GABAB receptor stimulation phase-shifts the mammalian circadian clock in vitro

The mammalian circadian clock in the suprachiasmatic nucleus (SCN) generates 24-h rhythms in vitro. Here we show that the GABAB agonist baclofen resets the SCN pacemaker in vitro in a phase-dependent manner: advances were induced at zeitgeber time (ZT) 6, and delays were induced at ZT 22. Both effects were blocked the GABAB antagonist, 2-hydroxysaclofen, while the GABAA antagonist, bicuculline was ineffective. Thus, the SCN pacemaker is sensitive to resetting by GABAB stimulation.     

Roles of suprachiasmatic nuclei and intergeniculate leaflets in mediating the phase-shifting effects of a serotonergic agonist and their photic modulation during subjective day

Serotonin (5-HT) has been implicated in the phase adjustment of the circadian system during the subjective day in response to nonphotic stimuli. Two components of the circadian system, the suprachiasmatic nucleus (SCN) (site of the circadian clock) and the intergeniculate leaflet (IGL), receive serotonergic projections from the median raphe nucleus and the dorsal raphe nucleus, respectively. Experiment 1, performed in golden hamsters housed in constant darkness, compared the effects of bilateral microinjections of the 5-HT1A/7 receptor agonist, 8-hydroxydipropylaminotetralin (8-OH-DPAT; 0.5 microgram in 0.2 microliter saline per side), into the IGL or the SCN during the mid-subjective day. Bilateral 8-OH-DPAT injections into either the SCN or the IGL led to significant phase advances of the circadian rhythm of wheel-running activity (p < .001). The phase advances following 8-OH-DPAT injections in the IGL were dose department (p < .001). Because a light pulse administered during the middle of the subjective day can attenuate the phase-resetting effect of a systemic injection of 8-OH-DPAT, Experiment 2 was designed to determine whether light could modulate 5-HT agonist activity at the level of the SCN and/or the IGL. Serotonergic receptor activation within the SCN, followed by a pulse of light (300 lux of white light lasting 30 min), still induced phase advances. In contrast, the effect of serotonergic stimulation within the IGL was blocked by a light pulse. These results indicate that the respective 5-HT projections to the SCN and IGL subserve different functions in the circadian responses to photic and nonphotic stimuli.     

Metabolic responses of postmenopausal women to supplemental dietary boron and aluminum during usual and low magnesium intake: boron, calcium, and magnesium absorption and retention and blood mineral concentrations

The regulation by neuropeptide Y of alpha2-adrenoceptors in the nucleus tractus solitarii was evaluated in the adult normotensive Wistar Kyoto rat and the adult spontaneously hypertensive rat. The microinjection of a submaximal dose of l-noradrenaline (800 pmol in 50 nl) alone into the nucleus tractus solitarii produced a significant reduction in the mean arterial blood pressure in either strain. The threshold dose (1 pmol in 50 nl) of neuropeptide Y(1-36) for the vasodepressor response in the Wistar Kyoto rat was five times higher than that (0.2 pmol in 50 nl) in the spontaneously hypertensive rat. Furthermore, neuropeptide Y(1-36) at 0.2 pmol in 50 nl could significantly counteract the vasodepressor response to l-noradrenaline (800 pmol in 50 nl) in the spontaneously hypertensive rat, but not in the Wistar Kyoto rat, in which 1 pmol in 50 nl of neuropeptide Y(1-36) must be employed to counteract the vasodepressor response to l-noradrenaline (800 pmol in 50 nl), although the vasodepressor responses are of a similar magnitude. The in situ hybridization and quantitative receptor autoradiographical experiments showed that the alpha2A-adrenoceptor messenger RNA levels and the B(max) value of the alpha2-adrenoceptor agonist [3H]p-aminoclonidine binding sites measured in the nucleus tractus solitarii of the spontaneously hypertensive rat were substantially lower than those in the Wistar Kyoto rat. The quantitative receptor autoradiographical results were consistent with the cardiovascular results and showed that in the spontaneously hypertensive rat, neuropeptide Y(1-36) at 1 nM led to a significant increase in the K(d) value of [3H]p-aminoclonidine binding sites. In the Wistar Kyoto rat, neuropeptide Y(1-36) produced this effect only at 10 nM. The present study provides evidence for an increase of the potency of neuropeptide Y(1-36) to antagonistically modulate alpha2-adrenoceptors in the nucleus tractus solitarii of the spontaneously hypertensive rat. This enhanced antagonistic action may partly be related to a reduction in the number of alpha2A-adrenoceptors in the nucleus tractus solitarii of the spontaneously hypertensive rat, since a decrease has been observed in the alpha2A-adrenoceptor messenger RNA levels and the alpha2-adrenoceptor binding sites in the spontaneously hypertensive rat. This increased potency of neuropeptide Y(1-36) to antagonize alpha2-adrenoceptor function in the nucleus tractus solitarii of the spontaneously hypertensive rat may contribute to the development of high blood pressure in this hypertensive strain.     

