Involvement of alpha7- and alpha4beta2-type postsynaptic nicotinic acetylcholine receptors in nicotine-induced excitation of dopaminergic neurons in the substantia nigra: a patch clamp and single-cell PCR study using acutely dissociated nigral neuron

摘要

The receptor subtypes, which mediate nicotine-induced excitation of dopaminergic neurons in the substantia nigra, were investigated by whole-cell patch clamp studies and single-cell RT-PCR using acutely dissociated nigral neurons. Three types of current were observed when acetylcholine (1 mM) was applied to the neurons in the presence of atropine (1 渭M) by the U-tube system, which allowed the rapid application of drugs. In 50% of neurons examined, acetylcholine (1 mM) plus atropine (1 渭M) evoked a current with a rapidly desensitizing decay phase (designated as type Ia current). In 14% of neurons tested, the current induced by acetylcholine plus atropine had a decay phase with slow desensitization (designated as type II current). The third type of response, which had both characteristics of type Ia and II currents, was evoked in 36% of neurons tested (designated as type Ib currents). Nicotine (1 mM) also induced three types of inward currents which were similar to those induced by acetylcholine (1 mM)plus atropine (1 渭M). In all three types of current, nicotine (0.1 渭M-1 mM)-evoked inward currents were dose-dependent. Type Ia and II currents were inhibited by methyllycaconitine (MLA, 0.01 渭M), a selective nicotinic alpha7 receptor antagonist, and dihydro-beta-erythroidine (DHbetaE, 0.1 渭M), an antagonist for alpha4beta2 receptor, respectively. In type Ib currents, a fast-decaying phase was inhibited by MLA (0.01 渭M), while a slow-decaying phase was blocked by DHbetaE (0.1渭M). After recording the type Ib current, single-cell RT-PCR analysis was performed using aspirated cytoplasm as total RNA templates. The results revealed that mRNAs for alpha7 nicotinic receptor subunit and tyrosine hydroxylase were detected in the same single neuron tested, which confirms the existence of alpha7-type nicotinic acetylcholine receptor in dopaminergic neurons of this area. These results suggest that nicotine directly acts on postsynaptic alpha7- and alpha4beta2-type nicotinic acetylcholine receptors and induces inward current, which result in the excitation of dopaminergic neurons in the substantia nigra.