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M H Whitnall

Publications and source records attributed to M H Whitnall.

At least 37 records · Page 2Linked to original sources
PubMed1989

Stress selectively activates the vasopressin-containing subset of corticotropin-releasing hormone neurons.

A functional ultrastructural assay was used to determine the response of corticotropin releasing hormone (CRH) neurosecretory cells to short-term stress. Depletion of neurosecretory vesicles from axonal swellings in the external zone of the rat median eminence was used as a measure of functional activity. One hour of immobilization or 5 h of insulin-induced hypoglycemia caused marked depletion of vesicles from the vasopressin (VP)-containing CRH axons, but had no effect on the VP-deficient subpopulation of CRH axons. Injection of colchicine (100 micrograms) into the lateral ventricle also resulted in selective depletion of vesicles from the VP-containing subpopulation over the course of 5 h, with no depletion from the VP-deficient axons. By 24 h after injection of 100 micrograms colchicine, however, both the VP-containing and the VP-deficient axons were severely depleted of neurosecretory vesicles. These data demonstrate for the first time that the CRH neurosecretory system contains functionally distinct components, and that the VP secreting component may specifically mediate the ACTH response to short-term stress.

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PubMed1989

Regulation of carboxypeptidase H gene expression in magnocellular neurons: response to osmotic stimulation.

Carboxypeptidase H (CPH) is a peptide-processing enzyme thought to be involved in the synthesis of many neuropeptides, including vasopressin (VP) and oxytocin (OT). In this study, employing in situ hybridization histochemistry, we have shown that CPH mRNA is abundantly expressed in the magnocellular paraventricular and supraoptic nuclei of the hypothalamus, the primary sites of OT and VP synthesis. Since this enzyme is copackaged in secretory vesicles and hence coreleased with the neurohypophysial hormones, enzyme stores are depleted in parallel with the peptide hormones during states of hypersecretion. Chronic osmotic stimulation, such as occurs in long-term salt-loading or in diabetes insipidus in the Brattleboro rat, causes depletion of neurohypophysial hormone stores and is accompanied by increased rates of neurohypophysial hormone transcription and translation. This study has shown that the expression of CPH mRNA is also significantly increased in oxytocin and vasopressin producing magnocellular neurons during chronic osmotic stimulation of the hypothalamic-neurohypophysial system. CPH mRNA levels in other peptidergic areas of the brain are not significantly changed by osmotic stimulation. These findings illustrate a coordinate regulation of the transcription of peptide hormones and an enzyme required for the hormones' posttranslational processing.

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PubMed1988

Distributions of pro-vasopressin expressing and pro-vasopressin deficient CRH neurons in the paraventricular hypothalamic nucleus of colchicine-treated normal and adrenalectomized rats.

The corticotropin-releasing hormone (CRH) neurosecretory system in normal rats consists of two major subpopulations of parvicellular neurons in the hypothalamic paraventricular nucleus distinguished by the presence or absence of coexistent vasopressin precursor (pro-AVP)-derived peptides. These neurons project to the external zone of the median eminence, where the two subtypes of axons (CRH +/AVP + and CRH+/AVP-) were previously found to be approximately equal in number. The present study was undertaken 1) to determine whether the relative numbers of pro-AVP expressing and pro-AVP deficient perikarya in the paraventricular nucleus corresponded to what we previously found for the axons in the median eminence, 2) to map the two cell types throughout the entire paraventricular nucleus to determine whether significant differences existed in their distributions, and 3) to ascertain whether or not the pro-AVP deficient subpopulation expressed pro-AVP after adrenalectomy. Postembedding electron microscopic immunocytochemistry on serial ultrathin sections was used to identify the peptide phenotypes of perikarya in the paraventricular nucleus in normal rats and 7 days after adrenalectomy with and without colchicine treatment. The peptide phenotypes of neuronal perikarya in the paraventricular nucleus were identified by using antibodies to CRH, AVP, neurophysin (NP), the C-terminal glycopeptide of pro-AVP (GP), and oxytocin-associated neurophysin (NPOT). Groups of three serial coronal ultrathin sections were analyzed at 200-micron intervals throughout the entire rostrocaudal extent of the paraventricular nucleus. The sections in each group were stained for CRH, a pro-AVP-derived peptide (AVP, NP, or GP), and NPOT, respectively. Parvicellular CRH neurons were defined as CRH-positive cells, approximately 10 micron in diameter, that did not contain detectable NPOT. Pro-AVP expressing cells were defined as staining positively for AVP, GP, or NP and negatively for NPOT. Approximately equal numbers of pro-AVP expressing ("NPAVP+") and pro-AVP deficient ("NPAVP-") parvicellular CRH neurons were found within the paraventricular nucleus of colchicine-treated normal rats, and the two subtypes were distributed differently within the paraventricular nucleus. Although the pro-AVP expressing CRH cells stained intensely for NP and GP, staining for AVP was quite variable and difficult to quantify in colchicine-treated normal animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenalectomy↗
PubMed1988

