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Background: Inhibition of NMDA receptor function in brain is believed to be an important action of ethanol (EtOH). To investigate EtOH inhibition of NMDA receptor responses in vivo, the interaction of these agents in brain after different routes of administration were investigated by using transcription factor Fos protein expression to follow NMDA receptor activation and EtOH inhibition of this response., Methods: The induction of Fos-like immunoreactivity (Fos-LI) in 38 regions of the rat brain was measured 2 hr after treatment with NMDA, EtOH, or both. To determine the relative contribution of abdominal drug effects on Fos induction, rats received either intraperitoneal (ip) or intragastric (ig) EtOH and ip or intravenous (iv) NMDA. Rats received EtOH (2.5 g/kg ip or 4 g/kg ig) or vehicle 15 min before NMDA (125 mg/kg ip or 60 mg/kg iv) or vehicle., Results: For the 38 forebrain regions examined, ip and iv NMDA significantly induced Fos-LI in 13 and 32 regions, respectively. These effects occurred without elicitation of tonic-clonic seizure activity and were strong after iv NMDA in the frontal, prefrontal, and cingulate cortices, supraoptic nucleus, anterior lateral septum, and dentate gyrus. For EtOH, prominent Fos-LI induction was found in the central amygdala, dorsolateral bed nucleus of the stria terminalis, Edinger-Westphal nucleus, and paraventricular hypothalamus. Despite ip and ig EtOH induction of Fos-LI in these regions, the major effect of EtOH was to block NMDA-induced Fos-LI in 8 of 13 (ip) and 27 of 32 (ig) of the NMDA-positive regions, respectively, including retrosplenial, cingulate, and medial prefrontal cortices, central amygdala, and taenia tecta., Conclusions: These results provide new evidence for the regionally specific functional interactions of EtOH on NMDA receptors in vivo. Moreover, these results support efforts to identify brain region-specific targets for EtOH and EtOH-induced changes in gene expression.

Background: Electrophysiological recording reveals that only a portion of cerebellar Purkinje neurons are sensitive to ethanol enhancement of gamma-aminobutyric acid (GABA) responses. Although activation of beta-adrenergic receptors permits ethanol enhancement of GABA function from some cerebellar Purkinje neurons, other neurons remain insensitive to ethanol. These findings are consistent with the finding that other external neural inputs are required to allow ethanol enhancement of GABA responses from Purkinje neurons. Because of a high expression of GABA(B) receptors on Purkinje cells, we tested whether activation of GABA(B) receptors might modulate the action of ethanol on GABA responsiveness., Methods: Extracellular single-unit electrophysiological recording was used to investigate the effects of ethanol on responses to GABA and muscimol (a GABA(A) agonist) from cerebellar Purkinje neurons. Drugs tested were baclophen (a GABA(B) agonist) and CGP35348 (a GABA(B) antagonist)., Results: Ethanol did not enhance responses to GABA and muscimol from all Purkinje neurons. Systemic administration of the GABA(B) agonist, baclophen (3 mg/kg intravenously), permitted ethanol to enhance GABA inhibition from approximately 75% of cerebellar Purkinje neurons not initially enhanced by ethanol. Local iontophoretic application of baclophen to Purkinje neurons also allowed ethanol to enhance GABA and muscimol responsiveness from a portion of neurons in which ethanol initially did not affect their actions. An inhibitory action of ethanol on responses to GABA and muscimol, which was also influenced by baclophen, was observed from some Purkinje neurons. From Purkinje neurons initially sensitive to ethanol enhancement of GABA and muscimol function, administration of CGP35348, a GABA(B) antagonist, diminished the effect of ethanol on the responsiveness of these agonists from the majority (9/15) of neurons., Conclusions: The present findings demonstrated that baclophen allows ethanol enhancement of GABA and muscimol responsiveness from some, but not all, cerebellar Purkinje neurons initially not sensitive to ethanol. Likewise, a GABA(B) antagonist can diminish ethanol enhancement of GABA and muscimol responses from some ethanol-sensitive neurons. Thus, these results emphasize that GABA(B) receptors on a portion of Purkinje neurons act as an auxiliary neural input that allows ethanol enhancement of GABA responses. Consequently, receptor structure alone does not account for the action of ethanol on GABA(A) receptor function on this cell type.

