Serotonin-2C receptor involved serotonin-induced Ca2+ mobilisations in neuronal progenitors and neurons in rat suprachiasmatic nucleus

The hypothalamic suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals, undergoes serotonergic regulation, but the underlying mechanisms remain obscure. Here, we generated a subclone of an SCN progenitor cell line expressing Ca2+ sensors (SCN2.2YC) and compared its 5-HT receptor signalling with that of rat SCN neurons in brain slices. SCN2.2YC cells expressed 5-HT1A/2A/2B/2C, but not 5A/7, while all six subtypes were expressed in SCN tissues. High K+ or 5-HT increased cytosolic Ca2+ in SCN2.2YC cells. The 5-HT responses were inhibited by ritanserin and SB-221284, but resistant to WAY-100635 and RS-127445, suggesting predominant involvement of 5-HT2C for Ca2+ mobilisations. Consistently, Ca2+ imaging and voltage-clamp electrophysiology using rat SCN slices demonstrated post-synaptic 5-HT2C expression. Because 5-HT2C expression was postnatally increased in the SCN and 5-HT-induced Ca2+ mobilisations were amplified in differentiated SCN2.2YC cells and developed SCN neurons, we suggest that this signalling development occurs in accordance with central clock maturations.

T he suprachiasmatic nucleus (SCN) of the hypothalamus functions as the circadian pacemaker in mammals 1,2 . The SCN pacemaker is maternally coupled in the foetus until birth 3 , and develops action potential firing rhythms and entrainability to environmental cues during early postnatal life [4][5][6] . However, the neuronal mechanisms underlying the development of the circadian clock are not well understood.
In adults, circadian rhythms in SCN neurons are entrained to the environmental light/dark cycle via the glutamatergic retinohypothalamic tract (RHT) 7 . In parallel with the postnatal development of the RHT, the number of astrocytes is increased and the number of neurons is decreased in the SCN [8][9][10] , suggesting dynamic reorganisation of the SCN circuits or contents in relation to RHT formation. In addition, c-amino-butyric acid (GABA)-A receptors mediate excitatory synaptic signal transduction in neonatal brains 11 , but are switched to reversible (i.e., excitatory and inhibitory) functions in SCN neurons during postnatal development 12 . The development of GABA-A receptor signalling and intracellular chloride homeostasis may also amplify the circadian action potential firing rhythms in these neurons 13 .
In addition to the above neuronal regulations, the SCN receives dense serotonergic innervations from the midbrain raphe nucleus 14 . The numbers of serotonin (5-HT)-containing axons are greatly increased in the SCN during postnatal life 15 . In adults, 5-HT has been shown to modulate the effects of light by inhibiting glutamatergic RHT synapses in the SCN 14 . However, c-Fos expression in the SCN induced by subcutaneous injection of a 5-HT2A/2C agonist (2,5-dimethoxy-4-iodoamphetamine; DOI) was increased in a slightly different time frame to RHT development in rats 16 , suggesting that differential developmental mechanisms may underlie these systems. In the mature SCN, significant diversity of 5-HT receptor subtypes has been reported for both pre-and postsynaptic sites [17][18][19][20][21][22][23][24][25][26][27][28][29] . However, none of the developmental processes of these 5-HT receptor subtypes have been determined in the SCN to our knowledge.
SCN2.2 cells are immortalised rat SCN progenitor cells created by infection with a retroviral vector encoding the adenovirus 12S E1A gene at embryonic day 18 30 . SCN2.2 cells display (i) extended growth potential without evidence of transformed or tumorigenic properties, (ii) expression of E1A protein within all cell nuclei and (iii) heterogeneous cell types in various stages of differentiation. A large proportion of SCN2.2 cells are characterised by glial cell-like morphologies, but show limited expression of corresponding cell type-specific antigens. Rather, it has been shown that a subpopulation of SCN2.2 cells exhibit neuronal characteristics. Because transplantation of SCN2.2 cells into SCN-lesioned rats recovered their behavioural rhythms 31 and indeed these cells contain diverse clock genes 32 , it has been proposed that SCN2.2 cells potentially function as substitutive circadian pacemakers, although the cellular component essential for their functions remains unclear. Thus, subcloning of SCN2.2 cells could provide useful tools for studying the development of the SCN and the manifestation of their distinct roles in mammalian circadian timekeeping.
