Ethanol-induced Motor Impairment Research Articles

The peripheral dopamine 2 receptor antagonist domperidone attenuates ethanol enhancement of dopamine levels in the nucleus accumbens.

Dopamine neuron firing in the ventral tegmental area (VTA) and dopamine release in the nucleus accumbens have been implicated in reward learning. Ethanol is known to increase both dopamine neuron firing in the VTA and dopamine levels in the nucleus accumbens. Despite this, some discrepancies exist between the dose of ethanol required to enhance firing in vivo and ex vivo. In the present study we investigated the effects of peripheral dopamine 2 subtype receptor antagonism on ethanol's effects on dopamine neurotransmission. Plasma catecholamine levels were assessed following ethanol administration across four different doses of EtOH. Microdialysis and voltammetry were used to assess the effects of domperidone pretreatment on ethanol-mediated increases in dopamine release in the nucleus accumbens. A place conditioning paradigm was used to assess conditioned preference for ethanol and whether domperidone pretreatment altered this preference. Open-field and loss-of-righting reflex paradigms were used to assess the effects of domperidone on ethanol-induced sedation. A rotarod apparatus was used to assess the effects of domperidone on ethanol-induced motor impairment. Domperidone attenuated ethanol's enhancement of mesolimbic dopamine release under non-physiological conditions at intermediate (1.0 and 2.0g/kg) doses of ethanol. Domperidone also decreased EtOH-induced sedation at 2.0g/kg. Domperidone did not alter ethanol conditioned place preference nor did it affect ethanol-induced motor impairment. These results show that peripheral dopamine 2 receptors mediate some of the effects of ethanol on nonphysiological dopamine neurotransmission, although these effects are not related to the rewarding properties of ethanol.

Synthetic cannabinoids enhanced ethanol-induced motor impairments through reduction of central glutamate neurotransmission

Marijuana or synthetic cannabinoids and alcohol are often used together, with these combinations causing motor impairments that can subsequently lead to motor vehicle accidents. This study investigated the combined use of both synthetic cannabinoids and ethanol and their effect on motor coordination in mice in addition to examining the neurochemical changes in the cerebellum. Ethanol (2 g/kg, i.p.) significantly induced motor impairment in the accelerating rotarod test in mice. Furthermore, ethanol-induced motor impairments were further accentuated when combined with the synthetic cannabinoid, JWH-018 or AB-CHMINACA. The enhancement effects of the synthetic cannabinoids were completely antagonized by pretreatment with the selective CB1 receptor antagonist AM251, but not by the selective CB2 receptor antagonist AM630. Neurochemical study results showed that ethanol caused a reduction in the extracellular glutamate levels in the cerebellum during periods of ethanol-induced motor impairment. In addition to the enhanced motor impairment seen when ethanol was combined with JWH-018, these combinations also enhanced the reduction of the extracellular glutamate levels in the cerebellum. We additionally used microelectrode array recordings to examine the effects of ethanol and/or JWH-018 on the spontaneous network activity in primary cultures from mouse cerebellum. Results showed that ethanol combined with JWH-018 significantly reduced spontaneous neuronal network activity in the primary cerebellar culture. Our findings demonstrate that ethanol-induced motor impairments are enhanced by synthetic cannabinoids, with these effects potentially mediated by CB1 receptors. An accentuated reduction of neurotransmissions in the cerebellum may play an important role in motor impairments caused by ethanol combined with synthetic cannabinoids.

Essential role for neuronal nitric oxide synthase in acute ethanol-induced motor impairment

The cerebellum is widely known as a motor structure because it regulates and controls motor learning, coordination, and balance. However, it is also critical for non-motor functions such as cognitive processing, sensory discrimination, addictive behaviors and mental disorders. The cerebellum has the highest relative abundance of neuronal nitric oxide synthase (nNos) and is sensitive to ethanol. Although it has been demonstrated that the interaction of γ-aminobutyric acid (GABA) and nitric oxide (NO) might play an important role in the regulation of ethanol-induced cerebellar ataxia, the molecular mechanisms through which ethanol regulates nNos function to elicit this behavioral effect have not been studied extensively. Here, we investigated the dose-dependent effects of acute ethanol treatment on motor impairment using the rotarod behavioral paradigm and the alterations of nNos mRNA expression in cerebellum, frontal cortex (FC), hippocampus and striatum. We also examined the link between acute ethanol-induced motor impairment and nNos by pharmacological manipulation of nNos function. We found that acute ethanol induced a dose-dependent elevation of ethanol blood levels which was associated with the impairment of motor coordination performance and decreased expression of cerebellar nNos. In contrast, acute ethanol increased nNos expression in FC but did not to change the expression for this enzyme in striatum and hippocampus. The effects of acute ethanol were attenuated by l-arginine, a precursor for NO and potentiated by 7-nitroindazole (7-NI), a selective inhibitor of nNos. Our data suggests that differential regulation of nNos mRNA expression in cerebellum and frontal cortex might be involved in acute ethanol-induced motor impairment.

