Diurnal variations in vasoactive intestinal polypeptide-like immunoreactivity in the suprachiasmatic nucleus of congenitally anophthalmic mice

The present study has combined recording of circadian locomotor rhythms with light microscopic immunocytochemistry for vasoactive intestinal polypeptide (VIP) in the suprachiasmatic nucleus (SCN) of congenitally anophthalmic mice. These mice, which never develop retinae or optic nerves and do not perceive light, are thus in constant darkness. Our data show a circadian rhythm in expression of VIP in the SCN of anophthalmic mice--expression is maximal during late subjective night/early subjective day and minimal in late subjective day/early subjective night. These observations support the hypothesis that expression of VIP is related to regulation of circadian rhythms by the SCN.     

M3 muscarinic receptor-mediated enhancement of NMDA-evoked adenosine release in rat cortical slices in vitro

Acetylcholine plays an important role in cortical arousal. Adenosine is released during increased metabolism and has been suggested to be a sleep-promoting factor. To understand the interaction of acetylcholine and adenosine in regulating cortical excitability, we examined the effect of carbachol on NMDA-evoked adenosine release and identified the muscarinic receptor subtype that mediated this effect in adult rat cortical slices in vitro. Carbachol (to 300 microM) alone did not affect the basal release of adenosine. However, carbachol (100 microM) induced a 253% increase in NMDA (20 microM)-evoked adenosine release in the presence of Mg2+. In the absence of Mg2+, carbachol's potentiating effect was less (60% increase). The nonselective muscarinic antagonist atropine (1.5 microM) blocked the facilitatory effect of carbachol on NMDA-evoked adenosine release, and this was mimicked by the M3-selective antagonist 4-diphenylacetoxy-N-methylpiperidine (1 microM). Neither an M1-selective dose of pirenzepine (50 nM) nor the M2-selective antagonist methoctramine (1 microM) affected carbachol's action on NMDA-evoked adenosine release. Carbachol had no effect on adenosine release evoked by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA). These results suggest that acetylcholine does not affect basal adenosine release but enhances NMDA receptor-mediated evoked adenosine release by acting at M3 receptors in the cortex. This interaction may have a role in regulating cortical neuronal excitability on a long-term basis.     

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.     

Glutamatergic antagonists do not attenuate light-induced fos protein in rat intergeniculate leaflet

Photic information that entrains circadian rhythms is transmitted to the suprachiasmatic nucleus (SCN) from the retina and from the retinorecipient intergeniculate leaflet (IGL). Expression of light-induced Fos protein in SCN neurons is correlated with the effectiveness of such light to induce phase shifts, and is prevented by pretreatment with glutamate receptor antagonists that prevent phase shifts as well. In the present study we demonstrate that treatments with N-methyl-d-aspartate (NMDA) and non-NMDA receptor antagonists prior to light pulses during the subjective night have no effect on light-induced Fos immunoreactivity (Fos-IR) in IGL neurons despite attenuating Fos-IR in the SCN. Transmission of photic information along retinogeniculate and retinohypothalamic pathways appears to be mediated by different mechanisms.     

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.     

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.     

Neuropeptide Y phase shifts the circadian clock in vitro via a Y2 receptor

The suprachiasmatic nuclei (SCN) contain a circadian clock whose activity can be recorded in vitro for several days. This clock can be reset by the application of neuropeptide Y. In this study, we focused on determination of the receptor responsible for neuropeptide Y phase shifts of the hamster circadian clock in vitro. Coronal hypothalamic slices containing the SCN were prepared from Syrian hamsters housed under a 14 h:10 h light:dark cycle. Tissue was bathed in artificial cerebrospinal fluid (ACSF), and the firing rates of individual cells were sampled throughout a 12 h period. Control slices received either no application or application of 200 nl ACSF to the SCN at zeitgeber time 6 (ZT6; ZT12 was defined as the time of lights off). Application of 200 ng/200 nl of neuropeptide Y at ZT6 resulted in a phase advance of 3.4 h. Application of the Y2 receptor agonist, neuropeptide Y (3-36), induced a similar phase advance in the rhythm, while the Y1 receptor agonist, [Leu31, Pro34]-neuropeptide Y had no effect. Pancreatic polypeptide (rat or avian) also had no measurable phase-shifting effect. Neuropeptide Y applied at ZT20 or 22 had no detectable phase-shifting effect. These results suggest that the phase-shifting effects of neuropeptide Y are mediated through a Y2 receptor, similar to results found in vivo.     

