Differential response of neutral endopeptidase 24.11 (“enkephalinase”), and cholinergic and opioidergic markers to hypoglossal axotomy

Neutral endopeptidase 24.11 (NEP; “enkephalinase”) may inactivate a number of centrally active neuropeptides including the enkephalins and substance P. In most areas of the central nervous system, the cell types which express NEP activity are not known. The hypoglossal nucleus (N. XII) was selected as a model system to characterize the cytochemical localization of NEP. The effect of hypoglossal nerve axotomy upon the distribution of NEP activity in the hypoglossal nucleus was compared to the effect upon cholinergic markers, the muopiate receptor, and the enkephalins. By use of a fluorescence histochemical method, NEP was localized at all levels of N.XII to the soma and proximal processes of the majority of the apparent motor neurons in the nucleus. Fluorescent double‐labeling studies revealed the presence of numerous enkephalinergic varicosities which localized to the neuropil surrounding NEP‐stained motor neurons. To determine whether NEP was synthesized by these motor neurons, 18 rats received a unilateral transection of the hypoglossal nerve. A pronounced decrease in NEP staining in N.XII was observed on the operated side as early as 3 days following axotomy. This decrease persisted at all levels of the nucleus for about 5 weeks. By 7 weeks, the staining between the control and operated sides was indistinguishable. By contrast, there was no apparent change in the density or distribution of enkephalin‐immunoreactive varicosities in five animals examined 6 to 32 days following axotomy. Radioligand binding of [³H]DAMGO to the μ‐opiate receptor in N.XII was studied in 20 animals by quantitative autoradiography at 2, 6, and 11 days after axotomy. No significant changes in the level of radioligand binding to the μ‐receptor were detected in response to axotomy. In contrast to the opiate system, the cholinergic enzymes choline acetyltransferase, acetylcholinesterase, and pseudocholinesterase showed a coordinate decrease in motor neuron‐associated staining on the operated side of N.XII at 3 ,6 , and 11 days following axotomy which paralleled the decrease in NEP staining. By contrast, the lysosomal enzyme marker, acid phosphatase, showed a pronounced increase in staining on the operated side. The results of this study are consistent with the synthesis of NEP by cholinergic N.XII motor neurons and indicates that the enkephalins and NEP in N.XII are closely associated, but derive from separate neuronal populations. The widespread overlap in the distribution of NEP‐stained motor neurons and enkephalinergic varicosities in N.XII provides additional anatomical support for a potential role for NEP in the inactivation of centrally active enkephalins. The apparently stable levels of the enkephalins and μ‐opiate receptor after axotomy, in contrast to the coordinate decrease in cholinergic enzyme staining, suggest a differential regulation of the opiate and cholinergic systems in response to axotomy. © 1994 Wiley‐Liss, Inc.