Ethanol-Induced Motor Impairment Mediated by Inhibition of α7 Nicotinic Receptors.

Nicotine and ethanol (EtOH) are among the most widely co-abused substances, and nicotinic acetylcholine receptors (nAChRs) contribute to the behavioral effects of both drugs. Along with their role in addiction, nAChRs also contribute to motor control circuitry. The α7 nAChR subtype is highly expressed in the laterodorsal tegmental nucleus (LDTg), a brainstem cholinergic center that contributes to motor performance through its projections to thalamic motor relay centers, including the mediodorsal thalamus. We demonstrate that EtOH concentrations just above the legal limits for intoxication in humans can inhibit α7 nAChRs in LDTg neurons from rats. This EtOH-induced inhibition is mediated by a decrease in cAMP/PKA signaling. The α7 nAChR-positive allosteric modulator PNU120596 [N-(5-chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl)-urea], which interferes with receptor desensitization, completely eliminated EtOH modulation of these receptors. These data suggest that EtOH inhibits α7 responses through a PKA-dependent enhancement of receptor desensitization. EtOH also inhibited the effects of nicotine at presynaptic α7 nAChRs on glutamate terminals in the mediodorsal thalamus. In vivo administration of PNU120596 either into the cerebral ventricles or directly into the mediodorsal thalamus attenuated EtOH-induced motor impairment. Thus, α7 nAChRs are likely important mediators of the motor impairing effects of moderate EtOH consumption. The motor-impairing effects of ethanol contribute to intoxication-related injury and death. Here we explore the cellular and neural circuit mechanisms underlying ethanol-induced motor impairment. Physiologically relevant concentrations of ethanol inhibit activity of a nicotinic receptor subtype that is expressed in brain areas associated with motor control. That receptor inhibition is mediated by decreased receptor phosphorylation, suggesting an indirect modulation of cell signaling pathways to achieve the physiological effects.

Oxytocin prevents ethanol actions at δ subunit-containing GABAA receptors and attenuates ethanol-induced motor impairment in rats

Even moderate doses of alcohol cause considerable impairment of motor coordination, an effect that substantially involves potentiation of GABAergic activity at δ subunit-containing GABA(A) receptors (δ-GABA(A)Rs). Here, we demonstrate that oxytocin selectively attenuates ethanol-induced motor impairment and ethanol-induced increases in GABAergic activity at δ-GABA(A)Rs and that this effect does not involve the oxytocin receptor. Specifically, oxytocin (1 µg i.c.v.) given before ethanol (1.5 g/kg i.p.) attenuated the sedation and ataxia induced by ethanol in the open-field locomotor test, wire-hanging test, and righting-reflex test in male rats. Using two-electrode voltage-clamp electrophysiology in Xenopus oocytes, oxytocin was found to completely block ethanol-enhanced activity at α4β1δ and α4β3δ recombinant GABA(A)Rs. Conversely, ethanol had no effect when applied to α4β1 or α4β3 cells, demonstrating the critical presence of the δ subunit in this effect. Oxytocin had no effect on the motor impairment or in vitro effects induced by the δ-selective GABA(A)R agonist 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol, which binds at a different site on δ-GABA(A)Rs than ethanol. Vasopressin, which is a nonapeptide with substantial structural similarity to oxytocin, did not alter ethanol effects at δ-GABA(A)Rs. This pattern of results confirms the specificity of the interaction between oxytocin and ethanol at δ-GABA(A)Rs. Finally, our in vitro constructs did not express any oxytocin receptors, meaning that the observed interactions occur directly at δ-GABA(A)Rs. The profound and direct interaction observed between oxytocin and ethanol at the behavioral and cellular level may have relevance for the development of novel therapeutics for alcohol intoxication and dependence.

