Journal of Neuroscience, Vol 10, 2804-2817, Copyright © 1990 by Society for Neuroscience
Electron microscopic localization of immunoreactive enkephalinase (EC 3.4.24.11) in the neostriatum of the rat
D Marcel, H Pollard, P Verroust, JC Schwartz and A Beaudet
Montreal Neurological Institute, Montreal, Quebec, Canada.
The fine structural distribution of the enzyme-neutral endopeptidase EC
3.4.24.11 (enkephalinase) was examined by immunoradioautography (using an
iodinated monoclonal antibody) and peroxidase immunocytochemistry (using
the same probe in nonradioactive form) in the neostriatum of the rat. At
the light microscopic level, both techniques revealed a heterogeneous
distribution of immunoreactive enkephalinase in the caudoputamen,
characterized by the presence of patches of intense immunolabeling
prominent against a relatively strong immunoreactive matrix, a pattern
reminiscent of mu opioid receptors radioautographically labeled in the same
region. Pilot experiments indicated that fixation of the brain with a
mixture of 4% paraformaldehyde, 0.05% glutaraldehyde, and 0.2% picric acid
did not modify the distribution and only slightly reduced the intensity of
striatal enkephalinase antigenicity, provided that the post-fixation period
did not exceed 1 hr. In the neostriatum of animals fixed according to this
protocol, enkephalinase immunoreactivity was found by electron microscopic
immunoradioautography to be exclusively confined to neuronal and glial
membrane interfaces. Immunoperoxidase cytochemistry confirmed the
association of immunoreactive enkephalinase with the plasma membrane of
neurons and, to a lesser extent, of astrocytes and oligodendrocytes. Both
immunoradioautographic and immunoperoxidase techniques revealed a
predominant association of the enzyme with neuronal perikarya and
dendrites. The morphological features of the labeled perikarya, together
with the presence of immunoreactive dendritic spines, suggested that some
of these neurons corresponded to striatofugal medium spiny neurons.
Immunoreactive enkephalinase was also detected at the level of myelinated
and unmyelinated axons and axon terminals. These axons could potentially
have originated from intrinsic striatal neurons or from the substantia
nigra. Statistical analysis of silver grain distribution in electron
microscopic immunoradioautographs indicated that immunoreactive
enkephalinase was not preferentially concentrated at the level of specific
membrane interfaces, but rather, was more or less uniformly distributed on
the surface of neurons and/or glial cells. A similarly diffuse localization
of the enzyme was apparent in peroxidase-reacted material, though the
latter technique also revealed a microheterogeneity in the deposition of
the reaction product along the labeled membranes. Finally, quantitative
analysis of immunoradioautographs clearly indicated an absence of
enkephalinase enrichment at the level of synaptic junctions. The similarity
between the light and electron microscopic distribution of enkephalinase
observed in the present study, and that previously reported for mu opioid
receptors, lends support to the concept that this ectoenzyme may be
involved in the inactivation of endogenous opioids in the mammalian
neostriatum.