Electrophysiological effects of melatonin on mouse Per1 and non-Per1 suprachiasmatic nuclei neurones in vitro.

The master circadian pacemaker in the suprachiasmatic nuclei (SCN) regulates the nocturnal secretion of the pineal hormone melatonin. Melatonin, in turn, has feedback effects on SCN neuronal activity rhythms via high affinity G protein-coupled receptors (MT(1) and MT(2) ). However, the precise effects of melatonin on the electrical properties of individual SCN neurones are unclear. In the present study, we investigated the acute effects of exogenous melatonin on SCN neurones using whole-cell patch-clamp recordings in brain slices prepared from Per1::d2EGFP-expressing transgenic mice. In current-clamp mode, bath applied melatonin, at near-physiological concentrations (1 nM), hyperpolarised the majority (63.7%) of SCN neurones tested at all times of the projected light/dark cycle. In addition, melatonin depolarised a small proportion of cells (11.0%). No differences were observed for the effects of melatonin between Per1::GFP or non-Per1::GFP SCN neurones. Melatonin-induced effects were blocked by the MT(1)/MT(2) antagonist, luzindole (1 µM) and the proportion of SCN neurones responsive to melatonin was greatly reduced in the presence of either tetrodotoxin (200 or 500 nM) or gabazine (20 µM). In voltage-clamp recordings, 1 nM melatonin increased the frequency of GABA-mediated currents. These findings indicate, for the first time, that exogenous melatonin can alter neuronal excitability in the majority of SCN neurones, regardless of whether or not they overtly express the core clock gene Per1. The results also suggest that melatonin acts mainly by modulating inhibitory GABAergic transmission within the SCN. This may explain why exogenous application of melatonin has heterogenous effects on individual SCN neurones.

Rhythm-promoting actions of exercise in mice with deficient neuropeptide signaling.

Daily exercise promotes physical health as well as improvements in mental and neural functions. Studies in intact wild-type (WT) rodents have revealed that the brain's suprachiasmatic nuclei (SCN), site of the main circadian pacemaker, are also responsive to scheduled wheel running. It is unclear, however, if and how animals with a dysfunctional circadian pacemaker respond to exercise. Here, we tested whether scheduled voluntary exercise (SVE) in a running wheel for 6 hours per day could promote neural and behavioral rhythmicity in animals whose circadian competence is compromised through genetically targeted loss of vasoactive intestinal polypeptide (VIP(-/-) mice) or its VPAC(2) receptor (Vipr2(-/-) mice). We report that in constant dark (DD), rhythmic VIP(-/-) and Vipr2(-/-) mice show weak free-running rhythms with a period of <23 hours and all wild-type mice are strongly rhythmic with approximately 23.5-hour periodicity. VIP(-/-) and Vipr2(-/-) mice rapidly (<7 days) synchronize to daily SVE, while WT mice take much longer (>35 days). Following 21 to 50 days of SVE, WT mice show small changes in their rhythms, and most Vipr2(-/-) mice now sustain robust near 24-hour behavioral rhythms, whereas very few VIP(-/-) mice do. This study demonstrates that scheduled daily exercise can markedly improve circadian rhythms in behavioral activity and raises the possibility that this noninvasive approach may be useful as an intervention in clinical etiologies in which there are dysfunctions of circadian time keeping.

Circadian oscillators in the epithalamus.

The habenula complex is implicated in a range of cognitive, emotional and reproductive behaviors, and recently this epithalamic structure was suggested to be a component of the brain's circadian system. Circadian timekeeping is driven in cells by the cyclical activity of core clock genes and proteins such as per2/PER2. There are currently no reports of rhythmic clock gene/protein expression in the habenula and therefore the question of whether this structure has an intrinsic molecular clock remains unresolved. Here, using videomicroscopy imaging and photon-counting of a PER2::luciferase (LUC) fusion protein together with multiunit electrophysiological recordings, we tested the endogenous circadian properties of the mouse habenula in vitro. We show that a circadian oscillator is localized primarily to the medial portion of the lateral habenula. Rhythms in PER2:: LUC bioluminescence here are visualized in single cells and oscillations continue in the presence of the sodium channel blocker, tetrodotoxin, indicating that individual cells have intrinsic timekeeping properties. Ependymal cells lining the dorsal third ventricle also express circadian oscillations of PER2. These findings establish that neurons and non-neuronal cells in the epithalamus express rhythms in cellular and molecular activities, indicating a role for circadian oscillators in the temporal regulation of habenula controlled processes and behavior.

