Cannabinoid Signaling Recruits Astrocytes to Modulate Presynaptic Function in the Suprachiasmatic Nucleus

WIN decreases mGPSC frequency in a CB1R and astrocyte-dependent manner. For all frequency and amplitude plots gray lines are individual cells, black lines indicate the group average. A, Individual cell and mean mGPSC frequencies before and after treatment with either DMSO (0.01%) or WIN (3 μM). B, Individual cell and mean amplitudes of mGPSCs before and after DMSO or WIN application. C, Representative mGPSC current recordings before and after treatment with DMSO or WIN. D, Individual cell and mean mGPSC frequency (top) and amplitude (bottom) pretreatment, with AM251 (5 μM), and with both AM251 (5 μM) and WIN (3 μM). E, Representative mGPSC recordings from a single neuron before and during treatment, with AM251, and with AM251 and WIN. F, Individual cell mGPSC frequency (top) and amplitude (bottom) before and during application of FC (1 μM), and with both FC (1 μM) and WIN (3 μM). G, Representative mGPSC recordings from a single neuron before and during treatment with FC, and with FC and WIN; *p < 0.001, repeated measures analysis.

Chloride sensor expression in GFAP-Cre+ animals is specific to GFAP expressing astrocytes and not neurons or glial precursor cells. A–F, GFAP labeling (left), Cl sensor YFP fluorescence (center), and colocalization (right) within the SCN of GFAP-Cre+/Cl sensor+ animals. G–L, NFHC neuronal marker (left), Cl sensor YFP (center), and lack of colocalization (right) within the SCN. Scale bars are all 50 μm, images A–C and G–I were taken at 20× and images D–F and J–L were taken at 40×.

WIN activates an astrocytic Ca²⁺ signaling pathway. A–D, top, Representative GCaMP6 images from slices treated with DMSO (0.01%), WIN (3 μM), WIN (3 μM) + TTX (1 μM) + CNQX (10 μM), and WIN (3 μM) + thapsigargin (1 μM; Thap). White scale bar is 50 μm, images taken at 40× and the inserts magnified 120×. Bottom, Representative traces showing the change in fluorescence from all regions measured (gray) and average response (black) from the slice depicted above. Black arrow indicates beginning of treatment. E–H, top, Pie charts depicting percentage of soma and non-soma regions across all slices responding with an increase (black), decrease (dark gray), variable response (light gray), or no response (white) to DMSO, WIN, WIN + TTX + CNQX, or WIN + Thap (left to right). Bottom, Cumulative average fluorescence increase during baseline and drug treatment for soma and non-soma regions across all slices.

Blockade of CB1R alters the frequency of spontaneous astrocytic Ca²⁺ events. A, Representative GCaMP6 image. White scale bar is 50 μm, image taken at 40×. White, numbered ellipses are the ROIs plotted in B. B, Representative traces of intensity 180 s before, during, and after AM251 treatment (indicated by black bar). Numbers 1–4 correspond to numbered regions in A. Note that the event examples were picked to reflect the varying responses to AM251 with traces 2 and 3 showing a clear reduction in frequency. C, Average event numbers across all experiments of regions before, during, and after AM251 treatment depicted as mean ± SEM (*p < 0.05, Friedman test). D, Average percentage of regions responding with a decrease, increase, or no change on treatment with AM251 across all experiments (mean ± SEM). E, Cumulative probability curves of amplitude for both soma (top) and non-soma (bottom) regions before, during, and after treatment with AM251.

Neurons utilize endocannabinoid signaling to activate astrocyte Ca²⁺ signaling pathways. A, Representative bright field (top) and GCaMP6 (bottom) images from a single slice. White scale bar is 50 μm, images taken at 40×. White arrow indicates the placement of the microelectrode tip. Numbered ellipses correspond to locations of traces in B. B, Representative intensity changes from the regions marked in A before and after depolarization of an SCN neuron (indicated by black bar) before (left) and during (right) AM251 (5 μM) treatment. C, Average number of events pre- and post-depolarization, before, during, and after treatment with AM251 across all experiments (mean ± SEM, *p < 0.05, Friedman test). D, Cumulative percentage of regions per change in event number from depolarization, before, during, and after AM251. E, Boxplot of event amplitudes pre- and post-depolarization, before, during, and after treatment with AM251.

Adenosine signaling is necessary for WIN-induced mGPSC frequency changes. A, top, Representative traces pretreatment (left), with ACPTII (middle), and with ACPTII+WIN (right) from an individual neuron. Bottom, Individual cell mGPSC frequency (left) and amplitude (right) before, during application of ACPTII (200 μM), and both ACPTII (200 μM) + WIN (3 μM; *p < 0.05, repeated measures analysis). B, top, Representative traces pretreatment (left), with CGS15943 (CGS, middle), and with CGS +WIN (right) from an individual neuron. Bottom, Individual cell mGPSC frequency and amplitude pretreatment, with CGS (50 μM), and both CGS (50 μM) and WIN (3 μM). C, top, Representative traces (left) pretreatment, with DPCPX (middle), and with DPCPX +WIN (right) from an individual neuron. Bottom, Individual cell and mean mGPSC frequency (left) and amplitude (right) pretreatment, with DPCPX (0.2 μM), and both DPCPX (0.2 μM) and WIN (3 μM). For all frequency and amplitude plots, gray lines indicate individual neuron responses, black lines represent group averages.

Adenosine decreases the mGPSC frequency. A, Representative mGPSC recordings from a single SCN neuron before and during adenosine (100 μM) application. Frequency (B) and amplitude (C) of mGPSCs before and during adenosine treatment. Gray lines are individual cells, black represents the group average; *p = 0.05.

Activation of Gq DREADDs increases intracellular Ca²⁺ in astrocytes. A, Representative SCN slice of a GFAP-Cre+ mouse injected with Gq DREADD and GCaMP6. Scale bar is 50 μm, images taken at 40× and the inserts magnified 120×. B, Fluorescent intensity over time of the slice shown in A. Black arrow indicates the CNO (10 μM) application. Gray lines are individual regions, the black line is the average response of regions. C, Pie charts depict the percentage of regions that increase (black), decrease (dark gray), have a variable response (light gray), or do not respond (white) to CNO treatment across all experiments. D, Average fluorescent intensity before and after CNO application displayed as mean ± SEM for soma and non-soma regions.

Astrocytic Gq DREADD activation decreases mGPSC frequency in an A1R-dependent manner. A, Representative mGPSC traces before and after CNO (10 μM) application from a single neuron. Frequency (B) and amplitude (C) of mGPSCs before and after CNO (10 μM) treatment (*p < 0.05, repeated measures analysis). Gray lines are individual neurons, the black line is the group average. D, Representative mGPSC recordings before treatment, with DPCPX (0.2 μM), and with both DPCPX (0.2 μM) + CNO (10 μM) from a single neuron. Frequency (E) and amplitude (F) of mGPSCs before treatment, with DPCPX, and DPCPX+CNO. Gray lines are individual neurons, the black line is the group average.