Astrocytes are active partners in neural information processing []. However, the roles of astrocytes in regulating behavior remain unclear []. Because astrocytes have persistent circadian clock gene expression and ATP release in vitro [], we hypothesized that they regulate daily rhythms in neurons and behavior. Here, we demonstrated that daily rhythms in astrocytes within the mammalian master circadian pacemaker, the suprachiasmatic nucleus (SCN), determine the period of wheel-running activity. Ablating the essential clock gene Bmal1 specifically in SCN astrocytes lengthened the circadian period of clock gene expression in the SCN and in locomotor behavior. Similarly, excision of the short-period CK1ε tau mutation specifically from SCN astrocytes resulted in lengthened rhythms in the SCN and behavior. These results indicate that astrocytes within the SCN communicate to neurons to determine circadian rhythms in physiology and in rest activity.

We next infected CK1εmice with a virus (AAV8-GFAP-Cre-GFP) to remove the tau mutation specifically in GFAP() astrocytes of the SCN ( Figure 4 E). Animals infected with the GFAP-Cre virus, like Aldh1L1- CK1εmice, had a lengthened period in locomotor activity ( Figures 4 F and 4H; 22.54 ± 0.14 hr versus 23.43 ± 0.25 hr, n = 4 per group, Student’s t test, p < 0.05) and in isolated SCN PER2::luc rhythms ( Figures 4 I–4J; 22.34 ± 0.12 hr versus 25.08 ± 0.41 hr, n = 4 per group, Student’s t test, p < 0.001). AAV infection was confirmed by GFP fluorescence in the SCN ( Figure 4 G). Taken together, these results show that two independent methods that increased the period of SCN astrocytes relative to the rest of the body lengthened the periods of SCN and behavioral rhythms.

Because BMAL1 is a transcription factor with circadian and non-circadian functions, we utilized the CK1ε tau mutation as an independent method to conditionally manipulate daily timing in astrocytes. This point mutation in exon 4 of the CK1ε gene shortens the period of wheel-running activity under constant darkness (CK1ε: ∼20 hr, CK1ε: ∼22 hr) []. The phenotype can be reversed when exon 4 is floxed out by Cre recombinase so that CK1εor CK1εmice have a 24-hr period []. We found that Crelittermates (termed CK1ε) showed an ∼22-hr period as previously reported, while Aldh1L1-Cre/; CK1ε; PER2::luc/(referred to as Aldh1L1- CK1ε) animals showed a significantly lengthened period ( Figures 4 A and 4B ; 22.21 ± 0.13 hr, n = 9 versus 23.42 ± 0.10 hr, n = 11, Student’s t test, p < 0.0001). The SCN of Aldh1L1- CK1εmice also showed lengthened PER2::luc rhythms ( Figures 4 C and 4D; 22.1 ± 0.18 hr versus 26.06 ± 0.68 hr, n = 5 SCN per group, Student’s t test, p < 0.01), indicating the behavioral period directly results from the lengthened rhythm within the SCN.

(F and G) Representative actograms of two CK1ε tau/+ mice injected with either AAV8-GFAP-GFP or AAV8-GFAP-Cre-GFP (F) with successful viral targeting to the SCN (G).

(E) Schematic of a second method to change circadian timing in astrocytes using GFAP AAV injection to CK1ε tau/+ SCN in vivo.

(C and D) Representative PER2::luc recordings from the isolated SCN of Aldh1L1- CK1ε tau/+ and CK1ε tau/+ littermates (C) with a longer circadian period in SCN where the CK1ε tau/+ mutation was removed from astrocytes (D) (p < 0.01, t test).

(A and B) Representative actograms of Aldh1L1- CK1ε tau/+ and CK1ε tau/+ littermates (A) showing how locomotor activity free-ran in constant darkness with a longer circadian period in mice where the CK1ε tau/+ mutation was removed from astrocytes (B) (p < 0.0001, t test).

