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.

Prenatal methadone exposure leads to long-term memory impairments and disruptions of dentate granule cell function in a sex-dependent manner.

Prenatal opioid exposures lead to extensive cognitive and emotion-regulation problems in children, persisting at least through school-age. Methadone, an opioid typically used for the treatment of opioid use disorder, has been approved for use in pregnant women for several decades. Importantly, however, the impacts of prenatal methadone exposure (PME), particularly on offspring as they progress into adulthood, has not been extensively examined. In recent years, children and young animal models have shown cognitive deficits related to PME, including evidence of hippocampal dysfunction. The present work aims to examine the persistent nature of these deficits, as well as determine how they may differ by sex. Pregnant Sprague-Dawley rats either received subcutaneous methadone or water injections twice daily from gestational days 3-20 or were left undisturbed. Following postnatal day 70, male and female offspring were behaviourally tested for impairments in recognition memory using the Novel Object Recognition task and working spatial memory through Spontaneous Alternation. Additionally, using whole-cell patch-clamp electrophysiology, hippocampal dentate granule cell function was examined in adult offspring. Results indicate that methadone-exposed females showed decreased excitability and increased inhibition of dentate granule cells compared to naïve controls, while males did not. These findings were accompanied by impairments in female working spatial memory and altered behaviour in the Object Recognition task. Overall, this work supports the continued investigation of the long-term effects of PME on adult male and female learning and memory, as well as promotes further exploration of adult hippocampal function as a neural mechanism impacted by this exposure.

Moderate prenatal alcohol exposure modifies sex-specific CRFR1 activity in the central amygdala and anxiety-like behavior in adolescent offspring.

Anxiety disorders are highly prevalent among individuals with a history of prenatal alcohol exposure (PAE), and adolescent rodents demonstrate anxiety-like behavior following moderate PAE on Gestational Day (G) 12. A likely systemic target of PAE is the stress peptide corticotropin-releasing factor (CRF), as activation of CRF receptor 1 (CRFR1) in the medial nucleus of the central amygdala (CeM) is known to increase anxiety-like behavior in adults. To determine if CRF-CRFR1 interactions underly PAE-induced anxiety, functional changes in CRF system activity were investigated in adolescent male and female Sprague Dawley rats following G12 PAE. Compared to air-exposed controls, PAE increased basal spontaneous (s) inhibitory postsynaptic current (IPSC) frequency in the CeM of males, but not females. Furthermore, PAE blunted CRFR1-regulated miniature (m) IPSCs in a sex- and concentration-specific manner, and only PAE males demonstrated tonic CRFR1 activity in the CeM. It was further determined that G12 PAE decreased CRFR1 mRNA in the CeM of males while increasing regional expression in females. Finally, infusion of a CRFR1 agonist into the CeM of adolescents produced a blunted expression of CRFR1-induced anxiety-like behavior exclusively in PAE males, mirroring the blunted physiology demonstrated by PAE males. Cumulatively, these data suggest that CRFR1 function within the CeM is age- and sex-specific, and PAE not only increases the expression of anxiety-like behavior, but may reduce the efficacy of treatment for PAE-induced anxiety through CRFR1-associated mechanisms. Therefore, future research will be necessary to develop targeted treatment of anxiety disorders in individuals with a history of PAE.

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.

Factors of sex and age dictate the regulation of GABAergic activity by corticotropin-releasing factor receptor 1 in the medial sub-nucleus of the central amygdala.

