Immunoautoradiographic and in situ hybridization analysis of corticotropin-releasing hormone biosynthesis in the hypothalamic paraventricular nucleus

doi:10.1016/0891-0618(96)00124-X

Journal of Chemical Neuroanatomy

Volume 11, Issue 1, July 1996, Pages 49-56

https://doi.org/10.1016/0891-0618(96)00124-X Get rights and content

Abstract

Corticotropin-releasing hormone (CRH) gene transcription and mRNA expression are keyed to stimuli activating the pituitary-adrenocortical axis, suggesting a connection between neuronal activation and synthesis of active peptide. However, the relationship between CRH mRNA and levels of CRH peptide remains to be definitively established. The present report characterizes an immunoautoradiographic (IAR) strategy to assess CRH peptide expression in an anatomical context. Non-fixed tissue sections through the rat hypothalamus were reacted with a primary antibody against rat CRH, followed by incubation with [³⁵S] or [¹²⁵I] labeled secondary antibody. Autoradiography performed on reacted sections revealed that CRH immunoreactivity could be detected in CRH-containing regions of the hypothalamus and amygdala. Generation of CRH signal was blocked by preabsorption of primary antibody with CRH peptide, demonstrating antibody specificity. IAR performed on nitrocellulose blotted with synthetic CRH peptide revealed a linear relationship between peptide quantity and intensity of autoradiographic signal, verifying that this method is appropriate for semi-quantitative analysis of CRH peptide regulation. Assessment of CRH peptide regulation revealed a significant increase in CRH content in adrenalectomized rats (ADX) relative to sham-adrenalectomized (SHAM) controls (196%). In situ hybridization performed on adjacent sections revealed a similar increase in CRH mRNA expression in ADX rats (256%), and a significant correlation between CRH peptide and mRNA measures (r = 0.68). No ADX induced changes were seen in median eminence, dorsomedial hypothalamus or central amygdaloid nucleus. The results of this study indicate that CRH biosynthesis appears to be driven by amount of available mRNA, rather than changes in translational efficacy. In addition, the IAR technique appears ideally suited to allow concomitant assessment of mRNA and protein expression within defined populations of CNS neurons.

