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.     

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.     

A TTX-sensitive local circuit is involved in the expression of PK2 and BDNF circadian rhythms in the mouse suprachiasmatic nucleus

The roles of a local circuit of electrophysiological activity were examined in the expression of circadian rhythms in the suprachiasmatic nucleus (SCN) of the adult mouse. The neuronal activity of cultured SCN was suppressed with tetrodotoxin (TTX), an Na⁺ channel blocker, and the circadian rhythms in mRNA level were assessed for 13 genes by in situ hybridization. SCN slices were cultured for 3 days and TTX was applied at the peak phase of Per1 expression rhythm. The SCN slices were examined at 4-h intervals up to 32 h after TTX application. The circadian rhythms in the expression of clock genes, Per1 , Per2 , Bmal1 and Cry1 , and of clock-associated genes, Dec1 , Dec2 , Rev-erb α, Rev-erb β and DBP , were not affected by TTX treatment. By contrast, TTX completely abolished the circadian rhythm in the BDNF mRNA level and substantially damped the rhythm in PK2 . The circadian rhythm in the AVP mRNA level was not changed significantly by TTX. These findings indicate that input through Na⁺-channel-dependent electrophysiological activity is not necessary for the expression of the circadian rhythms of clock and clock-associated genes, but necessary for full expression of the circadian rhythms of BDNF and PK2 in the SCN. A TTX-sensitive circuit is involved in the expression of BDNF and PK2 circadian rhythms in the mouse SCN.     

Role of the thalamic intergeniculate leaflet and its 5-HT afferences in the chronobiological properties of 8-OH-DPAT and triazolam in syrian hamster

The 5-HT(1A/7) receptor agonist 8-hydroxy-2-[di-n-propylamino]-tetralin (8-OH-DPAT) has chronobiological effects on the circadian system and, in the Syrian hamster, it is known that serotonergic (5-HT) projections connecting the median raphe nucleus to the suprachiasmatic nuclei (SCN) of the hypothalamus are a prerequisite for the expression of 8-OH-DPAT-induced phase advance of locomotor activity rhythm. We examined the possible involvement of the thalamic intergeniculate leaflet (IGL) in the phase-shifting properties of 8-OH-DPAT injections at CT7. Bilateral electrolytic lesions of the IGL blocked phase-shift responses to 8-OH-DPAT of the activity rhythm. Phase changes induced by injections of 8-OH-DPAT at CT7 and triazolam (Tz), a short-acting benzodiazepine, at CT6 were also studied after bilateral chemical lesion of the 5-HT fibres connecting the dorsal raphe nucleus (DR) to IGL. Destruction of 5-HT fibres within the IGL blocked the phase-shift response to Tz, but not the phase-shift response to 8-OH-DPAT. In conclusion, (a) IGL is essential for the phase-shifting effect of peripheral 8-OH-DPAT injections; (b) 5-HT fibres connecting DR to IGL are necessary for the expression of the phase-shifting effect of Tz but not of 8-OH-DPAT.     

Serotonergic activation potentiates light resetting of the main circadian clock and alters clock gene expression in a diurnal rodent

The main circadian clock, localized in the suprachiasmatic nuclei (SCN) in mammals, can be synchronized by light and non-photic factors such as serotonergic cues. In nocturnal rodents, injections during the subjective day of the 5-HT1A/7 receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) or its positive enantiomer, induce behavioral phase-advances in correlation with decreased expression of two clock genes, Per1/2. In addition, 8-OH-DPAT and the selective serotonin reuptake inhibitor fluoxetine reduce light-induced phase-shifts during the subjective night. Beside the chronobiotic effects of serotonin, changes of serotonergic activity in humans have been involved in mood disorders, that are often associated with alterations in circadian rhythmicity. To get insights into the circadian role of serotonin in diurnal species, we investigated its modulation of the SCN in Arvicanthis ansorgei housed in constant darkness. In striking contrast to nocturnal rodents, daily serotonin content in Arvicanthis SCN peaked during daytime while the sensitivity window of its SCN to (+)8-OH-DPAT occurred essentially during the subjective night. Moreover, fluoxetine produced behavioral phase-advances at circadian time (CT) 0 and CT12. Expression of Per1/2, Rev-erbalpha/beta and Roralpha/beta in the SCN was not modified after fluoxetine or (+)8-OH-DPAT injection. Furthermore, both treatments enhanced light-induced phase-advances and delays. Light responses of Per1 and Rorbeta expression at CT0 and those of Per2 and Rev-erbalpha at CT12 were markedly altered by serotonergic activation. The present findings demonstrate that the serotonergic modulation of the SCN clock appears to differ between nocturnal species and the diurnal Arvicanthis. The potentiating effects of fluoxetine on light resetting in a diurnal rodent may be clinically relevant.     

