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R E Harlan

Publications and source records attributed to R E Harlan.

At least 19 recordsLinked to original sources
PubMed1992

Regulation of glucocorticoid receptor immunoreactivity in the rat hippocampus by androgenic-anabolic steroids.

To determine whether androgenic-anabolic steroids (AAS) regulate glucocorticoid receptors (GR), the effects of a mixture of three commonly abused AAS (testosterone cypionate, nandrolone decanoate and boldenone undecylenate) on GR immunoreactivity (ir), were examined in hippocampi of adrenalectomized male rats. Treatment with AAS for 1 week increased nuclear GRir in pyramidal cells of CA1, and granule cells of the dentate gyrus. These findings suggest a role for GR in the mediation of some of the CNS effects of androgenic-anabolic steroids.

Adrenalectomy↗
PubMed1992

Glucocorticoid regulation of preproenkephalin gene expression in the rat forebrain.

The effects of glucocorticoids on the levels of preproenkephalin (PPE) mRNA in the rat forebrain were analyzed with in situ hybridization and dot blots. In adrenally-intact rats, high levels of PPE mRNA, as assessed by in situ hybridization, were localized in the caudate-putamen, nucleus accumbens, central amygdala, and ventrolateral ventromedial hypothalamus (VMHVL), and low levels in the hippocampus. After adrenalectomy, the density of PPE mRNA-positive cells and the level of PPE mRNA/cell were decreased in all regions except the hippocampus. Acute treatment with corticosterone (CORT) in adrenalectomized rats increased the level of PPE mRNA/cell in the caudate-putamen and VMHVL. In intact rats, chronic treatment with CORT increased the density of PPE mRNA-positive cells in the caudate-putamen and hippocampus, and the level of PPE mRNA/cell in the caudate-putamen and nucleus accumbens. The effect of chronic CORT treatment on PPE mRNA in the striatum, amygdala, hippocampus and mediobasal hypothalamus was assessed with dot blots. Chronic CORT treatment increased PPE mRNA levels in the caudate-putamen and hippocampus. There was a good correlation between results on the effect of chronic CORT treatment on PPE mRNA levels in intact rats, obtained from dot blots and in situ hybridization. Results from this study suggest that glucocorticoids are required for the maintenance of basal PPE mRNA levels in most regions of the rat forebrain. There is, however, considerable regional heterogeneity in the effect of glucocorticoid treatment on PPE mRNA levels in adrenalectomized and intact rats. Increased PPE mRNA levels in response to high circulating levels of glucocorticoids, e.g. in stress, may have important pathophysiological consequences.

Amygdala↗
PubMed1992

Localization of the tachykinin neurokinin B precursor peptide in rat brain by immunocytochemistry and in situ hybridization.

Tachykinins exert a broad range of actions in the mammalian nervous system. While much is known about the localization of peptides derived from one of the two mammalian tachykinin genes (substance P- and neurokinin A-encoding preprotachykinin), little has been reported on the localization of peptides derived from a second tachykinin gene encoding neurokinin B. Using an antiserum raised against a 30-residue peptide fragment (Peptide 2) of the protein precursor to neurokinin B, we have mapped the distribution of Peptide 2 by immunocytochemistry. Peptide 2 antiserum specificity was determined by western blot analysis (which showed antibody cross-reactivity to a neurokinin B fusion protein from a cloned neurokinin B-encoding complementary DNA) and by the elimination of immunoreactive product in brain tissue sections upon preabsorption with a 10 microM concentration of Peptide 2 peptide. In addition, we report on the distribution of neurokinin B-messenger RNA with a full-length complementary RNA probe to localize cells that express the neurokinin B precursor. Peptide 2 immunoreactivity and neurokinin B-messenger RNA-positive cells were found, in some instances, paralleling the distribution of substance P and in other cases existing separately from substance P. Peptide 2 immunoreactivity as well as neurokinin B-messenger RNA-positive cells were found in the main olfactory bulb, cortex, olfactory tubercle, nucleus accumbens, hippocampus, bed nucleus of the stria terminalis, amygdala, medial habenula, periaqueductal gray, superior and inferior colliculus, and nucleus of the spinal trigeminal tract. Whereas substance P is found throughout the rat brain, neurokinin B appears to be partitioned more to forebrain than to brainstem structures. The marked differences in the distribution of both tachykinins in the rat central nervous system suggests that neurokinin B may play an important role in olfactory, gustatory, visceral, and neuroendocrine processing of information.

Amino Acid Sequence↗
PubMed1992

Type II corticosteroid receptor-like immunoreactivity in the rat cerebellar cortex: differential regulation by corticosterone.

