Daily profiles of arginine vasopressin mRNA in the suprachiasmatic, supraoptic and paraventricular nuclei of the rat hypothalamus under various photoperiods

Daily rhythm of arginine vasopressin (AVP) mRNA levels in the suprachiasmatic nucleus (SCN) of rats maintained under a short, LD 8:16 photoperiod differed from that of rats maintained under a long, LD 16:8 photoperiod: under the short photoperiod the morning AVP rise occurred significantly later than under the long one. Daily profiles of AVP mRNA in the supraoptic and paraventricular nuclei were not rhythmic and AVP mRNA levels under LD 8:16 did not differ from those under LD 16:8. The data indicate that photoperiod affects selectively the clock driven AVP gene expression in the SCN.     

Calretinin is differentially localized in magnocellular oxytocin neurons of the rat hypothalamus. A double-labeling immunofluorescence study

By use of a double-labeling immunofluorescence method with a confocal laser scanning microscope, we have examined whether a calcium-binding protein, calretinin, is localized in magnocellular oxytocin and vasopressin neurons of the rat hypothalamus. In the supraoptic nucleus, all oxytocin-labeled cells were stained for calretinin. However, in the magnocellular part of the paraventricular nucleus, almost all oxytocin-stained cells were devoid of calretinin immunoreactivity. All vasopressin-positive cells of both the supraoptic nucleus and the magnocellular part of the paraventricular nucleus lacked calretinin immunoreactivity. No calretinin immunoreactivity was found in oxytocin-labeled cells of the the anterior commissural nucleus or in vasopressin-labeled cells of the suprachiasmatic nucleus. We previously showed that another calcium-binding protein, calbindin-D28k, was localized in magnocellular oxytocin neurons of the supraoptic nucleus but not in those of the paraventricular nucleus. These findings suggest that, in general, magnocellular oxytocin neurons of the supraoptic nucleus and those of the paraventricular nucleus can be chemically distinguished, that is, the former contain both calretinin and calbindin-D28k but the latter lack the two calcium-binding proteins.     

Expression of the proprotein convertases PC1 and PC2 mRNAs in thyrotropin releasing hormone neurons of the rat paraventricular nucleus of hypothalamus

PC1 and PC2 are subtilisin-like processing enzymes capable of cleaving thyrotropin releasing hormone (TRH) precursor (pro-TRH) at paired basic residues in vitro. In the paraventricular nucleus of the hypothalamus (PVN), pro-TRH is synthesized to control adenohypophysial thyrotropin and prolactin release. Biochemical and immunological approaches have shown that in the hypothalamus, pro-TRH is extensively cleaved at pairs of basic amino acids. We quantified, by two different approaches, in situ hybridization (ISH) on consecutive cryostat sections or double label ISH, the proportion of PVN TRH neurons containing either PC1 or PC2 mRNAs. Both techniques gave similar results: PC2 mRNA was present in 60–70% of TRH neurons, and PC1 mRNA in 37–46%. Values were similar in the anterior and medial parts of the parvocellular PVN. TRH neurons containing either PC1 or PC2 mRNA were found throughout the areas containing TRH cells without any evidence of anatomical segregation. These results suggest a biochemical heterogeneity in PVN TRH biosynthetic machinery.     

Progesterone receptor expression in the pregnant and parturient rat hypothalamus and brainstem

Oxytocin is synthesized by magnocellular neurons in the supraoptic and paraventricular nuclei (SON and PVN) and during pregnancy progesterone prevents premature activation of oxytocin neurons. Progesterone receptors (PR) are not detectable in SON oxytocin neurons of non-pregnant rats, so we sought to determine whether they are expressed during pregnancy and parturition. In addition, we examined PR expression in brainstem and hypothalamic regions that have known direct projections to the SON. Neuronal immunoreactive PR (irPR)-labeled nuclei were counted in sections from proestrous virgin, late pregnant (day 21) and parturient rats (90 min from birth onset). IrPR nuclei were not evident in the SON at any stage but irPR expression in the medial preoptic nucleus (MPA) significantly increased in pregnancy and parturition (159% and 189% of proestrous controls, respectively). Other hypothalamic areas did not exhibit a significant change in irPR expression. In the nucleus tractus solitarius (NTS) in the brainstem, there was no significant change in irPR in late pregnancy, but there was a significant reduction in irPR expression at parturition (22% of proestrous controls). Very few NTS neurons immunoreactive for tyrosine hydroxylase (irTH), and thus putatively noradrenergic, contained irPR. These findings taken with evidence that brainstem irTH neurons projecting to the SON are stimulated at parturition, whereas MPA cells projecting to the SON are not, suggest that any direct actions of progesterone or progesterone withdrawal on NTS or SON neurons are not mediated through the classical PR. Upregulation of PR expression in the MPA during pregnancy and parturition may relate to the onset of maternal behavior and/or regulation of GnRH neuronal activity.     