Spatiotemporal heterogeneity in the electrical activity of suprachiasmatic nuclei neurons and their response to photoperiod.

The coordinated activity of thousands of cellular oscillators in the suprachiasmatic nuclei (SCN) temporally regulates mammalian physiology to anticipate daily environmental changes across the seasons. The phasing of clock gene expression varies according to anatomical location in the SCN and is thought to encode photoperiodic information. However, it is unclear whether similar variations in phase occur in the electrical activity of SCN neurons, a measure of both intraSCN signaling and clock output. To address this, we recorded single-unit and multiunit activity (SUA/MUA) from dorsal and ventral subregions of the middle level of the rostrocaudal axis of the SCN in coronal brain slices prepared from mice housed under different photoperiods. We demonstrate that under a symmetrical (12 h light:12 h dark) photoperiod, cells in the dorsal SCN are less tightly synchronized than those in the ventral SCN. Comparison of recordings made from mice under short (8 h light:16 h dark) or long (16 h light:8 h dark) photoperiods shows that the phase distribution of ventral, but not dorsal, SCN neurons expands with increasing day length. Conversely, the duration that individual neurons are active increases in dorsal, but not ventral, SCN under long days. These data indicate that in the ventral SCN photoperiod is encoded at the network level, while this coding occurs at the level of individual cells in the dorsal SCN.

Electrophysiological actions of orexins on rat suprachiasmatic neurons in vitro.

The study of neural arousal mechanisms has been greatly aided by the discovery of the orexin peptides (orexin A and orexin B), the subsequent identification of the neurons that synthesize these peptides, their projections in the brain, and the distribution of orexin receptors in the central nervous system. Orexin neuron activation is partly controlled by circadian signals generated in the brain's main circadian pacemaker, the suprachiasmatic nuclei (SCN). The SCN clock is in turn reset by arousal-promoting stimuli and, intriguingly, orexin fibers and receptor expression are detected in the SCN region. It is unclear, however, if orexin can alter SCN neuronal activity. Here using a coronal brain slice preparation, we found that orexin A and orexin B (0.1-1 microM) elicited significant changes in the extracellularly recorded firing rate and firing pattern in approximately 80% of rat SCN cells tested; the most common response was suppression of firing rate. Co-application of orexin A with a cocktail of ionotropic GABA and glutamate receptor antagonists did not alter the actions of this peptide on firing rate, but did change some its effects on firing pattern. We conclude that orexins can alter SCN neurophysiology and may influence the transmission of information through the SCN to other CNS regions.

Angiotensin II regulates the activity of mouse suprachiasmatic nuclei neurons.

Neuropeptide signaling plays key roles in coordinating cellular activity within the suprachiasmatic nuclei (SCN), site of the master circadian oscillator in mammals. The neuropeptide angiotensin II (ANGII) and its cognate receptor AT1, are both expressed by SCN cells, but unlike other SCN neurochemicals, very little is known about the cellular actions of ANGII within this circadian clock. We used multi-electrode, multiunit, extracellular electrophysiology, coupled with whole-cell voltage and current clamp techniques to investigate the actions of ANGII in mouse SCN slices. ANGII (0.001-10 microM) dose dependently stimulated and inhibited extracellularly recorded neuronal discharge in many SCN neurons ( approximately 60%). Both actions were blocked by pre-treatment with the AT1 receptor antagonist ZD7155 (0.03 microM), while suppressions but not activations were prevented by pre-treatment with the GABA A receptor antagonist bicuculline (20 microM). AT1 receptor blockade itself suppressed discharge in a subset ( approximately 30%) of SCN neurons, and this action was not blocked by bicuculline. In voltage-clamped SCN neurons (-70 mV), AT1 receptor activation dose-dependently enhanced the frequency of action potential-driven, GABA A receptor-mediated currents, but did not alter their responses to exogenously applied GABA. In current-clamped SCN neurons perfused with tetrodotoxin, ANGII induced a membrane depolarization with a concomitant decrease in input resistance. In conclusion we show that AT1 receptor activation by ANGII depolarizes SCN neurons and stimulates action potential firing, leading to increased GABA release in the mouse SCN. Additionally we provide the first evidence that endogenous AT1 receptor signaling tonically regulates the activities of some SCN neurons.

