Demyelination and axonal dystrophy in alpha A-crystallin transgenic mice.

Homozygous mice transgenic for alphaA-crystallin, one of the structural eye lens proteins, developed hindlimb paralysis after 8 weeks of age. To unravel the pathogenesis of this unexpected finding and the possible role of alphaA-crystallin in this pathological process, mice were subjected to a histopathological and immunohistochemical investigation. Immunohistochemistry showed large deposits of alphaA-crystallin in the astrocytes of the spinal cord, and in the Schwann cells of dorsal roots and sciatic nerves. Additionally, microscopy showed dystrophic axons in the spinal cord and digestion chambers as a sign of ongoing demyelination in dorsal roots and sciatic nerves. Apart from a few areas with slight alphaA-crystallin-immunopositive structures, the brain was normal. Because the alphaA-crystallin protein expression appeared in specific cells of the nervous system (astrocytes and Schwann cells), the most plausible explanation for the paralysis is a disturbance of cell function caused by the excessive intracytoplasmic accumulation of the alphaA-crystallin protein. This is followed by a sequence of secondary changes (demyelination, axonal dystrophy) and finally arthrosis. In conclusion, alphaA-crystallin transgenic mice develop a peripheral and central neuropathy primarily affecting spinal cord areas at the dorsal side, dorsal root and sciatic nerve.

Demonstration of coexisting catecholamine (dopamine), amino acid (GABA), and peptide (NPY) involved in inhibition of melanotrope cell activity in Xenopus laevis: a quantitative ultrastructural, freeze-substitution immunocytochemical study.

This quantitative ultrastructural immunocytochemical study demonstrates the coexistence of a catecholamine [dopamine (DA)], an amino acid (GABA), and a neuropeptide [neuropeptide Y (NPY)] in axon varicosities innervating the pars intermedia of Xenopus laevis. The varicosities are assumed to control the pars intermedia melanotrope cells, which regulate skin color during the physiological process of background adaptation. Varicosity profiles appear to abut melanotrope cells and folliculostellate cells, star-shaped cells that intimately contact the melanotropes. All varicosity profiles contain two morphological types of vesicle. Monolabeling studies on routinely fixed and freeze-substituted tissues showed that the small, electron-lucent vesicles store GABA, whereas DA and NPY occur in larger, electron-dense ones. Double and triple labeling experiments, in which the degree of immunoreactivity was quantified per varicosity profile and per vesicle, led to the conclusion that (1) DA, GABA, and NPY coexist within almost all varicosity profiles and (2) DA and NPY are costored within electron-dense vesicles. Varicosity profiles that about melanotrope cells show a much higher ratio between the numbers of electron-lucent and electron-dense vesicles than varicosities contacting folliculostellate cells (15.8 and 3.3, respectively). This differential distribution is in line with the previous demonstration that, in contrast to GABA, NPY does not act directly on the melanotrope cells but indirectly, by controlling the activity of the folliculostellate cells.

Immunoblotting technique to study release of melanophore-stimulating hormone from individual melanotrope cells of the intermediate lobe of Xenopus laevis.

The melanotrope cells in the pars intermedia in the pituitary of Xenopus laevis synthesize and release the melanophore-stimulating hormone (alpha MSH), a small peptide that causes skin darkening during the process of background adaptation. Evidence has been found for a heterogeneity in biosynthetic activity of the melanotrope cells. In the present study two questions were addressed: (1) does the melanotrope cell population also show heterogeneous alpha MSH-release, and (2) can this heterogeneity be changed by extracellular messengers? Since dopamine is known to inhibit alpha MSH-release, this messenger is used to study the regulation of the heterogeneity. To quantify alpha MSH-release from individual cells, a cell blotting procedure has been developed for the binding and relative quantification of the small alpha MSH peptide. The immunoblotting procedure involves binding of the cells to a carrier slide and binding of released alpha MSH to a nitrocellulose filter. After immunostaining, the amount of alpha MSH per cell was quantitated by image analysis. Untreated melanotrope cells reveal a distinct variability in alpha MSH-release, some cells showing low secretory activity, whereas others are strongly secreting, indicating heterogeneity of alpha MSH-release. Dopamine treatment strongly inhibits alpha MSH-release from individual cells, resulting in a clearly less pronounced melanotrope cell heterogeneity. The effect of dopamine appears to be dose-dependent as a low dopamine concentration has only a moderate effect on the alpha MSH-release. It is proposed that dopamine is a physiological regulator of the degree of melanotrope cell heterogeneity in alpha MSH-release.

