conclusions: These data suggest that chronic ethanol does not facilitate tolerance to the aversive properties of ethanol but rather, attenuates incubation of ethanol-induced CTA. Loss of CTA incubation suggests that CIE exposure disrupts circuits encoding aversion.

A translational rodent model of individual differences in sensitivity to the aversive properties of ethanol.

Background: A strong relationship exists between individual sensitivity to the aversive properties of ethanol and risk for alcohol use disorder (AUD). Despite this, our understanding of the neurobiological mechanisms underlying subjective response to ethanol is relatively poor. A major contributor to this is the absence of preclinical models that enable exploration of this individual variability similar to studies performed in humans. Methods: Adult male and female Long-Evans rats were trained to associate a novel tastant (saccharin) with acute exposure to either saline or ethanol (1.5 g/kg or 2.0 g/kg i.p.) over three conditioning days using a standard conditioned taste aversion (CTA) procedure. Variability in sensitivity to ethanol-induced CTA was phenotypically characterized using a median split across the populations studied. Results: When examining group averages, both male and female rats that had saccharin paired with either dose of ethanol exhibited reduced saccharin intake relative to saline controls of ethanol-induced CTA. Examination of individual data revealed a bimodal distribution of responses uncovering two distinct phenotypes present in both sexes. CTA-sensitive rats exhibited a rapid and progressive reduction in saccharin intake with each successive ethanol pairing. In contrast, saccharin intake was unchanged or maintained after an initial decrease from baseline levels in CTA-resistant rats. While CTA magnitude was similar between male and female CTA-sensitive rats, CTA-resistant females were more resistant to the development of ethanol-induced CTA than their male counterparts. Phenotypic differences were not driven by differences in baseline saccharin intake. CTA sensitivity correlated with behavioral signs of intoxication in only a subset of rats. Conclusions: These data parallel work in humans by revealing individual differences in sensitivity to the aversive properties of ethanol that emerge immediately after initial exposure to ethanol in both sexes. This model can be leveraged in future studies to investigate the neurobiological mechanisms that confer risk for AUD.

Moderate prenatal alcohol exposure produces sex-specific social impairments and attenuates prelimbic excitability and amygdala-cortex modulation of adult social behaviour.

Lifelong social impairments are common in individuals with prenatal alcohol exposure (PAE), and preclinical studies have identified gestational day (G)12 as a vulnerable timepoint for producing social deficits following binge-level PAE. While moderate (m)PAE also produces social impairments, the long-term neuroadaptations underlying them are poorly understood. Activity of the projection from the basolateral amygdala to the prelimbic cortex (BLA â†’ PL) leads to social avoidance, and the PL is implicated in negative social behaviours, making each of these potential candidates for the neuroadaptations underlying mPAE-induced social impairments. To examine this, we first established that G12 mPAE produced sex-specific social impairments lasting into adulthood in Sprague-Dawley rats. We then chemogenetically inhibited the BLA â†’ PL using clozapine N-oxide (CNO) during adult social testing. This revealed that CNO reduced social investigation in control males but had no effect on mPAE males or females of either exposure, indicating that mPAE attenuated the role of this projection in regulating male social behaviour and highlighting one potential mechanism by which mPAE affects male social behaviour more severely. Using whole-cell electrophysiology, we also examined mPAE-induced changes to PL pyramidal cell physiology and determined that mPAE reduced cell excitability, likely due to increased suppression by inhibitory interneurons. Overall, this work identified two mPAE-induced neuroadaptations that last into adulthood and that may underlie the sex-specific vulnerability to mPAE-induced social impairments. Future research is necessary to expand upon how these circuits modulate both normal and pathological social behaviours and to identify sex-specific mechanisms, leading to differential vulnerability in males and females.

Moderate adolescent chronic intermittent ethanol exposure sex-dependently disrupts synaptic transmission and kappa opioid receptor function in the basolateral amygdala of adult rats.

Adolescent alcohol exposure is associated with many consequences in adulthood, including altered affective and reward-related behaviors. However, the long-term neurological disruptions underlying these behaviors are not fully understood. Shifts in the excitatory/inhibitory balance in the basolateral amygdala (BLA) relate to the expression of these behaviors and changes to BLA physiology are seen during withdrawal immediately following adolescent ethanol exposure, but no studies have examined whether these changes persist long-term. The kappa opioid receptor (KOR) neuromodulatory system mediates negative affective behaviors, and alterations of this system are implicated in behavioral changes following adult and adolescent chronic ethanol exposure. In the BLA, the KOR system undergoes functional changes across development, but whether BLA KOR function is disrupted by adolescent ethanol exposure is unknown. In this study, male and female Sprague-Dawley rats were exposed to a vapor model of moderate adolescent chronic intermittent ethanol (aCIE) and assessed for long-term effects on GABAergic and glutamatergic neurotransmission within the adult BLA and KOR modulation of these systems. aCIE exposure increased presynaptic glutamate transmission in females but had no effect in males or on GABA transmission in either sex. Additionally, aCIE exposure disrupted male KOR modulation of GABA release, with no effects in females or on glutamate transmission. These data suggest that aCIE produces sex-dependent and long-term changes to BLA physiology and KOR function. This is the first study to examine these persistent adaptations following adolescent alcohol exposure and opens a broad avenue for future investigation into other adolescent ethanol-induced disruptions of these systems.

Age and sex regulate kappa opioid receptor-mediated anxiety-like behavior in rats.

Anxiety occurs across ontogeny, but there is evidence that its etiology may vary across the lifespan. The kappa opioid receptor (KOR) system mediates some of the anxiogenic effects of stress and drug exposure, and is involved in aversive responses to environmental stimuli. However, much of this work has been conducted in adult males. Work assessing the effects of KOR activation in younger males has demonstrated that this system produces an anxiolytic/no response, indicating that that this system may be developmentally regulated. Despite these discrepancies, a direct comparison of KOR-induced anxiety in stress-naïve adolescents and adults has not been done. Additionally, the effects of KOR activation in females are poorly understood. Therefore, we assessed the impact of KOR activation on anxiety-like behavior in adolescent and adult male and female Sprague-Dawley rats. Animals were given an i.p. injection of the KOR agonist U69593 (0.01, 0.1, 1.0 mg/kg or vehicle) and were tested using the elevated plus maze. U69593 decreased open arm time in adult males, indicating increased anxiety-like behavior. Adolescents exhibited decreased stretch attend postures when collapsed across sex, suggesting reduced anxiety-like behavior. Adult females were not affected by U69593 administration. These data support studies that have identified age-dependent changes in the KOR system in males, and provide novel evidence that females may not exhibit this ontogenetic change. Given the prevalence of stress and drug exposure during the adolescent period, differences in how the KOR system may mediate the effects of these exposures across age and sex should be explored.