Biomedical subjects
Publications and source records attributed to J R Sladek.
Vasopressin neurons, transplanted from normal rat fetuses into the third ventricle of adult Brattleboro rats, alleviate the polydipsia and polyuria of the hosts. Determination of the antidiuretic activity of grafted neurons in hosts with congenital diabetes insipidus provides a convenient model for analyzing the development, plasticity, and function of transplanted central nervous system neurons in mammals.
The comparative morphology of catecholamine (CA) varicosities and neurophysin (NP)-containing perikarya of the supraoptic (SON) and paraventricular nuclei (PVN) was examined. The major CA innervation to the SON and PVN did not coexist with the major distribution of magnocellular perikarya, but was located peripheral to the nuclei. A dense distribution of CA varicosities was found ventral to the neurosecretory perikarya of the SON and overlapped numerous immunoreactive oxytocin- and vasopressin-containing neuritic profiles. Examination of Golgi-stained sections revealed that dendrites from SON perikarya projected to the CA zone and were likely candidates for the processes identified immunocytochemically. In addition, a heterogenous distribution of axosomatic contacts was found within the SON which suggested a preferential innervation of VP-containing neurons. The densest concentration of CA varicosities in the PVN occurred in the periventricular region adjacent to the third ventricle and in the contiguous parvocellular portion of the PVN. These CA varicosities overlapped scattered oxytocinerigic perikarya in both areas. In addition the ventromedial as well as the dorsolateral subnuclei of the PVN were contacted by CA varicosities; this heterogeneous distribution suggests that the each subnucleus of the PVN with its individual hypothalamic, neurohypophyseal, brainstem, or cortical projections may possibly receive a catecholaminergic innervation by a select group of CA cells or nuclear groups from the brain stem.
A method was developed that allows the analysis of neuropeptides and monoamines in a single tissue section by the application of the unlabeled antibody method for peptide staining to tissue sections freeze-dried for formaldehyde-induced monoamine histofluorescence. The hypothalamic magnocellular system of male albino rats served as a model for this study; neurons were stained with anti-neurophysin sera, which mark the vasopressin- and oxytocin-associated proteins. Neurophysin-containing perikarya appeared to be surrounded by catecholamine-containing varicosities. This phenomenon was seen to varying degrees within the supraoptic and paraventricular nuclei. The juxtaposition of varicosities and peptidergic neurons suggests an afferent fiber-target neuron relationship that might favor a functional interaction between monoamines and neuropeptides.
Blocks of the anterior hypothalamus containing vasopressin neurons were grafted from normal 17-day-old rat fetuses into the median eminence of adult female rats with a congenital deficiency of vasopressin neurons (Brattleboro strain rats). Immunocytochemical staining of the transplants 40 days after grafting demonstrated the presence of magnocellular neurons which stained positively for vasopressin and neurophysin. Axons from these neurons could be traced into the median eminence and the primary capillary plexus of the hypothalamo-hypophyseal portal system. Water consumption decreased by as much as 63% in animals carrying viable grafts. The observation that water consumption decreased and remained depressed in hosts carrying viable grafts along with the immunocytochemical data suggest that the transplanted neurons are synthesizing, storing, and releasing biologically active VP.
Adjacent tissue sections through the rat median eminence were examined for the distribution of gonadotropin-releasing hormone (GnRH) and catecholamines (CA). A simultaneous visualization technique was employed for this correlative neuroanatomical analysis. At rostral and mid-central levels of the median eminence the majority of GnRH terminals do not appear in coexistence with CA terminals; the latter were confined to the outer-most 10 micrometers of the median eminence while the densest concentration of GnRH terminals was located internal to this layer. However, individual GnRH fibers appeared to penetrate the outer CA zone wherein they were found in juxtaposition to portal capillaries. At caudal levels of the median eminence, there was an extensive overlap of CA and GnRH varicosities adjacent to the tubero-infundibular sulcus. In addition, numerous GnRH terminals were seen adjacent to portal vessels. The differences in the positions of CA and GnRH terminals between rostral and caudal median eminence may provide a morphological basis for the hypothesis of separate regulatory mechanisms for CA upon GnRH secretion at these two levels of the median eminence.
The ontogeny of the rat supraoptic (SON) and paraventricular (PVN) nuclei was studied using a combined fluorescence-immunocytochemical technique for the simultaneous localization of catecholamines (CA) and neurophysin (NP). NP neurons and CA varicosities were first detected in the SON and PVN at 1 days postcoitus. The development of NP neurons which included increases in immunoreactivity in both nuclei proceeded through fetal and neonatal stages, approaching maturity by 21-28 days postnatal; the maturation of the PVN lagged behind that of the SON. CA varicosities appear to make contact with NP neurons beginning at 21-22 days postnatal adult-like patterns were established. The prenatal dominance of NP stain relative to CA fluorescence may suggest a possible neurotrophic role for magnocellular neurons and/or their products upon ingrowing noradrenergic axons.