Major pro-vasopressin-expressing and pro-vasopressin-deficient subpopulations of corticotropin-releasing hormone neurons in normal rats. Differential distributions within the paraventricular nucleus.

Two approximately equal subpopulations of corticotropin-releasing hormone (CRH)-containing parvocellular axons can be identified in the external zone of the median eminence in normal (unadrenalectomized) rats: one that contains pro-vasopressin (AVP)-derived peptides (i.e. AVP, AVP-associated neurophysin and the carboxy terminal glycopeptide) copackaged with CRH in secretory vesicles, and another that contains no detectable pro-AVP-derived peptides. In this study, antibodies to pro-AVP-derived peptides were used to demonstrate for the first time that similar subpopulations of CRH-containing parvocellular perikarya exist in the paraventricular nucleus of the hypothalamus in normal rats treated with colchicine. Electron-microscopic immunocytochemistry was performed on serial ultrathin sections to identify neurosecretory cell perikarya containing CRH that also expressed pro-AVP peptides or pro-oxytocin-derived neurophysin. Of the CRH-positive neurons that were detected, more than half stained positively for two pro-AVP peptides: AVP-associated neurophysin and the carboxy-terminal glycopeptide. Many of these cells also stained for AVP, but staining was variable, making quantitation of AVP-positive cells difficult. The remaining CRH-positive neurons contained no detectable pro-AVP peptides, and less than 0.5% of these CRH perikarya contained oxytocin-associated neurophysin. In the neurons that stained positively for both CRH and the pro-AVP peptides, CRH and the pro-AVP peptides were localized in the same secretory vesicles. The pro-AVP expressing and pro-AVP-deficient CRH neurons were distributed differently within the paraventricular nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1987

Vasopressin coexists in half of the corticotropin-releasing factor axons present in the external zone of the median eminence in normal rats.

Electron-microscopic immunocytochemistry of serial thin sections showed that, in normal rats, approximately 50% of corticotropin-releasing factor (CRF) axons in the external zone of the median eminence contained vasopressin (AVP) and AVP precursor-derived peptides coexistent in the same secretory vesicles as CRF. The other 50% of the CRF axons contained little or no detectable AVP or AVP precursor-derived peptides. Differential activation of these subpopulations of CRF axons could provide a mechanism by which levels of CRF and AVP in portal plasma could be varied independently.

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PubMed1987

Vasopressin-containing and vasopressin-deficient subpopulations of corticotropin-releasing factor axons are differentially affected by adrenalectomy.

CRF-containing parvocellular axons in the external zone of the rat median eminence were classified as vasopressin-containing (CRF+/AVP+) and vasopressin-deficient (CRF+/AVP-) subpopulations based on post-embedding electron microscopic immunocytochemical staining of serial ultrathin sections for CRF, AVP and the other peptides derived from the AVP precursor: AVP-associated neurophysin (NP-AVP) and the C-terminal glycopeptide (GP). In normal animals, the CRF+/AVP+ and CRF+/AVP- subpopulations were approximately equal in terms of detectable axonal swellings. Three to 14 days after adrenalectomy (ADX), the CRF+/AVP+ and CRF+/AVP- subpopulations represented about 95% and 5%, respectively, of total CRF+ swellings. This change was due to a 90% decrease in the absolute number of detectable CRF+/AVP- swellings after ADX, whereas the absolute number of detectable CRF+/AVP+ swellings rose by less than 20%. These changes were completely blocked by administering the glucocorticoid agonist dexamethasone throughout the period after ADX. The results suggest that the CRF+/AVP+ and CRF+/AVP- subpopulations of neurosecretory axons in the external zone of the median eminence respond differently to ADX, indicating that they are independently regulated by glucocorticoids.

