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M C Bohn

Publications and source records attributed to M C Bohn.

At least 73 records · Page 4Linked to original sources
PubMed1988

Demonstration of glucocorticoid receptor-like immunoreactivity in glucocorticoid-sensitive vasopressin and corticotropin-releasing factor neurons in the hypothalamic paraventricular nucleus.

Many parvocellular neurons in the paraventricular nucleus of the hypothalamus express high levels of corticotropin releasing factor (CRF) or vasopressin following adrenalectomy. To determine whether glucocorticoids feed back directly on these neurons, a mouse monoclonal antibody directed against the rat liver glucocorticoid receptor was used in combination with polyclonal antisera directed against either vasopressin or CRF to permit simultaneous visualization of either peptide with glucocorticoid receptor-like immunoreactivity (IR). Rats were adrenalectomized (ADX) for 2 weeks to optimize numbers of vasopressin - and CRF-IR neurons. Six hours prior to sacrifice, a separate group of adrenalectomized rats was treated with corticosterone (40 mg/kg). This short-term replacement resulted in nuclear localization of glucocorticoid receptor-like-IR but did not attenuate the increased numbers of CRF- and vasopressin-IR neurons observed after adrenalectomy. It was therefore possible to visualize vasopressin- or CFR-IR and nuclear glucocorticoid receptor-like-IR simultaneously. Cell counts of double-labeled neurons in the paraventricular nucleus of the hypothalamus (PVH) demonstrated that glucocorticoid receptor-like-IR is colocalized in virtually all the CRF and vasopressin immunoreactive parvocellular neurons studied, which respond to adrenalectomy by increased peptide expression. These data suggest that a major feedback effect of glucocorticoids on the hypothalamic-pituitary-adrenal axis is exerted directly within nuclei of CRF and vasopressin neurons.

Adrenalectomy↗
PubMed1988

Distribution of the mineralocorticoid and the glucocorticoid receptor mRNAs in the rat hippocampus.

The cellular localization of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) gene expression in the rat hippocampus was studied by in situ hybridization using 35S-labeled RNA-probes, complementary to either 513 bases of the rat brain mineralocorticoid receptor (MR)-mRNA or 500 bases of the rat liver glucocorticoid receptor (GR)-mRNA. Neurons in CA1, CA2, and the dentate gyrus expressed both receptor genes at high levels. The MR-mRNA was demonstrated in all pyramidal cell fields (CA1-4) of the hippocampal formation and the granular neurons of the dentate gyrus. In contrast, GR-mRNA was mainly restricted to CA1 and CA2 pyramidal cell fields and the dentate gyrus. This pattern of hybridization was found to agree with the cellular distribution of the two types of corticosteroid receptors detected previously in the hippocampus by autoradiography of the radio-labeled receptors and by immunocytochemistry of the receptor protein. These observations suggest that the corticosteroid receptors described previously as type 1 and type 2 are encoded by MR- and GR-mRNA, respectively. Although both the MR and GR genes are co-expressed in some hippocampal neurons, the unique patterns of distribution of the two receptor mRNAs in the hippocampal formation suggest that the genes for these receptors are differentially regulated. Moreover, the microanatomy of MR and GR expression provides insight into molecular mechanisms underlying the characteristic action of various steroids on behaviors involved in stress and circadian regulation.

Animals↗
PubMed1987

Glucocorticoid effects on phenylethanolamine N-methyltransferase (PNMT) in explants of embryonic rat medulla oblongata.

