Toshihiro Imaki

Intracerebroventricular administration of corticotropin-releasing factor inducesc-fos mRNA expression in brain regions related to stress responses: comparison with pattern ofc-fos mRNA induction after stress

Centrally administered corticotropin-releasing factor (CRF) produces a number of physiological and behavioral changes akin to those elicited by exposure to acute stress. However, the specific brain site of action responsible for the centrally activating property of CRF has not been precisely determined. In this study, we used in situ hybridization histochemistry for c-fos mRNA to map potential neuronal structures activated after intracerebroventricular (i.c.v.) injection of CRF and compared the distribution of c-fos mRNA with that after stress. Wistar male rats were sacrificed 30, 60, 120 and 180 min after the i.c.v. injection of 1 microgram ovine CRF or vehicle alone. Another group of rats was exposed to immobilization stress for 60 min or electrical foot-shock stress (1.5 mA, 1-s duration, 30 x) for 15 min and sacrificed before and 30, 60, 120 and 180 min after the beginning of stress. Centrally administered CRF rapidly (30-60 min) induced c-fos mRNA expression in most of the areas that showed hybridization signals for c-fos after stress: the limbic structures, including the piriform cortex, cingulate cortex, the lateral septal nucleus, the hippocampus, the anterior corticomedial and the medial amygdaloid nuclei, the hypothalamic nuclei, such as the paraventricular nucleus, the supraoptic nucleus (SO) and the dorsomedial nucleus (DMD), and some brainstem nuclei like the pontine nucleus, the locus ceruleus (LC) and Barringtons nucleus. The granular layer of the cerebellum, some thalamic nuclei and the habenula also showed hybridization signals after i.c.v. injection of CRF and stress. However, c-fos induction in the bed nucleus of the stria terminalis, the central nucleus of the amygdala (CeA) and the nucleus tractus solitarius (SOL) was seen only after i.c.v. administration of CRF; in the septo-hypothalamic nucleus and the superior olive, however, c-fos mRNA expression was observed only after stress. There were no differences in the pattern of c-fos mRNA expression between the two stress paradigms. In contrast, i.c.v. injection of saline-induced expression of c-fos mRNA in the piriform cortex, neocortex, cingulate cortex and the amygdala was much less than that seen after i.c.v.-administered CRF as evident in the intensity of the signals. These results suggest that CRF produces c-fos mRNA expression in the brain areas related to stress response, and that CRF may induce behavioral and neuroendocrine responses through activating these brain structures, such as the limbic system and the hypothalamic nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)

Stress-induced activation of neuronal activity and corticotropin-releasing factor gene expression in the paraventricular nucleus is modulated by glucocorticoids in rats.

Intronic in situ hybridization methodology provides a means of determining the rate of gene transcription under basal and stimulated conditions. In the present study, we have used intronic in situ hybridization to the corticotropin-releasing factor (CRF) gene to measure hypothalamic CRF gene transcription after stress as well as its modulation by glucocorticoids. Using this and conventional exonic in situ hybridization we examined the time course of changes in c-fos mRNA, and CRF heteronuclear RNA (hnRNA) and mRNA concentrations in the paraventricular nucleus (PVN) of male Wistar rats after restraint stress. In addition, we determined the effects of adrenalectomy and dexamethasone administration on c-fos and CRF gene expression in the PVN. Restraint stress induced a rapid induction (within 5 min) of c-fos mRNA and CRF hnRNA expression in the PVN. Both RNA concentrations peaked at 30 min then decreased and were undetectable 2 h after stress onset. In contrast, the concentration of CRF mRNA increased gradually and a significant elevation was first detected 60 min after the beginning of stress. Adrenalectomy augmented and dexamethasone pretreatment inhibited c-fos mRNA, CRF hnRNA, and mRNA induction after stress. The data suggest that stress-induced activation of neurons, CRF gene transcription, and CRF synthesis in the PVN are modulated by glucocorticoids.

