Age- and stress-induced changes in corticotropin-releasing hormone mRNA expression in the paraventricular nucleus of the hypothalamus

In reaction to acute stress, prepubertal (25-28 days of age) animals demonstrate a prolonged adrenocorticotropic hormone (ACTH) and corticosterone response compared to adults (>65 days of age), while after chronic stress, prepubertal animals show a higher peak ACTH and corticosterone response, but a faster return to baseline compared to adults. Differential activation of corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus (PVN) of prepubertal and adult animals have been suggested to mediate these changes in stress responsiveness. The purpose of the present set of experiments was to further elucidate possible differences in PVN structure and function in prepubertal (28 days of age) and adult (77 days of age) male rats. The results indicate that PVN volume and somal size and cell number are similar in the parvocellular and magnocellular subdivision of the PVN before and after pubertal development. Furthermore, after a peripheral injection of the retrograde tracer Fluoro-Gold (FG), prepubertal and adult males demonstrate similar numbers of anterior pituitary projecting neurosecretory neurons in the parvocellular region of the PVN. Finally, using in situ hybridization we show that in response to acute stress, CRH mRNA in the PVN was affected by both age and stress such that prepubertal males have greater CRH expression than adults and both prepubertal and adult males show significant stress-induced increases in CRH mRNA. Interestingly, in response to repeated restraint, neither age nor stress significantly influence CRH expression. Together, these data indicate that both age and experience with stress interact to modulate CRH expression in the PVN.     

Vasopressin and vasoactive intestinal peptide infused in the paraventricular nucleus of the hypothalamus elevate plasma melatonin levels

Abstract: The connection between the suprachiasmatic nucleus (SCN) and the paraventricular nucleus of the hypothalamus (PVN) forms an important component of the melatonin rhythm-generating system. However, the chemical identity of this projection is not known. To test the possible implication of the SCN peptides vasopressin (VP) and vasoactive intestinal peptide (VIP) in this projection, we performed microinfusions in the PVN during the first half of the dark period and subsequently monitored resulting plasma melatonin levels. Infusions for 7 hr of either VP or VIP, but not oxytocin, caused increased plasma melatonin levels in the middle of the dark period. These observations confirm the role of the PVN in the melatonin rhythm-generating pathway and indicate that both VP and VIP released at the level of the PVN, and probably derived from the SCN, are able to influence peripheral plasma melatonin levels.     

Human plasma proopiomelanocortin N-terminal peptide and adrenocorticotropin: circadian rhythm, dexamethasone suppression, and corticotropin-releasing hormone stimulation

The circadian rhythm, suppression with dexamethasone, and stimulation by corticotropin-releasing hormone (CRH) of plasma immunoreactive (IR) proopiomelanocortin N-terminal (NT) and IR-ACTH were studied in nine normal subjects and two patients with Addison's disease. The RIA for human NT (hNT) used was specific for NT except for partial cross-reactivity with gamma 2MSH. In normal subjects, plasma IR-hNT and IR-ACTH had almost parallel circadian rhythms and were suppressed by dexamethasone. The mean plasma levels of IR-hNT and IR-ACTH at 0800 h were 140 +/- 23 (SD) and 23 +/- 5 pg/ml, respectively. Plasma IR-hNT increased in parallel with IR-ACTH 15 to 30 min after iv injection of 100 micrograms ovine CRH. Maximum percent increases in plasma IR-hNT and IR-ACTH were 185 +/- 47 and 235 +/- 10%, respectively. In Addison's disease, on the other hand, plasma levels of IR-hNT and IR-ACTH were markedly elevated and the circadian rhythms were parallel. The mean plasma IR-hNT and IR-ACTH levels at 0900 h were 4363 and 1750 pg/ml, respectively. These results suggest that plasma hNT and ACTH are produced from a common precursor in the pituitary gland and secreted concomitantly under various physiological conditions such as stimulation by CRH and inhibition by glucocorticoid.     

