Neuropeptide Y blocks serotonergic phase shifts of the suprachiasmatic circadian clock in vitro

The mammalian circadian pacemaker in the suprachiasmatic nuclei (SCN) can be reset in vitro by various neurochemical stimuli. This study investigated the phase-shifting properties of neuropeptide Y (NPY) and serotonin (5-HT) agonists when applied alone, as well as their combined effects on clock resetting. These neurotransmitters have both been shown to advance the SCN clock in vitro when applied during the daytime. By monitoring the SCN neuronal activity rhythm in vitro, I first confirm that the 5HT_1A/5HT₇ agonist (+)DPAT maximally advances the SCN clock when applied at zeitgeber time 6 (ZT6). Conversely, NPY only phase advances the neuronal activity rhythm when applied at ZT 10. This effect occurs through stimulation of Y₂ receptors. NPY, again acting through Y₂ receptors, blocks (+)DPAT-induced phase shifts at ZT 6, while neither (+)DPAT nor 5-HT affect NPY-induced phase shifts at ZT 10. NPY appears to block (+)DPAT-induced phase shifts by preventing increases in cyclic AMP. These data are the first to demonstrate in vitro interactions between daytime resetting stimuli in the rat, and provide critical insights into mechanisms controlling circadian clock phase.     

Blockade of the NPY Y5 receptor potentiates circadian responses to light: complementary in vivo and in vitro studies

Neuropeptide Y (NPY) is delivered to the suprachiasmatic nuclei (SCN) circadian pacemaker via an input from the thalamic intergeniculate leaflet. NPY can inhibit light-induced responses of the circadian system of Syrian hamsters. Here we studied whether an antagonist to NPY receptors can be used to potentiate photic phase shifts late in the subjective night. First we determined by in situ hybridization that both NPY Y1 and Y5 receptor mRNA are expressed in the SCN of Syrian hamsters. Second, similar to our previous findings at Zeitgeber time 14 (ZT 14, where ZT 12 was the time of lights off), we found that NPY applied at ZT 18.5 onto the SCN region of brain slices maintained in vitro could block NMDA-induced phase advances of the spontaneous firing rate rhythm, and this blocking effect was probably mediated by the Y5 receptor, since co-application of Y5 receptor antagonists completely reversed the effect of NPY, while application of a Y1 receptor antagonist had no effect under the same conditions. Third, we found that co-treatment with a Y5 receptor antagonist in vivo (s.c., 10 mg/kg) not only reversed the effect of NPY applied to the SCN in vivo through a cannula but also significantly potentiated the light-induced phase advance in the absence of NPY. This is the first report of a NPY receptor antagonist having such an effect, and indicates that NPY Y5 receptor antagonists could be clinically useful for potentiating circadian system responses to light.     

Tetrodotoxin blocks NPY-induced but not muscimol-induced phase advances of wheel-running activity in Syrian hamsters

During the middle of the subjective day, circadian activity rhythms in Syrian hamsters can be phase advanced by a variety of stimuli including microinjection of neuropeptide Y (NPY) or muscimol into the suprachiasmatic nucleus (SCN). It is not known, however, if these treatments shift activity rhythms by acting directly on pacemaker cells within the SCN. In the present study NPY and muscimol were microinjected with either tetrodotoxin or saline in order to determine whether classical synaptic transmission within the SCN is necessary for the phase advances produced by NPY or muscimol. Blockade of sodium-dependent action potentials within the SCN prevented NPY- but not muscimol-induced phase advances. These data, along with our previous finding that bicuculline blocks NPY-induced phase advances, suggest that NPY requires sodium-dependent action potentials within GABAergic neurons in order to phase-shift the circadian pacemaker.     

The bidirectional phase-shifting effects of melatonin on the arginine vasopressin secretion rhythm in rat suprachiasmatic nuclei in vitro

In vivo melatonin serves as a feedback signal to the circadian pacemaker located in the suprachiasmatic nuclei (SCN) and in vitro it phase advances the circadian rhythm of electrical activity in pacemaker cells. However, the occurrence and nature of phase shifting in secretion by cultured SCN neurons has not yet been established. Here we studied the effects of melatonin on the pattern of spontaneous arginine vasopressin (AVP) release in organotypic SCN slices. This culture mimicked the in vivo circadian AVP secretory rhythm, with low release during the subjective night and with peaks in secretion during the middle of subjective day. The endogenous period of the AVP secretory rhythm in organotypic culture ranged between 23 and 26 h, with the mean period of 24.1 +/- 0.3 h. Melatonin (10 nM) had variable effects on the pattern of AVP secretion depending on time of its application directly to the medium with organotypic SCN slices. When introduced at circadian time 22, 2 and 6 (the times corresponding to the late night and early day), melatonin delayed the AVP secretory rhythm by 1-4 h. When applied at circadian time 10 (late day), however, melatonin advanced the AVP secretory rhythm by about 2 h. At other circadian times, melatonin was ineffective. These results indicate that melatonin exhibits the bidirectional phase-shifting effects on circadian secretory rhythm clock, which depends on the time-window of its application.     

