Chronic cocaine causes long-term alterations in circadian period and photic entrainment in the mouse.

The disruptive effects of cocaine on physiological, behavioral and genetic processes are well established. However, few studies have focused on the actions of cocaine on the adult circadian timekeeping system, and none have explored the circadian implications of long-term (weeks to months) cocaine exposure. The present study was undertaken to explore the actions of such long-term cocaine administration on core circadian parameters in mice, including rhythm period, length of the nocturnal activity period and photic entrainment. For cocaine dosing over extended periods, cocaine was provided in drinking water using continuous and scheduled regimens. The impact of chronic cocaine on circadian regulation was evidenced by disruptions of the period of circadian entrainment and intrinsic free-running circadian period. Specifically, mice under a skeleton photoperiod (1-min pulse of dim light delivered daily) receiving continuous ad libitum cocaine entrained rapidly to the light pulse at activity onset. Conversely, water controls entrained more slowly at activity offset through a process of phase-delays, which resulted in their activity rhythms being entrained 147° out of phase with the cocaine group. This pattern persisted after cocaine withdrawal. Next, mice exposed to scheduled daily cocaine presentations exhibited free-running periods under constant darkness that were significantly longer than water controls and which also persisted after cocaine withdrawal. These cocaine-induced perturbations of clock timing could produce chronic psychological and physiological stress, contributing to increased cocaine use and dependence.

Cocaine modulates mammalian circadian clock timing by decreasing serotonin transport in the SCN.

Cocaine abuse disrupts reward and homeostatic processes through diverse processes, including those involved in circadian clock regulation. Recently we showed that cocaine administration to mice disrupts nocturnal photic phase resetting of the suprachiasmatic (SCN) circadian clock, whereas administration during the day induces non-photic phase shifts. Importantly, the same effects are seen when cocaine is applied to the SCN in vitro, where it blocks photic-like (glutamate-induced) phase shifts at night and induces phase advances during the day. Furthermore, our previous data suggest that cocaine acts in the SCN by enhancing 5-HT signaling. For example, the in vitro actions of cocaine mimic those of 5-HT and are blocked by the 5-HT antagonist, metergoline, but not the dopamine receptor antagonist, fluphenazine. Although our data are consistent with cocaine acting through enhanced 5-HT signaling, the nonselective actions of cocaine as an antagonist of monoamine transporters raises the question of whether inhibition of the 5-HT transporter (SERT) is key to its circadian effects. Here we investigate this issue using transgenic mice expressing a SERT that exhibits normal 5-HT recognition and transport but significantly reduced cocaine potency (SERT Met172). Circadian patterns of SCN behavioral and neuronal activity did not differ between wild-type (WT) and SERT Met172 mice, nor did they differ in the ability of the 5-HT1A,2,7 receptor agonist, 8-OH-DPAT to reset SCN clock phase, consistent with the normal SERT expression and activity in the transgenic mice. However, (1) cocaine administration does not induce phase advances when administered in vivo or in vitro in SERT Met172 mice; (2) cocaine does not block photic or glutamate-induced phase shifts in SERT Met172 mice; and (3) cocaine does not induce long-term changes in free-running period in SERT Met172 mice. We conclude that SERT antagonism is required for the phase shifting of the SCN circadian clock induced by cocaine.

Copper chelation and exogenous copper affect circadian clock phase resetting in the suprachiasmatic nucleus in vitro.