Indirect action of elevated potassium and neuropeptide Y on alpha MSH secretion from the pars intermedia of Xenopus laevis: a biochemical and morphological study.

A number of neurochemical messengers have been shown to act directly on the melanotrope cells of the pars intermedia of Xenopus laevis to regulate alpha MSH secretion. In the present study the possibility that the melanotropes are also indirectly controlled has been examined. For this purpose, the characteristics of alpha MSH release from superfused intact lobes, cultured lobe and isolated melanotropes were compared after treatment with elevated potassium. Isolated melanotropes responded with an increased secretion of alpha MSH, whereas intact lobes showed a profound inhibitory response, probably caused by potassium-induced release of inhibitory factors from nerve terminals. Cultured lobes displayed a biphasic response characterized by an initial activation followed by a strong inhibition; the stimulatory phase likely reflects a direct action of potassium on the melanotropes, before being overridden by an inhibitory mechanism. The inhibitory phase must originate from the action of nonneuroendocrine cells because the cultured lobes lack functionally active nerve terminals, as verified by immunocytochemistry and electron microscopy. The most likely candidates for this action are folliculo-stellate cells which are in intimate contact with the melanotropes and are innervated by neuropeptide Y-containing nerve terminals. Like elevated potassium, neuropeptide Y inhibited alpha MSH secretion from fresh and cultured lobes but not from isolated melanotropes. This indicates that NPY acts indirectly, in a nonpresynaptic way, to inhibit alpha MSH secretion.

[125I]Bolton-Hunter neuropeptide-Y-binding sites on folliculo-stellate cells of the pars intermedia of Xenopus laevis: a combined autoradiographic and immunocytochemical study.

It has previously been established that neuropeptide-Y (NPY) is a potent inhibitor of alpha MSH release from the pars intermedia of the amphibian Xenopus laevis. The location of binding sites for NPY in the pars intermedia of the pituitary has now been studied with light microscopic autoradiography, using a dispersed cell labeling method with the specific NPY receptor ligand [125I]Bolton-Hunter NPY. The majority of radioactive labeling was associated with folliculo-stellate cells; the percentage of labeling as well as the mean number of grains were approximately 5 times higher for folliculo-stellate cells than for melanotropes. An excess of nonlabeled NPY drastically reduced radiolabeling of folliculo-stellate cells, but had no effect on the degree of labeling of melanotropes. These results show that folliculo-stellate cells of X. laevis possess specific binding sites for NPY and indicate that NPY exerts its inhibitory action on the release of alpha MSH in an indirect fashion, by acting on the folliculo-stellate cells.

Demonstration of dopamine in electron-dense synaptic vesicles in the pars intermedia of Xenopus laevis, by freeze substitution and postembedding immunogold electron microscopy.

The presence of dopamine in the pituitary of the clawed toad Xenopus laevis was studied by light and electron microscope immunocytochemistry, using pre- and postembedding techniques. Light microscopy showed the presence of an intricate, anti-dopamine-positive fibre network throughout the pars intermedia. In preembedded stained material, dopamine appeared to occur in varicosities which make synaptic contacts with both folliculo-stellate cells and melanotrope cells. Post-embedding immunogold staining of freeze-substituted material permitted the localization of anti-dopamine reactivity in electron-dense vesicles in these varicosities. This finding supports the hypothesis that dopamine is involved in the (inhibitory) control of melanotrope cell activity in X. laevis.