Adolescents are phenotypically characterized with hyper-sensitivity to stress and inappropriate response to stress-inducing events. Despite behavioral distinctions from adults, investigations of developmental shifts in the function of stress peptide corticotropin-releasing factor (CRF) are generally limited. Rodent models have determined that CRF receptor 1 (CRFR1) activation within the central amygdala is associated with a stress response and induces increased GABAergic synaptic neurotransmission within adult males. To investigate age- and sex-specific function of this system, we performed whole-cell patch clamp electrophysiology in brain slices from naive adolescent (postnatal days (P) 40-49) and adult (>P70) male and female Sprague Dawley rats to assess GABAergic activity in the medial central amygdala (CeM). Our results indicate a dynamic influence of age and sex on neuronal excitability within this region, as well as basal spontaneous and miniature (m) inhibitory post-synaptic currents (IPSCs) in the CeM. In addition to replicating prior findings of CRFR1-regulated increases in mIPSC frequency in adult males, we found that the selective CRFR1 agonist, Stressin-1, attenuated mIPSC frequency in adolescent males, at a concentration that did not produce an effect in adult males. Importantly, this age-specific distinction was absent in females, as Stressin-1 attenuated mIPSC frequency in both adolescent and adult females. Finally, an increase in mIPSC frequency in response to the CRF1R antagonist, NBI 35965, was observed only in the CeM of adult males. Together, these data emphasize the robust influence of age and sex on neurophysiological function of a brain region involved in the production of the stress response.

Moderate Adolescent Ethanol Vapor Exposure and Acute Stress in Adulthood: Sex-Dependent Effects on Social Behavior and Ethanol Intake in Sprague-Dawley Rats.

Adolescent alcohol use can lead to numerous consequences, including altered stress reactivity and higher risk for later anxiety and alcohol use disorders. Many studies have examined the consequences of heavy ethanol exposure in adolescence, but far less is understood about lower levels of intoxication. The present study examined moderate adolescent ethanol exposure as a possible factor in increasing stress reactivity in adulthood, measured through general and social anxiety-like behaviors, as well voluntary ethanol intake. Male and female Sprague-Dawley rats underwent an adolescent chronic intermittent ethanol (aCIE) vapor exposure during early adolescence, reaching moderate blood ethanol concentrations. Animals then underwent two days of forced swim stress in adulthood. We found that ethanol-exposed males consumed more ethanol than their air counterparts and an interesting stress and ethanol exposure interaction in males. There were no significant effects on voluntary drinking in females. However, the social interaction test revealed increased play-fighting behavior in ethanol-exposed females and reduced social preference in females after two days of stress exposure. Overall, this work provides evidence for sex-specific, long-term effects of moderate aCIE and susceptibility to acute stress in adulthood.

Increased ethanol intake is associated with social anxiety in offspring exposed to ethanol on gestational day 12.

Prenatal alcohol exposure (PAE) can result in physical, cognitive, and neurological deficits termed Fetal Alcohol Spectrum Disorder (FASD). Deficits in social functioning associated with PAE are frequently observed and persist throughout the lifespan. Social impairments, such as social anxiety, are associated with increased alcohol abuse, which is also highly pervasive following PAE. Yet, the relationship between PAE-induced social alterations and alcohol intake later in life is not well understood. In order to test this relationship, we exposed pregnant female Sprague Dawley rats to a single instance of PAE on gestational day 12, a period of substantial neural development, and tested offspring in adulthood (postnatal day 63) in a modified social interaction test followed by alternating alone and social ethanol intake sessions. Consistent with our previous findings, we found that, in general, PAE reduced social preference (measure of social anxiety-like behavior) in female but not male adults. However, ethanol intake was significantly higher in the PAE group regardless of sex. When dividing subjects according to level of social anxiety-like behavior (low, medium, or high), PAE males (under both drinking contexts) and control females (under the social drinking context) with a high social anxiety phenotype showed the highest level of ethanol intake. Taken together, these data indicate that PAE differentially affects the interactions between social anxiety, ethanol intake, and drinking context in males and females. These findings extend our understanding of the complexity and persistence of PAE's sex-dependent effects into adulthood.

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.

Stress alters social behavior and sensitivity to pharmacological activation of kappa opioid receptors in an age-specific manner in Sprague Dawley rats.