Light stimulates specialized retinal ganglion cells to release glutamate (Glu) onto circadian clock neurons of the suprachiasmatic nucleus (SCN). Glu resets the phase of the SCN circadian clock by activating N-methyl-d-aspartate receptors (NMDAR) causing either delays or advances in the clock phase, depending on early- or late-night stimulation, respectively. In addition, these Glu-induced phase shifts require tropomyosin receptor kinase B (TrkB) receptor activity. Previous studies show that copper (Cu) released at hippocampal synapses can inhibit NMDAR activity, and application of exogenous Cu likewise inhibits NMDAR activity. We investigated the effects of Cu in acute SCN brain slices prepared from C57BL/6Nhsd adult, male mice using treatments that decrease or increase available Cu levels in vitro and recorded neuronal activity on the following day. When bath-applied for 10 min at zeitgeber time (ZT) 16 (where ZT0=lights-on in the donor animal colony), the Cu-specific chelators tetrathiomolybdate (TTM) and bathocuproine disulfonate each induce ∼2.5-3-h phase delays in circadian neuronal activity rhythms, similarly to Glu-induced phase delays. Co-application of 10 µM CuCl2, but not 10 µM CoCl2 blocks TTM-induced phase delays. Furthermore, TTM causes phase advances when applied at ZT23. At both application times, TTM-induced phase shifts are blocked by NMDA or TrkB receptor antagonists. Surprisingly, bath-application of 10 µM Cu alone also induces phase shifts in analogous experiments at ZT16 and ZT23. Inhibiting NMDAR does not block Cu-induced phase shifts. TrkB inhibition blocks Cu-induced phase delays but not phase advances. Thus, increasing and decreasing Cu availability appear to shift the SCN clock phase through different mechanisms, at least at the receptor level. We propose that Cu plays a role in the SCN circadian clock by modulating Glu signaling.

Ethanol modulates mammalian circadian clock phase resetting through extrasynaptic GABA receptor activation.

Ethanol modulates the actions of multiple neurotransmitter systems, including GABA. However, its enhancing effects on GABA signaling typically are seen only at high concentrations. In contrast, although GABA is a prominent neurotransmitter in the circadian clock of the suprachiasmatic nucleus (SCN), we see ethanol modulation of clock phase resetting at low concentrations (<50 mM). A possible explanation is that ethanol enhances GABAergic signaling in the SCN through activating GABA(A) receptors that contain the delta subunit (GABA(Adelta) receptors), which are sensitive to low ethanol concentrations. Therefore, we investigated whether ethanol acts on GABA(Adelta) receptors in the SCN. Here we show that acute application of the GABA(Adelta) receptor antagonist, RO15-4513, to mouse hypothalamic slices containing the SCN prevents ethanol inhibition of nighttime glutamate-induced (photic-like) phase delays of the circadian clock. Diazepam, which enhances activity of GABA(A) receptors containing the gamma subunit (GABA(Agamma) receptors), does not modulate these phase shifts. Moreover, we find that RO15-4513 prevents ethanol enhancement of daytime serotonergic (non-photic) phase advances of the circadian clock. Furthermore, diazepam phase-advances the SCN circadian clock when applied alone in the daytime, while ethanol has no effect by itself at that time. These data support the hypothesis that ethanol acts on GABA(Adelta) receptors in the SCN to modulate photic and non-photic circadian clock phase resetting. They also reveal distinct modulatory roles of different GABA(A) receptor subtypes in circadian clock phase regulation.

Acute ethanol modulates glutamatergic and serotonergic phase shifts of the mouse circadian clock in vitro.

Alcohol abuse is associated with sleep problems, which are often linked to circadian rhythm disturbances. However, there is no information on the direct effects of ethanol on the mammalian circadian clock. Acute ethanol inhibits glutamate signaling, which is the primary mechanism through which light resets the mammalian clock in the suprachiasmatic nucleus (SCN). Glutamate and light also inhibit circadian clock resetting induced by nonphotic signals, including 5-HT. Thus, we investigated the effects of acute ethanol on both glutamatergic and serotoninergic resetting of the mouse SCN clock in vitro. We show that ethanol dose-dependently inhibits glutamate-induced phase shifts and enhances serotonergic phase shifts. The inhibition of glutamate-induced phase shifts is not affected by excess glutamate, glycine or d-serine, but is prevented by excess brain-derived neurotrophic factor (BDNF). BDNF is known to augment glutamate signaling in the SCN and to be necessary for glutamate/light-induced phase shifts. Thus, ethanol may inhibit glutamate-induced clock resetting at least in part by blocking BDNF enhancement of glutamate signaling. Ethanol enhancement of serotonergic phase shifts is mimicked by treatments that suppress glutamate signaling in the SCN, including antagonists of glutamate receptors, BDNF signaling and nitric oxide synthase. The combined effect of ethanol with these treatments is not additive, suggesting they act through a common pathway. Our data indicate further that the interaction between 5-HT and glutamate in the SCN may occur downstream from nitric oxide synthase activation. Thus, acute ethanol disrupts normal circadian clock phase regulation, which could contribute to the physiological and psychological problems associated with alcohol abuse.

