Subunit composition of functional nicotinic receptors in dopaminergic neurons investigated with knock-out mice

Abstract

Nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic (DA) neurons have long been considered as potential therapeutic targets for the treatment of several neuropsychiatric diseases, including nicotine and cocaine addiction or Parkinson's disease. However, DA neurons express mRNAs coding for most, if not all, neuronal nAChR subunits, and the subunit composition of functional nAChRs has been difficult to establish. Immunoprecipitation experiments performed on mouse striatal extracts allowed us to identify three main types of heteromeric nAChRs (alpha4beta2*, alpha6beta2*, and alpha4alpha6beta2*) in DA terminal fields. The functional relevance of these subtypes was then examined by studying nicotine-induced DA release in striatal synaptosomes and recording ACh-elicited currents in DA neurons fromalpha4, alpha6, alpha4alpha6, and beta2 knock-out mice. Our results establish that alpha6beta2* nAChRs are functional and sensitive to alpha-conotoxin MII inhibition. These receptors are mainly located on DA terminals and consistently do not contribute to DA release induced by systemic nicotine administration, as evidenced by in vivo microdialysis. In contrast, (nonalpha6)alpha4beta2* nAChRs represent the majority of functional heteromeric nAChRs on DA neuronal soma. Thus, whereas a combination of alpha6beta2* and alpha4beta2* nAChRs may mediate the endogenous cholinergic modulation of DA release at the terminal level, somato-dendritic (nonalpha6)alpha4beta2* nAChRs most likely contribute to nicotine reinforcement.

Figure 1.

Subunit composition of striatal nAChRs. A, B, Solubilized striatal membrane extracts from α6-/-, α4-/-, α6+/+, and α4+/+ mice, preincubated with 2 nm³H-Epi, were immunoprecipitated with subunit-specific Abs. Results are expressed in femtomoles of immunoprecipitated ³H-Epi/mg of protein (n = 3-5; ^*p < 0.05 compared with matching Wt control; Student's t test). Total ³H-Epi and ¹²⁵I-αBgtX binding to striatal extracts is indicated. C, IPP experiments were conducted on extracts from the dorsal striatum of α6+/+ mice after 6-OHDA lesions. Each value is the mean of two to three independent determinations on different groups of animals (^*p < 0.05 compared with saline-lesioned groups; Student's t test). The extent of DA denervation was assessed by ³H-WIN35,428 binding, a ligand for DA transporter (204 ± 26 and 41 ± 3 fmol/mg protein in the saline- and 6-OHDA-lesioned groups, respectively). D, α6^* and α4^* nAChRs were purified from α6+/+ striatal extracts using an anti-α6 and an anti-α4 affinity column. (nonα6)α4^* nAChRs were purified on an anti-α4 affinity column using striatal extracts from α6-/- mice or from α6+/+ mice after α6 depletion. The subunit content of purified nAChRs was determined by IPP. The amount of ³H-Epi immunoprecipitated is expressed as percentage of total ³H-Epi bound to the purified material before IPP (n = 3).

Figure 2.

Displacement of [¹²⁵I]epibatidine binding to immunoimmobilized nAChRs. αCtxMII (A) and MLA (B) were used to displace ¹²⁵I-Epi binding to immunoimmobilized striatal nAChRs. Three types of nAChR preparation have been tested in these experiments: β2 immunoimmobilized nAChRs from α6-/- animals [β2(α6-/-)] and α6-immunoimmobilized nAChRs from α4-/- [α6 (α4-/-)] or α6+/+ [α6 (α6+/+)] animals. According to our IPP experiments, these three preparations are likely to contain primarily α4β2, α6β2, and a mix of α4α6β2 and α6β2 nAChRs, respectively. Data points were fitted to a one- or two-site model using the LIGAND program (see Table 1 for fitting parameter values). For both ligands, this analysis revealed the existence of a heterogeneous population of binding sites only in α6-immunoimmobilized nAChRs from Wt mice. This result is in agreement with the existence of α4α6β2 nAChRs. Each point is the mean ± SEM of at least four separate determinations.

Figure 3.

Nicotine-induced DA release in striatal synaptosomes. A, Striatal synaptosomes from α4-/-, α6-/-, β2-/-, and α4-/-α6-/- animals and their respective Wt controls were prepared and loaded with ³H-DA. The effect of 3 μm l-nicotine on DA release was determined in the presence or absence of 100 nm αCtxMII. Peak ³H-DA release values (expressed as percentage of release above baseline levels) represent the mean ± SEM of at least three determinations. ^*p < 0.01 compared with matching Wt control (Student's t test); °p < 0.01 compared with αCtxMII-free condition (paired Student's t test). B, C, Dose-response curves for nicotine-induced DA release. For each synaptosome preparation, data were normalized to the response obtained with 3 μm nicotine. Dose-response curves were fitted to the Hill equation: Release = R_max/(1+(EC₅₀/[Nic])ⁿ).

Figure 4.

ACh-elicited currents in SNc/VTA DA neurons. Effect of αCtxMII. Representative traces of ACh-elicited currents in DA neurons from α6+/+, α4-/-, α6-/-, and α4-/-α6-/- mice. Mean αCtxMII (100 nm) inhibition values are shown in Table 2. In α4-/-α6-/- mice, or inα4-/- animals after αCtxMII application, residual current exhibited the kinetic (fast rise and decay) and pharmacological characteristics (complete inhibition by 1 nm MLA) of α7 nAChR-mediated currents.

Figure 5.

Nicotine-elicited DA release in the ventral striatum of α6-/- mice. A-C, Mice (n = 4-7 animals per group) were injected intraperitoneally with a solution of 0.5 or 1 mg kg^-1 nicotine (free base), and extracellular DA levels were measured by microdialysis in the ventral striatum. Results are expressed as percentage of basal DA levels (mean of 5-8 samples collected immediately before treatment). Basal DA levels in perfusate did not differ between the two genotypes. D, AUC values, representing the amount of DA outflow collected during the 0-120 min post-treatment period, are expressed as percentage of basal values. (^*p < 0.05 and ^**p < 0.01 relative to the corresponding control NaCl-treated group; two-way ANOVA). No statistically significant difference between α6+/+ and α6-/- mice is observed.

Figure 6.

Subunit composition of functional nAChRs expressed by DA neurons. The relative proportion of the different nAChR subtypes mediating nicotine-induced DA release in striatal synaptosomes or ACh-elicited currents in SN/VTA are indicated (gray insets). The contribution of subunits in brackets to functional α6^* nAChRs remains to be fully established. In addition to its direct actions on nAChRs expressed by DA neurons, nicotine might increase DA concentration in the Nac by at least two other mechanisms. By acting on α7 nAChRs located on cortical glutamatergic terminals, nicotine stimulates intra-VTA glutamate release and alters the firing pattern of DA neurons via NMDA receptor activation (Nomikos et al., 2000). At concentrations close to those found in smokers' blood, nicotine desensitizes α4β2 nAChRs on GABAergic interneurons, thus relieving the inhibitory control they exert on DA neurons (Mansvelder et al., 2002). Both direct and indirect mechanisms are likely to play a role in nicotine reinforcement.