Ethanol-Induced Cerebellar Ataxia: Cellular and Molecular Mechanisms.

The cerebellum is an important target of ethanol toxicity given that cerebellar ataxia is the most consistent physical manifestation of acute ethanol consumption. Despite the significance of the cerebellum in ethanol-induced cerebellar ataxia (EICA), the cellular and molecular mechanisms underlying EICA are incompletely understood. However, two important findings have shed greater light on this phenomenon. First, ethanol-induced blockade of cerebellar adenosine uptake in rodent models points to a role for adenosinergic A1 modulation of EICA. Second, the consistent observation that intracerebellar administration of nicotine in mice leads to antagonism of EICA provides evidence for a critical role of cerebellar nitric oxide (NO) in EICA reversal. Based on these two important findings, this review discusses the potential molecular events at two key synaptic sites (mossy fiber-granule cell-Golgi cell (MGG synaptic site) and granule cell parallel fiber-Purkinje cell (GPP synaptic site) that lead to EICA. Specifically, ethanol-induced neuronal NOS inhibition at the MGG synaptic site acts as a critical trigger for Golgi cell activation which leads to granule cell deafferentation. Concurrently, ethanol-induced inhibition of adenosine uptake at the GPP synaptic site produces adenosine accumulation which decreases glutamate release and leads to the profound activation of Purkinje cells (PCs). These molecular events at the MGG and GPP synaptic sites are mutually reinforcing and lead to cerebellar dysfunction, decreased excitatory output of deep cerebellar nuclei, and EICA. The critical importance of PCs as the sole output of the cerebellar cortex suggests normalization of PC function could have important therapeutic implications.

Functional interaction and cross-tolerance between ethanol and Δ9-THC: possible modulation by mouse cerebellar adenosinergic A1/GABAergic-A receptors.

We have previously shown a functional motor interaction between ethanol and Δ(9)-tetrahydrocannabinol (Δ(9)-THC) that involved cerebellar adenosinergic A1 and GABAergic A receptor modulation. We now report the development of cross-tolerance between intracerebellar Δ(9)-THC and intraperitoneal ethanol using ataxia as the test response in male CD-1 mice. The drugs [Δ(9)-THC (20 µg), N(6)-cyclohexyladenosine, CHA (12 ng), muscimol (20 ng)] used in the study were directly microinfused stereotaxically via guide cannulas into the cerebellum except ethanol. Δ(9)-THC, infused once daily for 5 days followed 16 h after the last infusion by acute ethanol (2g/kg) and Rotorod evaluation, virtually abolished ethanol ataxia indicating development of cross-tolerance. The cross-tolerance was also observed when the order of ethanol and Δ(9)-THC treatment was reversed, i.e., ethanol injected once daily for 5 days followed 16 h after the last ethanol injection by Δ(9)-THC infusion. The cross-tolerance appeared within 24-48 h, lasted over 72 h and was maximal in 5-day ethanol/Δ(9)-THC-treated animals. Finally, tolerance in chronic ethanol/Δ(9)-THC/-treated animals developed not only to ethanol/Δ(9)-THC-induced ataxia, respectively, but also to the ataxia potentiating effect of CHA and muscimol, indicating modulation by cerebellar adenosinergic A1 and GABAA receptors. A practical implication of these results could be that marijuana smokers may experience little or no negative effects such as ataxia following alcohol consumption. Clinically, such antagonism of ethanol-induced ataxia can be observed in marijuana users thereby encouraging more alcohol consumption and thus may represent a risk factor for the development of alcoholism in this segment of population.

Tonic modulatory role of mouse cerebellar α- and ß-adrenergic receptors in the expression of ethanol-induced ataxia: role of AC-cAMP.