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.     

Autoradiographic quantification of serotonin1A receptors in rat brain following antidepressant drug treatment

There is growing evidence that the serotonergic (5-HT) system is involved in the pathogenesis and treatment of major depression. The 5-HT receptor subtype involved in the enhancing effect of antidepressant treatments, however, has not been identified. The present study was undertaken to quantify 5-HT1A sites in the rat brain by autoradiography and membrane binding, using the selective ligand [3H]8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT), following long-term antidepressant treatment. Following a 21-day treatment with amitriptyline (10 mg/kg/day), there was a significant increase of [3H]8-OH-DPAT binding measured by autoradiography in the dorsal hippocampus, but there was no change in the nucleus raphe dorsalis; whole brain membrane binding revealed an increase in the number of binding sites, with no change in the affinity for [3H]8-OH-DPAT. Conversely, fluoxetine (10 mg/kg/day), a selective blocker of 5-HT reuptake, and gepirone (10 mg/kg/day), a 5-HT1A agonist, both administered for 21 days, significantly reduced [3H]8-OH-DPAT binding measured by autoradiography in the nucleus raphe dorsalis without altering hippocampal binding sites. The control active treatment with diazepam (2 mg/kg/day) did not alter [3H]8-OH-DPAT binding in the hippocampus or in the nucleus raphe dorsalis. All groups were compared to a 21-day vehicle-treated control group. These results are fully consistent with previous electrophysiological and behavioral studies and suggest that alterations of 5-HT1A receptors might underlie the enhancement of 5-HT neurotransmission by antidepressant treatments.     

Effects of serotonergic agonists on firing rates of photically responsive cells in the hamster suprachiasmatic nucleus

Serotonergic neurons from the midbrain raphe nuclei innervate the suprachiasmatic nucleus (SCN) of the hypothalamus, which functions as the dominant pacemaker for mammalian circadian rhythms. We investigated the effects of serotonin (5-HT) on firing rates of light-activated SCN cells in urethane-anesthetized hamsters. Micro-iontophoretic application of 5-HT or 5-HT_1A agonists (8-OH-DPAT and 5-CT) causeda dose-dependent inhibition of spontaneous activity and photic responses in the majority of SCN cells tested. Application of metergoline alone, a non-selective 5-HT antagonist, slightly increased firing rates during darkness and light exposure, suggesting a tonic serotonergic suppression of SCN activity. Metergoline also effectively attenuated suppression induced by the three 5-HT agonists. In addition, the effects of 8-OH-DPAT were blocked by a 5-HT_1A antagonist, SDZ 216-525. However, other putative 5-HT antagonists were weak (propranolol and NAN-190) or ineffective (ketanserin) in blocking the action of 8-OH-DPAT. These results indicate that serotonin has a potent role in reducing photic effects on retinally activated SCN cells in hamsters, and that these effects are mediated by a receptor with properties similar to those of the 5-HT_1A subtype.     

Photoperiod regulates multiple gene expression in the suprachiasmatic nuclei and pars tuberalis of the Siberian hamster (Phodopus sungorus)

Photoperiod regulates the seasonal physiology of many mammals living in temperate latitudes. Photoperiodic information is decoded by the master circadian clock in the suprachiasmatic nuclei (SCN) of the hypothalamus and then transduced via pineal melatonin secretion. This neurochemical signal is interpreted by tissues expressing melatonin receptors (e.g. the pituitary pars tuberalis, PT) to drive physiological changes. In this study we analysed the photoperiodic regulation of the circadian clockwork in the SCN and PT of the Siberian hamster. Female hamsters were exposed to either long or short photoperiod for 8 weeks and sampled at 2-h intervals across the 24-h cycle. In the SCN, rhythmic expression of the clock genes Per1, Per2, Cry1, Rev-erbalpha, and the clock-controlled genes arginine vasopressin (AVP) and d-element binding protein (DBP) was modulated by photoperiod. All of these E-box-containing genes tracked dawn, with earlier peak mRNA expression in long, compared to short, photoperiod. This response occurred irrespective of the presence of additional regulatory cis-elements, suggesting photoperiodic regulation of SCN gene expression through a common E-box-related mechanism. In long photoperiod, expression of Cry1 and Per1 in the PT tracked the onset and offset of melatonin secretion, respectively. However, whereas Cry1 tracked melatonin onset in short period, Per1 expression was not detectably rhythmic. We therefore propose that, in the SCN, photoperiodic regulation of clock gene expression primarily occurs via E-boxes, whereas melatonin-driven signal transduction drives the phasing of a subset of clock genes in the PT, independently of the E-box.     