To test the necessity of astrocytes’ molecular clocks in behavioral rhythms, we injected AAV carrying sgBmal1 into the SCN of Aldh1L1-Cre/; LSL-Cas9/mice (Astro-Bmal1) and Crelittermate controls ( Figure 3 A). Before AAV injection, LSL-Cas9/and Aldh1L1-Cre/; LSL-Cas9/mice showed similar wheel-running periods in constant darkness (23.79 ± 0.05 hr, n = 9 and 23.75 ± 0.08 hr, n = 12, Student’s t test, not significant [n.s.]). Strikingly, CRISPR-mediated loss of Bmal1 in SCN astrocytes significantly lengthened their locomotor periodicity ( Figures 3 B and 3E; controls versus Aldh1L1-Bmal1: sgBmal1_E1: 23.76 ± 0.12 hr, n = 5 versus 24.22 ± 0.10 hr, n = 6; sgBmal1_E3: 23.75 ± 0.08 hr, n = 4 versus 24.38 ± 0.13 hr, n = 3; p < 0.01; one-way ANOVA with Sidak multiple comparison test). Loss of Bmal1 in SCN astrocytes was confirmed by immunohistochemistry ( Figures 3 C and 3D; 2.5% ± 1.2% of cells were BMAL1- and Aldh1L1-positive cells, and nearly 97.7% ± 0.1% of Aldh1L1-Cas9 cells were GFAP positive; Bmal1: n = 9; GFAP: n = 6). In Aldh1L1-Bmal1SCN, the period of PER2::luc rhythms was also significantly lengthened compared to Crelittermate controls ( Figures 3 F and 3G; 24.63 ± 0.14 hr, n = 6 versus 25.82 ± 0.41 hr, n = 5, p < 0.05, Student’s t test). Using a CRISPR-independent approach, we again found period lengthening of circadian rhythms in flox Bmal1 mice injected with AAV8-GFAP-Cre-GFP compared to flox Bmal1 mice injected with a control AAV8-GFAP-GFP (23.7 ± 0.09 hr, n = 5 versus 23.07 ± 0.07 hr, n = 7; p < 0.01, t test). We conclude that loss of Bmal1 in SCN astrocytes lengthens circadian rhythms in SCN neurons and behavior.

(F) Representative PER2::luc traces from the cultured SCN of a Aldh1L1-Cre/ + ; LSL-Cas9/ + ; PER2::luc/ + mouse (red) and a LSL-Cas9/ + ; PER2::luc/ + littermate control (blue) both injected with sgBmal1_E3 into bilateral SCN in vivo.

(E) Bmal1 ablation in Aldh1L1 cells by either of two independent guide RNAs (E1 or E3) in the SCN increased the circadian period of locomotor activity compared to Cre – controls. ∗ p < 0.05; ∗∗ p < 0.01, one-way ANOVA with Sidak multiple comparison test.

(C) Coronal brain sections immunostained for Bmal1 (red) and GFP (green) with insets showing the abundant BMAL1 staining in the control and lack of BMAL1 in Aldh1L1 cells in mouse with targeted deletion.

(B) Representative locomotor activity of a LSL-Cas9/ + littermate control and an Aldh1L1-Cre/ + ; LSL-Cas9/ + mouse both injected with AAV-sgBmal1_E3 into bilateral SCN. Each line shows wheel running (black ticks) over 2 days with the second day’s data replotted on the line below. The mice were less active in the light (yellow) in the 12-hr:12-hr light:dark cycle of the first 25 days of recording and showed free-running rhythms in constant darkness.

Using a recently developed gene editing technique [], we ablated the Bmal1 gene from targeted cells. In this strategy, short guide RNAs (sgRNAs) designed to direct the disruption of the Bmal1 gene were delivered to cells expressing the CRISPR machinery under the control of cell-type-specific promoters. As a proof of concept, we injected a mixture of adeno-associated virus (AAV) carrying ubiquitously expressed pCBh-Cre and sgRNA against either LacZ (control, sgLacZ) or Bmal1 (sgBmal1; E1 and E3 denote independent sequences targeting exons 1 or 3 of the Bmal1 gene) into the SCN of LSL-Cas9/; PER2::luc/mice. We found that Cas9 was subsequently expressed throughout the SCN from a GFP fusion protein co-expressed with Cas9 (data not shown). Animals that received sgBmal1 lost daily rhythms in both behavior (n = 2 of 2 mice, one for each Bmal1 sgRNA vector) and whole SCN PER2::luc (n = 12 of 12 SCN, n = 6 for each Bmal1 sgRNA vector) as predicted for global Bmal1 knockouts [], while those that received sgLacZ (n = 2 of two mice and 5 of five SCN) remained circadian ( Figure S2 ).