Aldosterone Breakthrough During Angiotensin II Receptor Antagonist Therapy in Stroke-Prone Spontaneously Hypertensive Rats

Aldosterone breakthrough during ACE inhibitor therapy has been reported. This study investigates changes in plasma aldosterone concentration (PAC) and its mechanism and effects on target organ damage during long-term angiotensin II type 1 (AT1) receptor antagonist (AT1A) therapy in hypertensive rats. An AT1A (candesartan, 1 mg/kg per day PO) was administered in stroke-prone spontaneously hypertensive rats from 4 weeks of age for 34 weeks. PAC was significantly decreased during the first 4 weeks but showed aldosterone breakthrough after 8 weeks of AT1A administration. Plasma angiotensin II concentration was significantly elevated, whereas no change was seen in plasma ACTH or serum potassium. The mechanism(s) of aldosterone breakthrough were investigated by giving high doses of candesartan (3 mg/kg per day PO), dexamethasone (200 &mgr;g/kg per day IP), or the AT2 antagonist (PD123319, 10 mg/kg per day SC) during the last week of the 24-week AT1A treatment period. Dexamethasone and AT2 antagonist but not high-dose AT1A produced a significant decrease in PAC, with a larger decrease produced by the AT2 antagonist. To clarify the effects of the residual aldosterone, effects of coadministration of low-dose spironolactone (10 mg/kg per day SC), an aldosterone antagonist, on left ventricular hypertrophy and expression of brain natriuretic peptide mRNA were determined. Low-dose spironolactone further improved left ventricular hypertrophy and brain natriuretic peptide mRNA expression despite no additional depressor effect. These results suggest that aldosterone breakthrough occurs during long-term AT1A therapy, mainly by an AT2-dependent mechanism. Residual aldosterone may attenuate the cardioprotective effects of AT1A.

Involvement of corticotropin-releasing factor in restraint stress-induced anorexia and reversion of the anorexia by somatostatin in the rat

The mechanism by which restraint stress induces suppression of food intake and the influence of intracerebroventricular (icv) administration of somatostatin on the anorexia induced by restraint stress were examined in the rat. Ninety minutes of restraint stress reduced food intake of rats to approximately 60% that of control. Anorexia induced by 90 min restraint stress was partially reversed by icv administration of alpha-helical CRF (9-41), a corticotropin-releasing factor (CRF) antagonist, and completely reversed by anti-CRF gamma-globulin. These results provide further evidence in support of the theory that CRF is involved in the inhibitory mechanism of food intake in restraint stress. ICV administration of somatostatin 14 and SMS 201-995, an analog of somatostatin, also reversed restraint stress-induced anorexia. It is, therefore, suggested that somatostatin may counteract the suppressive action of CRF on food intake in stress.

Distinct distribution and time-course changes in neuronal nitric oxide synthase and inducible NOS in the paraventricular nucleus following lipopolysaccharide injection

Nitric oxide (NO) is known to be involved in the modulation of neuroendocrine function. To clarify the role of different isoforms of NO synthase (NOS) in the neuroendocrine response to immune challenge, the expressions of neuronal NOS (nNOS) and inducible NOS (iNOS) genes in the hypothalamus following lipopolysaccharide (LPS) injection were examined using in situ hybridization. NOS activity was also determined by NADPH-diaphorase (NADPH-d) histochemistry. LPS (25 mg/kg) or sterile saline was injected intraperitoneally to male Wistar rats and the rats sacrificed 30 min, or 1, 2, 3, 5, 12 or 24 h after injection. nNOS mRNA expression in the paraventricular nucleus (PVN) was significantly increased 2 h after LPS injection. iNOS mRNA, which was not detected until 2 h after LPS injection, was significantly increased in the PVN 3 h after LPS injection. Both RNA expressions had returned to basal levels by 12 h after LPS injection. The number of NADPH-d positive cells was significantly increased 5 h after LPS injection. iNOS expression was more robust in parvocellular PVN, while nNOS was distributed mainly in the magnocellular PVN. Double in situ hybridization histochemistry revealed that some of the iNOS- (48.4%) or nNOS-positive cells (34. 3%) in the parvocellular PVN expressed CRF mRNA. The results demonstrate that LPS-induced sepsis causes significant increases in nNOS and iNOS gene expression with different time-courses and distributions, and that iNOS mRNA was more frequently co-localized with CRF-producing parvocellular neurons in the PVN. Thus, NO produced by iNOS and nNOS may play an important role in the neuroendocrine response to an immune challenge. Distinct differences in the distribution and time-course changes of iNOS and nNOS suggest different roles for the hypothalamic-pituitary-adrenal axis and/or neurohypophyseal system.