The dynorphin/kappa opioid receptor (DYN/KOR) system has been identified as a primary target of stress due to behavioral effects, such as dysphoria, aversion, and anxiety-like alterations that result from activation of this system. Numerous adaptations in the DYN/KOR system have also been identified in response to stress. However, whereas most studies examining the function of the DYN/KOR system have been conducted in adult rodents, there is growing evidence suggesting that this system is ontogenetically regulated. Likewise, the outcome of exposure to stress also differs across ontogeny. Based on these developmental similarities, the objective of this study was to systematically test effects of a selective KOR agonist, U-62066, on various aspects of social behavior across ontogeny in non-stressed male and female rats as well as in males and females with a prior history of repeated exposure to restraint (90 min/day, 5 exposures). We found that the social consequences of repeated restraint differed as a function of age: juvenile stress produced substantial increases in play fighting, whereas adolescent and adult stress resulted in decreases in social investigation and social preference. The KOR agonist U-62066 dose-dependently reduced social behaviors in non-stressed adults, producing social avoidance at the highest dose tested, while younger animals displayed reduced sensitivity to this socially suppressing effect of U-62066. Interestingly, in stressed animals, the socially suppressing effects of the KOR agonist were blunted at all ages, with juveniles and adolescents exhibiting increased social preference in response to certain doses of U-62066. Taken together, these findings support the hypothesis that the DYN/KOR system changes with age and differentially responds and adapts to stress across development.

Moderate Maternal Alcohol Exposure on Gestational Day 12 Impacts Anxiety-Like Behavior in Offspring.

Among the numerous consequences of prenatal alcohol exposure (PAE) is an increase in anxiety-like behavior that can prove debilitating to daily functioning. A significant body of literature has linked gestational day 12 (G12) heavy ethanol exposure with social anxiety, evident in adolescent males and females. However, the association between non-social anxiety-like behavior and moderate alcohol exposure, a more common pattern of drinking in pregnant women, is yet unidentified. To model moderate PAE (mPAE), we exposed pregnant Sprague-Dawley rats to either room air or vaporized ethanol for 6 h on G12. Adolescent offspring were then tested on postnatal days (P) 41-47 in one of the following four anxiety assays: novelty-induced hypophagia (NIH), elevated plus maze (EPM), light-dark box (LDB) and open-field (OF). Our findings revealed significant increases in measures of anxiety-like behavior in male PAE offspring in the NIH, LDB and OF, with no differences observed in females on any test. Additionally, male offspring who demonstrated heightened anxiety-like behavior as adolescents demonstrated decreased anxiety-like behavior in adulthood, as measured by a marble-burying test (MBT), while females continued to be unaffected in adulthood. These results suggest that mPAE leads to dynamic changes in anxiety-like behavior exclusively in male offspring.

Acute prenatal exposure to ethanol on gestational day 12 elicits opposing deficits in social behaviors and anxiety-like behaviors in Sprague Dawley rats.

Our previous research has shown that in Long Evans rats acute prenatal exposure to a high dose of ethanol on gestational day (G) 12 produces social deficits in male offspring and elicits substantial decreases in social preference relative to controls, in late adolescents and adults regardless of sex. In order to generalize the observed detrimental effects of ethanol exposure on G12, pregnant female Sprague Dawley rats were exposed to ethanol or saline and their offspring were assessed in a modified social interaction (SI) test as early adolescents, late adolescents, or young adults. Anxiety-like behavior was also assessed in adults using the elevated plus maze (EPM) or the light/dark box (LDB) test. Age- and sex-dependent social alterations were evident in ethanol-exposed animals. Ethanol-exposed males showed deficits in social investigation at all ages and age-dependent alterations in social preference. Play fighting was not affected in males. In contrast, ethanol-exposed early adolescent females showed no changes in social interactions, whereas older females demonstrated social deficits and social indifference. In adulthood, anxiety-like behavior was decreased in males and females prenatally exposed to ethanol in the EPM, but not the LDB. These findings suggest that social alterations associated with acute exposure to ethanol on G12 are not strain-specific, although they are more pronounced in Long Evans males and Sprague Dawley females. Furthermore, given that anxiety-like behaviors were attenuated in a test-specific manner, this study indicates that early ethanol exposure can have differential effects on different forms of anxiety.