Dorsal raphe nuclear stimulation of SCN serotonin release and circadian phase-resetting

Serotonin (5-HT) is strongly implicated in the regulation of mammalian circadian rhythms. However, little is known of the functional relationship between the circadian clock located in the suprachiasmatic nucleus (SCN) and its source of serotonergic innervation, the midbrain raphe nuclei. In previous studies, we reported that electrical stimulation of the dorsal or median raphe nuclei (DRN and MRN, respectively) induced 5-HT release in the SCN. Notably, DRN- but not MRN-stimulated 5-HT release was blocked by the 5-HT(1,2,7) antagonist, metergoline, suggesting that the DRN signals to the SCN indirectly via the activation of a 5-HT-responsive multisynaptic pathway. In the present study, pretreatment with the 5-HT(2,7) antagonist, ritanserin, also significantly inhibited DRN-electrically stimulated SCN 5-HT release. However, pretreatment with the 5-HT(1A) antagonist, NAN-190, or the 5-HT(2) antagonists ketanserin and cinanserin had little suppressive effect on this DRN-stimulated 5-HT release. In complementary behavioral trials, electrical stimulation of the DRN during subjective midday caused a 1.3-h advance in the free-running circadian activity rhythm under constant darkness, which was inhibited by metergoline. Collectively, these results are evidence that: (1) DRN-stimulated 5-HT release in the SCN requires the activation of an intermediate target with receptors having 5-HT(7) pharmacological characteristics; (2) electrical stimulation of the DRN induces phase-resetting of the circadian activity rhythm; and (3) activation of 5-HT receptors is necessary for this DRN-stimulated circadian phase-resetting. In view of the dynamic changes in DRN neuronal activity incumbent with the daily sleep-activity cycle, and its functional linkages to the SCN and intergeniculate leaflet, the DRN could serve to provide behavioral/arousal state information to various sites comprising the brain circadian system.     

MDMA and fenfluramine alter the response of the circadian clock to a serotonin agonist in vitro

The substituted amphetamine drugs, 3,4-methylenedioxymethamphetamine (MDMA or ‘Ecstasy’) and fenfluramine, are known to damage 5-HT neurons in the brain of animals. However, little is known about the drugs’ effects on circadian rhythmicity which is known to be influenced by serotonergic input to the suprachiasmatic nuclei. In the present study, we tested the ability of MDMA and fenfluramine treatment to alter the ability of the circadian clock to reset in response to an agonist of the 5-HT1A and 5-HT7 receptor subtypes soon after treatment with the drugs, and then again at 20 weeks. Coronal hypothalamic slices containing the suprachiasmatic nuclei (SCN) were prepared from rats and 3-min recordings of the firing rate of individual cells were performed throughout a 12-h period. The ability of the 5-HT agonist, 8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT), to cause a phase advance in the firing pattern of SCN neurons was assessed in slices from control animals and those pretreated with MDMA or fenfluramine (10, 15 and 20 mg/kg administered on successive days) 6–10 days or 20 weeks previously. Phase advances to 8-OH-DPAT in the slice were attenuated by pretreatment with MDMA or fenfluramine at both drug-test intervals. Our study demonstrates that repeated exposure to MDMA or fenfluramine may interfere with the ability of serotonin to phase shift the circadian clock in the rat. It is possible that such an effect may be responsible for some of the clinical changes, such as sleep disorders and mood changes, sometimes reported by human users of the substituted amphetamines.     

