Exaggerated response of arginine vasopressin-enhanced green fluorescent protein fusion gene to salt loading without disturbance of body fluid homeostasis in rats

We examined the effects of chronic salt loading on the hypothalamic expressions of the enhanced green fluorescent protein (eGFP), arginine vasopressin (AVP) and oxytocin (OXT) genes in AVP-eGFP transgenic rats that expressed eGFP in the hypothalamic AVP-containing neurones. In these rats, salt loading for 5 days caused a marked increase of the eGFP fluorescence in the magnocellular divisions of the paraventricular nucleus (PVN), the supraoptic nucleus (SON) and the internal layer of the median eminence. Expression of the eGFP gene was increased seven- to eight-fold in the PVN and SON of salt-loaded rats in comparison with euhydrated rats. By contrast, none of these changes were observed in the suprachiasmatic nucleus. The expression of the AVP and OXT genes was increased 1.5- to two-fold in the PVN and SON of salt-loaded nontransgenic (control) and transgenic rats. There were no differences in the expression levels of the AVP and OXT genes in the PVN and SON between nontransgenic (control) and transgenic animals under normal conditions and after salt loading. In the posterior pituitary gland, the intensity of the eGFP fluorescence did not change after salt loading for 5 days, but increased after 10 days of salt loading. Upon salt loading, significant increases in the plasma AVP concentrations, plasma osmolality and plasma Na+ were observed. Furthermore, there were no significant differences in changes of water intake, food intake, urine volume, urine osmolality, urine Na+ concentrations, and the body weights in both models under normal or salt-loaded conditions. Our results show that the response of the AVP-eGFP fusion gene to chronic salt loading is exaggerated, and humoral responses such as AVP and OXT and the body fluid homeostasis are maintained in AVP-eGFP transgenic rats. The AVP-eGFP transgenic rat gives us a new opportunity to study the dynamics of the AVP system in vivo.     

Peptide GEGLSS-Like Immunoreactivity in the Rat Central Nervous System

A rabbit antiserum was raised against the N-terminal fragment peptide, GEGLSS (Gly-Glu-Gly-Leu-Ser-Ser) of bovine neuropeptide AF (NPAF, A18Famide). NPAF is an octadecapeptide isolated from the bovine brain together with neuropeptide FF (NPFF). GEGLSS-like immunoreactivity was localized with immunofluorescence technique in colchicine-treated rats in neuronal cell bodies of the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. A few neurons were also observed in the retrochiasmatic part of the SON. GEGLSS-like immunoreactivity was also localized to nerve terminals of the posterior pituitary. No GEGLSS-ir neuronal cell bodies were observed in the medial hypothalamus, in an area that contains NPFF-ir neurons. GEGLSS immunoreactivity was also seen in the fibers and terminals of nucleus of the solitary tract. We injected a retrograde tracer, fluorogold, to the posterior pituitary gland and visualized GEGLSS-ir neuronal cell bodies double-labeled with the tracer in SON, PVN, and SOR. The pituitary stalk transsection totally abolished the GEGLSS-ir structures from the posterior pituitary. Our results suggest that GEGLSS immunoreactivity in the rat brain has a more limited distribution than NPFF immunoreactivity. GEGLSS immunoreactivity was partially colocalized with arginine-vasopressin and oxytocin in neuronal cell bodies in the SON and PVN. Considering the fact that the known rat NPFF-NPAF precursor does not contain GEGLSS structure, the detected GEGLSS immunoreactivity may be derived from a previously unknown precursor.     

Neuronal expression of Fos protein in the hypothalamus of rats with heart failure