Although glucocorticoid hormones have important roles in the development of neurotransmitter systems in cells derived from the neural crest, it is not known whether they have parallel effects on neuronal development in the brain. To address this issue, we have established an in vitro system of fetal medulla oblongata (MO) to follow development of the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT). Embryonic MO was explanted from E13 or E18 embryos and maintained for up to 3 weeks. Successful culture of adrenergic neurons was possible only in explants taken from young embryos, since E18 explants failed to develop. In E13 explants, immunoreactivity to both PNMT and tyrosine hydroxylase, the rate limiting enzyme in catecholamine synthesis, was observed. PNMT catalytic activity which was barely detectable at the time of explanation increased markedly during the first week in vitro. To study the effects of glucocorticoids on PNMT development in central neurons, MO explants were grown in glucocorticoid deficient medium in which rat serum from adrenalectomized rats was substituted for human placental serum. Addition of natural glucocorticoids, cortisol or corticosterone, or the mineralcorticoid, deoxycorticosterone, during the third culture week had no effect on PNMT activity. Dexamethasone (DEX), a synthetic glucocorticoid, also had no effect on PNMT during the first or second weeks in culture. However, addition of DEX during the third culture week resulted in a doubling of PNMT activity. However, attempts to block the DEX effect during the third week or to block the increase in PNMT activity during the first week in control cultures with the glucocorticoid receptor antagonist, dexamethasone 21-mesylate, were unsuccessful. These results suggest that PNMT in central neurons does not require glucocorticoids for ontogeny during the embryonic period. This is in contrast to PNMT in adrenal medulla which requires glucocorticoids for normal development during both the embryonic and postnatal periods. More generally, these studies suggest that development of the same neurotransmitter phenotype in brain and periphery may be differentially regulated.

Animals↗
PubMed1987

Adrenal medulla grafts enhance recovery of striatal dopaminergic fibers.

The drug, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP), depletes striatal dopamine levels in primates and certain rodents, including mice, and produces parkinsonian-like symptoms in humans and nonhuman primates. To investigate the consequences of grafting adrenal medullary tissue into the brain of a rodent model of Parkinson's disease, a piece of adult mouse adrenal medulla was grafted unilaterally into mouse striatum 1 week after MPTP treatment. This MPTP treatment resulted in the virtual disappearance of tyrosine hydroxylase-immunoreactive fibers and severely depleted striatal dopamine levels. At 2, 4, and 6 weeks after grafting, dense tyrosine hydroxylase-immunoreactive fibers were observed in the grafted striatum, while only sparse fibers were seen in the contralateral striatum. In all cases, tyrosine hydroxylase-immunoreactive fibers appeared to be from the host rather than from the grafts, which survived poorly. These observations suggest that, in mice, adrenal medullary grafts exert a neurotrophic action in the host brain to enhance recovery of dopaminergic neurons. This effect may be relevant to the symptomatic recovery in Parkinson's disease patients who have received adrenal medullary grafts.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1987

Ultrastructural localization of glucocorticoid receptor (GR) in hypothalamic paraventricular neurons synthesizing corticotropin releasing factor (CRF).

Corticotropin releasing factor (CRF) synthesizing neurons, located in the hypothalamic paraventricular nucleus (PVN), are the main central regulators of the pituitary-adrenal cortex endocrine axis. The hormone production and release of CRF-synthesizing neurons is regulated by neuronal messages and feedback action(s) of glucocorticoids secreted by the adrenal gland. In order to characterize the latter mechanism, glucocorticoid receptor (GR)-immunoreactive (IR) sites were studied in hypothalamic paraventricular neurons of intact, long-term adrenalectomized, and adrenalectomized plus glucocorticoid treated animals, by means of ultrastructural immunocytochemical labelling. In intact animals, glucocorticoid receptor immunoreactivity was found predominantly in the nuclei of parvocellular neurons. Following adrenalectomy GR-immunoreactivity was localized in the cytoplasm of the cells, and there was a concomitant disappearance of the label from the nuclei. After corticosterone administration to adrenalectomized animals, GR-IR sites were again concentrated within the cell nuclei. Immunocytochemical double labelling studies performed on adrenalectomized plus corticosterone-replaced animals demonstrated glucocorticoid receptor-IR sites in the cell nuclei of parvocellular paraventricular neurons that expressed CRF-immunoreactivity in their cytoplasm. These ultrastructural data indicate that the intracellular location of glucocorticoid receptor is dependent on the availability of glucocorticoids by the neurons. The simultaneous expression of GR- and CRF-immunoreactivity in parvocellular paraventricular neurons supports the concept of a direct feedback action of glucocorticoids upon CRF-synthesizing neurons.

Animals↗
PubMed1987

Division of small intensely fluorescent cells in neonatal rat superior cervical ganglion is inhibited by glucocorticoids.

