J. Guy

Localization and identification of Neuropeptide Y (NPY)-like immunoreactivity in the frog brain

The distribution of neuropeptide Y (NPY) in the central nervous system of the frog Rana ridibunda was determined by immunofluorescence using a highly specific antiserum. NPY-like containing perikarya were localized in the infundibulum, mainly in the ventral and dorsal nuclei of the infundibulum, in the preoptic nucleus, in the posterocentral nucleus of the thalamus, in the anteroventral nucleus of the mesencephalic tegmentum, in the part posterior to the torus semicircularis, and in the mesencephalic cerebellar nucleus. Numerous perikarya were also distributed in all cerebral cortex. Important tracts of immunoreactive fibers were found in the infundibulum, in the preoptic area, in the lateral amygdala, in the habenular region, and in the tectum. The cerebral cortex was also densely innervated by NPY-like immunoreactive fibers. A rich network of fibers was observed in the median eminence coursing towards the pituitary stalk. Scattered fibers were found in all other parts of the brain except in the cerebellum, the nucleus isthmi and the torus semicircularis, where no immunoreactivity could be detected. NPY-immunoreactive fibers were observed at all levels of the spinal cord, with particularly distinct plexus around the ependymal canal and in the distal region of the dorsal horn. At the electron microscope level, NPY containing perikarya and fibers were visualized in the ventral nuclei of the infundibulum, using the peroxidase-antiperoxidase and the immunogold techniques. NPY-like material was stored in dense core vesicles of 100 nm in diameter. A sensitive and specific radioimmunoassay was developed. The detection limit of the assay was 20 fmole/tube. The standard curves of synthetic NPY and the dilution curves for acetic acid extracts of cerebral cortex, infundibulum, preoptic region, and mesencephalon plus thalamus were strictly parallel. The NPY concentrations measured in these regions were (pmole/mg proteins) 163 +/- 8, 233 +/- 16, 151 +/- 12 and 60 +/- 13, respectively. NPY was not detectable in cerebellar extracts. After Sephadex G-50 gel filtration of acetic acid extracts from whole frog brain, NPY-like immunoreactivity eluted in a single peak. Reverse phase high performance liquid chromatography (HPLC) and radioimmunoassay were used to characterize NPY-like peptides in the frog brain. HPLC analysis revealed that infundibulum, preoptic area and telencephalon extracts contained a major peptide bearing NPY-like immunoreactivity. The retention times of frog NPY and synthetic porcine NPY were markedly different. HPLC analysis revealed also the existence, in brain extracts, of several other minor components cross-reacting with NPY antibodies.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of a second category of α-melanocyte-stimulating hormone (α-MSH) neurons in the rathypothalamus

Summary An immunocytochemical localization of α-melanocyte-stimulating hormone (α-MSH), as well as ACTH and a fragment (16K) of the common precursor of ACTH and β-lipotropin (β-LPH) was performed in rat brain. Two different groups of neuronal cell bodies showing α-MSH-like immunoreactivity (α-MSH-LI) were observed in the hypothalamus. One group of neurons located in the arcuate nucleus was shown to contain not only α-MSH-LI, but also ACTH and the 16K fragment. A second category of α-MSH-LI-containing neurons was characterized by the complete absence of staining for ACTH and 16K fragment. These neurons were mainly located in the dorsal-lateral portion of the hypothalamus. Immunoelectron microscopy showed that immunostaining for α-MSH was restricted to dense core vesicles in the positive perikarya. Nerve fibers staining for α-MSH (but not for ACTH and 16K fragment) were also observed outside the ACTH-β-LPH pathway, especially in the cortex, caudate-putamen nucleus and hippocampus. These findings strongly suggest the presence of two different neuronal systems reacting with antibodies to α-MSH.

