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. 2020 Feb 13;11:869. doi: 10.1038/s41467-019-14154-6

Fig. 3. Replication of the GABAAR subunit switch through BDNF-TrkB signaling, but not by blocking KCC2.

Fig. 3

a Low magnification confocal acquisitions of IB4 and KCC2 immunostainings in sham SDH and their intensity profiles in the SDH b id. in PNI rats. c From left to right, averaged pixel KCC2 immunostaining intensities in the SDH of the L4-L5 lumbar segments of: ipsilateral SDH of shams, contralateral SDH of PNIs, in the surrounding (´out´) and exactly in the region defined by the loss in IB4 labeling that delineates the core of the central projection of lesioned afferents in PNI rats (´in´). d Parallel intensity quantifications of the IB4 and KCC2 immunostaining in the SDH of PNI rats in the same two last regions defined by the loss of IB4 terminals (outside vs. inside). e Currents evoked by muscimol puffs in dorsal horn neurons from control rats or in presence of the KCC2 antagonist (VU0240551 at 15 µM) applied at increasing 12.5 mV voltage steps ranging from −93 to −18 mV. In red the muscimol response recorded at –43 mV. f EGABA recorded from superficial dorsal horn neurons of naive rats in control and in the presence of VU0240551 (-44.1 ± 1.7 mV, CTL; -36.1 ± 1 mV, VU; n = 6 cells). g Antagonizing KCC2 did not modify the synaptic location at inhibitory sites of GABAARs and GlyRs. h Blocking KCC2 did not affect the number of clusters of the α1, α2 or β2,3 GABAAR nor of the α1 GlyR or gephyrin. i Example of the synaptic α2 GABAAR expression increase in control spinal cord slices incubated in 50 ng·ml-1 BDNF in artificial cerebrospinal fluid (ACSF) for 3 h. j The quantification of the synaptic GABAAR subunits showed an increase in synaptic α2, α3 and β2,3 GABAAR subunits in BDNF-treated slices. The synaptic expressions in α1, α5 GABAAR and α1 GlyR remained unchanged with BDNF treatment. k BDNF however did not modify the number of inhibitory synapses by itself. l Examples of GABAA mIPSC traces recorded from control lumbar spinal cord slices incubated in ACSF with or without BDNF and averaged mIPSC aligned by rise time and y-scaled by amplitude. The incubation in a BDNF-containing ACSF significantly prolonged the decay time of the GABAA mIPSC currents in dorsal horn neurons but did not significantly modify their mean amplitude. m Blocking BDNF-TrkB signaling with an anti-TrkB antibody in PNI spinal cord paired-sections (2 μg ml−1 in ACSF for 3 h) was able to reverse the GABAAR synaptic over-expression and phenotypic switch in PNI rats, n but did not modify the number in GABAAR, GlyR or gephyrin clusters. Numbers of rats are indicated at the bottom of each bar; CTL, control rat. (*P < 0.05; **P < 0.01; ***P < 0.001). Source data is available as a Source Data file.