Effects of immunoneutralization of corticotropin-releasing hormone on ultradian rhythms of plasma adrenocorticotropin

ACTH, like other anterior pituitary peptide hormones, is secreted episodically and demonstrates both circadian and ultradian rhythms. CRH is the major regulator of ACTH release from the pituitary corticotroph. To determine the dependence of ACTH ultradian rhythms on CRH, passive immunoneutralization was used to block the activity of endogenous CRH in rats with indwelling venous catheters. Blood was sampled at 2- and 15-min intervals while blood volume was replaced. Plasma ACTH was measured by RIA. Time-series analysis of plasma ACTH concentrations was performed with PULSAR and Cluster Analysis. The 2 min data demonstrated secretory bursts approximately every 20 min. CRH immunoneutralization had no effect on the frequency of these pulses, but significantly reduced their amplitude. This was the case for raw data as well as data in which lower frequency variation had been filtered out. The 15 min data demonstrated pulsatile secretion, with a secretory episode approximately every 100 min. This lower frequency rhythm was also observed when high frequency components were filtered out of the 2 min data series. Analysis of the 15 min and the filtered 2 min time series showed this rhythm to be almost totally ablated by CRH immunoneutralization. These results suggest that CRH is responsible for amplitude modulation of an underlying CRH-independent rhythm and that through intermittent amplitude modulation of this rhythm a lower frequency rhythm is generated. Comparison between treatment groups of pulses identified by PULSAR or Cluster Analysis yielded similar results, but the programs were discordant with each other. This is the first in vivo evidence of pulsatile ACTH secretion independent of CRH, the first report demonstrating that different ultradian rhythms of ACTH may be regulated by different mechanisms, and the first comparison of PULSAR and Cluster Analysis on plasma ACTH time series.     

Expression of corticotropin-releasing hormone type 1 receptor in paraventricular nucleus after acute stress

We have previously proposed the existence of ultrashort loop-positive feedback regulation of corticotropin-releasing hormone (CRH) in the hypothalamus. To gain a better understanding of this effect, we performed double-label in situ hybridization to identify the neurons in the paraventricular nucleus (PVN) that express CRH type 1 receptor (CRH-R1) following stress. We also conducted immunohistochemistry to determine whether CRH-R1 mRNA was translated to CRH-R1 protein in the PVN. Thirty-minute restraint stress given to male Wistar rats increased c-fos mRNA expression primarily in the CRH-producing neurons of the parvocellular PVN. Small numbers of vasopressin and oxytoxin-producing cells were also labeled by c-fos probes. Approximately 70% of CRH-R1 positive neurons exhibited CRH mRNA 2 h after the beginning of stress, while only a small percentage of the vasopressin and oxytocin-producing cells coexpressed CRH-R1 mRNA. CRH-R1 immunoreactivity, which was detected in the perikarya and fibers of PVN neurons, appeared to increase in response to stress, though this was not statistically significant. Pretreatment with a selective CRH-R1 antagonist, CP-154,526, significantly attenuated stress-induced corticotropin (ACTH) secretion as well as c-fos mRNA expression in the PVN. These results demonstrate that acute stress increases neuronal activation and CRH-R1 mRNA expression primarily in CRH-producing neurons of the parvocellular PVN, that CRH-R1 message is translated to CRH-R1 protein, and that PVN neurons are activated at least in part through CRH-R1 under acute stress. The data further support the possibility of feedback regulation of CRH itself in CRH-producing neurons.     

Stress-induced alterations in corticotropin-releasing hormone and vasopressin gene expression in the paraventricular nucleus during ontogeny

From postnatal day (PND) 4 to 14, neonates display a minimal pituitary-adrenal response to mild stress, the so-called 'stress hyporesponsive period' (SHRP). During the SHRP, maternal deprivation (MD) alters the pituitary-adrenal system, enabling neonates to become endocrine responsive to specific stimuli. We have previously reported that during the SHRP, mild stress enhances corticotropin-releasing hormone (CRH) messenger RNA (mRNA) expression in the paraventricular nucleus (PVN). Insofar as elevated CRH mRNA was observed both in the presence and absence of adrenocorticotropin (ACTH) release, we hypothesized that other ACTH secretagogues may participate in the pituitary stress response. During the SHRP, does arginine vasopressin (AVP) complement the actions of CRH which might be reflected centrally by the enhanced biosynthesis of both neuropeptides? To test this hypothesis we examined the time course of stress-induced CRH and AVP mRNA in the PVN at PND 6, 12, and 18. As an index of neural activity, c-fos mRNA in the PVN was also examined. Restraint was used as the stressor and MD was employed to enable an endocrine response during the SHRP. Despite the absence of stress-induced ACTH, in nondeprived pups during the SHRP, CRH mRNA was rapidly enhanced. In their maternally deprived (DEP) counterparts, ACTH levels were increased, and a significant induction of CRH mRNA was only observed at day 12. AVP mRNA levels were elevated in DEP 12-day-old pups at 15, 30 and 60 min. In rats beyond the SHRP, plasma ACTH levels, CRH and AVP mRNA were all enhanced following restraint. At PND 18, elevated CRH mRNA was not observed until 4 h after stimulus. Following restraint, c-fos mRNA was increased at all three ages, although the magnitude of c-fos response was less during the SHRP. These results demonstrate that when restraint elicits prototypical ACTH release, the neonatal central response is to enhance the biosynthesis of both AVP and CRH. If nucleic acid changes correlate with release, the increased synthesis of both neuropeptides may indicate the potential for AVP to synergize with CRH during the neonatal stress response.     

