Abstract

To assess the density and distribution of functional nicotinic acetylcholine receptors (nAChRs) and gamma-aminobutyric acid (GABA) receptors on hippocampal neurons, we have combined infrared videomicroscopy with a nanorobotic micromanipulator system and studied the receptor-mediated currents by using the whole-cell patch-clamp technique. Acetylcholine or GABA was applied by pressure ejection onto a small segment of either cell soma or dendrite, and the resulting current was measured in cultured hippocampal neurons by using a whole-cell pipette positioned at the cell soma. Type IA nicotinic currents, sensitive to blockade by methyllycaconitine (1 nM) and alpha-bungarotoxin and associated with alpha 7-subunit-containing nAChRs, type II currents, sensitive to blockade by dihydro-beta-erythroidine (100 nM) subserved by alpha 4 beta 2 nAChRs, and GABA-mediated currents were evoked when the agonists were applied to either cell soma or the dendrites. Analysis of the current amplitude with respect to the membrane area covered by the applied agonist provided an estimation of the receptor density along the somato-dendritic axis of the hippocampal neurons. Such analysis revealed: 1) a nonuniform distribution for the receptor types studied; 2) a higher density of nAChR and GABA receptors at the dendrites than at the soma; and 3) an increasing density for both nAChR subtypes with distance from the center of the cell soma, but increasing and then decreasing density for the GABA receptor. Exposure of cultured hippocampal neurons to colchicine (100 nM for 3 days) produced a dramatic reduction in the dendritic branching, and this morphological feature was associated with a significant decrease in the receptor density, such an effect being more prominent for nAChRs than for GABA receptors. Given the high Ca+2 permeability of nAChRs, the dendritic localization of nAChRs suggest that they are involved in modulating the synaptic efficacy at the level of the dendrites.