Different chronic ethanol exposure regimens in adolescent and adult male rats: Effects on tolerance to ethanol-induced motor impairment

Findings are mixed regarding the expression of tolerance after repeated ethanol exposure, perhaps in part due to dose/frequency variations in exposure regimens. The present study compared age-related differences in tolerance development following 10 days of 1 g/kg twice daily, 2 g/kg once daily, or intermittent 4 g/kg ethanol exposure regimens. To measure expression of chronic tolerance and acute tolerance, ethanol-induced motor impairment was assessed on day 12, with functionally equivalent ethanol doses administered across age (2 g/kg – adolescents; 1.5 g/kg – adults). Subsequent challenge doses resulted in lower brain ethanol concentrations in both age groups as a function of the chronic ethanol regimens. Expected age-related differences emerged in acute tolerance expression in non-manipulated animals, with adolescents, but not adults showing acute tolerance. Regimens sufficient to induce alterations in ethanol metabolism did not result in chronic functional tolerance at either age, although chronic injections were sufficient to induce acute tolerance in adults.

Effect of the Selective NMDA NR2B Antagonist, Ifenprodil, on Acute Tolerance to Ethanol-Induced Motor Impairment in Adolescent and Adult Rats

Adolescent rats have been observed to be less sensitive than adults to a number of acute ethanol effects, including ethanol-induced motor impairment. These adolescent insensitivities may be related in part to the more rapid emergence of within session (acute) tolerance in adolescents than adults. Adolescent-related alterations in neural systems that serve as ethanol target sites, including changes in NMDA receptor subunit expression, may influence the responsiveness of adolescents to acute ethanol effects. This study explored the role of NMDA NR2B receptors in the development of acute tolerance to ethanol-induced motor impairment in male adolescent [postnatal day (P)28-30] and adult (P68-70) Sprague-Dawley rats. Motor-impairing effects of ethanol on the stationary inclined plane and blood ethanol concentrations (BECs) were examined following challenge at each age with a functionally equivalent ethanol dose (adolescents: 2.25 g/kg; adults: 1.5 g/kg). Data were collected at two postinjection intervals (10 or 60 minutes) to compare rate of recovery from ethanol intoxication with BEC declines using the Radlow approach (Radlow, 1994) and changes in motor impairment/BEC ratios over time for assessing acute tolerance. Both vehicle-treated adolescent and adult animals showed similar acute tolerance development to the motor-impairing effects of ethanol at these functionally equivalent doses on the stationary inclined plane, as indexed by an increasing time-dependent dissociation between BECs and ethanol-induced motor impairment, with motor impairment declining faster than BECs, as well as by significant declines in motor impairment/BEC ratios over time. Acute tolerance development was reliably blocked by administration of the NR2B antagonist, ifenprodil, (5.0 mg/kg), in adult rats, whereas adolescents were affected by a higher dose (10.0 mg/kg). These data support the suggestion that alterations in NMDA receptor systems occurring during adolescence may contribute to reduced sensitivity to ethanol by enhancing the expression of acute tolerance development in adolescents relative to adults.

The role of connexin‐36 gap junctions in alcohol intoxication and consumption

Ventral tegmental area (VTA) GABA neurons appear to be critical substrates underlying the acute and chronic effects of ethanol on dopamine (DA) neurotransmission in the mesocorticolimbic system implicated in alcohol reward. The aim of this study was to examine the role of midbrain connexin-36 (Cx36) gap junctions (GJs) in ethanol intoxication and consumption. Using behavioral, molecular, and electrophysiological methods, we compared the effects of ethanol in mature Cx36 knockout (KO) mice and age-matched wild-type (WT) controls. Compared to WT mice, Cx36 KO mice exhibited significantly more ethanol-induced motor impairment in the open field test, but less disruption in motor coordination in the rotarod paradigm. Cx36 KO mice, and WT mice treated with the Cx36 antagonist mefloquine (MFQ), consumed significantly less ethanol than their WT controls in the drink-in-the-dark procedure. The firing rate of VTA GABA neurons in WT mice was inhibited by ethanol with an IC₅₀ of 0.25 g/kg, while VTA GABA neurons in KO mice were significantly less sensitive to ethanol. Dopamine neuron GABA-mediated sIPSC frequency was reduced by ethanol (30 mM) in WT mice, but not affected in KO mice. Cx36 KO mice evinced a significant up-regulation in DAT and D2 receptors in the VTA, as assessed by quantitative RT-PCR. These findings demonstrate the behavioral relevance of Cx36 GJ-mediated electrical coupling between GABA neurons in mature animals, and suggest that loss of coupling between VTA GABA neurons results in disinhibition of DA neurons, a hyper-DAergic state and lowered hedonic valence for ethanol consumption.