Diurnal variation in the response of plasma adrenocorticotropin and cortisol to intravenous ovine corticotropin-releasing hormone

To determine whether the plasma immunoreactive ACTH (IR-ACTH) and IR-cortisol responses to ovine corticotropin-releasing hormone (oCRH) depend on the time of day, we administered 1 microgram/kg BW synthetic oCRH as an iv bolus dose to five normal men at their usual time of awakening between 0530-0740 h, at 1600 h, and at 2300 h. Mean basal plasma IR-ACTH and IR-cortisol levels were highest upon awakening, intermediate at 1600 h, and lowest at 2300 h, reflecting the diurnal rhythm of ACTH secretion. There was no significant difference in the plasma IR-ACTH response to oCRH at different times of the day. In contrast, the mean maximum plasma IR-cortisol increment and mean integrated response were 2- and 2.6-fold greater (P less than 0.05), respectively, at 2300 h than upon awakening. In another study, oCRH was given in the morning (0700-0900 h) to 22 normal men and in the late afternoon (1600-1800 h) to 24 normal men. Mean basal plasma IR-ACTH and IR-cortisol levels were significantly higher (P less than 0.001) in the morning [24 +/- 3 pg/ml (mean +/- SEM) and 10.6 +/- 0.8 micrograms/dl, respectively] than in the afternoon (13 +/- 2 pg/ml and 5.6 +/- 0.6 micrograms/dl, respectively). Mean peak plasma IR-ACTH was slightly greater in the morning (60 +/- 5.5 pg/ml) than in the afternoon (47 +/- 5.5 pg/ml), the mean maximum plasma IR-ACTH increments were the same (35 +/- 4 and 34 +/- 5 pg/ml, respectively), and the mean integrated IR-ACTH response was slightly less in the morning (2036 +/- 414 vs. 2365 +/- 358 pg . min/ml), but none of these differences was statistically significant. Mean peak plasma IR-cortisol concentrations in the morning and afternoon were similar (18.7 +/- 0.7 and 17.3 +/- 0.9 micrograms/dl, respectively), but the mean maximum plasma IR-cortisol increments (8.1 +/- 0.8 and 11.7 +/- 0.9 micrograms/dl, respectively; P less than 0.005), and the mean integrated IR-cortisol responses (588 +/- 115 and 976 +/- 95 micrograms . min/dl, respectively; P less than 0.01) were greater in the afternoon. There was an inverse correlation between basal plasma IR-cortisol concentration and the integrated IR-ACTH response (P less than 0.05), the maximum IR-cortisol increment (P less than 0.001), and the integrated IR-cortisol response (P less than 0.001).(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of ovine corticotropin-releasing hormone administered during insulin-induced hypoglycemia on plasma adrenocorticotropin and cortisol

