Ultrastructural localization of adrenocorticotrophic hormone and the phosphoprotein B-50/growth-associated protein 43 in freeze-substituted, Lowicryl HM20-embedded mesencephalic central gray substance of the rat.

Previous studies have shown that the endogenous phosphorylation of the neuron-specific protein B-50 in isolated synaptic plasma membranes is inhibited by adrenocorticotrophic hormone(1-24). The aim of this study is to examine if there is a specific neuroanatomical interaction of adrenocorticotrophic hormone and B-50 in the mesencephalic central gray substance of the rat. With light microscopy, high B-50 immunoreactivity was detected throughout the mesencephalic central gray substance, overlapping with those areas where adrenocorticotrophic hormone-immunoreactive fibres were present. To study the ultrastructural localization of B-50 and adrenocorticotrophic hormone, we employed a method of immunogold labelling on ultrathin sections of freeze-substituted and Lowicryl HM20-embedded fixed brain tissue. This offered optimal morphological preservation together with high retention of antigenicity. At the electron microscopic level, adrenocorticotrophic hormone immunoreactivity was detected in dense-core secretory granules present in non-junctional regions of axoinal varicosities. This suggests a non-synaptic release of adrenocorticotrophic hormone from the axons. Using double immunolabelling techniques we showed that in adrenocorticotrophic hormone-innervated areas of the mesencephalic central gray substance B-50 immunoreactivity was present at plasma membranes of all unmyelinated axons and axonal varicosities and virtually absent in dendrites. The result on B-50 localization agrees well with previous studies in the hippocampus [Van Lookeren Campagne et al. 1990 J. Neurocytol. 19, 948-961] and in the pyramidal tract [Gorgels et al. 1989 J. Neurosci. 9, 3861-3869] of the rat and suggests that in the mature rat central nervous system, B-50 expression in axons is a general phenomenon. For the adrenocorticotrophic hormone-innervated areas, we discuss the proposal that non-synaptically released adrenocorticotrophic hormone modulates B-50 phosphorylation in axons and axon terminals.