To further study neurochemical basis of ethanol-induced ataxia (EIA), we investigated role of cerebellar α and ß-adrenergic receptors. Male CD-1 mice received intracerebellar microinfusion of adrenergic drugs to evaluate their effect on EIA (2g/kg; ip) by Rotorod. Isoproterenol, phenylephrine (4, 8, 16 ng each), methoxamine (8 ng), and atenolol (2, 4, 8 ng), propranolol (4, 8, 16 ng), markedly attenuated and accentuated, respectively, EIA indicating the tonic nature of modulation. The attenuation of EIA by isoproterenol is ß(1)-receptor mediated because it is blocked by atenolol. Tonic ß(1) modulation is functionally correlated with EIA potentiation by atenolol and propranolol. The prazosin-induced attenuation of EIA, initially thought of α(1)-receptor mediated, appeared instead ß(1)-receptor modulated because of: (i) blockade by atenolol; and (ii) phosphodiesterase inhibition by prazosin. The phenylephrine/methoxamine-induced attenuation of EIA seems paradoxical as the response is similar to antagonist prazosin. However, functionally the attenuation seems ß(1) receptor-mediated since atenolol blocked it but prazosin did not. Also norepinephrine (NE) attenuated EIA that was inhibited by atenolol suggesting role of ß(1) receptors. Similarly yohimbine and rauwolscine attenuated EIA that indicates α(2)-receptor modulation associated with stimulation of AC-cAMP pathway. The results of study support the hypothesis that attenuation and potentiation of EIA is mediated by activation and inhibition of AC-cAMP pathway, respectively, in agreement with our previous reports, via direct and/or indirect activation of ß-receptor.

Sustained antagonism of acute ethanol-induced ataxia following microinfusion of cyclic AMP and cpt-cAMP in the mouse cerebellum.

Ataxia is a conspicuous physical manifestation of alcohol consumption in humans and laboratory animals. Previously we reported possible involvement of cAMP in ethanol-induced ataxia. We now report a sustained antagonism of ataxia due to multiple ethanol injections following intracerebellar (ICB) cAMP or cpt-cAMP microinfusion. Adenylyl cyclase drugs cAMP, cpt-cAMP, Sp-cAMP, Rp-cAMP, adenosine A1 agonist, N6-cyclohexyladenosine (CHA) and GABA(A) agonist muscimol were directly microinfused into the cerebellum of CD-1 male mice to evaluate their effect on ethanol (2 g/kg; i.p.) ataxia. Drug microinfusions were made via stereotaxically implanted stainless steel guide cannulas. Rotorod was used to evaluate the ethanol's ataxic response. Intracerebellar cAMP (0.1, 1, 10 fmol) or cpt-cAMP (0.5, 1, 2 fmol) 60 min before ethanol treatment, dose-dependently attenuated ethanol-induced ataxia in general agreement with previous observations. Intracerebellar microinfusion of cAMP (100 fmol) or cpt-cAMP (2 fmol) produced a sustained attenuation of ataxia following ethanol administration at 1, 4, 7 and 25 h or 31 h post-cAMP/cpt-cAMP microinfusion. At 31 h post-cAMP, the ataxic response of ethanol reappeared. Additionally, marked antagonism to the accentuation of ethanol-induced ataxia by adenosine A1 and GABA(A) agonists, CHA (34 pmol) and muscimol (88 pmol), respectively, was noted 24h after cAMP and cpt-cAMP treatment. This indicated possible participation of AC/cAMP/PKA signaling in the co-modulation of ethanol-induced ataxia by A1 adenosinergic and GABAergic systems. No change in normal motor coordination was noted when cAMP or cpt-cAMP microinfusion was followed by saline. Finally, Rp-cAMP (PKA inhibitor, 22 pmol) accentuated ethanol-induced ataxia and antagonized its attenuation by cAMP whereas Sp-cAMP (PKA activator, 22 pmol) produced just the opposite effects, further indicating participation of cAMP-dependent PKA downstream. Overall, the results support a role of AC/cAMP/PKA signaling in the expression of ethanol-induced ataxia and its co-modulation by adenosine A1 and GABA(A) receptors.

Role of mouse cerebellar nicotinic acetylcholine receptor (nAChR) α(4)ß(2)- and α(7) subtypes in the behavioral cross-tolerance between nicotine and ethanol-induced ataxia.