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.     

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.     

Anterograde tracing of retinal afferents to the tree shrew hypothalamus and raphe

The anterograde neuronal transport of Cholera toxin B subunit (CTB) was used in this study to label the termination of retinal afferents in the hypothalamus of the tree shrew Tupaia belangeri. Upon pressure-injection of the substance into the vitrous body of one eye, a major projection of the retinohypothalamic tract (RHT) was found to the hypothalamic suprachiasmatic nuclei (SCN). Although the innervation pattern was bilateral, the ipsilateral SCN received a somewhat stronger projection. Labeling was also found in the supraoptic nucleus and its perinuclear zone, respectively, mainly ipsilaterally as well as in the bilateral para- and periventricular hypothalamic regions without lateral predominance. In the raphe region, scattered fibers and terminals were seen in the dorsal and median raphe nuclei. CTB-immunoreactive structures were observed neither in the locus ceruleus nor in vagal nuclei. Our results, partly in contradiction to earlier studies using different tracing techniques in another tree shrew species (Tupaia glis), reveal that hypothalamic nuclei, in particular the SCN, are contacted by retino-afferent fibers which are thought to mediate the effects of light to the endogenous ‘clock’ and to parts of the neuroendocrine system.     

The role of the anteromedial hypothalamus in Dahl hypertension

Radiofrequency lesions were placed in the anteromedial hypothalamus in Dahl rats to investigate the role of this area in salt-induced hypertension. Lesions of the paraventricular and suprachiasmatic nuclei and intervening tissue (PVN-SCN lesions) prevented the rise in blood pressure (mmHg) in salt-sensitive (DS) rats fed a high-salt (8% NaCl) diet for 15 weeks (controls: 195 +/- 9, lesioned: 128 +/- 11, p less than 0.01). Similar lesions in salt-resistant (DR) rats did not alter long-term blood pressure (controls: 121 +/- 6 mmHg, lesioned: 131 +/- 5). Lesions sparing the PVN had no effect on blood pressure in DS rats, while lesions primarily confined to the PVN delayed the rise of blood pressure in DS fed a high salt diet. Round-the-clock determinations demonstrated that the blood pressure of DS rats with PVN-SCN lesions was reduced relative to controls at all times of the day throughout the study. No differences were observed between the angiotensin-induced drinking and plasma sodium concentrations of rats with PVN-SCN lesions and those of controls. Twenty-four hour mean heart rate was decreased by 10% in DS rats with PVN-SCN lesions. The anteromedial hypothalamus may participate in the initiation of Dahl hypertension.     

Hypovolemia upregulates the expression of neuronal nitric oxide synthase gene in the paraventricular and supraoptic nuclei of rats

We have examined the effects of isotonic hypovolemia on the expression of the neuronal nitric oxide synthase (nNOS) gene in the paraventricular (PVN) and supraoptic nuclei (SON) of the rat, using in situ hybridization histochemistry with a ³⁵S-labelled oligodeoxynucleotide probe complementary to nNOS mRNA. Intraperitoneal (i.p.) administration of polyethylene glycol (PEG) (MW 4000, 20 ml/kg body weight) dissolved in 0.9% saline (20% w/v) induced isotonic hypovolemia. The expression of the nNOS gene in the PVN and SON 6 h after i.p. administration of PEG was increased significantly in comparison with controls. The dual staining for NADPH diaphorase activity and Fos-like immunoreactivity (Fos-LI) showed that at 3 and 6 h after i.p. administration of PEG, a subpopulation of NADPH diaphorase-positive cells in the PVN and SON exhibited nuclear Fos-LI. These results suggest that NO in the PVN and SON may be involved in the neuroendocrine and autonomic responses to non-osmotic hypovolemia.     

