Selective effects of ethanol exposure on metabotropic glutamate receptor and guanine nucleotide stimulated phospholipase C activity in primary cultures of astrocytes

The effects of acute and chronic ethanol exposures on the stimulation of inositol specific phospholipase C by metabotropic glutamate receptor activation were determined in primary cultures of rat cortical astrocytes. Phospholipase C activity was monitored by the formation of [³H]inositol phosphates in the presence of lithium in cells prelabelled with [³H]inositol. Acute exposure to 200 mM ethanol had no significant effect on either basal or l-glutamate stimulated [³H]inositol phosphate formation. In cells chronically exposed to ethanol for 4 days, the [³H]inositol phosphate responses to l-glutamate, quisqualate, and the selective metabotropic receptor agonist, 1S,3R-1-amino-cyclopentane-1,3 dicarboxylic acid (trans-ACPD), were significantly inhibited when compared to control (untreated) cells. In contrast, chronic ethanol exposure had no significant effect on the [³H]inositol phosphate response to endothelin-1, a peptide structurally and functionally unrelated to l-glutamate. Similarly, the stimulation of [³H]inositol phosphate formation by the stable GTP analog, guanine 5′-(γ-thiotrisphosphate), was also unaffected by chronic ethanol exposure. The results suggest that chronic ethanol exposure does not affect the coupling of GTP binding proteins to phospholipase C, but rather acts in a selective manner to either alter the metabotropic receptor number or to disrupt the normal coupling of this receptor to its GTP binding protein, which may in turn affect receptor affinity.     

Ethanol effects on bradykinin-stimulated phosphoinositide hydrolysis in NG 108-15 neuroblastoma-glioma cells

The effect of short-and long-term ethanol exposure on bradykinin-stimulated hydrolysis of phosphatidylinositol 4.5-bisphosphate (PIP2) was investigated in neuroblastoma X glioma hybrid cells (NG 108-15). Acute exposure of 50-150 mM ethanol neither influenced the bradykinin-stimulated accumulation of [3H]-inositol phosphates (IP1, IP2, IP3) nor the hydrolysis of PIP2 in cells labelled with [3H]-inositol. Furthermore, ethanol (100 mM) added in the absence of agonist did not influence these parameters. However, in cells cultivated for 4 days in 100 mM ethanol, PIP2 hydrolysis and IP1, IP2 and IP3 formation after stimulation by 10(-6)-10(-5) M bradykinin was markedly inhibited while there was no effect on the basal levels or on the levels found after stimulation with low concentrations of bradykinin. The inhibitory effect of ethanol on IP accumulation became significant after 2-3 days of ethanol.     

Chronic ethanol inhibits receptor-stimulated phosphoinositide hydrolysis in rat liver slices.

The effects of chronic ethanol feeding on norepinephrine (NE)- and arginine-vasopressin (AVP)-stimulated phosphoinositide (PI) hydrolysis in rat liver slices was determined. The maximum NE-stimulated PI response was significantly reduced by 40% in liver slices from 8-month-old rats which had been treated for 5 months with a liquid diet containing ethanol compared to pair-fed controls. The maximum AVP-stimulated PI response was decreased by 39% in liver slices from the ethanol-fed rats compared to control. EC50 values for NE- and AVP-stimulated PI hydrolysis in liver slices were not affected by the chronic ethanol treatment. Similar reductions in the maximal NE- and AVP-stimulated PI hydrolysis (28% and 27%, respectively) were found in 22-month-old rats which had been maintained on an ethanol containing diet for 5 months compared to pair-fed controls. The binding of [3H]prazosin and [3H]AVP to liver plasma membranes from 8-month-old ethanol-fed rats was not significantly different from binding to liver membranes from sucrose-fed controls. Our data suggest that chronic ethanol ingestion may lead to a reduction in PI-linked signal transduction in liver.     

