Effects of Microinjections of Substance P Into the Suprachiasmatic Nucleus Region on Hamster Wheel-Running Rhythms

The suprachiasmatic nucleus (SCN) receives a direct retinal projection, which in rats includes substance P (SP)-immunoreactive retinal ganglion cells. While SP has been shown to have neurophysiological effects on SCN cells in Syrian hamsters and rats, it is not known what effects SP in the SCN has on circadian rhythms in hamsters. We examined this question using male Syrian hamsters that were implanted with cannulas aimed at the SCN region and maintained in constant dim red lighting conditions. Hamsters received 0.5

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microinjections of saline or SP (500 pmol in saline) at a variety of circadian times (CT). Saline injections had little or no phase-shifting effects at any phase tested. SP had no significant effects at CT4–8, 16–20, or 20–24, but did cause small phase delays of −23.7 ± 7 min (mean ± sem) at CT12–16. In order to examine the dose-response relations of this effect, hamsters were also microinjected with 50 and 2500 pmol of SP at CT12–16. Both the 50 and 2500 pmol doses induced very small phase delays (−14.2 ± 7 min and −18.2 ± 5 min, respectively), indicating no obvious dose dependence within this range. These results do not suggest that SP alone in the SCN mimics light effects on circadian rhythms or is a key neurotransmitter involved in photic entrainment. It remains to be determined whether SP interacts with other transmitters in the SCN to modulate their effects on rhythm phase.     

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.     

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.     

A nonphotic stimulus causes instantaneous phase advances of the light-entrainable circadian oscillator of the Syrian hamster but does not induce the expression of c-fos in the suprachiasmatic nuclei.

The study investigated whether nonphotic cues that alter the phase of overt circadian rhythms do so by causing instantaneous shifts in the underlying, light-sensitive clock. Wheel-running activity in Syrian hamsters was studied under free-running conditions of constant dim red light as an overt marker of circadian phase, the daily onset of activity being defined as circadian time 12 (CT 12). Exposure to a 15 min pulse of bright light at CT 12.20 caused a phase delay in activity onset, whereas pulses delivered at CT 11.20 had no effect upon the overt rhythm. Correlated with their effect on behavior, light pulses delivered at CT 12.20 induced expression of c-fos-like immunoreactivity in the retinorecipient regions of the suprachiasmatic nuclei of the hypothalamus (SCN), whereas pulses delivered at CT 11.20 had no effect upon the expression of c-fos. Expression of this immediate-early gene therefore provided a second marker of circadian phase, because its induction by light is closely correlated with the onset of subjective night (CT 12). To establish a suitable protocol for nonphotic shifts of the activity rhythm, animals were handled and received a subcutaneous injection of saline at different circadian phases. Injections at CT 8 or CT10 caused an immediate bout of wheel-running activity, and a consequent phase advance in the activity rhythm as assessed by the earlier onsets of activity in successive days. Handling and injections at other circadian phases were without effect. Despite shifting the overt rhythm, these procedures at CT 10 did not lead to the expression of c-fos in the SCN.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of Circadian Neuropeptides in the Hypothalamus of Adult Mice is Affected by Postnatal Light Experience

The suprachiasmatic nuclei (SCN) of the hypothalamus are the principal pacemaker in mammals, controlling daily, circadian rhythms in physiology and behaviour. Environmental light during development has long-term effects on circadian behaviour, but it is still unclear what the relevant adaptations within the brain are. In the present study, we examined the manifestation of the circadian rhythm of locomotor activity, and the expression of arginine-vasopressin (AVP) and vasointestinal polypeptide (VIP) in the SCN of adult mice reared under different light environments during the suckling period, and synchronised to light/dark cycles after weaning. We found that animals reared under constant light had higher amplitude and more stable activity rhythms, together with lower levels of VIP- and AVP-immunostaining in the SCN, compared to mice reared under light/dark cycles or constant darkness. Differences in AVP expression were also found in the paraventricular nucleus and the supraoptic nucleus, two brain areas which receive SCN projections. These results indicate that the postnatal light experience may affect clock function and clock output, and suggest implications for the control of hormonal homeostasis and circadian behaviour.     