Adrenalectomy↗
PubMed1986

Improved visualization of the immunoreactive hypothalamo-neurohypophysial system by use of immuno-gold techniques.

Ultrastructural post-embedding immuno-gold techniques were applied to the supraoptic nucleus and the neurohypophysis of mice and rats. The primary antibodies were three different monoclonal antineurophysins, used in protein A-gold and immunoglobulin-gold procedures. Conventional plastic embedding as well as hydrophilic media (L.R. White) were used; non-osmicated and osmicated tissues were immunolabeled; sodium metaperiodate oxidation was used, but was not essential for immunolabeling. Vasopressinergic and oxytocinergic NSGs were identified by the specific immunoreactivity of their respective neurophysins on adjacent thin sections, and by sequential double labeling on the same thin section using two different antibodies associated with gold probes of different diameters. The immunoidentification indicates that vasopressin NSGs can additionally be differentiated as larger, with more electron-dense matrix, and susceptible to damage by sodium metaperiodate. The only organelles consistently labeled were neurosecretory granules (NSGs), either intact or within lysosomal configurations. Some lysosomal dense bodies were immunoreactive even when discrete NSGs were no longer morphologically recognisable within them. Labeled NSGs were located within neuronal cell bodies, along axonal shafts and within axonal swellings and endings; occasionally immunoreactive NSGs were observed within synaptic boutons. Labeling intensity was semi-quantitatively gauged by counting gold particles in relation to numbers of NSGs per axonal varicosity. The precise localisation achieved with particulate immunogold labeling surpasses that previously obtained with diffuse electron-dense immunoreaction products.

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PubMed1985

Ultrastructural immunolocalization of vasopressin and neurophysin in neurosecretory cells of dehydrated rats.

It has been proposed that neurosecretory material may be transported in a non-vesicular compartment in magnocellular neurons in the hypothalamus of osmotically stimulated rats. We have reexamined this issue using postembedding electron microscopic immunoperoxidase labeling of the secreted peptides (vasopressin and neurophysin) found in these neurons. Reaction product was found exclusively over neurosecretory vesicles in cell bodies, and in axons in the median eminence and neurohypophysis. The findings are consistent with an exclusive secretory vesicle location of the neuropeptides in the hypothalamo-neurohypophysial system of both normal and dehydrated rats.

Animals↗
PubMed1985

Neurophysin in the hypothalamo-neurohypophysial system. I. Production and characterization of monoclonal antibodies.

Seven mouse monoclonal antibodies (IgGs) were produced against rat neurophysins (NPs). Three were specifically directed against vasopressin-associated NP (NP-AVP), and four were specific for oxytocin-associated NP (NP-OT). These specificities were observed in liquid phase assays, immunoblot, and immunoprecipitation experiments. Homozygous Brattleboro rat tissues and extracts, which do not contain vasopressin or NP-AVP, did not react with the anti-NP-AVP antibodies but reacted with high affinity to the anti-NP-OT antibodies. In immunoprecipitation assays the antibodies brought down the appropriate NPs as well as their precursor molecules synthesized in vivo with no detectable cross-reactivity. In solid phase assays where the antigens were presented in a different manner, there was a significant cross-reactivity of the anti-NP-AVP antibodies with NP-OT. The extent of this cross-reactivity in solid phase correlated with the cross-reactivities of the antibodies observed in immunocytochemical studies. These solid phase (and immunocytochemical) data demonstrated that liquid phase specificities and absorption controls of antibodies are inadequate to assess their immunocytochemical (solid phase) specificities. Posterior pituitary extracts from the mouse and frog, as well as purified NPs from the rat, cow, and human were studied for their cross-reactivities to two of the antibodies, PS 36 and PS 45. In liquid phase assays the anti-rat NP-OT antibody, PS 36, reacted only with rat and mouse NPs and did not cross-react with NPs from any of the other species. In contrast, the anti-rat NP-AVP antibody, PS 45, was cross-reactive across species lines including an NP-like antigen extracted from frog posterior pituitaries. Immunoblot staining with these antibodies showed heterogeneity of NP-AVP and NP-OT in the rat posterior pituitary. Analysis of the epitopes for PS 36 and PS 45 indicated the antigenic determinants were located near amino acid positions 80 to 81 in NP-OT and 75 to 86 in NP-AVP, respectively.