We sought to identify the areas that have altered neuronal activity within the hypothalamus of rats with heart failure (HF) by mapping neuronal staining of c-Fos protein (Fos) 6-8 weeks following coronary artery ligation (HF group; n=17) or sham surgery (sham-operated control group, n=15). Fos-like immunoreactivity was observed in the paraventricular nucleus (PVN), supraoptic nucleus (SON), median preoptic nucleus (MnPO), anterior hypothalamus (AH) and posterior hypothalamus (PH) using a standard ABC immunocytochemical protocol. The rats in the HF group displayed infarcts averaging 34+/-2% of the outer circumference and 41+/-1% of the inner circumference of the left ventricular wall. Sham-operated control rats had no observable damage to the myocardium. Rats with chronic heart failure (n=5) but no manipulation (no surgery) had a similar number of Fos-staining cells in PVN SON, MnPO, AH and PH compared to sham-operated rats. Acute surgery for isolation of vagus nerves and anesthesia for 90 min increased the number of Fos positive cells in PVN, SON and MnPO of both sham-operated rats and rats with HF. Furthermore, rats with heart failure (n=5) had significantly higher number of Fos-staining cells in PVN (four times), SON (4.5 times) and MnPO (1.5 times) compared to sham-operated rats after acute surgery for isolation of the vagus. The number of Fos-staining cells remained unaltered in AH and PH in both groups of rats. However, in a third series of experiments vagotomy reduced the number of Fos-staining cells in the PVN, SON or MnPO of rats with HF (n=5) to those observed in sham-operated vagotomized rats. This study shows that: (1) there is augmented neuronal activity as indicated by increased number of Fos staining neurons in the PVN, SON and MnPO due to acute surgical stress in rats with HF, and (2) vagal afferents are responsible for the increased neuronal activity in PVN, SON and MnPO of rats with HF during acute surgical stress. These data support the conclusion that vasopressin producing neurons and autonomic areas within the hypothalamus influenced by vagal afferents are activated during HF and are sensitive to 'acute surgical stress' and may contribute to the elevated levels of vasopressin and sympatho-excitation commonly observed in heart failure.     

Effects of Cholecystokinin in the Supraoptic Nucleus and Paraventricular Nucleus are Negatively Modulated by Leptin in 24‐h Fasted Lean Male Rats

Cholecystokinin (CCK) and leptin are two important satiety factors that are considered to act in synergy to reduce meal size. Peripheral injection of CCK activates neurones in several hypothalamic nuclei, including the supraoptic (SON) and paraventricular (PVN) nuclei and neurones in the brainstem of fed rats. We investigated whether peripheral leptin would modulate the effects of CCK on neuronal activity in the hypothalamus and brainstem of fasted rats by investigating Fos expression in the PVN, SON, arcuate nucleus, ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), area postrema (AP) and the nucleus tractus solitarii (NTS). Male rats, fasted for 24 h, received either one i.p. injection of vehicle, leptin or CCK‐8 alone, or received one injection of vehicle or leptin before an i.p. injection of CCK‐8. We found that CCK increased Fos expression in the PVN and SON as well as in the NTS and AP, but had no effect on Fos expression in the arcuate nucleus, VMH or DMH compared to vehicle. Leptin injected alone significantly increased Fos expression in the arcuate nucleus but had no effect on Fos expression in the VMH, DMH, SON, PVN, AP or NTS compared to vehicle. Fos expression was significantly increased in the AP in rats injected with both leptin and CCK compared to rats injected with vehicle and CCK. Unexpectedly, there was significantly less Fos expression in the PVN and SON of fasted rats injected with leptin and CCK than in rats injected with vehicle and CCK, suggesting that leptin attenuated CCK‐induced Fos expression in the SON and PVN. However, Fos expression in the NTS was similar in fasted rats injected with vehicle and CCK or with leptin and CCK. Taken together, these results suggest that leptin dampens the effects of CCK on Fos expression in the SON and PVN, independently from NTS pathways, and this may reflect a direct action on magnocellular neurones.     

3-Methoxy-4-hydroxyphenylethyleneglycol concentrations in discrete hypothalamic nuclei reflect the activity of noradrenergic neurons

