Felino Ramon A. Cagampang

Circadian variation of arginine-vasopressin messenger RNA in the rat suprachiasmatic nucleus.

Arginine-vasopressin (AVP) gene expression in the rat suprachiasmatic nucleus (SCN) is subject to daily rhythmic changes. To determine whether this variation is endogenously generated, temporal changes in the SCN AVP mRNA level in constant dark (DD) condition was compared with changes occurring under the light-dark (LD) condition. In both lighting conditions, the presence of a rhythm in AVP mRNA level was observed in the SCN. In LD condition, peak level of AVP mRNA was found during the latter part of the day (zeitgeber time or ZT 8) and trough value during the night at ZT 20. Correspondingly, peak level of AVP mRNA under DD condition was observed during the latter part of the subjective day (circadian time or CT 8) and a trough during the subjective night (CT 20). Under both lighting conditions, a rapid increase and decrease of mRNA around the peak time was also observed. On the other hand, no significant daily variation in AVP mRNA was found in the supraoptic nucleus in both LD and DD conditions. These results provide evidence that a rhythmic change in AVP mRNA level is regulated by a circadian clock intrinsic to the SCN. The phase relationship of AVP mRNA rhythm to peptide rhythm in the SCN is discussed.

Circadian changes in the expression of vasoactive intestinal peptide 2 receptor mRNA in the rat suprachiasmatic nuclei.

The suprachiasmatic nuclei (SCN) in the hypothalamus function as the primary circadian pacemaker. A receptor for vasoactive intestinal peptide (VIP), denoted as VIP2, is abundantly expressed in the SCN. Since the rodent circadian clock demonstrates phase-dependent sensitivity to exogenous VIP, we investigated the possibility that VIP2 receptor mRNA is differentially expressed in the SCN across the 24 h cycle. To establish whether VIP2 receptor mRNA levels change across the 12:12 h light-dark (LD) cycle (lights on designated as Zeitgeber time (ZT)O), rats were killed at ZT 0, 2, 6, 10, 12, 14, 18 and 22. To determine if variation in this mRNA occurs in the absence of LD entrainment cues, lights were not turned on at the time of transition from dark to light (designated as CT O); the animals in this group were killed in constant darkness (DD) at CT 0, 2, 6, 10, 12, 14, 18 and 22. In situ hybridization histochemistry indicated no variations in VIP2 receptor mRNA in the cingulate cortex under either LD or DD conditions. There was, however, significant variation in the expression of VIP2 receptor mRNA within the SCN during the LD cycle, with one peak at ZT 6 and at ZT 22. A comparable biphasic pattern of mRNA expression was observed in DD animals with peaks at CT 10 and another at CT 22. The results suggest that the phase-dependent actions of VIP on the clock may involve phase-specific changes in the availability of VIP2 receptor within the SCN.

Diurnal and circadian changes of serotonin in the suprachiasmatic nuclei : regulation by light and an endogenous pacemaker

Daily variations of serotonin (5-HT) in the suprachiasmatic nuclei (SCN) were measured in rats kept under various lighting conditions to elucidate the serotonergic contribution to the mechanism underlying SCN function on circadian rhythmicity. Animals kept in 12-h light-12-h dark (LD) cycles showed a peak 5-HT level during the light period and a trough during the dark period. In constant darkness (DD), rhythmic 5-HT variation was out of phase to changes observed in LD. Rats that have been kept in DD and then exposed to constant light (LL) showed transitory increases in 5-HT just after lights on. Taken together, these results show that 5-HT variation in the SCN is generated by an endogenous pacemaker and is also influenced by photic cues.

Serotonin in the raphe nuclei: regulation by light and an endogenous pacemaker

TEMPORAL changes of serotonin (5-HT) content in the median (MRN) and dorsal (DRN) raphe nuclei were measured in rats kept under various lighting conditions. Serotonin content in the MRN and DRN under light-dark (LD) condition showed diurnal rhythmicity, with a peak during early light wphase and a trough during the dark phase. In constant dark (DD) condition, a single peak was observed and was out of phase to the 5-HT peak found under LD condition. Animals exposed to constant light (LL) after 2 days in DD showed marked increase in 5-HT after lights on. These results suggest that changes in 5-HT in the MRN and DRN are regulated by an endogenous pacemaker and by light.

Circadian changes in PACAP type 1 (PAC1) receptor mRNA in the rat suprachiasmatic and supraoptic nuclei.

A receptor for pituitary adenylate cyclase activating polypeptide (PACAP), denoted as PAC1, is expressed in the suprachiasmatic nuclei (SCN). Since the circadian clock demonstrates phase-dependent sensitivity to PACAP, we have used in situ hybridization histochemistry to examine whether PAC1 mRNA is differentially expressed in the rat SCN across the 24-h cycle. There was a significant variation in PAC1 mRNA within the SCN and supraoptic nuclei during the light-dark cycle and in constant darkness, with peaks at the middle of both the real and subjective day and night; no significant variation was observed in the cingulate cortex. The results suggest that the phase-dependent actions of PACAP on the clock may involve phase-specific changes in the availability of PAC1 receptors within the SCN.