GABAergic modulation of optic nerve-evoked field potentials in the rat suprachiasmatic nucleus

The suprachiasmatic nuclei (SCN) at the base of the hypothalamus are known to be the site of the endogenous circadian pacemaker in mammals. The SCN are innervated by the retinohypothalamic tract, which conveys photic information to the SCN. GABA is one of the most abundant neurotransmitters in the SCN, and has been implicated in the modulation of photic responses of the SCN circadian pacemaker. This study sought to examine the effect of GABAergic compounds on optic nerve-evoked SCN field potentials recorded in rat horizontal hypothalamic slices. The GABAA agonist muscimol (10 microM) potentiated SCN field potentials by 23%, while application of the GABAA antagonist bicuculline (10 microM) inhibited SCN field potentials by a similar amount, (22%). Conversely, the GABA, agonist baclofen (1.0 microM) inhibited SCN field potentials by 48%, while the GABAB antagonist phaclofen (0.5 mM) augmented SCN field potentials by 62%. Recordings performed at both day and night times indicate that there were no qualitative day-night differences in GABAergic activity on SCN field potentials. This study concludes that, in general, GABAA activity tends to increase, and GABAB activity tends to decrease the response of SCN neurons to optic nerve stimulation.     

Family coping during critical illness

The suprachiasmatic nuclei (SCN) of the anterior hypothalamus contain the master circadian pacemaker in mammals. On the occasion of the 25th anniversary of the discovery of the SCN as the circadian clock, Charles A. Czeisler and Steven M. Reppert organized a meeting to review milestones and recent developments in the study of the SCN. The discovery that the SCN contain tissue necessary for generation of circadian rhythmicity was established by lesion studies published in 1972. The second phase of study demonstrated unequivocally that the SCN contain an autonomous circadian pacemaker. The principal studies in this period showed the presence of metabolic and electrical activity rhythms in the SCN in vivo and progressed to studies showing that the SCN maintain rhythmicity in vitro, demonstrating that the transplanted SCN can restore circadian function following destruction of the host SCN and ultimately showing that single SCN "clock cells" exhibit independent rhythms in firing rate. The third phase of study, aimed at identifying the biochemical and molecular mechanisms responsible for rhythmicity within the SCN, has begun with the identification of circadian mutants (tau mutant hamsters and Clock mutant mice) and the isolation of the Clock gene. This report traces the important steps forward in our understanding of the suprachiasmatic circadian clock by recounting the information presented at the SCN Silver Anniversary Celebration.     

Immortal time: circadian clock properties of rat suprachiasmatic cell lines

Cell lines derived from the rat suprachiasmatic nucleus (SCN) were screened for circadian clock properties distinctive of the SCN in situ. Immortalized SCN cells generated robust rhythms in uptake of the metabolic marker 2-deoxyglucose and in their content of neurotrophins. The phase relationship between these rhythms in vitro was identical to that exhibited by the SCN in vivo. Transplantation of SCN cell lines, but not mesencephalic or fibroblast lines, restored the circadian activity rhythm in arrhythmic, SCN-lesioned rats. Thus, distinctive oscillator, pacemaker, and clock properties of the SCN are not only retained but also maintained in an appropriate circadian phase relationship by immortalized SCN progenitors.     