Corticotropin-releasing factor up-regulates its own receptor mRNA in the paraventricular nucleus of the hypothalamus

We investigated the role of CRF in regulating receptor expression in the paraventricular nucleus (PVN). First, to clarify the effect of exogenously administered CRF, 1 microgram of ovine CRF was injected into rat lateral ventricle and changes in concentration of the CRF type 1 receptor (CRF1-R) and CRF mRNA in the PVN were semiquantified after in situ hybridization. Second, we determined the effect of stress, as a stimulant of endogenous CRF secretion, on mRNA accumulation. While CRF1-R mRNA expression was low to be undetectable in the PVN of controls, both intracerebroventricular administration of CRF and restraint significantly increased CRF1-R and CRF signals in the parvocellular PVN. Thus CRF may modulate CRF production and release from the PVN, by regulating CRF1-R expression.

Melanocortin 2 receptor is required for adrenal gland development, steroidogenesis, and neonatal gluconeogenesis

ACTH (i.e., corticotropin) is the principal regulator of the hypothalamus–pituitary–adrenal axis and stimulates steroidogenesis in the adrenal gland via the specific cell-surface melanocortin 2 receptor (MC2R). Here, we generated mice with an inactivation mutation of the MC2R gene to elucidate the roles of MC2R in adrenal development, steroidogenesis, and carbohydrate metabolism. These mice, the last of the knockout (KO) mice to be generated for melanocortin family receptors, provide the opportunity to compare the phenotype of proopiomelanocortin KO mice with that of MC1R–MC5R KO mice. We found that the MC2R KO mutation led to neonatal lethality in three-quarters of the mice, possibly as a result of hypoglycemia. Those surviving to adulthood exhibited macroscopically detectable adrenal glands with markedly atrophied zona fasciculata, whereas the zona glomerulosa and the medulla remained fairly intact. Mutations of MC2R have been reported to be responsible for 25% of familial glucocorticoid deficiency (FGD) cases. Adult MC2R KO mice resembled FGD patients in several aspects, such as undetectable levels of corticosterone despite high levels of ACTH, unresponsiveness to ACTH, and hypoglycemia after prolonged (36 h) fasting. However, MC2R KO mice differ from patients with MC2R-null mutations in several aspects, such as low aldosterone levels and unaltered body length. These results indicate that MC2R is required for postnatal adrenal development and adrenal steroidogenesis and that MC2R KO mice provide a useful animal model by which to study FGD.

Injection of anti-neuropeptide Y γ-globulin into the hypothalamic paraventricular nucleus decreases food intake in rats

The effect of immunoneutralization of hypothalamic neuropeptide Y (NPY) on food intake was examined to clarify the involvement of NPY in the control mechanism of feeding behavior. Injection of anti-NPY gamma-globulin into the bilateral hypothalamic paraventricular nuclei of 24-h food-deprived rats significantly inhibited food intake for 1 h after the injection compared with rats that received normal rabbit gamma-globulin. This result suggests that NPY in the paraventricular nucleus plays a role in stimulating feeding behavior.

In vitro release of growth hormone-releasing factor from rat hypothalamus: effect of insulin-like growth factor-1

The release of growth hormone-releasing factor (GHRF) from rat hypothalamus was investigated in vitro. After 60 min preincubation the released GHRF from sliced rat hypothalamic fragments during 60 min incubation was detected by a highly specific and sensitive radioimmunoassay for rat GHRF. The release of GHRF was Ca2+-dependent and enhanced by high concentration of K+. Insulin-like growth factor-1 (IGF-1) significantly decreased GHRF release to 65% and 84% of the control at concentrations of 10(-8) M and 10(-7) M, respectively. These results suggest that this in vitro system is useful for the investigation of the mechanism of GHRF release from the hypothalamus and that IGF-1 is probably involved in the feedback inhibition of growth hormone secretion by attenuating GHRF release from the hypothalamus besides countering the effect of GHRF on the pituitary.

Urocortin mRNA is expressed in the enteric nervous system of the rat

The expression of the urocortin gene in the gastrointestinal tract was investigated using reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization histochemistry. PCR demonstrated the presence of urocortin mRNA in the rat brain, duodenum, small intestine, and colon. By in situ hybridization, urocortin-containing cells were exclusively localized to the submucosal plexus and myenteric plexus in the duodenum, small intestine and colon. These results suggest that urocortin may play an important role in the regulation of gastrointestinal motor function throughout the enteric nervous system.