Postnatal genesis of small, intensely fluorescent cells was studied in the rat superior cervical ganglion by combining immunocytochemistry of tyrosine hydroxylase with tritiated thymidine autoradiography. After injection of tritiated thymidine during the first postnatal week, silver grains were observed over the nuclei of many small cells with intense staining for tyrosine hydroxylase, suggesting that SIF cells are dividing postnatally. Cell counts in ganglia of rats sacrificed 2 h after tritiated thymidine showed that the rate of SIF cell proliferation was highest during the first postnatal week with approximately 20% of SIF cells dividing and that the rate declined thereafter. Counts of labeled SIF cells at 30 days in rats injected with tritiated thymidine on days 0, 2, 4, 6, 8, 10 or 14 revealed a peak of SIF cell birthdays on day 8. In these long-survival experiments, many labeled SIF cells were present in adult superior cervical ganglions. In contrast, only one labeled principal neuron was observed in a total of 450 sections. Glucocorticoid treatment of the rats during the first postnatal week paradoxically increased the number of SIF cells, but inhibited the rate of SIF cell proliferation. Dividing SIF cells immunoreactive for both tyrosine hydroxylase and phenylethanolamine N-methyltransferase were observed in glucocorticoid-treated rats. These observations suggest that many SIF cells are dividing during the first postnatal week. After cessation of division, these cells either remain SIF cells or die, but do not differentiate into principal neurons. Since glucocorticoids do not stimulate SIF cell proliferation, they must increase the number of SIF cells by biasing the differentiation of precursor cells in the superior cervical ganglion and/or enhancing SIF cell survival.

Age Factors↗
PubMed1987

Catecholaminergic horizontal and amacrine cells in the ferret retina.

Enzymes involved in the synthesis of catecholamines were detected in amacrine and what appeared to be a specific class of horizontal cells in the ferret retina. Antisera directed against the enzymes tyrosine hydroxylase (TH), which converts tyrosine to DOPA, and phenylethanolamine N-methyl-transferase (PNMT), which converts norepinephrine to epinephrine, were used with conventional immunohistochemical techniques. A population of perikarya located at the outer margin of the inner nuclear layer (INL) exhibited TH-like immunoreactivity. The cell bodies were 9-12 micron in diameter and gave rise to stout dendrites that tapered rapidly after emergence from the somata. The processes formed a planar array in the inner half of the outer plexiform layer (OPL) slightly external to the cells of origin. We could not detect any inwardly directed processes. A population of PNMT-positive cells was also observed in the outer tier of cells in the INL. These cells were very similar to those exhibiting TH immunoreactivity. An apparent difference between the 2 populations was that there were areas of intense, somewhat punctate PNMT immunoreactivity in the outer OPL. These were not observed in the TH-stained sections. Examination of horizontal sections showed that each TH-positive cell body gave rise to 4-5 major dendrites that branched to form a roughly circular dendritic field. In the periphery of the retina, an individual cell's dendrites encompassed an area up to 170 micron in diameter. The dendritic fields of cells near the center of the retina were substantially smaller.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Expression and development of phenylethanolamine N-methyltransferase (PNMT) in rat brain stem: studies with glucocorticoids.

To study the differentiation of adrenergic (epinephrine-synthesizing) neurons in brain, the initial appearance and ontogeny of phenylethanolamine N-methyltransferase (PNMT), a specific marker of the adrenergic phenotype, were studied with immunocytochemistry and catalytic assay. The appearance of immunoreactivity to dopamine beta-hydroxylase (DBH-IR), an enzyme common to the noradrenergic and adrenergic phenotypes, was also studied. DBH-IR was initially observed on embryonic Day 13 (E13) in cells located on the ventrolateral floor and wall of the rhombencephalon. A day later (E14), PNMT-IR cells and PNMT catalytic activity were observed in the rhombencephalon suggesting that, as in the adrenal gland, noradrenergic expression precedes adrenergic expression. The PNMT-IR cells were presumed to be precursors of C1 neurons since they were located in the ventrolateral medulla oblongata. Cells located in the wall of the medulla which appeared to be migrating ventrally to the C1 group also contained PNMT-IR. On E15, cells which had PNMT-IR processes coursing through the germinal zone were observed dorsally near the fourth ventricle. Although the location of the C1 cell group was apparent when PNMT was initially expressed, the dorsal C2 and C3 adrenergic cell groups were not evident until late in gestation on E19. Even in the term embryo there appeared to be PNMT-IR cells which had not yet reached their final destination. On E14 and E15, PNMT-IR cells were also observed on the floor of the pons just rostral to the pontine flexure. However, these were not observed in older embryos, suggesting that transient expression of PNMT occurs in brain, as well as in the periphery. To determine whether glucocorticoids regulate brain PNMT, we examined the effects of altered glucocorticoid levels. In contrast to PNMT in the sympathetic nervous system, PNMT activity in medulla oblongata was not affected in neonates or adults by the decrease in glucocorticoids following adrenalectomy or hypophysectomy. Conversely, elevation of glucocorticoids by hormonal treatment did not alter PNMT in neonates. Notably, however, treatment of pregnant rats with dexamethasone on E18-E21, but not earlier, increased PNMT activity in the fetal brain stem. These observations suggest that PNMT expression and development is regulated by different factors in cells derived from neural crest and tube. PNMT is expressed earlier in brain than in adrenal and sympathetic ganglia. Further, the development of PNMT in the periphery, but not in the brain, is dependent on maintenance of physiological levels of glucocorticoids.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenalectomy↗
PubMed1984