Type II corticosteroid receptor-like immunoreactivity (type II-ir) was localized at the light- and electron-microscopic levels in the rat cerebellar cortex using BUGR2 monoclonal antibody. In intact rats, type II-ir was observed in the nuclei of basket, stellate, Golgi and Purkinje cells. After 1 week of adrenalectomy, type II-ir was barely resolvable in basket, stellate, Golgi and most Purkinje cells. Vermal Purkinje cells showed intense nuclear and cytoplasmic type II-ir. After 4 weeks of adrenalectomy, type II-ir was markedly reduced in most vermal Purkinje cells, however a few in lobules 1-3 and 9 and 10 retained diffuse immunoreactivity. Acute treatment with corticosterone restored nuclear type II-ir to basket, stellate, Golgi and Purkinje cells in the cerebellar hemispheres, flocculi and paraflocculi. In the vermis most Purkinje cells showed nuclear type II-ir following corticosterone treatment. Diffuse type II-ir was, however, retained in a few Purkinje cells in vermal lobules 1-3, 9 and 10. The distribution of GABA-like immunoreactivity (GABA-ir) in the cerebellar cortex was not altered by adrenalectomy or acute corticosterone treatment. 30-60% of type II-ir Purkinje cells in intact rats were GABA-ir. In adrenalectomized and corticosterone-treated rats, a similar proportion of type II-ir Purkinje cells were GABA-ir. The differential regulation of neuronal type II-ir in the cerebellum by corticosterone may account for some of the known effects of glucocorticoids on motor coordination.

Adrenal Glands↗
PubMed1992

Postnatal development of corticosteroid receptor immunoreactivity in the rat cerebellum and brain stem.

The postnatal development of corticosteroid receptor immunoreactivity in the rat cerebellum and related brainstem nuclei was studied using a type I receptor antiserum, MINREC4, and a type II receptor monoclonal antibody, BUGR2. Type I receptor immunoreactive (ir) Purkinje cells were first observed at postnatal day 5 (P5), and increased to adult levels by P20. Type I-ir cells, presumably migrating granule cells, were observed in the developing molecular layer of the cerebellum at P5. By P30, the density of type I-ir cells in the definitive molecular and granular layers was still less than adult levels. In contrast, type II-ir Purkinje cells were first observed at P15 and increased to adult levels by P20. No type II-ir cells were observed in the proliferative and migratory zones of the molecular layer. By P30, the density of type II-ir cells in the molecular and granular layers was far less than adult levels. In the deep cerebellar nuclei and most brain stem nuclei type I-ir was observed at P5 and developed to adult levels by P30. Type II-ir was observed in the deep cerebellar nuclei, red and medial vestibular nuclei by P15. The pontine and inferior olivary nuclei showed type II-ir cells by P10. Type II-ir in these regions developed to adult levels by P30. The earlier development of type I-ir suggests that the type I receptor may mediate the actions of corticosteroids in the cerebellum and related brain stem nuclei during early postnatal life.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Glucocorticoid receptors in LHRH neurons.

Luteinizing hormone-releasing hormone (LHRH)-producing neurons constitute the final pathway for regulation of reproductive endocrine function by the central nervous system. Chronically elevated levels of glucocorticoids exert an inhibitory effect on reproductive function. Although this is thought to be mediated in part via modulation of classical transmitters and peptides which regulate LHRH synthesis and release, it is also possible that glucocorticoids may regulate LHRH neurons directly. We localized glucocorticoid receptors (GR) in LHRH neurons in the rat central nervous system using immunocytochemistry. In males and randomly cycling females 10-24% of LHRH neurons in the medial septum-diagonal bands of Broca, and preoptic regions colocalized nuclear GR. Ovariectomy increased the percentage of GR/LHRH neurons at the level of the organum vasculosum of the lamina terminalis to 34%, half of which showed both nuclear and cytoplasmic GR. Treatment with estradiol reversed this effect. We suggest that the actions of glucocorticoids on reproductive endocrine function are mediated partly through direct modulation of LHRH gene expression and/or release by activated GR. Moreover, GR in LHRH neurons may provide a mechanism by which the gonadal steroid progesterone can affect LHRH neurons directly, despite a lack of progesterone receptors in these neurons.

Animals↗
PubMed1992

Sexual dimorphism in regulation of type II corticosteroid receptor immunoreactivity in the rat hippocampus.

