Circadian PER2::LUC rhythms in the olfactory bulb of freely moving mice depend on the suprachiasmatic nucleus but not on behaviour rhythms

The temporal order of physiology and behaviour in mammals is regulated by the coordination of the master circadian clock in the suprachiasmatic nucleus (SCN) and peripheral clocks in various tissues outside the SCN. Because the circadian oscillator(s) in the olfactory bulb (OB) is regarded as SCN independent, we examined the relationship between the SCN master clock and the circadian clock in the OB. We also examined the role of vasoactive intestinal peptide receptor 2 in the circadian organization of the OB. We continuously monitored the circadian rhythms of a clock gene product PER2 in the SCN and OB of freely moving mice by means of a bioluminescence reporter and an optical fibre implanted in the brain. Robust circadian rhythms were detected in the OB and SCN for up to 19 days. Bilateral SCN lesions abolished the circadian behaviour rhythms and disorganized the PER2 rhythms in the OB. The PER2 rhythms in the OB showed more than one oscillatory component of a similar circadian period, suggesting internal desynchronization of constituent oscillators. By contrast, significant circadian PER2 rhythms were detected in the vasoactive intestinal peptide receptor 2‐deficient mice, despite the substantial deterioration or abolition of circadian behavioural rhythms. These findings indicate that the circadian clock in the OB of freely moving mice depends on the SCN master clock but not on the circadian behavioural rhythms. The circadian PER2::LUC rhythm in the cultured OB was as robust as that in the cultured SCN but reset by slice preparation, suggesting that culturing of the slice reinforces the circadian rhythm.     

Differential responses of circadian Per2 rhythms in cultured slices of discrete brain areas from rats showing internal desynchronisation by methamphetamine

Chronic methamphetamine (MAP) treatment desynchronises the behavior rhythms of rats from light–dark cycles. Our previous study (Masubuchi et al., 2000) demonstrated the phase reversal of circadian rhythms in clock gene expression in several brain areas of rats treated with MAP. However, for technical reasons, it was not clear whether the phase shifts were the consequence of phase‐shifted behavior rhythms or reflected phase shifts of extra‐suprachiasmatic nucleus (SCN) oscillators in these areas. In the present study, circadian gene expression rhythms in discrete brain areas were continuously monitored in slice cultures of MAP‐treated rats. Methamphetamine was given to rats carrying a Period2‐dLuciferase reporter system via the drinking water for more than 2 weeks. When behavior rhythms were completely phase reversed, the brain was sampled for slice cultures and circadian bioluminescence rhythms were measured for 5 days in the SCN and four areas of the dopaminergic system, the olfactory bulb, caudate putamen, parietal cortex and substantia nigra. The circadian rhythms in the SCN and caudate putamen were not significantly phase shifted, whereas those in the parietal cortex and substantia nigra showed significant phase‐delay shifts of 6–8 h and that in the olfactory bulb showed phase‐advance shifts of ca. 8 h. Neither the period nor the amplitude of the circadian rhythm was changed by MAP treatment. These findings indicate that the extra‐SCN oscillators in several brain areas are desynchronised from the SCN circadian pacemaker by MAP treatment in parallel with the desynchronisation of behavior rhythms in rats. As the direction and extent of phase shifts of circadian rhythms were different among the areas examined, the brain extra‐SCN oscillators responded differentially to MAP.     

Depression‐like behaviour in mice is associated with disrupted circadian rhythms in nucleus accumbens and periaqueductal grey

An association between circadian rhythms and mood regulation is well established, and disturbed circadian clocks are believed to contribute to the development of mood disorders, including major depressive disorder. The circadian system is coordinated by the suprachiasmatic nucleus (SCN), the master pacemaker in the hypothalamus that receives light input from the retina and synchronizes circadian oscillators in other brain regions and peripheral tissues. Lacking the tight neuronal network that couples single‐cell oscillators in the SCN, circadian clocks outside the SCN may be less stable and more susceptible to disturbances, for example by clock gene mutations or uncontrollable stress. However, non‐SCN circadian clocks have not been studied extensively in rodent models of mood disorders. In the present study, it was hypothesized that disturbances of local circadian clocks in mood‐regulating brain areas are associated with depression‐like behaviour in mice. Using the learned helplessness procedure, depression‐like behaviour was evoked in mice bearing the PER2::LUC circadian reporter, and then circadian rhythms of PER2 expression were examined in brain slices from these mice using luminometry and bioluminescence imaging. It was found that helplessness is associated with absence of circadian rhythms in the nucleus accumbens and the periaqueductal grey, two of the most critical brain regions within the reward circuit. The current study provides evidence that susceptibility of mice to depression‐like behaviour is associated with disturbed local circadian clocks in a subset of mood‐regulating brain areas, but the direction of causality remains to be determined.     