Serotonergic pre-treatments block in vitro serotonergic phase shifts of the mouse suprachiasmatic nucleus circadian clock.

The suprachiasmatic nucleus (SCN) contains a circadian clock that maintains its time-generating and phase-modulating capacities in vitro. Previous studies report clear differences in the ability of serotonergic stimuli to phase-shift the SCN clock when applied directly to the SCN either in vivo or in vitro: while mice and rat circadian clocks are readily phase-shifted by serotonin (5-HT) or 5-HT agonists applied in vitro, hamster and mice circadian clocks respond inconsistently to 5-HT agonists injected directly into the SCN in vivo. Here we have investigated one possible explanation for these differences: that the SCN isolated in vitro experiences reduced endogenous 5-HT signaling, which increases clock sensitivity to subsequent 5-HT stimulation. For these experiments we treated mouse SCN brain slices with low concentrations of compounds that increase serotonin signaling: 5-HT, a 5-HT agonist (8-OH-DPAT), the 5-HT precursor, l-tryptophan, or the 5-HT re-uptake inhibitor, fluoxetine. Pretreatment with each of these substances completely blocked subsequent phase-shifts induced by mid-subjective day treatment with either 5-HT or 8-OH-DPAT, while they did not block phase-shifts induced by the adenylate cyclase activator, forskolin. Time-course data on l-tryptophan-induced inhibition are consistent with this treatment inducing receptor internalization, while timing of the recovery from inhibition is consistent with receptor reinsertion. Together these data support the hypothesis that SCN clock sensitivity to serotonergic phase modulation is affected by the amount of prior serotonin signaling present in the SCN, and that this signaling alters the density of surface 5-HT receptors on SCN clock neurons.

Glutamate blocks serotonergic phase advances of the mammalian circadian pacemaker through AMPA and NMDA receptors.

The phase of the mammalian circadian pacemaker, located in the suprachiasmatic nucleus (SCN), is modulated by a variety of stimuli, most notably the environmental light cycle. Light information is perceived by the circadian pacemaker through glutamate that is released from retinal ganglion cell terminals in the SCN. Other prominent modulatory inputs to the SCN include a serotonergic projection from the raphe nuclei and a neuropeptide Y (NPY) input from the intergeniculate leaflet. Light and glutamate phase-shift the SCN pacemaker at night, whereas serotonin (5-HT) and NPY primarily phase-shift the pacemaker during the day. In addition to directly phase-shifting the circadian pacemaker, SCN inputs have been shown to modulate the actions of one another. For example, 5-HT can inhibit the phase-shifting effects of light or glutamate applied to the SCN at night, and NPY and glutamate inhibit phase shifts of one another. In this study, we explored the possibility that glutamate can modulate serotonergic phase shifts during the day. For these experiments, we applied various combinations of 5-HT agonists, glutamate agonists, and electrical stimulation of the optic chiasm to SCN brain slices to determine the effect of these treatments on the rhythm of spontaneous neuronal activity generated by the SCN circadian pacemaker. We found that glutamate agonists and optic chiasm stimulation inhibit serotonergic phase advances and that this inhibition involves both AMPA and NMDA receptors. This inhibition by glutamate may be indirect, because it is blocked by both tetrodotoxin and the GABA(A) antagonist, bicuculline.

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.

Melatonin inhibits in vitro serotonergic phase shifts of the suprachiasmatic circadian clock.