Resynchronisation of a diurnal rodent circadian clock accelerated by a melatonin agonist

Using 'jet lag' paradigms involving phase shifts in the light-dark (LD) cycle, we studied the effects of S-20098 on the circadian clock of a diurnal rodent. Arvicanthis mordax, entrained to a regular LD cycle, were subjected to advance shifts (i.e. 4, 6 or 8 h) in the LD cycle and injected with vehicle or the melatonin agonist S-20098 (20 mg/kg) the day of the shift (and also on subsequent days in the 6 h or 8 h shift paradigms). In each condition, S-20098 accelerated by about 30% resynchronization to the new LD cycle. These data, which are the first to demonstrate the chronobiotic effects of a melatonin agonist in a diurnal rodent, provide new insights for the design of human chronopharmacological protocols.     

Circadian Phase Alteration by GABA and Light Differs in Diurnal and Nocturnal Rodents During the Day.

These studies investigated the circadian effects of light and gamma aminobutyric acid-A (GABAA) receptor activation in the suprachiasmatic nucleus (SCN) of the diurnal unstriped Nile grass rat (Arvicanthis niloticus). Microinjection of the GABAA agonist muscimol into the SCN during the day produced phase shifts that were opposite in direction to those previously reported in nocturnal rodents. In addition, light had no significant effect on the magnitude of muscimol-induced phase delays during the daytime. Injection of muscimol during the night, however, significantly inhibited light-induced phase delays and advances in a manner similar to that previously reported in nocturnal rodents. Therefore, the circadian effects of GABAA receptor activation are similar in diurnal and nocturnal species during the night but differ significantly during the day. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Destruction of serotonergic neurons in the median raphe nucleus blocks circadian rhythm phase shifts to triazolam but not to novel wheel access

Systematic treatment of hamsters with triazolam (TRZ) or novel wheel (NW) access will yield PRCs similar to those for neuropeptide Y. Both TRZ and NW access require an intact intergeniculate leaflet (IGL) to modulate circadian rhythm phase. It is commonly suggested that both stimulus types influence rhythm phase response via a mechanism associated with drug-induced or wheel access-associated locomotion. Furthermore, there have been suggestions that one or both of these stimulus conditions require an intact serotonergic system for modulation of rhythm phase. The present study investigated these issues by making serotonin neuron-specific neurotoxic lesions of the median or dorsal raphe nuclei and evaluating phase response of the hamster circadian locomotor rhythm to TRZ treatment or NW access. The expected effect of TRZ injected at CT 6 h on the average phase advance was virtually eliminated by destruction of serotonin neurons in the median, but not the dorsal, raphe nucleus. No control or lesioned animal engaged in substantial wheel running in response to TRZ. By contrast, all median raphe-lesioned hamsters that engaged in substantial amounts of running when given access to a NW had phase shifts comparable to control or dorsal raphe-lesioned animals. The results demonstrate that serotonergic neurons in the median raphe nucleus contribute to the regulation of rhythm phase response to TRZ and that it is unlikely that these neurons are necessary for phase response to NW access. The data further suggest the presence of separate pathways mediating phase response to the two stimulus conditions. These pathways converge on the IGL, a nucleus afferent to the circadian clock, that is necessary for the expression of phase response to each stimulus type.     

Atherosclerosis in the human brachial artery

The hypothalamic suprachiasmatic nucleus (SCN) of the mammal is the circadian pacemaker responsible for generation of circadian rhythms. Several immediate-early genes are expressed in the SCN by light stimuli which induce phase shifts of animal activity rhythms. In the present study, we investigated whether Homer, a PDZ-like protein which is rapidly induced following synaptic activation, mRNA expression is regulated by light in rat SCN. Homer mRNA expression in the SCN of rat killed at 4 h after onset of the light and dark phases was very low. One hour light stimuli during the subjective night dramatically induced Homer mRNA expression in the ventrolateral portion of the SCN, whereas light stimuli during the subjective light phase did not. This finding implies that Homer may be involved in the photic entrainment of the circadian clock.     

