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Circadian functions of the suprachiasmatic nuclei (SCN) are influenced by cyclic AMP (cAMP). Adenylyl cyclase type II (AC-II) is a cAMP-generating enzyme which, in the context of activation by Gsalpha, is further stimulated by protein kinase C or G protein betagamma subunits. Using in situ hybridization we have found a biphasic variation in AC-II mRNA within the rat SCN during the light-dark cycle (peaks at Zeitgeber time 6 and 18) and also in constant darkness (peaks at circadian time 2 and 14). The cingulate cortex showed no such variation. These findings suggest that circadian changes in AC-II expression may be pertinent to the rhythmic functions of the SCN., (Copyright 1998 Elsevier Science B.V.)

We investigated the effects of neuropeptide Y and related analogues on the extracellularly recorded spontaneous firing rate activity of adult Syrian hamster suprachiasmatic nucleus neurones in vitro. Sixty-seven neurones were tested with neuropeptide Y: 45% were suppressed, 4% were activated, and the remaining 51% were unresponsive. These responses were not blocked by the GABA receptor antagonist bicuculline, indicating that neuropeptide Y-evoked responses did not appear to be dependent on GABA(A) receptor activation. We tested the effects of the neuropeptide Y Y1 receptor agonist [Leu31, Pro34]neuropeptide Y and the neuropeptide Y Y2 receptor agonist neuropeptide Y-(13-36) on nine cells suppressed by neuropeptide Y in order to determine the receptor subtype(s) mediating the effects of neuropeptide Y. Four of nine cells were suppressed by [Leu31, Pro34]neuropeptide Y only, one of nine was suppressed by neuropeptide Y-(13-36) only, two of nine were suppressed by both compounds, while the remaining two cells did not respond to either compound. These data suggest that neuropeptide Y can modulate suprachiasmatic nucleus function directly, without recruitment of GABA(A) interneurones. Further, our results indicate that neuropeptide Y may act on more than one receptor subtype within the adult hamster suprachiasmatic nucleus.

Previous studies have suggested a role for histamine in mediating effects of light on rodent circadian systems. We combined behavioural and neuroanatomical studies to analyze the potential role of histamine in circadian rhythm regulation of Syrian hamsters. The effects of histamine and the selective H2 receptor agonist dimaprit on free-running locomotor activity rhythms were monitored. In addition, histamine projections were mapped immunohistochemically. Histamine (0.5 microl of 1 mM) microinjected into the region of the suprachiasmatic nucleus induced very small delay phase-shifts, which were significantly greater than those elicited by vehicle at circadian time (CT) 18. No other significant differences were observed. Dimaprit (0.5 microl of 1 mM) microinjected into the region of the suprachiasmatic nucleus at CT 6 and CT 18, induced very small phase-shifts which were significantly different from the effects of vehicle, but did not resemble those of light. Numerous cell bodies immunopositive for histamine were found in the tuberomammillary nucleus of the posterior hypothalamus, while histamine immunoreactive fibres were seen in the periventricular nucleus and paraventricular nucleus of the anterior hypothalamus. The suprachiasmatic nuclei were largely devoid of immunostaining. Finally, preliminary electrophysiological studies demonstrated that ionophoretic application of histamine and the H2 receptor agonist dimaprit elicit inconsistent and weak responses in the hamster suprachiasmatic nucleus in vitro. The results presented here show that microinjections of HA or dimaprit into the SCN region do not mimic the effects of light on hamster circadian rhythms, and the SCN of the Syrian hamster is almost devoid of histaminergic fibres. These studies suggest that histamine does not play a prominent role in circadian rhythm regulation in this species.

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.

Glutamate is thought to mediate the effects of light on the circadian pacemaker contained in the suprachiasmatic nucleus. Glutamate can reset this pacemaker both in vivo and in vitro while glutamate antagonists can reduce photically induced phase shifts in activity rhythms and c-fos expression in the suprachiasmatic nucleus. Most behavioural and gene expression experiments investigating circadian rhythms use hamsters, but the majority of the anatomical data on the presence and distribution of selected glutamate receptor subunits in the suprachiasmatic nucleus has been collected from rat. In the present study, we examined the distribution of ionotropic glutamate receptor subunits in the hamster suprachiasmatic nucleus using mono- and polyclonal antibodies directed against these subunits. In addition, we examined the distribution of immunostaining for these subunits in a second structure of the mammalian circadian system, the intergeniculate leaflet of the thalamus since it also is thought to receive glutamatergic input from the retina and is important in the entrainment of circadian rhythms. The results indicated that all of the subunits investigated (GluR1, GluR2/3, GluR4, GluR5/6/7, and NMDAR1) were present in the suprachiasmatic nucleus and that all but GluR4 were present in the intergeniculate leaflet. Each of the subunits investigated had a unique pattern of distribution and intensity of staining. The distribution of immunoreactivity for these subunits in the hamster suprachiasmatic nucleus and intergeniculate leaflet differed from that reported in the rat. The presence of these subunits in the suprachiasmatic nucleus and intergeniculate leaflet implies the presence of functional NMDA and non-NMDA receptors in these structures that may have a role in photic entrainment of the circadian pacemaker.