We have developed a method for transfecting yellow cameleon (YC) genes into cultured SCN neurons, thereby enabling monitoring of the circadian cytosolic Ca 21 waves in these neurons 33 . In the present study, we generated subclones of SCN2.2 cells expressing YC3.6 and monitored their cytosolic Ca 21 . Since rhythmic expression of voltage-gated Ca 21 channels is a proposed physiological output from SCN2.2 cells 34 , we retrieved a clone using a high-potassium (high K 1 )-induced Ca 21 increase as a marker. Here, we report the characteristics of one subclone (SCN2.2YC) with special interest in its 5-HT receptor expressions and functions. The predominant 5-HT receptor subtypes linked to intracellular Ca 21 signalling were comparatively examined in SCN2.2YC cells and rat SCN neurons.

Results
Profiles of 5-HT receptor expressions in rat SCN punch-outs, SCN astrocytes and SCN2.2 cells. The expressions of various 5-HT receptor subtypes were analysed in punch-outs of the SCN prepared at four different times of the day. The relative expression levels of most of the 5-HT receptor subtypes showed steady transcriptional levels (Fig. 1). The only exception was 5-HT2A receptors, whose transcriptional activities were significantly lower at the time of dark onset (ZT12; Fig. 1b) and showed anti-phased expression rhythms against the clock gene Per2 transcription rhythms (Fig. 1f). It should be emphasised that there were large variations in the expression levels of the individual 5-HT receptor subtypes. Of the receptor subtypes examined, 5-HT2C showed significantly higher (6-68 times) expression levels than the other subtypes (F 5,24 5 170; P , 0.01 by one-way ANOVA; Fig. 2a). In addition, the 5-HT2C receptor expression levels increased during postnatal development; the signal intensity was five times higher in tissues at postnatal day 15 than in tissues of neonates (F 4,16 5 36; P , 0.01 by one-way ANOVA; Fig. 1g). Since RHT projections are known to be increased during a similar postnatal period, we analysed the possible interaction with retinal inputs to the SCN. However, neonatal enucleation surgery did not modulate the transcriptional levels of 5-HT2C in the SCN (Fig. 1g), indicating that the developmental increase in 5-HT2C receptors occurs regardless of the presence or absence of the RHT.
The expression patterns of diverse 5-HT receptor subtypes were also examined in primary cultures of rat SCN astrocytes. The SCN astrocytes displayed expression of the 2A and 2B subtypes, with higher levels of the 2B subtype (Fig. 2b). This expression pattern did not fit with either the total SCN tissues or SCN2.2 cells (Fig. 2c). The original (i.e., heterogeneous) SCN2.2 cells displayed relatively low expression levels of 5-HT receptors, with only the 2A subtype showing equivalent levels to the SCN punch-outs (Fig. 2c). However, the SCN2.2YC subclone showed higher expression levels of the 1A subtype (P , 0.05 by two-tailed Student's t-test) and 2C  Developmental differences in the expression levels of 5-HT receptor subtype 2C were analysed in SCN punch-outs of rats that underwent neonatal enucleation surgery (white circles) or a sham operation (black circles). n 5 4-7 rats per group. *P , 0.05, **P , 0.01 (black asterisks for intact rats and grey asterisks for eyeless rats) versus the day 0 levels by oneway ANOVA followed by Duncan subtype (P , 0.05 by two-tailed Student's t-test) compared with the original SCN2.2 cells (Fig. 2d). Differentiation of SCN2.2YC cells with five factors-supplemented (5F) medium increased the transcriptional levels of these 5-HT receptor subtypes, but the differences were not statistically significant (Fig. 2e). The characteristics of the SCN2.2YC clone were more like those of neuronal progenitors than glial progenitors, because SCN2.2YC cells expressed a neuronal cell marker, microtubule-associated protein 2 (MAP2), but not a glial cell marker, glial fibrillary acidic protein (GFAP) (Fig. 3).