To determine whether there are sex differences in the distribution of type II corticosteroid receptor-immunoreactive (type II-ir) cells in the rat hippocampus, we carried out a quantitative morphometric immunocytochemical study using a mouse monoclonal antibody, BUGR2. We report that in adrenally intact male and female rats, high densities of cells with nuclear type II-ir were observed in the pyramidal layer of field CA1 and the granular layer of the dentate gyrus. In intact males very few cells, presumably glia, in the stratum oriens showed type II-ir. In contrast, in females, interneurons with diffuse or cytoplasmic type II-ir were observed in the stratum oriens of CA1 and CA3. There were also sex differences in the regulation of type II-ir by corticosterone, the predominant glucocorticoid, and female sex steroids. In male rats the density of cells with nuclear type II-ir in all parts of Ammon's horn and the dentate gyrus was decreased significantly after adrenalectomy (adx). In contrast, in females such reductions were observed only in the pyramidal layer of CA1 and the granular layer of the dentate gyrus. In both sexes, cells with intense diffuse or mainly cytoplasmic type II-ir were observed in the pyramidal layer and stratum oriens after adx. The loss of nuclear type II-ir in the hippocampus of adx females was not affected significantly by ovariectomy. In adx males, nuclear Type II-ir was restored in CA1 and the dentate gyrus after treatment with corticosterone or progesterone. Cells in CA3 were, however, unresponsive to treatment with either hormone. In contrast, in adx females, treatment with either corticosterone or progesterone restored nuclear type II-ir to cells in all regions of the hippocampus. In both adx males and females, cytoplasmic type II-ir observed in some cells in the pyramidal layer and stratum oriens, was abolished completely by corticosterone, and partially by progesterone treatment. In both adx males and females, estradiol treatment did not affect significantly the pattern of type II-ir. Sex differences in the distribution of type II-ir interneurons in intact rats and the regulation of the intracellular location of type II-ir of adx rats by corticosterone and progesterone, may be important determinants of sex differences in the modulation of hippocampal function by glucocorticoids.

Adrenalectomy↗
PubMed1991

Absence of pituitary prolactin epitopes in immunoreactive prolactin of rat brain.

Immunoreactive prolactin (ir-PRL) in rat brain has been consistently documented. However, the identity of this ir-PRL is controversial. Ir-PRL is defined by its ability to bind to PRL antibodies. All previous studies of brain ir-PRL have used polyclonal antibodies, at least one of which apparently crossreacts with a portion of the proopiomelanocortin molecule. To begin to define the epitopes comprising ir-PRL in the brain, we utilized two monoclonal antibodies (MAb) that recognize pituitary PRL in a variety of species, including rat. Immunocytochemistry was performed on rat brains and pituitary glands using two monoclonal and one polyclonal PRL antibody. Although both MAb immunostained lactotrophs of the rat pituitary gland, neither antibody immunostained cell bodies or neuronal processes in the brain. However, the polyclonal antiserum immunostained lactotrophs and a system of neuronal cell bodies and processes in the brain. Thus, epitopes found in pituitary PRL from several species are not found in ir-PRL in rat brain.

Animals↗
PubMed1991

Differential corticosteroid regulation of type II glucocorticoid receptor-like immunoreactivity in the rat central nervous system: topography and implications.

Neuronal type II glucocorticoid receptor-like immunoreactivity in the central nervous system shows heterogeneity in intensities and relative densities. This might predict variations in the regional responses of neuronal immunoreactivity to corticosteroids. We investigated changes in the intracellular location of immunoreactivity in the rat central nervous system after adrenalectomy and corticosteroid treatment, and carried out detailed statistical analysis of changes in neuronal nuclear immunoreactivity in the hippocampus and caudateputamen. Three types of responses were observed. The majority of neurons, classified type A, showed a predominant nuclear immunoreactivity in intact rats, lost nuclear and eventually cytoplasmic immunoreactivity after adrenalectomy, and regained nuclear immunoreactivity within 5 min of corticosterone and 2 h of aldosterone treatment, respectively. A subgroup of neurons in the hippocampus, striatum, septum, and habenula, classified type B, were not immunoreactive in intact rats, showed intense cytoplasmic immunoreactivity after adrenalectomy, and disappeared rapidly after corticosterone treatment and later in response to aldosterone. A subgroup of vermal cerebellar Purkinje neurons, classified type C, developed an intense cytoplasmic immunoreactivity after adrenalectomy, increased in number in response to corticosterone, and did not respond to aldosterone.

Adrenal Cortex Hormones↗
PubMed1991

Immunocytochemical localization of chloramphenicol acetyltransferase as a single-cell marker of transfected and transgenic cells.

A technique is described for immunocytochemical localization of the bacterial gene product chloramphenicol acetyltransferase, which is a commonly used reporter gene in transfected and transgenic cells. The described procedure is capable of localizing the enzyme in individual cells, providing a means of determining the cell type(s) expressing a foreign construct in complex cultures or in tissue sections of transgenic mice.