Phase-resetting effect of 8-OH-DPAT, a serotonin1A receptor agonist, on the circadian rhythm of firing rate in the rat suprachiasmatic nuclei in vitro.

The 5-HTergic neurons in the mesencephalic raphe nuclei provide a robust projection to the hypothalamic suprachiasmatic nucleus (SCN), the site of a putative neuronal circadian pacemaker. Although it has been suggested that 5-HT neurons may play a role in the circadian timing system, this role has not yet been specified. Prosser et al. (Brain Res., 534 (1990) 336-339) reported that 1 h treatments with quipazine induce robust phase shifts in vitro, and that this effect depends upon the circadian time of treatment. However, quipazine is a non-specific 5-HT agonist. Besides, it is reported that the 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino)tetraline hydrobromide (8-OH-DPAT) affected a circadian rhythm of hamster wheel-running activity. In the present study we investigated whether the 5-HT1A agonist 8-OH-DPAT can reset the phase of the SCN clock when it is isolated in vitro. The present results show that 1 h treatments with 8-OH-DPAT induce robust phase advances in vitro when it was administered during the subjective day. This result suggests that 5HT1A receptor functioning may play a role in modulating the phase of SCN clock, especially during the subjective day.     

Serotonin phase-shifts the mouse suprachiasmatic circadian clock in vitro

The mammalian circadian clock in the suprachiasmatic nucleus (SCN) receives multiple afferent signals that could potentially modulate its phase. One input, the serotonin (5-HT) projection from the raphe nuclei, has been extensively investigated in rats and hamsters, yet its role(s) in modulating circadian clock phase remains controversial. To expand our investigation of 5-HT modulation of the SCN clock, we investigated the phase-shifting effects of 5-HT and its agonist, (+)8-hydroxy-2-(di-n-propylamino)tetralin (DPAT), when applied to mouse SCN brain slices. 5-HT induced 2-3 h phase advances when applied during subjective day, while non-significant phase shifts were seen after 5-HT application at other times. These phase shifts were completely blocked by the 5-HT antagonist, metergoline. DPAT also induced phase shifts when applied during mid-subjective day, and this effect appeared dose-dependent. Together, these results demonstrate that the mouse SCN, like that of the rat, is directly sensitive to in vitro phase-resetting by 5-HT.     

Short-term exposure to constant light promotes strong circadian phase-resetting responses to nonphotic stimuli in Syrian hamsters

Behavioral (nonphotic) stimuli can shift circadian rhythms by serotonin (5-HT) and/or neuropeptide Y (NPY) inputs to the suprachiasmatic nucleus (SCN) circadian clock. Based on the idea that behavioral phase resetting is modulated by endogenous changes in postsynaptic sensitivity to such transmitters, hamsters were exposed to constant light (LL; approximately 250 lx) for 1-3 days, which suppresses locomotor activity and eliminates the daily rhythm of SCN 5-HT release measured by microdialysis. Groups subjected to brief LL or maintained under a light/dark cycle (LD) received phase-resetting treatments with the 5-HT(1A,7) agonist (+/-)-2-dipropyl-amino-8-hydroxyl-1,2,3,4-tetrahydronapthalene (8-OH-DPAT) or sleep deprivation (SD). Animals were released to constant darkness at the start of the treatments. Phase advances to 8-OH-DPAT and SD during the day were 11 and 3 h for LL vs. 2 and 1 h for LD, respectively. Phase delays during the night were -12 and -5 h for LL vs. no responses for LD, respectively. Phase-transition curves for both LL treatments had slopes approximating 0, indicative of Type 0 phase resetting. For all treatments, the degree of locomotor suppression by LL was not correlated with the phase shift magnitude. Re-establishing locomotor activity by overnight food deprivation did not prevent potentiated shifting to SD. However, re-establishing peak extracellular 5-HT levels by intra-SCN 5-HT reverse microdialysis perfusion in LL did significantly reduce potentiated 8-OH-DPAT phase advances. Constant light also enhanced intra-SCN NPY-induced phase advances during the day (6 vs. 2 h for LD). These results suggest that LL promotes Type 0 phase resetting by supersensitizing 5-HT and/or NPY postsynaptic responses and possibly by attenuating the amplitude of the circadian pacemaker, thus enhancing circadian clock resetting nonspecifically.     

