Upregulation of [3H]methyllycaconitine binding sites following continuous infusion of nicotine, without changes of alpha7 or alpha6 subunit mRNA: an autoradiography and in situ hybridization study in rat brain.

It is well established that exposure of experimental animals to nicotine results in upregulation of the alpha4beta2-subtype of neuronal nicotinic acetylcholine receptors (nAChRs). The aim of this study was to determine the effect of nicotine on the levels of alpha7-nAChRs in rat brain, for which only partial information is available. Rats were infused with nicotine (3 mg/kg/day) or saline for 2 weeks and their brains processed for receptor autoradiography with [3H]methyllycaconitine (MLA), a radioligand with nanomolar affinity for alpha7-nAChRs. In control rats binding was high in hippocampus, intermediate in cerebral cortex and hypothalamus, and low in striatum, thalamus and cerebellum. There was high correlation between the distribution of [3H]MLA binding sites and alpha7 subunit mRNA (r = 0.816). With respect to saline-treated controls, nicotine-treated rats presented higher [3H]nicotine binding in 11 out of 15 brain regions analysed (average increase 46 +/- 6%). In contrast, only four regions showed greater [3H]MLA binding, among which the ventral tegmental area (VTA) and cingulate cortex (mean increase 32 +/- 3%). No changes in alpha7 mRNA levels were observed after nicotine treatment. Similarly, there was no variation of alpha6 subunit transcript in the VTA, a region which may contain MLA-sensitive (non-alpha7)-alpha6*-nAChRs (Klink et al., 2001). In conclusion, nicotine increased [3H]MLA binding, although to a smaller extent and in a more restricted regional pattern than [3H]nicotine. The enhancement of binding was not paralleled by a significant change of alpha7 and alpha6 subunit transcription. Finally, the present results provide the first anatomical description of the distribution of [3H]MLA binding sites in rat brain.

Chronic nicotine treatment changes the axonal distribution of 68 kDa neurofilaments in the rat ventral tegmental area.

Region-specific decreases of neurofilament proteins (NF) were described in the ventral tegmental area (VTA) of rats treated chronically with morphine, cocaine or alcohol. In a previous study, we demonstrated that NF levels were also changed in the VTA after chronic treatment with nicotine. The aim of this study was to clarify the submicroscopic basis of decreased immunoreactivity for NF-68, NF-160 and NF-200, as determined by using NR4, BF10 and RT97 antibodies, respectively. Microdensitometric analysis of brain sections showed that immunoreactivity for all NF was reduced in the VTA of animals exposed chronically to nicotine (0.4 mg/kg per day, 6 days of treatment), when compared to rats exposed to saline. Reduction in immunoreactivity was significant for NF-68 (P < 0.05), NF-160 (P < 0.01) and NF-200 (P < 0.05), showing a relative reduction of 34%, 42% and 38%, respectively, when compared to saline-treated rats. No difference was observed for any of the NF under study when immunoreactivity measurements in the substantia nigra were compared. Ultrastructural analysis was applied to evaluate changes in NF-68, NF-160 and NF-200 immunoreactivity in regions of the VTA that contain dopaminergic neurons following chronic nicotine treatment. At the electron microscopic level, no degenerative changes were found in neurons or glial cells of the VTA. With ultrastructural immunohistochemistry, evaluation of the homogeneity parameter of NF distribution showed a loss of homogeneity for NF-68 linked to the nicotine treatment. In areas in which NF organization appeared well preserved, analysis of the numerical density of NF revealed no significant difference for NF-68 (897/ micro m2 vs. 990/ micro m2), NF-160 (970/ micro m2 vs. 820/ micro m2) and NF-200 (1107/ micro m2 vs. 905/ micro m2) in nicotine-treated rats when compared to saline-treated rats. These results confirm that nicotine shares the same properties with cocaine and morphine in reducing NF in the VTA, a key brain structure of the rewards system, and that chronic nicotine treatment changes the axonal distribution of 68 kDa neurofilaments in the rat VTA.