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.     

The effects of GABA and benzodiazepines on neurones in the suprachiasmatic nucleus (SCN) of Syrian hamsters

Administration of benzodiazepines at appropriate times in the circadian cycle induce phase-shifts in circadian locomotor activity. The possibility that benzodiazepine-induced shifts are mediated at the level of the suprachiasmatic nuclei (SCN), identified as the circadian pacemaker in mammals, was examined electrophysiologically. Extracellular recordings were made from Syrian hamster (Mesocricetus auratus) hypothalamic SCN neurones in vitro to assess (1) the effects of gamma-aminobutyric acid (GABA) on SCN neuronal activity and (2) the effects of benzodiazepines (chlordiazepoxide and flurazepam) on GABA-evoked responses. Of 93 SCN cells tested, 86 were suppressed by iontophoresed GABA (20 mM) in a current(dose)-dependent manner, while 6 were unaffected; suppression was found during both the projected light and dark phases of the circadian cycle. Application of bicuculline methiodide alone elevated mean discharge activity, while GABA-evoked suppressions were blocked by bicuculline (n = 9/11 cells). Iontophoresis of chlordiazepoxide or flurazepam (20 mM; 1-10 nA) alone produced a current(dose)-dependent prolonged suppression of cell firing which was antagonised by bicuculline. These results indicate that benzodiazepine/GABA-evoked responses are at least partially mediated by GABAA receptors within the SCN and suggest that SCN may be a possible locus for the action of benzodiazepines in their induction of phase-shifts in circadian function.     

损毁中缝背核或注射对氯苯丙氨酸对小鼠踏转轮运动昼夜节律的影响

哺乳动物下丘脑视交叉上核（ＳＣＮ）在昼夜节律的发生和调节中起着非常重要的作用。它大量接受来自中缝背核（ＤＲ）神经元的神经支配。本实验结果表明，不论在正常光照条件下，或者在连续光照和连续黑暗条件下，损毁ＤＲ后，小鼠踏转轮运动的昼夜节律消失；注射５—ＨＴ台成抑制剂对氯苯百氨酸后，踏转轮运动的节律也消失，但在注射后第７天起开始恢复。结果提示，从ＤＲ到ＳＣＮ的５－ＨＴ能神经传递可能参与踏转轮活动的昼夜节律调节。     

Regulation of the vgf gene in the golden hamster suprachiasmatic nucleus by light and by the circadian clock

By using in situ hybridization in the golden hamster brain, we have found that vgf mRNA levels are induced as a response to light stimulation in the suprachiasmatic nuclei (SCN), the site of the mammalian circadian pacemaker. The induction exhibits delayed kinetics relative to known light-induced immediate early genes: induction of vgf mRNA occurs over a period of 3 to 9 hours after light exposure. Photic induction of vgf expression does not occur in the paraventricular nucleus (PVN) of the hypothalamus, though this nucleus expresses vgf at the mRNA and protein levels. Photic induction of vgf in the SCN occurs only at circadian times when light also causes a phase shift of the circadian system. The irradiance threshold of vgf induction in the SCN closely matches that of the behavioral phase shifting response. In addition, basal expression of vgf in the SCN, but not in the PVN, exhibits a circadian rhythm in constant darkness. The photic induction and circadian rhythm of vgf expression are anatomically separated in the caudal and rostral portions of the SCN, respectively. These results represent the first example of a delayed response to light relative to light-induced immediate early genes at the mRNA level in the SCN. J. Comp. Neurol. 378:229–238, 1997. © 1997 Wiley-Liss, Inc.     

