Localization of acetylcholine receptors on isolated CNS neurons: cellular and subcellular differentiation

Abstract

Significant progress has been made in determining regional expression of neurotransmitter receptors within the CNS, but little information is available at the neuronal level. In the current study, to begin characterizing cellular and subcellular aspects of receptor differentiation, we have localized ACh receptors on neurons isolated from the chicken CNS. Localization was determined autoradiographically using 2 cholinergic receptor probes: 3H-propylbenzilylcholine mustard (PrBCM) a muscarinic antagonist, and 125I-alpha-bungarotoxin (BTX), a putative nicotinic ligand in the avian CNS. To isolate neurons, we incubated embryonic chicken retinas (E13-E19) in Ca2+/Mg2+-free buffer containing 8 units/ml papain for 20 min and then gently agitated the tissue by trituration. Large numbers of dendrite-rich neurons, belonging to recognizable morphological subpopulations (e.g., multipolar neurons of various sizes, small bipolar and unipolar neurons), were collected on slides for autoradiography. Cell isolation had no effect on ligand binding levels, and a high ratio of specific to nonspecific binding allowed us to associate silver grains with receptor position. Muscarinic-receptor-positive cells comprised a recognizable subpopulation that had small rounded cell bodies (6-7 micron) and a single emergent arbor. The cells had an axial or elongated appearance. Muscarinic receptors were abundant over dendrites but absent from cell bodies. Segregation to dendrites was complete by E13, the age when synapses reportedly first begin to appear. Cells labeled with 125I-BTX were more heterogeneous in morphology. The most striking BTX-positive cells comprised neurons with large cell bodies (approximately 15 micron) and multiple processes. Dendrites were profusely labeled, but only sporadic labeling was seen on cell bodies, and often this was at sites crossed by labeled dendrites. Maximum labeling occurred in the distal, smallest-caliber ends of the dendritic arbors. All limbs of BTX-positive multipolar neurons expressed abundant receptors. Occasionally, a thin uniform-caliber process was seen branching from a primary dendrite, and such processes, which may have been axons, were never labeled. Very small neurons with bipolar morphology also showed minimal or no labeling on one process, despite dense labeling on the other. Photoreceptors and Mueller cells were never labeled with BTX or PrBCM. The data show that, within a discrete CNS region, specific subpopulations of neurons independently regulate expression of ACh receptors and that, even early in development, control mechanisms segregate receptors to physiologically appropriate regions of the cell surface.