The cerebellar GABA AR α6-R100Q polymorphism alters ligand binding in outbred Sprague–Dawley rats in a similar manner as in selectively bred AT and ANT rats

The alcohol-tolerant AT and alcohol-nontolerant ANT rat lines have been selectively bred for innate sensitivity to ethanol-induced motor impairment. The cerebellar GABA A receptor (GABA AR) α6 subunit alleles α6-100R and α6-100Q are segregated in the AT and ANT rats, respectively. This α6 polymorphism might explain various differences in pharmacological properties and density of GABA ARs between the rat lines. In the present study, we have used nonselected outbred Sprague–Dawley rats homozygous for the α6-100RR (RR) and α6-100QQ (QQ) genotypes to show that these RR and QQ rats display similar differences between genotypes as AT and ANT rat lines. The genotypes differed in their affinity for [ 3H]Ro 15-4513 and classic benzodiazepines (BZs) to cerebellar “diazepam-insensitive” (DZ-IS) binding sites, in density of cerebellar [ 3H]muscimol binding and in the antagonizing effect of furosemide on GABA-induced inhibition of [ 3H]EBOB binding. The results suggest the involvement of α6-R100Q polymorphism in these line differences and in the differences previously found between AT and ANT rats. In addition, the α6-R100Q polymorphism induces striking differences in [ 3H]Ro 15-4513 binding kinetics to recombinant α6β3γ2s receptors and cerebellar DZ-IS sites. Association of [ 3H]Ro 15-4513 binding was ∼10-fold faster and dissociation was ∼3–4-fold faster in DZ-IS α6βγ2 receptors containing the α6-100Q allele, with a resulting change of ∼2.5-fold in equilibrium dissociation constant ( K D). The results indicate that in addition to the central role of the homologous α6-100R/Q (α1-101H) residue in BZ binding and efficacy, this critical BZ binding site residue has a major impact on BZ binding kinetics.

Ontogeny of ethanol-induced motor impairment following acute ethanol: Assessment via the negative geotaxis reflex in adolescent and adult rats

Adolescent rats have been observed to be less sensitive than adults to a number of ethanol effects that may serve as feedback cues to reduce further ethanol intake. Among these findings are a few reports of attenuated sensitivities of adolescents to ethanol-induced motor impairment. The purpose of the present study was to further explore potential age-related differences in ethanol-induced motor impairment in both male and female adolescent (postnatal day [P]28–32), and adult (P68–72) Sprague–Dawley rats using an inclined plane assessment of the negative geotaxis reflex. Adult males displayed significant motor impairment at 1.5 g/kg, whereas adolescent males required higher doses, showing significant motor impairment only at doses of 2.25 g/kg ethanol or greater. Intoxicated practice did not significantly influence level of motor impairment at either age. When female rats of both ages were separately analyzed in terms of their response to ethanol, a dose of 1.5 g/kg ethanol was found to significantly impair adults, whereas adolescent females showed significant motor impairment when challenged with 2.25 g/kg but not 1.5 g/kg ethanol. Yet when the 1.5 g/kg data of females at the two ages were directly compared, no significant age difference was seen at this dose. These data document an attenuated sensitivity of adolescent relative to adult rats to the motor impairing effects of ethanol using a stationary inclined plane test, an effect particularly robust in male animals, and demonstrates the utility of this test for assessment of motor coordination in adolescent and adult rats.