Lesions of the paraventricular nucleus area of the hypothalamus disrupt the suprachiasmatic→ spinal cord circuit in the melatonin rhythm generating system

The circadian rhythm in melatonin production in mammals is regulated by a suprachiasmatic (SCN) leads to spinal cord leads to pineal circuit. In the present investigation the possible participation of the paraventricular nucleus of the hypothalamus (PVN) in the SCN leads to spinal cord segment of this circuit was investigated in the rat. Bilateral lesions of the PVN area were produced and one to two weeks later melatonin production was evaluated by measuring the activities of the two pineal enzymes required for the formation of melatonin from serotonin, indoleamine N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), and urinary 6-hydroxymelatonin, the major melatonin metabolite. In some cases pineal melatonin was also measured. Control animals received sham-PVN lesions. Histological examination of the lesions indicated that the PVN were bilaterally destroyed 100% in 12 animals. The nighttime pineal melatonin and urinary 6-hydroxymelatonin values in this group were reduced about 90%, nighttime pineal NAT activity was reduced about 98%, and HIOMT activity about 75%. The urinary 6-hydroxymelatonin values of PVN-lesioned animals and animals with denervated pineal glands were similar. In animals with hypothalamic lesions involving less than 30% of the PVN, nighttime values of NAT, HIOMT, and urinary 6-hydroxymelatonin were normal; in animals with 30 to 95% PVN damage these parameters were altered to a small degree. These studies, together with histochemical observations, indicate the SCN neurons responsible for pineal circadian rhythms project to the PVN area of the hypothalamus.     

Inter- and intra-nuclear differences in galanin expression between the hypothalamic paraventricular and supraoptic nuclei in colchicine-untreated rats

Not much is known of the topography of galanin expression in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei neurons in colchicine (an axoplasmic inhibitor)-untreated animals. Insight into the biological implication(s) of galanin expression in the PVN and SON will depend, at least in part, on the nature of its distribution in colchicine-untreated animals. In this study therefore, the topographical distribution of galaninergic profiles was examined in the PVN and SON of colchicine-untreated rats. Staining in the parvocellular PVN (PVN(p)) was predominantly as varicose thin galanin fiber processes while the magnocellular PVN (PVN(m)) contained large cell soma and fiber processes. The relative fiber density was higher in the anterior, periventricular and medial PVN(p) than in the dorsal, lateral and posterior subdivisions. Large-sized cells and thick fibers were limited to the posterior PVN(m) while the anterior and medial PVN(m) contained varicose profiles. Light- and intensely-stained galanin-positive cells as well as large- and small-diameter (varicose or non-varicose) fibers were observed in the SON. The large and thin fibers exhibit preferential ventral and dorsal distribution, respectively. Together with the complexity of specific afferent and efferent connections within the PVN and SON, these observations underscore heterogeneous galanin expression and raise potential implications for understanding the biological role of galanin by physiologically challenging stimuli.     

A retinal projection to the paraventricular nuclei of the hypothalamus in the Syrian hamster (Mesocricetus auratus)

The retino-hypothalamic tract of the Syrian hamster (Mesocricetus auratus) was examined using intraocular injections of horseradish peroxidase conjugated to cholera toxin. In addition to the retinal input to the suprachiasmatic nuclei (SCN), a retinal projection to the caudal paraventricular nuclei was identified. This projection may mediate some effects of light upon pineal gland physiology independently of the SCN and the circadian-rhythm generating system.     

Neurofibrillary pathology in the human paraventricular and supraoptic nuclei

Severe neurofibrillary changes were identified in the paraventricular and supraoptic nuclei of elderly individuals using markers for Alzheimer’s disease-related intraneuronal pathology. This neurofibrillary pathology is remarkable in that the magnocellular paraventricular and supraoptic nuclei are particularly resistant to Alzheimer’s disease. Moreover, the changes were observed even in non-demented controls, indicating that they develop independently of Alzheimer’s disease. The alterations in the paraventricular and supraoptic nuclei were consistently accompanied by neurofibrillary changes in the mediobasal hypothalamus. Received: 29 October 1996 / Revised, accepted: 24 January 1997     

Corticotropin-releasing factor and systemic capsaicin-sensitive afferents are involved in abdominal surgery-induced Fos expression in the paraventricular nucleus of the hypothalamus

