Extended Data Fig. 6: Chemogenetic manipulation of astrocyte Ca²⁺ via hM3Dq-actuation.

a, In vivo astrocyte Ca²⁺ fibre photometry measuring effects of 3 mg/kg CNO in animals expressing hM3Dq or a control mCherry virus over 10 min post-CNO injection, as shown by population traces (left) and AUC quantification (right) (2-way repeated measures ANOVA and Fisher’s LSD post hoc tests, *P = 0.0022, Control n = 7, hM3Dq n = 6 mice). b, Effects of 3 mg/kg CNO on CS-related astrocyte Ca²⁺ activity tested beginning 3 min after CNO-injection, as shown by population traces (left) and AUC quantification (right) (2-way repeated measures ANOVA and Fisher’s LSD post hoc test on AUC values, *P < 0.05, Control n = 7, hM3Dq n = 6 mice). c, In vivo astrocyte on Ca²⁺ fibre photometry measuring effects of 3 mg/kg CNO in animals expressing hM3Dq or a control mCherry virus over 20 min post-CNO injection, as shown by population traces (left) and AUC quantification (right) (2-way repeated measures ANOVA and Šídák’s post hoc tests on AUC values, at 10 min *P < 0.0001, 20 min *P = 0.0306, Control n = 7, hM3Dq n = 6 mice). d, Effects of 3 mg/kg CNO on CS-related astrocyte Ca²⁺ activity tested 10 min post-CNO injection, as shown by population traces (left) and AUC quantification (right) (2-way repeated measures ANOVA and Fisher’s LSD post hoc test on AUC values, *P = 0.0069, Control n = 7, hM3Dq n = 6 mice). e, In vivo astrocyte Ca²⁺ fibre photometry measuring effects of 3 mg/kg CNO on Ca²⁺ activity in animals expressing hM3Dq or a mCherry control virus over 30 min post-CNO injection, as shown by population traces (left) and AUC quantification (centre) and Ca²⁺ events (right) (2-way repeated measures ANOVA and Šídák’s post hoc tests for AUC values at 10 min *P < 0.0001, 20 min *P = 0.0013, and Ca²⁺ transients at 10 min *P = 0.0002, 20 min *P = 0.0138, 30 min *P = 0.0200, Control n = 7, hM3Dq n = 6 mice). f, Effects of 3 mg/kg CNO on 0.4 mA shock-related Ca²⁺ activity tested 30 min post-CNO injection, as shown by population traces (left) and AUC quantification (right) (2-way repeated measures ANOVA and Fisher’s LSD post hoc test for AUC values, *P = 0.0006, Control n = 4, hM3Dq n = 3 mice). g, Effects of 3 mg/kg CNO on 1.0 mA shock-related Ca²⁺ activity tested 30 min post-CNO injection, as shown by population traces (left) and AUC quantification (right) (2-way repeated measures ANOVA and Fisher’s LSD post hoc test for AUC values, *P < 0.0001, Control n = 3, hM3Dq n = 3 mice). h, Optical fibre tip placement for in vivo astrocyte Ca²⁺ fibre photometry assessment of Ca²⁺ activity in animals expressing a diluted 1:8 concentration of hM3Dq-expressing virus (hM3Dq 1:8), undiluted hM3Dq-expressing virus (hM3Dq), or a mCherry control virus (Control) administered 0.1 or 3 mg/kg CNO. i, Effects of a lower (0.1 mg/kg) CNO dose on Ca²⁺ activity in animals expressing hM3Dq or a mCherry control virus over 30 min post-CNO injection, as shown by population traces (left) and AUC quantification (centre) and Ca²⁺ events (right) (2-way repeated measures ANOVA and Šídák’s post hoc tests for AUC values at 20 min *P = 0.0026 and Ca²⁺ transients at 10 min *P = 0.0058, 20 min *P = 0.0003, 30 min *P = 0.0458, Control n = 4, hM3Dq n = 5 mice). j, Effects of 0.1 mg/kg CNO on 0.4 mA shock-related Ca²⁺ activity tested 30 min post-CNO injection, as shown by population traces (left) and AUC quantification (centre) and Ca²⁺ events (right) (2-way repeated measures ANOVA and Fisher’s LSD post hoc test, *P = 0.0133, Control n = 4, hM3Dq n = 5 mice). k, Effects of 3 mg/kg CNO on Ca²⁺ in animals expressing a diluted 1:8 concentration of hM3Dq-expressing virus or a mCherry control virus over 30 min post-CNO injection, as shown by population traces (left) and AUC quantification (centre) and Ca²⁺ events (right) (2-way repeated measures ANOVA and Šídák’s post hoc tests for AUC values at 10 min *P = 0.0459 and Ca²⁺ transients at 10 min *P = 0.0448, Control n = 4, hM3Dq 1:8 n = 4 mice). l, Effects of 3 mg/kg CNO on 0.4 mA shock-related Ca²⁺ activity in animals expressing a diluted 1:8 concentration of hM3Dq-expressing virus or a mCherry control virus tested 30 min post-CNO injection, as shown by population traces (left) and AUC quantification (centre) and Ca²⁺ events (right) (2-way repeated measures ANOVA and Fisher’s LSD post hoc tests, P > 0.05, Control n = 4, hM3Dq 1:8, n = 4 mice). m, Effects of 0.1 mg/kg CNO injected prior to extinction training on freezing levels during F-Con, Ext, E-Ret, and F-Ren in animals expressing hM3Dq or a mCherry control virus (2-way repeated measures ANOVA and Šídák’s post hoc tests, Ext block 3 *P = 0.0165, block 4 *P = 0.0432, Control n = 12, hM3Dq n = 9 mice). n, Effects of 0.1 mg/kg CNO on novel open field total distance and centre time (2 tailed unpaired t-tests, P > 0.05, Control n = 12, hM3Dq n = 9 mice). o, Effects of 3 mg/kg CNO injected prior to extinction training on freezing levels in animals expressing a diluted 1:8 concentration of hM3Dq-expressing virus or a mCherry control virus (2-way repeated measures ANOVA and Šídák’s post hoc tests, P > 0.05, Control n = 11, hM3Dq 1:8 n = 12 mice). p, Effects of 3 mg/kg CNO on novel open field total distance and centre time in animals expressing a diluted 1:8 concentration of hM3Dq-expressing virus or a mCherry control virus (2-tailed unpaired t-tests, P > 0.05, Control n = 11, hM3Dq 1:8 n = 12 mice). Horizontal lines above traces in b,d,f,g,j,l denote permutation test-determined significant difference from chance for hM3Dq (yellow) and Control (black) or significant difference between groups (orange). Fluorescence values were z-scored to either the 30-min or 3-min pre-CNO period, the 5-s pre-CS period, or the 5-s pre-shock period. Data mean ± SEM.

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