The functions of 5-HT receptors at the glutamatergic synapses of SCN neurons were further analysed by monitoring miniature excitatory postsynaptic currents (EPSCs) using voltage-clamp electrophysiology in acute SCN slices of postnatal days 12-16. In a subpopulation of SCN neurons that displayed spontaneous miniature EPSCs (6 of 28 neurons; average amplitude 5 18.2 6 0.2 pA), DOI (1 mM) magnified the miniature EPSC amplitude in half of the neurons (average amplitude 5 21.3 6 0.3 pA; P , 0.01 by a two-tailed Student's t-test; n 5 3; Fig. 4d), but had no effects on the frequency (Fig. 4e). In addition, co-perfusion of SB-221284 (500 nM) antagonised the DOI actions on the EPSC amplitudes in these neurons. These results suggest the presence of 5-HT2C receptors in SCN neurons at post-synaptic sites of glutamate synapses.     antagonist for 5-HT2B, RS-127445 (Fig. 7b,c). These series of pharmacological examinations indicate that the 5-HT-induced Ca 21 increases are predominantly mediated by 5-HT2C receptors in SCN2.2YC cells.
Circadian oscillatory profiles of SCN2.2YC cells. In the present study, we evaluated whether circadian rhythmic components are present in SCN2.2YC cells. First, chemiluminescence imaging of Per1-luciferase was used to visualise the presence of Per1 transcriptional rhythms in undifferentiated SCN2.2YC cells (Fig. 8a). The circadian phase of each cellular rhythm did not depend on the time of cell division (Fig. 8b), consistent with the characteristic clock gene transcriptional rhythms. In addition, the cell differentiation by 5F medium did not modulate the Per1-luciferase rhythms, although the basal transcription levels tended to be higher in the medium (Fig. 8b). These results demonstrate the presence of clock gene transcriptional rhythms in both undifferentiated and differentiated SCN2.2YC cells. Conversely, voltage-dependent sodium channels were not expressed or not functional in undifferentiated (n 5 6) and 4F-differentiated (n 5 8) SCN2.2YC cells, since the voltage steps failed to evoke typical inward currents (Fig. 8c). These findings demonstrate the lack of action potential firings in SCN2.2YC cells under our cell culture conditions. In addition, long-term ratiometric fluorescence measurements were used to visualise the cytosolic Ca 21 levels for 6 days in SCN2.2YC cells (n 5 8 for undifferentiated cells; n 5 10 for 4F-differentiated cells), but there were no circadian oscillations in the baseline Ca 21 levels (Fig. 8d).
These results demonstrate few neuronal output rhythms in SCN2.2YC cells under the cell culture conditions examined.

Discussion
The SCN is a tightly packed neuronal nucleus composed of approximately 10,000 heterogeneous neurons, and the receptor expression profile in each neuron is generally almost inaccessible. The 5-HT system is not an exception, and diversity of 5-HT receptor subtypes has been reported for both pre-and post-synaptic sites [17][18][19][20][21][22][23][24][25][26][27][28][29] . The involvement of 5-HT2C receptors in circadian clock regulation has been shown at the system level 16,17 , but there was no evidence regarding the receptor localisations involved. The present study demonstrated significant expression of 5-HT2C receptors in the rat SCN, which increased during postnatal development but was independent of RHT development. The 5-HT2C receptors were post-synaptically expressed in SCN neurons because the EPSC amplitudes, but not frequencies, were increased following receptor stimulations. In addition, stimulation of 5-HT2C receptors mobilised intracellular Ca 21 in SCN cells in rat brain slices with a larger response in juveniles than in neonates. Consistently, a monoclonal rat SCN progenitor cell line, SCN2.2YC, presented Ca 21 mobilisations predominantly via 5-HT2C receptors with a larger response in differentiated cells. Taken together, we suggest that the development of 5-HT2Cmediated cellular responses occurs in parallel with the maturation of the central circadian clock. On the other hand, since neither functional voltage-dependent sodium channels nor circadian cytosolic Ca 21 waves were present in differentiated SCN2.2YC cells, it is also suggested that the expression of 5-HT2C receptors is not a determinant for the development of physiological rhythms in SCN neurons.