Background: A majority of alcoholics also smoke, suggesting that alcohol and nicotine share a common action on nicotinic cholinergic receptors., Methods: Extracellular single-unit recording was used to investigate the effects of ethanol on responses to nicotine from rat cerebellar interneurons and medial septal neurons., Results: Nicotine produced inhibition from medial septal neurons, but increased neural activity of cerebellar interneurons. When ethanol was applied locally to cerebellar interneurons, the excitatory response to nicotine was enhanced in a dose-related manner. Nicotine-induced inhibition from medial septal neurons was reduced by ethanol from the majority of neurons, but a dose relationship for this inhibition by ethanol was not observed. Ethanol affected responses to nicotine from over 90% of all neurons investigated at these sites. Initially, it was established that the nicotinic antagonists, methyllycaconitine (MLA) and alpha-bungarotoxin, which affect a nicotinic cholinergic (nACh) receptor with an alpha7 subunit, had similar actions on responses to nicotine from individual medial septal cells and cerebellar interneurons. When MLA was tested against responses to nicotine from neurons in the two brain regions, MLA antagonized responses to nicotine from only 27% of the neurons rather than the 90% found for ethanol. This latter observation provided evidence that ethanol was affecting neurons with MLA-insensitive receptors. When the actions of ethanol on responses to nicotine were compared directly with the action of MLA on the same medial septal neurons, both ethanol and MLA caused a greater than 50% antagonism of the response to nicotine, indicative that nACh receptors with the alpha7 subunit were sensitive to ethanol., Conclusions: Collectively, these data provide evidence that ethanol affects responses to nicotine not only from nACh receptors on medial septal cells and cerebellar interneurons containing an alpha7 subunit (i.e., MLA-sensitive receptors), but also from nACh receptor subtypes without this specific nACh receptor subunit (i.e., MLA-insensitive receptors).

The observation that cerebellar Purkinje cells contain type-I benzodiazepine-sensitive GABA(A) receptors is consistent with findings in the present work that the majority of Purkinje neurons are sensitive to enhancement of GABA by the type-1 benzodiazepine agonist, zolpidem. Previous work has demonstrated a relation between zolpidem and ethanol enhancement of GABA responses in several brain regions, but had not tested Purkinje neurons. Therefore, given that a majority of Purkinje neurons were found to be sensitive to zolpidem, ethanol would have been expected to enhance GABA responses from this cell type. However, in agreement with earlier electrophysiological studies, ethanol enhanced GABA inhibitory responses from only a small proportion of these cerebellar Purkinje neurons. Rather than enhancement of GABA, local application of ethanol either inhibited or did not affect responses to GABA from a majority of cerebellar-Purkinje neurons. Nonetheless, as previously reported, a portion of the Purkinje neurons initially insensitive to ethanol enhancement of GABA became sensitive to this action of ethanol with co-application of the beta-adrenergic agonist, isoproterenol. Thus, these results collectively implicate a beta-adrenergic input dependency for ethanol enhancement of GABA from some, but not all, cerebellar Purkinje neurons sensitive to zolpidem. Because a beta-adrenergic input did not allow ethanol enhancement of GABA from all Purkinje neurons, future studies should explore the possibility that other auxiliary neural inputs to zolpidem-sensitive cerebellar Purkinje neurons may be required for ethanol enhancement of GABA responsiveness when a beta-adrenergic input does not have this action. Likewise, knowing that the action of zolpidem can predict ethanol enhancement of GABA in other brain regions, the present findings suggest that a future determination be made concerning whether zolpidem-sensitive neurons in these other regions of brain require a beta-adrenergic or an alternative neural input for ethanol enhancement of GABA responses.

Previous research has suggested that rats tested at 28 to 30 days of age show a marked subsensitivity to the sedative effects of ethanol. In the present study, rats of different ages were tested for aerial righting following acute ethanol (3 g/kg) treatment. These results were compared with the effects of the atypical benzodiazepine zolpidem (3 and 5 mg/kg) and pentobarbital (10 and 15 mg/kg). Animals tested at 25, 28, or 35 days of age were significantly less impaired by ethanol than preweanling rats (age 20 days) or older rats (age 65 to 75 days), whereas animals tested at 25 or 28 days of age were less impaired by the higher dose of zolpidem. With pentobarbital, the most distinct age-related trend was greater impairment in 20-day-old rats. Because ethanol may be active at the same type I GABA(A) receptor site selectively labeled by [3H]zolpidem, levels of [3H]zolpidem binding were determined for rats of different ages. Although some brain regions showed progressive increases in binding of [3H]zolpidem across development, other regions demonstrated increased binding from day 12 or 17 to day 20, then a plateau of binding levels across days 20, 25, and 28, with further increases occurring by day 36 or day 60. This pattern was observed in the cingulate cortex, medial septal nucleus, globus pallidus, inferior colliculus, red nucleus, and cerebellum. Overall, the results indicate that the period of subsensitivity to the sedative effects of ethanol is coincident with a change in the developmental pattern of GABA(A) receptor sites targeted by [3H]zolpidem.