Animals↗
PubMed1985

Neurophysin in the hypothalamo-neurohypophysial system. II. Immunocytochemical studies of the ontogeny of oxytocinergic and vasopressinergic neurons.

Two anti-neurophysin monoclonal antibodies (MABs), PS 36 and PS 41, described in the preceding paper (Ben-Barak, Y, J.T. Russell, M.H. Whitnall, K. Ozato, and H. Gainer (1985) J. Neurosci. 5:000-000), allowed us to specifically stain for oxytocin-associated neurophysin (NP-OT) or vasopressin-associated neurophysin (NP-AVP) in the hypothalamus of developing rats. Staining with these MABs specific for NP-OT or NP-AVP showed that both types of neurophysin appeared in cells in the developing hypothalamus as early as embryonic day (E16) and continued to increase in immunoreactivity throughout fetal life. The literature indicated that oxytocin appears in the system between E20 and E22, much later than vasopressin (E16 to E17), which we confirmed in immunocytochemical experiments using affinity-purified antisera to these hormones. Since the MABs recognize the specific prohormones as well as the specific mature neurophysins (Ben-Barak, Y., J. T. Russell, M.H. Whitnall, K. Ozato, and H. Gainer (1985) J. Neurosci. 5: 81-97), we conclude that there is a developmental delay between the synthesis of the oxytocin prohormone (pro-oxyphysin) and its processing to form oxytocin and NP-OT. The delay in prohormone processing in the oxytocin cells was correlated with a delay in immunocytochemically detectable neurites as compared to the vasopressin cells. This reduced level of axonal and dendritic immunoreactivity was still obvious in the oxytocin cells at 9 days after birth. In contrast, the clustering of cells to form adult-like hypothalamic nuclei appeared to follow similar time courses for the two types of cells. Adult-like distributions of cells staining for NP-OT and NP-AVP were already apparent in the supraoptic and paraventricular nuclei by E17.

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PubMed1983

Dynorphin-A-(1-8) is contained within vasopressin neurosecretory vesicles in rat pituitary.

Dynorphin-A-(1-8), an opioid peptide widely distributed in the rat central nervous system, is present in vasopressin-containing neurosecretory cells terminating in the neural lobe of the pituitary. Electron microscopic immunocytochemistry reveals that dynorphin-A-(1-8) is contained within the same neurosecretory vesicles as vasopressin and vasopressin-associated neurophysin in the neural lobe of the rat. The results indicate that dynorphin may be released in the pituitary concomitantly with vasopressin during the antidiuretic response.

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PubMed1982

Perikaryal routing of newly synthesized proteins in regenerating neurons: quantitative electron microscopic autoradiography.

Intracellular transport of newly synthesized proteins through organelles in the perikarya of regenerating goldfish retinal ganglion cells was studied using electron microscopic autoradiography. Retinas were removed 14 or 30 days after optic tract cut or sham operation, pulse-labeled in [3H]proline-containing medium for 5 min, and then chase-incubated in medium containing unlabeled proline for various times up to 55 min before fixation. Fourteen days after axotomy, during rapid growth of the regenerating axons, the time course of change of relative grain density (% grains/% area) in the rough endoplasmic reticulum in regenerating cells was almost identical to that in control cells. However, the grain distribution analysis revealed an increased delivery of newly synthesized proteins to the Golgi apparatus, perikaryal plasma membrane and nucleus in regenerating cells. Thirty days after axotomy, during synaptogenesis, Golgi apparatus labeling in the regenerating cells became significantly higher than control, but the increase was delayed compared to the increase seen 14 days after axotomy. Labeling of the plasma membrane and nucleus did not rise above control in 30-day regenerating cells chase-incubated for up to 55 min. Thus the pattern of intracellular transport of newly synthesized proteins varies with the stage stage of axonal regeneration.

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PubMed1981

The relationship between extracellular amino acids and protein synthesis is altered during axonal regeneration.

The incorporation of [3H]proline into proteins of goldfish retinal ganglion cells was measured by light microscopic autoradiography of isolated retinas that had been incubated in labeled medium under pulse-chase conditions. It was found that the composition of the immediate precursor pool for protein synthesis is more directly influenced by extracellular amino acids in regenerating cells 14 d after axotomy than in sham-operated controls.

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