Development of cross tolerance between repeated intracerebellar nicotine and acute δ9-THC-induced cerebellar ataxia: role of cerebellar nitric oxide

M. Saeed Dar, Aaron Smith. Brody School of Medicine, East Carolina University, Greenville, North Carolina, United States.

We have previously shown that acute intracerebellar (ICB) nicotine and RJR, a nAChR α4β2-selective subtype agonist, dose-dependently attenuated THC ataxia which was functionally correlated with cerebellar nitric oxide (NO) (Smith & Dar, 2006, 2007). Now we report the effect of repeated ICB nicotine and RJR on acute ICB THC ataxia and NOx.

Male CD-1 mice, weighing 24-28 g, received nicotine, RJR and THC directly within the culmen of the anterior lobe of cerebellum. Rotorod was used to evaluate the ataxia. Drugs were microinfused via stereotaxically implanted [from bregma (in mm) AP -6.4, ML + 0.8, DV -1.0] guide cannulas (22ga. 10mm). NOx (total nitrite + nitrate) was measured by a fluorometric assay based on reaction with 2, 3-diaminonaphthalene (DAN) reagent (Rao et al., 1998). Rotorod and NOx data were analyzed by 2-way (with repeated measures) and 1-way ANOVA (with Bonferroni post hoc analysis), respectively.

Results indicate that ICB nicotine (1.25, 2.5, 5 ng) or RJR (250, 500, 750 ng) microinfused once daily for 5 days, significantly (P<0.5) attenuated THC ataxia dose-dependently. The results also suggest development of cross tolerance between nicotine/RJR and THC and an absence of desensitization of nAChRs. When RJR (750 ng) was microinfused for 1, 2, 3, 5, and 7 days (once daily), it attenuated (P<0.5) THC ataxia in 2, 3, 5, and 7-day treatment groups with optimal cross tolerance noted on day-5. Desensitization of nAChRs may explain lower degree of cross tolerance in 7-day nicotine/RJR-2403 exposure group vs.5-day group. Cross tolerance lasted till 36 h after the last RJR microinfusion, a period interestingly comparable to 48 h needed for the onset of cross tolerance.

Hexamethonium (nAChR antagonist; 1μg ICB) or DHβE (α4β2 nAChR antagonist; 500 ng ICB) treatment for 5 days, 10 min prior to daily nicotine or RJR microinfusion, virtually blocked the development of cross tolerance, suggesting participation of nAChRs and absence of their desensitization. On the contrary, when hexamethonium and DHβE were microinfused 10 min after daily ICB nicotine or RJR, there was no effect on cross tolerance, suggesting involvement of downstream second messenger mechanisms.

Finally, cerebellar NOx was significantly (P<0.5) reduced by acute ICB THC which explains the THC-induced cerebellar ataxia and indicates an inverse relationship between cerebellar NOx level and ataxia. Nicotine or RJR alone significantly (P<0.5) increased cerebellar NOx (32% and 29% over basal levels) associated with no change in normal motor coordination of animals. THC microinfusion in nicotine/RJR treated mice resulted in a reduction of NOx towards basal level only which explains lack of THC-induced ataxia in the presence of nicotine/RJR. The inability of THC to produce ataxia in the presence of nicotine/RJR correlates with its inability to lower NOx levels below basal levels. The NOx data, therefore, points towards a pivotal role of cerebellar NOx in acute THC-induced cerebellar ataxia as well as in the cross tolerance between nicotine/RJR and THC. The NOx data also provide yet another evidence that neither nAChRs nor CB1 receptor undergo desensitization during 5-day repeated nicotine and RJR exposure.