We have demonstrated that nicotine attenuated ethanol-induced ataxia via nicotinic-acetylcholine-receptor (nAChR) subtypes α(4)ß(2) and α(7). In the present study, ethanol (2g/kg; i.p.)-induced ataxia was assessed by Rotorod performance following repeated intracerebellar infusion of α(4)ß(2)- and α(7)-selective agonists. Localization of α(4)ß(2) and α(7) nAChRs was confirmed immunohistochemically. Cerebellar NO(x) (nitrite+nitrate) was determined flurometrically. Repeated intracerebellar microinfusion of the α(4)ß(2)-selective agonist, RJR-2403 (for 1, 2, 3, 5 or 7 days) or the α(7)-selective agonist, PNU-282987 (1, 2, 3 or 5 days), dose-dependently attenuated ethanol-induced ataxia. These results suggest the development of cross-tolerance between ethanol-induced ataxia and α(4)ß(2) and α(7) nAChR agonists. With RJR-2403, the cross-tolerance was maximal after a 5-day treatment and lasted 48h. Cross-tolerance was maximal after a 1-day treatment with PNU-282987 and lasted 72h. Pretreatment with α(4)ß(2)- and α(7)-selective antagonists, dihydro-ß-erythroidine and methyllycaconitine, respectively, prevented the development of cross-tolerance confirming α(4)ß(2) and α(7) involvement. Repeated agonist infusions elevated cerebellar NO(x) 16h after the last treatment while acute ethanol exposure decreased it. Pretreatment with repeated RJR-2403 or PNU-282987 reversed ethanol-induced decrease in NOx. The NO(x) data suggests the involvement of the nitric oxide (NO)-cGMP signaling pathway in the cross-tolerance that develops between α(4)ß(2)- and α(7)-selective agonists and ethanol ataxia. Both α(4)ß(2) and α(7) subtypes exhibited high immunoreactivity in Purkinje but sparse expression in molecular and granular cell layers. Our results support a role for α(4)ß(2) and α(7) nAChR subtypes in the development of cross-tolerance between nicotine and ethanol with the NO signaling pathway as a potential mechanism.

Behavioral cross-tolerance between repeated intracerebellar nicotine and acute Delta(9)-tetrahydrocannabinol-induced cerebellar ataxia: role of cerebellar nitric oxide.

We have previously demonstrated that acute intracerebellar nicotine or N-methyl-4-(3-pyridinyl)-3-buten-1-amine (RJR-2403), a selective alpha(4)beta(2) nicotinic acetylcholine receptor (nAChR) agonist, dose dependently attenuates Delta(9)-tetrahydrocannabinol (Delta(9)THC)-induced ataxia. Presently, we have shown that intracerebellar nicotine (1.25, 2.5, and 5 ng; once daily for 5 days) and RJR-2403 (250, 500, and 750 ng; once daily for 5 days) significantly attenuate cerebellar Delta(9)-THC-induced ataxia dose dependently, suggesting the development of cross-tolerance between nicotine or RJR-2403 with Delta(9)-THC in male CD-1 mice. Intracerebellar RJR-2403 (750 ng) microinfused for 1, 2, 3, 5, and 7 days (once daily) significantly attenuated Delta(9)-THC-induced ataxia in the 3-, 5-, and 7-day treatment groups; optimal cross-tolerance was evident at day 5 and persisted till 36 h after the last RJR-2403 microinfusion. Intracerebellar microinfusion of hexamethonium (nAChR antagonist; 1 microg) or dihydro-beta-erythroidine hydrobromide (alpha(4)beta(2) nAChR antagonist; 500 ng) for 5 days 10 min before daily intracerebellar nicotine or RJR-2403 microinfusion virtually abolished cross-tolerance between nicotine or RJR-2403 and Delta(9)-THC, indicating nAChR participation. In addition, microinfusion of antagonists 10 min after daily intracerebellar nicotine or RJR-2403 failed to alter the cross-tolerance, suggesting possible involvement of downstream cerebellar second-messenger mechanisms. Finally, the cerebellar concentration of nitric oxide products [total sum of nitrite + nitrate (NO(x))] was increased after 5 days of intracerebellar nicotine or RJR-2403 treatment, which was decreased by acute intracerebellar Delta(9)-THC treatment. The "nicotine or RJR-2403 + Delta(9)-THC" treatments significantly increased cerebellar NO(x) levels compared with treatment with Delta(9)-THC alone, supporting a functional correlation between cerebellar nitric oxide production and cerebellar Delta(9)-THC-induced ataxia and suggesting participation of nitric oxide in the observed cross-tolerance between nicotine/RJR-2403 and Delta(9)-THC.