Simultaneous expression of the SP-peptidergic and noradrenergic phenotypes in rat sympathetic neurons.

We previously observed that substance P (SP) levels in the rat superior cervical ganglion (SCG) rise sharply when the ganglion is maintained in vitro. To define the cellular localization of SP, sections of cultured SCG were stained for the possible dual presence of SP and tyrosine hydroxylase (TOH). Immunoreactivity to both SP and TOH was present in the majority of principal neurons. This observation suggests that principal sympathetic neurons can express simultaneously both the noradrenergic and SP-peptidergic phenotypes.

Animals↗
PubMed1984

Glucocorticoid regulation of phenylethanolamine N-methyltransferase (PNMT) in organ culture of superior cervical ganglia.

Glucocorticoid regulation of the adrenergic enzyme, phenylethanolamine N-methyltransferase (PNMT) was studied in organ cultures of the superior cervical ganglion (SCG) from newborn rats. Although PNMT catalytic activity was present in control ganglia, enzyme levels were too low to allow visualization of PNMT immunofluorescent cells. Addition of dexamethasone (DEX) or corticosterone to the medium resulted in a large increase in PNMT activity and bright PNMT immunoreactive (PNMT-IR) staining in cells resembling small, intensely fluorescent (SIF) cells. Addition of non-glucocorticoid steroids was ineffective. Exposure to a brief, 2-hr pulse of DEX (10(-6) M) in vitro elicited the same increase in PNMT as continual exposure to DEX. Studies using metabolic inhibitors demonstrated that the steroid-dependent increase in PNMT activity required both protein and RNA synthesis. Furthermore, the increase was inhibited by cytochalasin B and by the glucocorticoid receptor antagonists, DEX 21-mesylate and cortisol 21-mesylate. These observations suggest that glucocorticoids increase PNMT protein in SIF cells by interacting with specific steroid receptors that undergo translocation to the nucleus.

Animals↗
PubMed1984

Development and characterization of glucocorticoid receptors in rat superior cervical ganglion.

Glucocorticoids have many effects on the development and regulation of catecholamine systems in sympathetic neurons. Although it has been suggested that some of these effects may be mediated by cytoplasmic steroid receptors, the presence of high affinity glucocorticoid receptors in sympathetic cells has not been established. To determine whether such receptors are present and whether receptor levels change during development, glucocorticoid binding was measured in the superior cervical ganglion (SCG) of perfused rats 24 h after adrenalectomy using [3H]dexamethasone [( 3H]Dex). Binding was optimal when [3H]Dex was added directly to tissue homogenates in the presence of 20 mM molybdate prior to preparing cytosol fractions. In the SCG of adult rats, 5.3 +/- 0.9 X 10(-15) mol of [3H]Dex was bound/ganglion. The affinity and specificity of the SCG receptors were characteristic of cytosolic glucocorticoid receptors in other tissues. Binding of [3H]Dex was completely inhibited by dexamethasone and corticosterone, partially inhibited by progesterone, and not inhibited by estradiol or testosterone. The Kd was estimated to be 1-2.5 nM from IC50 values. The ontogeny of glucocorticoid receptor binding was measured at 2, 10 and 60 days of age. Receptor concentration was highest at 2 days, 99 +/- 9 X 10(-15) mol/mg protein, and decreased to 29 +/- 4 X 10(-15) mol/mg protein at 60 days. This decrease was entirely accounted for by the developmental increase in ganglion protein, whereas total binding remained constant at approximately 5 X 10(-15) mol/ganglion at all ages.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1983

Appearance of enkephalin-immunoreactivity in rat adrenal medulla following treatment with nicotinic antagonists or reserpine.