The suprachiasmatic nucleus (SCN) receives a direct retinal projection, which in rats includes substance P (SP)-immunoreactive retinal ganglion cells. While SP has been shown to have neurophysiological effects on SCN cells in Syrian hamsters and rats, it is not known what effects SP in the SCN has on circadian rhythms in hamsters. We examined this question using male Syrian hamsters that were implanted with cannulas aimed at the SCN region and maintained in constant dim red lighting conditions. Hamsters received 0.5 microl microinjections of saline or SP (500 pmol in saline) at a variety of circadian times (CT). Saline injections had little or no phase-shifting effects at any phase tested. SP had no significant effects at CT4-8, 16-20, or 20-24, but did cause small phase delays of -23.7 +/- 7 min (mean +/- sem) at CT12-16. In order to examine the dose-response relations of this effect, hamsters were also microinjected with 50 and 2500 pmol of SP at CT12-16. Both the 50 and 2500 pmol doses induced very small phase delays (-14.2 +/- 7 min and -18.2 +/- 5 min, respectively), indicating no obvious dose dependence within this range. These results do not suggest that SP alone in the SCN mimics light effects on circadian rhythms or is a key neurotransmitter involved in photic entrainment. It remains to be determined whether SP interacts with other transmitters in the SCN to modulate their effects on rhythm phase.

Pituitary adenylate cyclase activating polypeptide (PACAP) is found in two forms of 27 and 38 amino acids (PACAP-27 and PACAP-38 respectively) in the mammalian central nervous system. Using antibodies to these two forms of PACAP, we examined the distribution of PACAP immunoreactivity in the rat hypothalamus and a number of extrahypothalamic areas. The patterns of immunostaining for PACAP-27 and PACAP-38 were similar: prominent terminal labelling was present in the retrochiasmatic area, median eminence, and posterior periventricular nucleus of the hypothalamus as well as the bed nucleus of the stria terminalis and amygdaloid complex. After colchicine treatment, immunopositive cell bodies were found in the preoptic region of the periventricular zone of the hypothalamus, the suprachiasmatic and paraventricular hypothalamic nuclei, neural structures adjacent to the median eminence (including the retrochiasmatic area, arcuate nucleus, ventromedial hypothalamus, and tuber cinereum), and the lateral mammillary and supramammillary nuclei. In all these areas, immunolabelling appeared specific since it was abolished by preabsorption of primary antisera with the appropriate PACAP peptide. However, the number of immunopositive cells in the suprachiasmatic nucleus was also reduced by preabsorption of PACAP-27/38 antisera with vasoactive intestinal polypeptide, suggesting that a subpopulation of cells in the suprachiasmatic nucleus express a peptide which has significant sequence homology with both PACAP-27/38 and vasoactive intestinal polypeptide. The distribution of PACAP immunoreactivity throughout the hypothalamus, bed nucleus of the stria terminalis, and amygdala suggests the involvement of PACAP in a number of processes including limbic, autonomic, and neuroendocrine functions as well as regulation of the circadian pacemaker.

ionophoretic ejection of substance P (SP) activated 31% and suppressed 9% of hamster suprachiasmatic nucleus (SCN) cells in vitro. Hamster SCN cells did not demonstrate variation in sensitivity to SP across the circadian phases tested. SP modulated the response of 47% of hamster SCN cells to the excitatory amino acid (EAA) agonists glutamate and N-methyl-D-aspartate (NMDA). The results indicate that SP can alter both the spontaneous and EAA-evoked firing rate characteristics of hamster SCN neurons.

Ionophoresis of the smaller bombesin-like peptides (gastrin releasing peptide [GRP]-(18-27), neuromedin B, and bombesin) evoked responses from 30-60% of hamster suprachiasmatic nucleus cells recorded in a hypothalamic slice preparation, depending on the circadian phase. We also demonstrated for the first time that the putative bombesin-like peptide receptor antagonists [D-F5,D-Phe6,D-Ala11]bombesin-(6-13)methyl ester (BIM 26226) and [D-Phe6,Des-Met14]bombesin-(6-14)ethyl amide can be applied ionophoretically to block physiological responses to bombesin-like peptides. Together with earlier findings, these results show that bombesin-like peptides administered by several methods can potently alter the firing rates of hamster suprachiasmatic nucleus neurons in vitro. These results indicate that bombesin-like peptides affect suprachiasmatic nucleus cells and could play a role in modulating suprachiasmatic nucleus-mediated circadian rhythm entrainment.