Behavioral responses of Vipr2-/- mice to light.

Vasoactive intestinal polypeptide and its receptor, VPAC2, play important roles in the functioning of the dominant circadian pacemaker, located in the hypothalamic suprachiasmatic nuclei (SCN). Mice lacking VPAC2 receptors (Vipr2-/-) show altered circadian rhythms and impaired synchronization to environmental lighting cues. However, light can increase phosphoprotein and immediate early gene expression in the Vipr2-/- SCN demonstrating that the circadian clock is readily responsive to light in these mice. It is not clear whether these neurochemical responses to light can be transduced to behavioral changes as seen in wild-type (WT) animals. In this study we investigated the diurnal and circadian wheel-running profile of WT (C57BL/6J) and Vipr2-/- mice under a 12-h light:12-h complete darkness (LD) lighting schedule and in constant darkness (DD) and used 1-h light pulses to shift the activity of mice in DD. Unlike WT mice, Vipr2-/- mice show grossly altered locomotor patterns making the analysis of behavioral responses to light problematic. However, analyses of both the onset and the offset of locomotor activity reveal that in a subset of these mice, light can reset the offset of behavioral rhythms during the subjective night. This suggests that the SCN clock of Vipr2-/- mice and the rhythms it generates are responsive to photic stimulation and that these responses can be integrated to whole animal behavioral changes.

Adaptive switch from infanticide to parental care: how do beetles time their behaviour?

In species where parents may commit infanticide, temporal kin recognition can help ensure parents kill unrelated young but care for their own offspring. This is not true recognition, but rather depends on accurate timing of the arrival of young and a behavioural switch from killing to caring for offspring. Mistakes have clear fitness consequences; how do species that use temporal kin recognition ensure accurate timing? We manipulated photic cues and show that the switch from infanticide to parental care in the burying beetle Nicrophorus vespilloides depends on day-length inputs. Extending the light period after carcass discovery influenced timing of both oviposition and the cessation of infanticide. Manipulation of the light : dark cycle after oviposition also influenced timing of the switch to parental care. The timing mechanism is therefore sensitive to photic cues and access to a carcass and is not triggered by oviposition. The behavioural switch is directly related to the timing mechanism rather than changes in reproductive physiology. Given the conserved nature and extensive homology of genetic influences on biological timing, we speculate that the molecular mechanisms regulating circadian behaviour may have been co-opted to allow beetles to determine how much time has passed after carcass discovery even though this is over 50 h.

Disrupted neuronal activity rhythms in the suprachiasmatic nuclei of vasoactive intestinal polypeptide-deficient mice.

Vasoactive intestinal polypeptide (VIP), acting via the VPAC(2) receptor, is a key signaling pathway in the suprachiasmatic nuclei (SCN), the master clock controlling daily rhythms in mammals. Most mice lacking functional VPAC(2) receptors are unable to sustain behavioral rhythms and lack detectable SCN electrical rhythms in vitro. Adult mice that do not produce VIP (VIP/PHI(-/-)) exhibit less severe alterations in wheel-running rhythms, but the effects of this deficiency on the amplitude, phasing, or periodicity of their SCN cellular rhythms are unknown. To investigate this, we used suction electrodes to extracellularly record multiple- and single-unit electrical activity in SCN brain slices from mice with varying degrees of VIP deficiency, ranging from wild-type (VIP/PHI(+/+)) to heterozygous (VIP/PHI(+/-)) and VIP/PHI(-/-) animals. We found decreasing proportions of rhythmic cells in SCN slices from VIP/PHI(+/+) ( approximately 91%, n = 23) through VIP/PHI(-/+) ( approximately 71%, n = 28) to VIP/PHI(-/-) mice (62%; n = 37) and a parallel trend toward decreasing amplitude in the remaining rhythmic cells. SCN neurons from VIP/PHI(-/-) mice exhibited a broad range in the period and phasing of electrical rhythms, concordant with the known alterations in their behavioral rhythms. Further, treatment of VIP/PHI(-/-) slices with a VPAC(2) receptor antagonist significantly reduced the proportion of oscillating neurons, suggesting that VPAC(2) receptors still become activated in the SCN of these mice. The results establish that VIP is important for appropriate periodicity and phasing of SCN neuronal rhythms and suggest that residual VPAC(2) receptor signaling promotes rhythmicity in adult VIP/PHI(-/-) mice.