Various neuroendocrine factors known to be important in the regulation of adrenal catecholamine biosynthesis were investigated for possible effects on enkephalin-like immunoreactivity (Enk-IR) in the adrenal medulla of the rat. In normal rats, the adrenal chromaffin cells were not stained for either methionine (met-) or leucine (leu-) Enk-IR. Staining for Enk-IR appeared in many chromaffin cells following denervation of the adrenal or treatment of rats with the nicotinic receptor antagonists chlorisondamine or pempidine. These observations suggest that splanchnic nerve activity normally depresses the levels of enkephalin-like peptides in chromaffin cells through a trans-synaptic mechanism involving acetylcholine release and nicotinic receptor stimulation. Paradoxically, treatment with reserpine also increased Enk-IR in chromaffin cells. However, this increase did not appear to result from the well known effect of reserpine to increase presynaptic nerve firing and tyrosine hydroxylase (TOH) activity, since no increase in Enk-IR was observed following treatment with phenoxybenzamine or 6-hydroxydopamine, drugs which also increase TOH activity through trans-synaptic mechanisms. The reserpine effect also did not appear to be mediated by a stress-induced increase in glucocorticoid hormones since glucocorticoid therapy alone did not increase adrenal Enk-IR. It is suggested that the increase in adrenal Enk-IR following reserpine may result from a direct action of reserpine on chromaffin cells.

Adrenal Medulla↗
PubMed1982

Development of the adrenergic phenotype: increase in adrenal messenger RNA coding for phenylethanolamine-N-methyltransferase.

Mechanisms regulating the developmental increase in the activity of adrenal phenylethanolamine-N-methyltransferase (PNMTase), an index of the adrenergic phenotype, were examined. Immunotitration indicated that the increase in catalytic activity in rat adrenal from birth to adulthood was attributable to increased numbers of PNMTase molecules, not enzyme activation. To determine whether the ontogenetic increase in PNMTase protein was associated with elevation of mRNA coding for PNMTase, cell-free translation was performed on total cellular mRNA obtained from adrenals at different ages. Translation in wheat-germ and reticulocyte lysate systems, followed by immunoprecipitation of the PNMTase product, NaDodSO4 gel electrophoresis, and fluorography, showed an 8-fold increase in the proportion of specific PNMTase mRNA relative to total mRNA in rat adrenals from birth to adulthood. Moreover, bovine adrenal medullae exhibited a 100-fold increase in PNMTase mRNA levels between embryonic life and adulthood. Consequently, the ontogenetic increase in adrenal PNMTase appears to be due to a developmental rise in specific mRNA coding for the protein.

Adrenal Medulla↗
PubMed1982

Recovery of myelination in rat optic nerve after developmental retardation by cortisol.

Oligodendrocyte formation and myelination in the optic nerve of rats treated with cortisol on postnatal days 7 and 18 were investigated at 21 and 60 days by autoradiographic and morphometric methods. At 21 days, the amount of myelin in the optic nerve of treated rats was reduced by 42%. This reduction was accompanied by a 39% decrease in the number of myelinated axons. In addition, the myelin/axon area ratio was reduced 22% for those axons which were myelinated. At 60 days, the amount of myelin, the number of axons myelinated, the average axonal size, and the average amount of myelin per axon had completely recovered to normal values. Tritiated thymidine-labeling experiments showed that the genesis of oligodendrocytes was enhanced following the cessation of treatment. Since the number of oligodendrocytes in the optic nerve of treated rats was normal at 60 days, it is suggested that the retarding effect of cortisol on myelination in the optic nerve may be mediated, at least in part, by inhibited formation of oligodendrocytes during treatment.

Aging↗