Elsevier

Neuroscience

Volume 91, Issue 4, July 1999, Pages 1483-1497
Neuroscience

Endothelin converting enzyme-1-, endothelin-1-, and endothelin-3-like immunoreactivity in the rat brain

https://doi.org/10.1016/S0306-4522(98)00692-7Get rights and content

Abstract

Neurons likely to use endothelin as a neurotransmitter/neurohormone were mapped in the rat brain using polyclonal antibodies directed against endothelin-converting enzyme-1, endothelin-1, and endothelin-3. Anti-endothelin-converting enzyme-1 antibodies produced the most robust staining, permitting the best visualization of the distribution and morphology of neurons. Labeled neurons were found in the dorsal thalamic nuclei and reticular thalamic nuclei, medial preoptic area, pontine nucleus, and locus coeruleus. Localization of endothelin-converting enzyme-like immunoreactivity in the locus coeruleus and in the reticular nucleus of the thalamus suggests that endothelin is co-localized with norepinephrine and GABA, respectively. Additionally, endothelin-converting enzyme-like immunoreactivity was found in the globus pallidus, septal nuclei, and in both the vertical and horizontal limbs of the nucleus of the diagonal band of Broca, and the ventrolateral area of the caudate–putamen. Strong endothelin-converting enzyme-like immunoreactivity was found in a continuous band of pyramidal neurons throughout the neocortex primarily in layer V, extending into the cingulate gyrus and piriform cortex. Motor nuclei, including oculomotor, facial, and trigeminal nuclei, were also endothelin-converting enzyme-immunoreactive. In the cerebellum, Purkinje cells were stained. Non-neuronal cells such as oligodendroglia, microglia, and astrocytes generally were not endothelin-converting enzyme-immunoreactive, although astrocytes were rarely stained. Endothelin-converting enzyme-, endothelin-1-, and endothelin-3-like immunoreactivities were generally found co-existing in given nuclei.

The diversity of neurons immunostained for endothelin suggests multiple roles of endothelin in the CNS.

Section snippets

Experimental procedures

Adult male Wistar rats (250–400 g; Charles River, Wilmington, MA and Harlan Sprague Dawley, Indianapolis, IN) were perfused transaortically with 4.0% paraformaldehyde, under sodium pentobarbital anesthesia. The brain was immediately removed and placed in 4.0% paraformaldehyde/20% sucrose in 0.01 M sodium phosphate-buffered normal saline (PBS) pH 7.4, at 4°C overnight. Eighty-micrometer brain sections were obtained using a freezing, sliding microtome. The sections were placed in PBS, for three

Results

In general, the pattern of ECE-1-like immunoreactivity was similar to endothelin-1-like and endothelin-3-like immunoreactivity. The distribution of ECE-1- and endothelin-1-like immunoreactivity in serial coronal sections is illustrated in Fig. 1. The immunoreactivities to endothelin-1 and endothelin-3 were less robust than those obtained with antibodies to ECE-1. These markers for putative endothelinergic neurons were distributed in all parts of the neuraxis, including neurons in neo-, archi-,

Discussion

Some of the most striking and robust ECE-1-like immunoreactivity was seen in the reticular nucleus of the thalamus, medial preoptic nucleus, pontine nucleus, locus coeruleus, and cranial motor nuclei. Very intense ECE-1-like immunoreactivity was displayed by the Purkinje cells in the cerebellum. Also, pyramidal neurons throughout the neocortex, primarily in layer V, in addition to a subset of neurons in layers III, IV, and VI were ECE-1-immunoreactive.

Previous studies using antibodies directed

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