Previous work has shown that cortical astrocyte cultures and an astrocyte-like cell line (SCN2.2) have circadian rhythms in clock gene expression and release of ATP, a potent gliotransmitter []. To determine whether SCN astrocytes possess daily rhythms in clock genes, we stained Aldh1L1-GFPbrain sections for BMAL1 (also called ARNTL or MOP3). We found that 85.9% ± 5.6% (n = 6 mice perfused at zeitgeber time [ZT] 2) of Aldh1L1 cells in the SCN expressed BMAL1 ( Figure 1 C). We next infected Aldh1L1-Cre/organotypic SCN slices with a novel adeno-associated virus carrying a Cre-activated bioluminescent reporter of Bmal1 transcription (AAV2/10-Bmal1ext-DIO-luc; Figure 2 A). These Aldh1L1-Bmal1SCN slices were rhythmic with a circadian period (23.6 ± 0.2 hr, n = 6; Figure 2 C) and ∼10-fold higher light emission than a Crelittermate SCN infected with the same virus ( Table S1 ). For comparison, we found similar amplitude Bmal1-luc rhythms in SCN vasoactive intestinal polypeptide (VIP) neurons ( Table S1 ). To measure coordination among SCN astrocytes, we imaged Aldh1L1-Bmal1with an ultracooled charge-coupled device (CCD) camera (n = 3 SCN). We reliably detected astrocyte-shaped cells expressing the reporter of Bmal1 activity throughout the SCN ( Figure 2 B; data not shown). Bioluminescence in 72.8% ± 4.0% of cell-sized regions of interest (ROIs) was circadian and peaked at a similar time each day ( Figure 2 D; p < 0.0001, Rayleigh’s test, mean vector length = 0.85 ± 0.02). The remaining ROIs were considered arrhythmic. We conclude that astrocytes function as synchronous circadian oscillators within the SCN.

(D) Raster plot of Bmal1 reporter expression across a representative Aldh1L1-Bmal1 luc SCN slice from (B). The bioluminescence in each ROI peaked at approximately the same time daily over the 3 days of recording with an ultracooled CCD camera. Bioluminescence for each ROI was normalized to its maximum and pseudocolored (color bar at right).

(B) Left: representative frame from a movie of bioluminescence recorded from an Aldh1L1-Bmal1 luc SCN slice. Note the glowing astroglial cells throughout the bilateral SCN. Scale bar, 400 μm. Right: cell-sized regions of interest (ROIs) were used to track Bmal1 expression from astrocytes within the SCN.

We targeted astrocytes using Aldh1L1-Cre BAC transgenic mice [] because Aldh1L1 expression in the brain is high, broad, and specific to astrocytes []. To test the specificity and expression pattern of Aldh1L1 in the SCN, we crossed Aldh1L1-Cre/mice to mice carrying Cre-activated nuclear GFP transgene (LSL-GFP[nucleus-localization signal]; Table S2 details the genotypes and treatments for all experiments). Immunofluorescence staining showed that Aldh1L1-GFPlabels 10.9% ± 1.1% of SCN cells (n = 6 mice, one brain section each, mean ± SEM). The astrocyte marker glial fibrillary acidic protein (GFAP) labeled 96.4% ± 1.7% of the Aldh1L1-GFP-positive cells ( Figure 1 A; n = 1 section each from four mice). None of the Aldh1L1-GFPcells expressed the neuronal markers FOX2 (a homolog of NeuN []; Figure 1 B; n = 3 brains, 762 FOX2cells counted) or arginine vasopressin (AVP) ( Figure S1 ; n = 6 brains, 220 AVPcells counted). In summary, the Aldh1L1-Cre mouse line provides one way to specifically label astrocytes in the SCN.