Animals↗
PubMed1990

The fos proto-oncogene protein:regulation by morphine in the rat hypothalamus.

Mechanisms by which opiates alter neuronal functions, including neuroendocrine functions, are not well defined. We have previously demonstrated that morphine rapidly and transiently increases expression of the proto-oncogene c-fos in the rat caudate-putamen. This regulation of the c-fos gene by morphine may represent a portion of the intracellular cascade coupling activation of opiate receptors on the cell surface to subsequent alterations in neuropeptide gene expression. In the present study, we have focussed on effects of morphine on c-fos expression in the ventromedial hypothalamus, which contains estrogen-concentrating neurons and a large number of neurons expressing the opioid proenkephalin and Proopiomelanocortin. The hypothalamus has been identified as a "final common pathway" between the remainder of the central nervous system and the pituitary gland. As a marker for c-fos expression, we have detected pp50 c-fos (FOS) protein immunocytochemically, using a polyclonal antibody to the M peptide of FOS, and revealed an intense nuclear stain in many neurons. Labeled nuclei were drawn by camera lucida from 12 matched sections (one side only) covering the rostral and middle levels of the ventromedial nucleus of six rats given morphine and six given phosphate buffered saline. Morphine treatment significantly increased the number and density of immuno-labeled nuclei in the ventromedial nucleus, but not in the arcuate nucleus. These results suggest effects of morphine (directly or indirectly) on neurons in the ventromedial hypothalamic nucleus, despite the relative absence of morphine receptors in this nucleus. These results may also provide an anatomical basis for neuroendocrine alterations following morphine treatment.

Animals↗
PubMed1990

Charting of type II glucocorticoid receptor-like immunoreactivity in the rat central nervous system.

The rat brain and spinal cord have been mapped for Type II glucocorticoid receptor-like immunoreactivity in neurons and glia, using a monoclonal antibody, BUGR2, which recognizes an epitope close to the DNA-binding domain of the rat Type II receptor. The study revealed a widespread distribution of Type II-like immunoreactive neurons and glia, and a heterogeneity of densities and intensities of immunoreactive elements. Our results corresponded to a large extent with previous immunocytochemical mapping using Ig2a, a monoclonal antibody against a different epitope in the variable domain, with some notable differences in the hippocampus, hypothalamus and cerebellum. There was also a good correlation between immunocytochemical mapping and binding studies, [3H]steroid autoradiography and mRNA localization of the Type II receptor.

Animals↗
PubMed1990

Chemical characterization of neuroendocrine targets for progesterone in the female rat brain and pituitary.

The secretory products of some of the cell types which respond directly to actions of progesterone in the female rat brain and pituitary were determined by combining immunocytochemistry with autoradiography following systemic administration of the synthetic progestin ligand [3H]-R5020. Four major findings are reported: (1) Approximately 90% of the tyrosine hydroxylase (TH)-immunoreactive neurons in the hypothalamic arcuate nucleus have progesterone receptors, while TH-immunoreactive neurons in other portions of the hypothalamus (e.g. the periventricular region and the zona incerta) do not. (2) Approximately 30% of the beta-endorphin neurons in the hypothalamus have progesterone receptors. (3) None of the luteinizing hormone-releasing hormone neurons examined have progesterone receptors. (4) Approximately 98% of the cells in the anterior pituitary that have progesterone receptors contain luteinizing hormone. Lactotrophs do not contain progesterone receptors. Many progestin targets in the brain remain to be characterized chemically. The implications for progesterone-inducible genes and neuroendocrine control systems are discussed.

Animals↗
PubMed1990

Gonadal steroid regulation of substance P (SP) and SP-encoding messenger ribonucleic acids in the rat anterior pituitary and hypothalamus.