An analytical technique is described which permits the quantitation of picogram concentrations of 3-methoxy-4-hydroxyphenylethylene-glycol (MHPG) in acid hydrolyzed extracts of microdissected regions of the rat brain, and this procedure is used to determine if alterations in the activity of noradrenergic neurons are reflected by changes in the concentrations of MHPG in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the rat hypothalamus. MHPG was not detected in non-hydrolyzed samples of either the PVN or SON, but following acid hydrolysis (heating of samples at 94 degrees C for 5 min in 0.16 M perchloric acid) MHPG was detected in both of these regions. These results indicate that MHPG exists primarily as a conjugate in the PVN and SON. Neurotoxin-induced lesions of the ventral noradrenergic bundle decreased norepinephrine (NE) and MHPG concentrations in the PVN and SON, demonstrating that tissue levels of MHPG in these brain regions are dependent upon the presence of noradrenergic neurons. Electrical stimulation of the locus coeruleus increased MHPG concentrations in the PVN, but not in the SON, whereas electrical stimulation of the medial forebrain bundle increased MHPG concentrations in both of these regions. The alpha 2-adrenergic receptor antagonist idazoxan increased, while the alpha 2-adrenergic receptor agonist clonidine decreased MHPG concentrations in both the PVN and SON, but neither idazoxan nor clonidine altered NE concentrations in these regions. Immobilization of rats in the supine position increased MHPG concentrations in the PVN and SON, and this was accompanied by a decrease in NE concentrations in the SON.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased levels of hypothalamic neuronal nitric oxide synthase and vasopressin in salt-loaded Dahl rat

The plasma concentration of arginine vasopression (AVP) and the expression level of the neuronal nitric oxide synthase (nNOS) gene in the paraventricular nucleus (PVN) and the Supraoptic nucleus (SON) of Sprague-Dawley (SD). Dahl salt-sensitive (S) and Dahl salt-resistant (R) rats on a high salt diet were examined by radioimmunoassay for AVP and in situ hybridization histochemistry for nNOS. The high salt diet containing 8.0% NaCl was given for 4 weeks. The concentrations of AVP in hypertensive Dahl S rats were significantly increased in comparison with those in SD rats and Dahl R rats on a high salt diet. The levels of nNOS mRNA and NADPH-diaphorase activity in the PVN and SON of hypertensive Dahl S rats were greater than those in Dahl R rats on a high salt diet. The antihypertensive drugs, either nicardipine or captopril were administered to the Dahl S rats for 2 weeks beginning 2 weeks after the start of the high salt diet The nNOS mRNA in the PVN and SON of Dahl S rats given a high salt diet was not upregulated by treatment with nicardipine, while the nNOS mRNA in salt loaded Dahl S rats was greater upregulated by treatment with captopril to that greater than without the antihypertensive drug. Our results suggest that the increased NO production in the PVN and SON of hypertensive Dahl S rats may be ineffective in decreasing blood pressure or inhibiting AVP secretion.     

万拉法新对强迫游泳大鼠下丘脑和海马c-fos及c-jun蛋白表达的下调作用

目的　探索新一代抗抑郁药万拉法新对大鼠下丘脑和海马内ｃｆｏｓ 和ｃｊｕｎ 蛋白表达的影响。方法　采用特异性抗体的原位免疫细胞化学方法,在强迫游泳大鼠抑郁模型上,观察万拉法新慢性给药（ 腹腔内注射每日１ 次,连续７ 次）对大鼠游泳不动时间和下丘脑及海马核团ｃｆｏｓ 和ｃｊｕｎ 表达的影响;用图像分析技术对大鼠下丘脑室旁核（ Ｐ Ｖ Ｎ） 、视上核（ Ｓ Ｏ Ｎ） 和海马齿状回（ Ｄ Ｇ） 内的ｆｏｓ 和ｊｕｎ 阳性细胞的相对切面面积比和平均目标灰度进行分析。结果　强迫游泳可使大鼠下丘脑和海马内多个核团的ｃｆｏｓ 和ｃｊｕｎ 蛋白表达水平增加,而万拉法新明显缩短了强迫游泳大鼠的不动时间。图像分析结果提示,万拉法新使强迫游泳大鼠下丘脑 Ｐ Ｖ Ｎ 和 Ｓ Ｏ Ｎ 及海马 Ｄ Ｇ 内ｆｏｓ 和ｊｕｎ 阳性细胞相对切面面积比明显降低（ Ｐ＜００５） ,而平均目标灰度显著增加（ Ｐ＜ ００１） 。结论　下丘脑 Ｐ Ｖ Ｎ、 Ｓ Ｏ Ｎ 和海马 Ｄ Ｇ 可能是介导抗抑郁药抑制大鼠绝望行为的重要中枢核团,ｆｏｓ 和ｊｕｎ 蛋白可能是抗抑郁药发挥受体后作用的传导物质。     

Expression of corticotropin releasing factor (CRF), urocortin and CRF type 1 receptors in hypothalamic-hypophyseal systems under osmotic stimulation