Hypoglycaemia-Induced Inhibition of Pulsatile Luteinizing Hormone Secretion in Female Rats: Role of Oestradiol, Endogenous Opioids and the Adrenal Medulla

Oestradiol (E2) has been shown to exacerbate the inhibitory effect of hypoglycaemic stress on gonadotrophin‐releasing hormone pulse generator (GnRH) activity in primates. The mechanism by which this is mediated is not yet known. We therefore aimed to establish whether there is a sensitizing influence of E2 on the suppression of LH pulsatility in response to hypoglycaemia in the female rat, thus providing a more amenable model in which to study this phenomenon. In ovariectomized Wistar rats with E2 replacement, insulin‐induced hypoglycaemia (0.5 U/kg iv) resulted in an interruption of pulsatile LH secretion. Induction of the same degree of hypoglycaemia in ovariectomized rats without E2 replacement was without effect on LH pulsatility. Naloxone administration prevented the hypoglycaemia‐induced inhibition of LH pulses. Because hypoglycaemia is a potent activator of the sympathetic nervous system, we also tested the hypothesis that the adrenal medulla is involved in this suppression of LH pulses in the rat. Adrenomedullectomy completely prevented this inhibitory response to hypoglycaemic stress. These data are consistent with the hypothesis that E2 sensitizes the GnRH pulse generator to the inhibitory influences of hypoglycaemic stress in the rat. Furthermore, a clear role for both endogenous opioid peptides and the adrenal medulla in the stress‐induced suppression of LH pulsatility is identified.

Vasoactive intestinal polypeptide precursor mRNA exhibits diurnal variation in the rat suprachiasmatic nuclei.

Vasoactive intestinal polypeptide (VIP), which is synthesized in the ventrolateral subdivision of the suprachiasmatic nucleus (SCN), the circadian pacemaker in the mammalian brain, is suggested to mediate information on environmental light. Diurnal rhythms of VIP precursor mRNA in the SCN was determined in the present study by Northern blot hybridization with an antisense RNA probe in rats kept under light-dark conditions. VIP precursor mRNA was found most abundant during the dark phase, as light exposure suppressed VIP precursor mRNA. This suppression, however, did not last throughout the light phase and VIP precursor mRNA returned to the level before the onset of light after 8 h in light, suggesting that VIP mRNA responds to photic cues and not to light per se. This is in contrast to the sustained suppression of VIP peptide level in the SCN. It indicates that VIP mRNA is acting at a stage of light information processing upstream to VIP peptide in the circadian pacemaker.

Estrogen receptor immunoreactivity is present in the majority of central histaminergic neurons: Evidence for a new neuroendocrine pathway associated with luteinizing hormone-releasing hormone-synthesizing neurons in rats and humans

The central regulation of the preovulatory LH surge requires a complex sequence of interactions between neuronal systems that impinge on LH-releasing hormone (LHRH)-synthesizing neurons. The reported absence of estrogen receptors (ERs) in LHRH neurons indicates that estrogen-receptive neurons that are afferent to LHRH neurons are involved in mediating the effects of this steroid. We now present evidence indicating that central histaminergic neurons, exclusively located in the tuberomammillary complex of the caudal diencephalon, serve as an important relay in this system. Evaluation of this system revealed that 76% of histamine-synthesising neurons display ERα-immunoreactivity in their nucleus; furthermore histaminergic axons exhibit axo-dendritic and axo-somatic appositions onto LHRH neurons in both the rodent and the human brain. Our in vivo studies show that the intracerebroventricular administration of the histamine-1 (H1) receptor antagonist, mepyramine, but not the H2 receptor antagonist, ranitidine,...

Circadian changes of glutamate decarboxylase 65 and 67 mRNA in the rat suprachiasmatic nuclei

Gaba has been implicated in the regulation of the circadian clock within the suprachiasmatic nuclei (SCN). In the present study in situ hybridization histochemistry was used to assess expression of mRNA for the two isoforms of the enzyme governing GABA synthesis, glutamic acid decarboxylase (GAD) 65 and 67, within the rat SCN during a 24 h cycle in constant darkness. GAD 65 mRNA exhibited a monophasic rhythm, with a peak at the beginning of the subjective day and a nadir early in the subjective night. In contrast, there was a biphasic variation in GAD 67 expression, with peaks at the beginning of the subjective day and early in the subjective night. No significant variation in the mRNAs for GAD 65 or GAD 67 was observed in the cingulate cortex. These results suggest that the clock intrinsic to the SCN controls the expression of the mRNAs encoding GAD 65 and GAD 67 in both a coordinated and a differential manner.

α2-adrenergic receptors are involved in the suppression of Luteinizing hormone release during acute fasting in the ovariectomized estradiol-primed rats

It has been previously reported that the adrenergic system is involved in the control of feeding behavior and LH release. In the present study, the role of the adrenergic receptors in the suppression of LH release during acute fasting are examined by injecting the alpha 1-antagonist (prazosin), alpha 2-antagonists (idazoxan, SKF 86466-A, piperoxan), or beta-antagonist (propranolol) into the third ventricle of unfasted and 48 h fasted ovariectomized estradiol-treated rats. Blood samples were collected every 6 min for 3 h and the drugs were administered after the first hour of the sampling period. Prazosin caused a significant suppression of LH release in the unfasted animals while idazoxan and propranolol had no significant effects. In contrast, all alpha 2-antagonists blocked the inhibitory effect of fasting on LH release and significantly reinstated the suppressed LH release while prazosin and propranolol had no significant effects. We conclude from these results that the suppression of LH release during acute fasting is mediated by alpha 2-adrenergic receptors but not alpha 1- or beta-adrenergic receptors.