Resetting of the hamster circadian system by dark pulses.

Circadian rhythms of animals are reset by exposure to light as well as dark; however, although the parameters of photic entrainment are well characterized, the phase-shifting actions of dark pulses are poorly understood. Here, we determined the tonic and phasic effects of short (0.25 h), moderate (3 h), and long (6-9 h) duration dark pulses on the wheel-running rhythms of hamsters in constant light. Moderate- and long-duration dark pulses phase dependently reset behavioral rhythms, and the magnitude of these phase shifts increased as a function of the duration of the dark pulse. In contrast, the 0.25-h dark pulses failed to evoke consistent effects at any circadian phase tested. Interestingly, moderate- and long-dark pulses elevated locomotor activity (wheel-running) on the day of treatment. This induced wheel-running was highly correlated with phase shift magnitude when the pulse was given during the subjective day. This, together with the finding that animals pulsed during the subjective day are behaviorally active throughout the pulse, suggests that both locomotor activity and behavioral activation play an important role in the phase-resetting actions of dark pulses. We also found that the robustness of the wheel-running rhythm was weakened, and the amount of wheel-running decreased on the days after exposure to dark pulses; these effects were dependent on pulse duration. In summary, similarly to light, the resetting actions of dark pulses are dependent on both circadian phase and stimulus duration. However, dark pulses appear more complex stimuli, with both photic and nonphotic resetting properties.

Ageing and the diurnal expression of mRNAs for vasoactive intestinal peptide and for the VPAC2 and PAC1 receptors in the suprachiasmatic nucleus of male rats.

Ageing alters fundamental aspects of circadian rhythmicity in mammals; the effects include reduced rhythm amplitude and alterations in period length and in entrainment to the light/dark cycle. Such changes may reflect disruptions in cellular function within the suprachiasmatic nucleus (SCN), the site of the predominant circadian pacemaker. In the SCN, vasoactive intestinal peptide (VIP)-synthesizing neurones receive various inputs, including retinohypothalamic projections containing pituitary adenylate cyclase activating peptide (PACAP). SCN VIP cells establish connections with local neurones and send efferents beyond the nucleus. Considerable evidence implicates VIP and PACAP in circadian rhythm maintenance and/or entrainment to photic Zeitgebers. These actions involve members of a distinct family of receptors; mRNAs for two such receptors, VPAC2 and PAC1, are present in the SCN. This study used isotopic in situ hybridization to examine the effects of ageing on expression of mRNAs for VIP, VPAC2 and PAC1 in the SCN of male rats under a 12 : 12 h light/dark cycle. Analysis of film autoradiographs from young adult (2-3 months) or aged (19-20 months) rats, at eight time points across the light/dark cycle, showed loss of diurnal rhythmicity and reduced levels for VIP mRNA in the aged group. A diurnal rhythm of VPAC2 receptor mRNA was present in both groups, but its levels were reduced in the aged rats. There were no differences between the two groups for PAC1 receptor mRNA expression. The present results indicate that ageing reduces VIP and VPAC2 receptor mRNA and eliminates diurnal expression of VIP mRNA within the SCN of aged male rats.

The roles of vasoactive intestinal polypeptide in the mammalian circadian clock.

