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Astrocyte Deletion of Bmal1 Alters Daily Locomotor Activity and Cognitive Functions via GABA Signalling

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Astrocyte Deletion of Bmal1 Alters Daily Locomotor Activity and Cognitive Functions via GABA Signalling

Olga Barca-Mayo et al. Nat Commun.

Abstract

Circadian rhythms are controlled by a network of clock neurons in the central pacemaker, the suprachiasmatic nucleus (SCN). Core clock genes, such as Bmal1, are expressed in SCN neurons and in other brain cells, such as astrocytes. However, the role of astrocytic clock genes in controlling rhythmic behaviour is unknown. Here we show that ablation of Bmal1 in GLAST-positive astrocytes alters circadian locomotor behaviour and cognition in mice. Specifically, deletion of astrocytic Bmal1 has an impact on the neuronal clock through GABA signalling. Importantly, pharmacological modulation of GABAA-receptor signalling completely rescues the behavioural phenotypes. Our results reveal a crucial role of astrocytic Bmal1 for the coordination of neuronal clocks and propose a new cellular target, astrocytes, for neuropharmacology of transient or chronic perturbation of circadian rhythms, where alteration of astrocytic clock genes might contribute to the impairment of the neurobehavioural outputs such as cognition.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Glast-Cre-ERt2 mediates astrocyte-specific deletion of Bmal1 in the SCN.
(a) Glast:CreERT2 mice drive the expression of reporter-Td-TOMATO in GFAP-positive SCN astrocytes. Representative micrographs of GFAP immunostaining in the SCN (dashed line) of control mice (4,6-diamidino-2-phenylindole (DAPI) in blue, TOMATO in red and GFAP in green). Scale bar, 50 μm. Quantification of the percentage of Td-TOMATO-positive cells that co-localized with GFAP in control (Glast-Cre-Td-Tomato) or Bmal1cKO-Td-Tomato animals is shown in the right panel. The value express mean±s.e.m. (n=4 animals per group). (b) Glast:CreERT2-driven Td-TOMATO-positive SCN astrocytes express BMALl1. Representative micrographs of BMAL1 immunostaining in the SCN of control or Bmal1cKO-Td-Tomato animals in 12:12 h LD cycles (DAPI in blue, TOMATO in red and BMAL1 in green). Scale bars, 50 μm and 20 μm in the higher magnification images. (c) A 50% reduction of BMAL1 positive cells was observed in the SCN of Bmal1cKO mice compared with control animals (Y axis represents the percentage of total BMAL1-positive cells in the SCN). A 70% reduction of BMAL1-positive cells was observed in the population of Td-TOMATO-positive cells of Bmal1cKO compared with control animals (red, paired t-test, ***P<0.001 versus control animals). A 51% reduction of BMAL1-positive cells in the population of Td-TOMATO-negative cells was found in Bmal1cKO compared with control animals (green, paired t-test *P<0.05 versus control animals). The value express the means±s.e.m. (n=4 animals per group). (d,e) Percent of BMAL1-positive cells was significantly reduced in GFAP (d) or S100β (e) astrocytes in Bmal1cKO-Td-Tomato mice compared with control animals (paired t-test, ***P<0.001 and **P<0.01 versus control animals). The value express mean±s.e.m. (n=4 animals per group).
Figure 2
Figure 2. Circadian locomotor alteration and cognitive impairments on astrocyte-specific Bmal1 deletion.
(a) Bmal1cKO and control mice were treated with TM and 6–8 weeks after treatment, circadian locomotor activity and cognitive tests were evaluated. (b) Representative actograms of control and Bmal1cKO mice during the 12:12 h LD, DD and the re-entrainment to a new LD cycle (rLD). Time of light is indicated by yellow shaded areas in the LD or rLD periods. The Lomb–Scargle periodograms (right panels) show the bimodal pattern of Bmal1cKO mice. (c) Activity waveforms under the LD, DD and rLD are shown for controls (n=8) and Bmal1cKO (n=7) mice. Activity counts are expressed as the average amount of activity in 5-minute bins. For LD and rLD, data are plotted with nighttime hours from 7 to 19 and given in Zeitgeber