Fig. 2: Postsynaptic scaling and GABA_AR subunit switch after peripheral nerve injury (PNI).

a Illustration of the approach used to analyze immunostaining at gephyrin(+) postsynaptic sites, from left to right: gephyrin immunostaining; its binarization (gephyrin mask); β2,3 GABA_AR immunostaining in gray scale; the product of the gephyrin mask with the image of the β2,3 GABA_AR immunostaining allowing the quantification of intensity of immunolabelled GABA_ARs specifically at inhibitory postsynaptic sites. b Profile plot examples of the β2,3 GABA_AR immunolabelling intensity in sham vs. PNI animals. c Bar graph summarizing the results obtained for GABA_AR and α1 GlyR subunits at synapses defined by gephyrin. d qRT-PCR in the ipsilateral SDH of Gabra1, Gabra2, Gabra3, GPHN (gephyrin), SLC12A5 (KCC2) and SLC12A2 (NKCC1) mRNAs in shams vs. PNIs. e The house-keeping gene TFRC compared to three other house-keeping genes known to be stable after PNI (ACTB, RPL13a and GAPDH). f Violin plot of Gabra2 mRNA copy index detected by RNAscope in 5 μm-thick z-stack spinal cord sections, in inhibitory SLC32A1(+) and excitatory SLC17A6(+) neurons of sham and PNI rats. g Spatial Intensity distribution analysis (SpIDA) of the α1 and α2 GABA_AR monomers or dimers within gephyrin clusters in sham vs. PNI rats. h Total number of the α1 and α2 GABA_ARs (-mers) in shams vs. PNIs. i Schematic representation of the distribution of oligomerization states for these two subunits in sham vs. PNI rats. j Quantitative analysis of the decay time-constant (τ_D) and amplitude of GABA_AR mIPSCs in sham vs. PNI rats. Insets: representative electrophysiological traces of GABA_A mIPSC recordings along with superimposed averaged traces (of the 100 first detected mIPSCs). k Computer simulations showing the fraction of open GABA_A channels as a function of GABA concentration in the synaptic cleft in sham and PNI rats. For comparison, these relations for pure α1β3γ2 (blue) and α2β3γ2 (orange) GABA_AR stoichiometries^{39, 41} are also shown. As illustrated by the orange arrows, α2β2γ2 channels are supposed to have slower kinetics than α2β3γ2 channel⁴¹. l Computer simulations of Cl⁻ conductance during a synaptic event in sham (black) and PNI (red) rats as well as with the assumption of a conductance increase without a change in affinity (i.e., no synaptic switch in GABA_AR subunit stoichiometry modifying the affinity for GABA, dashed purple line). i.u., intensity unit. (*P < 0.05; **P < 0.01; ***P < 0.001). Source data is available as a Source Data file.