Neuropeptide Y in the intermediate lobe of the frog pituitary acts as an α-MSH-release inhibiting factor

The presence of neuropeptide tyrosine (NPY) in the intermediate lobe of the frog pituitary was demonstrated using indirect immunofluorescence, the immunogold technique and a specific radioimmunoassay combined with high pressure liquid chromatography (HPLC). A high density of NPY-containing fibers, was found among the parenchymal cells of the intermediate lobe. These fibers originated from the ventral infundibular nucleus, travelled via the median eminence to the pars intermedia. At the electron microscopic level, NPY-like material was found exclusively in nerve fibers where the product of the immunoreaction was associated to dense-core vesicles. High concentrations of NPY-like peptide were found in neurointermediate lobe extracts. After Sephadex G-50 gel filtration the major peak of immunoreactive material appeared to co-elute with synthetic porcine NPY. Conversely, HPLC analysis revealed that the NPY-like peptide of the frog pituitary had a retention time shorter than the porcine NPY. The localization of NPY-like material in the pars intermedia suggested a possible role of NPY in the regulation of melanotropic cell secretion. In fact, graded concentrations of synthetic NPY induced a dose-dependent inhibition of alpha-melanotropin (alpha-MSH) release in vitro. The lack of effect of a dopaminergic antagonist on NPY-induced alpha-MSH release inhibition demonstrated that the local dopaminergic system could not account for the NPY action. These results indicate that NPY located in the hypothalamo-hypophyseal system of the frog may act as a melanotropin-release inhibiting factor.

Localization and identification of α-melanocyte-stimulating hormone (α-MSH) in the frog brain

Abstract The distribution of α-melanocyte-stimulating hormone (α-MSH) in the central nervous system of the frog Rana ridibunda was determined by immunofluorescence using a highly specific antiserum. α-MSH-like containing perikarya were localized in the infundibular region, mainly in the ventral hypothalamic nucleus. A rich plexus of immunoreactive fibers directed towards the ventral telencephalic region was detected. Reverse-phase high-performance liquid chromatography and radioimmunoassay were used to characterize α-MSH-like peptides in the frog brain. Chromatographic separation revealed that immunoreactive α-MSH coeluted with synthetic des-Nα-acetyl α-MSH, authentic α-MSH and their sulfoxide derivatives. The heterogeneity of α-MSH-like material in the frog brain was in marked contrast with the figure observed in the intermediate lobe of the pituitary gland where only des-Nα-acetyl α-MSH is present. These findings support the existence of discrete α-MSH immunoreactive neurons in the frog brain containing both desacetyl and authentic α-MSH.

Differential projections of two immunoreactive α-melanocyte stimulating hormone (α-MSH) neuronal systems in the rat brain

Abstract Immunocytochemical localization of α-MSH was performed in the brain of rats of which the arcuate nucleus has been destroyed by treatment with monosodium glutamate in the neonatal period. In these animals, α-MSH cell bodies normally found in the arcuate nucleus were almost completely absent. The reactive fibers found in the ACTH-β-LPH pathway were also markedly decreased. On the other hand, α-MSH cell bodies located in the dorsolateral hypothalamus as well as fibers located outside the ACTH-β-LPH pathway were not decreased. These results strongly suggest that α-MSH cell bodies in dorsolateral hypothalamus have projections completely different from those located in the arcuate nucleus.

Serotonin innervation of neuropeptide Y-containing neurons in the rat arcuate nucleus

Structural non-synaptic appositions between serotonin (5-HT) nerve endings and neuropeptide Y (NPY)-containing neurons were demonstrated in the rat arcuate nucleus by means of a combined radioautographic and immunocytochemical detection of [3H]5-HT uptake sites and NPY-immunoreactivity. Such cellular relationships are proposed to constitute a morphological substrate for putative 5-HT/NPY interactions in neuroendocrine hypothalamus.

Biological and immunological characterization of α-melanocyte-stimulating hormone (α-MSH) in two neuronal systems of the rat brain