Astrocyte nuclei in the SCN were labeled in green by Cre-mediated recombination of Aldh1L1-Cre/;LSL-GFPmice. Coronal brain sections were immunostained for an astroglial marker GFAP (A), a neuronal marker FOX2 (B), or a circadian clock protein BMAL1 (C) in red, and all nuclei were counterstained with DAPI. Cells within the yellow boxes (left) were magnified (right). Filled arrowheads indicate double-labeled cells, and arrows point to cells that showed no red and green colocalization. Note that Aldh1L1-positive cells reliably express the astrocyte marker GFAP and the circadian clock protein BMAL1, but not the neuronal marker FOX2. Scale bar, 75 μm. See also Figure S1

Discussion

18 Evans J.A. Collective timekeeping among cells of the master circadian clock. 19 Jackson F.R. Glial cell modulation of circadian rhythms. 6 Burkeen J.F.

Womac A.D.

Earnest D.J.

Zoran M.J. Mitochondrial calcium signaling mediates rhythmic extracellular ATP accumulation in suprachiasmatic nucleus astrocytes. 7 Womac A.D.

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Zoran M.J. Circadian rhythms of extracellular ATP accumulation in suprachiasmatic nucleus cells and cultured astrocytes. 20 Yamaguchi S.

Isejima H.

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Beaulé C. Circadian regulation of ATP release in astrocytes. 8 Prolo L.M.

Takahashi J.S.

Herzog E.D. Circadian rhythm generation and entrainment in astrocytes. 21 Ng F.S.

Tangredi M.M.

Jackson F.R. Glial cells physiologically modulate clock neurons and circadian behavior in a calcium-dependent manner. 22 Zerr D.M.

Hall J.C.

Rosbash M.

Siwicki K.K. Circadian fluctuations of period protein immunoreactivity in the CNS and the visual system of Drosophila. 23 Inouye S.T.

Kawamura H. Persistence of circadian rhythmicity in a mammalian hypothalamic “island” containing the suprachiasmatic nucleus. 24 Jin X.

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Reppert S.M. A molecular mechanism regulating rhythmic output from the suprachiasmatic circadian clock. 25 Schwartz W.J.

Gainer H. Suprachiasmatic nucleus: Use of 14C-labeled deoxyglucose uptake as a functional marker. Here, four independent manipulations of clock genes in astrocytes similarly changed daily rhythms in the SCN and in behavior. Although previous publications implicated astrocytes in SCN function (reviewed in []), we lacked the tools to target these glial cells directly. Using promoters for GFAP and Aldh1L1 to drive reporters and manipulate clock genes, we were able to control astrocytes specifically within the SCN in vivo and in vitro. A conditional, real-time reporter of Bmal1 transcription revealed synchronous circadian rhythms in astrocytes within SCN slice cultures. This is consistent with the reported rhythms in extracellular ATP attributed to astroglia in the isolated SCN [] but differs from a study that did not find daily cycles in Per1-luc expression in SCN astrocytes []. That report was based on an absence of immunohistochemical evidence for colocalization of GFAP and luciferase and may have been confounded by low or anti-phase levels of the two proteins. Furthermore, circadian rhythms have been reported in Per1-luc, PER2::luc, Bmal1-luc, and ATP release in cultured cortical astrocytes [] and in PER levels in Drosophila glial cells []. We conclude that astrocytes are functional circadian oscillators within the SCN. It will be important to investigate phase differences between daily rhythms in SCN neurons and astrocytes and whether oscillations of SCN neuronal firing [], neuropeptide release [], or metabolic demand [] are coupled to astrocyte rhythms.

26 Mieda M.

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Hasegawa E.

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Honma S.

Sakurai T. Cellular clocks in AVP neurons of the SCN are critical for interneuronal coupling regulating circadian behavior rhythm. 27 Hong H.K.

Chong J.L.

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Schook A.C.

Ko C.H.

Takahashi J.S. Inducible and reversible Clock gene expression in brain using the tTA system for the study of circadian behavior. 28 Izumo M.

Pejchal M.

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Takahashi J.S. Differential effects of light and feeding on circadian organization of peripheral clocks in a forebrain Bmal1 mutant. 29 Low-Zeddies S.S.

