Figure 2: E_GABA shift after CCI is due to the elevated intracellular chloride regulated by NKCC1.

(a) Two-photon images of Clomeleon in acutely dissected DRGs from intact (left) and 2 days post injury (right) animals. (b) Decrease in Clomeleon ratio (F_YFP/F_CFP) in intact DRG compared with 2 days after injury, corresponding to an increase in intracellular chloride (unpaired t-test, P<0.0001). (c) Representative staining of NKCC1 (green) on the cell membrane of DRG from control and 2 d.p.i. animals. Nuclei (blue) were stained with DAPI (4,6-diamidino-2-phenylindole). (d) Quantification of immunofluorescence signals for cell surface NKCC1 protein (n=3 DRGs from 3 animals for each; unpaired t-test, P<0.05). (e) Bright-field and Fura-2 ratiometric images of DRG neurons labelled as 1–4. (f) Representative traces evoked by GABA (1 mM) in the calcium imaging experiment from the four neurons labelled in e. ΔF₃₄₀=F₃₄₀–B₃₄₀, ΔF₃₈₀=F₃₈₀–B₃₈₀. (g) The proportion of neurons displaying a GABA-evoked in [Ca²⁺]_i was not different between control and injured animals (control: large neuron: n=37, small n=207; 2 d.p.i.: large neuron: n=19, small n=127; Fisher’s test, P>0.05). (h) Left, representative traces recorded by perforated patch under current clamp. Right, the amplitude of depolarization induced by brief GABA application was not different between control and injured animals (unpaired t-test, P>0.05). Error bars indicate s.e.m. Scale bars, (a) 50 μm; (c) 10 μm; (e) 30 μm. *P<0.05; ***P<0.001.