We previously reported that abdominal surgery induces Fos expression in specific hypothalamic and medullary nuclei and also causes gastric stasis. The gastric ileus is reduced by systemic capsaicin and abolished by central injection of corticotropin-releasing factor (CRF) antagonist. We studied the influence of systemic capsaicin and intracerebroventricular (i.c.v.) injection of the CRF antagonist, α-helical CRF_9–41, on Fos expression in the brain 1 h after abdominal surgery in conscious rats using immunocytochemical detection. In control groups (vehicle s.c. or i.c.v.), abdominal surgery (laparotomy with cecal manipulation) performed under 7–8 min of enflurane anesthesia induced Fos staining in neurons of the spinal trigeminal, C1/A1 group, ventrolateral medulla, central amygdala, parabrachial nucleus, cuneate nucleus, nucleus tractus solitarii (NTS), paraventricular nucleus of the hypothalamus (PVN) and supraoptic nucleus (SON). Capsaicin (125 mg/kg s.c., 2 weeks before) or α-helical CRF_9–41 (50 μg i.c.v., before surgery) reduced the number of Fos-positive cells by 50% in the PVN while not modifying the number of Fos-labelled cells in the other nuclei. These results indicate that capsaicin-sensitive primary afferents and brain CRF receptors are part of the pathways and biochemical coding through which abdominal surgery activates PVN neurons 1 h post surgery.     

Prepro-thyrotropin releasing hormone 178-199 immunoreactivity is altered in the hypothalamus of the Wistar–Kyoto strain of rat

The rat prepro-thyrotropin releasing hormone (TRH) 178-199 is derived from prepro-TRH by the actions of the endopeptidases, prohormone convertase 1 (PC1) and PC2. PPTRH 178-199 attenuates the synthesis and secretion of adrenocorticotropic hormone (ACTH) from the anterior pituitary both in vitro and in vivo, suggesting an inhibitory action on hypothalamic–pituitary–adrenal (HPA) axis function. This peptide also acts centrally to increase activity and decrease anxiety related behaviors. To elucidate the involvement of this peptide in these functions, we have compared the expression of PPTRH 178-199, PPTRH mRNA, and PC1 and PC2 mRNAs in the Wistar–Kyoto (WKY) and Wistar strains of rat. WKY rats have been shown to possess neuroendocrine abnormalities (HPA hyper-activity) and hyper-emotional behavioral characteristics. Immunohistochemical analysis of PPTRH 178-199 demonstrated significant strain differences in the paraventricular nucleus (PVN) of the hypothalamus and the parastrial nucleus (PSN). WKY rats had significantly greater numbers of immunoreactive (IR) cell body profiles (P<0.0005) than Wistar rats in the PVN and a significantly lower fiber density (P<0.002) in the PSN. Levels of PPTRH, PC1, and PC2 mRNA were not different between strains in any brain region examined. These data suggest that altered levels of PPTRH 178-199 in WKY rats could cause, at least in part, the hyper-activity of the HPA axis and the hyper-emotional behavioral characteristics seen in this rat strain. Such data fit with the hypothesis that PPTRH 178-199 is involved in the regulation of the HPA axis and behavior.     

Polysynaptic neural pathways between the hypothalamus, including the suprachiasmatic nucleus, and the liver

The suprachiasmatic nucleus of the hypothalamus is responsible for a 24-h rhythm in basal glucose levels in the rat. The neural pathways used by the suprachiasmatic nucleus to mediate this rhythm in plasma glucose have not yet been identified. In the present study we examined whether there are any connections between hypothalamic centers, including the suprachiasmatic nucleus, and the liver, which is the main site for glucose production and storage. Transneuronal virus tracing from the liver showed that after injection of pseudorabies virus, specific neuronal cell populations in the central nervous system were labeled retrogradely, suggesting that specific sites in the central nervous system may control liver metabolism. First-order neurons belonged to the sympathetic and parasympathetic system, while second-order and third-order neurons were present in both the brainstem and hypothalamus. The presence of third-order neurons in the suprachiasmatic nucleus suggests an involvement of the biological clock in the neural control of the liver.     

Regulation of proopiomelanocortin gene expression by neuropeptide Y in the rat arcuate nucleus

In the arcuate nucleus which is richly innervated by both proopiomelanocortin (POMC) and neuropeptide Y (NPY) neurons, it has been shown that NPY fibers are in synaptic contact with POMC cell bodies. In order to determine whether NPY could influence POMC neuronal activity, we have studied the effects of NPY and some NPY analogs on POMC gene expression using quantitative in situ hybridization. The following peptides NPY, [Leu³¹, Pro³⁴]-NPY (a Y₁ receptor agonist), and NPY_13–36 (a Y₂ receptor agonist) were injected into the left lateral cerebral ventricle of adult male rats 4 h before being perfused for histological procedures. The intracerebroventricular injection of NPY and NPY_13–36 induced a significant decrease in the number of grains overlying the labelled neurons. On the other hand, the Y₁ receptor agonist [Leu31, Pro34]-NPY did not modify POMC mRNA levels. These data then strongly suggest that NPY negatively regulates the genetic expression of POMC neurons via the Y₂ NPY receptor subtype.