The suprachiasmatic (SCN) circadian pacemaker generates 24 h rhythms of spontaneous neuronal activity when isolated in an acute brain slice preparation. The isolated pacemaker also retains its capacity to be reset, or phase-shifted by exogenous stimuli. For example, serotonin (5-HT) agonists advance the SCN pacemaker when applied during mid subjective day, while neuropeptide Y (NPY) agonists and melatonin advance the pacemaker when applied during late subjective day. Previous work has demonstrated interactions between NPY and 5-HT agonists, such that NPY can block 5-HTergic phase advances, while 5-HT agonists do not prevent NPY-induced advances. Due to a number of similarities in the actions of melatonin and NPY in the SCN, it seemed possible that melatonin and 5-HT might interact in the SCN as well. Therefore, in this study potential interactions between melatonin and 5-HT agonists were explored. Melatonin inhibited phase advances by the 5-HT agonist, (+)DPAT, and this inhibition was decreased by co-application of tetrodotoxin. Conversely, melatonin was unable to block phase advances by the cyclic AMP analog, 8BA-cAMP. Finally, neither 5-HT agonists nor 8BA-AMP were able to block melatonin-induced phase advances. These results demonstrate a clear interaction between melatonin and 5-HT in the SCN, and suggest that melatonin and NPY may play similar roles with respect to modulating the phase of the SCN circadian pacemaker in rats.

Neuropeptide Y blocks GABAB-induced phase-shifts of the suprachiasmatic circadian clock in vitro.

The mammalian circadian clock in the suprachiasmatic nucleus (SCN) generates 24-h rhythms of neuronal activity in vitro. We have previously shown that the GABAB agonist baclofen resets the SCN pacemaker in vitro in a phase-dependent manner: advances are induced at zeitgeber time (ZT) 6 and delays are induced at ZT 22. We have also previously shown that neuropeptide Y (NPY) phase-shifts the SCN clock when applied at ZT 10 but not at other times. Here, we show that NPY blocks the baclofen-induced phase-shifts at ZT 6 and ZT 22. The inhibition by NPY appears dose-dependent, and a higher concentration of NPY is required for complete inhibition of the baclofen-induced phase-advances than the phase-delays. Conversely, NPY-induced phase-shifts at ZT 10 are unaffected by co-application of baclofen. These results are consistent with previous findings showing that NPY blocks in vitro phase-shifts induced by a variety of neuromodulators during both the daytime and nighttime.

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 5HT1A/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.

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.

In vitro circadian rhythms of the mammalian suprachiasmatic nuclei: comparison of multi-unit and single-unit neuronal activity recordings.

The circadian clock in the mammalian suprachiasmatic nuclei (SCN) expresses 24-h rhythms when isolated in vitro. Numerous studies have demonstrated that recordings of SCN single-unit neuronal activity (SUA), when expressed as a population rhythm, can be used to reliably estimate SCN circadian clock phase in vitro. The main disadvantage of this technique is its laborious nature. Thus, the present experiments were designed to investigate whether in vitro multi-unit neuronal activity (MUA) recordings from the SCN could reliably substitute for SUA recordings. The results show that an MUA rhythm can be recorded from rat SCN for 3 days in vitro but that this rhythm is extremely variable; times of peak MUA in control experiments vary by 7 to 9 h each day. They also show that several serotonergic agents previously shown to consistently advance the SUA rhythm 2 to 3 h when applied during the day induce apparent advances in the MUA rhythm in some experiments; in other cases, however, there appears to be a delay or no change in the phase of the rhythm. Thus, the mean change in time of peak seen after these treatments was an advance of about 1 h. Finally, the results show that glutamate and optic chiasm stimulation applied during early subjective night can induce apparent delays in the MUA rhythm. The results of these experiments were less variable, so that the overall effect was a delay in peak MUA of 2.5 to 3.5 h. Nevertheless, these experiments still exhibited more variability than that generally seen in SUA experiments. Taken together, these results indicate that MUA recordings of the SCN exhibit significantly more variability than do SUA recordings. The extent of this variability leads to the conclusion that, using the techniques and equipment outlined here, MUA recordings are not an adequate substitute for SUA recordings when trying to estimate the phase of the SCN circadian clock.

c-fos mRNA in the suprachiasmatic nuclei in vitro shows a circadian rhythm and responds to a serotonergic agonist.