Dual regulation of clock gene Per2 expression in discrete brain areas by the circadian pacemaker and methamphetamine‐induced oscillator in rats

Behavioral rhythms induced by methamphetamine (MAP) treatment in rats are independent of the circadian pacemaker in the suprachiasmatic nucleus (SCN). To know the site and mechanism of an underlying oscillation (MAP‐induced oscillator; MAO), extra‐SCN circadian rhythms in the discrete brain areas were examined in rats with and without the SCN. To fix the phase of MAO, MAP was supplied in drinking water at a restricted time of day for 14 days (R‐MAP) and subsequently given ad libitum (ad‐MAP). Plain water was given to the controls at the same restricted time (R‐Water). Clock gene Per2 expression was measured by a bioluminescence reporter in cultured brain tissues. In SCN‐intact rats, MAO was induced by R‐MAP and behavioral rhythms were phase‐delayed from the restricted time under ad‐MAP with relative coordination. Circadian Per2 rhythms in R‐MAP rats were not affected in the SCN but were slightly phase‐advanced in the olfactory bulb (OB), caudate–putamen (CPU) and substantia nigra (SN) as compared with R‐Water rats. Following SCN lesion, R‐MAP‐induced MAO phase‐shifted more slowly and did not show a sign of relative coordination. In these rats, circadian Per2 rhythms were significantly phase‐shifted in the OB and SN as compared with SCN‐intact rats. These findings indicate that MAO was induced by MAP given at a restricted time of day in association with phase‐shifts of the extra‐SCN circadian oscillators in the brain dopaminergic areas. The findings also suggest that these extra‐SCN oscillators are the components of MAO and receive dual regulation by MAO and the SCN circadian pacemaker.     

Urokinase‐type plasminogen activator modulates mammalian circadian clock phase regulation in tissue‐type plasminogen activator knockout mice

Glutamate phase shifts the circadian clock in the mammalian suprachiasmatic nucleus (SCN) by activating NMDA receptors. Tissue‐type plasminogen activator (tPA) gates phase shifts by activating plasmin to generate m(ature) BDNF, which binds TrkB receptors allowing clock phase shifts. Here, we investigate phase shifting in tPA knockout (tPA^?/?; B6.129S2‐Plat^tm1Mlg/J) mice, and identify urokinase‐type plasminogen activator (uPA) as an additional circadian clock regulator. Behavioral activity rhythms in tPA^?/? mice entrain to a light‐dark (LD) cycle and phase shift in response to nocturnal light pulses with no apparent loss in sensitivity. When the LD cycle is inverted, tPA^?/? mice take significantly longer to entrain than C57BL/6J wild‐type (WT) mice. SCN brain slices from tPA^?/? mice exhibit entrained neuronal activity rhythms and phase shift in response to nocturnal glutamate with no change in dose‐dependency. Pre‐treating slices with the tPA/uPA inhibitor, plasminogen activator inhibitor‐1 (PAI‐1), inhibits glutamate‐induced phase delays in tPA^?/? slices. Selective inhibition of uPA with UK122 prevents glutamate‐induced phase resetting in tPA^?/? but not WT SCN slices. tPA expression is higher at night than the day in WT SCN, while uPA expression remains constant in WT and tPA^?/? slices. Casein‐plasminogen zymography reveals that neither tPA nor uPA total proteolytic activity is under circadian control in WT or tPA^?/? SCN. Finally, tPA^?/? SCN tissue has lower mBDNF levels than WT tissue, while UK122 does not affect mBDNF levels in either strain. Together, these results suggest that either tPA or uPA can support photic/glutamatergic phase shifts of the SCN circadian clock, possibly acting through distinct mechanisms.     

