Fig. 5

Rescuing Effect of GABA and Rapamycin on depdc5 Knockout Zebrafish

(A) Swimming activity of 8-dpf larvae, with the activity of depdc5^+/+ larvae normalized to 100%, shows that treatment with GABA (100 μM) can completely rescue hypoactivity; N = 2, n = 20/genotype/treatment. Rapamycin treatment (100 nM from day 0 to 2, 300 nM from day 3 onward) was used as a positive control.

(B) Spontaneous coiling activity in 20-hpf embryos shows that the hypoactivity of depdc5^−/− embryos is rescued by treating with 100 nM rapamycin or with 100 μM GABA from the single-cell stage. N = 2, n = 20/genotype/treatment; Student’s t test, ^∗∗∗∗p(control WT versus HM) < 0.0001. Coiling activity of depdc5^+/+ was set at 100% for each sample, and the relative activity of the depdc5^−/− larvae is represented.

(C) Western blotting of 9-dpf larval lysates to examine phospho-S6 levels in depdc5^+/+ and depdc5^−/− larvae treated with DMSO or rapamycin (100 nM from day 0 to 2, 300 nM from day 3 onward) shows inhibition of mTOR signaling upon rapamycin treatment.

(D) Distance swam by 2-dpf larvae treated with GABA (100 μM) or rapamycin (100 nM) upon PTZ exposure (3 mM). N = 3, n > 10.

(E) Representative swimming tracks of 2-dpf embryos (untreated or treated with GABA or rapamycin) during a 10-min period following PTZ exposure (3 mM).

(F) Images of transverse sections of 7-dpf GABA-treated depdc5^+/+ and depc5^−/− larval brains immunostained with α-GAD65/67 and counterstained with DAPI. Regions indicated in red boxes have been magnified to show the branching defects. GABA treatment (100 μM) was begun at 8 hpf; n = 3 embryos/genotype. Scale bar, 10 μm.

(G) Quantification of the number of GAD65/67-positive neurofilaments in GABA-treated depdc5^+/+ and ^−/− larval brain sections showing that GABA treatment does not rescue the defects in depdc5^−/−.

(H) Survival curve showing that treatment of depdc5^−/− larvae with rapamycin (100 nM from day 0 to 2, 300 nM from day 3 onward) first prolongs lifespan as long as rapamycin is supplemented in the water (until day 25; shaded). Withdrawal of rapamycin results in a drop in survival, and all the larvae die by day 35. On the contrary, treatment with 100 μM GABA from 8 hpf does not extend survival. Neither rapamycin nor GABA affected depdc5^+/+.