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. 2020 Dec 23;9:e59545. doi: 10.7554/eLife.59545

Figure 1. Nlgn3 is predominantly expressed in VGT3+ and CB1+ inhibitory synapses.

(A) Single immunofluorescence of Nlgn3 in the hippocampal CA1 region shows a basket-like clustering of Nlgn3 immunofluorescent signals around the somata of pyramidal cells. Py, st. pyramidale; Ra, st. radiatum. (B–E) Triple immunofluorescence for Nlgn3, VIAAT, and interneuron markers: VGT3 (B), CB1 (C), Pv (D), and Sst (E). The boxed area in low magnification images is enlarged in lower panels. Arrowheads indicate Nlgn3 immunofluorescent puncta associated with (yellow) or distant from (white) interneuron markers. (F and G) Summary of the relative intensity (F) and co-localization frequency (G) of Nlgn3 immunofluorescent signals at different inhibitory synapses. Plots are obtained from each synapse for intensities (F) or image for co-localizations (G). Noise levels for the intensity and co-localization were obtained from images with the Nlgn3 channel rotated 90° (90° rotation). ***p<0.001; n.s. not significant; One-way ANOVA with Sidak’s post hoc test. Bars on each column represent mean ± SEM. Scale bars, 100 μm (A) and 2 μm (B–E).

Figure 1.

Figure 1—figure supplement 1. Validation of anti-Nlgn3 antibody.

Figure 1—figure supplement 1.

Single immunofluorescent images of Nlgn3 (green) in the whole brain (A and D), hippocampal formation (B and E), and CA1 region (C and F) of P21 wild-type (A–C, WT) and Nlgn3 knockout (D–F, Nlgn3-KO) mice. Nissl staining is magenta. Higher levels are detected in the cortex (A, Cx), striatum (A, St), olfactory bulb (A), (OB), and hippocampal formation (A–C, Hi). In the hippocampus, intense labeling is noted in the st. lacunosum-moleculare (LM) of Ammon's horn and in the molecular layer (Mo) of dentate gyrus (B). This staining is not detected in Nlgn3-KO mice (D–F). (G) The noise levels for intensities and co-localizations of Nlgn3 signals obtained from immunofluorescent images for Nlgn3 (green), VIAAT (blue), and interneuron markers (red) including VGT3, CB1, Pv, and Sst with the Nlgn3 channel rotated 90°. Arrows indicate inhibitory synapses co-labeled with VIAAT and different interneuron markers. CA1–3, CA1–3 regions of the Ammon's horn; Cb, cerebellum; Cx, cerebral cortex; DG, dentate gyrus; Gr, granule cell layer; Hi, hippocampus; Mb, midbrain; MO, medulla oblongata; OB, olfactory bulb; Or, st. oriens; Py, pyramidale; Ra, st. radiatum; St, striatum; Th, thalamus. Scale bars, 1 mm (A and D), 500 µm (B and E), 5 µm (C and F), and 2 µm (G).