The ethanol withdrawal syndrome includes anxiety as a prominent symptom. Because the extent that specific regions of brain are critical to the generation of this emotional state is unknown, Fos-like immunoreactivity (Fos-LI) was used to associate specific regions of the rat brain with the anxiety component of the ethanol withdrawal syndrome exacerbated by an air puff challenge in rats. Chronic ethanol liquid diet was administered intragastrically for 4 days or by having the rats consume the diet for 14 days. During withdrawal from either treatment protocol, Fos-LI was induced most prominently in forebrain areas, although the midbrain and hindbrain were also represented. Included in these Fos-LI positive regions were many cortical regions, septum, accumbens, claustrum, amygdala, paraventricular nucleus of the thalamus and hypothalamus, hippocampus, locus coeruleus, and central gray. Fos-LI expression differed mostly in intensity between the two treatment and withdrawal protocols, with the gastric protocol producing the greatest Fos-LI induction in most brain regions. The threshold for air puff-induced ultrasonic vocalizations was decreased, and the number of vocalizations was increased and the period of vocalization was extended. These behavioral data indicate that aversively motivated responding in rats during ethanol withdrawal can be readily quantified with the ultrasonic vocalizations test without precipitating convulsive activity. Furthermore, a comparison of the effects of the air puff challenge versus withdrawal on Fos-LI indicated that the behavioral state induced in these two situations share functional neuroanatomical features. Some regions--such as the accumbens core, medial septum, subregions of the amygdala, hippocampus, substantia nigra, and cerebellum--exhibited little Fos-LI during withdrawal and also did not exhibit strong increases after the addition of the air puff challenge. However, other regions-such as the cerebral cortex (medial prefrontal, frontal, cingulate and ventrolateral orbital, claustrum, and tenia tecta), hypothalamus, and locus ceoruleus- exhibited Fos-LI at levels higher than that seen after either the ethanol withdrawal or puff challenge alone. These overlapping patterns of Fos-LI in specific regions of the brain, activated by both ethanol withdrawal and an anxiety provoking behavioral challenge, suggest that specific neuroanatomical sites in brain are associated with the symptom of anxiety observed during the "ethanol withdrawal syndrome."

To further understand the relationship between emotional state and alcohol intake in rats, the tendency to emit ultrasonic vocalizations in response to an aversive, but nonpainful, air puff stimulus was tested in several rat lines. Included in this group were Maudsley Reactive (MR) and Non-Reactive (MNR) rats, and several lines of rats with either high ethanol preference or a low ethanol preference: Preferring, (P), Alko-Alcohol (AA), and Fawn-Hooded (FH) animals; and Non-Preferring (NP), Alko-Non-Alcohol (ANA), and Flinders Resistant Line (FRL). MR rats emitted fewer ultrasonic vocalizations (USVs) and showed less preference for ethanol than did MNR animals. An overall analysis that included the P, NP, FH, FRL, AA, and ANA groups demonstrated a significant negative correlation between the total number of USVs emitted and ethanol consumption. NP, FRL, and especially ANA rats (low ethanol-preferring) emitted the most USVs--to an extent similar to that typically found for normal rats. The duration of vocalizing was higher only in the NP and the FRL rats the relative to their P and FH comparison groups, respectively. In the ethanol-preferring and nonpreferring lines, the numbers of USVs emitted correlated positively with the duration of vocalizing, but not with the latency to vocalize, which in turn did not correlate strongly with ethanol intake. The latency to vocalize did not correlate significantly with ethanol intake across all drinking lines or MR or MNR rats, but was found to be higher in FH and AA rats relative to their nondrinking comparison groups. These associations suggest that the relationship between emotional state and ethanol drinking is complex and cannot be attributed to a simple elevated state of anxiety or emotionality. Further examination of the central nervous system mechanisms mediating the difference in USVs between paired lines of ethanol-preferring and nonpreferring rats may identify neurochemical factors that predict ethanol preference.