Biological oscillations with an endogenous period of near 24 h (circadian rhythms) are generated by the master circadian pacemaker or clock located in the suprachiasmatic nuclei (SCN) of the hypothalamus. This clock is synchronised to recurring environmental signals conveyed by selective neural pathways. One of the main chemical constituents of SCN neurones is vasoactive intestinal polypeptide (VIP). Such neurones are retinorecipient and activated by light. Exogenous application of VIP resets the SCN circadian clock in a light-like manner, both in vivo and in vitro. These resetting actions appear to be mediated through the VPAC2 receptor (a type of receptor for VIP). Unexpectedly, genetically ablating expression of the VPAC2 receptor renders the circadian clock arrhythmic at the molecular, neurophysiological and behavioural levels. These findings indicate that this intrinsic neuropeptide acting through the VPAC2 receptor participates in both resetting to light and maintenance of ongoing rhythmicity of the SCN.

Effects of vasoactive intestinal polypeptide on neurones of the rat suprachiasmatic nuclei in vitro.

The suprachiasmatic nuclei (SCN) of the hypothalamus house the main circadian pacemaker in mammals. Vasoactive intestinal polypeptide (VIP) is the most abundant neuropeptide in the SCN and has been shown to phase-shift the electrical activity rhythm of SCN cells in vitro. However, the effects of VIP on the cellular activity of rat SCN neurones are unknown. In this study, we examined the acute effects of VIP on the extracellularly recorded spontaneous firing rate of SCN neurones in an in-vitro hypothalamic slice preparation. Furthermore, with the use of receptor-selective agonists and antagonists, we determined which receptors might mediate the effects of VIP in the SCN. Approximately 50% of cells responded to VIP; the main type of response was suppression in firing rate, although a few cells were activated. Suppression responses to VIP were mimicked by the VPAC(2) receptor agonist Ro 25-1553 and blocked by the selective VPAC(2) receptor antagonist PG 99-465. The PAC(1) receptor agonist maxadilan evoked responses from 40% of SCN cells, and activations to this agonist were not altered by PG 99-465. Responses to VIP were not blocked by antagonists to ionotropic glutamate receptors, but the duration of suppression was modulated by the GABA(A) receptor antagonist bicuculline. Our data indicate that VIP alters the electrical activity of rat SCN neurones in vitro, via both VPAC(2) and PAC(1) receptors.

Distribution of substance P and neurokinin-1 receptor immunoreactivity in the suprachiasmatic nuclei and intergeniculate leaflet of hamster, mouse, and rat.

The circadian pacemaker in the hypothalamic suprachiasmatic nuclei (SCN) receives photic information directly via the retinohypothalamic tract (RHT) and indirectly from retinally innervated cells in the thalamic intergeniculate leaflet (IGL) that project to the SCN. Using standard immunohistochemical methods, we examined the presence and distribution of substance P (SP) and the neurokinin-1 receptor (NK-1) in the SCN and IGL of rat and determined whether the patterns of immunostaining generalized to the SCN and IGL of Syrian hamster, Siberian hamster, and mouse. Terminals immunoreactive for SP were sparse within the SCN of Siberian and Syrian hamsters and mouse but were intense in the ventral, retinally innervated portion of the rat SCN. Immunostaining for the NK-1 receptor was mainly absent from the SCN of hamster and mouse. In contrast, a plexus of NK-1-ir cells and processes that was in close proximity to SP-ir terminals was found in the ventral SCN of the rat. Substance P-ir terminals were observed in the IGL of all four species, as were NK-1-ir cells and fibres. Double-labelled IGL sections of hamster or rat revealed SP-ir terminals in close apposition to NK-1-immunostained cells and/or fibres. These data indicate that SP could be a neurotransmitter of the RHT in rat, but not in hamster or in mouse, and they highlight potential species differences in the role of SP within the SCN circadian pacemaker. Such species differences do not appear to exist at the level of the IGL, where SP-ir and NK-1-ir were similar in all species studied.

Orexin A-like immunoreactivity in the hypothalamus and thalamus of the Syrian hamster (Mesocricetus auratus) and Siberian hamster (Phodopus sungorus), with special reference to circadian structures.