Paradoxical effects of NPY in the suprachiasmatic nucleus

The circadian clock in the suprachiasmatic nucleus (SCN) is synchronized by the 24 h, light : dark cycle, and is reset by photic and non-photic cues. The acute effects of light in the SCN include the increase of mRNA levels of the circadian clock gene Per1 and a dramatic reduction of pineal melatonin. Neuropeptide Y (NPY), which appears to mediate the phase-resetting effects of non-photic stimuli, prevents the ability of light, and stimuli that mimic light, to phase shift the circadian clock when injected into the SCN. The purpose of the present study was to determine if NPY inhibits the ability of light to suppress pineal melatonin. Surprisingly, NPY injected into the SCN of hamsters mimicked the effects of light by suppressing pineal melatonin levels. To confirm that NPY inhibited the effects of light on the induction of Per1 mRNA levels, Per1 mRNA levels in the SCN were measured in these same animals. NPY significantly reduced Per1 mRNA levels induced by the light pulse. The suppression of melatonin by NPY appears to be mediated by the same subtype of NPY receptors in the SCN that mediate the modulation of phase shifts. Injection of Y5 receptor agonists mimicked the effects of NPY on pineal melatonin, while injection of a Y2 agonist did not. Thus, these data are the first to demonstrate the paradoxical effects of NPY within the SCN. NPY mimics the effects of light on pineal melatonin and inhibits the effects of light on the induction of Per1 mRNA.     

Melatonin influences Fos expression in the rat suprachiasmatic.

Administration of the pineal hormone melatonin to rats induces expression of Fos, the protein product of the c-fos proto-oncogene, in the suprachiasmatic nucleus (SCN), the putative biological clock of mammals. Expression of the Fos protein is dependent on circadian phase: injections in the late subjective night (circadian time (CT) 22) induce Fos expression in cells within the ventral SCN whereas injections during the subjective day are ineffective. Since melatonin injections in the late subjective day have previously been shown to phase advance circadian rhythms, these results indicate that phase-advances of the circadian system can occur without increased expression of Fos protein in the SCN, at least at levels detectable by immunohistochemistry. In support of in situ hybridization histochemical evidence obtained previously, immunocytochemical data from vehicle-injected control rats suggest that the Fos protein undergoes an endogenous fluctuation with peak levels in the SCN occurring during the subjective night. These observations indicate that melatonin can affect immediate early gene expression within the SCN.     

TTX blocks baclofen-induced phase shifts of the mammalian circadian pacemaker in vitro

The mammalian circadian pacemaker, located in the suprachiasmatic nucleus (SCN), expresses 24-h rhythms when isolated in vitro. The GABA_A agonist, muscimol, induces phase advances during the mid-subjective day, while the GABA_B agonist, baclofen, induces both daytime phase advances and nighttime phase delays. Here, we present evidence that tetrodotoxin (TTX) completely blocks baclofen-induced phase shifts in vitro, but does not block in vitro phase advances induced by muscimol. These results suggest that GABA_A, but not GABA_B, receptors are located on SCN pacemaker cells.     

A serotonin agonist phase-shifts the circadian clock in the suprachiasmatic nuclei in vitro

The mammalian circadian pacemaker in the suprachiasmatic nuclei (SCN) receives a large serotonergic (5-HTergic) projection from the raphe nuclei. Whether the SCN pacemaker can be modulated by this afferent projection is a question of considerable theoretical and practical interest. In this study we investigated whether the 5-HT agonist, quipazine, can reset the phase of the SCN clock when it is isolated in vitro. Our results show that 1 h treatments with quipazine induce robust phase shifts in vitro, and that this effect depends upon the circadian time of treatment. We further show that the ability of quipazine to induce phase shifts is dose-dependent. These results suggest that the SCN circadian pacemaker is sensitive to 5-HTergic stimulation, and therefore that the 5-HTergic projection to the SCN may play a role in modulating the phase of the SCN clock in the intact animal.     