Inhibitors of Calcium-Activated Anion Channels Modulate Hypnotic Ethanol Responses in Adult Sprague Dawley Rats.

BACKGROUND: Ethanol is widely known for its depressant effects; however, the underlying neurobiological mechanisms are not clear. Calcium-activated anion channels (CAACs) contribute to extracellular chloride levels and thus may be involved in regulating inhibitory mechanisms within the central nervous system. Therefore, we hypothesized that CAACs influence ethanol behavioral sensitivity by altering CAAC expression. METHODS: We assessed the role of CAACs in ethanol-induced loss of righting reflex (LORR) and locomotor activity using intracerebroventricular infusions of several nonselective CAAC blockers. CAAC expression was determined after ethanol exposure. RESULTS: Ethanol-induced LORR (4.0 g/kg, intraperitoneally [i.p.]) was significantly attenuated by all 4 CAAC blockers. Blocking CAACs did not impact ethanol's low-dose (1.5 g/kg, i.p.) locomotor-impairing effects. Biochemical analysis of CAAC protein expression revealed that cortical Bestrophin1 (Best1) and Tweety1 levels were reduced as early as 30 minutes following a single ethanol injection (3.5 g/kg, intraperitoneally [i.p.]) and remained decreased 24 hours later in P2 fractions. Cortical Best1 levels were also reduced following 1.5 g/kg. However, CAAC expression was unaltered in the striatum following a single ethanol exposure. Ethanol did not affect Tweety2 levels in either brain region. CONCLUSIONS: These results suggest that CAACs are a major target of ethanol in vivo, and the regulation of these channels contributes to select behavioral actions of ethanol.

Sensitivity of GABAergic Tonic Currents to Acute Ethanol in Cerebellar Granule Neurons is Not Age- or δ Subunit-Dependent in Developing Rats.

BACKGROUND: The age of first exposure to ethanol (EtOH), as well as reduced sensitivity to its motor-impairing effects, are associated with a future predisposition to abuse EtOH. In adolescence, acute EtOH potentiates GABA transmission, including tonic inhibition mediated by δ-containing extrasynaptic GABAA receptors (GABAA Rs) in cerebellar granule neurons (CGNs), an effect that likely contributes to EtOH-induced motor impairment. Prenatal EtOH exposure is strikingly prevalent and is associated with increased EtOH abuse later in life; however, the acute effects of EtOH on GABA transmission in developing CGNs are unknown. METHODS: Using whole-cell patch-clamp electrophysiological techniques in acute brain slices, we examined the acute effects of EtOH on GABA transmission and functionally assessed the role of δ-containing GABAA Rs in CGNs of preweanling (postnatal day [P] 12 to 14) and postweanling (P28 to 30) male Sprague-Dawley rats. RESULTS: The magnitude of basal tonic currents were similar at both ages. However, 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride, an agonist with preferential affinity for δ-containing GABAA Rs, significantly potentiated tonic currents to a larger magnitude in CGNs from postweanlings compared to preweanlings. Conversely, acute application of EtOH (80 mM) significantly increased tonic currents and the frequency of spontaneous inhibitory postsynaptic currents to a similar extent in CGNs from pre- and postweanlings. CONCLUSIONS: These findings highlight the sensitivity of the developing cerebellum to EtOH. Furthermore, this study demonstrates age-dependent functional changes in a well-characterized circuitry that may contribute to the short- and long-term effects of prenatal exposure to EtOH.

Third trimester-equivalent ethanol exposure increases anxiety-like behavior and glutamatergic transmission in the basolateral amygdala.