The expression of corticotropin releasing factor (CRF) and urocortin in hypothalamic magnocellular neurones increases in response to osmotic challenge. To gain a better understanding of the physiological roles of CRF and urocortin in fluid homeostasis, CRF, urocortin and CRF type 1 receptor (CRFR-1) gene expression was examined in the hypothalamic-hypophyseal system usingin situ and double-label in situ hybridization following chronic salt loading. CRFR-1 expression was further examined by immunohistochemistry and receptor binding. Ingestion of hypertonic saline by Sprague-Dawley rats for 7 days induced CRF mRNA exclusively in the oxytocin neurones of the magnocellular paraventricular nucleus (PVN) and the supraoptic nucleus (SON), but induced CRFR-1 mRNA in both oxytocin and vasopressin-containing magnocellular neurones. Hypertonic saline treatment also increased urocortin mRNA expression in the PVN and the SON. In the SON, urocortin was localized to vasopressin and oxytocin neurones but was rarely seen in CRF-positive cells. Changes in CRFR-1 mRNA expression in magnocellular neurones by hypertonic saline treatment were accompanied by changes in CRFR-1 protein levels and receptor binding. Hypertonic saline treatment increased CRFR-1-like immunoreactivity in the magnocellular PVN and SON, and decreased it in the parvocellular PVN. CRF receptor binding in the PVN and SON was also increased in response to osmotic stimulation. Finally, hypertonic saline treatment increased CRFR-1 mRNA, CRFR-1-like immunoreactivity and CRF receptor binding in the intermediate pituitary. These results demonstrate that the increase in the expression of CRF and urocortin message in magnocellular neurones induced by salt loading is accompanied by an increase in CRF receptor levels and binding in the hypothalamus and intermediate pituitary. Thus, CRF and urocortin may exert modulatory effects locally within magnocellular neurones as well as at the pituitary gland in response to osmotic stimulation.     

Cyclic nucleotide dynamics in the rat hypothalamus during osmotic stimulation: in vivo and in vitro studies

The molecular mechanisms which regulate expression of vasopressin (AVP)- and oxytocin (OT)-encoding genes are unknown. We have investigated the regulatory role of one class of second messenger, the cyclic nucleotides, by examining levels of both adenosine 3',5'-monophosphate (cAMP) and guanosine 3',5'-monophosphate (cGMP) in hypothalamic nuclei of rats during osmotic stimulation. In vivo studies, in which rats were given 2% saline to drink for different periods (salt loading), demonstrated elevated levels of cAMP in the supraoptic nucleus (SON) after 2 days. Raised levels were also evident at 3 and 7 days. A similar (less marked) pattern was observed in the paraventricular nucleus (PVN) but not in the suprachiasmatic nucleus (SCN). cGMP was present at much lower levels than cAMP and did not exhibit parallel dynamics during salt loading; however, significant changes in cGMP levels were found in the SON and PVN. In vitro studies, in which explant cultures of punched hypothalamic nuclei were challenged with hypertonic media, demonstrated that increasing medium osmolality from 290 to 310 mOsm/kg doubled the level of cAMP in the SON but did not change levels in the PVN or SCN. A greater stimulus, 325 mOsm/kg, caused a 4-fold increase in SON cAMP, and small cAMP responses in the PVN and SCN. Marked cGMP responses were also observed in the SON following stimulation at 310 and 325 mOsm/kg, smaller responses being found in the PVN and SCN. These results are consistent with previous demonstrations of SON neuron osmosensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Importance of endogenous nitric oxide synthase in the rat hypothalamus and amygdala in mediating the response to capsaicin