Resetting the circadian clock in cultured Xenopus eyecups: regulation of retinal melatonin rhythms by light and D2 dopamine receptors.

A circadian oscillator is located within the eye of Xenopus laevis. This oscillator regulates retinal melatonin synthesis, stimulating it at night. The primary goal of the studies reported here was to define input pathways to this circadian oscillator as a step toward identification of circadian clock mechanisms. A flow-through superfusion culture system was developed to monitor circadian rhythms of melatonin release from individual eyecups. This system was used to determine the effects of light and dopaminergic agents on melatonin production and on the phase of the circadian oscillator. Six hour light pulses suppressed melatonin production and reset the phase of the free-running melatonin rhythm. Light pulses caused phase delays when applied during the early subjective night, phase advances when applied during the late subjective night, and no phase shift when applied during the subjective day. Dopamine receptor agonists mimicked light in suppressing melatonin release and resetting the phase of the circadian rhythm. The phase-response relationship for phase shifts induced by quinpirole, a D2 dopamine receptor agonist, was similar to that for phase shifts induced by light. Pharmacological analysis with selective catecholamine receptor agonists and antagonists indicated that there are pathways to the melatonin-generating system and the circadian oscillator that include D2 dopamine receptors. A D2 receptor antagonist, eticlopride, completely blocked the effects of dopamine on melatonin release and on circadian phase. However, eticlopride did not alter similar effects induced by light, indicating that dopamine-independent pathways exist for light input to these systems. The effects of light and quinpirole on melatonin release and circadian phase were not additive, indicating that the pathways converge. These pathways to the circadian oscillator in the retina present new avenues for pursuit of cellular circadian clock mechanisms.     

Serotonin antagonists do not attenuate activity-induced phase shifts of circadian rhythms in the Syrian hamster

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-HT_1/2 antagonist; i.p.), (+)-WAY100135 (5-HT_1A postsynaptic antagonist, which may also reduce 5-HT release by an agonist effect at 5-HT_1A raphe autoreceptors; i.p.), NAN-190 (5-HT_1A postsynaptic antagonist, which also reduces 5-HT release via an agonist effect at 5-HT_1A raphe autoreceptors; i.p.) and ritanserin (5-HT_2/7 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.     

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.     

Hamster circadian rhythms are phase-shifted by electrical stimulation of the geniculo-hypothalamic tract

The suprachiasmatic nuclei (SCN) contain the major pacemaker for mammalian circadian rhythms. The SCN receive photic input both directly, via the retinohypothalamic tract (RHT), and indirectly, via the geniculohypothalamic tract (GHT), which originates in cells in the intergeniculate leaflet (IGL) and anterior portions of the ventral lateral geniculate nucleus (vLGN). We tested whether electrical stimulation of the GHT would induce phase shifts in wheel-running activity rhythms of Syrian hamsters housed in continuous darkness or continuous illumination. In both lighting conditions, electrical stimulation of the GHT induced mainly phase advances when given during the late subjective day and small phase delays when given during the late subjective night and early subjective day. Stimulation in the thalamus outside the GHT failed to produce similar phase shifts. Repeated daily stimulation had only a weak entraining effect on the activity rhythm. Activation of GHT neurons appears to influence the pacemaker for activity rhythms in a phase-dependent manner.     

Tetrodotoxin resets the clock

In mammals, circadian rhythms are driven by a pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus. We measured the rhythm of arginine vasopressin release in rat organotypic SCN slices following application of tetrodotoxin (TTX) or N-methyl-D-aspartate (NMDA) at various times throughout the circadian cycle. TTX resets the clock in a manner similar to dark pulses. A 4-h application of TTX starting in mid subjective day, at around circadian time (CT) 7.0, induced phase advances, while TTX treatment started in early subjective morning, at about CT 2.0, induced phase delays. On the other hand, NMDA resets the clock in a manner similar to a light pulse; that is, NMDA treatment in the early evening induced phase delays while treatment in the late night induced phase advances. The data indicate that deprivation of neuronal firing changes the circadian rhythm.     

SNC 80, a delta-opioid agonist, elicits phase advances in hamster circadian activity rhythms

Non-photic stimuli administered to hamsters during the subjective day can cause phase advances in circadian wheel running activity. It is believed that afferent projections from the intergeniculate leaflet of the thalamus to circadian pacemaker cells within the suprachiasmatic nucleus mediate the phase shifting effects of some non-photic stimuli. In hamsters, many of the intergeniculate leaflet afferents contain enkephalin, yet the role of opioids in producing non-photic phase shifts in hamsters has not been reported. In the present study, we show that SNC 80, an agonist for the delta opioid receptor subtype, will phase advance hamster wheel running activity rhythms when administered late in the subjective day. These results indicate that opioids may be involved in modulating the circadian pacemaker in hamsters.     