Takahashi J.S. Chimera analysis of the Clock mutation in mice shows that complex cellular integration determines circadian behavior. In this study, we found evidence that circadian clocks in SCN astrocytes, like in SCN neurons, are modulators of daily rhythms in the SCN and behavior. Loss of rhythm in SCN astrocytes through Bmal1 deletion resulted in lengthened circadian period of rest-activity rhythms. A similar 1-hr increase in period was recently reported for mice in which AVP neurons had Bmal1 knocked out []. Thus, in contrast to the arrhythmicity produced by ablation of the clock in many SCN neurons [], Bmal1 deletion in a small proportion of SCN cells appears to change the period of the SCN and behavior. Specifically, loss of rhythmicity in the 20% of SCN cells that express AVP or the 10% cells that express Aldh1L1 or GFAP suffices to lengthen SCN periodicity. This is consistent with the results of genetic chimera mice where the circadian phenotype scaled with the fraction of SCN cells homozygous for the dominant-negative form of CLOCK []. We conclude that astrocytes likely play as important a role as any neuronal cell class in circadian timekeeping in the SCN and behavior.

28 Izumo M.

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Schook A.C.

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Walisser J.A.

Sato T.R.

Wang X.

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Takahashi J.S. Differential effects of light and feeding on circadian organization of peripheral clocks in a forebrain Bmal1 mutant. 30 Chen R.

Schirmer A.

Lee Y.

Lee H.

Kumar V.

Yoo S.H.

Takahashi J.S.

Lee C. Rhythmic PER abundance defines a critical nodal point for negative feedback within the circadian clock mechanism. 31 Lee I.T.

Chang A.S.

Manandhar M.

Shan Y.

Fan J.

Izumo M.

Ikeda Y.

Motoike T.

Dixon S.

Seinfeld J.E.

et al. Neuromedin s-producing neurons act as essential pacemakers in the suprachiasmatic nucleus to couple clock neurons and dictate circadian rhythms. 27 Hong H.K.

Chong J.L.

Song W.

Song E.J.

Jyawook A.A.

Schook A.C.

Ko C.H.

Takahashi J.S. Inducible and reversible Clock gene expression in brain using the tTA system for the study of circadian behavior. 31 Lee I.T.

Chang A.S.

Manandhar M.

Shan Y.

Fan J.

Izumo M.

Ikeda Y.

Motoike T.

Dixon S.

Seinfeld J.E.

et al. Neuromedin s-producing neurons act as essential pacemakers in the suprachiasmatic nucleus to couple clock neurons and dictate circadian rhythms. 32 Mieda M.

Okamoto H.

Sakurai T. Manipulating the cellular circadian period of arginine vasopressin neurons alters the behavioral circadian period. Δ19 in SCN VIP neurons (∼10% of SCN neurons) does not lengthen behavioral period [ 31 Lee I.T.

Chang A.S.

Manandhar M.

Shan Y.

Fan J.

Izumo M.

Ikeda Y.

Motoike T.

Dixon S.

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et al. Neuromedin s-producing neurons act as essential pacemakers in the suprachiasmatic nucleus to couple clock neurons and dictate circadian rhythms. 33 Smyllie N.J.

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Hastings M.H. Temporally chimeric mice reveal flexibility of circadian period-setting in the suprachiasmatic nucleus. Are circadian rhythms in astrocytes sufficient or necessary for daily rhythms in the SCN or behavior? Several recent studies have found that Bmal1 deletion or PER2 overexpression in SCN neurons abolished circadian rhythms in locomotion []. We therefore posit that having a molecular clock in astrocytes is not sufficient to sustain behavioral rhythms. Our finding that mice remain circadian after loss of Bmal1 in astrocytes also argues against the necessity of an astrocyte circadian clock. However, increasing the intrinsic period of SCN astrocytes by deletion of CK1ε reliably lengthens behavioral period. This is a striking result because increasing period in a subpopulation of SCN neurons does not necessarily drive behavioral rhythms to longer periods. For example, using drivers that include SCN AVP neurons (AVP, neuromedin-S [NMS], or Scg2) to alter clock gene expression can increase locomotor period [], but overexpression of Clockin SCN VIP neurons (∼10% of SCN neurons) does not lengthen behavioral period []. Furthermore, manipulations that would be predicted to increase the cell-intrinsic period by at least 4 hr, even when targeted to 40% of SCN cells (e.g., with Drd1a), also tend to have smaller and less reliable effects on behavioral period []. Remarkably, we found that deletion of CK1ε in astrocytes, which has the cell-autonomous effect of increasing period by about 2 hr, sufficed to increase the behavioral period by about 1.5 hr. Thus, our data argue that clocks in SCN astrocytes likely play a more important role than some SCN neurons in determining behavioral periodicity.

