β2- and β4-Subunit Composition of Nicotinic Acetylcholine Receptors in the Guinea Pig Inferior Mesenteric Ganglion: Immunohistochemical and Electrophysiological Studies

Abstract

1 Bogomolets Institute of Physiology, National Academy of Sciences of Ukraine, Kyiv, Ukraine. Correspondence should be addressed to L. P. Voitenko (e-mail: lvoit@serv.biph. kiev.ua). Affinity-purified rabbit antibodies (Ab) against extracellular fragments 193-204 of nicotinic acetylcholine receptor (nAChR) β2 and β4 subunits were used in the experiments with immunoperoxidase labeling of guinea pig inferior mesenteric ganglion (IMG) neurons and intracellular recording of excitatory postsynaptic potentials (EPSP) from these units. Since the epitopes of these Ab are close to an acetylcholine-binding site located at the interface of α and β subunits, we expected that the Ab can block acetylcholine-mediated EPSP responses of IMG neurons, thus enabling us to determine the involvement of β2and β4-containing nAChR in synaptic transmission through intermesenteric pathways. Microscopic examination revealed the presence of two cell types in IMG preparations: principal ganglionic neurons (PGN), 48-60 μm in diameter, and small cells of unknown origin, 6-18 μm in diameter. The β4-specifi c Ab labeled 59.8 ± 7.5% of PGN, while the β2-specifi c Ab labeled less than 1% of these cells, the β4-positive label being much more intensive than the β2-positive one. The β4-specifi c labeling was similar to that revealed with the α3-specifi c Ab. It should be noted that the percentage of α5-immunoreactive IMG neurons differed insignifi cantly from that of α3-labeled PGN. The somata of small cells were labeled with the β4-specifi c Ab too, and a group of β2-positive small cells was also found. Both β4and β2-positive neurons were localized near the origins of the main nerve trunks branching from the IMG (mesenteric, colonic, and hypogastric nerves). In electrophysiological experiments, the β4-specific Ab decreased the EPSP amplitudes by 25.5 ± 1.8% in all 10 PGN tested. In contrast, the β2-specific Ab did not suppress the synaptic responses under study. Our findings showed that α3-, α5-, and β4-containing nAChR are present in the guinea pig IMG neurons and are involved in synaptic transmission through the intermesenteric pathways. Probably, the α3β4 nAChR subtype is more widespread that is consistent with the generally recognized nature of ganglionic nAChR.