The orexins are recently discovered neuropeptides that reportedly play a role in energy homeostasis, in addition to various other physiological processes. The synthesis of orexin A undergoes diurnal variation in certain areas of the brain, while the mutation of the orexin receptor 2 gene has been implicated in canine narcolepsy. Since the circadian pacemaker in the suprachiasmatic nucleus modulates the sleep/wake cycle, there is a putative role for orexins in the mammalian circadian system. In this study, immunohistochemical techniques were used to determine the distribution of orexin A in the structures of the hypothalamus and thalamus of Syrian and Siberian hamsters. In both species, the pattern of immunoreactivity was similar. Cells immunoreactive for orexin A were noted in the lateral hypothalamic area. Immunoreactive varicose orexin A fibres were found throughout the hypothalamus. The suprachiasmatic nucleus possessed little or no immunoreactive orexin A fibres in its core, but had fibres at its periphery. The thalamus of both species contained comparatively few immunoreactive fibres, which were mainly localised around the midline. The thalamic intergeniculate leaflet contained a plexus of immunoreactive orexin A fibres throughout its rostro-caudal extent. Three areas of the brainstem, the dorsal and median raphe nuclei and the locus coeruleus, were also investigated owing to their relevance to the circadian system and all were found to contain immunoreactive orexin A fibres. The presence of orexin A-immunoreactive fibres in the neural architecture of the mammalian circadian system suggests an important role for orexin A in circadian timekeeping processes.

Phase-shifting effects of pituitary adenylate cyclase activating polypeptide on hamster wheel-running rhythms.

Pituitary adenylate cyclase activating polypeptide (PACAP38) is a putative neurochemical of the main retinal input to the mammalian circadian pacemaker housed in the suprachiasmatic nucleus (SCN). We assessed the phase-resetting effects of microinjection of PACAP38 into the SCN region on hamster wheel-running rhythms. When administered during the middle of the subjective day, PACAP38 evoked large but transient phase advances ( approximately 60 min), that were followed by small, steady-state phase delays. During the early subjective night, PACAP38 elicited small to moderate phase delays without any detectable concentration-dependence. Late in the subjective night, PACAP38 had no significant effects. Saline microinjection had no effect at any phase tested. These findings show that PACAP38 has small to moderate effects on the phase of the hamster SCN circadian pacemaker, including significant phase delays early in the subjective night.

Effects of neurotensin on discharge rates of rat suprachiasmatic nucleus neurons in vitro.

The neuropeptide neurotensin and two classes of its receptors, the neurotensin receptor-1 and 2, are present in the suprachiasmatic nucleus of the mammalian hypothalamus. The suprachiasmatic nucleus houses the mammalian central circadian pacemaker, but the effects of neurotensin on cellular activity in this circadian pacemaker are unknown. In this study, we examined the effects of neurotensin on the spontaneous discharge rate of rat SCN cells in an in vitro slice preparation. Neurotensin (1-10 microM) increased cell firing rate in approximately 50% of cells tested, while approximately 10% of suprachiasmatic cells tested showed a decrease in firing rate in response to neurotensin. These effects of neurotensin were not altered by the GABA receptor antagonist bicuculline (20 microM) or the glutamate receptor antagonists, D-aminophosphopentanoic acid (50 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (20 microM). The neurotensin receptor selective antagonists SR48692 and SR142948a (10 microM) failed to antagonise neurotensin responses in the majority of cells examined. Compounds that function as agonists selective for the neurotensin-receptor subtypes 1 and 2, JMV-510 and JMV-431 respectively, elicited neurotensin-like responses in approximately 90% of cells tested. Six out of seven cells tested responded to both JMV-510 and JMV-431. Neuropeptide Y (100nM) treatment of suprachiasmatic nucleus slices was found to elicit profound suppression of neuronal firing rate. Co-application of neurotensin with neuropeptide Y significantly (P<0.05) reduced the duration of the response, as compared to that elicited with neuropeptide Y alone. Together, these results demonstrate for the first time the actions of neurotensin in the suprachiasmatic nucleus and raise the possibility that this neuropeptide may play a role in modulating circadian pacemaker function.