Daily exposure to cold phase‐shifts the circadian clock of neonatal rats in vivo

Maternal rhythms entrain the prenatal and neonatal circadian clock in the suprachiasmatic nucleus (SCN) before light entrainment is established. However, the responsible time cues for maternal entrainment are not identified. To examine the role of cyclic changes of ambient temperature in maternal entrainment, blind neonatal rats carrying a clock gene (Per2) bioluminescence reporter were exposed to either of three ambient temperatures (10, 20 or 30 °C) during 6‐h maternal separation in the early light phase. Cold exposure was performed from postnatal day 1 (P1) to P5. On P6, the SCN was harvested and cultured for photometric monitoring of the circadian rhythm in Per2 expression. Here we demonstrate that the daily cold exposure phase‐delayed the circadian Per2 expression rhythms at P6 in a temperature‐dependent manner. Exposure to 10 °C produced the largest phase‐shift of 12.7 h, and exposure to 20 and 30 °C yielded moderate shifts of 4.1 and 4.5 h, respectively. There was no significant difference in the phase‐shifts between the latter two temperatures, indicating that ambient temperature is not the sole factor for the phase‐shift. Behavioral rhythms that developed after weaning reflected the phase‐shift of clock gene expression rhythm in the SCN. These findings indicate that a daily exposure to an ambient temperature of 10 °C during the neonatal period is capable of resetting the circadian clock in the SCN, but other factors yet unidentified are also involved in maternal entrainment.     

Circadian rhythms in the mouse reproductive axis during the estrous cycle and pregnancy

Molecular and behavioral timekeeping is regulated by the circadian system which includes the brain's suprachiasmatic nucleus (SCN) that translates environmental light information into neuronal and endocrine signals aligning peripheral tissue rhythms to the time of day. Despite the critical role of circadian rhythms in fertility, it remains unexplored how circadian rhythms change within reproductive tissues during pregnancy. To determine how estrous cycle and pregnancy impact phase relationships of reproductive tissues, we used PER2::Luciferase (PER2::LUC) circadian reporter mice and determined the time of day of PER2::LUC peak (phase) in the SCN, pituitary, uterus, and ovary. The relationships between reproductive tissue PER2::LUC phases changed throughout the estrous cycle and late pregnancy and were accompanied by changes to PER2::LUC period in the SCN, uterus, and ovary. To determine if the phase relationship adaptations were driven by sex steroids, we asked if progesterone, a hormone involved in estrous cyclicity and pregnancy, could regulate Per2‐luciferase expression. Using an in vitro transfection assay, we found that progesterone increased Per2‐luciferase expression in immortalized SCN (SCN2.2) and arcuate nucleus (KTAR) cells. In addition, progesterone shortened PER2::LUC period in ex vivo uterine tissue recordings collected during pregnancy. As progesterone dramatically increases during pregnancy, we evaluated wheel‐running patterns in PER2::LUC mice. We confirmed that activity levels decrease during pregnancy and found that activity onset was delayed. Although SCN, but not arcuate nucleus, PER2::LUC period changed during late pregnancy, onset of locomotor activity did not correlate with SCN or arcuate nucleus PER2::LUC period.     

Regional pacemakers composed of multiple oscillator neurons in the rat suprachiasmatic nucleus

Regional specificities of the dorsal and ventral regions of the suprachiasmatic nucleus (SCN) were examined to elucidate the structure of multioscillator circadian organization. The circadian rhythms of arginine vasopressin (AVP) and vasoactive intestinal polypeptide (VIP) release, and of electrical activity of individual neurons were measured in an organotypic, static slice culture of the SCN obtained from neonatal rats. Five days after the start of culture, robust circadian rhythms were detected in AVP release with a peak located consistently at the middle of the original light phase, while the 24 h profiles of VIP release were either arrhythmic or rhythmic. In the latter case, a phase delay of 5-7 h was observed in the circadian peak from the AVP rhythm. Multi-channel, extracellular recording revealed that 51 (76.1%) out of 67 firing neurons, examined in the SCN, showed circadian rhythms in their firing rate. The percentage of rhythmic neurons was significantly larger in the dorsal (86.8%) than in the ventral (62.1%) region of the SCN, where the AVP and VIP containing neurons predominate, respectively. Twenty-seven percent of the firing rhythms were almost antiphasic from the majority of rhythms. There was no regional specificity in the distribution of the antiphasic rhythm. These findings, that the dorsal and ventral regions of the SCN both contain circadian pacemakers with different properties that regulate the AVP and VIP release separately, is probably due to differences in the number and, hence, the coupling strength of oscillating neurons.     