Involvement of the alpha4beta2 nicotinic receptor subtype in nicotine-induced attenuation of delta9-THC cerebellar ataxia: role of cerebellar nitric oxide.

We have recently reported that mediation of intracerebellar nicotine-induced attenuation of cerebellar delta9-THC ataxia was via the alpha4beta2 nAChR. The present study was meant to investigate the role of cerebellar nitric oxide (NO)-guanylyl cyclase (GC) signaling in the alpha4beta2-mediated attenuation in CD-1 male mice. Drugs were given via intracerebellar microinfusion using stereotaxically implanted guide cannulas, with ataxia evaluated by Rotorod. Intracerebellar microinfusion of SNP (sodium nitroprusside, NO donor; 15, 30, 60 pg) and SMT (S-methylisothiourea, inhibitor of inducible NO synthase; 70, 140, 280 fg) significantly enhanced and reduced, respectively, intracerebellar RJR-2403 (selective alpha4beta2 agonist)-induced attenuation of delta9-THC ataxia dose-dependently. Intracerebellar isoliquiritigenin (GC-activator; 1, 2, 4 pg) and ODQ (1H[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, GC inhibitor; 200, 400, 800 fg), significantly enhanced and reduced, respectively, intracerebellar RJR-2403-induced attenuation of delta9-THC ataxia dose-dependently. Further support for the role of NO was evidenced via increases in cerebellar NO(x) (nitrate+nitrite) levels following microinfusion of nicotine or RJR-2403 as compared to control, whereas delta9-THC significantly decreased NO(x) levels. "Nicotine/RJR-2403+delta9-THC" treated mice had cerebellar NO(x) levels significantly increased as compared to mice infused with delta9-THC alone. Results of the present investigation support the role of cerebellar NO-GC signaling in alpha4beta2 nAChR subtype-mediated attenuation of delta9-THC ataxia.

Mouse cerebellar nicotinic-cholinergic receptor modulation of Delta9-THC ataxia: role of the alpha4beta2 subtype.

In spite of widespread association of nicotine and cannabinoids in humans, very few studies in which nicotine and cannabinoids are co-administered have been reported. Previously, we have reported that intracerebellar (ICB) Delta(9)-tetrahydrocannabinol (Delta(9)-THC) produces dose-dependent cerebellar ataxia. The present study investigated the functional consequences of ICB microinfusion of nicotine on ICB Delta(9)-THC ataxia in CD-1 male mice. Nicotine (0.625, 1.25, 2.5, 5 ng; ICB) markedly attenuated Delta(9)-THC ataxia dose dependently, which was abolished by ICB hexamethonium (5 microg), thus suggesting that the attenuation by nicotine occurred via the nicotinic acetylcholine receptor (nAChR). To further investigate which specific nAChR subtype was involved, ICB microinfusion of RJR-2403 (250, 375, 500, 750 ng), a alpha(4)beta(2) selective nAChR agonist, markedly attenuated Delta(9)-THC ataxia. DHbetaE (500 ng), a alpha(4)beta(2) selective nAChR antagonist, virtually abolished RJR-2403-induced attenuation of Delta(9)-THC ataxia. ICB microinfusion of MLA, a alpha(7) selective nAChR antagonist (1, 5 microg) failed to antagonize nicotine or RJR-2403-induced attenuation of Delta(9)-THC ataxia. This suggested a lack of a role of the alpha(7) subtype and further reinforced the significance of alpha(4)beta(2). Additionally, ICB treatment with DHbetaE virtually abolished nicotine-induced attenuation of Delta(9)-THC ataxia that suggested alpha(4)beta(2) as the primary cerebellar nAChR subtype. Lack of effect of ICB DHbetaE or MLA alone on Delta(9)-THC ataxia ruled out a tonic effect of the alpha(4)beta(2) subtype. The results of the present investigation, therefore, strongly support involvement of the cerebellar alpha(4)beta(2), but not alpha(7), nicotinic receptor subtype in the mediation via nicotine and RJR-2403 on attenuation of Delta(9)-THC ataxia.