Melatonin directly resets the rat suprachiasmatic circadian clock in vitro.

The environmental photoperiod regulates the synthesis of melatonin by the pineal gland, which in turn induces daily and seasonal adjustments in behavioral and physiological state. The mechanisms by which melatonin mediates these effects are not known, but accumulating data suggest that melatonin modulates a circadian biological clock, either directly or indirectly via neural inputs. The hypothesis that melatonin acts directly at the level of the suprachiasmatic nucleus (SCN), a central mammalian circadian pacemaker, was tested in a rat brain slice preparation maintained in vitro for 2-3 days. Exposure of the SCN to melatonin for 1 h late in the subjective day or early subjective night induced a significant advance in the SCN electrical activity rhythm; at other times melatonin was without apparent effect. These results demonstrate that melatonin can directly reset this circadian clock during the period surrounding the day-night transition.     

Serotonin‐induced phase advances of SCN neuronal firing in vitro: A possible role for 5‐HT5A receptors?

Spontaneous firing rates of neurons in the suprachiasmatic nuclei (SCN) follow a consistent pattern, peaking near the midpoint of the light phase in a 12:12 light/dark schedule, and repeating this brief period of increased activity in subsequent circadian cycles. These carefully timed fluctuations reflect the output signal of the SCN, long recognized as the site of the endogenous biological clock in mammals. In rat hypothalamic slices, bath incubations of 8-OH-DPAT had previously been shown to elicit phase advances when applied at ZT6 (or 6 h following the onset of light), an action that could readily be attributed to 5-HT7 receptor activation. The present studies set out with the simple goal of establishing that the same receptor mechanism was responsible for the phase-shifting actions of 5-HT itself. Surprisingly, the phase advances elicited by 5-HT (0.5 microM, 1 h) at ZT6 were reduced by one 5-HT7 antagonist, ritanserin (10 microM), but not by another, mesulergine (10 microM). Receptor binding studies demonstrated a 25-fold greater affinity of ritanserin for h5-HT5A sites compared to mesulergine (Ki = 71 nM vs. 1,800 nM), an observation suggestive of a 5-HT5A mechanism for 5-HT and consistent with earlier observations of robust labeling of 5-HT5A sites in the SCN. 5-HT generated by the addition of L-tryptophan (10 microM, 1 h) to the slices displayed the same pattern of sensitivity, that is, blockade by ritanserin but not by mesulergine. Rp-cAMPS, a cAMP antagonist, failed to block the phase shifts elicited by 5-HT at a concentration (1 microM) previously shown to be effective against 8-OH-DPAT-induced phase shifts, in keeping with the proposed negative coupling of 5-HT5A receptors to cAMP production. Taken together, these results suggest that activation of both 5-HT5A and 5-HT7 receptors can produce phase advances of the circadian clock in vitro when they occur during mid-subjective day.     

Different populations of cells in the suprachiasmatic nuclei express c-fos in association with light-induced phase delays and advances of the free-running activity rhythm in hamsters

Circadian rhythmicity is controlled by a light-entrainable pacemaker located in the suprachiasmatic nuclei (SCN) of the mammalian hypothalamus. Brief light exposure during the subjective night causes phase shifts of the free-running activity rhythm and expression of c-fos-related proteins (Fos) among a population of cells in the hamster SCN. Light exposure (30 lux for 15 min) during the early subjective night (CT13) causes phase delays (-60 +/- 12 min), while exposure at mid-subjective night (CT18) causes phase advances (114 +/- 48 min) of the free-running activity rhythm. Light exposure at mid-subjective day (CT6) does not cause phase alterations of the rhythm. Similarly, only light exposure at CT13 or CT18 induces Fos expression in the SCN. The distribution of Fos-immunoreactive cells in the SCN is more widespread in animals stimulated with light at CT18. In addition, a group of cells located dorsal and anterior to the SCN express Fos only after stimulation at CT18. The data are consistent with the hypothesis that Fos expression represents an event in the signal transduction pathway leading to light-induced alterations in circadian pacemaker function. Furthermore, the data raise the possibility that different populations of cells in the suprachiasmatic hypothalamus may participate in light-induced phase advances and delays of the circadian pacemaker.