Morphology of the pars intermedia and the melanophore-stimulating cells in Xenopus laevis in relation to background adaptation.

The melanophore-stimulating hormone (MSH) cells of the amphibian pars intermedia secrete the peptide alpha-melanophore-stimulating hormone (alpha-MSH), which induces pigment dispersion in dermal melanophores. The purpose of the present study was to determine (1) whether prolonged activation of the secretory activity of the pars intermedia is associated with hypertrophy, hyperplasia, or both and (2) whether the MSH cells function as a homogeneous or heterogeneous population in meeting the physiological demand for MSH. The demand for MSH was manipulated by adapting animals for at least 3 weeks to white, two shades of grey, or black backgrounds. Morphometric analysis showed that the intermediate lobe volume was positively correlated with the degree of pigment dispersion in the melanophores. The number of MSH cells per lobe was not affected by the degree of pigment dispersion. Therefore, we conclude that enlargement of the tissue associated with MSH cell activation involves hypertrophy rather than hyperplasia. Ultrastructural examination indicated that the majority of MSH cells in black-adapted animals are biosynthetically active, whereas the cells of white-adapted animals are relatively inactive and show granule storage. The pars intermedia of grey-adapted toads contained both active and inactive cells, indicating that MSH cells respond as a heterogeneous cell population in meeting the endocrine demand imposed by background.

The effect of chronic apomorphine treatment on the ultrastructure of the prolactin cells and on plasma prolactin levels in young and aged male Wistar rats.

Effects of two doses of apomorphine on the plasma prolactin (PRL) levels and on the ultrastructure of PRL cells in young and aged male Wistar rats were investigated. In young and aged control rats no significant differences were found between the plasma PRL levels. Immunocytochemical staining with anti-r-PRL revealed significant differences between young and aged control rats; in young rats the number of PRL cells with polymorphic granules exceeded the number of cells with round granules, whereas in aged rats almost exclusively cells with round granules were found. In young rats, chronic treatment with a low dose (0.01 mg/kg/day) of apomorphine did not result in a significant change in plasma PRL level or cell morphology. However, high dose (0.25 mg/kg/day) of apomorphine resulted in a significant decrease in plasma PRL levels, a decrease of number of cells with polymorphic granules and an increase of cells with round granules. The occurrence of PRL cells with round granules and plasma levels was negatively correlated. In aged rats, apomorphine (0.01 or 0.25 mg/kg/day) treatment did not affect plasma levels nor did it affect the distribution of the cell types. We conclude that in young rats PRL cells are sensitive to apomorphine and that their ultrastructure reflects a phase of the secretory cycle. In aged rats, the cells appear to have lost their sensitivity to apomorphine. The fact, that the distribution over the different cell types in control aged rats is similar to that of the apomorphine-treated young rats, suggests a strong influence of endogenous dopamine on PRL cell physiology in the aged rat.

GABA and neuropeptide Y co-exist in axons innervating the neurointermediate lobe of the pituitary of Xenopus laevis--an immunoelectron microscopic study.

The neural innervation of the neurointermediate lobe of the pituitary of the amphibian Xenopus laevis has been studied at the light and electron microscopic level. In the pars intermedia melanotropes and stellate cells are abutted by varicosities originating from GABA- and neuropeptide Y-producing neurons. The varicosities contain two types of vesicle: electron-lucent vesicles (mean diameter 50 nm) which are immunopositive for GABA and larger (80 nm) electron-dense vesicles which are immunopositive for neuropeptide Y. Double immunogold labeling established that GABA and neuropeptide Y co-exist within the varicosities. In the pars nervosa similar varicosities, though low in number, occur. They are associated with neurosecretory nerve terminals, pituicytes and blood vessels. The possible significance of GABA and neuropeptide Y for the neural regulation of melanophore stimulating hormone-release from the pars intermedia is discussed.