Although capsaicin has been shown to activate certain neuronal groups in the hypothalamus and amygdala, the neurotransmitters involved and the exact mechanism of action are not clearly understood at present. The aim of this study was to examine the hypothesis that the effect of capsaicin in the rat hypothalamus and amygdala primarily involves direct activation of the endogenous nitric oxide synthase (NOS) neurons responsible for the synthesis of nitric oxide (NO). Subcutaneous capsaicin injection in male rats, compared with vehicle, caused a significant increase in Fos expression in the paraventricular nucleus (PVN), supraoptic nucleus (SON), and medial and cortical amygdala. The expression of nicotinamide adenine dinucleotide phosphate diaphorase, a histochemical marker for NOS, was also increased in these brain areas in addition to the periventricular and lateral hypothalamic area and central amygdaloid nucleus. Also, capsaicin significantly increased the expression of neuronal NOS messenger RNA and protein in the PVN, SON, and medial amygdala as demonstrated by in situ hybridization and immunohistochemistry, respectively. A higher proportion of the NOS neurons in the PVN, periventricular region, SON and amygdala showed Fos expression in response to capsaicin than vehicle injection. There was little, if any, Fos activation in the NOS-positive neurons in the lateral hypothalamic area. The capsaicin-induced activation of the hypothalamic PVN and SON neurons and the medial amygdaloid nucleus was attenuated in the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) -pretreated animals in comparison with the inactive enantiomer D-NAME. These observations indicate that activation of the endogenous NOS system and production of NO constitute a major pathway through which capsaicin exerts its effect within the hypothalamus and amygdala.     

Galanin-R1 receptor in anterior and mid-hypothalamus: Distribution and regulation

The distribution and regulation of galanin-R1 receptor (GAL-R1-R) mRNA has been studied in the anterior and mid-diencephalon by using in situ hybridization. Moreover, possible colocalization of GAL-R1-R mRNA and prepro-galanin or vasopressin mRNAs has been analyzed at the cellular level using double in situ hybridization methodology. Many nuclei in the hypothalamus expressed GAL-R1-R mRNA, including the paraventricular nucleus (PVN) and the supraoptic nucleus (SON). Strong expression was also seen in the same sections in various areas outside of the diencephalon. The distribution patterns are similar to those described in earlier studies. Double labeling experiments showed GAL-R1-R mRNA in vasopressin neurons in the PVN and SON. Moreover, GAL-R1-R mRNA and prepro-galanin mRNA were colocalized in several hypothalamic nuclei. GAL-R1-R mRNA levels showed a high degree of plasticity. Thus, salt loading resulted in a marked increase in GAL-R1-R mRNA levels in the PVN and SON and a moderate decrease was seen during lactation. In contrast, hypophysectomy caused a decrease in GAL-R1-R mRNA levels. Differential effects of colchicine were recorded with a decrease of GAL-R1-R mRNA in the magnocellular hypothalamic neurons. After salt loading or during lactation, GAL-R1-R mRNA and prepro-galanin mRNA were regulated in parallel, whereas their levels changed in opposite directions after hypophysectomy and colchicine injection. In conclusion, GAL-R1-Rs are present in several hypothalamic nuclei, partly in neurons synthesizing galanin. The receptors are regulated in a specific fashion in the various nuclei, depending on the stimulus applied. The results suggest that the effect of galanin in the hypothalamus partly depends on the state of receptor expression. J. Comp. Neurol. 399:321–340, 1998. © 1998 Wiley-Liss, Inc.     

Response of Arginine Vasopressin-Enhanced Green Fluorescent Protein Fusion Gene in the Hypothalamus of Adjuvant-Induced Arthritic Rats

Arginine vasopressin (AVP) and corticotrophin-releasing hormone (CRH) in the parvocellular neurosecretory cells of the paraventricular nucleus (PVN) play a major role in activating the hypothalamic-pituitary-adrenal axis, which is the main neuroendocrine response against the many kinds of stress. We examined the effects of chronic inflammatory/nociceptive stress on the expression of the AVP-enhanced green fluorescent protein (eGFP) fusion gene in the hypothalamus, using the adjuvant arthritis (AA) model. To induce AA, the AVP-eGFP rats were intracutaneously injected heat-killed Mycobacterium butyricum (1 mg/rat) in paraffin liquid at the base of their tails. We measured AVP, oxytocin and corticosterone levels in plasma and changes in eGFP and CRH mRNA in the hypothalamus during the time course of AA development. Then, we examined eGFP fluorescence in the PVN, the supraoptic nucleus (SON), median eminence (ME) and posterior pituitary gland (PP) when AA was established. The plasma concentrations of AVP, oxytocin and corticosterone were significantly increased on days 15 and 22 in AA rats, without affecting the plasma osmolality and sodium. Although CRH mRNA levels in the PVN were significantly decreased, eGFP mRNA levels in the PVN and the SON were significantly increased on days 15 and 22 in AA rats. The eGFP fluorescence in the SON, the PVN, internal and external layers of the ME and PP was apparently increased in AA compared to control rats. These results suggest that the increases in the concentrations of ACTH and corticosterone in AA rats are induced by hypothalamic AVP, based on data from AVP-eGFP transgenic rats.     