Ethanol does not affect serotonin receptor binding in rodent brain

The effects of ethanol on serotonin (5-hydroxytryptamine, 5-HT) receptor binding in rat and mouse brain were determined under in vitro conditions and in mouse brain following seven days of ethanol ingestion. 5-HT1A receptor characteristics were measured utilizing the agonist [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([ 3H]DPAT), and 5HT2 receptor-binding studies utilized the antagonist [3H]ketanserin. At the highest concentration of ethanol tested in vitro (680 mM), there was only 25% inhibition of [3H]DPAT binding in rat and mouse brain and 14% inhibition of [3H]ketanserin binding in rat brain. Effects of an anesthetic concentration of ethanol (100 mM) on agonist binding in the presence and absence of the guanine nucleotide GTP were also evaluated in vitro in mouse brain. In no case did ethanol (100 mM) significantly affect 5-HT1A or 5-HT2 receptor-binding characteristics. When 5-HT receptor characteristics were measured after mice consumed ethanol for seven days, there was no change in either 5-HT1A or 5-HT2 receptor-binding properties in any of the brain areas examined.     

Lipopolysaccharide-stimulated nitric oxide production and inhibition of cell proliferation is antagonized by ethanol in a clonal macrophage cell line.

Both chronic and acute ethanol exposure have been shown to be cytotoxic and also to disrupt normal cell function or responses in a variety of cell types. Macrophage function has specifically been shown to be disrupted by chronic ethanol exposure by mechanisms that have not been elucidated. It is known that exposure of macrophages to lipopolysaccharide (LPS) from gram-negative bacteria will decrease the number of cells. Since increased exposure to endotoxin is often associated with chronic alcoholism, this may be one mechanism to account for loss of macrophages in alcoholic patients. The loss of macrophages, as a consequence of endotoxin treatment, appears to be linked to cell activation and, in particular, LPS-stimulated synthesis of nitric oxide which has been suggested to cause an increase in apoptosis. Ethanol also increases apoptosis in some cell types but, in general, ethanol inhibits activation of macrophages. Thus, the overall effect on cell numbers and cell proliferation elicited by treating macrophages concomitantly with ethanol and LPS depends on the balance between inhibiting LPS-mediated activation and the actions of ethanol. The interaction between ethanol and LPS was investigated in a macrophage cell line (RAW 264.7 cells) by measuring nitric oxide production and cell proliferation. A 24-h exposure to ethanol (100 mM) decreased [3H]-thymidine incorporation significantly. LPS treatment elicited a concentration-dependent decrease in [3H]-thymidine incorporation at LPS concentrations of 0.1 ng/ml to 1000 ng/ml and stimulated nitric oxide production at concentrations above 1 ng/ml. LPS-stimulated nitric oxide production was inhibited by ethanol (20 to 100 mM) and the nitric oxide synthesis inhibitor, N(G)Nitro-L-arginine methyl L-NAME) ester (100 and 500 microM). However, LPS-inhibited [3H]-thymidine incorporation was not be totally reversed by ethanol- or L-NAME-treatment. A direct correlation between nitric oxide production and inhibition of cell proliferation could not be demonstrated. However, it appears that ethanol and LPS do affect some common mechanism(s) in this cell line.     

Protein kinase C modulation of the ethanol effect on serotonin2 receptor transduction in astrocytes

Acute ethanol exposure stimulated serotonin2 receptor signalling in cultured astrocytes. Pretreatment with the protein kinase C inhibitor H7 significantly increased the ethanol-induced potentiation of [3H]-inositol phosphates accumulation. The increase could be explained by an augmented activation of phospholipase C. The results indicate a role of PKC for the modulation of ethanol effects on cellular signalling.     

Ethanol exposure alters K+- but not bradykinin-induced dopamine release in PC12 cells

The effects of ethanol on [3H]dopamine release were investigated in cultured PC12 cells using two methods to stimulate dopamine release: exposure to depolarizing concentrations of extracellular K+ and incubation with the highly active secretagogue, bradykinin. Both K+ and bradykinin dose-dependently increased [3H]dopamine release. The mean +/- S.E.M. EC50 for K+ was 35.8 +/- 1.2 mM; for bradykinin it was 1.07 +/- 0.23 x 10(-7) M. The characteristics of the bradykinin-stimulated dopamine release showed it to be dependent on extracellular Ca2+ and was attenuated by 1 mM Co2+ or 1 mM Ni2+. However, release was unaffected by either the voltage-dependent Ca2+ channel antagonist, verapamil, or the dihydropyridine (DHP) Ca2+ channel agonist, BAY K 8644. In contrast, 1 mM Co2+ completely blocked, verapamil inhibited and BAY K 8644 augmented K+-stimulated [3H]dopamine release. PC12 cells acutely exposed to ethanol (100 and 200 mM) showed diminished K+-stimulated [3H]dopamine release but an unaltered bradykinin-stimulated response. Cells exposed to 200 mM ethanol for 6 days showed significantly enhanced [3H]dopamine release in response to high concentrations of K+ but no changes were observed in their response to bradykinin. These data provide evidence that ethanol, within the same cell, can differentially affect neurotransmitter release, dependent upon the secretagogue used.     