Muscarinic regulation of Ca2+ oscillation frequency in GH3 cells

The circadian timekeeping system exhibits many functional changes with aging, including a loss of sensitivity to time cues such as systemic injections of the serotonergic agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). In order to elucidate the neurochemical mechanisms responsible for this age-related loss of sensitivity of the circadian pacemaker to serotonin agonists, the present study used quantitative autoradiography to determine whether aging decreases serotonin receptor populations in male Syrian hamsters. Four neuroanatomical regions that regulate circadian timekeeping were studied (the suprachiasmatic nuclei [SCN], the lateral geniculate nuclei [LGN], and the median raphe nucleus [MRN] and dorsal raphe nucleus [DRN]). The specific binding of [3H]8-OH-DPAT to serotonin7 (5-HT7) and serotonin1A (5-HT1A) receptors was investigated by competitive inhibition with ritanserin and pindolol, respectively. The results showed that the SCN, IGL, MRN, and DRN of the male Syrian hamster exhibited specific binding of [3H]8-OH-DPAT to both the 5-HT7 and 5-HT1A receptors, and that the latter receptor subtype is more abundant in all of these regions. At 17-19 months of age, a 50% decrease in 5-HT7 receptors was found in the DRN but not in any other regions. No significant age-related changes in 5-HT1A receptors were observed in any regions examined. The finding that a marked decrease in 5-HT7 receptors occurs in the DRN at the age previously characterized by loss of sensitivity to 8-OH-DPAT suggests that this region and this receptor subtype play important roles in 8-OH-DPAT induction of circadian phase shifts in vivo and that they constitute an important locus of aging in the circadian timing system.     

Modification of 5-HT neuron properties by sustained administration of the 5-HT1A agonist gepirone: electrophysiological studies in the rat brain

The sustained administration of the 5-HT1A agonist gepirone (15 mg/kg/day, s.c.) in the rat produced an initial decrease of the firing activity of dorsal raphe 5-HT neurons which was followed by a progressive recovery to normal after 14 days of treatment. At this point in time, the effect of intravenous lysergic acid diethylamide (LSD) on the firing activity of 5-HT neurons was markedly reduced, whereas those of 8-hydroxy-2-N,N-propylamino-tetralin (8-OH-DPAT) and of gepirone were unchanged; however, the responsiveness of 5-HT neurons to direct microiontophoretic application of 5-HT, LSD, 8-OH-DPAT, and gepirone, but not of GABA, was reduced. The responsiveness of postsynaptic dorsal hippocampus pyramidal neurons to 5-HT, 8-OH-DPAT, and gepirone was not altered by the 14-day gepirone treatment. The effectiveness of the electrical stimulation of the ascending 5-HT pathway in reducing pyramidal neuron firing activity was not significantly modified in rats treated with gepirone for 14 days. Furthermore, this treatment did not alter the function of the terminal 5-HT autoreceptor. It is concluded that the progressive restoration of the firing activity of 5-HT neurons, due to a desensitization of the somatodendritic 5-HT autoreceptor, combined with the direct activation of normosensitive postsynaptic 5-HT1A receptor by gepirone, should result in an augmented tonic activation of postsynaptic 5-HT1A receptors. The progressive appearance of this phenomenon would be consistent with the time course of the clinical anxiolytic, and possibly antidepressant, effects of gepirone.     

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.     

Light and melatonin inhibit in vivo serotonergic phase advances without altering serotonergic-induced decrease of Per expression in the hamster suprachiasmatic nucleus

In the Syrian hamster a serotonergic (5-HTergic) stimulation during daytime acts on the circadian timing system by inducing behavioral phase advances and by decreasing Per1 and Per2 (Period) mRNA levels in the suprachiasmatic nuclei, containing the main circadian clock in mammals. The present study was conducted in Syrian hamsters, housed in constant darkness, to investigate the interactions between light or melatonin with serotonergic stimulation in terms of phase resetting and clock gene expression. Both light exposure and systemic administration of melatonin prior to the injection of a 5-HT(1A/7) receptor agonist, 8-OH-DPAT, in the middle of the day blocked behavioral phase advances. In contrast, neither light nor melatonin treatment during daytime prevented serotonergic-induced down-regulation of Per1 and/or Per2 mRNA levels in the suprachiasmatic nuclei. Taken together, the results show that interactions between afferent cues to the suprachiasmatic nuclei differentially modulate phase adjustment and clock gene expression during daytime.     

Differential responsiveness of the rat dorsal and median raphe 5-HT systems to 5-HT1 receptor agonists and p-chloroamphetamine