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Citation Excerpt :
Statistical analysis showed in average a twofold increase in the total number of CRH neurons and about fivefold increase in the amount of CRH mRNA in the hypertensive Pa compared with the controls (Fig. 24.2). Thus the results of our immunohistochemical analysis agree very well with changes seen in CRH mRNA expression determined by in situ hybridization on adjacent sections and are consistent with a relationship between increased mRNA and peptide synthesis (Herman and Morrison, 1996). It was shown previously that increased peptide synthesis in the hypothalamic Pa proportionally stimulates axonal transport of peptide (Roberts et al., 1991) and is itself upregulated by increased peptide release (Majzoub, 1985; Zingg et al., 1986; Robinson et al., 1990; Roberts et al., 1991; Robinson and Fitzsimmons, 1993; Herman and Morrison, 1996).
For the majority of hypertensive patients, the etiology of their disease is unknown. The hypothalamus is a central structure of the brain which provides an adaptive, integrative, autonomic, and neuroendocrine response to any fluctuations in physiological conditions of the external or internal environment. Hypothalamic insufficiency leads to severe metabolic and functional disorders, including persistent increase in blood pressure. Here, we discuss alterations in the neurochemical organization of the paraventricular and suprachiasmatic nucleus in the hypothalamus of patients who suffered from essential hypertension and died suddenly due to acute coronary failure. The changes observed are hypothesized to contribute to the pathogenesis of disease.
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The objective of this research was to evaluate the effect of stimulation of receptors CRH2 in the paraventricular nucleus of the hypothalamus on food intake and expression of behavioral satiety sequence (BSS) in adrenalectomized rats. Eight independent groups of Wistar rats were utilized; four adrenalectomized groups (ADX) and four were false surgery. All subjects were implanted with a cannula in the paraventricular nucleus of the hypothalamus and were administered with one of the four treatments: vehicle, urocortin-2 (UCN2, CRH2 agonist), antisauvagine-30 (CRH2 antagonist) or antisauvagine-30 + UCN2 (pretreatment). UCN2 administration reduced intake of carbohydrates and fats in ADX rats due to the interruption of the BSS. In rats with false surgery it decreased fat intake due to the advancement of the BSS. Pretreatment prevented the effects induced by UCN2 in ADX rats but not in rats with false surgery. These results suggest that receptors CRH2 modulated intake and BSS in ADX rats, contributing with relevant information for the understanding of the anorexic ethiology and the behavioral pattern associated to it.
El objetivo de la presente investigación fue evaluar el efecto de la estimulación de los receptores CRH2 del núcleo paraventricular hipotalámico sobre la ingesta de alimento y la expresión de la secuencia de saciedad conductual (SSC) en ratas adrenalectomizadas. Se trabajó con ocho grupos independientes de ratas Wistar, cuatro grupos adrenalectomizados (ADX) y cuatro con falsa cirugía. A todos los sujetos se les implantó una cánula en el núcleo paraventricular hipotalámico, y se les administró uno de cuatro tratamientos: vehículo, urocortina 2 (UCN2, agonista de CRH2), antisauvagina 30 (antagonista de CRH2), y antisauvagina-30 + UCN2 (pretratamiento). La administración de UCN2 redujo la ingesta de hidratos de carbono y de grasas en las ratas ADX, debido a la interrupción de la SSC; mientras que en las ratas con falsa cirugía, la UCN2 solo disminuyó la ingesta de grasas, debido al adelanto de la SSC. El pretratamiento previno los efectos inducidos en las ratas ADX, pero no en las ratas con falsa cirugía. Estos resultados indican que los receptores CRH2 modularon la ingesta y la SSC en las ratas ADX, lo que constituye un aporte importante en la comprensión de la etiología de la anorexia y del patrón conductual asociado a esta.
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Inflammatory and infectious processes evoke neuroendocrine and behavioral changes known as acute-phase response that includes activation of the hypothalamo-pituitary-adrenal (HPA) axis and reduction of food intake. Besides its action as the most important ACTH secretagogue, corticotrophin-releasing factor (CRF), synthesized in the paraventricular nucleus (PVN), is also involved in the control of food intake. Alpha-melanocyte stimulating hormone (α-MSH) in the arcuate nucleus also plays a role in the energy homeostasis, possessing anorexigenic effects. To investigate the participation of neuropeptides involved in the regulation of food intake during endotoxemia, we administrated lipopolysaccharide (LPS) in sham-operated and adrenalectomized (ADX) male Wistar rats to evaluate food intake, hormone responses and Fos-CRF and Fos-α-MSH immunoreactivity in the PVN and arcuate nucleus, as well as CRF and POMC mRNA expression in these hypothalamic nuclei. In sham-operated rats, treatment with LPS (100 µg/kg) showed lower food intake, higher plasma ACTH and corticosterone levels, as well as an increase in Fos-CRF double labeled neurons and CRF mRNA expression in the PVN, with no changes in Fos-α-MSH immunoreactivity and POMC mRNA expression in the arcuate nucleus, compared to saline treated rats. After LPS treatment, ADX rats showed further increase in plasma ACTH levels, marked decrease of food intake, higher Fos-CRF immunoreactive neurons in the PVN and CRF mRNA expression, as well as an increase in Fos-α-MSH immunoreactivity and POMC mRNA expression in the arcuate nucleus, compared to sham-operated rats treated with LPS. In conclusion, the present data indicate that the marked hypophagia during endotoxemia following ADX is associated with an increased activation of CRF and POMC neurons in the hypothalamus and an increased mRNA expression of these neuropeptides.
Postnatal handling does not normalize hypothalamic corticotropin-releasing factor mRNA levels in animals prenatally exposed to ethanol
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Postnatal handling has been shown to attenuate some of the deficits in developmental outcome observed following prenatal ethanol exposure (E) although it appears to be ineffective at ameliorating the hypothalamic–pituitary–adrenal (HPA) hyperresponsiveness to stressors that has been observed in adult E animals. However, the effects of postnatal handling on central regulation of HPA activity in E animals, particularly with regard to alterations in steady-state hypothalamic corticotropin-releasing factor (CRF) activity, have not been examined. In the present study, offspring from E, pair-fed (PF), and ad-libitum-fed control (C) groups were exposed to daily handling during the first 2 weeks of life (H) or were left entirely undisturbed until weaning (NH). Basal CRF and arginine vasopressin (AVP) mRNA in the parvocellular portion of the paraventricular nucleus (pPVN) of the hypothalamus were assessed at 90–110 days of age. Prenatal ethanol exposure resulted in elevated basal pPVN CRF mRNA levels compared to those in ad-libitum-fed controls. Handling altered CRF mRNA levels in a sex-specific and prenatal treatment-specific manner. Females showed no significant effects of handling. In contrast, handling decreased CRF mRNA levels in PF and C but not E males compared to their NH counterparts. There were no effects of prenatal ethanol or postnatal handling on AVP mRNA levels. These findings indicate that prenatal ethanol exposure results in elevated basal CRF mRNA levels in adulthood and that handling appears to be ineffective in normalizing those elevations, supporting the suggestion that altered basal HPA regulation in E animals may, at least in part, underlie their HPA hyperresponsiveness to stressors.
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Single exposure to the proinflammatory cytokine interleukin-1 induces sensitization of the adrenocorticotropin hormone and corticosterone responses to stressors weeks later (hypothalamus-pituitary-adrenal sensitization). Hypothalamus-pituitary-adrenal responses are controlled by corticotropin-releasing hormone and arginine-vasopressin secreted from parvocellular corticotropin-releasing hormone neurons of the hypothalamic paraventricular nucleus and may involve autoexcitatory feedback mechanisms. Therefore, we studied the temporal relationship between resting levels of corticotropin-releasing hormone, corticotropin-releasing hormone-R1 and arginine-vasopressin receptor (V1a, V1b) mRNAs in the paraventricular nucleus and the development of hypothalamus-pituitary-adrenal sensitization to an emotional stressor (novelty). The adrenocorticotropin hormone precursor molecule proopiomelanocortin hnRNA in the pituitary gland served as an index for acute activation. Single administration of interleukin-1 induced sensitization of the hypothalamus-pituitary-adrenal to novelty from 3 to 22 days later, but not after 42 days. Single administration of interleukin-1 induced biphasic increases in corticotropin-releasing hormone and corticotropin-releasing hormone-R1 mRNAs in the paraventricular nucleus: an early peak within 24 h, followed by a delayed (>7 days) increase that peaked after 22 days. Hypothalamic V1a and V1b mRNA levels were unaffected. In contrast, in the pituitary gland, there was an early decrease in corticotropin-releasing hormone-R1 mRNA (from 10.5 to 3 h after interleukin-1) and V1b receptor mRNA (3 to 6 h), which returned to control levels from 24 h onwards. Thus, interleukin-1–induced long-lasting hypothalamus-pituitary-adrenal sensitizationis associated with prolonged activation of corticotropin-releasing hormone and corticotropin-releasing hormone-R1 mRNA expression in the paraventricular nucleus, but not with changes in the expression of proopiomelanocortin hnRNA or V1b receptor or corticotropin-releasing hormone R1 mRNAs in the pituitary gland. We propose that transient exposure to immune events can induce long-lasting hypothalamus-pituitary-adrenal sensitization, which at least in part involves long-term hypothalamic adaptations that enhance central corticotropin-releasing hormone signaling.
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Immunoautoradiographic and in situ hybridization analysis of corticotropin-releasing hormone biosynthesis in the hypothalamic paraventricular nucleus