Managed care and medication compliance: implications for chronic depression

Syrian hamsters, Mesocricetus auratus, were confined to novel running wheels for a 3-h period, starting at approximately circadian time (CT) 4.5 (i.e., approaching the middle of their subjective day). It can be reliably predicted from the amount of running in this situation whether or not there will be a subsequent phase-shift. Expression of the immediate early genes c-fos and fosB was examined by immunocytochemistry in the suprachiasmatic nucleus (SCN), the intergeniculate leaflet (IGL) of the thalamus, and the medial pretectal area of hamsters that ran vigorously in the novel wheel and would have phase-shifted. c-Fos was increased, compared to levels in a control group left in their home cages, in the IGL, and the pretectum (PT), but decreased in the SCN. No significant changes in FosB were detected in any region examined. An additional experiment argued against the possibility that the changes in c-Fos could be attributed to a rapid advance of the pacemaker to a different phase in the circadian cycle. Counts of c-Fos-positive cells in the IGL were similar in animals given pulses of running starting at CT 4.5 and starting at CT 12.5-16 (i.e., in the subjective night when they would have been active anyway). Altogether the results support the view that activation of the IGL is important in nonphotic clock resetting, and raise the possibility that the PT may also be involved in nonphotic resetting. However, the results also indicate that novelty-induced running does not alter c-Fos induction in a phase-specific manner in the IGL. The inhibition of c-Fos in the SCN by nonphotic phase-shifting events contrasts with the well-known inducing effects of light pulses. These different effects might underlie some of the interactions between nonphotic and photic zeitgebers when both act together on the circadian system.     

Dynamics of spontaneous beating of cardiomyocytes in vitro depend on the number of involved cells

The hypothalamic suprachiasmatic nucleus (SCN) is the predominant pacemaker of the mammalian brain that generates and controls circadian rhythms of various endocrine and behavioral processes. Different lines of evidence suggest that stress interferes with the maintenance of such rhythms. As a first approach to investigate whether the neuropeptide arginine vasopressin (AVP), which shows circadian rhythms of synthesis and release within the SCN, might contribute to this stress-induced alterations in circadian rhythms, we monitored acute effects of swim stress on the intra-SCN release of AVP in male rats by means of the microdialysis technique. A 10-min forced swimming session triggered a marked but relatively short-lasting increase in the intranuclear release of AVP (to approx. 440%). This effect was restricted to the area containing predominantly somata and dendrites of vasopressinergic neurons, since no changes in AVP release could be measured in one of their major projection areas, the nucleus of the dorsomedial hypothalamus. Our data provide evidence that the amount of AVP released within the SCN can vary widely not only in accordance with AVP's intrinsically regulated circadian rhythm but also in response to a physiologically relevant stressor. In this way, the neuropeptide may contribute to the regulation of endocrine and behavioral rhythms particularly in challenging situations associated with resettings of the endogenous clock.     

Serotonergic innervation of the hypothalamic suprachiasmatic nucleus and photic regulation of circadian rhythms

Converging lines of evidence have firmly established that the hypothalamic suprachiasmatic nucleus (SCN) is a light-entrainable circadian oscillator in mammals, critically important for the expression of behavioral and physiological circadian rhythms. Photic information essential for the daily phase resetting of the SCN circadian clock is conveyed directly to the SCN from retinal ganglion cells via the retinohypothalamic tract. The SCN also receives a dense serotonergic innervation arising from the mesencephalic raphe. The terminal fields of retinal and serotonergic afferents within the SCN are co-extensive, and serotonergic agonists can modify the response of the SCN circadian oscillator to light. However, the functional organization and subcellular localization of 5HT receptor subtypes in the SCN are just beginning to be clarified. This information is necessary to understand the role 5HT afferents play in modulating photic input to the SCN. In this paper, we review evidence suggesting that the serotonergic modulation of retinohypothalamic neurotransmission may be achieved via at least two different cellular mechanisms: 1) a postsynaptic mechanism mediated via 5HT1A or 5ht7 receptors located on SCN neurons; and 2) a presynaptic mechanism mediated via 5HT1B receptors located on retinal axon terminals in the SCN. Activation of either of these 5HT receptor mechanisms in the SCN by specific 5HT agonists inhibits the effects of light on circadian function. We hypothesize that 5HT modulation of photic input to the SCN may serve to set the gain of the SCN circadian system to light.     

