Nicotinic receptor subtypes in rat subfornical organ neurons and glial cells

It is unclear which nicotinic acetylcholine receptor (nAChR) subtypes are involved in the nicotinic activation of cells in the subfornical organ (SFO). We investigated the nAChR subtype using molecular biological, electrophysiological, pharmacological and immunohistochemical techniques. The use of reverse transcription-polymerase chain reaction in rats demonstrated the presence of mRNAs for the alpha2, alpha3, alpha4, alpha6, alpha7, beta2 and beta4 subunits in the SFO. The characteristics and dose-dependency of nicotine-induced inward currents in many dissociated SFO neurons were similar to those induced by acetylcholine in the presence of atropine. The nicotine-induced currents were larger than those induced by cytisine in most responding cells, suggesting the predominance of the beta2- rather than the beta4-containing nAChR. NIC-induced currents were significantly inhibited by dihydro-beta-erythroidine (a relatively selective antagonist for alpha4-containing nAChRs, and a partial antagonist for alpha2 or alpha3) at 300 nM in all responding cells. Additionally, the currents were significantly inhibited by alpha-conotoxin MII at 10 nM (a selective antagonist for alpha3- and/or alpha6-containing nAChRs) in some but not all responding cells. Methyllycaconitine at 10 nM (a selective alpha7-nAChR antagonist) reduced the nicotine-induced current significantly, but to a lesser extent. Fluorescence-labeled alpha-bungarotoxin (a homomeric alpha7 subtype selective binding drug) binding and immunofluorescence for the alpha7 subunit showed that positive images almost overlapped with those immunopositive for an astrocyte marker. These results suggest that the alpha4beta2 subtype is the main functional receptor in SFO neurons while alpha2, alpha3, alpha6, and beta4 subunits have some effect, and homomeric the alpha7 subtype exists in SFO astrocytes.