The factors that mediate the hypothalamic-pituitary response to hypoglycemia in man are unknown. To investigate the role of CRH in the plasma ACTH response to hypoglycemia, two different doses of ovine CRH (oCRH) were given to normal men during insulin-induced hypoglycemia. We hypothesized that if the endogenous CRH response to hypoglycemia were less than maximally stimulating, administration of oCRH during hypoglycemia would result in a greater peak plasma immunoreactive (IR) ACTH response. Six normal men were given 1) 0.15 U/kg regular insulin, iv; 2) insulin plus 1 microgram/kg oCRH, iv, 5 min after serum glucose fell to 40 mg/dL or less; and 3) oCRH alone. The degree and duration of hypoglycemia were the same when insulin was given alone or with oCRH. Plasma IR-ACTH after insulin alone and insulin plus oCRH rose at the same rate to similar peaks of 226 +/- 37 (mean +/- SEM) and 213 +/- 53 pg/mL, respectively, both of which were greater (P less than 0.05) than the peak plasma IR-ACTH after oCRH alone (61 +/- 19 pg/mL). The peak plasma IR-cortisol levels after insulin alone (24 +/- 4 micrograms/dL), insulin plus oCRH (27 +/- 3 micrograms/dL), and oCRH alone (18 +/- 2 micrograms/dL) were not significantly different. In a second study, six normal men were given 0.15 U/kg regular insulin, iv; insulin plus 10 micrograms/kg oCRH, iv; and 10 micrograms/kg oCRH alone. Administration of oCRH 5 min after serum glucose fell to 40 mg/dL or less did not affect the degree or duration of hypoglycemia. Plasma IR-ACTH after insulin alone and insulin plus oCRH rose at the same rate to similar peaks of 258 +/- 14 and 290 +/- 33 pg/mL, respectively, both of which were greater (P less than 0.01) than the peak (54 +/- 6 pg/mL) after oCRH alone. After insulin alone, plasma IR-ACTH declined to baseline by 3 h. However, after insulin plus oCRH, plasma IR-ACTH fell gradually until 2 h, rose to a second peak at 2.5-3 h, and remained greater (P less than 0.01) than after insulin or oCRH alone for the 4-h duration of the study. The mean peak plasma IR-cortisol level after insulin plus oCRH (33 +/- 4 micrograms/dL) was similar to that after insulin alone (28 +/- 3 micrograms/dL), but was greater (P less than 0.05) than that after oCRH alone (18 +/- 2 micrograms/dL).(ABSTRACT TRUNCATED AT 400 WORDS)     

The corticotropin-releasing hormone test in normal short children: comparison of plasma adrenocorticotropin and cortisol responses to human corticotropin-releasing hormone and insulin-induced hypoglycemia

The human corticotropin-releasing hormone (hCRH) tests were performed in twelve normal short children, and the responses of plasma ACTH and cortisol to iv administration of 1 micrograms/kg hCRH were compared with those to insulin-induced hypoglycemia. After administration of hCRH, the mean plasma ACTH level rose from a basal value of 3.3 +/- 0.4 pmol/l (mean +/- SEM) to a peak value of 9.2 +/- 0.8 pmol/l at 30 min, and the mean plasma cortisol level rose from a basal value of 231 +/- 25 nmol/l to a peak value of 546 +/- 30 nmol/l at 30 min. The ACTH response after insulin-induced hypoglycemia was greater than that after hCRH administration; the mean peak level (P less than 0.01), the percent maximum increment (P less than 0.01), and the area under the ACTH response curve (P less than 0.01) were all significantly greater after insulin-induced hypoglycemia than those after hCRH administration. Although the mean peak cortisol level after insulin-induced hypoglycemia was about 1.3-fold higher than that after hCRH administration (P less than 0.01), neither the percent maximum increment in plasma cortisol nor the area under the cortisol response curve after insulin-induced hypoglycemia was significantly different from that after hCRH administration. Consequently, the acute increases in plasma ACTH after the administration of 1 microgram/kg hCRH stimulated the adrenal gland to almost the same cortisol response as that obtained with a much greater increase in plasma ACTH after insulin-induced hypoglycemia. These results suggest that a plasma ACTH peak of 9-11 pmol/l produces near maximum acute stimulation of adrenal steroidogenesis.     

Neuropeptide Y in the paraventricular nucleus of the hypothalamus increases ethanol intake in high- and low-alcohol-drinking rats

BACKGROUND: The behavioral effects of neuropeptide Y (NPY) attributed to its actions in the hypothalamus are complex and include effects on feeding, sedation, and the hypothalamic-pituitary-adrenal axis. NPY infused into the paraventricular nucleus (PVN) increases ethanol intake in unselected rats. High-alcohol-drinking (HAD1) and low-alcohol-drinking (LAD1) rats differ in basal NPY levels in the PVN, and HAD1, but not LAD1, rats exhibit decreases in ethanol intake after infusion of NPY into the ventricles. This study examined whether NPY infused into the PVN alters ethanol intake in HAD1 and LAD1 rats. METHODS: Female HAD1 (n = 14) and LAD1 (n = 18) rats were given 24-hr free-choice continuous access to 15% (v/v) ethanol and water for 6 weeks and then implanted bilaterally with cannulas aimed at the PVN. Two weeks later, rats received a series of microinfusions, each separated by 1 week, that included four doses of NPY (0.0, 0.25, 0.5, and 1.0 microg). Ethanol, water, and food were available ad libitum after infusions. All rats received a final microinfusion of 1.0 microg of NPY, after which ethanol and water, but no food, were made available for 2 hr. RESULTS: During the 2 hr after infusion, NPY yielded dose-dependent increases in both water and food consumption. With food concurrently available, the 0.25- and 1.0-microg doses of NPY did not alter baseline ethanol intake, whereas the 0.5-microg dose increased ethanol intake. Infusion of 1.0 microg of NPY in the absence of food yielded a decrease in water intake and an increase in ethanol intake relative to the same dose in the presence of food. Twenty-four hours after infusion, there were no effects of NPY on water and food intake, and increases in ethanol intake were no longer apparent. CONCLUSIONS: Increases in ethanol intake after infusion of NPY into the PVN may depend on NPY dose and whether food is concurrently available.     