Alterations in serotonin receptor subtypes in ethanol-dependent rats.

Serotonin (5-HT) receptor subtypes were investigated during severe ethanol intoxication and withdrawal. Ethanol was administered intragastrically five times a day for 4 days (12 g/kg per day). 5-HT receptor subtypes were studied: (1) in severely intoxicated animals (mean blood ethanol concentration (BEC) = 4.7 g/l); (2) during the withdrawal reaction; and (3) in a control group. The maximal density of [3H] 8-hydroxy-2-(di-n- propylamino)tetralin [( 3H] 8-OH-DPAT) binding (Bmax) to 5-HT1a receptors was decreased by 25 and 17% in the hippocampus during chronic ethanol intoxication and withdrawal, respectively. [3H]Ketanserin binding to 5-HT2 receptors in the cortex, (-)[125I]-iodo-cyanopindolol [( 125I]CYP) binding to 5-HT1b receptors in the striatum and hypothalamus, and [3H] 8-OH-DPAT binding in the cortex were not affected by chronic ethanol administration. Previous in vitro experiments have shown that 5-HT1a receptors in the hippocampus are inhibitory. The down-regulation of these receptors may play a role in physical ethanol dependence, by inducing hyperexcitability of the hippocampus.     

Ethanol-induced alterations in the function of cerebral GABAA receptor complex: effect on GABA-dependent 36Cl- influx into cerebral membrane vesicles.

Effect of addition in vitro of ethanol on the functions of the GABAA receptor complex was investigated using synaptic membrane preparation obtained from mouse brain. Ethanol (50-200 mM) had no significant effect on the specific bindings of [3H]muscimol to GABAA receptor and [3H]flunitrazepam to benzodiazepine receptor in cerebral particulate preparations. However, ethanol induced an inhibition of [3H]TBOB binding to Cl- channel and a suppression of flunitrazepam-induced enhancement of [3H]muscimol binding and of salsolinol-induced accentuation of [3H]flunitrazepam binding to cerebral particulate fraction. In contrast, ethanol facilitated GABA-dependent 36Cl- influx but eliminated the stimulatory effects of flunitrazepam and salsolinol on GABA-dependent 36Cl- influx into cerebral membrane vesicles. These results suggest that ethanol may facilitate the function of GABA-gated chloride channel in spite of inducing deteriorations of antagonist binding capacity of chloride channel as well as of the functional coupling between GABAA receptor and benzodiazepine receptor in the brain.     

Effect of ethanol in vitro and in vivo on Ca2+-activated metabolism of membrane phospholipids in rat synaptosomal and brain slice preparations

Phospholipase C (PLC) activity was measured by the incorporation of [3H]-inositol into lipids and by the breakdown of [3H]-inositol-labelled phosphatidylinositols (PI) and polyphosphatidylinositols (PPI) to [3H]-inositol phosphates; phospholipase A2 (PLA2) activity by the breakdown of [3H]oleic acid-labelled phosphatidylcholine [( 3H]PC) to [3H]oleic acid and the enzymes of phospholipid base exchange (PLBE) by the incorporation of [14C]serine into membrane lipids. The activities of these enzymes in rat brain preparations were all increased by procedures which increase intracellular Ca2+, and were all inhibited to a varying extent by the presence of ethanol, 50 mM, in vitro. In contrast, the activities of PLA2 and PLBE enzymes were markedly increased in preparations from animals which had received ethanol chronically in vivo. Similarly, although the basal activity of PLC was only slightly increased in such preparations, depolarization induced the breakdown of a significantly greater fraction of radiolabelled PI than that which was obtained in control preparations. The results suggest compensatory alterations in the activity of Ca2+-activated enzymes of phospholipid metabolism in brain tissue during the continued presence of ethanol in vivo.     

Prenatal ethanol exposure during the last third of gestation in rat reduces hippocampal NMDA agonist binding site density in 45-day-old offspring.