Distinct changes in peptide YY binding to, and mRNA levels of, Y1 and Y2 receptors in the rat hippocampus associated with kindling epileptogenesis

Electrical kindling of the rat dorsal hippocampus induced significant changes in the binding of 125I-peptide YY to Y1 and Y2 subtypes of neuropeptide Y receptors and in their mRNA levels in the area dentata as assessed by quantitative receptor autoradiography and in situ hybridization histochemistry. Binding to Y1 receptor sites decreased by 50% (p < 0.05) in the molecular layer of the stimulated dentate gyrus, 2 days after preconvulsive stage 2 and 1 week or 1 month after generalized stage 5 seizures compared with sham-stimulated rats. Binding to Y2 receptor sites increased bilaterally by 36-87% (p < 0.05) in the hilus at stage 2 and 1 week or 1 month after stage 5. No significant changes were observed after one afterdischarge or in the other hippocampal subfields or in the cortex. Y1 receptor mRNA signal decreased bilaterally by 50-64% (p < 0.01) in the granule cell layer, 6 h but not 24 h after stages 2 and 5. The Y2 receptor mRNA signal was enhanced by 283% (p < 0.01) in the stimulated granule cell layer 24 h after stage 2. At 6 and 24 h after stage 5, mRNA levels were increased both ipsilaterally (283 and 360%, respectively; p < 0.01) and contralaterally (190 and 260%, respectively; p < 0.05). No significant changes in level of either mRNA was found following one afterdischarge. These modifications, and the enhanced neuropeptide Y release previously shown in the hippocampus, suggest that kindling is associated with lasting changes in neuropeptide Y-mediated neurotransmission.     

Cholinergic regulation of the suprachiasmatic nucleus circadian rhythm via a muscarinic mechanism at night

In mammals, the suprachiasmatic nucleus (SCN) is responsible for the generation of most circadian rhythms and for their entrainment to environmental cues. Carbachol, an agonist of acetylcholine (ACh), has been shown to shift the phase of circadian rhythms in rodents when injected intracerebroventricularly. However, the site and receptor type mediating this action have been unknown. In the present experiments, we used the hypothalamic brain-slice technique to study the regulation of the SCN circadian rhythm of neuronal firing rate by cholinergic agonists and to identify the receptor subtypes involved. We found that the phase of the oscillation in SCN neuronal activity was reset by a 5 min treatment with a carbachol microdrop (1 microliter, 100 microM), but only when applied during the subjective night, with the largest phase shift (+ 6 hr) elicited during the middle of the subjective night. This effect also was produced by ACh and two muscarinic receptor (mAChR) agonists, muscarine and McN-A-343 (M1-selective), but not by nicotine. Furthermore, the effect of carbachol was blocked by the mAChR antagonist atropine (0.1 microM), not by two nicotinic antagonists, dihydro-beta-erythroidine (10 microM) and d-tubocurarine (10 microM). The M1-selective mAChR antagonist pirenzepine completely blocked the carbachol effect at 1 microM, whereas an M3-selective antagonist, 4,2-(4,4'-diacetoxydiphenylmethyl)pyridine, partially blocked the effect at the same concentration. These results demonstrate that carbachol acts directly on the SCN to reset the phase of its firing rhythm during the subjective night via an M1-like mAChR.     