Substance P-like immunoreactivity (SP-LI) is present in the rat anterior pituitary (AP) and in hypothalamic neurons that may be involved in the control of AP secretion and/or reproductive function. The presence of multiple SP-encoding mRNAs and tachykinin peptides and their regulation by steroid hormones were examined in APs and hypothalami from normal, gonadectomized, and steroid-treated male and female rats. SP-encoding mRNAs were identified by nuclease protection assays of RNA, and tachykinin peptides were identified by combined HPLC-RIA of tissue extracts, beta- and gamma-preprotachykinin (PPT) mRNAs and SP, neurokinin A, and neuropeptide gamma peptides were identified in the AP. The alpha-, beta-, and gamma-PPT mRNAs and SP, neurokinin A, neuropeptide gamma, neuropeptide K, and neurokinin B peptides were present in hypothalamic tissue. Previous studies have established that in the AP, SP is differentially regulated by gonadal steroids; estrogen decreases and androgen increases AP SP. Steroid effects were further analyzed in experiments using RIAs to measure SP levels in the AP and median eminence (ME) of steroid- and oil-treated gonadectomized rats. To assess whether steroids alter steady state PPT mRNA levels and presumably SP synthesis in these tissues, potential effects on AP and hypothalamic SP-encoding mRNAs were determined. Ovariectomized rats treated for 10 days with estradiol benzoate showed a 50% decrease in AP SP and a 90% decrease in AP beta- and gamma-PPT mRNAs compared to ovariectomized oil-treated controls. Estradiol benzoate replacement had no effect on SP levels in the isolated ME, but did cause a 50% increase in alpha-, beta-, and gamma PPT mRNAs in the hypothalamus. Although there was no significant effect of testosterone propionate on AP SP levels in castrated males, 10 days of testosterone propionate replacement did cause a significant increase in beta- and gamma PPT mRNAs in the AP. No androgen effects were seen on either ME SP or hypothalamic SP-encoding mRNAs. These data demonstrate that estrogen up-regulates SP-encoding mRNAs in the hypothalamus, whereas it down-regulates SP-encoding mRNAs in the pituitary. These results implicate SP and other tachykinins derived from the SP gene as steroid-regulated modulators of AP secretion and possibly reproductive function.

Animals↗
PubMed1989

Cellular localization of substance P- and neurokinin A-encoding preprotachykinin mRNA in the female rat brain.

To determine the locations of neurons in the rat brain expressing substance P and neurokinin A mRNA, we performed in situ hybridization with a radiolabeled cRNA probe that was complementary to alpha-, beta-, and gamma-preprotachykinin mRNA. Several types of controls indicated specificity of the labeling. Brain regions containing many labeled neurons include the anterior olfactory nucleus, layer II of the olfactory tubercle, the islands of Calleja, the nucleus accumbens, the caudate-putamen, portions of the amygdala and hypothalamus, the medial habenular nucleus, nuclei of the pontine tegmentum, several raphe nuclei, several portions of the reticular formation, and the nucleus of the solitary tract. Less frequent labeled neurons were also found in many other regions of the brain. These results extend many previous immunocytochemical studies of the locations of neurons containing immunoreactive substance P, neurokinin A, and neuropeptide K.

Animals↗
PubMed1989

Distribution and partial characterization of immunoreactive prolactin in the rat brain.

Immunoreactive (IR) prolactin was localized immunocytochemically in cell bodies in the mediobasal hypothalamus and in fibers in many regions of the rat brain. The cell bodies were found in the arcuate nuclei and the adjacent areas ventral to the ventromedial nuclei. Fiber projections extended rostrally to and/or through the anterior hypothalamus, preoptic area, nucleus accumbens, septum, diagonal bands of Broca, caudate-putamen, frontal cortex and accessory olfactory bulb; laterally to the amygdala, especially the central nucleus and some parts of the medial nucleus; caudally to and/or through the midbrain central gray, reticular formation, parabrachial region, and several portions of the lower brain stem and spinal cord extending to sacral levels. The system appears to be essentially identical to that containing proopiomelanocortin (POMC) and its processed peptides, as shown by double immunocytochemistry. Preabsorption of the antiprolactin antiserum with either prolactin or the 16,000-dalton N-terminus of POMC eliminated immunoreactivity in the brain. Preabsorption with other POMC-derived peptides, including beta-lipotropic hormone, beta-endorphin, met-enkephalin, adrenocorticotrophic hormone (1-24), corticotropin-like intermediate lobe peptide, alpha- and gamma-melanocyte-stimulating hormones and an octapeptide region of the N-terminus of POMC bearing some homology with prolactin, did not eliminate immunoreactivity in the brain. Similarly, preabsorption with growth hormone, luteinizing hormone, follicle-stimulating hormone, motilin or fetuin did not eliminate immunoreactivity in the brain. The antiprolactin antiserum also recognized all cells in the intermediate lobe and a subset of cells in the anterior lobe of the Snell dwarf mouse pituitary. This immunoreactivity was eliminated by preabsorption of the antiserum with prolactin or with the 16,000-dalton N-terminus of POMC. These results suggest that IR prolactin in the brain may be related to the N-terminus of POMC. Additional results based on one- and two-dimensional gel electrophoresis and immunoblotting indicate that the antiprolactin antiserum used in the majority of the immunocytochemical studies recognized a number of proteins.

Animals↗