The effect of ethanol exposure during different periods of prenatal or postnatal development on hippocampal N-methyl-D-aspartate (NMDA) receptor binding was studied in rat. Fetal rat pups were exposed to ethanol for different periods of time during gestation via maternal consumption of a 3.35% ethanol liquid diet. In a separate experiment, neonatal pups were fed 2.51 g ethanol/kg body weight/day from Postnatal Day (PD) 4 to PD 10 via intragastric feeding tube. These two ethanol administration paradigms produced average peak maternal and pup blood ethanol concentrations of 39 mg/dl and 57 mg/dl, respectively. At 45 days of age, offspring from each treatment group were sacrificed for measurements of hippocampal NMDA-sensitive [3H]-glutamate binding site density using in vitro radiohistochemical techniques. As observed previously, prenatal ethanol exposure throughout gestation resulted in NMDA-sensitive [3H]-glutamate binding site reductions in the apical dendritic field regions of dentate gyrus, hippocampal CA1 and subiculum of dorsal hippocampal formation compared to the ad lib or pair-fed control groups. NMDA-sensitive [3H]-glutamate binding was not different than control in rats exposed to ethanol during the first half of gestation only. Prenatal ethanol exposure during the last half or the last third of gestation resulted in NMDA-sensitive [3H]-glutamate binding site reductions comparable to the binding site reductions observed in rats exposed to ethanol throughout gestation. Hippocampal NMDA-sensitive [3H]-glutamate binding site density in postnatal ethanol-exposed rats was not different than the suckling or gastrostomy control groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prenatal ethanol exposure decreases hippocampal NMDA-sensitive [3H]-glutamate binding site density in 45-day-old rats.

The effect of prenatal ethanol exposure on N-methyl-D-aspartate (NMDA)-sensitive [3H]-glutamate receptor binding site density was studied in rat brain. Pregnant Sprague-Dawley rats were fed a liquid diet containing 3.35% ethanol throughout gestation. This diet produced maternal peak blood ethanol levels of about 39 mg/dl eight hours after the administration of the liquid diet. Pair-fed dams received an isocalorically matched liquid diet and an ad lib lab chow group served as control for the paired feeding technique. At 45 days of age, offspring from each of the three diet groups were sacrificed and brain NMDA-sensitive [3H]-glutamate binding site density measured using in vitro radiohistochemical techniques. NMDA-sensitive [3H]-glutamate binding site density was reduced significantly by 19 to 29% in the apical dendritic field regions of dentate gyrus, hippocampal CA1 and subiculum of dorsal hippocampal formation of fetal alcohol rats compared to pair-fed and ad lib controls. NMDA-sensitive [3H]-glutamate binding site density was not significantly different among the three groups in the ventral hippocampal formation, posterior neocortex, lateral entorhinal cortex or cerebellum. These results are consistent with our previous observations of a reduction in total [3H]-glutamate receptor binding site density in the dorsal hippocampal formation of fetal alcohol rats, as well as more recent electrophysiological observations of a decrease in the sensitivity of fetal alcohol hippocampal slices to NMDA.     

Effects of ethanol exposure on neuropeptide-stimulated calcium mobilization in N1E-115 neuroblastoma

The effects of acute and chronic (100 mM for 7 days) ethanol exposures on resting intracellular free calcium, [Ca²⁺]_i, as well as bradykinin and neurotensin mediated [Ca²⁺]_i mobilization were determined in intact N1E-115 neuroblastoma. [Ca²⁺]_i was monitored fluorometrically with the calcium indicator, fluo-3/AM. Acute exposure to ethanol resulted in an inhibition of bradykinin mediated [Ca²⁺]_i mobilization with significant effects observed only at 400 mM ethanol. Neurotensin mediated [Ca²⁺]_i mobilization was not significantly affected by any of the ethanol concentrations tested. Similarly, resting [Ca²⁺]_i (64 ± 2 nM) was unaffected by either chronic or acute ethanol as high as 400 mM. However, chronic exposure to ethanol significantly reduced the magnitude of bradykinin mediated [Ca²⁺]_i mobilization both in the absence and presence of extracellular [Ca²⁺]. In contrast, [Ca²⁺]_i mobilization in the presence of various concentrations of neurotensin was not significantly affected by chronic ethanol exposure. The results suggest that neuropeptide mediated [Ca²⁺]_i mobilization is relatively insensitive to the acute presence of ethanol. In addition, chronic ethanol exposure appears to have selective effects on receptor mediated [Ca²⁺]_i mobilization because this response to bradykinin, but not neurotensin, was significantly reduced in cells exposed to ethanol. The results also suggest that the reduction in bradykinin stimulated [Ca²⁺]_i mobilization in chronically exposed cells is due in part to an inhibition of the release of intracellularly bound [Ca²⁺].     