Effect of transplantation of embryonic anterior hypothalamic tissue from spontaneously hypertensive rats to normotensive Wistar rats on circadian rhythms of systolic arterial pressure and heart rate

The anterior hypothalamus (AH) participates in the regulation of arterial pressure. The suprachiasmatic nuclei (SCN) of the AH are a major circadian oscillator necessary for the generation and/or the entrainment of circadian rhythms. Circadian rhythms of systolic arterial pressure (SAP) and heart rate (HR) were investigated in spontaneously hypertensive rats (SHR) and in normotensive Wistar rats (NWI) with intact SCN, grafted with SHR embryonic AH tissue containing the SCN. Prominent circadian rhythms for SAP and HR in both NWI and SHR with acrophases during dark were found. The elevation of the MESOR (midline-estimated statistic of rhythm) of the SAP in normotensive rats grafted with AH embryonic tissue obtained from SHR was accompanied by disappearance of the circadian rhythm of SAP. This result suggests an interaction between the grafted tissue containing the SCN on the one hand, and the host SCN on the other hand. Our data ascribe a role for the SCN in the entrainment of the circadian rhythm of arterial pressure. The circadian rhythm of HR was not eliminated by the SCN graft in spite of the amplitude decrease and the phase delay observed. It seems that the entrainment of the circadian rhythm of HR is probably not crucially dependent on the SCN in rats. The circadian rhythms of SAP and HR in rats were differently affected by the grafts, thus suggesting a multioscillatory system for circadian regulation in rats.     

Rapid resetting of the mammalian circadian clock

The suprachiasmatic nuclei (SCN) contain the principal circadian clock governing overt daily rhythms of physiology and behavior. The endogenous circadian cycle is entrained to the light/dark via direct glutamatergic retinal afferents to the SCN. To understand the molecular basis of entrainment, it is first necessary to define how rapidly the clock is reset by a light pulse. We used a two-pulse paradigm, in combination with cellular and behavioral analyses of SCN function, to explore the speed of resetting of the circadian oscillator in Syrian hamster and mouse. Analysis of c-fos induction and cAMP response element-binding protein phosphorylation in the retinorecipient SCN demonstrated that the SCN are able to resolve and respond to light pulses presented 1 or 2 hr apart. Analysis of the phase shifts of the circadian wheel-running activity rhythm of hamsters presented with single or double pulses demonstrated that resetting of the oscillator occurred within 2 hr. This was the case for both delaying and advancing phase shifts. Examination of delaying shifts in the mouse showed resetting within 2 hr and in addition showed that resetting is not completed within 1 hr of a light pulse. These results establish the temporal window within which to define the primary molecular mechanisms of circadian resetting in the mammal.     

Effects of muscarinic agents on cultured human trabecular meshwork cells

Intracellular calcium measurements were performed in cultured human trabecular meshwork cells preloaded with the cell permeant dye fura 2-AM. Fluctuations in calcium levels were then monitored with microscope-based ratio fluorometry. Carbachol increased intracellular calcium in a dose-dependent manner; as did oxotremorine-M, aceclidine, and pilocarpine. Carbachol's effect was blocked by the non-selective muscarinic antagonist atropine, as well as by muscarinic receptor subtype-selective antagonists such as pirenzepine (M1-selective), p-fHHSiD (M3-selective), and 4-DAMP (M1, M3 subtypes). Rank order of potencies for the antagonists' effects was atropine = 4-DAMP > p-fHHSiD > pirenzepine, a profile suggesting that the M3 receptor subtype is essential in the carbachol effect. Phospholipase C activity was estimated via measurement of total production of inositol phosphates in cultured human trabecular meshwork cells pre-exposed to 3H-myoinositol. In these cells, carbachol also stimulated phosphoinositide production in a dose-dependent manner, and an antagonist profile similar to that seen for calcium response was obtained when carbachol was used as the effector. The data indicate that muscarinic effects on cultured human trabecular meshwork calcium mobilization and phospholipase C activity are mediated by an M3-like receptor subtype. Therefore, the muscarinic M3 receptor may play a role in trabecular meshwork cell function(s).     

