Ultrastructural localization of [125I]neurotensin binding sites to cholinergic neurons of the rat nucleus basalis magnocellularis

Abstract

The distribution of specifically-labeled neurotensin binding sites was examined in relation to that of cholinergic neurons in the rat nucleus basalis magnocellularis at both light and electron microscopic levels. Lightly prefixed forebrain slices were either labeled with [125I](Tyr3) neurotensin alone or processed for combined [125I]neurotensin radioautography and acetylcholinesterase histochemistry. In light microscopic radioautographs from 1-microns-thick sections taken from the surface of single-labeled slices, silver grains were found to be preferentially localized over perikarya and proximal processes of nucleus basalis cells. The label was distributed both throughout the cytoplasm and along the plasma membrane of magnocellular neurons all of which were found to be cholinesterase-positive in a double-labeled material. Probability circle analysis of silver grain distribution in electron microscopic radioautographs confirmed that the major fraction (80-89%) of specifically-labeled binding sites associated with cholinesterase-reactive cell bodies and dendrites was intraneuronal. These intraneuronal sites were mainly dispersed throughout the cytoplasm and are thus likely to represent receptors undergoing synthesis, transport and/or recycling. A proportion of the specific label was also localized over the nucleus, suggesting that neurotensin could modulate the expression of acetylcholine-related enzymes in the nucleus basalis. The remainder of the grains (11-20%) were classified as shared, i.e. overlied the plasma membrane of acetylcholinesterase-positive neuronal perikarya and dendrites. Extrapolation from light microscopic data, combined with the observation that shared grains were detected at several contact points along the plasma membrane of cells which also exhibited exclusive grains, made it possible to ascribe these membrane-associated receptors to the cholinergic neurons themselves rather than to abutting cellular profiles. Comparison of grain distribution with the frequency of occurrence of elements directly abutting the plasma membrane of neurotensin-labeled/cholinesterase-positive perikarya indicated that labeled cell surface receptors were more or less evenly distributed along the membrane as opposed to being concentrated opposite abutting axon terminals endowed or not with a visible junctional specialization. The low incidence of labeled binding sites found in close association with abutting axons makes it unlikely that only this sub-population of sites corresponds to functional receptors. On the contrary, the dispersion of labeled receptors seen here along the plasma membrane of cholinergic neurons suggests that neurotensin acts primarily in a paracrine mode to influence the magnocellular cholinergic system in the nucleus basalis.