The dorsal and median raphe 5-HT neurons give rise to projections that differ in axon morphology and in vulnerability to certain amphetamine derivatives. The present study was undertaken to determine if these two 5-HT systems possess different functional properties. To this end, we studied the effects of selective 5-HT1A or 5-HT1A/5-HT1B receptor agonists and of p-chloroamphetamine on extracellular levels of indoleamines, as measured by differential pulse voltammetry with extracellular levels of indoleamines, as measured by differential pulse voltammetry with electrochemically pretreated carbon fiber electrodes, in cell body and nerve terminal regions of these subsets of 5-HT neurons in the rat brain. The selective 5-HT1A agonist 8-OH-DPAT produced a gradual decrease in the height of the 300 mV oxidation peak in the dorsal raphe and in the frontal cortex, reaching a maximum of 60% 3 h after the i.v. injection of 30 micrograms/kg. However, the same dose of 8-OH-DPAT was ineffective in the median raphe and in the dentate gyrus that receives its 5-HT innervation exclusively from the median raphe. A higher dose of 8-OH-DPAT (150 micrograms/kg, i.v.) produced a 60% decrease in the height of the 300 mV oxidation peak in the median raphe, whereas only a 20% decrease was obtained in the dentate gyrus. In contrast, the non-selective 5-HT1 agonist RU 24,969 (10 mg/kg, i.p.) caused a 70% reduction of the 300 mV peak height in both the dorsal and median raphe and a 50% decrease in both the frontal cortex and the dentate gyrus. Moreover, although a high dose of 8-OH-DPAT (150 micrograms/kg, i.v.) given alone reduced by 20% the amplitude of the oxidative peak in the dentate gyrus, subsequent administration of RU 24,969 (10 mg/kg, i.p.) caused a further 30% diminution of the oxidative peak height. The greater responsiveness of dorsal as compared to median raphe 5-HT systems to 5-HT1A receptor agonists was confirmed in two further series of experiments. First, the microiontophoretic application of 8-OH-DPAT directly onto 5-HT neurons was three times more potent in suppressing the firing rate of dorsal raphe 5-HT neurons than that of their median raphe congeners. Second, 8-OH-DPAT and buspirone were ten and four times, respectively, more potent in decreasing 5-HT synthesis in the frontal cortex than in the hippocampus.(ABSTRACT TRUNCATED AT 400 WORDS)     

5-HT7 receptors mediate serotonergic effects on light-sensitive suprachiasmatic nucleus neurons

Serotonin (5-HT) has been shown to phase shift circadian rhythms in mammals and to affect responses of the circadian system to light, but it is not clear which receptors are involved in these actions. We found that drugs which act as 5-HT_1A receptor agonists suppressed photic responses of hamster SCN cells, but these drugs also exhibit high affinity for the recently cloned 5-HT₇ receptor. We therefore studied the effects of 5-HT agonists and antagonists with differential affinities for 5-HT₇ and 5-HT_1A receptors on responses of hamster SCN cells to retinal illumination. We confirmed that the 5-HT receptor agonists 5-HT, 8-OH-DPAT and 5-CT, dose-dependently reduced photic activation of SCN cells. These effects could be blocked by co-application of antagonists with high affinities for 5-HT₇ receptors: ritanserin or clozapine. The 5-HT_1A/B/D antagonist, cyanopindolol, which is inactive at 5-HT₇ receptors, did not antagonize the actions of 8-OH-DPAT. Selective 5-HT_1A antagonists, WAY100635 and p-MPPI, had weak or no antagonist effects on the responses to 8-OH-DPAT in the SCN, but they effectively antagonized the actions of 8-OH-DPAT in the hippocampus. In the cerebellar cortex where few 5-HT₇ receptors are present, ritanserin failed to antagonize the effects of 8-OH-DPAT. Our results indicate that the 5-HT₇ receptor subtype plays a major role in mediating the effects of 5-HT on photic responses of SCN cells in the hamster.     

Nonphotic entrainment by 5-HT1A/7 receptor agonists accompanied by reduced Per1 and Per2 mRNA levels in the suprachiasmatic nuclei.

In mammals, the environmental light/dark cycle strongly synchronizes the circadian clock within the suprachiasmatic nuclei (SCN) to 24 hr. It is well known that not only photic but also nonphotic stimuli can entrain the SCN clock. Actually, many studies have shown that a daytime injection of 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH DPAT), a serotonin 1A/7 receptor agonist, as a nonphotic stimulus induces phase advances in hamster behavioral circadian rhythms in vivo, as well as the neuron activity rhythm of the SCN in vitro. Recent reports suggest that mammalian homologs of the Drosophila clock gene, Period (Per), are involved in photic entrainment. Therefore, we examined whether phase advances elicited by 8-OH DPAT were associated with a change of Period mRNA levels in the SCN. In this experiment, we cloned partial cDNAs encoding hamster Per1, Per2, and Per3 and observed both circadian oscillation and the light responsiveness of Period. Furthermore, we found that the inhibitory effect of 8-OH DPAT on hamster Per1 and Per2 mRNA levels in the SCN occurred only during the hamster's mid-subjective day, but not during the early subjective day or subjective night. The present findings demonstrate that the acute and circadian time-dependent reduction of Per1 and/or Per2 mRNA in the hamster SCN by 8-OH DPAT is strongly correlated with the phase resetting in response to 8-OH DPAT.