An in vitro model for studying the effects of continuous ethanol exposure on N-methyl-d-aspartate receptor function

Long-term ethanol exposure has deleterious effects on both glial and neuronal function. We assessed alterations in both astrocytic and neuronal viability, and alterations in N-methyl-d-aspartate receptor (NMDAR) function, in cocultures of rat cerebellar granule cells (CGCs) and astrocytes after continuous ethanol exposure (CEE). Treatment of cells with 100 mM EtOH once every 24 h for 4 days resulted in a mean ethanol concentration of 57.3 ± 2.1 mM. Comparisons between control and post-ethanol-treated cells were made 4 days after the last ethanol treatment. CEE did not alter glial cell viability, as indicated by the absence of either changes in astrocytic morphology, actin depolymerization, or disruption of astrocytic intracellular mitochondrial distribution at any day postethanol treatment. The CGCs were healthy and viable after CEE, as indicated by phase-contrast microscopy and the trypan-blue exclusion method. Whole-cell patch-clamp experiments indicated that NMDA-induced currents (I_NMDA) were altered by CEE treatment. Similar to previous results obtained during the withdrawal phase from chronic ethanol exposure, I_NMDA from CEE-treated cells were significantly larger than I_NMDA from NMDARs in control CGCs, but returned to control values by the fourth day post-CEE. However, after the last ethanol dosing and during a time when ethanol concentrations remained high, I_NMDA were significantly smaller than control values. Identical results were observed in CGCs expressing the NR2A or NR2B subunit. In summary, both neurons and astrocytes remained healthy following exposure to CEE with no signs of neurotoxicity at the cellular level, and modulation of NMDAR function is consistent with findings from prior experiments. Thus, we conclude that the CEE paradigm in glial-neuronal cocultures readily lends itself to long-term in vitro studies of ethanol effects that include glial-neuronal interactions and the ability to study ethanol withdrawal-induced neurotoxicity.     

Repeated ethanol treatment in adolescent rats alters cortical NMDA receptor.

Earlier we have reported that repeated ethanol treatment during adolescence causes long-lasting impairments in spatial learning and memory. The present study was undertaken to determine the cellular mechanisms underlying the persistent ethanol-induced cognitive dysfunction in adolescent male rats. Since in adult animals ethanol is known to affect the N-methyl-d-aspartate (NMDA) receptor-gated ion channel, the hypothesis tested here was that adolescent ethanol exposure modulates NMDA receptor (NR) regulation in the brain. Adolescent male rats were injected daily with ethanol (2g/kg intraperitoneally) for 5 consecutive days. Control rats received isovolumetric saline for the same number of days. Groups of control and experimental rats were sacrificed 7 days after the last ethanol/saline administration, and NR activity was measured in specific brain regions (frontal cortex, hippocampus) using the [(3)H]MK-801 binding assay. In addition, some rats were sacrificed and their brains were used to investigate changes in NR pharmacology by measuring specific NR2 subunits immunohistochemically. Compared to saline-treated controls, ethanol-treated rats showed significant increases in [(3)H]MK-801 maximal binding in the frontal cortex. This was associated with increased cortical NR2B subunit protein. [(3)H]MK-801 binding in the hippocampus was minimally affected. These results indicate that ethanol exposure during the adolescent period produces brain region-specific alterations in NR activity. These changes are different from those reported in literature for ethanol administration during the perinatal period or adulthood. Together, these data suggest that adolescence represents a unique stage in brain development in its long-term sensitivity to ethanol.     