Differential responses of circadian Per2 expression rhythms in discrete brain areas to daily injection of methamphetamine and restricted feeding in rats

Behavioral rhythms induced by methamphetamine (MAP) and daily restricted feeding (RF) in rats are independent of the circadian pacemaker in the suprachiasmatic nucleus (SCN), and have been regarded to share a common oscillatory mechanism. In the present study, in order to examine the responses of brain oscillatory systems to MAP and RF, circadian rhythms in clock gene, Period2, expression were measured in several brain areas in rats. Transgenic rats carrying a bioluminescence reporter of Period2‐dLuciferase were subjected to either daily injection of MAP or RF of 2 h at a fixed time of day for 14 days. As a result, spontaneous movement and wheel‐running activity were greatly enhanced following MAP injection and prior to daily meal under RF. Circadian Per2 rhythms were measured in the cultured brain tissues containing one of the following structures: the olfactory bulb; caudate‐putamen; parietal cortex; substantia nigra; and SCN. Except for the SCN, the circadian Per2 rhythms in the brain tissues were significantly phase‐delayed by 1.9 h on average in MAP‐injected rats as compared with the saline‐controls. On the other hand, the circadian rhythms outside the SCN were significantly phase‐advanced by 6.3 h on average in rats under RF as compared with those under ad libitum feeding. These findings indicate that the circadian rhythms in specific brain areas of the central dopaminergic system respond differentially to MAP injection and RF, suggesting that different oscillatory mechanisms in the brain underlie the MAP‐induced behavior and pre‐feeding activity under RF.     

Immortalized cell lines for real-time analysis of circadian pacemaker and peripheral oscillator properties

In the mammalian circadian system, cell‐autonomous clocks in the suprachiasmatic nuclei (SCN) are distinguished from those in other brain regions and peripheral tissues by the capacity to generate coordinated rhythms and drive oscillations in other cells. To further establish in vitro models for distinguishing the functional properties of SCN and peripheral oscillators, we developed immortalized cell lines derived from fibroblasts and the SCN anlage of mPer2 ^Luc knockin mice. Circadian rhythms in luminescence driven by the mPER2::LUC fusion protein were observed in cultures of mPer2 ^Luc SCN cells and in serum‐shocked or SCN2.2‐co‐cultured mPer2 ^Luc fibroblasts. SCN mPer2 ^Luc cells generated self‐sustained circadian oscillations that persisted for at least four cycles with periodicities of ≈24 h. Immortalized fibroblasts only showed circadian rhythms of mPER2::LUC expression in response to serum shock or when co‐cultured with SCN2.2 cells. Circadian oscillations of luminescence in mPer2 ^Luc fibroblasts decayed after 3–4 cycles in serum‐shocked cultures but robustly persisted for 6–7 cycles in the presence of SCN2.2 cells. In the co‐culture model, the circadian behavior of mPer2 ^Luc fibroblasts was dependent on the integrity of the molecular clockworks in co‐cultured SCN cells as persistent rhythmicity was not observed in the presence of immortalized SCN cells derived from mice with targeted disruption of Per1 and Per2 (Per1^ldc/Per2 ^ldc). Because immortalized mPer2 ^Luc SCN cells and fibroblasts retain their indigenous circadian properties, these in vitro models will be valuable for real‐time comparisons of clock gene rhythms in SCN and peripheral oscillators and identifying the diffusible signals that mediate the distinctive pacemaking function of the SCN.     

Recurring circadian disruption alters circadian clock sensitivity to resetting

A single phase advance of the light:dark (LD) cycle can temporarily disrupt synchrony of neural circadian rhythms within the suprachiasmatic nucleus (SCN) and between the SCN and peripheral tissues. Compounding this, modern life can involve repeated disruptive light conditions. To model chronic disruption to the circadian system, we exposed male mice to more than a month of a 20‐hr light cycle (LD10:10), which mice typically cannot entrain to. Control animals were housed under LD12:12. We measured locomotor activity and body temperature rhythms in vivo, and rhythms of PER2::LUC bioluminescence in SCN and peripheral tissues ex vivo. Unexpectedly, we discovered strong effects of the time of dissection on circadian phase of PER2::LUC bioluminescent rhythms, which varied across tissues. White adipose tissue was strongly reset by dissection, while thymus phase appeared independent of dissection timing. Prior light exposure impacted the SCN, resulting in strong resetting of SCN phase by dissection for mice housed under LD10:10, and weak phase shifts by time of dissection in SCN from control LD12:12 mice. These findings suggest that exposure to circadian disruption may desynchronize SCN neurons, increasing network sensitivity to perturbations. We propose that tissues with a weakened circadian network, such as the SCN under disruptive light conditions, or with little to no coupling, for example, some peripheral tissues, will show increased resetting effects. In particular, exposure to light at inconsistent circadian times on a recurring weekly basis disrupts circadian rhythms and alters sensitivity of the SCN neural pacemaker to dissection time.     