A strong association has been observed between [3H]zolpidem binding and the presence of gamma-aminobutyric acid (GABAA) receptor mRNA for alpha 1-, beta 2-, and gamma 2-subunits in specific brain regions. This correlates with observed sensitivity of individual neurons to zolpidem and ethanol in these same regions. Previous studies using homogenate binding approaches showed small alterations in [3H]zolpidem binding levels after chronic ethanol exposure. This study was undertaken to ascertain if there is regional specificity of the effects of chronic ethanol administration on [3H]zolpidem binding levels. Chronic ethanol administration induced small, but significant alterations in [3H]zolpidem (5 nM) binding in the inferior colliculus, substantia nigra, and the medial septum. [3H]Zolpidem binding was increased in the inferior colliculus and substantia nigra, and decreased in the medial septum. No significant differences in [3H]zolpidem binding were noted in any other brain area analyzed, including the cortex and cerebellum. These findings show that chronic ethanol administration has small effects on [3H]zolpidem binding, although they occur in a site-specific and bidirectional manner. Moreover, there is no correlation between changes in [3H]zolpidem binding and alterations in GABAA receptor subunit expression.

Several lines of research have suggested a link between the reward value of a drug and its ability to stimulate locomotion. One goal of the present study was to determine whether ethanol preferentially stimulates locomotor activity in lines of rat that show a preference for ethanol. A secondary goal was to determine the extent to which the benzodiazepine-like and NMDA antagonistic action of ethanol accounted for its effect on locomotor activity. To meet these goals, the effects of varying doses of ethanol (0.125-1.0 g/kg), MK-801 (0.1-0.3 mg/kg), and chlordiazepoxide (0.3-3 mg/kg) on locomotor activity were studied in several lines of rats that had been habituated to the testing procedure. The effect of low doses of ethanol on motor activity in the Alcohol-Preferring (P) and Fawn-Hooded rats, which show a strong ethanol preference, were similar to those of the alcohol-nonpreferring (NP), Flinders Sensitive Line, and Flinders Resistant Line rats. Only the Flinder Resistant Line rats showed a small, but significant increase in locomotor activity after the administration of ethanol. The highest dose of ethanol (1.0 g/kg) produced locomotor depression in all lines except the P and NP lines, which were not tested at this dose. These findings do not support a link between locomotor stimulation by ethanol and ethanol preference. In contrast, all lines exhibited locomotor stimulation after moderate (0.1-0.3 mg/kg) doses of MK-801, but did not exhibit increases in activity following any dose of chlordiazepoxide. These data indicate that the profiles of activity after MK-801 and chlordiazepoxide were distinct from that of ethanol in the various rat lines.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of ethanol, chlordiazepoxide, and MK-801 on performance in the elevated-plus maze and on activity measured in a circular activity monitor were compared in Sprague-Dawley rats to determine whether these effects of ethanol could be explained by its action on either GABAA or NMDA receptors. Both ethanol and chlordiazepoxide produced an increase in the time spent in the open arms of the elevated-plus maze and in the ratio of open arm to total arm entries, indicative of an anxiolytic action of these drugs. MK-801 did not alter either the time spent in the open arms or the ratio of open to total arm entries. Chlordiazepoxide and MK-801 produced an increase in total arm entries that suggested that these compounds were increasing locomotor activity. Ethanol also increased total arm entries, but the effect was not statistically reliable. Following habituation to an activity monitor, neither ethanol nor chlordiazepoxide increased activity in this task, whereas MK-801 produced a robust increase in locomotion. Additionally, neither ethanol nor chlordiazepoxide blocked the MK-801-induced locomotor stimulation. The latter finding suggests that the effects of ethanol on GABAA receptors was not blocking an increased activity level produced by its antagonism of NMDA. Additionally, these results indicate that the anxiolytic and locomotor action of ethanol in rats parallel the effects of a benzodiazepine and not those of an NMDA antagonist. Finally, these results suggest that the consequence of ethanol's antagonism of NMDA receptor function is more restricted than that produced by MK-801.

Because multiple withdrawals from chronic ethanol treatment facilitate the rate of kindling from the inferior collicular cortex, the following studies sought to identify potential sources of this long-term change in seizure sensitivity. When rats received 6 or 10 withdrawals from a 5-day ethanol liquid diet, a significant decrease was found in the threshold frequency for seizure genesis, 6-7 days post-withdrawal. The magnitude of this change was related to the number of withdrawals (6 withdrawals, -2.0 +/- 0.3 Hz; 10 withdrawals, -3.2 +/- 1.2 Hz). Thus, multiple ethanol withdrawals increased seizure sensitivity within the inferior collicular cortex. On the following day in the same animals, changes in inhibitory or excitatory function were evaluated within the inferior collicular cortex. We found a withdrawal-related increase in the effectiveness of bicuculline to reduce the seizure threshold current within the inferior collicular cortex. Seizure sensitivity to collicular N-methyl-D-aspartic acid (NMDA) microinjection was decreased after 6 ethanol withdrawals, and increased after 10 withdrawals, but control liquid diet animals exhibited similar responses to collicular NMDA microinjection. Therefore, multiple withdrawals from ethanol alters the seizure sensitivity within the inferior collicular cortex. One possible contribution to this change is a local decrease in GABA inhibitory function.