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.     

Can a population of suprachiasmatic nucleus neurons with different period lengths produce a stable circadian rhythm?

The firing rate of a population of SCN neurons in vivo exhibits stable circadian oscillations, but the period length of individual neurons is not known and may be different or similar to the population rhythm. To address this question we used published data from Bos and Mirmiran [Brain Res., 511 (1990) 158-162] that reported different period lengths and amplitudes for individual neurons recorded in explant cultures of the SCN. We reconstructed the individual rhythms for several cycles, calculated the population rhythm, and then tested its stability. The period and amplitude of the rhythm of groups of neurons with different period lengths were unstable. Furthermore, the stability of the rhythm was reduced as the number of sampled neurons increased. These results suggest that the stable circadian rhythm reported for neuron populations in the intact SCN emerges from the identical period length of individual neurons. The possible intercellular interactions in the SCN that may underlie the stable circadian rhythm are discussed.     

Reentrainment of motor activity and spontaneous neuronal activity in the suprachiasmatic nucleus of Djungarian hamsters

Neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus exhibit a daily rhythm in spontaneous electrical activity. Essentially two methods have been employed to record this circadian rhythm: (1) an in vitro brain slice technique and (2) in vivo multiunit recordings. Reentrainment of a circadian output to a shifted light:dark cycle commonly takes several cycles (depending on the amount of shift) until completed. Such a resetting kinetic has also been shown to be valid for SCN electrical activity if recorded in vivo. In an in vitro slice preparation, however, pharmacologically induced resetting is much faster and lacks transients; that is, a shift is completed within one cycle. This study was designed to probe for the presence of transients in the neuronal activity of the SCN in a brain slice preparation. The authors exposed Djungarian hamsters to an 8-h advanced or delayed light:dark cycle and monitored wheel-running activity during reentrainment. Additional groups of identically treated hamsters were used to record the pattern of spontaneous neuronal activity within the SCN using the brain slice preparation. Neuronal activity exhibited the usual rhythm with high firing rates during the projected day and low firing rates during the projected night. However, following 1 day of exposure to the 8-h advanced light:dark cycle, this rhythm disappeared in 6 of 7 slices. Rhythmicity was still absent following 3 days of exposure to the advanced light:dark cycle (n = 4). By contrast, 3 of 7 slices prepared from hamsters exposed to a delayed light:dark cycle for 3 days exhibited a daily rhythm in electrical activity. Although pharmacological agents reset the in vitro SCN neuronal activity almost instantaneously and in in vivo studies a stable phase relationship to a shifted light:dark cycle occurs gradually over several cycles, the authors did not detect either of these patterns. Such differences in resetting kinetics (e.g., rapid resetting, gradual reentrainment, temporary lack of measurable rhythmicity) may be due to (a) application of a resetting stimulus in vivo versus in vitro, (b) duration of the resetting stimulus, (c) the nature of the resetting stimulus, or (d) the recording technique employed.     

Light responsiveness of the suprachiasmatic nucleus: long-term multiunit and single-unit recordings in freely moving rats

The suprachiasmatic nuclei (SCN) of the hypothalamus contain a pacemaker that generates circadian rhythms in many functions. Light is the most important stimulus that synchronizes the circadian pacemaker to the environmental cycle. In this paper we have characterized the baseline neuronal firing patterns of the SCN as well as their response to light in freely moving rats. Multiunit and single-unit recordings showed that SCN neurons increase discharge during daytime and decrease discharge at night. Discharge levels of individual neurons that were followed throughout the circadian cycle appeared in phase with the population and were characterized by low discharge rates (often below 1 Hz), with a twofold increase during the day. The effect of light on the multiunit response was dependent on the duration of light exposure and on light intensity, with light thresholds of approximately 0.1 lux. The light response level showed a strong dependency on time of day, with large responsiveness at night and low responsiveness during day. At both phases of the circadian cycle, the response level could be raised by an increase in light intensity. Single-unit measurements revealed that the time-dependent light response of SCN neurons was present also at the level of single units. The results show that the basic light response characteristics that were observed at the multiunit level result from an integrated response of similarly behaving single units. Research at the single-unit level is therefore a useful approach for investigating the basic principles of photic entrainment.     