Circadian rhythm of Arg–vasopressin contents in the suprachiasmatic nucleus in relation to corticosterone

Circadian rhythms of locomotor activity and adrenal glucocorticoid are controlled by the suprachiasmatic nucleus (SCN), the center of a biological clock, in mammals. Arg–vasopressin (AVP) contents in the SCN play a role in endogenous circadian rhythm during the absence of time cues. The AVP-containing neurons in the SCN are considered to transmit a circadian signal to the other parts of the brain. The circadian rhythms of AVP in the SCN in relation to the plasma corticosterone and locomotor activity were investigated. Under the light–dark cycle, plasma corticosterone levels were reciprocally correlated with the AVP content in the SCN. Under free-running conditions with constant dim light, AVP rhythms were reciprocally synchronized with the locomotor activity. The correlation of AVP with plasma corticosterone is different at different times of the day both under the LD cycle and constant dim light. Dexamethasone (i.p., 0.1 mg/100) increased the AVP contents, and this tendency was significantly greater during the dark period. These results indicate that corticosterone in the blood may regulate the circadian rhythm through AVP variation in the SCN.     

Differential contributions of intra‐cellular and inter‐cellular mechanisms to the spatial and temporal architecture of the suprachiasmatic nucleus circadian circuitry in wild‐type,cryptochrome‐null and vasoactive intestinal peptide receptor 2‐null mutant mice

To serve as a robust internal circadian clock, the cell‐autonomous molecular and electrophysiological activities of the individual neurons of the mammalian suprachiasmatic nucleus (SCN) are coordinated in time and neuroanatomical space. Although the contributions of the chemical and electrical interconnections between neurons are essential to this circuit‐level orchestration, the features upon which they operate to confer robustness to the ensemble signal are not known. To address this, we applied several methods to deconstruct the interactions between the spatial and temporal organisation of circadian oscillations in organotypic slices from mice with circadian abnormalities. We studied the SCN of mice lacking Cryptochrome genes (Cry1 and Cry2), which are essential for cell‐autonomous oscillation, and the SCN of mice lacking the vasoactive intestinal peptide receptor 2 (VPAC2‐null), which is necessary for circuit‐level integration, in order to map biological mechanisms to the revealed oscillatory features. The SCN of wild‐type mice showed a strong link between the temporal rhythm of the bioluminescence profiles of PER2::LUC and regularly repeated spatially organised oscillation. The Cry‐null SCN had stable spatial organisation but lacked temporal organisation, whereas in VPAC2‐null SCN some specimens exhibited temporal organisation in the absence of spatial organisation. The results indicated that spatial and temporal organisation were separable, that they may have different mechanistic origins (cell‐autonomous vs. interneuronal signaling) and that both were necessary to maintain robust and organised circadian rhythms throughout the SCN. This study therefore provided evidence that the coherent emergent properties of the neuronal circuitry, revealed in the spatially organised clusters, were essential to the pacemaking function of the SCN.     

Not only vasopressin,but also the intracellular messenger protein kinase Calpha in the suprachiasmatic nucleus correlates with expression of circadian rhythmicity in voles

The suprachiasmatic nucleus (SCN) is the locus of the main pacemaker for circadian behavioral rhythms. In common voles, variation in circadian behavioral rhythmicity correlates with vasopressin (AVP) immunoreactive cells in the SCN. Here we studied the immunostaining of four AVP linked Ca(2+)-dependent protein kinase C (PKC) isoforms (PKCalpha, PKCbeta1, PKCbeta2, and PKCgamma) at the beginning of the light period, and conclude that PKCalpha is highly expressed in the vole SCN compared to the other isozymes. Voles, characterized as strongly circadian rhythmic showed circadian variation in numbers of PKCalpha immunoreactive SCN neurons, while voles with weak or no circadian rhythmicity did not reveal such a circadian profile. PKCalpha immunoreactivity in acute SCN slices that were treated with a physiological dose of AVP was significantly lowered when compared with control slices. The intracellular messenger PKCalpha may reflect variation in locomotor behavior via the AVP system in the vole SCN. This system could play a key role in the vole SCN by mediating output of its circadian clock.     

