Journal: bioRxiv
Article Title: A systematic cross-modal approach identifies astrocytic VCAM1 as a regulator of hippocampal synapse development
doi: 10.64898/2026.03.17.712320
Figure Lengend Snippet: (A) Schematic of the workflow used to assess the effect of constitutive CRISPR/Cas9-mediated KO of Vcam1 or Gpr37l1 on hippocampal synapse development. SO: stratum oriens, SR: stratum radiatum, SLM: stratum lacunosum-moleculare, SL: stratum lucidum, DG: dentate gyrus, ML: molecular layer. Created with BioRender.com. (B) Quality control of Vcam1 KO by immunohistochemistry for VCAM1 and DAPI. Insets show CA1 region of both hemispheres. (C) Quality control of Gpr37l1 KO by immunohistochemistry for GPR37L1 and DAPI. Insets show CA1 region of both hemispheres. (D) Heatmap showing the log2 fold change in synaptic metrics comparing LacZ-gRNA and VCAM1-gRNA hemispheres across hippocampal laminae. For each synaptic metric and lamina, 3 FOVs (sections) per brain from 4 brains were analyzed. Log2-transformed metrics were analyzed using a linear mixed-effects model with gRNA, hippocampal lamina and their interaction as fixed effects to test gene- and lamina-specific effects, and brain as a random intercept to account for inter-brain variability. Asterisks denote significant differences after false discovery rate (FDR) correction for each metric across hippocampal laminae (FDR < 0.05). (E) Heatmap showing the log2 fold change in synaptic metrics comparing LacZ-gRNA and GPR37L1-gRNA hemispheres. Same sampling, plotting and analyses as in (G). (F) Representative processed Airyscan images of VGLUT1 and PSD95 in CA1 SR from LacZ-gRNA- and VCAM1-gRNA-injected hemispheres, with quantification of local peak max and presynapse MFI normalized to the LacZ-gRNA hemisphere. Each small data point represents a single FOV; larger linked data points represent hemisphere means. Statistical significance was assessed using a linear mixed effects model as in (G), followed by Tukey’s post hoc tests and FDR corrected significance. (G) Representative processed Airyscan images of VGLUT1 and PSD95 in CA3 SR from LacZ-gRNA- and VCAM1-gRNA-injected hemispheres, with quantification of local peak max and presynapse MFI normalized to the LacZ-gRNA hemisphere. Same sampling, plotting and analyses as in (F). (H) Schematic of the workflow used to assess the effect of VCAM1-Fc protein treatment on hippocampal synapses in vitro . Created with BioRender.com. (I) Representative images of hippocampal neurons treated with recombinant protein from DIV8 to DIV14. Fc or VCAM1-Fc (2 µg/mL) was added at DIV8 and replenished at DIV11. Neurons were fixed at DIV14 and stained for VGLUT1 (cyan), PSD95 (magenta), and MAP2 (yellow). Insets show the merge followed by the isolated VGLUT1-PSD95 signal. (J) Quantification of excitatory synaptic puncta density along MAP2+ dendrites. VGLUT1+PSD95+ colocalized puncta, as well as VGLUT1+ and PSD95+ puncta separately, were measured per 100 μm dendrite and normalized to the Fc condition. Each FOV contained two neuronal somas for standardized sampling; 9-11 FOVs per condition were analyzed in N = 4 independent experiments. Each data point represents one experiment is color-shaded by experiment. A linear mixed-effects model on log2 transformed data was used to account for inter-experimental variability. Post-hoc pairwise comparisons were conducted using Tukey’s HSD test. Individual FOV data points are shown in .
Article Snippet: Upon arrival, lyophilized recombinant mouse VCAM1-Fc (R&D Systems, 643-VM) was briefly centrifuged and reconstituted in sterile Dulbecco’s phosphate-buffered saline (DPBS).
Techniques: CRISPR, Control, Immunohistochemistry, Transformation Assay, Sampling, Injection, In Vitro, Recombinant, Staining, Isolation