During the course of the Ca 21 imaging analyses of SCN2.2YC cells in the present study, we found 5-HT-induced Ca 21 transients in these cells. Similar 5-HT-induced Ca 21 responses were reported in fura-2AM-stained rat SCN slices 35 , although the receptor subtypes involved were not analysed in that study. Therefore, to further analyse the coherency with the rat SCN, the expression profiles of the 5-HT receptor subtypes were examined in detail using real-time RT-PCR. Although 5-HT1B receptors are known to be involved in photic regulation of circadian behavioural rhythms, 5-HT1B receptors have been shown to be localised at pre-synaptic terminals of the RHT or other axon terminals 23,36,37 . Since the principal aim of this study was to characterise the expression of the 5-HT receptor subtypes in SCN cells, we focused on all other 5-HT receptor subtypes known to be expressed in SCN neurons or to regulate circadian behavioural rhythms. Within the rat SCN punch-outs, 5-HT1A, 2A, 2B, 2C, 5A and 7 subtypes were all expressed, but showed little circadian variations in their expression levels. Notably, we observed significant expression of the 2C subtype among these subtypes. This It has also been shown that activation of 5-HT2C receptors acutely induces Per gene expression in the rat SCN in vitro and in vivo 38,39 .
Conversely, 5-HT-induced phase-shifts of the action potential firing rhythms were observed in SCN slices of 5-HT7 receptor knockout mice, which were reduced by either WAY-100635 or ritanserin 40 . These observations indicate the co-contributions of 1A and 2 receptors to the production of circadian phase-shifts in mice. Furthermore, it has been shown that a 5-HT1A receptor agonist (MKC-242) potentiated, while 8-OH-DPAT inhibited, the photic entrainment of circadian behaviours in hamsters 18,41 . Since the effect of MKC-242 on the photic entrainment was blocked by WAY-100635, but not by ritanserin, in the hamster SCN, it was suggested that 5-HT1A receptors, but not 5-HT2 subtypes, are involved in the photic regulation of the hamster circadian clock 18 . Thus, it should be noted that the differences in involvement of each 5-HT receptor subtype or expression at pre-or post-synaptic sites to control the system-wide clock effects may be variable among animal species.
In SCN2.2YC cells, expression of 5-HT5A and 7 receptors was not detected. Because SCN2.2YC cells are monocloned cells, these results may simply arise through subtraction of a characteristic cell population from the heterogeneous SCN cells. However, it was to our surprise that SCN2.2YC cells still showed diversity in their expressions of 5-HT receptor subtypes. These results suggest that the expressions of 5-HT receptor subtypes may not be pre-programmed at the progenitor stages and could be modified during developmental processes or by cell culture conditions. In this regard, the present study also demonstrated gradual increases in 5-HT2C receptor expression in rats during early postnatal development (postnatal days 3-11), regardless of the presence or absence of the RHT. These observations are consistent with a former report showing that c-Fos expression evoked by subcutaneous injection of DOI was increased during postnatal development in a slightly different time frame to RHT development in rats 16 . Since 5-HT-containing axons are greatly increased in the SCN during the corresponding period in early postnatal life 15 , the increases in 5-HT2C receptor expression and maturation of other neuronal functions may arise through interaction with the developmental process of 5-HT-containing axons projecting from the midbrain raphe nucleus.