Neuronal expression of fos protein in the forebrain of diabetic rats

We sought to identify the areas that have altered neuronal activity within the hypothalamus of diabetic rats by mapping neuronal expression of c-fos protein (Fos) and Fos-related antigens. After a standard PAP immunocytochemical protocol, Fos-like immunoreactivity was observed in the paraventricular nucleus (PVN), supraoptic nucleus (SON), median preoptic area (MnPO), anterior hypothalamus (AH) and posterior hypothalamus (PH) of control (vehicle; n=6) and diabetic rats (Sprague-Dawley rats injected with STZ 65 mg/kg/ip 4 weeks prior to the experiment; n=6). Blood glucose levels were significantly elevated in the diabetic group (370+/-8 mg/dl) compared to control group (104+/-3 mg/dl). Diabetic rats had a significantly higher number of Fos-positive cells in PVN (2.5x), SON (7x) and MnPO (2x) compared to the control rats. However, diabetic rats had significantly fewer Fos-positive cells in the AH (0.3x) and no difference was observed in the PH between the diabetic and control rats. Despite the elevated number of Fos-positive cells in the diabetic rats, dehydration (water withdrawal for 24 h) or hypertonic challenge (1.5 ml of 0.1 M NaCl i.p. injection) produced a further increase in the number of Fos-positive cells in the PVN, SON and MnPO. Dehydration did not alter the number of Fos-positive cells in the AH or PH, but hypertonic challenge produced a significant increase in the Fos-positive cells in both the AH and PH of diabetic rats. This study demonstrates that: (1) there is increased basal neuronal activity in the PVN, SON and MnPO, a decrease in neuronal activity in the AH and no change in neuronal activity in the PH as indicated by Fos staining in diabetic rats; and (2) dehydration or hypertonic challenge produces a further increase in the number of Fos-positive cells in the PVN, SON, and MnPO which is comparable to control rats. These data support the conclusion that vasopressin producing neurons in the PVN and SON and autonomic areas within the lamina terminalis and hypothalamus are activated during diabetes and may contribute to the elevated levels of vasopressin and autonomic dysfunction during diabetes.     

Increased nitric oxide synthase activity and expression in the hypothalamus of hindlimb unloaded rats

Upon return from spaceflight or resumption of normal posture after bed rest, individuals often exhibit cardiovascular deconditioning. Although the mechanisms responsible for cardiovascular deconditioning have yet to be fully elucidated, alterations within the central nervous system have been postulated to be involved. The paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus are important brain regions in control of sympathetic outflow and body fluid homeostasis. Nitric oxide (NO) modulates the activity of PVN and SON neurons, and alterations in NO transmission within these brain regions may contribute to symptoms of cardiovascular deconditioning. The purpose of the present study was to examine nitric oxide synthase (NOS) activity and expression in the PVN and SON of control and hindlimb unloaded (HU) rats, an animal model of cardiovascular deconditioning. The number of neurons exhibiting NOS activity as assessed by NADPH-diaphorase staining was significantly greater in the PVN but not SON of HU rats. Western blot analysis revealed that neuronal NOS (nNOS) but not endothelial NOS (eNOS) protein expression was higher in the PVN of HU rats. In the SON, there was a strong trend for an increase in nNOS (p=0.052) and a significant increase in eNOS expression in HU rats. Our results suggest that increased nNOS in the PVN contributes to autonomic and humoral alterations following cardiovascular deconditioning. In contrast, the functional significance of increases in nNOS and eNOS protein in the SON may be related to alterations in vasopressin release observed previously in HU rats.     

