Extended Data Fig. 13: Astrocytic iβARK2 validation in the amygdala.

a, Schematics showing the Gαq GPCR signalling cascade and the blocking of which with iβARK. Left: iβARK, a 122-residue inhibitory peptide from β-adrenergic receptor kinase²⁶, directly binds to Gαq subunits and thus sequesters them to block subsequent Gαq-GPCR-induced signalling cascades. Right: iβARK2 with Lck allows it to localize to the plasma membrane, thus being more proximal to and binding Gαq subunits more efficiently. b, Top: AAV2/5 constructs for astrocytic iβARK or iβARKmut. Bottom: AAV2/5 constructs for iβARK2 and iβARK2mut. c, Schematic for Ca²⁺ imaging of iβARK-positive LA/B astrocytes. d, Representative images showing expression of iβARK and iβARK2 in LA/B. iβARK2 has improved expression near plasma membrane (iβARK: n = 5, iβARK2: n = 5 biological replicates). e, Representative pseudo-coloured images showing the high fluorescence of GCaMP6f in LA/B astrocytes (representative of n = 28 iβARK2mut and 13 iβARK2 astrocytes). f, dF/F traces of Ca²⁺ responses in a iβARK(D110A)- or iβARK-expressing astrocyte evoked by PE (phenylephrine), an agonist of Gαq-coupled α1A-adrenoceptor (coded by Adra1a) analyzed for soma and processes separately. g, Scatter graph showing the average Ca²⁺ responses to 0, 1, and 3 µM PE, where iβARK significantly reduced the PE-induced Ca²⁺ responses. iβARKmut: n = 29 cells, iβARK: n = 33 cells, iβARK2mut: n = 28 cells, iβARK2: n = 13 cells, 3-4 mice per group. Two-way ANOVA, Tukey’s multiple comparisons test. Data are mean ± SEM. **p < 0.01; ns, not significantly different. Sample sizes and replicates in Supplementary Table 1.