To address the above hypothesis, we preliminarily examined 5-HT (1 mM) supplementation in the culture medium to observe the changes in the 5-HT receptor expression levels in SCN2.2YC cells, but did not detect any changes in these levels. Therefore, we used a conventional strategy to differentiate these cells. We initially examined differentiation methods with dibutyryl-cAMP or retinoic acid, but failed to differentiate SCN2.2 cells using these reagents. Thus, we used the 4F medium method that successfully differentiated neural progenitor cell lines derived from the cerebellum of a p53-deficient mouse 42 . Addition of chorea toxin, an activator of G s -mediated signalling, may be critical for the cell differentiation, because the cell divisions were greatly inhibited, while the cell polarisations were greatly enhanced, by chorea toxin. In addition, we added phorbol-12-myristate 13-acetate (PMA), an activator of protein kinase C signalling, into the 4F medium (5F) and observed further enhancements of the 5-HT responses. However, it should be noted that these artificial cell differentiation protocols still failed to increase the 5-HT2C receptor gene expression levels up to the level observed in the adult SCN. Furthermore, these protocols failed to produce voltagedependent sodium currents, which are essential for the production of action potential firings. Thus, we speculate that other critical factors may be needed for more complete neural differentiation of SCN progenitor cell lines.
To analyse the oscillatory profiles of SCN2.2YC cells in the present study, we examined Per1-luciferase and Ca 21 imaging. The Per1-luciferase imaging in SCN2.2YC cells demonstrated circadian transcriptional rhythms of Per1 in individual cells and each phase synchronised in the culture dish, but not with the time of cell division. Phase-uncoupling between Per1 transcriptional rhythms and cell division cycles is consistent with the characteristics of clock gene transcriptional rhythms in NIH3T3 fibroblasts 43 . Notably, our Per1luciferase imaging demonstrated that the circadian amplitude was small and the waveform did not obey a typical sinusoidal wave in each SCN2.2YC cell, unlike the fibroblast models. These observations may not simply be caused by the technical limitations of our imaging system, because the same system demonstrated robust circadian waves in the Per-luciferase signals in single cells in Drosophila culture models 44 . Thus, we suggest that the clock gene transcriptional rhythms are weaker in SCN2.2YC cells than in fibroblasts or in cells located in the real clock system. The circadian amplitude and phase of the Per1-luciferase rhythms in SCN2.2YC cells were not significantly modified by cell differentiation. Thus, although functional 5-HT receptors are present, it seems likely that SCN2.2YC cells may be immature as circadian pacemakers. The majority of cultured SCN neurons display robust circadian cytosolic Ca 21 waves 33,[45][46][47] , but this did not occur in SCN2.2YC cells. Because Ca 21 triggers clock gene transcriptions 48 , circadian cytosolic Ca 21 waves could represent feedback signals to the clock gene transcriptional cycles [49][50][51] . Therefore, the relatively weak Per1 rhythms observed in SCN2.2YC cells may be explained by the lack of physiological feedback in these cells. Since the original SCN2.2 cells could synchronise the fibroblast rhythms in coculture models 52  One ultimate goal of studying SCN2.2 cells is to determine the key factors that enable the recovery of system-wide rhythms in SCNlesioned rats. Thus, the issue of whether or not implantation of SCN2.2YC cells is sufficient for the recovery of locomotor activity rhythms in SCN-lesioned rats is an important aspect for investigation, albeit beyond the scope of the present study.
Because SCN2.2 cells are resistant to neomycin, the YC3.6 gene was ligated to the multiple cloning site of a zeocin-resistant vector (pcDNA3.1/zeo; Invitrogen) and transfected into SCN2.2 cells using Lipofectamine 2000 (Invitrogen). Subsequently, the cells were cultured in medium containing zeocin (400-800 mg/ml) for cell selection. Four colonies grown from single cells were picked up by a cloning ring for further subcloning using 96-well dishes. Finally, one clone showing the highest high-K 1 -induced Ca 21 increase was stored and used for the following experiments.
