Ethanol Acutely Inhibits Ionotropic Glutamate Receptor‐Mediated Responses and Long‐Term Potentiation in the Developing CA1 Hippocampus

Background: Developmental ethanol (EtOH) exposure damages the hippocampus, causing long‐lasting alterations in learning and memory. Alterations in glutamatergic synaptic transmission and plasticity may play a role in the mechanism of action of EtOH. This signaling is fundamental for synaptogenesis, which occurs during the third trimester of human pregnancy (first 12 days of life in rats). Methods: Acute coronal brain slices were prepared from 7‐ to 9‐day‐old rats. Extracellular and patch‐clamp electrophysiological recording techniques were used to characterize the acute effects of EtOH on α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate receptor (AMPAR)‐ and N‐methyl‐d ‐aspartate receptor (NMDAR)‐mediated responses and long‐term potentiation (LTP) in the CA1 hippocampal region. Results: Ethanol (40 and 80 mM) inhibited AMPAR‐ and NMDAR‐mediated field excitatory postsynaptic potentials (fEPSPs). EtOH (80 mM) also reduced AMPAR‐mediated fEPSPs in the presence of an inhibitor of Ca²⁺ permeable AMPARs. The effect of 80 mM EtOH on NMDAR‐mediated fEPSPs was significantly greater in the presence of Mg²⁺. EtOH (80 mM) neither affected the paired‐pulse ratio of AMPAR‐mediated fEPSPs nor the presynaptic volley. The paired‐pulse ratio of AMPAR‐mediated excitatory postsynaptic currents was not affected either, and the amplitude of these currents was inhibited to a lesser extent than that of fEPSPs. EtOH (80 mM) inhibited LTP of AMPAR‐mediated fEPSPs. Conclusions: Acute EtOH exposure during the third‐trimester equivalent of human pregnancy inhibits hippocampal glutamatergic transmission and LTP induction, which could alter synapse refinement and ultimately contribute to the pathophysiology of fetal alcohol spectrum disorder.     

Behavioral Effects of Ethanol in Cerebellum Are Age Dependent: Potential System and Molecular Mechanisms

Background: Adolescent rats are less sensitive to the motor‐impairing effects of ethanol than adults. However, the cellular and molecular mechanisms underlying this age‐dependent effect of ethanol have yet to be fully elucidated. Method: Male rats of various ages were used to investigate ethanol‐induced ataxia and its underlying cellular correlates. In addition, Purkinje neurons from adolescent and adult rats were recorded both in vivo and in vitro. Finally, protein kinase C (PKCγ) expression was determined in 3 brain regions in both adolescent and adult rats. Results: The present multi‐methodological investigation confirms that adolescents are less sensitive to the motor‐impairing effects of ethanol, and this differential effect is not because of differential blood ethanol levels. In addition, we identify a particular cellular correlate that may underlie the reduced motor impairment. Specifically, the in vivo firing rate of cerebellar Purkinje neurons recorded from adolescent rats was insensitive to an acute ethanol challenge, while the firing rate of adult cerebellar Purkinje neurons was significantly depressed. Finally, it is demonstrated that PKCγ expression in the cortex and cerebellum mirrors the age‐dependent effect of ethanol: adolescents have significantly less PKCγ expression compared to adults. Conclusions: Adolescents are less sensitive than adults to the motor‐impairing effects of ethanol, and a similar effect is seen with in vivo electrophysiological recordings of cerebellar Purkinje neurons. While still under investigation, PKCγ expression mirrors the age effect of ethanol and may contribute to the age‐dependent differences in the ataxic effects of ethanol.     

Purkinje Cell Vulnerability to Developmental Ethanol Exposure in the Rat Cerebellum

BACKGROUND: Ethanol exposure is a consistent and reliable producer of neuronal toxicity, especially during periods of enhanced neuronal vulnerability. For rat cerebellar Purkinje cells, the postnatal period during the time of the brain growth spurt exhibits the greatest vulnerability to ethanol. Analyses of studies completed over more than 20 years provides sufficient detail to allow for the determination of the specific vulnerable window for ethanol-induced loss of Purkinje cells. METHODS: Data reporting Purkinje cell counts after ethanol exposure were compiled from 18 studies published since 1975. We conducted linear regression analysis between peak blood ethanol concentration (BEC) and percent reduction in Purkinje cells for the following individual postnatal (PN) days: PN4, PN5. PN6, PN7, and PN8 or beyond (+). The slope of the regression and the coefficients of determination (r2) were the primary factors of interest. Analysis of variance of the regressions was conducted to identify whether the slopes were significantly different from zero, or from each other. RESULTS: Exposures involving the PN4-6 period demonstrated the greatest significance in the relationship between BEC and reduction of Purkinje cell number. No significant differences were identified between different ethanol exposure techniques or for different Purkinje cell counting techniques. In addition, the initial day of exposure and the duration of exposure were not identified as critical variables. CONCLUSIONS: The literature database, developed over the past 20 years is clear in its direction that studies designed to identify the ethanol-specific mechanisms of Purkinje cell death are best designed to involve ethanol exposure during the vulnerable window of postnatal days 4-6.     