Luciferase expression controlled by a viral gene promoter in a mammalian circadian pacemaker

Retinal light exposure induces several immediate-early genes in the hypothalamic suprachiasmatic nucleus (SCN), which contains the major circadian pacemaker of mammals. Clock-controlled and light-induced genes expressed in the SCN such as c- and contain upstream regulatory elements similar to those of the major immediate-early gene (IE-1) of the human cytomegalovirus. IE-1 expression is critical for viral reactivation from latency and increases in response to agents acting through depolarization or the cAMP response element. To test whether IE-1 could be under circadian control, bioluminescence was imaged in individual SCN cells of brain slice cultures from transgenic mice containing the IE-1 enhancer/promoter upstream from the firefly luciferase gene. A small percentage of the cells in neonatal and adult cultures displayed circadian transgene expression, particularly ones near the dorsomedial edge of the SCN. Single-cell bioluminescence imaging revealed that the circadian pacemaker can regulate exogenous viral genes and could play a role in viral diseases.     

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.     

Swim stress triggers the release of vasopressin within the suprachiasmatic nucleus of male rats

The hypothalamic suprachiasmatic nucleus (SCN) is the predominant pacemaker of the mammalian brain that generates and controls circadian rhythms of various endocrine and behavioral processes. Different lines of evidence suggest that stress interferes with the maintenance of such rhythms. As a first approach to investigate whether the neuropeptide arginine vasopressin (AVP), which shows circadian rhythms of synthesis and release within the SCN, might contribute to this stress-induced alterations in circadian rhythms, we monitored acute effects of swim stress on the intra-SCN release of AVP in male rats by means of the microdialysis technique. A 10-min forced swimming session triggered a marked but relatively short-lasting increase in the intranuclear release of AVP (to approx. 440%). This effect was restricted to the area containing predominantly somata and dendrites of vasopressinergic neurons, since no changes in AVP release could be measured in one of their major projection areas, the nucleus of the dorsomedial hypothalamus. Our data provide evidence that the amount of AVP released within the SCN can vary widely not only in accordance with AVP's intrinsically regulated circadian rhythm but also in response to a physiologically relevant stressor. In this way, the neuropeptide may contribute to the regulation of endocrine and behavioral rhythms particularly in challenging situations associated with resettings of the endogenous clock.     

The clock in the dorsal suprachiasmatic nucleus runs faster than that in the ventral

In mammals, circadian rhythms are driven by a pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. The pacemaker is composed of an ensemble of multiple, single-cell oscillators in the SCN. We measured arginine-vasopressin (AVP) release in organotypic SCN slices. The SCN slice culture showed circadian oscillation of AVP release with a period length (+/- SEM) of 23.84 +/- 00.03 h. This period is very similar to the one we previously reported in dispersed SCN cultures and is also close to that of behavioural rhythms. When the ventral part was removed by a surgical cut across the slice in the horizontal plane, however, the period became shorter (23.22 +/- 00.08 h). On the other hand, the removal of the dorsal part did not affect period length. These results suggest that the oscillators in ventral and dorsal cells contribute differently to period length and that the dorsal oscillators are entrained by the ventral ones to form a single integrated oscillator.     

Real-time analysis of rhythmic gene expression in immortalized suprachiasmatic nucleus cells

Immortalized cells derived from the suprachiasmatic nucleus (SCN) retain many properties of the SCN including the capacity to generate circadian rhythms. Stably transfected SCN2.2 cells expressing the human c- promoter linked to a luciferase reporter gene ( /luc) were examined for evidence of transgene responses to stimuli known to induce c- expression and of endogenous rhythmic variation. Bioluminescence-reported transgene expression was induced in SCN2.2 /luc cells following stimulation with fetal bovine serum or KCl. SCN2.2 /luc cells showed 24 h rhythms of bioluminescence with a 9- to 19-fold difference between peak and minimum levels. These results demonstrate that the regulation of /luc transgene expression in SCN2.2 cells is similar to that of the endogenous c- gene in the SCN.