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. 2023 Mar 20;2(4):pgad088. doi: 10.1093/pnasnexus/pgad088

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© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Fig. 3. — Trans-synaptic labeling identifies postsynaptic targets of commissural GCs. A) Experimental design (also see main text). B) Confocal images show mRuby+ cells in the contralateral hippocampus after ipsilateral saline or KA injection. The CA3 and CA1 areas are shown in higher magnification in both panels. SO, stratum oriens; SP, stratum pyramidale; SL, stratum lucidum; SR, stratum radiatum. C) Quantification of mRuby+ cells in contralateral hippocampal DG, CA3-CA2, and CA1-subiculum areas after ipsilateral saline or KA injection. Each point represents data from one animal. Data were normalized by subtracting mRuby+ cell counts obtained from wild-type animals [see main text; for nonnormalized data, see Fig. S4; two-way ANOVA, F_area(2, 16) = 56, P < 0.0001; F_treatment(1, 8) = 38, P = 0.0003; F_{area×treatment}(2, 16) = 24, P < 0.0001; adjusted P-values (FDR) of post hoc analyses are indicated in the figure]. D) Confocal images show immunostaining for mRuby and DAPI as well as immunostaining for either GluR2/3 (left, in contralateral CA3), Wfs1 (middle, in contralateral CA1; note that Wfs1 immuno-positivity was mostly apparent in the superficial, but not deep, CA1 pyramidal cells), or PV (right, in contralateral CA3 and CA1) in 50-µm-thick sections after saline (top row) and KA injections (bottom row). Insets show magnification of the highlighted areas (the three staining methods are shown separately). E) Bar plots show the coexpression of mRuby with GluR2/3 (in CA3), or Wfs1 (in CA1), or PV (in CA3 and CA1). F) Reconstruction of trans-synaptically labeled mRuby+ neurons after saline and KA injections reveals pyramidal cell morphology. Dendrites and axons are shown in black and red or magenta, respectively. G) The upper left image shows reconstruction of anterogradely labeled EGFP+ axons in the contralateral hippocampus in a 50-µm-thick section after ipsilateral saline injection. The electron microscopy images show EGFP+ synapses (green and red arrowheads) in the CA3 (stratum oriens) and CA1 (stratum radium) area, as well as EGFP nonlabeled synapses (yellow and blue arrowheads), for comparison. In the lower right image, the asterisk labels a putative interneuron dendrite; synapses between interneuron dendrites and adjacent EGFP+ processes were not present.