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. 2023 Mar 30;14:1771. doi: 10.1038/s41467-023-36872-8

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a, b Experimental design of in vivo Nrxn3 deletions and rescues. The OB was stereotactically infected with AAVs expressing ΔCre (control), Cre, or Cre with additional AAVs encoding Nrxn3-LNS2 rescue constructs (a, schematic of stereotactic injections; b, representative fluorescence image of an OB section that was infected with AAVs expressing tdTomato fused to Cre, with subsequent patching of mitral cells that were filled with neurobiotin (blue)). Neuron-specific expression of ΔCre/Cre and rescue constructs was achieved using the synapsin-1 promoter. Note that AAVs infect granule cells more efficiently than mitral cells (see panel b). c Minimal Nrxn3α-LNS2 rescue proteins are localized to synaptic layers in the OB after expression via AAVs, as visualized by immunocytochemistry for the N-terminal HA-epitope contained in the constructs (white, HA-Nrxn3α-LNS2 proteins; red, tdTomato). d, e Conditional Nrxn3 deletion and rescue with minimal Nrxn3α-LNS2 constructs does not alter inhibitory synapse numbers in vivo. Sections from mice (infected as shown in A) were analyzed by quantitative immunocytochemistry for the presynaptic marker synaptoporin (a.k.a. synaptophysin-2; light blue) that is specific for granule cell→mitral cell synapses in the OB, and for the postsynaptic inhibitory synapse marker gephyrin (green) (d, sample images; e, summary graphs of puncta densities). Data are means ± SEM; n’s (cells/experiments) indicated in summary graph bars apply to all graphs in an experimental series. Statistical analyzes using one-way ANOVA with Tukey’s multiple comparison test (e). Appropriate HA labeling in c was confirmed in tissue from all animals quantified in e. Puncta densities in e are analyzed both per animal (top) and per region-of-interest (bottom); statistical significance is observed for gephyrin staining but not the other parameters when regions-of-interest are used as n’s because pseudo-replicates in this analysis boost statistical significance independent of the actual number of experiments. Source data and statistical results for all experiments are provided within the Source Data file.