Influence of epipregnanolone on the modulation of rapid tolerance to ethanol by neurosteroids

The objective of the present study was to investigate the effect of epipregnanolone on the influence of neurosteroids on the development of rapid tolerance to the motor impairing and hypothermic effects of ethanol. Experiment 1: on Day 1 groups of mice were pretreated with saline or with epipregnanolone. After 30 min each group was further divided in subgroups that received ethanol or saline. Thirty, 60 and 90 min after the injections the animals were tested on the rota-rod or the body temperature was measured. On Day 2 all groups received ethanol and a similar procedure was followed to evaluate rapid tolerance. Experiment 2 and 3: On Day 1 groups of mice were treated with epipregnanolone and after 15 min each group was divided into three groups in order to receive pregnenolone sulfate, dehydroepiandrosterone sulfate or saline. Thirty minutes later, each group was further divided into two subgroups in order to receive ethanol or saline, respectively, and 30, 60 and 90 min later the animals were tested as in the experiment 1. On Day 2 all groups received ethanol and a similar procedure was followed to evaluate rapid tolerance. Pretreatment with epipregnanolone (0.10-0.30 mg/kg) significantly blocked the development of tolerance to the motor impairing and hypothermic effects induced by ethanol in mice. Considering tolerance to ethanol-induced motor impairment, epipregnanolone (0.15 mg/kg) reversed the stimulatory action of dehydroepiandrosterone sulfate (0.15 mg/kg), but did not affect the actions of pregnenolone sulfate (0.08 mg/kg). Moreover, epipregnanolone prevented the inhibitory action of allotetrahydrodeoxycorticosterone (0.10 mg/kg). In relation to ethanol-induced hypothermia, the results showed that pretreatment with epipregnanolone (0.30 mg/kg) significantly prevented the stimulatory action of dehydroepiandrosterone sulfate and pregnenolone sulfate, as well as the inhibitory action of allotetrahydrodeoxicorticosterone (0.20 mg/kg), on tolerance to this effect. The results suggest a differential interaction between neurosteroids that might modulate the development of rapid tolerance to ethanol.

The Role of Protein Kinase A in Acute Ethanol-Induced Neurobehavioral Actions in Rats

cAMP-dependent protein kinase (PKA) signaling pathways are involved in the regulation of ethanol-induced sedative effects in knockout mouse models. In the present study, we examined the role of PKA on the behavioral action caused by ethanol in Sprague Dawley rats. A loss of righting reflex (LORR) test was used to study the acute sedative effects of intraperitoneally injected ethanol. Rotarod performance was used to study the motor impairment caused by ethanol. Convulsions induced by intracerebroventricular (ICV) N-methyl-d-aspartate (NMDA) were used to evaluate ethanol's effect on NMDA receptors. Western blot analysis was used to assay protein levels for NR1 and phosphoserine 897 on NR1 subnuits. ICV pretreatment with H-9 (a nonspecific PK inhibitor) or KT 5720 (a specific PKA inhibitor) dose-dependently attenuated ethanol-induced sleeping time as assessed by LORR. ICV KT 5720 did not reduce ketamine or pentobarbital-induced sleeping time. Pretreatment with forskolin (an activator of adenylyl cyclase) or chelerythrine (a selective PKC inhibitor) had no effect on ethanol-induced LORR. Ethanol-induced motor impairment was also attenuated after pretreatment with KT 5720. Ethanol significantly inhibited NMDA-induced convulsions; the inhibitory effects of ethanol were reduced by prior ICV KT 5720, which had no significant effects on the levels of phosphoserine 897 on NMDA NR1 subunits in the several brain areas we examined. Our results suggest that the PKA pathway may participate in ethanol-induced neurobehavioral changes and that NMDA receptors may be involved in the PKA regulation of ethanol's actions.

Age-Dependent Variation in Behavior Following Acute Ethanol Administration in Male and Female Adolescent Rhesus Macaques (Macaca mulatta)