Nitric oxide synthase inhibitor blocks light-induced phase shifts of the circadian activity rhythm, but not c-fos expression in the suprachiasmatic nucleus of the Syrian hamster

Circadian rhythms in mammals are entrained to the environmental light cycle by daily adjustments in the phase of the circadian pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Brief exposure of hamsters maintained under constant darkness to ambient light during subjective nighttime produces both phase shifts of the circadian activity rhythm and characteristic patterns of c-fos protein (Fos) immunoreactivity in the SCN. In this study, we demonstrate that light-induced phase shifts of the circadian activity rhythm are blocked by intracerebroventricular (i.c.v.) injection of the competitive nitric oxide synthase (NOS) inhibitor,N-nitro-l-arginine methyl ester (l-NAME), but not by the inactive isomer,d-NAME. The effects ofl-NAME are reversible and dose-related, and are countered by co-injection of arginine, the natural substrate for NOS. While effects on behavioral rhythms are pronounced, similar treatment does not alter the pattern of light-induced Fos immunoreactivity in the SCN. These results suggest that nitric oxide is a component of the signal transduction pathway that communicates photic information to the SCN circadian pacemaker, and that nitric oxide production is either independent of, or downstream from, pathways involved in induction of c-fos expression.     

Stimulation of BIT induces a circadian phase shift of locomotor activity in rats

Circadian rhythms of mammals are generated by a circadian oscillation of master pacemaker genes in the suprachiasmatic nucleus of the hypothalamus (SCN), and entrained by environmental factors such as 24-h light-dark cycles. We have previously shown that light exposure during the dark period enhanced tyrosine phosphorylation of brain immunoglobulin-like molecule with tyrosine-based activation motifs (BIT) in the rat SCN. To elucidate the functional roles of BIT in the circadian clock, we stimulated BIT using an anti-BIT monoclonal antibody (mAb) 1D4, which reacts with its extracellular region and induces phosphorylation of its intracellular tyrosine residues. Administration of mAb 1D4 into the third cerebral ventricle induced tyrosine phosphorylation of BIT in the SCN. Behavioral analyses showed that the SCN-injection of the antibody at CT15 induced a phase delay of the circadian rhythm of locomotor activity, and that at CT20 induced a phase advance. Pretreatment with MK801, a non-competitive antagonist of NMDARs, diminished the 1D4-induced phase shift at CT20, but not at CT15. These results suggest that BIT is involved in the entrainment of circadian rhythms through the function of NMDARs and non-NMDARs.     

Regional Distribution of Iodomelatonin Binding Sites within the Suprachiasmatic Nucleus of the Syrian Hamster and the Siberian Hamster