Abstract

Brain Res.

Neurosci. Lett.

Brain Res.

Peptides

Brain Res.

Brain Res.

Prog. Neurobiol.

Hypothalamic control of adrenocorticotropin secretion: advances since the discovery of 41-residue corticotropin-releasing factor

Endocrine Rev.

Hypoglycemia enhances turnover of corticotropin-releasing factor and of vasopressin in the zona externa of the rat median eminence

Endocrinology

Cellular quantification of tyrosine hydroxylase in the rat brain by immunoautoradiography

J. Neurochem.

Repeated stress-induced activation of corticotropin-releasing factor neurons enhances vasopressin stores and colocalization with corticotropin-releasing factor in the median eminence of rats

Neuroendocrinology

mRNA structure, in situ, as assessed by microscopic techniques

Microsc. Res. Tech.

Effects of adrenalectomy and glucocorticoids on the peptides CRF-41, AVP and oxytocin in rat hypophysial portal blood

J. Physiol. (Lond.)

Radioimmunocytochemical localization of corticotropin-releasing factor and adrenocorticotropin in the hypothalamo-hypophyseal system of the rat: effects of adrenalectomy

J. Histochem. Cytochem.

Rapid regulation of CRH gene expression in vivo

Mol. Endocrinol.

Regulation of basal corticotropin-releasing hormone and arginine vasopressin messenger ribonucleic acid expression in the paraventricular nucleus: effects of selective hypothalamic deafferentations

Endocrinology