Corticotropin-releasing factor type-1 receptor mRNA is not induced in mouse hypothalamus by either stress or osmotic stimulation

In rats, acute stress substantially increases corticotropin-releasing factor (CRF) type 1 receptor (CRFR-1) mRNA expression in the paraventricular nucleus (PVN) and osmotic stimulation induces both CRF and CRFR-1 mRNA in magnocellular PVN and supraoptic nucleus (SON). However, these phenomena have not been analysed in other species. We compared CRF and CRFR-1 expression in rat and mouse hypothalamus. Male C57BL/6 mice and Wistar rats were exposed to acute restraint stress for 3 h, or to hypertonic saline ingestion for 7 days. Restraint stress increased CRF and c-fos mRNA expression in both rat and mouse PVN. CRFR-1 mRNA was barely detectable in controls, whereas restraint stress substantially increased CRFR-1 mRNA in rat PVN, but not in mouse. Hypertonic saline ingestion induced CRF mRNA in magnocellular PVN and SON of the rat, but did not alter CRF mRNA levels in mouse hypothalamus. CRFR-1 mRNA was also induced in magnocellular PVN and SON of the rat in response to osmotic stimulation, but not in mouse. Immunohistochemistry demonstrated that CRFR-1-like immunoreactivity (ir) was distributed within parvocellular and magnocellular PVN of mouse and rat. CRFR-1-ir in rat PVN was increased by acute stress and osmotic stimulation. By contrast, these treatments did not alter CRFR-1-ir in mouse PVN. Combined immunohistochemistry and in situ hybridization revealed that CRFR-1-ir was most frequently colocalized to CRF in mouse PVN, whereas only a small percentage of oxytocin and vasopressin-producing cells coexpressed CRFR-1-ir. These results indicate that (i) by contrast to rats, neither acute stress nor osmotic stimulation induces CRFR-1 mRNA expression in the mouse PVN; (ii) osmotic stimulation does not alter CRF mRNA expression in parvocellular and magnocellular neurones of mouse PVN; and (iii) acute stress increases c-fos and CRF mRNA to a similar degree in mouse and rat PVN. Thus, differences may exist between mouse and rat in the regulation of CRF and CRFR-1 gene expression in hypothalamus following stress and osmotic stimulation.     

Cold and novel environment stress affects AVP mRNA in the paraventricular nucleus, but not the supraoptic nucleus: An in Situ hybridization study

The distribution and area of label for arginine vasopressin (AVP) mRNA or peptides were studied in rats exposed to cold or novel environments. In situ hybridization histochemistry was employed to detect AVP mRNA in hypothalamic frozen sections with a 45-mer photobiotinylated oligonucleotide probe. The storage of the peptide in both the hypothalamus and the pituitary was determined by immunohistochemistry. Label for mRNA or peptide was then quantified by the Cue-3 color image analysis system. Exposure to 4°C for 30 min caused a 3.5-fold increase in the label for AVP mRNA in the paraventricular nucleus (PVN) compared with that of control rats. This was correlated with a 2-fold elevation in serum ACTH. In addition, rats exposed to 30 min of a novel, thermoneutral (24°C) environment showed a 1.2- to -2.3-fold enhancement of the label for AVP mRNA in the PVN. In contrast, no changes were seen in the supraoptic nucleus (SON) following exposure to either cold or novel environments. Furthermore, neither stress caused significant changes in the storage of AVP peptide in the PVN, SON, median eminence, and posterior lobe of pituitary. This in vivo study demonstrates that PVN and SON neurons respond differentially to cold and novel environment exposures. The elevation of serum ACTH is correlated with the increased level of label for AVP mRNA in the rat hypothalamus, which suggests that AVP may play a role in the regulation of pituitary—adrenal responses to cold and novel environment stresses.     

cGMP-induced presynaptic depression and postsynaptic facilitation at glutamatergic synapses in visual cortex