Ethanol consumption during pregnancy produces a wide range of morphological and behavioral alterations known as fetal alcohol spectrum disorder (FASD). Among the behavioral deficits associated with FASD is an increased probability of developing anxiety disorders. Studies with animal models of FASD have demonstrated that ethanol exposure during the equivalent to the 1(st) and 2(nd) trimesters of human pregnancy increases anxiety-like behavior. Here, we examined the impact on this type of behavior of exposure to high doses of ethanol in vapor inhalation chambers during the rat equivalent to the human 3rd trimester of pregnancy (i.e., neonatal period in these animals). We evaluated anxiety-like behavior with the elevated plus maze. Using whole-cell patch-clamp electrophysiological techniques in brain slices, we also characterized glutamatergic and GABAergic synaptic transmission in the basolateral amygdala, a brain region that has been implicated to play a role in emotional behavior. We found that ethanol-exposed adolescent offspring preferred the closed arms over the open arms in the elevated plus maze and displayed lower head dipping activity than controls. Electrophysiological measurements showed an increase in the frequency of spontaneous and miniature excitatory postsynaptic currents in pyramidal neurons from the ethanol group. These findings suggest that high-dose ethanol exposure during the equivalent to the last trimester of human pregnancy can persistently increase excitatory synaptic inputs to principal neurons in the basolateral amygdala, leading to an increase in anxiety-like behaviors.

Construction of vapor chambers used to expose mice to alcohol during the equivalent of all three trimesters of human development.

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.

Repeated intermittent alcohol exposure during the third trimester-equivalent increases expression of the GABA(A) receptor δ subunit in cerebellar granule neurons and delays motor development in rats.

Exposure to ethanol (EtOH) during fetal development can lead to long-lasting alterations, including deficits in fine motor skills and motor learning. Studies suggest that these are, in part, a consequence of cerebellar damage. Cerebellar granule neurons (CGNs) are the gateway of information into the cerebellar cortex. Functionally, CGNs are heavily regulated by phasic and tonic GABAergic inhibition from Golgi cell interneurons; however, the effect of EtOH exposure on the development of GABAergic transmission in immature CGNs has not been investigated. To model EtOH exposure during the 3rd trimester-equivalent of human pregnancy, neonatal pups were exposed intermittently to high levels of vaporized EtOH from postnatal day (P) 2 to P12. This exposure gradually increased pup serum EtOH concentrations (SECs) to ∼60 mM (∼0.28 g/dl) during the 4 h of exposure. EtOH levels gradually decreased to baseline 8 h after the end of exposure. Surprisingly, basal tonic and phasic GABAergic currents in CGNs were not significantly affected by postnatal alcohol exposure (PAE). However, PAE increased δ subunit expression at P28 as detected by immunohistochemical and western blot analyses. Also, electrophysiological studies with an agonist that is highly selective for δ-containing GABA(A) receptors, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol (THIP), showed an increase in THIP-induced tonic current. Behavioral studies of PAE rats did not reveal any deficits in motor coordination, except for a delay in the acquisition of the mid-air righting reflex that was apparent at P15 to P18. These findings demonstrate that repeated intermittent exposure to high levels of EtOH during the equivalent of the last trimester of human pregnancy has significant but relatively subtle effects on motor coordination and GABAergic transmission in CGNs in rats.

Ethanol untangles the amygdala-anxiety circuit through tonic GABA inhibition.

The central amygdala (CeA) has a unique role in integrating stress and the rewarding effects of ethanol (EtOH) and plays a major role in the development of EtOH dependence via signaling of corticotropin-releasing factor (CRF). A recent report by Herman and colleagues (2013) entitled "Novel Subunit-Specific Tonic GABA Currents and Differential Effects of Ethanol in the Central Amygdala of CRF Receptor-1 Reporter Mice" is the first study to investigate inhibitory tonic currents in relation to CRF signaling in the CeA. The findings of that study significantly enhance our understanding of inhibitory tonic currents in the CeA and give insight into how EtOH may differentially affect CRF signaling within the CeA, leading to the development of EtOH dependence. This commentary will focus on the recent findings of Herman and colleagues and will discuss the effects of EtOH on the entire anxiety/emotion circuitry.

Na+/K+-ATPase inhibition partially mimics the ethanol-induced increase of the Golgi cell-dependent component of the tonic GABAergic current in rat cerebellar granule cells.