Central activin administration modulates corticotropin-releasing hormone and adrenocorticotropin secretion

A broad and diffuse neuronal network conveys information reflecting the state of the internal and external environment to the neurosecretory hypothalamus. Recently, we identified an inhibin-beta A- (I beta A) immunoreactive terminal field within the CRF-rich portion of the dorsomedial paraventricular nucleus which originates from a cell group in the commissural portion of the nucleus of the solitary tract (NTS). The NTS receives baroreceptor input, somatosensory input via the spinosolitary tract, and sensory information from the oral, thoracic, and abdominal cavities and, thus, is positioned to serve as a primary relay for visceral sensory inputs to neurons critical to the function of the hypothalamic-pituitary-adrenal (HPA) axis. Although these NTS cells contain multiple putative transmitters, we present evidence that activin, an inhibin-beta A dimer, plays a modulatory role in HPA axis function via facilitation of CRF release. First, intraventricular injection of activin-A (0-3 nmol), but not the related inhibin heterodimer, evoked dose-related 1.7- to 2.8-fold elevations of circulating ACTH levels in male rats. Second, analysis of hypophysial-portal plasma after bilateral paraventricular nucleus microinfusion of activin-A revealed a dose-related facilitation of CRF secretion up to 4-fold above preinjection levels which was unaccompanied by changes in arginine vasopressin levels. Finally, activin-A also enhanced CRF secretion from neonatal hypothalamic cells in primary culture with an EC50 dose of approximately 0.25 nM. Overall, these observations provide evidence of both an anatomical and a pharmacological substrate for activin-mediated central modulation of HPA axis function.     

Relation between corticotropin-releasing hormone neuron number in the hypothalamic paraventricular nucleus and depressive state in Alzheimer's disease

BACKGROUND/AIMS: Depression occurs in 20-50% of the Alzheimer disease (AD) patients. It is not known whether depression in AD shares its pathophysiology with depressive disorder. Previously we found a fourfold increase of corticotropin-releasing hormone (CRH)-immunoreactive (IR) neurons in the hypothalamic paraventricular nucleus in depression. The objective of the present study was to find out whether in depression in AD the same phenomenon of an increased number of CRH-IR neurons could be observed. METHODS: Post-mortem brain tissue was obtained from a cohort of 23 AD patients prospectively studied using the Cornell Scale for Depression in Dementia to measure depressive symptoms. The number of CRH-IR neurons was determined using immunocytochemistry and the Image Pro Plus analysis program. RESULTS: A significant positive correlation was found between the Cornell scores and the number of CRH-IR neurons (p = 0.039) in AD patients. CONCLUSION: These results suggest that depressive disorder and depression in AD share, at least partly, their pathophysiology.     

Glucocorticoids decrease thyrotropin-releasing hormone messenger ribonucleic acid expression in the paraventricular nucleus of the human hypothalamus

The way glucocorticoids affect TRH mRNA expression in the paraventricular nucleus of the hypothalamus is still unclear. In view of its relevance for Cushing's syndrome and depression, we measured TRH mRNA expression in human hypothalami obtained at autopsy by means of quantitative TRH mRNA in situ hybridization. In corticosteroid-treated subjects (n = 10), TRH mRNA hybridization signal was decreased as compared with matched control subjects (n = 10) (Mann-Whitney U test, P = 0.02). By inference, hypercortisolism as present in patients with Cushing's syndrome or major depression may contribute to lower serum TSH or symptoms of depression by lowering hypothalamic TRH expression.     