There has been considerable focus on the adolescent stage of development in the study of alcohol use and the etiology of alcohol-related problems. Because adolescence is a process of dynamic change rather than a discrete or static stage of development, it is important to consider ontogenetic changes in the response to ethanol within the adolescent time period. In rodents, levels of ethanol-induced motor impairment have been shown to increase from early to late adolescence. This study investigated associations between behavior following acute ethanol administration and age, rearing condition (mother-reared vs nursery-reared), and serotonin transporter (rh5-HTTLPR) genotype in a sample of alcohol-naïve adolescent rhesus macaques. Rhesus macaques (n=97; 41 males, 56 females), ranging in age from 28 to 48 months, were administered intravenous (IV) doses of ethanol (2.2 g/kg for males, 2.0 g/kg for females) twice in 2 separate testing sessions. A saline/ethanol group (n=16; 8 males, 6 females) was administered saline in 1 testing session and ethanol in the second session. Following each IV injection, subjects underwent a 30-minute general motor behavioral assessment. Behavior in the saline/ethanol group was compared between the saline and ethanol-testing sessions using analysis of variance. Behavioral data for the larger study sample were averaged between the 2 testing sessions and summarized using factor analysis. Rotated factor scores were used as dependent variables in multiple regression analyses to test for relationships between behavior and age, rearing condition, and rh5-HTTLPR genotype. During the ethanol-testing session, behaviors indicative of motor impairment (stumbles, falls, sways, bumping the wall, and unsuccessful jumps) were frequently observed in the saline/ethanol group, while they did not occur under the saline-testing session. Factor analysis of behavior following ethanol administration in the larger study sample yielded 3 factors: Ataxia, Impaired Jumping Ability, and Stimulation. Significant negative correlations between age and Ataxia were found for both males and females. Females also exhibited positive correlations between age and Impaired Jumping Ability and age and Stimulation. No significant correlations were found with either rearing condition or rh5-HTTLPR genotype. These findings suggest that ontogenetic changes during adolescence in the behavioral response to ethanol differ between rodents and primates. Furthermore, sex differences in the behavioral response to ethanol appear to develop during adolescence.

Co-modulation of acute ethanol-induced motor impairment by mouse cerebellar adenosinergic A 1 and GABA A receptor systems

We have previously demonstrated that cerebellar adenosine modulates ethanol ataxia. Using Rotorod method, we investigated the role of cerebellar GABA A receptors in the adenosinergic modulation of ethanol ataxia in mice. Direct cerebellar microinfusion of GABA A agonist, muscimol (2.5, 5 and 10 ng) and antagonist, bicuculline (50, 100 and 200 ng), via permanently implanted guide cannulas, produced a marked and dose-dependent accentuation and attenuation, respectively, of ethanol (2 g/kg; IP) ataxia. The accentuation of ethanol ataxia by intracerebellar muscimol was through GABA A receptor because intracerebellar pretreatment with bicuculline virtually abolished muscimol effect. Intracerebellar microinfusion of adenosine A 1 agonist, N 6-cyclohexyladenosine (CHA: 4 ng), and antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX: 100 ng) markedly accentuated and attenuated, respectively, ethanol ataxia consistent with our previously published data. Intracerebellar microinfusion of CHA (4 ng) or DPCPX (100 ng) markedly enhanced and reduced, respectively, muscimol (10 ng)-induced accentuation of ethanol ataxia suggesting co-modulation of ethanol ataxia by cerebellar adenosinergic A 1 and GABA A receptors. Similarly, intracerebellar bicuculline (200 ng) pretreatment not only prevented CHA-induced accentuation of ethanol ataxia, but caused further decrease in ethanol ataxia. No change in the normal coordination was observed when microinfusion of the highest dose of muscimol, bicuculline, DPCPX or CHA alone or in combination was followed by saline injection instead of ethanol. The results of the present study suggest a functional similarity between GABA A and adenosine A 1 receptors even though both receptor types are known to couple to different signaling system and their location is on the opposite ends of the cerebellar granule cells, axons and axonal terminals (i.e., GABA A at the granule cells and adenosine A 1 on axons and axonal terminals of the granule cells) and act as co-modulators of ethanol ataxia.

Possible role of mouse cerebellar nitric oxide in the behavioral interaction between chronic intracerebellar nicotine and acute ethanol administration: Observation of cross-tolerance