Serotonin Modulates Ethanol-Induced Depression in Cerebellar Purkinje Neurons

In the present study, we found that local application of serotonin (5-HT) potentiated ethanol-induced depressions of the spontaneous activity of Purkinje neurons in urethane-anesthetized rats. 5-HT also potentiated depressions induced by γ-aminobutyric acid; however, this modulatory response was quantitatively smaller than 5-HT-induced potentiation of ethanol depression. Previous reports suggested that the release of 5HT can be regulated by presynaptic 5-HT autoreceptors. We found that local application of methiothepin, which may induce 5-HT overflow through the inhibition of presynaptic autoreceptors, facilited ethanol-mediated responses. This methiothepin effect was greatly diminished in neonatally 5,7-dihydroxyhyptamine-lesioned animals, suggesting a presynaptic mechanism was involved. We also found that the 5-HT_IAantagonist UH301 did not attenuate 5-HT-facilitated ethanol reactions. On the other hand, local application of SHT_IB agonist CGS12066B potentiated ethanol-induced depression. Taken together, our data suggest that 5-HT can modulate ethanol-mediated electrophysiological depression, possibly mediated through CHT, receptors in the cerebellum.     

Electrophysiological Interactions of Ethanol with GABAergic Mechanisms in the Rat Cerebellum in Vivo

Biochemical studies indicate that ethanol (EtOH) will facilitate the activation of the GABA_A/CI^– channel, and behavioral studies demonstrate that EtOH-induced sedative and incoordinating effects can be potentiated by GABA mimetics and blocked by GABA antagonists. It has been difficult, however, to demonstrate an EtOH-induced potentiation of the depressant electrophysiological effects of locally applied GABA in mammalian brain in vivo. Similarly, in this study, local EtOH applications only infrequently caused potentiations of the depressant effects of microiontophoretically applied GABA on cerebellar Purkinje neurons, and this interaction was modest when present. The predominant interaction of locally applied EtOH was an antagonism of GABA-induced depressions of neuronal activity. However, the GABA_A receptor antagonist bicuculline reversibly and apparently competitively blocked the depressant effects of locally applied EtOH on single cerebellar Purkinje neurons. Our data suggest that EtOH potentiation of GABA responses alone is insufficient to account for EtOH-induced depressions of cerebellar Purkinje neurons. However, these data clearly imply that activation of a GABA_A receptor is required for the expression of EtOH-induced depressions of neuronal activity in this brain area. It is less clear how lower, nondepressant doses of EtOH interact with GABA mechanisms. We hypothesize that either the GABA_A receptor mechanism must be sensitized to the potentiative effects of EtOH through the influences of neuromodulatory and/or hormonal regulation, or that EtOH interacts directly with these regulatory processes.     

Sensation Seeking in Long‐Term Abstinent Alcoholics,Treatment‐Naïve Active Alcoholics,and Nonalcoholic Controls

Background: Elevated sensation seeking is associated with the development of alcohol dependence; however, it has not been studied in long‐term abstinent alcoholics. In the current study, we examine sensation seeking in middle‐aged long‐term abstinent alcoholics (LTAA) and in younger actively drinking treatment‐naïve alcoholics (TxN). Methods: A modified version of the Sensation Seeking Scale (SSS) was administered to 52 middle‐aged LTAA (average age = 46.6 years) and 86 younger TxN (average age = 31.2 years), each study with its own age and gender comparable nonalcoholic controls (NAC). The SSS was modified to remove items specifically associated with alcohol or drug use. The associations between the SSS and alcohol use and deviance proneness were examined. Results: The 2 NAC samples did not differ on the SSS, allowing the 2 NAC samples to be combined into a single control group (NAC = 118), and the LTAA and TxN samples to be directly compared without concern for cohort effects. LTAA did not differ from NAC on the SSS; however, the TxN group had higher SSS scores compared with NAC on all subscales except Boredom Susceptibility. Sensation seeking was comparably associated with lower socialization in each group. Conclusions: The results suggest that either sensation seeking normalizes with long‐term abstinence or that relatively normal levels of sensation seeking predict the ability to achieve long‐term abstinence. In either case, the results have important implications for our understanding of long‐term abstinence.     

Ethanol Decreases Glial Derived Neurotrophic Factor (GDNF) Protein Release but Not mRNA Expression and Increases GDNF-Stimulated Shc Phosphorylation in the Developing Cerebellum

BACKGROUND: Ethanol exposure during development leads to substantial neuronal loss in multiple regions of the brain. Although differentiating Purkinje cells of the cerebellum are particularly vulnerable to ethanol exposure, the mechanisms underlying ethanol-induced Purkinje cell loss have not been well defined. Our previous research indicated that exogenous Glial-Derived Neurotrophic Factor (GDNF) attenuated ethanol-induced Purkinje cell loss in cerebellar explant cultures, which suggests that ethanol, in turn, may decrease endogenous trophic factor-mediated survival mechanisms. METHODS: The present experiments used an explant culture model of the developing rat cerebellum to test the hypothesis that ethanol decreases endogenous trophic support by limiting the availability of trophic factors, such as GDNF, or by altering the activation of key adapter proteins such as Shc (Src homology domain carboxy-terminal) that couple GDNF binding to multiple intracellular signaling pathways. GDNF mRNA and protein levels were measured by reverse northern blot analysis and sandwich enzyme-linked immunosorbent assay respectively, whereas Shc phosphorylation was measured by immunoprecipitation/western immunoblot analysis. RESULTS: The developing cerebellum expresses both GDNF mRNA and protein in vitro. Ethanol exposure (68, 103, or 137 mM) had no effect on cerebellar levels of GDNF mRNA. However, ethanol (68 and 137 mM) decreased levels of GDNF protein released into culture medium. In addition, ethanol itself had no effect on She phosphorylation. However, in the presence of the highest dose of ethanol (137 mM) GDNF did stimulate Shc phosphorylation. CONCLUSIONS: Together, these results suggest that ethanol decreases GDNF-mediated trophic support of Purkinje cells in the developing cerebellum. However, GDNF in turn activates intracellular signaling pathways throughout the developing cerebellum as part of its Purkinje cell-selective neuroprotective response to ethanol exposure.