The suprachiasmatic nuclei (SCN) of the rodent hypothalamus function as a light entrainable circadian pacemaker. The SCN contain moderate to high concentrations of a number of neuropeptides including peptides showing structural homology with the amphibian derived tetradecapeptide, bombesin (BN), called bombesin-like peptides (BNLPs). BNLPs include the 27 amino acid peptide, gastrin releasing peptide (GRP1-27), a smaller decapeptide (GRP18-27) and another decapeptide with less structural homology, neuromedin B (NmB). Immunoreactivity for BN and receptors for BNLPs have been demonstrated in the region of the rat SCN receiving photic input. We studied the effects of local pressure ejections of BNLPs dissolved in saline/1% bovine serum albumin (BSA) vehicle on the extracellularly recorded firing rates of Syrian hamster SCM neurons in a hypothalamic slice preparation. In one study, an ejecting electrode containing BN (10(-8) to 10(-4) M) was positioned 20 to 60 microm from a recording electrode. Of 74 cells tested with BN, 50 (67.6%) showed significant increases in firing rate, while 3 of 29 cells (15.8%) tested with vehicle ejections were activated. In a second study, a single electrode was used for both recordings and pressure ejections. Of 48 cells tested, BN (10(-6) to 10(-4) M) activated 30 (62.5%) and suppressed firing in 4 (8.3%). Of 208 cells tested with GRP1-27 (10(-9) to 10(-4) M), 105 (50.5%) were activated and 2 (1.0%) were suppressed. The percentage of cells responding increased with the concentration of GRP1027 used in the electrode. No circadian variation in responsiveness to GRP1-27 was detected. GRP18-27 (5 x 10(-5) to 10(-4) M) activated 10 out of 18 cells tested (55.6%), while NmB (10(-4) M) activated 2 out of 30 cells tested (6.7%) and vehicle ejections activated 1 out of 36 cells tested (2.8%). GRP1-27, GRP18-27 and BN, the BNLPs showing the greatest degree of structural homology, activate approximately 50% of SCN cells, apparently via the BN/GRP-preferring receptor subtype, and may play a role in photic entrainment.

Long-term consequences of neonatal blockade of bombesin (BN) receptors were examined in the present study. Rat pups were injected twice daily with [D-Phe6, phi Leu13-Cpa14]BN(6-14), a BN receptor antagonist, at either high (10 mg/kg; HD group) or low (5 mg/kg; LD group) doses from postnatal day 1 through 8. Their behavioral responses to a variety of conditions were compared to those of rats neonatally injected with saline (SAL group) or animals handled but not injected during infancy (UNT group). Adult HD rats entered and spent more time on the open arms of the elevated plus maze than LD, SAL, or UNT animals. Under the conditions of a water deprivation schedule, neither central nor peripheral injections of BN differentiated the neonatally pretreated groups as determined by measures of grooming, feeding, and drinking behaviors. These results indicate that at the dosage regimen employed, neonatal injections of [D-Phe6, phi Leu13-Cpa14]BN(6-14) had little effect on adult sensitivity to BN, but that such treatments could alter activity on the elevated plus maze through as yet unknown mechanisms.

Rhythms of spontaneous firing rate and of responsiveness to pressure ejection of melatonin were recorded from neurons in the Syrian hamster suprachiasmatic nuclei (SCN) in a slice preparation. In animals taken from light-dark cycles (LD 14:10), SCN cells had high firing rates during the projected day and lower rates during the projected night. The proportion of melatonin-suppressed cells (35% overall) was also high during the day and fell during the night, while melatonin activated approximately 23% of cells at all phases. To assess the source of the melatonin-responsiveness rhythm, hamsters were exposed for approximately 48 h to constant illumination (LL) to suppress melatonin secretion. LL exposure before slice preparation altered both firing-rate and melatonin-responsiveness rhythms. Firing rates failed to show a morning peak and remained at low levels, with no indication of daily rhythmicity. Melatonin responsiveness also failed to show the usual rhythm and even tended to rise at night. Overall melatonin responsiveness rose after LL exposure so that 50% of cells were suppressed and 21% activated. LL exposure also increased the proportions of cells which showed regular baseline firing rates. Control studies indicated that pressure artifacts did not account for either suppression or activation by melatonin, while the composition of the saline vehicle appeared to be responsible for the activations recorded. The results indicate that brief LL exposure alters SCN sensitivity to melatonin and SCN rhythmicity in Syrian hamsters, perhaps as a result of the loss of the daily melatonin secretion rhythm. Physiological melatonin patterns may have important effects on the rodent circadian pacemaker.

We review recent studies in our laboratory that have investigated the neural mechanisms underlying photic entrainment of the mammalian circadian system. The results from studies of extracellular single-unit recordings and of photic induction of Fos-like immunoreactivity (Fos-lir) indicate that excitatory amino acid (EAA) transmission, and particularly activation of the N-methyl-D-aspartate (NMDA) receptor subtype, is important for conveying photic information to suprachiasmatic nucleus (SCN) cells. We have also found that a subregion of the SCN still shows Fos-lir after blockade of EAA receptors, and we have evidence suggesting that these cells are innervated by a distinct subdivision of the retinal projection to the SCN. In addition, we have found that photic responses of cells in the intergeniculate leaflet (which projects to the SCN) and of SCN cells are modulated by serotonin (5-HT) via a receptor that resembles the 5-HT1A subtype.