time (ZT), such that ZT0 (lights on)=hour 19. For DD, units on the abscissa are given in circadian time (CT) and mean activity was expressed as the average amount of activity in 5 min bins over each animal's circadian cycle. The value express the means+s.e.m. (d) Quantification of the activity onset in DD (left panel) indicated that Bmal1cKO mice significantly delayed their active phase compared with control animals (paired t-test, *P<0.05 versus control animals). Bmal1cKO mice also showed an activity onset and offset advance in rLD cycles (middle and right panels) (paired t-test, *P<0.05 versus control animals). The value express the means+s.e.m. (e) Diagram of the experimental design for the NOR and SOL tasks. (f) Performance on the NOR during 1 h retention and 24 h recall session and SOL in Bmal1cKO (n=9) and control mice (n=10). Paired t-test revealed a significant reduction in the DI between familiar and new object in Bmal1cKO in NOR tests and in the location of the object in the SOL test (paired t-test, ***P<0.001 and ****P<0.0001 versus control animals). The value express means+s.e.m.
Figure 3
Figure 3. Altered VIP expression in the SCN and impaired cortical and hippocampal oscillations in Bmal1cKO mice.
(a) Representative micrographs of VIP immunostaining in the SCN of Bmal1cKO and control mice in 12:12 h LD cycles at ZT12. Quantification of fluorescence intensity demonstrates higher VIP levels in Bmal1cKO at ZT12 compared with control animals (paired t-test *P<0.05 versus ZT0 and #P<0.05 versus control animals). The value express the mean±s.e.m. (n=3 animals per group). Scale bar, 40 μm. (b) Analysis of Bmal1 and Per2 in the cortex (upper panels) and hippocampus (lower panels) of control (blue) and Bmal1cKO (red) mice, showing impaired rhythmic expression in mutant mice. Experimental data were cosine fitted. Samples were collected from mice under 12:12 h LD cycles. It is noteworthy that the ZT24 time point is the ZT0 time point, shown again. Means±s.e.m. of five animals per group at each time point (paired t-test; *P<0.05, **P<0.01 and ***P<0.001 versus control animals). (c) Representative images of cortical BMAL1 and PER2 western blottings, showing no oscillation of those proteins in Bmal1cKO as compared ith control mice (n=3 animals per group and time point).
Figure 4
Figure 4. Bmal1 knockdown in astrocytes suppresses entrainment of co-cultured cortical neurons in vitro.
(a) Primary cortical astrocytes were transfected with scramble (Scrbl) or Bmal1 siRNAs. After 48 h, astrocytes were synchronized with 100 nM of Dexamethasone for 2 h (Astro Dexa). After washing, astrocytes were placed in co-culture with asynchronous cortical neurons without physical contact, but sharing the same culture media. (b) Bmal1, Cry1, Per2 and BMAL1 target Dbp were analysed in astrocytes (upper panels) and neurons (lower panels) at the indicated time points by quantitative PCR. Graphs show the mean±s.e.m. of the cosine-fitted curves from three experiments performed in triplicate. (c) Representative images of western blottings for BMAL1 in primary astrocytes (left panels) or CRY1 in co-cultured neurons (right panels), showing expression of BMAL1 in Dexamethasone-treated astrocytes in isolated cultures (top) or Dexamethasone-treated astrocytes in co-culture with asynchronous neurons (middle and bottom), on transfection with scramble siRNAs (CSA Scrbl) or on transfection with Bmal1 siRNAs (CSA KD); (right panels) entrainment of CRY1 in cortical neurons after co-culture with Scrbl transfected synchronous astrocytes (Neu-CSA Scrbl) is not observed when co-culture is performed with arrhythmic astrocytes (Neu-CSA KD) (n=2 independent experiments).
Figure 5
Figure 5. GABAA receptor signalling is sufficient and required to synchronize cortical neurons in vitro.