The present investigation tested the hypothesis that multiple withdrawals from chronic ethanol treatment "kindles" seizure activity. Two animal models of kindled seizure activity--electrical stimulation of the inferior collicular cortex or the amygdala--were used to evaluate this hypothesis. Four withdrawals from a 12-day ethanol-liquid diet regimen facilitated the seizure kindling rate in the inferior collicular cortex, when the stimulation was initiated 7 days after the last withdrawal. In contrast, four withdrawals from this chronic ethanol regimen significantly attenuated the rate of amygdaloid kindling. When the withdrawals were increased to six or 10 using a 5-day chronic ethanol treatment schedule, the kindling rate in the inferior collicular cortex proved directly proportional to the withdrawal number. Continuous ethanol exposure over the same period as the 10 withdrawal group also facilitated the inferior collicular kindling rate, but not to the extent found in the 10 withdrawal group. A before, 10 withdrawals from the 5-day chronic ethanol liquid diet treatment attenuated the rate of amygdaloid kindling. Thus, this kindling action of repeated ethanol withdrawals appears specific to seizures originating from the inferior collicular cortex, not the limbic system. These findings support a previous hypothesis for a kindling etiology of alcoholism related seizures.

The effects of four putative ethanol antagonists [thyrotropin releasing hormone (TRH), naloxone, d-amphetamine, and fenmetozole] on two distinct behavioral actions of ethanol were compared. TRH (20-40 mg/kg) reduced ethanol-induced impairment of the aerial righting reflex (ARR) but enhanced the ethanol-induced increase in punished drinking (anticonflict effect). Naloxone antagonized both actions of ethanol but only at high doses (20-60 mg/kg). Amphetamine (1-4 mg/kg) abolished the ethanol effect on punished drinking but did not alter its impairment of the ARR (1-8 mg/kg). Conversely, fenmetozole antagonized the ethanol impairment of the ARR (15-30 mg/kg) but not ethanol's anticonflict action. The inconsistent pattern of "antagonist" interactions of these drugs with the behavioral actions of ethanol suggests that ethanol alters several neurochemical systems to produce its behavioral effects.

A conflict task that does not require food or water deprivation is described. Drugs with known anticonflict action including chlordiazepoxide, methysergide, ethanol, and TRH were active in this test while amphetamine, buspirone, and morphine were not. Because this procedure did not require food or water deprivation, the effect of chronic ethanol administered in a liquid diet on the anticonflict actions of ethanol and chlordiazepoxide could be examined. Seven or 12 days of chronic ethanol treatment resulted in tolerance to the acute anticonflict action of ethanol whereas 3 days of treatment did not. Tolerance was not observed to a dose of ethanol administered 3 hr after a previous ethanol treatment. Following a chronic ethanol liquid diet, cross-tolerance to the anticonflict action of chloridazepoxide was observed. This latter observation is consistent with the view that ethanol influences a neural mechanism associated with benzodiazepine function. The importance of tolerance to the anticonflict action of ethanol is discussed.

Both thyrotropin-releasing hormone (TRH) and RO 15-4513 antagonize ethanol-induced depression, but this common property does not infer that both compounds share similar mechanisms of action. In the present studies, both TRH (30 mg/kg, i.p.) and RO 15-4513 (10 mg/kg, i.p.) reversed ethanol-induced depression of locomotor activity, in accord with previous reports. However, the benzodiazepine antagonist, RO 15-1788, blocked this action of RO 15-4513, while exerting no effect on the analeptic action of TRH. Using a model of seizure activity electrically elicited from the inferior colliculus, ethanol exerted a dose-related attenuation of seizure activity. This anticonvulsant action of ethanol was not altered by TRH (30 mg/kg, i.p.), but RO 15-4513 (3 mg/kg) reversed the effect of the 0.5, but not the 1.0 g/kg, dose of ethanol. In addition, pretreatment with RO 15-4513 (1 or 3 mg/kg, i.p.), but not TRH (30 mg/kg, i.p.), caused seizure generalization into the forebrain following inferior collicular stimulation, further verifying the proconvulsant properties of RO 15-4513. In conclusion, the analeptic action of TRH appears independent of benzodiazepine activity, and in contrast to RO 15-4513, TRH does not exhibit proconvulsant properties. Furthermore, because TRH did not antagonize both depressant actions of ethanol studied, it appears unlikely that TRH directly interacts with the molecular basis of ethanol action.