Figure 1
EphA7 is required for the stabilisation of GABAergic synaptic markers at proximal dendrites and somata of granule cells in vivo.
(a–g) Lentiviral vectors were stereotaxically injected into the dentate gyrus of adult female rats. For the identification of transduced principal neurons, lentiviral constructs expressed EGFP under the control of the CamKII promoter. Likewise, EphA7-specific shRNA 1737 or sh392 were coexpressed from the same vector as indicated. Brain slices were stained for pre- and postsynaptic, inhibitory and excitatory synapse markers VGAT, gephyrin, VGluT, PSD95 and the γ2 subunit of GABAA receptors, respectively. Example micrographs are shown in a-f. Insets in (a–f) are enlarged in (a’–f’). All images were rendered using IMARIS software. Scale bars, 10 μm. (g) Quantification of synaptic marker densities on different compartments of granule cells as indicated. Proximal dendritic segments: gephyrin, n = 140, ***p < 0.0001; GABAA γ2, n = 40, ***p < 0.0001; VGAT, PSD95, VGluT, n = 90, ***p < 0.0001; ns, not significant. Distal dendritic segments: gephyrin, PSD95, n = 70; ns, not significant. Soma: gephyrin, n = 60, **p < 0.01. Axon initial segment (AIS): gephyrin, n = 60; ns, not significant. ANOVA, Fisher-PLSD; error bars: S.E.M. (h,i) Representative images of granule cells transduced with control or EphA7 knockdown lentivirus coexpressing EGFP and stained for parvalbumin. (k) Parvalbumin-stained voxels representative for basket cell terminals were identified and quantified as objects in confocal z-stacks. The volume of parvalbumin-positive structures per contacted soma were determined as a measure for basket cell innervation after EphA7 knockdown (65%) compared to control (100%). n = 26, **p < 0.01; ANOVA, Fisher-PLSD; error bars: S.E.M. scale bars: 10 μm. (l) Representative image of EGFP fluorescence indicating lentiviral infection of the dentate gyrus. Scale bar 200 μm.
