Extended Data Fig. 9: Additional data related to the GFAPcreERT2Slc17a7fl/fltdTomlsl/lsl mouse model, the electrophysiology and the behavioural studies presented in Fig. 3.
From: Specialized astrocytes mediate glutamatergic gliotransmission in the CNS
a, Representative fluorescence activated cell sorting (FACS) of tdTomato-positive (Tom+) astrocytes in cerebral cortex samples of VGLUT1GFAP-WT mice (sorted ≥2 x 105 Tom+ cells per experiment, n = 2 independent experiments, 5 mice per experiment), VGLUT1GFAP-KO mice (sorted ≥2 x 105 Tom+ cells per experiment, n = 2 independent experiments, 5 mice per experiment) and GFAPCreERT2tdTomlsl/lsl mice (sorted ≥2 x 105 Tom+ cells per experiment, n = 2 independent experiments, 5 mice per experiment). b, Representative images, here acquired with confocal microscope (n = 2), confirming no leakage in the absence of TAM-induced cre recombination, i.e., lack of any Tom+ cells (red) in the hippocampus of VGLUT1GFAP-WT and VGLUT1WT-TAM control mice also stained with the astrocyte markers GS and S100β (green), and the nuclear marker, DAPI (blue), n = 8 images from 4 independent experiments, 2 mice per group. Scale bar: 50 µm.c, Left, table presenting the total number per mm2 of Tom+ cells and the relative numbers of the same Tom+ cells co-labelled with astrocyte (GS+S100β), neuron (NeuN), oligodendrocyte (Olig2) or microglia (Iba1) markers, counted in two hippocampal regions (CA1 and DG) and in the visual cortex of VGLUT1GFAP-KO mice upon TAM-induced cre recombination. Data are presented as means ± s.e.m. Right, Confocal images confirming lack of any co-labelling of Tom+ cells with microglia (Iba1, green) oligodendrocyte (Olig2, white) or neuronal (NeuN, green) markers in the DG of VGLUT1GFAP-KO mice. n = 8 images from 4 independent experiments, 2 mice per group. Scale bar: 50 µm. d, ϴ-LTP recorded in DGML of wild-type mice by 3 local field potential (LFP) electrodes positioned along the same bundle of PP fibres at an average distance of 200 µm (electrode 1), 300 µm (electrode 2) and 400 µm (electrode 3) from the stimulation pipette (STIM). ϴ-LTP magnitude is the same at all tested locations (two-way ANOVA repeated measures (P = 0.78, n = 6 slices, 3 mice). Data are means ± s.e.m. e, Setting for ϴ-LTP induction and measure in GFAPcreERT2Slc17a7fl/fltdTomlsl/lsl mice undergone short TAM treatment (Methods). Top, bright-field (BF) and fluorescence images (Tom) show positioning of the stimulation pipette (STIM) and of the two LFP recording electrodes in the DGML, with about 200 µm interdistance. Scale bar: 200 µm. Bottom, higher zoom images show the position of electrode 1, proximal to a non-fluorescent astrocyte (astro), and of electrode 2, proximal to a fluorescent astrocyte (astro Tom+). n = 16 slices, 12 mice. Scale bar: 50 µm. f, Basal input-output curves (left) and basal fEPSP amplitudes (right) recorded in two DGML fields containing, respectively, a VGLUT1GFAP-WT (grey) and a VGLUT1GFAP-KO astrocyte (orange), show no significant differences (data mean ± s.e.m.; paired Student’s t test, two tails, P = 0.337). Thin lines connect individual LFP electrode pairs (n = 16 slices, 12 mice). g–l, Astrocyte Ca2+ dynamics during low and high-frequency stimulation of MPP in VGLUTGFAP-WT and VGLUT1GFAP-KO mice. g, Top, in control experiments, Slc17a7fl/f mice are injected with AAV5-GFAP-mCherry virus (control virus) and AAV5-GFAP-GCaMP6f virus to report astrocyte Ca2+ dynamics. Bottom, multiple astrocytes present in the same FOV as in h and i display both mCherry (red) and GCaMP6f (green) fluorescence. Scale bar: 50 µm. n = 2 FOVs, 2 mice. h, Left, mean time projection over 70 frames (21 s) of the GCaMP6f signal in astrocytes in the period before ϴ-LTP induction (Pre). Right, representative Ca2+ traces from selected single-cell ROIs during the same Pre period. Astrocytes show small asynchronous local Ca2+ activity and a few larger responses to single MPP stimulations. i, Left, mean time projection of the GCaMP6f signal in astrocytes as in h but during MMP stimulation inducing ϴ-LTP (ϴ-LTP). Right, representative Ca2+ traces from single astrocyte ROIs during ϴ-LTP induction. Multiple astrocytes show very large Ca2+ elevation (note the scale is 10-fold larger than in the Pre period) almost synchronously at the start of the ϴ-LTP protocol (red vertical line). j, Top, in experiments in VGLUTGFAP-KO mice, Slc17a7fl/fl mice are injected with AAV5-GFAP-mCherry-iCre virus to delete VGLUT1 selectively in astrocytes, and with AAV5-GFAP-GCaMP6f virus to report astrocyte Ca2+ dynamics. Bottom, multiple astrocytes, present in the same FOV as in k and l, display both mCherry fluorescence (red) indicating Cre recombination and GCaMP6f fluorescence (green). Scale bar: 50 µm. n = 2 FOVs, 2 mice. k Left, mean time projection over 80 frames (24 s) of the GCaMP6f signal in astrocytes in the Pre period. Right, representative Ca2+ traces from selected single-cell ROIs in the same Pre period. Astrocyte Ca2+ dynamics in VGLUT1GFAP-KO mice in the Pre period are comparable to those in controls mice (h). l, Left: mean time projection of the GCaMP6f signal in astrocytes as in k but in the ϴ-LTP induction period. Right, representative Ca2+ traces from selected single-cell ROIs during ϴ-LTP induction. The very large synchronous astrocyte Ca2+ responses in VGLUT1GFAP-KO mice are comparable to those in controls. m, Open field (O.F.) and activity tests (A.T.) performed on VGLUT1GFAP-KO (orange, n = 9 mice), VGLUT1GFAP-WT (light grey, n = 9 mice) and VGLUT1WT-TAM (dark grey, n = 10 mice) mice. Left, top, histograms reporting parameters index of locomotor activity (total distance travelled, P = 0.102; and velocity, P = 0.086) and anxiety (time spent immobile, P = 0.28; and time in the inner zone of the arena, P = 0.28) do not show group differences. Bottom, example traces of locomotor activity in the three mouse groups placed in the O.F. for 20 min. Right, top: Histograms reporting parameters index of exploratory activity (total number of rearings measured during the 5 min activity test preceding fear conditioning) do not show group differences (P = 0.89). Right, bottom, histograms reporting parameters index of pain sensitivity (mean total distance moved during 2s e-shocks repeated 6 times during the fear conditioning test) do not show group differences (P = 0.84). Data presented as mean ± s.e.m. One-way ANOVA with Tukey test.