Many studies have reported cross-tolerance between nicotine and ethanol. Previously we demonstrated that intracerebellar nicotine attenuates ethanol-induced motor impairment. In this study, intracerebellar nicotine (0.625, 2.5, 5 ng; once daily for five days) significantly attenuated ethanol-induced motor impairment in a dose-dependent fashion suggesting the development of cross-tolerance between nicotine and ethanol in male CD-1 mice. Using the same paradigm, intracerebellar nicotine (5 ng) microinfused for 1, 2, 3, 5, 7 days significantly attenuated ethanol-induced motor impairment in all groups except the 1-day treatment group. Cross-tolerance, which developed optimally in 5-day nicotine treatment group, was reversible and detectable up to 40 h post-nicotine microinfusion. Intracerebellar microinfusion of hexamethonium (1 μg once daily for 5 days): (i) did not alter ethanol-induced motor impairment indicating no tonic nicotine receptor involvement; (ii) 10 min prior to daily intracerebellar nicotine treatment virtually abolished the cross-tolerance between nicotine and ethanol indicating nicotinic acetylcholine receptor participation; (iii) when microinfused 10 min after daily intracerebellar nicotine treatment, failed to abolish the cross-tolerance which suggested possible participation of downstream second messenger mechanisms. Chronic intracerebellar microinfusion of nicotine: (i) failed to attenuate acute pentobarbital (25mg/kg i.p.)-induced motor impairment; and (ii) produced no change in normal motor coordination when followed by saline injection. Finally, the cerebellar concentration of total nitric oxide products (nitrite+nitrate; NO x); (i) was increased after 5-day intracerebellar nicotine; (ii) was decreased by acute ethanol administration; and (iii) decreased due to acute ethanol administration which was opposed by chronic intracerebellar nicotine treatment. These results support a functional correlation between the cerebellar nitric oxide production and ethanol-induced motor impairment and suggest possible participation of nitric oxide as a factor in the observed cross-tolerance between nicotine and ethanol.

Blockade of adenosine and dopamine receptors inhibits the development of rapid tolerance to ethanol in mice

Several reports have suggested the involvement of brain adenosine and dopamine receptors in different actions produced by ethanol such as motor incoordination or anxiolytic, hypnotic and reinforcing effects. The co-localization and interaction between adenosine and dopamine receptors in different brain regions has also been well documented. However, few studies have demonstrated the involvement of these mechanisms in the tolerance induced by ethanol. The aim of the present study was to evaluate the role of adenosine and dopamine receptors in the development of rapid tolerance to ethanol-induced motor incoordination in mice. In connection with the rota-rod apparatus, the effects of acute administration of the adenosine receptor antagonists caffeine (non-selective), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, adenosine A1 receptor antagonist) and 4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo-{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)phenol (ZM241385, adenosine A2A receptor antagonist), together with R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390, dopamine D1 receptor antagonist) and sulpiride (dopamine D2 receptor antagonist), alone or in combination with ethanol (2.25 g/kg, i.p.), were studied. Twenty-four hours after, all animals were re-tested on the rota-rod after receiving the same dose of ethanol. The repeated administration of ethanol promoted a significant reduction of motor impairment on day 2 (i.e. rapid tolerance). This effect was blocked by caffeine (3.0-30.0 mg/kg, i.p.), DPCPX (3.0-6.0 mg/kg, i.p.) or SCH23390 (0.01-0.03 mg/kg, s.c.), but not with ZM241385 (0.5-1.0 mg/kg, i.p.) or sulpiride (1.0-3.0 mg/kg, i.p.). Our results suggest that the rapid tolerance to ethanol-induced motor impairment in mice may be modulated by adenosine A1 receptors and dopamine D1 receptors.

Characterizing the ontogeny of ethanol-associated increases in corticosterone

The following experiments were conducted as adjuncts to previous work in an effort to characterize the ontogenetic profile of the elevations in corticosterone after ethanol challenge. In Experiment 1, female and male Sprague–Dawley rats were administered intraperitoneally either a 1.5- or a 4.5-g/kg dose of ethanol on postnatal day (PND) 16, 26, 36, or 56. Blood samples were collected at 40, 80, or 160 min after ethanol injection and analyzed by means of radioimmunoassay for corticosterone levels and correlated with brain alcohol levels (BrALs) determined from brain samples collected at the same time intervals. In Experiment 2, the ethanol dose was varied ontogenetically to equate functional impairment across age, with the use of intraperitoneal doses of ethanol of 3.2, 2.6, or 2.2 g/kg, to induce equivalent amounts of ethanol-induced motor impairment in infant (PND 22), adolescent (PND 28), or adult (PND 60) rats, respectively. Animals were tested on a swim task 15 min after injection, with blood and brain samples collected immediately after the swim and analyzed for corticosterone levels and BrALs as in Experiment 1. Reminiscent of previous reports of an age-related increase in sensitivity to the hypnotic and motor-impairing effects of ethanol, the corticosterone response to an ethanol challenge increased at least through adolescence, with sex differences emerging by PND 26 and becoming more pronounced in adulthood. To the extent that corticosterone release is involved in the reinforcing effects of drugs, ontogenetic differences in the response of the hypothalamic–adrenal–pituitary (HPA) axis to ethanol could contribute to the excessive alcohol consumption often observed during adolescence.

