Fig. 2: Convergence of neural signals evokes astrocyte Fos.

a, Schematic of pharmacological screening. b, NA increased c-Fos intensity in GFAP⁺ astrocytes (n = 27–50 cells per condition). One-way ANOVA, Dunnett’s test versus vehicle (Veh). 5-HT, serotonin; a.u., arbitrary units; ACh, acetylcholine; DA, dopamine; GABA, γ-aminobutyric acid. c, Image showing NA (1 µM)-induced c-Fos increase (vehicle: n = 40; NA 1 μM: n = 41 cells). Scale bar, 10 μm. d, NA-induced c-Fos was blocked by inhibition of β-receptors or cAMP but not by Ca²⁺ mobilization (n = 28–55 cells per condition). One-way ANOVA, Dunnett’s or Tukey’s test. AC, adenylate cyclase; Atip, atipamezole; Frsk, forskolin; Isop, isoproterenol; Praz, prazosin; Prop, propranolol. e, Schematic of AAV-mediated Grm3 (encoding mGluR3) expression. f,g, Convergent NA and glutamate stimulation enhanced c-Fos in mGluR3⁺ astrocytes, but not in mGluR3⁻ astrocytes (n = 98–131 cells per condition). One-way ANOVA, Tukey’s test. Scale bar, 20 μm. h, Schematic of signalling cascade. α₁R, α₁-receptor; βR, β-receptor. i, Ionomycin (Iono)-induced increase in Ca²⁺ increased c-Fos when combined with NA, isoproterenol (β-receptor agonist) or forskolin (n = 41–59 cells per condition). One-way ANOVA, Tukey’s test. j, Schematic of AAV injection into amygdala (LA/B) and LC in TRAP2 mice. Right, hM3Dq expression. Scale bars, 1 mm. k–m, Dual stimulation of LA/B and LC increased astrocyte density (LC–/LA/B–: n = 5; LC+/LA/B–: n = 4; LC–/LA/B+: n = 6; LC+/LA/B+: n = 5 mice). k, Images of hM3Dq-expressing cells. Scale bar, 200 μm. l, Fos co-expression in hM3Dq⁺ cells. Scale bar, 10 μm. m, Quantification of Fos-tagged astrocytes (mNG⁺) localized near hM3Dq⁺ LA/B neurons and NA fibres in l (representative of five biological replicates from the LC+/LA+ group). One-way ANOVA, Dunnett’s test. n–q, FR-BAE tagging with β-receptor blockade (propranolol, 10 mg kg⁻¹) (n), LC silencing (o), amygdala (Amy) silencing (p) or RAM-based engram silencing (q) reduced BAE density. Scale bars (n,o–q, top and middle), 1 mm. o–q, Bottom image, magnified view showing local hM4Di-mCherry in neurons. Scale bars, 40 μm. Dox, doxycycline. r, FR-BAE density was reduced in all perturbation groups (Prop–/hM4Di–: n = 4; Prop+/hM4Di–: n = 4; LC: n = 4; amygdala (Amy): n = 5; engram: n = 4 mice). One-way ANOVA, Dunnett’s test. Data are mean ± s.e.m. Sample sizes and replicates in Supplementary Table 1.