(a) Asynchronous cortical neurons were treated with a short pulse (2 h) of GABA (100 μM), glutamate (10 μM) or with vehicle as control. Cells were harvested at indicated time points and Bmal1, Cry1, Per2 and Dbp were analysed by quantitative PCR (qPCR) and fitted to a cosinor curve. Each point reflects the means±s.e.m. of three experiments performed in triplicate. (b) Primary cortical astrocytes were synchronized with Dexamethasone (100 nM) treatment for 2 h. After washing, astrocytes were placed in co-culture with asynchronous cortical neurons in the presence of Bicuculline (30 μM) or vehicle. Bmal1, Cry1, Per2 and Dbp were analysed in neurons at indicated time points by qPCR and fitted to a cosinor curve. Graphs show the means±s.e.m. of three independent experiments performed in triplicate.
Figure 6
Figure 6. Alteration of GATs and GABA uptake on deletion of astrocytic Bmal1.
(a,b) Left panels: Gat1 (a) and Gat3 (b) in the cortex of control (blue) and Bmal1cKO (red) mice, showing reduced expression in Bmal1cKO. Samples were collected from mice under 12:12 h LD cycles. It is noteworthy that the ZT24 time point is the ZT0 time point, shown again. Means±s.e.m. of five animals per group at each time point (paired t-test *P<0.05 versus control animals). (a,b) Right panels: quantification of fluorescence intensity of GAT1 (a) and GAT3 (b) demonstrates decreased expression of GAT1 and GAT3 in Bmal1cKO-Td-Tomato or Bmal1cKO, respectively, at ZT0 compared with control animals. Means±s.e.m. of four animals per group (paired t-test; *P<0.05 versus control animals). (c) Primary cortical astrocytes were transfected with scramble (Scrbl) or Bmal1 siRNAs (Bmal1 KD) and after 48 h, were treated with 5, 10 or 40 μM of GABA for 15 min. GABA concentration in the extracellular medium was measured by enzyme-linked immunosorbent assay (ELISA). Graph shows the means±s.e.m. of two independent experiments performed in triplicate (paired t-test; *P<0.05 versus control astrocytes). (d) Determination of GABA levels CSF of Bmal1cKO and control animals at ZT0 by ultra performance LC–MS/MS. Bmal1cKO mice showed significantly higher levels of GABA in the CSF than control animals. A total number of 14 controls and 10 Bmal1cKO animals were used for this experiment. Two or three individual animals were pooled into final samples (n=5). Graph shows the means±s.e.m. (paired t-test; *P<0.05 versus control animals). (e) Quantification of fluorescence intensity of GAT3 in the SCN, demonstrates lower levels in the dorsal part and increased levels in the ventral SCN in Bmal1cKO at ZT0 compared with control animals. Graph shows the means±s.e.m. (n=4 per group, paired t-test; *P<0.05 versus control animals).
Figure 7
Figure 7. GABAA receptor antagonists rescue the behavioural phenotypes of Bmal1cKO mice.
(a) Bmal1cKO and control mice received, 6–8 weeks after TM treatment, one daily injection of (bd) PTZ or (e) PTX at ZT6 for 10 days, before being subjected to (bd) wheel-running activity analysis or (e) cognitive tests. (b) Upper panel: representative actograms of control and Bmal1cKO mice during the 12:12 h LD, DD and rLD cycles. Time of light is indicated by yellow shaded areas in the LD or rLD periods. (b) Lower panels: the Lomb–Scargle periodograms show the rescue of the bimodal pattern of Bmal1cKO mice by PTZ treatment. (c) Activity waveforms under the LD, DD and rLD are shown for controls (n=9) and Bmal1cKO (n=10) mice. Activity counts are expressed as indicated in Fig. 2c. The value express the means+s.e.m. (d) Quantification of the onset of activity in DD (left panel), in rLD (middle panel) and offset in rLD (right panel), indicating no differences among PTZ-treated Bmal1cKO and PTZ-treated control animals (paired t-test). The value express the means+s.e.m. (e) Performance on the NOR test during 1 h retention and 24 h recall session, and SOL task in Bmal1cKO (n=10) and control mice (n=9). No significant differences were found in the DI between familiar and new object in PTZ- or PTX-treated Bmal1cKO mice in NOR tests and in the location of the object in the SOL test, compared with control animals. The value express the means+s.e.m. (two-way analysis of variance, ###P<0.001 versus control animals: *P<0.05 and ***P<0.001 versus untreated animals (that is, with neither PTZ or PTX).

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