Cerebellar granule cells (CGNs) are one of many neurons that express phasic and tonic GABAergic conductances. Although it is well established that Golgi cells (GoCs) mediate phasic GABAergic currents in CGNs, their role in mediating tonic currents in CGNs (CGN-I(tonic)) is controversial. Earlier studies suggested that GoCs mediate a component of CGN-I(tonic) that is present only in preparations from immature rodents. However, more recent studies have detected a GoC-dependent component of CGN-I(tonic) in preparations of mature rodents. In addition, acute exposure to ethanol was shown to potentiate the GoC component of CGN-I(tonic) and to induce a parallel increase in spontaneous inhibitory postsynaptic current frequency at CGNs. Here, we tested the hypothesis that these effects of ethanol on GABAergic transmission in CGNs are mediated by inhibition of the Na(+)/K(+)-ATPase. We used whole-cell patch-clamp electrophysiology techniques in cerebellar slices of male rats (postnatal day 23-30). Under these conditions, we reliably detected a GoC-dependent component of CGN-I(tonic) that could be blocked with tetrodotoxin. Further analysis revealed a positive correlation between basal sIPSC frequency and the magnitude of the GoC-dependent component of CGN-I(tonic). Inhibition of the Na(+)/K(+)-ATPase with a submaximal concentration of ouabain partially mimicked the ethanol-induced potentiation of both phasic and tonic GABAergic currents in CGNs. Modeling studies suggest that selective inhibition of the Na(+)/K(+)-ATPase in GoCs can, in part, explain these effects of ethanol. These findings establish a novel mechanism of action of ethanol on GABAergic transmission in the central nervous system.

Moderate prenatal alcohol exposure reduces plasticity and alters NMDA receptor subunit composition in the dentate gyrus.

Although it is well documented that heavy consumption of alcohol during pregnancy impairs brain development, it remains controversial whether moderate consumption causes significant damage. Using a limited access, voluntary consumption paradigm, we recently demonstrated that moderate prenatal alcohol exposure (MPAE) is associated with dentate gyrus-dependent learning and memory deficits that are manifested in adulthood. Here, we identified a novel mechanism that may underlie this effect of MPAE. We found that MPAE mice exhibit deficits in NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) in the dentate gyrus. Further, using semiquantitative immunoblotting techniques, we found that the levels of GluN2B subunits were decreased in the synaptic membrane, while levels of C2'-containing GluN1 and GluN3A subunits were increased, in the dentate gyrus of MPAE mice. These data suggest that MPAE alters the subunit composition of synaptic NMDARs, leading to impaired NMDAR-dependent LTP in the dentate gyrus.

Thalamic glutamatergic afferents into the rat basolateral amygdala exhibit increased presynaptic glutamate function following withdrawal from chronic intermittent ethanol.

Amygdala glutamatergic neurotransmission regulates withdrawal induced anxiety-like behaviors following chronic ethanol exposure. The lateral/basolateral amygdala receives multiple glutamatergic projections that contribute to overall amygdala function. Our lab has previously shown that rat cortical (external capsule) afferents express postsynaptic alterations during chronic intermittent ethanol exposure and withdrawal. However, thalamic (internal capsule) afferents also provide crucial glutamatergic input during behavioral conditioning, and they have not been studied in the context of chronic drug exposure. We report here that these thalamic inputs express altered presynaptic function during withdrawal from chronic ethanol exposure. This is characterized by enhanced release probability, as exemplified by altered paired-pulse ratios and decreased failure rates of unitary events, and increased concentrations of synaptic glutamate. Quantal analysis further implicates a withdrawal-dependent enhancement of the readily releasable pool of vesicles as a probable mechanism. These functional alterations are accompanied by increased expression of vesicle associated protein markers. These data demonstrate that chronic ethanol modulation of glutamate neurotransmission in the rat lateral/basolateral amygdala is afferent-specific. Further, presynaptic regulation of lateral/basolateral amygdala thalamic inputs by chronic ethanol may be a novel neurobiological mechanism contributing to the increased anxiety-like behaviors that characterize withdrawal.