Reduction of arthritis and pneumonitis in motheaten mice by soluble tumor necrosis factor receptor

A variety of observations from several rodent species suggest that a serotonin (5-HT) input to the suprachiasmatic nucleus (SCN) circadian pacemaker may play a role in resetting or entrainment of circadian rhythms by non-photic stimuli such as scheduled wheel running. If 5-HT activity within the SCN is necessary for activity-induced phase shifting, then it should be possible to block or attenuate these phase shifts by reducing 5-HT release or by blocking post-synaptic 5-HT receptors. Animals received one of four serotonergic drugs and were then locked in a novel wheel for 3 h during the mid-rest phase, when novelty-induced activity produces maximal phase advance shifts. Drugs tested at several doses were metergoline (5-HT1/2 antagonist; i.p.), (+)-WAY100135 (5-HT1A postsynaptic antagonist, which may also reduce 5-HT release by an agonist effect at 5-HT1A raphe autoreceptors; i.p.), NAN-190 (5-HT1A postsynaptic antagonist, which also reduces 5-HT release via an agonist effect at 5-HT1A raphe autoreceptors; i.p.) and ritanserin (5-HT2/7 antagonist; i.p. and i.c.v.). Mean and maximal phase shifts to running in novel wheels were not significantly affected by any drug at any dose. These results do not support a hypothesis that 5-HT release or activity at 5HT1, 2 and 7 receptors in the SCN is necessary for the production of activity-induced phase shifts in hamsters.     

Altered neuropeptide Y Y1 responses in mesenteric arteries in rats with congestive heart failure

The aim of the present study was to elucidate if the potentiating effect of neuropeptide Y on various vasoactive agents in vitro is (1) altered in mesenteric arteries from rats with congestive heart failure and (2) mediated by the neuropeptide Y Y1 receptor. The direct vascular effects of neuropeptide Y and its modulating effects on the contractions induced by endothelin-1-, noradrenaline-, 5-hydroxytryptamine (5-HT)-, U46619-(9,11-dideoxy-11alpha, 9alpha-epoxymethano-prostaglandin F2alpha) and ATP, and acetylcholine-induced dilatations were studied in the presence and absence of the neuropeptide Y Y1 antagonist, BIBP3226 (BIBP3226?(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl ]-D-arginine-amide?). Neuropeptide Y, per se, had no vasoactive effect in the arteries. The potency of endothelin-1 was significantly decreased in congestive heart failure rats. Neuropeptide Y and neuropeptide Y-(13-36) potentiated the endothelin-1-induced contraction in congestive heart failure mesenteric arteries. In 20% of the congestive heart failure rats, sarafotoxin 6c induced a contraction of 31+/-4%. Neuropeptide Y also potentiated U46619- and noradrenaline-induced contractions but not 5-HT-induced contractions in congestive heart failure arteries. In sham-operated animals neuropeptide Y potentiated noradrenaline- and 5-HT-induced contractions. These potentiations were inhibited by BIBP3226. Acetylcholine induced an equipotent relaxation in both groups which was unaffected by neuropeptide Y. In conclusion, neuropeptide Y responses are altered in congestive heart failure rats. The potentiating effect differs between vasoactive substances. Neuropeptide Y Y1 and non-neuropeptide Y1 receptors are involved.     

Genetic resistance to parasitic infection

Intraventricular administration of carbachol can induce phase shifts in wheel-running activity in rodents, which depend on circadian phase and are mediated via muscarinic cholinergic receptors in Syrian hamsters. We studied the circadian variation in binding of [3H]-N-methylscopolamine ([3H]NMS), a hydrophilic muscarinic receptor antagonist, in micropunches obtained from the anterior hypothalamus and occipital cortex of Syrian hamsters housed in a 14:10 light:dark cycle. Binding sites were characterized on cells contained within 1 mm punches (obtained from slices 300 microm thick), using a method to selectively detect cell surface (functional) receptors. Atropine sulphate was used to determine nonspecific binding. Cortex showed a significant daily rhythm in [3H]NMS binding with a peak occurring late in the light phase and a trough at lights on, while the hypothalamus showed no detectable rhythm. Following suprachiasmatic nucleus (SCN) ablation or maintenance in constant darkness, the rhythm in the cortex was abolished. These findings suggest that photic information conveyed via the SCN is responsible for the receptor binding rhythm in the cortex. Autoradiographic studies ([3H]NMS; 2 nM, 3 weeks exposure) clearly revealed both M1 and M2 subtypes of muscarinic receptors in the region of the SCN and the visual cortex.