Photic responses of suprachiasmatic area neurons in diurnal degus (Octodon degus) and nocturnal rats (Rattus norvegicus)

Photic sensitivity of cells in the suprachiasmatic nuclei (SCN), the principal pacemaker of the mammalian circadian system, has been documented in several species. In nocturnal rodents, the majority of photically responsive SCN cells are activated by retinal illumination. One report identified mostly photic suppressions among SCN cells in a diurnal rodent, studied under somewhat different conditions. We examined photic sensitivity of SCN cells in a predominantly diurnal rodent, the degu, studied in vivo under identical conditions to rats, and found that a large majority of photic SCN cells were suppressed by light. In both rats and degus, SCN cells were more responsive to light during the subjective night than during the subjective day. Light-responsive cells did not show a daily rhythm in baseline firing rates in either species, but rat SCN cells that did not respond to light were more active spontaneously during the subjective day. Light-unresponsive SCN cells in degus did not show a similar pattern. There are substantial differences in the neurophysiological activity and photic responsiveness of SCN cells in diurnal degus and nocturnal rats.     

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.     

Effects of ACh on the electric activity of rostral ventrolateral medullary neurons of rat in vitro

Extracellular single-unit discharges were obtained from 165 spontaneously active neurons within the region of the rostral ventrolateral medulla (RVLM) by glass microelectrode from 89 brain slices of the Sprague-Dawley rats. The units could be divided into three types: regular (61.8%), irregular (24.2%) and silent (14%). Acetylcholine (ACh, 0.1, 0.3 mumol/L) showed four kinds of effects on spontaneous discharges of RVLM neurons: excitatory, inhibitory, biphasic and non-responsive, counting respectively 41.8%, 20%, 3% and 35.2% of the neurons tested. The excitatory effect of ACh was dose-dependent. The effects, either excitatory or inhibitory, of ACh (n = 49) were mostly blocked by atropine (0.3 mumol/L, n = 42). The excitatory effect of ACh (n = 14) could be blocked mainly by selective antagonist of M1 receptor, pirenzepine (PZ, 30 nmol/L, n = 9), but not by selective antagonist of M2 receptor, methoctramine (MT) and AFDX-116. The inhibitory effect of ACh (n = 10) could be blocked mostly by M2 receptor antagonist MT (30 nmol/L, n = 7); and this inhibitory effect (n = 9) could be blocked mostly by another M2 receptor antagonist AFDX-116 (30 nmol/L, n = 6), but not by M1 receptor antagonist PZ.     

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.     

Cyclic AMP resets the circadian clock in cultured Xenopus retinal photoreceptor layers

The Xenopus retinal photoreceptor layer contains a circadian oscillator that regulates melatonin synthesis in vitro. The phase of this oscillator can be reset by light or dopamine. The phase-response curves for light and dopamine are similar, with transitions from phase delays to phase advances in the mid-subjective night. Light and dopamine each can inhibit adenylate cyclase in retinal photoreceptors, suggesting cyclic AMP as a candidate second messenger for entrainment of the circadian oscillator. We report here that treatments that increase intracellular cyclic AMP reset the phase of the photoreceptor circadian oscillator, and that the phase-response curves for these treatments are 180 degrees out of phase with the phase-response curves for light and dopamine. Activation of adenylate cyclase by forskolin during the late subjective day or early subjective night caused phase advances. The same treatment during the late subjective night or early subjective day caused phase delays. Similar phase shifts were induced by 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) or 8-(4-chlorophenylthio)cyclic AMP. All of these treatments also acutely increased melatonin release. Forskolin and 3-isobutyl-1-methylxanthine increased the accumulation of intracellular cyclic AMP, but not cyclic GMP, in photoreceptor layers. The results indicate that cyclic AMP-dependent pathways regulate the photoreceptor circadian oscillator and suggest that a decrease in cyclic AMP may be involved in circadian entrainment by light and/or dopamine.