The pineal hormone melatonin is a potent regulator of seasonal and circadian rhythms in vertebrates. In order to characterize potential target tissues of melatonin, the distribution of iodomelatonin (IMEL)-binding sites was examined within neurochemically and anatomically defined subdivisions of the suprachiasmatic nucleus (SCN), a structure necessary for seasonal and circadian rhythms in mammals. Studies were carried out in both the adult Syrian (Mesocricetus auratus) and Siberian (Phodopus sungorus) hamster. The retinoreceptive zone of the SCN was identified anatomically by immunocytochemical (ICC) visualization of cholera toxin B subunit tracer (ChTB-ir) following its intra-ocular injection. Photically-responsive SCN cells were identified by immunostaining for the protein product of the immediate-early gene c-fos (Fos-ir) following exposure of the animal to light. The non-photoresponsive zone of the SCN was identified using in situ hybridization (ISH) for arginine vasopressin (AVP) mRNA, whilst sites of IMEL-binding in the SCN were identified by in vitro film autoradiography using the specific ligand 2-[¹²⁵l]-iodomelatonin. To compare directly the distribution of IMEL-binding sites and one of the functional zones of the nucleus, alternate serial coronal sections through the SCN were processed for autoradiography for IMEL and one of the following: ICC for ChTB-ir or Fos-ir, or ISH for AVP mRNA. Overall, the regional distribution of the various markers within the SCN was comparable in the two species. The retinorecipient (ChTB-ir) and photically-responsive (Fos-ir) zones of the SCN mapped together to the middle and caudal thirds of the nucleus, predominantly in its ventro-lateral division. IMEL-binding was present throughout the full rostro-caudal extent of the SCN, but by far the most extensive area of IMEL-binding was in the rostral half of the nucleus, leading to a clear dissociation along the rostro-caudal axis of the principal zone of IMEL-binding and the retinorecipient zone of the nucleus. In the Syrian hamster, in coronal sections of the caudal SCN which did contain significant amounts of both IMEL-binding and Fos-ir, IMEL-binding was confined to the medial zone, distinct from the Fos-ir region of the ventro-lateral SCN. The segregation was less clear-cut in the Siberian hamster where the area of IMEL-binding was more extensive. The dissociation of IMEL-binding and photically-responsive cells in the Syrian hamster was confirmed in a series of sagittal sections which were processed alternately for Fos-ir and IMEL-binding. Whereas Fos-ir was confined to the ventro-lateral SCN, IMEL-binding was concentrated in the medial zone of the nucleus. In both species, mRNA for AVP was found throughout the rostro-caudal extent of the SCN, but the peak area was located in the rostral half, and so was segregated from the principal retinorecipient zone. The distribution of mRNA for AVP along the rostro-caudal and medio-lateral axes was in direct register with the IMEL-binding in both species. These studies suggest that melatonin acts upon pathways within the SCN different to those addressed by light, and that it may influence directly the efferent activity of the nucleus, possibly via an effect on vasopressinergic cells.     

Development of the mammalian circadian clock

The mammalian circadian system is composed of a central clock situated in the hypothalamic suprachiasmatic nucleus (SCN) and peripheral clocks of each tissue and organ in the body. While much has been learned about the pre‐ and postnatal development of the circadian system, there are still many unanswered questions about how and when cellular clocks start to tick and form the circadian system. Most SCN neurons contain a cell‐autonomous circadian clock with individual specific periodicity. Therefore, the network of cellular oscillators is critical for the coherent rhythm expression and orchestration of the peripheral clocks by the SCN. The SCN is the only circadian clock entrained by an environmental light–dark cycle. Photic entrainment starts postnatally, and the SCN starts to function gradually as a central clock that controls physiological and behavioral rhythms during postnatal development. The SCN exhibits circadian rhythms in clock gene expression from the embryonic stage throughout postnatal life and the rhythm phenotypes remain basically unchanged. However, the disappearance of coherent circadian rhythms in cryptochrome‐deficient SCN revealed changes in the SCN networks that occur in postnatal weeks 2–3. The SCN network consists of multiple clusters of cellular circadian rhythms that are differentially integrated by the vasoactive intestinal polypeptide and arginine vasopressin signaling depending on the period of postnatal development.     

NMDA receptor binding in rodent suprachiasmatic nucleus

NMDA receptors are thought to mediate effects of light on circadian rhythms and on immediate-early gene expression in the suprachiasmatic nucleus (SCN), the primary circadian pacemaker in mammals. The present study characterized NMDA receptors in autoradiographs of SCN incubated with the NMDA antagonist [³H]MK-801. In both rat and hamster, [³H]MK-801 binding did not delineate the SCN and was fairly uniformly distributed across the SCN region. Binding levels were unaffected by circadian time, light vs. dark conditions, or enucleation. Scatchard analyses revealed species differences in both receptor number and affinity in the SCN. The [³H]MK-801 binding sites characterized in this study could mediate the NMDA antagonist-sensitive effects of light on the SCN and circadian rhythms.     