This study was aimed to examine the neuronal and glial response in the hypothalamus and neurohypophysis of rats with streptozotocin-induced diabetes. At various time intervals after induction of diabetes the neurons in the paraventricular- (PVN) and supraoptic- (SON) nucleus showed upregulated arginine vasopressin (AVP) and oxytocin (OXT) immunoexpression, being most pronounced at 2 weeks. Concomitant to this was the hypertrophy of PVN and SON neurons. NMDAR1, which was constitutively and moderately expressed in normal rats, was markedly augmented, being most intense at 4 months. This coincided with the expression of neuronal nitric oxide synthase (nNOS). Contrary to this, the expression of GluR2/3 was progressively downregulated, so that it was hardly detected at 4 months. Both astrocytes and microglia marked by anti-GFAP and OX-42, respectively, appeared activated. In pars nervosa, the projection target of the axon terminals of PVN and SON neurons, massive axons and terminals (Herring bodies) laden with neurosecretions were observed in diabetic rats. Colocalization study showed that the neurosecretions were internalized by activated pituicytes and microglia associated with the axons. The present results suggest that the neurosecretion of PVN and SON neurons is enhanced in diabetes. This is coupled by upregulation of NMDAR1 and nNOS but downregulation of GluR2/3. It is speculated that the glutamate receptors and NO are linked to overactivation of PVN and SON neurons leading ultimately to cell death of some of them. The pituicytes and microglia in pars nervosa would help to modulate the release of neurosecretion.     

Distribution and morphometric characteristics of oxytocin- and vasopressin-immunoreactive neurons in the rabbit hypothalamus

The distribution, morphological features, and morphometric characteristics of cell bodies producing oxytocin (OT) and vasopressin (AVP) were studied in the rabbit hypothalamus by means of a conventional immunoperoxidase method. The aim of the present study was to determine the existence or not of a species-specific OT-cell group that might be involved in the dense OT innervation of the intermediate lobe in the leporidae. No OT-cell group clearly distinct from the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei was found, even in colchicine-treated animals. Most immunoreactive perikarya were found within these nuclei. In addition, small AVP neurons occurred in the suprachiasmatic nucleus. In the SON, the predominant, tightly packed AVP cells occupied the ventral part of the nucleus, whereas OT neurons were dorsolaterally located. The PVN presented a loose organization without any obvious subdivision. OT cells, which predominated, occupied the medial part of the nucleus. The PVN had a prominent rostral anterobasal extension composed mainly of OT cells. Laterally to the nucleus, numerous large AVP neurons, with few and smaller OT cells, dispersed along the neurosecretory tract without forming definite cell clusters. AVP cell bodies had a rough granular aspect contrasting with the smooth and fine one of OT cells. Spinelike processes were rarely observed on the perikarya, except on large scattered AVP neurons, but frequently covered the proximal dendrites of both types of neurons. Throughout the hypothalamus, OT neurons had definitely smaller mean somal areas and were more homogeneous in size than AVP cells.     

Vasopressin and oxytocin gene expression in intact rats and under catecholamine deficiency during ontogenesis

The development of the hypothalamic vasopressin (VP) and oxytocin (OT) systems has been studied in rats from the 16th embryonic day (E16) until the 11th postnatal day (P11). The VP and OT mRNA-producing neurons were identified on cryostat sections by in situ hybridization using oligonucleotide probes Iabeled by [³⁵S], [³H] or digoxigenin. Moreover, VP and OT gene expressions were evaluated either at E21 or at P11 following chronic depletion of catecholamines (CA). For this purpose, pregnant rats were daily injected with α-methyl-m(p)-tyrosine from gestational day 13 to 20, while neonates were daily injected with a-methyl-m(p)-tyrosine and neurotoxin 6-hydroxydopamine from postnatal day 2 to 10. No VP mRNA- or OT mRNA-expressing cells were observed in the hypothalamus of intact fetuses at E16, while 2 days later rather numerous VP and OT neurons occupied the anterior hypothalamus. One major bilateral group of VP and OT neurons was located in the supreoptic nucleus (SON). Less numerous labeled cab were found in the developing paraventricular nucleus (PVN). Some VP and OT neurons were also spread along the ventrolateral surface of the hypothalamus from the level of the median eminence, caudally, to the level of the optic nerves, rostrally. From E18 until birth, the OT neurons were localized in the dorsal portion of the SON, while its ventral portion was occupied by the VP neurons. The VP mRNA- and OT mRNA-expressing cells seemed to increase both in size and in number over the perinatal period. Frequent relatively long neuronal processes contained VP and OT mRNAs in fetuses and in newborns. When performed during the second half of the fetal life, the chronic depletion of CA did not cause any change in the VP and OT mRNA concentrations in the SON and PVN of fetuses. By contrast, similar treatment of neonates resulted in a significant increase of both mRNA levels in the SON. These data suggest that at least in the SON VP and OT gene expressions might be under the inhibitory control of CA during the neonatal period.