Behavioral interaction between nicotine and ethanol: possible modulation by mouse cerebellar glutamate.

BACKGROUND: Epidemiological studies show that people who drink alcoholic beverages also smoke cigarettes and vice versa. Furthermore, animal studies provide circumstantial evidence for ethanol and nicotine interaction. Previously, we demonstrated that intracerebellar nicotine attenuates ethanol ataxia. This study investigated the possible role of glutamate in modulating the interaction of nicotine and ethanol. METHODS: Glutamate drugs N-methyl-d-aspartate (NMDA) and (+)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrate (AMPA) as well as their antagonists were directly microinfused into the cerebellum of CD-1 male mice to evaluate their effect on ethanol (2 g/kg i.p.) ataxia. Drug microinfusions were made via stereotaxically implanted stainless-steel guide cannulas. Rotorod was used to evaluate the ataxic response of ethanol. RESULTS: Microinfusion of nicotine (0.3125, 1.25, 5 ng) significantly attenuated ethanol ataxia dose-dependently, confirming the functional interaction between nicotine and ethanol as reported earlier. Intracerebellar pretreatment with hexamethonium, a nicotinic receptor (nAChR) antagonist, significantly blocked nicotine-induced attenuation of ethanol ataxia suggesting participation of nAChRs. When ethanol was injected before nicotine microinfusion, nicotine failed to attenuate ethanol ataxia, indicating the critical importance of initial activation of nAChRs by nicotine. Intracerebellar microinfusion of NMDA (30, 60, 125 ng) and its antagonist, (+)-MK-801 (50, 100, 200 ng), significantly increased and decreased, respectively, the nicotine-induced attenuation of ethanol ataxia in a dose-related manner, suggesting participation of the NMDA receptor. Similarly, intracerebellar microinfusion of AMPA (7.5, 15, 30 ng) and its antagonist, nitro -2, 3-dioxobenzoquinoxaline-sulfonamide (NBQX; 25, 50, 100 ng), significantly increased and decreased, respectively, the nicotine-induced attenuation of ethanol ataxia in a dose-dependent manner. This suggests participation of the AMPA receptor and further supports involvement of the glutamate system in the ethanol-nicotine interaction. Intracerebellar nicotine failed to attenuate sodium-pentobarbital (25 mg/kg i.p.) ataxia, suggesting the relative specificity of the nicotine-ethanol interaction. CONCLUSIONS: The results suggested that glutamate modulates the functional interaction between nicotine and ethanol because NMDA and AMPA enhanced the nicotine-induced attenuation of ethanol ataxia, whereas (+)-MK-801 and NBQX reduced the attenuation.

Antagonism of ethanol ataxia by intracerebellar nicotine: possible modulation by mouse cerebellar nitric oxide and cGMP.