Abstract Recent immunocytochemical studies have demonstrated the existence of two different neuronal systems containing α-MSH-like material in the brain: one originating from the arcuate nucleus and the other one from the dorsolateral hypothalamus. The aim of the present study was to further characterize α-MSH in these two discrete regions of the rat diencephalon. Intracerebroventricular administration of colchicine resulted in a marked decrease in the number of ACTH and β-endorphin nerve fibers and a significant reduction in ACTH and β-endorphin content in the dorsolateral hypothalamus. Conversely, colchicine treatment did not alter α-MSH, ACTH or β-endorphin content in the arcuate nucleus and did not significantly affect α-MSH concentration in the dorsal region. Reverse-phase high-performance liquid chromatography showed that the major α-MSH-like compound localized in the dorsal hypothalamus co-migrated exactly with synthetic α-MSH, whereas the arcuate nucleus contained 5 peptides cross-reacting with α-MSH antibodies, 4 of them being different from standard α-MSH. Significant amounts of biologically active melanotropin, which migrated on Sephadex G-25 columns like synthetic α-MSH, were also detected in both the arcuate nucleus and dorsolateral hypothalamus. Taken together, these data demonstrate that the α-MSH cell bodies located in the dorsolateral hypothalamus specifically produce authentic α-MSH, whereas the α-MSH cell bodies in the arcuate nucleus also contain ACTH, β-endorphin and several peptides immunologically related but not identical to α-MSH.

Neuronal interactions between neuropeptide Y (NPY) and catecholaminergic systems in the rat arcuate nucleus as shown by dual immunocytochemistry.

Several recent studies have suggested interactions between catecholamine (CA) and neuropeptide Y (NPY) neuronal systems in the rat brain. In order to obtain morphological evidence for such CA/NPY interactions in the arcuate nucleus, we have used a double immunostaining procedure using an anti-tyrosine hydroxylase (TH) antiserum as a marker for catecholamine neurons and an anti-NPY antiserum. This double staining, where the first staining is silver-gold intensified, was detectable at both light and electron microscopic levels. In semi-thin sections, a substantial overlap and close proximity of TH-immunopositive neurons and NPY neuronal elements could be seen within the arcuate nucleus. At the electron microscopic level, direct appositions between TH- and NPY-immunoreactive structures could be detected. These appositions were of axosomatic, axodendritic or axoaxonic types without any synaptic membrane differentiation. Moreover, direct appositions between NPY-immunoreactive structures have also been observed. This morphological study showing appositions between TH and NPY neuronal systems suggest direct interactions between these two systems in the arcuate nucleus.

Further studies on the identification of neurons containing immunoreactive alpha-melanocyte-stimulating hormone (α-MSH) in the rat brain

In order to identify more accurately the organelles containing immunoreactive alpha-melanocyte-stimulating hormone (alpha-MSH) in the rat brain, attempts were made to improve the ultrastructural preservation of the neural tissue used for immunostaining of the peptide. With the post-embedding staining technique, the quality of the preservation was not adequate since only low concentrations (0.2-0.5%) of OsO4 could be used for postfixation. Higher concentrations of OsO4 completely destroyed the antigenicity of alpha-MSH. With the pre-embedding technique in which the immunostaining is performed prior to postfixation with OsO4, a good preservation could be obtained making possible the identification of organelles and classification of endings containing immunoreactive alpha-MSH. In the dendrites, the staining was rather diffuse without any clear association with organelles. In the endings, the staining was mostly restricted to dense core vesicles with some degree of diffusion in the cytoplasm. None of the positive endings were seen making synaptic contact. These results support the hypothesis that alpha-MSH could be released in sites other than the classical synaptic junction and act as a local hormone.

Immunocytochemical localization of somatostatin-281–12 in the rat hypothalamus

Somatostatin-28 (SS28) is considered as a precursor of somatostatin-14 (SS14) and also of the remaining NH2 terminus of SS28, the dodecapeptide SS28 which has been recently characterized. In order to study the cellular and subcellular localization of SS28 in the rat hypothalamus, we have conducted a light and electron microscopic immunocytochemical study, using both the peroxidase-antiperoxidase and the immunogold technique. Several antisera which selectively recognize one or more of these 3 somatostatin-related peptides were used. In serial paraffin sections, it was observed that SS28 was contained in the same neuronal cell bodies which also contain SS28. These neurons were exclusively located in the periventricular nucleus. All the antisera used also produced a strong staining in the external zone of the median eminence. At the electron microscopic level, the 3 peptides were exclusively localized in all the dense core vesicles in cell bodies and axons in the periventricular nucleus and terminals in the median eminence. These results strongly suggest that SS28 and SS14 originate from the precursor SS28 and that processing of the precursor occurs in the cell body. They also support the hypothesis that the 3 peptides are released simultaneously under appropriate stimulation.