The mammalian suprachiasmatic nuclei (SCN) contain a circadian clock that produces approximately 24 h rhythms of physiology and behavior even during constant dark. Under such conditions, light stimuli applied during the subjective night induce phase shifts of circadian rhythms and increase immediate early gene expression (c-fos) in the SCN. In vitro preparations of the SCN continue to show circadian rhythms of metabolic rate and neuronal firing rates, which can be phase shifted by non-photic stimuli. This study was designed to investigate whether the SCN display a rhythm of c-fos mRNA levels in vitro and whether quipazine, which phase-shifts the SCN circadian clock, induces c-fos expression in vitro. Levels of c-fos mRNA were found to be significantly higher in the subjective day than subjective night in the SCN in vitro. This rhythm parallels other in vivo and in vitro rhythms in SCN metabolic and neuronal activity and is consistent with previous in vivo work showing higher daytime levels of Fos-like immunoreactivity in animals maintained under constant dark conditions. Quipazine treatment during the subjective day (which phase-advances the circadian rhythm of neuronal firing in the SCN) decreased c-fos mRNA levels in the dorsomedial but not ventrolateral SCN, but quipazine did not affect c-fos levels when administered at night. This effect is consistent with serotonergic agonists inhibiting SCN neuronal activity and is the first evidence that a non-photic phase-shifting stimulus alters c-fos in the SCN at a phase-appropriate time.

A possible glial role in the mammalian circadian clock.

The primary mammalian circadian clock is located within the suprachiasmatic nuclei (SCN), but the cellular organization of the clock is not yet known. We investigated the potential role of glial cells in the clock mechanism by determining whether disrupting glial activity affects the in vitro circadian rhythm of neuronal activity and the in vivo circadian activity rhythm in rats. We used two agents (octanol and halothane) that block gap junctions, and one (fluorocitrate) that inhibits glial metabolism. All three agents disrupted the circadian pattern of neuronal activity. Octanol flattened the rhythm at the highest concentration (200 microM) and induced a small phase delay at a lower concentration (66 microM). Halothane and fluorocitrate induced ultradian rhythmicity. Fluorocitrate injected into the SCN of an intact rat induced arrhythmicity for about 1 week, after which the rhythm reappeared with a 1.6 h delay. These results suggest that glia play an important role in the SCN circadian clock.

Serotonergic phase advances of the mammalian circadian clock involve protein kinase A and K+ channel opening.

The mammalian circadian clock located in the suprachiasmatic nuclei (SCN) continues to oscillate when isolated in a brain slice preparation, and can be phase shifted in vitro by a variety of serotonergic (5-HTergic) agents. We have previously shown that 5-HT and a 5-HT agonist, quipazine, induce phase advances in the daytime and phase delays at night; the phase advances are mimicked by the 5-HT1A-selective agonist 8-OH-DPAT, by analogs of cyclic AMP, and by treatments that increase endogenous levels of cyclic AMP. Here we investigated the intracellular pathway through which these daytime phase advances occur. We find that quipazine- and 8-OH-DPAT-induced phase advances are blocked by two inhibitors of the cyclic AMP-dependent protein kinase, PK-A (H8 and Rp-cAMPS) as well as by a variety of K+ channel blockers (BaCl2, apamin, and charybdotoxin). Furthermore, we confirm previous work showing that a cyclic AMP analog induces phase advances in the daytime, and show that these phase advances are also blocked by BaCl2 and apamin. Finally, we show that a K+ ionophore induces similar phase advances in the subjective day, and these phase advances are blocked by Rp-cAMPS. These results indicate that both activation of PK-A and opening of K+ channels are necessary for 5-HT-induced phase advances of the SCN circadian clock. We propose a model that can account for our results.

A novel adenylyl cyclase-activating serotonin receptor (5-HT7) implicated in the regulation of mammalian circadian rhythms.

We report the cloning and characterization of a novel serotonin receptor, designated as 5-HT7, which is coupled to the stimulation of adenylyl cyclase. 5-HT7 mRNA is expressed discretely throughout the CNS, predominantly in the thalamus and hypothalamus. 5-HT7 has a unique pharmacological profile that redefines agonist and antagonist classification of ligands previously thought to be "selective." The circadian phase of spontaneous neuronal activity of the rat suprachiasmatic nucleus of the hypothalamus advances in response to serotonin ligands with a pharmacological profile consistent exclusively with that of 5-HT7. These findings suggest a physiological role in the regulation of circadian rhythms for one subtype of serotonin receptor, 5-HT7, and provide a pharmacological test to evaluate its role in other neuronal systems.

Serotonin and the mammalian circadian system: I. In vitro phase shifts by serotonergic agonists and antagonists.