We have reported previously that intracerebellar nicotine attenuates ethanol ataxia via nicotinic-cholinergic receptors. We report now that attenuation of ethanol ataxia by intracerebellar nicotine is modulated by cerebellar nitric oxide-guanylyl cyclase (GC) messenger system. Intracerebellar microinfusion of SNP (sodium nitroprusside, a nitric oxide donor; 15, 30, and 60 pg) and SMT (S-methylisothiourea; 70, 140, and 280 fg; an inhibitor of inducible nitric oxide synthase), significantly enhanced and reduced, respectively, intracerebellar nicotine-induced attenuation of ethanol ataxia in a dose-related manner. Similarly, intracerebellar isoliquiritigenin (an activator of GC; 1, 2, and 4 pg) and ODQ (1H [1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, an inhibitor of GC; 375, 750, and 1500 fg), significantly enhanced and reduced, respectively, intracerebellar nicotine-induced attenuation of ethanol ataxia in a dose-related fashion. These results suggest that the functional interaction between nicotine and ethanol may involve modulation by cerebellar nitric oxide and cGMP. Intracerebellar microinfusion of isoliquiritigenin (4, 8, and 16 pg) in the absence of nicotine significantly attenuated ethanol ataxia dose-dependently indicating a tonic involvement of cGMP in ethanol ataxia. Finally, intracerebellar nicotine (5 ng) significantly increased and ethanol 2 g/kg i.p. decreased levels of total cerebellar nitrite+nitrate (NOx) which were functionally correlated with ethanol ataxia and its attenuation by intracerebellar nicotine. The ethanol-induced decrease in NOx was significantly antagonized by intracerebellar nicotine. The NOx data further supported an involvement of nitric oxide in the behavioral interaction between nicotine and ethanol. Overall, the results of the present investigation demonstrate a functional correlation between cerebellar nitric oxide messenger system and the behavioral interaction between nicotine and ethanol.

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 mug 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.

Acute ethanol-induced adenosine diphosphate ribosylation regulates the functional activity of rat striatal pertussis toxin-sensitive g proteins.

BACKGROUND: We demonstrated previously that striatal adenosine modulates ethanol-induced motor incoordination (EIMI) via adenosine A1 receptors coupled to pertussis toxin (PT)-sensitive G protein and adenylyl cyclase-cyclic adenosine monophosphate (cAMP). Additionally, intrastriatal (IST) PT antagonizes EIMI and its potentiation by the adenosine A1 agonist N-cyclohexyladenosine; it also inhibits cAMP concentration. METHODS: Guide cannulas were stereotaxically implanted for IST pretreatment with PT followed 5 days later by IST of N-cyclohexyladenosine and intraperitoneal ethanol. The adenosine diphosphate (ADP) ribosylation reaction involved PT-catalyzed [P]nicotinamide adenine dinucleotide (NAD) labeling of rat striatal membranes. Antagonism of EIMI (Rotorod method) after IST microinfusion of PT was investigated to determine whether it was due to a decrease in the functional activity of G proteins due to ADP ribosylation of the Gialpha subunit caused it. RESULTS: Striatal membranes from IST PT (0.5 microg)-treated animals exhibited significantly attenuated (up to 90%) in vitro ADP ribosylation with [P]NAD. Striatal membranes from animals injected with ethanol (1.5 g/kg intraperitoneally) exhibited statistically significant increase (11%) in in vitro ADP ribosylation. Similarly, ethanol (50 mM) added to striatal membranes from untreated animals produced significant stimulation of in vitro ADP ribosylation. The decrease in the functional activity of G proteins due to ADP ribosylation of the Gialpha subunit after IST PT was functionally correlated with marked attenuation in EIMI, as observed previously. This finding suggests a blockade of functional activity of PT-sensitive striatal Gi/Go proteins (i.e., fewer available sites for labeled NAD incorporation). The in vivo ethanol results indicate that it must have caused an increase in the ribosylation capacity of Gialpha in vivo (i.e., increased Gi activity). Increased ADP ribosylation by in vitro ethanol increases Gi/Go activity, consistent with EIMI, as previously reported. CONCLUSIONS: The results provide biochemical evidence of an ethanol-induced increase in ADP ribosylation of Gialpha causing a decrease in the functional activity of G proteins coupled via Gi/Go to adenylyl cyclase-cAMP. These results confirm the previously observed antagonism of EIMI by PT (IST).