21 Ng F.S.

Tangredi M.M.

Jackson F.R. Glial cells physiologically modulate clock neurons and circadian behavior in a calcium-dependent manner. 34 Jackson F.R.

Ng F.S.

Sengupta S.

You S.

Huang Y. Glial cell regulation of rhythmic behavior. 35 Suh J.

Jackson F.R. Drosophila ebony activity is required in glia for the circadian regulation of locomotor activity. 2+ storage can lead to behavioral arrhythmicity. This suggests that dysregulation of glial physiology can interfere with daily rhythms in physiology and behavior in flies. Future studies should test whether and which cellular functions of SCN astrocytes are necessary for rhythmicity in the SCN and behavior. Our findings also highlighted that glia in mammals and Drosophila may play fundamentally different roles in their circadian circuits. In contrast to our findings in SCN astrocytes, glial-specific knockdown of the essential clock gene, Per, did not change PER expression in neurons or locomotor activity rhythms in flies []. Since PER expression was absent from glia cells in those flies, it is possible that clock-less astrocytes in the fly brain can be driven to oscillate by circadian neurons so that behavior remains intact. Alternatively, it may be that loss of PER in flies is not equivalent to loss of Bmal1 in mice. The consequence of the loss of the Bmal1 ortholog, Cyc, is yet to be tested in glial cells in flies. Notably, in flies, loss-of-function mutation in the glial-specific, clock-controlled gene ebony [] or glial-specific perturbations of membrane potential, vesicular release, or intracellular Castorage can lead to behavioral arrhythmicity. This suggests that dysregulation of glial physiology can interfere with daily rhythms in physiology and behavior in flies. Future studies should test whether and which cellular functions of SCN astrocytes are necessary for rhythmicity in the SCN and behavior.

Since our experiments ablating BMAL1 or rescuing the tau mutation in SCN astrocytes all resulted in a remarkably similar period lengthening in vitro and in vivo, it is likely that any genetic or environmental disruptions of daily rhythms in astroglia will alter daily rhythms in behavior. That is, it is unlikely that the period lengthening phenotype reflects functions of BMAL1 and CK1ε outside of circadian rhythms. This is supported by our prediction and finding that removal of the CK1ε tau mutation from SCN astrocytes would lengthen the period close to 24 hr. Thus, our data strongly argue that the astrocyte circadian clock regulates daily behaviors.

Why do genetic manipulations predicted to abolish or increase period in SCN astrocytes both result in a similar long period in the SCN and behavior? It could be that loss of BMAL1 or CK1ε similarly impacts a signal (or signals) from astrocytes (e.g., a diffusible factor, structural change, or a metabolic precursor) that influences daily rhythms in SCN neurons. Based on our data, this signal is not normally necessary for rhythmicity in neurons, but its levels are likely clock controlled and accelerate the period of the neuronal clock either directly or by modulating neuron-neuron interactions. Several testable predictions can be made with this proposed model: (1) arrhythmicity in astrocytes caused by disruption of either the positive (e.g., BMAL1 or CLOCK) or negative limb (e.g., PERs or CRYs) of the circadian transcription-translation feedback loop will always lengthen period; (2) combining SCN astrocytes with intrinsically different circadian periods with wild-type (WT) neurons will draw behavioral period toward the astrocyte period; (3) blocking signaling from SCN neurons to astrocytes will alter daily behavior and the phase relationship between rhythms in SCN neurons and astrocytes and/or between SCN neurons.