Characterizing the ontogeny of ethanol-associated increases in corticosterone

The following experiments were conducted as adjuncts to previous work in an effort to characterize the ontogenetic profile of the elevations in corticosterone after ethanol challenge. In Experiment 1, female and male Sprague–Dawley rats were administered intraperitoneally either a 1.5- or a 4.5-g/kg dose of ethanol on postnatal day (PND) 16, 26, 36, or 56. Blood samples were collected at 40, 80, or 160 min after ethanol injection and analyzed by means of radioimmunoassay for corticosterone levels and correlated with brain alcohol levels (BrALs) determined from brain samples collected at the same time intervals. In Experiment 2, the ethanol dose was varied ontogenetically to equate functional impairment across age, with the use of intraperitoneal doses of ethanol of 3.2, 2.6, or 2.2 g/kg, to induce equivalent amounts of ethanol-induced motor impairment in infant (PND 22), adolescent (PND 28), or adult (PND 60) rats, respectively. Animals were tested on a swim task 15 min after injection, with blood and brain samples collected immediately after the swim and analyzed for corticosterone levels and BrALs as in Experiment 1. Reminiscent of previous reports of an age-related increase in sensitivity to the hypnotic and motor-impairing effects of ethanol, the corticosterone response to an ethanol challenge increased at least through adolescence, with sex differences emerging by PND 26 and becoming more pronounced in adulthood. To the extent that corticosterone release is involved in the reinforcing effects of drugs, ontogenetic differences in the response of the hypothalamic–adrenal–pituitary (HPA) axis to ethanol could contribute to the excessive alcohol consumption often observed during adolescence.

Effects of ethanol on the accumbal output of dopamine, GABA and glutamate in alcohol-tolerant and alcohol-nontolerant rats

Effects of ethanol on the accumbal extracellular concentrations of dopamine, as well as of the amino acid transmitters gamma-amino butyric acid (GABA), glutamate and taurine, were studied in the alcohol-insensitive (alcohol-tolerant, AT) and alcohol-sensitive (alcohol-nontolerant, ANT) rats selected for low and high sensitivity to ethanol-induced motor impairment. Ethanol (2 or 3 g/kg ip) enhanced the output of dopamine and its metabolites in freely moving rats of both lines as measured by in vivo microdialysis. The effect of ethanol on the metabolites of dopamine tended to be stronger in the ANT rats. The smaller dose of ethanol decreased the output of GABA only in the AT rats, whereas the larger dose of ethanol decreased the output of GABA in rats of both lines to a similar degree. Ethanol at the dose of 2 g/kg slightly, but statistically, significantly decreased the output of glutamate in rats of both lines, but the larger dose of ethanol decreased the output of glutamate only in the AT rats. Ethanol at the dose of 2 g/kg induced a small transient increase in the output of taurine within 2 h after its administration in rats of both lines, but the larger dose of ethanol was without significant effect. These results confirm the previous findings that ethanol suppresses the release of GABA more in the AT than ANT rats. Thus, among the neurotransmitter systems we studied, the effects of ethanol might be the most relevant on GABAergic transmission regarding the sensitivity towards ethanol. However, our findings suggest that glutamate is also involved in this respect.

Low Brain Histamine Content Affects Ethanol-Induced Motor Impairment

The effect of ethanol on motor performance in humans is well established but how neural mechanisms are affected by ethanol action remains largely unknown. To investigate whether the brain histaminergic system is important in it, we used a genetic model consisting of rat lines selectively outbred for differential ethanol sensitivity. Ethanol-sensitive rats had lower levels of brain histamine and lower densities of histamine-immunoreactive fibers than ethanol-insensitive rats, although both rat lines showed no changes in histamine synthesizing neurons. Lowering the high brain histamine content of the ethanol-insensitive rats with α-fluoromethylhistidine before ethanol administration increased their ethanol sensitivity in a behavioral motor function test. Higher H3 receptor ligand binding and histamine-induced G-protein activation was detected in several brain regions of ethanol-naive ethanol-sensitive rats. Brain histamine levels and possibly signaling via H3 receptors may thus correlate with genetic differences in ethanol-induced motor impairment.