The primary mammalian circadian clock, located in the suprachiasmatic nuclei (SCN), receives a major input from the raphe nuclei. The role of this input is largely unknown, and is the focus of this research. The SCN clock survives in vitro, where it produces a 24-hr rhythm in spontaneous neuronal activity that is sustained for at least three cycles. The sensitivity of the SCN clock to drugs can therefore be tested in vitro by determining whether various compounds alter the phase of this rhythm. We have previously shown that the nonspecific serotonin (5-HT) agonist quipazine resets the SCN clock in vitro, inducing phase advances in the daytime and phase delays at night. These results suggest that the 5-HT-ergic input from the raphe nuclei can modulate the phase of the SCN circadian clock. In this study we began by using autoradiography to determine that the SCN contain abundant 5-HT1A and 5-HT1B receptors, very few 5-HT1C and 5-HT2 receptors, and no 5-HT3 receptors. Next we investigated the ability of 5-HT-ergic agonists and antagonists to reset the clock in vitro, in order to determine what type or types of 5-HT receptor(s) are functionally linked to the SCN clock. We began by providing further evidence of 5-HT-ergic effects in the SCN. We found that 5-HT mimicked the effects of quipazine, whereas the nonspecific 5-HT antagonist metergoline blocked these effects, in both the day and night. Next we found that the 5-HT1A agonist 8-OH-DPAT, and to a lesser extent the 5-HT1A-1B agonist RU 24969, mimicked the effects of quipazine during the subjective daytime, whereas the 5-HT1A antagonist NAN-190 blocked quipazine's effects. None of the other specific agonists or antagonists we tried induced similar effects. This suggests that quipazine acts on 5-HT1A receptors in the daytime to advance the SCN clock. None of the specific agents we tried were able either to mimic or to block the actions of 5-HT or quipazine at circadian time 15. Thus, we were unable to determine the type of 5-HT receptor involved in nighttime phase delays by quipazine or 5-HT. However, since the dose-response curves for quipazine during the day and night are virtually identical, we hypothesize that the nighttime 5-HT receptor is a 5-HT1-like receptor.

Serotonin and the mammalian circadian system: II. Phase-shifting rat behavioral rhythms with serotonergic agonists.

The suprachiasmatic nuclei (SCN) receive primary afferents from the median and dorsal raphe, but the role of these projections in circadian timekeeping is poorly understood. Studies of the SCN in vitro suggest that quipazine, a general serotonin (5-HT) receptor agonist, can produce circadian time-dependent phase advances and phase delays in circadian rhythms of neuronal activity. The present study addresses whether quipazine and the selective 5-HT1A receptor agonist 8-OH-DPAT are similarly effective in vivo. Drinking and wheel-running patterns of male Wistar rats individually housed in constant darkness were monitored before and after subcutaneous administration of quipazine (5-10 mg/kg) at either circadian time (CT) 6 or CT 18, with and without running wheels available. Dose-dependent phase advances (20-180 min) were produced at CT 6. Significant phase shifts were not observed at CT 18. CT 6 quipazine-treated animals also showed a sustained and significant shortening of rhythm period (tau) following treatment (-0.28 hr; p < 0.002). tau shortening was inconsistently observed in CT 18 quipazine-treated rats. Neither quipazine-induced phase shifts nor tau effects were dependent on wheel-running activity per se. 8-OH-DPAT delivered via intracerebral ventricular treatment into the third ventricle (5 microliters at 100 microM in saline) produced slightly smaller phase advances (20-90 min) at CT 6, but did not produce phase delays at CT 18 or changes in tau. These findings support in vitro evidence that 5-HT-ergic agonists can phase-shift the circadian pacemaker.

Serotonergic phase shifts of the mammalian circadian clock: effects of tetrodotoxin and high Mg2+.

The mammalian circadian clock in the suprachiasmatic nuclei (SCN) can be phase-shifted in vitro by the serotonin agonist quipazine. Here we show that quipazine resets the SCN clock in the presence of tetrodotoxin or 10 mM Mg2+, treatments that block Na+ action potentials and Ca2+ channels, respectively. These results support the hypothesis that quipazine resets the clock by stimulating receptors located on clock elements rather than on cells afferent to the clock.