Vasoactive intestinal polypeptide (VIP) phase-shifts the rat suprachiasmatic nucleus clock in vitro.

In mammals, the principal circadian pacemaker is housed in the hypothalamic suprachiasmatic nuclei (SCN). The SCN exhibit high levels of vasoactive intestinal polypeptide (VIP) immunoreactivity and two of the three VIP receptors, VPAC(2) and PAC(1), are found in the rat SCN. However, the role of VIP in the SCN remains unclear. In this study, we examined the phase-resetting actions of VIP and selective VIP receptor agonists on the electrical activity rhythm of rat SCN neurons in vitro. Application of VIP during the subjective day did not shift the peak in the firing rate rhythm. However, VIP treatment during the early or late subjective night evoked a small phase delay or a large phase advance, respectively. The phase-advancing effect of VIP was reproduced by the novel VPAC(2) receptor agonist RO 25-1553, but not by pituitary adenylate cyclase-activating peptide (a potent PAC(1) receptor agonist), or by [K15,R16,L27]VIP(1-7)/GRF(8-27), a novel, selective VPAC(1) receptor agonist. These data show that VIP phase-dependently phase-resets the rodent SCN pacemaker in vitro, presumably via the VPAC(2) receptor. As the pattern of phase-shifting evoked by VIP and RO 25-1553 resembles the phase-resetting actions of light on rodent behavioural rhythms, these data support a role for VIP and the VPAC(2) receptor in photic entrainment of the rodent circadian pacemaker.

Gastrin-releasing peptide phase-shifts suprachiasmatic nuclei neuronal rhythms in vitro.

The main mammalian circadian pacemaker is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Gastrin-releasing peptide (GRP) and its receptor (BB(2)) are synthesized by rodent SCN neurons, but the role of GRP in circadian rhythm processes is unknown. In this study, we examined the phase-resetting actions of GRP on the electrical activity rhythms of hamster and rat SCN neurons in vitro. In both rat and hamster SCN slices, GRP treatment during the day did not alter the time of peak SCN firing. In contrast, GRP application early in the subjective night phase-delayed, whereas similar treatment later in the subjective night phase-advanced the firing rate rhythm in rat and hamster SCN slices. These phase shifts were completely blocked by the selective BB(2) receptor antagonist, [d-Phe(6), Des-Met(14)]-bombesin 6-14 ethylamide. We also investigated the temporal changes in the expression of genes for the BB(1) and BB(2) receptors in the rat SCN using a quantitative competitive RT-PCR protocol. The expression of the genes for both receptors was easily detected, but their expression did not vary over the diurnal cycle. These data show that GRP phase-dependently phase resets the rodent SCN circadian pacemaker in vitro apparently via the BB(2) receptor. Because this pattern of phase shifting resembles that of light on rodent behavioral rhythms, these results support the contention that GRP participates in the photic entrainment of the rodent SCN circadian pacemaker.

Expression of mt(1) melatonin receptor subtype mRNA in the entrained rat suprachiasmatic nucleus: a quantitative RT-PCR study across the diurnal cycle.

Melatonin acts on specific receptors in the suprachiasmatic nuclei (SCN) to phase-dependently regulate the phase of the circadian clock. How the gating of melatonin's effect is restricted to particular times of day is not known, but may be related to temporal differences in receptor availability. In the present study, we used a competitive reverse transcription-polymerase chain reaction (RT-PCR) method to determine if the expression of mt(1) melatonin receptor subtype mRNA in rat SCN varied across the 12:12 light-dark (LD) cycle. Measurement of core body temperature using radiotelemetry confirmed that the male Wistar rats used exhibited a robust diurnal rhythm. mt(1) receptor mRNA was readily detected in reduced SCN slices at all times of day. However, there was no significant variation in the amount of mt(1) mRNA with time of day. Expression of MT(2) melatonin receptor subtype mRNA in reduced SCN slices was confirmed by nested PCR. These results indicate that changes in the level of mt(1) mRNA do not underlie the diurnal and/or circadian variation in the response of the SCN circadian clock to the phase-resetting effects of melatonin.