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. 2017 Apr 3;27(7):1055-1061.
doi: 10.1016/j.cub.2017.02.037. Epub 2017 Mar 23.

Astrocytes Regulate Daily Rhythms in the Suprachiasmatic Nucleus and Behavior

Chak Foon Tso  1 , Tatiana Simon  1 , Alison C Greenlaw  1 , Tanvi Puri  1 , Michihiro Mieda  2 , Erik D Herzog  3
Affiliations

Astrocytes Regulate Daily Rhythms in the Suprachiasmatic Nucleus and Behavior

Chak Foon Tso et al. Curr Biol. .

Abstract

Astrocytes are active partners in neural information processing [1, 2]. However, the roles of astrocytes in regulating behavior remain unclear [3, 4]. Because astrocytes have persistent circadian clock gene expression and ATP release in vitro [5-8], 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.

Keywords: Aldh1l1; Bmal1; GABA; GFAP; Per2; SCN; astroglia; casein kinase 1; circadian oscillator; glia.

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Figures

Figure 1
Figure 1. Astrocytes targeted in the SCN with the Aldh1L1-Cre/+ mouse line
Astrocyte nuclei in the SCN were labeled in green by Cre-mediated recombination of Aldh1L1-Cre/+;LSL-GFPNLS/+ mice. 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 counter stained 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.
Figure 2
Figure 2. SCN astrocytes are functional circadian oscillators
(A) Schematic of AAV-Bmal1ext-DIO-luc vector that provides a real-time, cell-type specific report of Bmal1 transcription. (B) (Left) Representative frame from a movie of bioluminescence recorded from an Aldh1L1-Bmal1luc SCN slice. Note the glowing astroglial cells throughout the bilateral SCN. Scale bar = 400μm (Right) Cell-sized regions of interest (ROIs) were used to track Bmal1 expression from astrocytes within the SCN. (C) Representative bioluminescence traces from PMT recording of two Aldh1L1-Bmal1luc SCN slices. (D) Raster plot of Bmal1 reporter expression across a representative Aldh1L1-Bmal1luc SCN slice from (B). The bioluminescence in each ROI peaked at approximately the same time daily over the three days of recording with an ultracooled CCD camera. Bioluminescence for each ROI was normalized to its maximum and pseudocolored (color bar at right). See also Table S1.
Figure 3
Figure 3. Loss of Bmal1 in SCN astrocytes lengthens circadian period in vitro and in vivo
(A) Schematic showing how CRISPR was used to delete the Bmal1 gene in SCN astroglia. (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 two days with the second day’s data replotted on the line below. The mice were less active in the light (yellow) in the 12h: 12h light:dark cycle of the first 25 days of recording and showed free-running rhythms in constant darkness. (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. (D) GFAP staining (red) reliably colocalized with Cre-activated GFP (green) in this representative SCN of an Aldh1L1-Cas9 mouse. (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; 1-way ANOVA with Sidak multiple comparison test. (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. (G) Loss of Bmal1 lengthened circadian period in the isolated SCN. * : p < 0.05; t-test. Scale bars = 75 μm. Error bars: mean ± sem. See also figure S2.
Figure 4
Figure 4. Loss of CK1ε in SCN astrocytes lengthens circadian period in vivo and in vitro
(A) Representative actograms of Aldh1L1- CK1εtau/+ and CK1εtau/+ littermates showing how locomotor activity free-ran in constant darkness with (B) a longer circadian period in mice where the CK1εtau/+ mutation was removed from astrocytes (p < 0.0001, t-test). (C) Representative PER2::luc recordings from the isolated SCN of Aldh1L1- CK1εtau/+ and CK1εtau/+ littermates with (d) a longer circadian period in SCN where the CK1εtau/+ mutation was removed from astrocytes (p < 0.01, t-test). (E) Schematic of a second method to change circadian timing in astrocytes using GFAP AAV injection to CK1εtau/+ SCN in vivo. (F) Representative actograms of two CK1εtau/+ mice injected with either AAV8-GFAP-GFP or AAV8-GFAP-Cre-GFP with (G) successful viral targeting to the SCN. (H) The circadian period of locomotor activity of AAV8-GFAP-Cre injected CK1εtau/+ mice was approximately 1 h longer than controls (p < 0.05, t-test). (I) Representative whole SCN PER2::luc recording from CK1εtau/+ animals injected with either AAV8-GFAP-GFP or AAV8-GFAP-Cre-GFP. (J) Period of PER2::luc recordings from AAV8-GFAP-Cre; CK1εtau/+ SCN was approximately 2 h longer than controls (p < 0.001, t-test). Error bars: mean ± sem.
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