Fig. 3: Abluminal expression of GluN1 in mouse and primate cerebral aSMCs.

a, RNA scope images of Grin1 mRNA in hippocampus (left). Airyscan image of a single slice of p-arteriole in barrel cortex (right) from a SMACreERCreER:Ai14 mouse. Grin1 mRNA foci (white arrows) are shown in aSMCs. Experiments were repeated with four mice. b,c, Grin1 mRNA and protein in cortex and sorted cerebral aSMCs from littermate control (SMACreER:Ai47) and aSMC-cKO^Grin1 (SMACreER:Ai47:Grin1^fl/fl) mice. N = 4 mice. d, Immunofluorescence images of anti-GluN1. Top, isolated arterioles from control (Grin1^fl/fl) and aSMC-cKO^Grin1 (SMACreER:Grin1^fl/fl) mice, aSMC marker smoothelin and DAPI. Bottom, cortex of an aSMC-cKO^Grin1 mouse. N = 6 p-arterioles from two mice of each genotype. e, Membrane GluN1 puncta (red, indicated by white arrows) in the subcellular region of primary aSMCs from control (top) and aSMC-cKO^Grin1 (bottom) mice, in both genotypes; aSMCs were labeled with EGFP. N = 3 repeated experiments. f, Colocalization analysis of GluN1 with PSD95 in primary EGFP⁺ aSMCs. n = 16 cells from three independent experiment replicates. g, Superimposed image of wide-field and fluorescence signals of brain slices from SMACreER:Ai47 mice. The region (white box) underwent EM scanning. h–j,l,o, GluN1-gold beads in asymmetric synapses of mouse (h), monkey (i) and human (j), in aSMCs (magenta arrows), and endothelia (blue arrows) of p-arterioles from control mice (l, left, SMACreER:Ai47), aSMC-cKO^Grin1 mice (l, right, SMACreER:Ai47:Grin1^fl/fl), monkey (o, left) and human brain slices (o, right). N = 3 mice; one sample each for monkey and human. k, Histogram reflecting distances of GluN1 beads from presynaptic and postsynaptic membranes. m, Percentages of GluN1 immunoreactivity with abluminal or luminal distribution in aSMCs and endothelia of p-arterioles from control mice. n,p, Manual counting of GluN1 immunoreactivity in synapses, aSMCs and endothelia in brain slices from control and aSMC-cKO^Grin1 mice (n), monkey and human (p). N = 4 mice,10 brain slices from one monkey and one human. In a, d, g, h and k–n, tamoxifen was intragastrically administered in adult mice at the age of 2 to 3 months, followed by euthanasia 1 month later. Data are the mean ± s.e.m.; nested, unpaired, two-tailed t-test (b, c, f, m, n and p).

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