Acute ethanol/cannabinoid-induced ataxia and its antagonism by oral/systemic/intracerebellar A1 adenosine receptor antisense in mice.

Previous reports from our laboratory have demonstrated that ethanol- and cannabinoid-induced ataxia is modulated by cerebellar adenosine A(1) receptor because intracerebellar (i.c.b.) adenosine A(1) agonists potentiated and A(1) antagonist attenuated ataxia by these psychoactive drugs. In this study, the novel approach involving pretreatment with adenosine A(1) antisense oligodeoxynucleotide via multiple routes provided further direct evidence of mouse cerebellar A(1) modulation of ethanol- and cannabinoid-induced ataxia. Animal groups were pretreated with A(1) antisense and its mismatch by oral (p.o.) (3.12, 6.25, 12.5, 50 microg/12 h; total three treatments/each dose), intraperitoneal (i.p.) (3.12, 5, 10, 50 microg/12 h; total three treatments/each dose), and i.c.b. (2 microg/12 h; total three treatments) routes. Based on our standard rotorod test, marked antagonism to ethanol (2 g/kg; i.p.) and delta(9)-THC (15 microg; i.c.b)-induced ataxia was observed 12 h after the last antisense treatment. Pretreatment with A(1) receptor mismatch was without an effect. The antagonism following systemic (p.o.; i.p.) antisense pretreatment was dose-dependent. No change in the normal motor coordination was observed when the animals were pretreated with antisense or its mismatch followed by vehicle. Results of Western blotting using commercially available antibodies and cerebellar membranes from various animal groups which received antisense and its mismatch via three routes confirmed a significant decrease in the A(1) adenosine receptor protein. These results, for the first time, demonstrated an oral and systemic effectiveness of A(1) antisense towards adenosine receptors in the central nervous system.

Mouse cerebellar adenosine-glutamate interactions and modulation of ethanol-induced motor incoordination.

BACKGROUND: It was demonstrated previously that cerebellar adenosine modulates ethanol-induced motor incoordination via A(1) subtype of adenosine receptors. Several reports suggest the involvement of brain glutamate mechanisms, in particular N-methyl-d-aspartate (NMDA) receptor sites, in the central nervous system (CNS) actions of ethanol. Mutually antagonistic functional responses as a result of glutamate and adenosine within the brain regions have also been well documented. METHODS: With the use of rotorod performance as the test response, this study was conducted to evaluate possible functional interactions between cerebellar adenosine and glutamate and its consequence on ethanol-induced motor incoordination. Except for ethanol, which was injected intraperitoneally, all drugs used were microinfused directly into the cerebellum. RESULTS: Direct intracerebellar microinfusion of glutamate (125, 250, and 500 ng) and the antagonist l-glutamic acid diethyl-ester (125, 250, and 500 ng) markedly and dose-dependently attenuated and accentuated, respectively, ethanol-induced motor incoordination, suggesting an involvement of glutamate. Subsequently, intracerebellar microinfusions of NMDA (125, 250, and 500 ng) and its antagonists AP-5 [(+)-2-amino-5-phosphoropentanoic acid; 125, 250, and 500 ng] and (+)-MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclo-hepten-5,10-imine hydrogen maleate; 25, 50, and 100 ng] significantly attenuated and accentuated, respectively, ethanol-induced motor incoordination in a dose-related manner, indicating participation of NMDA receptor. The attenuation of ethanol-induced motor incoordination by glutamate and NMDA was receptor mediated as it was antagonized by their receptor antagonists. Adenosine A(1) -selective agonist N(6) -cyclohexyladenosine and antagonist 8-cyclopentyl-1,3-dipropylxanthine functionally opposed the attenuation by glutamate and NMDA and the accentuation by L-glutamic acid diethyl-ester, AP-5, and (+)-MK-801, respectively, of ethanol-induced motor incoordination. CONCLUSIONS: These results suggest a functional antagonism between glutamate NMDA and adenosine A(1) receptors exhibiting a co-modulation of ethanol-induced motor incoordination within the cerebellum.