Ethanol promotes hydrolysis of 3H-labeled sialoconjugates from brain of mice in vitro

Acute administration of ethanol reportedly decreases total sialic acid in brain. Here, we tested the hypothesis in brain and liver that the decrement is due to increased hydrolysis of sialoglycoconjugates. Mouse tissue slices were pulse-labeled with N-[³H]acetyl-D-mannosamine, the precursor of sialic acid. Incorporation was linear for up to 4 hr of incubation. When the labeled slices were incubated with three concentrations of ethanol (0.1, 0.5, and 1 M) for 5 hr, labeled liver sialoconjugates were significantly affected only at 0.5 and 1 M ethanol, whereas labeled brain sialoconjugates were markedly decreased even at 100 mM ethanol. Sialidase activity decreased steadily with increasing concentration of ethanol, indicating that the increased hydrolysis was not attributable to an enhanced sialidase activity. n-Propanol and t-butanol had the same degradative effect as ethanol on sialocompounds; and 3 mM pyrazole, an inhibitor of alcohol dehydrogenase (ADH), had no effect on ethanol-induced degradation of sialocompounds. The protein/DNA ratio in liver showed a steady decrease with increasing ethanol. The data thus confirm the in vivo reports of ethanol-enhanced cleavage and rule out any increase in sialidase activity as a major cause.     

Alterations in Ca2+-dependent and Ca2+-independent release of catecholamines in preparations of rat brain produced by ethanol treatment in vivo

Compared to preparations from control animals, superfused striatal slice preparations from brains of rats treated chronically with ethanol released a significantly greater fraction of stored [3H] dopamine on depolarisation in 40 mM K+. Similarly, the electrically-evoked release of [3H]-norepinephrine from cortical slices and of [3H]-dopamine from striatal slices is also increased, although with this mechanism of depolarisation the change is significant only in the case of [3H] norepinephrine release. In contrast to this tendency to enhancement of Ca2+-dependent depolarisation-induced release, a reduced fraction of stored [3H]-catecholamines was released from these preparations by the indirect sympathomimetics tyramine and (+)-amphetamine. The catecholamine release induced by these indirect sympathomimetics is largely independent of external Ca2+ and the results are interpreted as suggesting that chronic alcohol treatment changes the distribution of catecholamine neurotransmitters between storage pools in the nerve terminal which do or do not require Ca2+ entry for release.     

Chronic ethanol exposure alters MK-801 binding sites in the cerebral cortex of the near-term fetal guinea pig.

The mechanism of ethanol central nervous system (CNS) teratogenesis, resulting from chronic maternal ingestion of high-dose ethanol during pregnancy, is not clearly understood. One of the target sites for ethanol-induced damage in the developing brain is the cerebral cortex. It has been proposed that chronic prenatal ethanol exposure alters NMDA receptors in the developing cerebral cortex. To test this hypothesis, timed pregnant guinea pigs were administered one of the following oral treatments throughout gestation: 4 g ethanol/kg maternal body weight/day; isocaloric sucrose/pair-feeding; water; or no treatment (ad lib). Near-term fetuses were studied at gestational day (GD) 63 (term, about GD 68). This ethanol regimen produced a maternal blood ethanol concentration of 66+/-4 mM (304+/-19 mg/dl) at 1 h after the daily dose on GD 58. The chronic ethanol regimen decreased near-term fetal body weight (12-26% decrease), brain weight (23% decrease), and cerebral cortical weight (21% decrease), compared with the isocaloric sucrose/pair-feeding, and combined water/ad lib experimental groups. Saturation analysis of near-term fetal cerebral cortical membranes using a [3H]MK-801 radioligand binding assay demonstrated a decreased affinity and increased number of MK-801 binding sites for the chronic ethanol regimen compared with the control treatments. These data support the suggestion that upregulation of NMDA receptors in the cerebral cortex after chronic prenatal ethanol exposure could lead to NMDA receptor-mediated excitotoxicity in this brain region.     

Long-term ethanol treatment elicits changes in nicotinic receptor binding in only a few brain regions

Chronic nicotine treatment will produce an upregulation of brain nicotinic receptors, and rats treated for 5 months with ethanol had increased [³H]nicotine binding in two of the three brain regions that were studied. However, several studies using short-term treatment did not detect an effect of ethanol on mouse brain nicotinic receptor numbers. Therefore, LS and SS mice were force-fed ethanol (15%, v/v) in the drinking water for 6 months. The LS mice developed tolerance to ethanol as measured by Y-maze crossing and rearing activity, body temperature, and sleep time. No evidence for tolerance to ethanol was seen in the SS mice. However, the SS mice showed increases in [³H]nicotine binding in thalamus and an increase in [¹²⁵I]α-bungarotoxin binding in the cerebellum and superior colliculus. LS mice had reduced levels of hippocampal [¹²⁵I]α-bungarotoxin binding. Thus, long-term ethanol treatment may affect brain nicotinic receptor binding but the effect is limited to only a few brain regions and may be influenced by genetic factors.