Differentiation of SCN2.2YC cells. For differentiation of SCN2.2YC cells, the culture medium was switched from the above standard medium to serum-free Ham's F12/DMEM. Initially, we examined the addition of the following three factors (3F): 10 mg/ml insulin, 10 nM sodium selenite and 10 mg/ml transferrin. Since SCN2.2YC cells exhibited cell divisions even after culture with 3F supplemented serum-free medium, 10 ng/ml cholera toxin was supplemented in the 3F medium (4F). In addition, 10 ng/ml PMA was supplemented in the 4F medium (5F). The cell division was significantly reduced and the cell shape was polarised by the 4F medium, and the cells survived for more than 10 days after replacement of the culture medium with 5F. Isolated astrocyte cultures from the SCN. The experimental procedures below regarding the use of animals were approved by the Institutional Animal Care and Use Committee of the University of Toyama and Korea University. Primary cultures of isolated astrocytes were prepared from Sprague-Dawley rats at postnatal days 2-3 by the following method. Under deep anaesthesia with an intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight), the brains were removed and coronal hypothalamic slices (400-mm) containing the SCN were prepared using a vibrating blade microtome in ice-cold high-Mg 21 artificial cerebrospinal fluid (ACSF), as described previously 12 . Subsequently, trimmed SCN tissues from eight pups were pooled in DMEM containing 100 mg/mL penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO) and gently agitated in DMEM supplemented with 0.025% (w/v) trypsin (Sigma) for 15 min at 37uC in a water bath shaker. The trypsinisation was stopped by adding an equal volume of standard DMEM. A single-cell suspension of the tissue was prepared by gentle trituration and passage through a 40-mm nylon mesh cell strainer (BD Falcon, Bedford, MA), and seeded into standard culture flasks. The resultant primary cultures of SCN neurons and astrocytes were placed in a humidified CO 2 incubator at 37uC. The medium was changed every 4-5 days until the cells reached confluency (approximately 15 days). Subsequently, the cultures were treated with a high-K 1 buffered salt solution (BSS) consisting of (in mM) 73 NaCl, 60 KCl, 2.7 CaCl 2 , 1.2 MgCl 2 , 1 Na 2 HPO 4 , 10 glucose and 10 HEPES/NaOH (pH 7.3) for 1 h to induce neuronal cell death. Adhesive cells were passaged onto new flasks to generate www.nature.com/scientificreports SCIENTIFIC REPORTS | 4 : 4106 | DOI: 10.1038/srep04106 SCN astrocytes. Finally, the cultures were characterised by immunostaining for GFAP.
Real-time RT-PCR assay. Adult Sprague-Dawley rats (300-450 g; 60-70 days of age) were maintained on a 12-h/12-h light/dark cycle at a constant ambient temperature (25 6 1uC). Food and water were available ad libitum. Male newborn pups underwent enucleation surgery or a sham operation under hypothermic anaesthesia on ice (10 min). A total of 40,000 U of penicillin G potassium (Sigma) was locally applied to the incisions. Finally, the pups were odorised with nesting chips and returned to their dam. For brain sampling, animals were deeply anesthetised with an intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight). The whole brains were then removed and directly frozen on dry ice. The frozen brains were transferred to a cryostat chamber at 230uC, mounted with OCT compound at 215uC and sectioned at 100-mm thickness. The frozen sections were transferred onto glass slides, punched out using flat-top stainless pipettes (inner diameter, 0.33 mm; hand-made from 23 G disposable syringe tips) on ice and homogenised using a bio-masher (Funakoshi, Tokyo, Japan) in 600 ml of RLT buffer (RNeasy Kit; Qiagen, Chatsworth, CA) at 2500 rpm for 30 s. After addition of 600 ml of 70% ethanol, the samples were stored at 280uC. SCN2.2YC cells or SCN astrocytes in 25 mm 2 flasks at 90% confluence were rinsed, suspended in PBS, transferred to 1.5 ml RNase-free tubes and centrifuged for 5 min at 1000 rpm at room temperature. The cell pellets were transferred to 350 ml of RLT buffer and homogenised using an ultrasonic cell disruptor. The resultant cell lysates were diluted with an equivalent volume of 70% ethanol and stored at 280uC until RNA extraction.
Total RNA (4 mg/sample) was extracted from tissue homogenates using the