N-Methyl-D-aspartate microinjected into the suprachiasmatic nucleus mimics the phase-shifting effects of light in the diurnal Nile grass rat (Arvicanthis niloticus)

Mammals exhibit circadian rhythms in behavior generated by the suprachiasmatic nucleus (SCN). Exposure to light synchronizes the circadian clock to the environmental light:dark cycle through the release of glutamate into the SCN. In nocturnal animals such as Syrian hamsters, direct application of NMDA to the SCN results in phase shifts similar to those produced by exposure to light. This study was designed to determine if light phase shifts the circadian pacemaker of diurnal Nile grass rats (Arvicanthis niloticus) housed in constant darkness by acting on NMDA-type glutamate receptors in the suprachiasmatic nucleus (SCN). N-Methyl-D-aspartate (NMDA; 0, 1, 10, 50, and 100 mM) was administered through guide cannulae aimed at the SCN at circadian times when light induces phase shifts. Maximal phase delays were attained with 50 mM NMDA, and maximal phase advances were seen after 100 mM NMDA. A phase-response curve (PRC) for NMDA, determined by administering NMDA at each hour over the circadian cycle, resembled the PRC to light in this species. These data support the hypothesis that NMDA-type glutamate receptors play a critical role in mediating the phase shifting effects of light in diurnal, as well as nocturnal, animals. In addition, these data suggest that diurnal grass rats may be less sensitive to the phase shifting properties of NMDA than nocturnal rodents.     

Circadian rhythm photic phase shifts are not altered by histamine receptor antagonists

Histamine may play a role in synchronizing endogenous circadian rhythms with exogenous photic cues. Direct application of histamine to the suprachiasmatic nucleus, the site of the mammalian circadian pacemaker, phase shifts the circadian rhythm in neural activity [7]. Intraventricular injections of histamine also phase shift circadian rhythms [14]. The magnitude and direction of the phase shifting effects of histamine depend on circadian phase in a manner similar to light [7,14]. Depletion of brain histamine levels by inhibition of histamine synthesis reduces phase shifts to light [10].     

Twelve-hour days in the brain and behavior of split hamsters

Hamsters will spontaneously ‘split’ and exhibit two rest–activity cycles each day when housed in constant light (LL). The suprachiasmatic nucleus (SCN) is the locus of a brain clock organizing circadian rhythmicity. In split hamsters, the right and left SCN oscillate 12 h out of phase with each other, and the twice‐daily locomotor bouts alternately correspond to one or the other. This unique configuration of the circadian system is useful for investigation of SCN communication to efferent targets. To track phase and period in the SCN and its targets, we measured wheel‐running and FOS expression in the brains of split and unsplit hamsters housed in LL or light–dark cycles. The amount and duration of activity before splitting were correlated with latency to split, suggesting behavioral feedback to circadian organization. LL induced a robust rhythm in the SCN core, regardless of splitting. The split hamsters’ SCN exhibited 24‐h rhythms of FOS that cycled in antiphase between left and right sides and between core and shell subregions. In contrast, the medial preoptic area, paraventricular nucleus of the hypothalamus, dorsomedial hypothalamus and orexin‐A neurons all exhibited 12‐h rhythms of FOS expression, in‐phase between hemispheres, with some detectable right–left differences in amplitude. Importantly, in all conditions studied, the onset of FOS expression in targets occurred at a common phase reference point of the SCN oscillation, suggesting that each SCN may signal these targets once daily. Finally, the transduction of 24‐h SCN rhythms to 12‐h extra‐SCN rhythms indicates that each SCN signals both ipsilateral and contralateral targets.     

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.     

Microinjection of NMDA into the SCN region mimics the phase shifting effect of light in hamsters

Although there is considerable data that glutamate is the primary transducer of photic information to the circadian clock in the suprachiasmatic nucleus (SCN), the ability of glutamate to mimic the phase-shifting effects of light has yet to be demonstrated in vivo. In the present study, microinjections of the glutamate agonist NMDA directly into the SCN of Syrian hamsters induced significant phase delays at circadian time (CT) 13.5 and phase advances at CT 19. These results support the hypothesis that glutamate is the primary neurotransmitter responsible for the transduction of photic information to the SCN.