The distribution of dopamine, somatostatin and LHRH was examined in young and aged male rats of the Fisher 344 strain. Dopamine histofluorescence and peptide immunocytochemical staining were performed together in each animal by the use of a stimultaneous visualization technique. Comparative analysis or rats at 3, 12, 20, and 30 months of age revealed a general decrease in somatostatin and LHRH in the median eminence; dopamine fluorescence intensity also was depressed in the median eminence although dopaminergic perikarya of the arcuate nucleus of the hypothalamus appeared to increase in intensity with age. The age-related decline in median eminence LHRH may point to a central locus of reproductive senescence in the rat.
Histopharmacological and microspectrofluorometric analysis was applied to the rat hypothalamus for the identification of serotonin. Neuronal perikarya, especially certain of those of the endocrinologically important arcuate nucleus, were seen to contain a yellow histofluorescence which possessed spectral characteristics consistent with the serotonin fluorophor. Serotonin was localized also within varicosities of various hypothalamic nuclei, as well as within an ependymally associated network of fibers. Pharmacological administration of the serotonin precursor, tryptophan, resulted in the identification of serotonin histofluorescence and spectra within hypothalamic neurons yielding evidence that these neurons possess the capacity to synthesize serotonin. These newly described cells may be of importance to the regulatin of hypothalamically mediated endocrine function due to their neuroanatomical placement in the medical basal hypothalamus.
Gonadotropin-releasing hormone and dopamine were identified simultaneously in the same block of tissue from the median eminence of the rat brain. Two distinct bands of dopamine terminals were found in the lateral median eminence: an inner band which overlapped the gonadotropin-releasing hormone terminals and an outer band which appeared juxtaposed to portal capillaries.
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Formaldehyde and glyoxylic acid histochemical methods were employed to examine monoamine fluorescence of the rat median eminence. Tanycytes of the median eminence contained a yellow histofluorescence which was verified with microspectrofluorometry as due to the presence of serotonin. Catecholamine-containing varicosities, arranged in linear profiles throughout the depth of the median eminence, were observed. These linear profiles appeared to follow the contours of serotonin-containing tanycytes. Organ-culture experiments supported the hypothesis that the serotonin associated with tanycytes is localized within the tanycytes and does not arise from an extrahypothalamic source of nerve terminals. These data provide evidence that a tanycytic catecholamine-indoleamine morphological juxtaposition occurs in a manner reminiscent of that of another circumventricular organ, the pineal.
The relative content of monoamines within identified brain stem neurons in 4 and 20-year old monkeys (Macaca nemestrina) was determined with microspectrofluorometric techniques. Intraneuronal monoamine content was found to decrease with age in the locus coeruleus, substantia nigra and raphe and was found to be reduced further in the locus coeruleus by the presence of lipofuscin granules. These data indicate that intraneuronal monoamine content is reduced in the aged macaque.
A new method for the simultaneous visualization of brain peptides and monoamine neurotransmitters was employed to analyze the integrated morphology of hypothalamic catecholamines and neurophysins in young and old monkeys. Immunocytochemical analysis using bovine neurophysin revealed a dual papulation of light and dark stained cells in the paraventricular nucleus in young and old macaques. In general, both populations of neurons stained with less density in old macaques indicating the possibility of a reduced content of neurophysin. Further analysis using specific neurophysin antisera for vasopressin or oxytocin revealed an appreciable decrease in the number of vassopressin-containing perikarya in the 20 year monkey whereas oxytocin-synthesizing neurons did not show a similar change in numbers with age. Qualitatively, terminal innervation patterns of hypothalamic catecholamines remained strikingly constant in spite of marked reductions in dark-stained neurophysin perikarya of the paraventricular nucleus.
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Primary cultures of dispersed cells were prepared from 3-5 mg pieces of basal hypothalami of 10-12-day-old rats. The tissue included median eminence, arcuate nucleus and variable amounts of adjacent hypothalamus and preoptic area. The dispersion procedure consisted basically of tissue trypsinization and mechanical dissociation of cells. They were cultured in a modified L-15 medium in an air atmosphere. Neurons survived approximately 3 months. On the basis of morphological characteristics, two basic cell types could be distinguished. One was a larger (50 mum diameter) multipolar cell; microspectrofluorometric analysis revealed that a small percentage of these neurons contained a catecholamine. A second type was smaller, fusiform or ovoid and generally bipolar; a significant number of these were immunoreactive for the releasing hormone LHRH.
Microspectrofluorometry, fluorescence histochemistry and light and electron microscopic autoradiography have established the presence of sub-populations of neurons in the arcuate-periventricular region of the rat hypothalamus that sequester both radiolabeled dopamine and demonstrate formaldehyde-induced fluorescence. These characteristics are consistent with a catecholaminergic function. Selective sequestration of 3H-dopamine at the light and ultrastructural level is discussed in the context of an ultrashort loop autoregulatory mechanism for this neuronal population.
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The histochemical fluorescence technique of Falck and Hillarp was used to examine the distribution of catecholamine-containing varicostities within the hypothalamus of the rhesus monkey (Macaca mulatta). Varicosities were seen in heaviest concentrations within the periventricular area and the following nuclei: lateral and madial preoptic, supraoptic, paraventricular, arcuate and dorsomedial. Patterns observed presented striking similarities and dissimilarities to catecholamine distribution reported in the rat and car. Catecholaminergic perikarya were not observed routinely.