Figure 4: Association of Shrm4 with dynein/dynactin drives GABA_BRs dendritic transport.

(a) Images of dendrites (d) and axons (ax) of hippocampal neurons at 14DIV, transfected with GFP (control), knockdown (shRNA#1), rescue, or GFP-p150-cc1 at 8DIV. The mean fluorescence intensities of GABA_BRs are shown with a polarity index (PI=(I_d−I_a)/(I_d+I_a)). For uniformly distribution PI=0, whereas for dendritic polarization PI>0, and for axonic polarization PI<0. GABA_BR immunostaining was reduced in dendrites in knockdown and GFP-p150-cc1-expressing neurons compared with those for control and rescue neurons (Left histograms, n=10, 10, 15, 10; ***P<0.0001; F(3,41)=21.91; one-way ANOVA-Mann–Whitney). In addition, the mean PI of shRNA#1- and GFP-p150-cc1-expressing neurons is also reduced (right histograms, n=10, 10, 15, 10; ***P<0.0001; F(3,41)=14.72; One-way ANOVA-Mann–Whitney). Scale bar, 10 μm. (b) Images, mean fluorescences and PIs for dendrites (d) and axons (ax) of 14DIV hippocampal neurons co-transfected with GFP (control), knockdown- (shRNA#1) or GFP-p150-cc1 together with either GABA_B1a-myc or GABA_B1b-myc at 8DIV. GABA_B1 was detected with anti-myc and brevican identified the axons. Histograms show mean±s.e.m.; GabaB1a: n=10–15; ***P<0.0001; F(2,27)=49.75; one-way ANOVA-Mann–Whitney; GABAB1b: n=10–15; ***P=0.0001; F(2, 27)=12.53; one-way ANOVA-Mann–Whitney. Scale bar, 10 μm. (c) Fluorescence recovery after photobleaching (FRAP) of cultured hippocampal neurons expressing GABA_B1-RFP together with GFP, shRNA#1 or GFP-p150-cc1. The mean half-time (t_1/2) for fluorescence recovery (fluorescent recovery post-bleach corrected to 0%) is significantly longer in knockdown and GFP-p150-cc1-expressing neurons than GFP control. Histograms show mean±s.e.m.; n=7; ***P=0.0002; F(2,15)=16.43; one-way ANOVA-Mann–Whitney. (d) Whole-cell GIRK currents recorded in response to GABA_BR activation by 10 (top) or 100 (bottom) μM baclofen, from rat hippocampal neurons at 14DIV, transfected either with scrambled, knockdown (shRNA#1) or GFP-p150-cc1 at 8DIV. Baclofen-activated (10 and 100 μM) GIRK current densities in Shrm4-silenced, dynein-inhibited neurons and scrambled shRNA neurons are shown. Histograms show mean±s.e.m.; 10 μM: n=10, 10, 11; ***P=0.0003; F(2,28)=10.96, one-way ANOVA-Mann–Whitney; 100 μM: n=10, 9, 12; ***P=0007; F(2,28)=9.466). (e) Schematic of injection of AAV5-scrambled#1 (or #2) and AAV5-shRNA#1 (or #2) into opposing CA1 areas of rat, with time line for recovery and experiments. (f) Representative traces and histograms showing changes in GIRK and current densities (pA/pF) from CA1 pyramidal neurons evoked by baclofen (100 μM, horizontal bars) and blocked by the selective GABA_BR antagonist, CGP54626 (5 μM). Histograms show mean±s.e.m.; n=10; (left histogram shRNA#1: *P=0.0298; t-test; right histogram shRNA#2: *P=0.0471, t-test).