Thyroid hormones selectively regulate the posttranslational processing of prothyrotropin-releasing hormone in the paraventricular nucleus of the hypothalamus

Over the last few years, our laboratory has demonstrated that different physiological conditions or stressors affect the posttranslational processing of hypophysiotropic and nonhypophysiotropic proTRH and, consequently, the output of TRH and other proTRH-derived peptides. These alterations in proTRH processing are generally associated with parallel changes in the levels of two members of the family of prohormone convertases 1/3 and 2 (PC1/3 and PC2). An important regulator of proTRH is thyroid hormone, which is the peripheral end product of the hypothalamic (TRH)-pituitary (TSH)-thyroid (T3/4) (HPT) axis. In this study we investigated the effect of thyroid status on the processing of proTRH inside and outside the HPT axis. Our data showed that high levels of thyroid hormone down-regulated PC1/3 and PC2 and TRH synthesis, which led to an accumulation of intermediate forms of proTRH processing. Conversely, low levels of thyroid hormone up-regulated proTRH synthesis and PC1/3 and PC2 levels. Control of the activity of PCs and proTRH processing occurred specifically in the paraventricular nucleus, whereas no change due to thyroid status was found in the lateral hypothalamus or preoptic area. The posttranslational regulation of proTRH processing in the paraventricular nucleus by thyroid status is a novel aspect of the regulation of the HPT axis, which may have important implications for the pathophysiology of hypo- and hyperthyroidism.     

Mitogen-activated protein kinase contributes to lipopolysaccharide-induced activation of corticotropin-releasing hormone synthesizing neurons in the hypothalamic paraventricular nucleus

To determine whether the p44/p42 MAPK (ERK1/2) signaling pathway is involved in the activation of CRH-containing neurons in the hypothalamic paraventricular nucleus (PVN) after bacterial lipopolysaccharide (LPS) administration, Sprague Dawley rats were injected with LPS, and studied after 2, 6, 9, and 12 h. In saline-treated controls, isolated weak phosphorylated (phospho)ERK1/2 immunoreactive neurons were observed in the PVN. However, a dramatic increase in phospho-ERK1/2 immunoreactivity was apparent in the PVN 2 h after LPS administration, and gradually declined to baseline levels 9-12 h after injection. By double-labeling immunofluorescence, all CRH-containing neurons in the PVN contained phospho-ERK1/2 2 h after LPS. Intracerebroventricular administration of the MAPK inhibitor, PD98059, prevented LPS-induced ERK1/2 phosphorylation, c-fos activation, and the increase of CRH gene expression in the PVN but had no effect on c-fos activation in brainstem A2-C1/C2 regions. We conclude that LPS rapidly increases the phospho-ERK1/2 in CRH-containing neurons in the PVN and that activation of MAPKs is necessary for LPS-induced activation of the hypothalamic-pituitary-adrenal axis.     

Influence of human corticotropin-releasing hormone and adrenocorticotropin upon spontaneous growth hormone secretion.

Hormones of the hypothalamo-pituitary-adrenocortical (HPA-) axis are considered to be of physiological and clinical relevance in regulating spontaneous growth hormone (GH) secretion. To further investigate interdependencies between both systems, we studied the effects of adrenocorticotropin [ACTH(1-24)] and human corticotropin-releasing hormone (h-CRH) upon spontaneous GH secretion in 10 male volunteers. Administration of 1 microgram ACTH (1-24), 10 micrograms h-CRH or saline (control: CTL) every hour from 9.00 to 6.00 p.m. resulted in significant differences of cortisol secretion during the entire observation period (8.00 a.m.-3.00 a.m.) between the three groups (p less than 0.001, Friedman two-way ANOVA). Mean area under the time course curve (AUC) values (+/- SEM) for cortisol expressed as ng x 1,000 x min/ml showed also significant differences between the three treatments from 8.00 a.m. to 3.00 a.m.: CTL 64.0 +/- 6.4, ACTH(1-24) 178.5 +/- 9.4 (p less than 0.01, Wilcoxon test), h-CRH 88.5 +/- 5.6 (p less than 0.01). The main portion of cortisol was released during daytime from 8.00 a.m. to 11.00 p.m., where the most significant differences in the AUC values emerged: CTL 59.6 +/- 5.8, ACTH(1-24) 171.5 +/- 8.8 (p less than 0.01, Wilcoxon test), h-CRH 80.2 +/- 5.1 (p less than 0.01). With regard to GH secretion, significant differences became obvious between the three treatments during daytime from 8.00 a.m. to 11.00 p.m. and the sleep-related period from 11.00 p.m. to 3.00 a.m. (p less than 0.01 and p less than 0.02, Friedman two-way ANOVA).(ABSTRACT TRUNCATED AT 250 WORDS)     

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)