Fig. 5

Activation of Radial Astrocytes Reduces Swimming

(A) Experimental design. Rat TRPV1 was expressed in ∼1% of radial astrocytes in Tg(gfap:jRGECO1b) fish. Fish were exposed to the TRPV1 agonist capsaicin.

(B) Example fish with increased Ca²⁺ transients in L-MO and concurrent increase in passivity after capsaicin treatment. The effect diminished after ∼1 h, maybe due to TRPV1 desensitization and internalization (Sanz-Salvador et al., 2012), showing that fish remain healthy, as they can swim normally. Dashed line (passive) means the fish is passive 100% of the time; dashed line (active) means it is passive 0% of the time (both measured in 2 min bins).

(C) Population data, showing increased passivity after activation of TRPV1. n = 7 fish.

(D) Population data of L-MO astrocytic Ca²⁺ increase and passivity with TRPV1 activation. Two-tailed paired t test, behavior: p = 0.0029, n = 7 fish. ΔF/F: p = 0.027, n = 5 fish.

(E) Astrocytic Ca²⁺ in L-MO near passivity onset. Average signal increased just before passivity onset and peaked just after, as in futility-induced passivity (Figures 2G–2I). Shading, SEM.

(F) Control, example. No reduction of swimming or increase in astrocytic Ca²⁺ when saline was added to fish expressing TRPV1.

(G) Control, population data. No change in behavior when saline was added to fish expressing TRPV1 (n = 6 fish) or capsaicin to fish without TRPV1 (n = 5 fish).

(H) Summary of controls. Experiments as in (F) and (G) were pooled. Two-tailed paired t test, behavior: p = 0.48, n = 11 fish. ΔF/F: p = 0.51, n = 7 fish.

(I) Normalized mean L-MO glial Ca²⁺ signals centered on the onset of passivity, induced by TRPV1 in closed loop (top) or by motor futility in open loop (bottom). Surges appear similar. Ca²⁺ in TRPV1+ animals did not saturate.

(J) Experimental design. Local optogenetic excitation of glial processes in fish expressing CoChR or Opto-α1-AR increases glial Ca²⁺ in L-MO (CoChR shown).

(K) Optogenetic (CoChR) excitation of L-MO glia reduced swimming for ∼10 s. Blue light outside the brain caused a much milder and briefer reduction.

(L and M) Population data show reduced swimming relative to controls in fish expressing CoChR (L) or Opto-α1-AR (M) in radial astrocytes. Two-tailed paired t test, CoChR, p = 0.00012, n = 9 fish; Opto-α1-AR, p = 0.0065, n = 10 fish.

(N–P) Controls for (K)–(M) show no effect in fish without CoChR or Opto-α1-AR. Two-tailed paired t test, CoChR: p = 0.89, n = 6 fish. Opto-α1-AR, p = 0.50, n = 8 fish.

(Q) Summary of Figures 4 and 5, showing significant increases in passivity after increases in astrocytic Ca²⁺, decreases after astrocytic silencing, and no effects in control experiments. Effects are normalized by maximal possible change (control passive % for decreases; control active % for increases) for each fish.

(R) Optogenetic activation of L-MO radial astrocytes activates GABAergic neurons at edge of L-MO in Tg(gfap:CoChR-eGFP);Tg(elavl3:jRGECO1b) fish. Left: maximum-intensity projection of ΔF/F after optogenetic activation of radial astrocytes (magenta) and anatomy (cyan) in example fish. Inset: Ca²⁺ in GABAergic neurons after astrocytic activation; stimulation time in cyan. Top right: Tg(gad1b:RFP);Tg(elavl3:H2B-GCaMP6f) used to label GABAergic neurons; bottom right: Tg(gad1b:Gal4);Tg(UAS:CoChR-eGFP) to sparsely label GABAergic neurons.

(S) Activation of GABAergic cells with laser light in Tg(Gad1b:Gal4);Tg(UAS:CoChR-eGFP) suppresses swimming. Two-tailed paired t test, p = 0.0074, n = 7 fish.