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. 2018 Apr 18;98(2):306–319.e7. doi: 10.1016/j.neuron.2018.03.010

Figure 1.

Figure 1

Kir4.1 Upregulation in Ventral Horn AS around Large FαMN MNs

(A and B) Kir4.1 mRNA is enriched in spinal cord compared to other CNS regions in humans (GTEx-Consortium, 2015) (A) and mice (Kasukawa et al., 2011) (B). Data are represented as mean ± SEM.

(C and D) Perineuronal enrichment of Kir4.1 expression in human and mouse ventral spinal cord.

(C) KIR4.1 is expressed around SMI32+ neurons in adult human ventral spinal cord (scale bars, 1 mm, left; 50 μm, right). Representative image of n = 3 control human spinal cord. Patient data are provided in Table S1.

(D) Kir4.1 is expressed around ChAT+ MNs in mouse ventral lumbar spinal cord at P16 (scale bars, 200 μm, left; 50 μm, right). Dotted line denotes gray/white matter boundary. WM, white matter. Arrowheads denote Kir4.1 enrichment around ventral horn neurons. High-resolution images are single-plane confocal images.

(E) Increased ventral (V) compared to dorsal (D) Kir4.1 protein by western blot from mouse lumbar spinal cord at P30 (n = 4 mice, mean ± SEM, Welch’s t test).

(F) Left: fold change of Kir4.1 mRNA levels between ventral and dorsal samples from cultured (n = 6 mice, mean ± SEM, one-sample t test) neonatal mouse spinal cord AS. Right: FACS-purified AS from P5 Aldh1l1-GFP+ (n = 3 mice, mean ± SEM, one-sample t test) mouse spinal cord.

(G) Kir4.1 mRNA levels in P5 Aldh1l1-GFP+ FACS-purified AS compared to Aldh1l1-GFP− non-AS cells from mouse ventral spinal cord (n = 3 mice, mean ± SEM, one-sample t test).

(H) Kir4.1 protein is preferentially found around larger MMP-9+ FαMNs (white arrowheads) compared to smaller MMP-9− SαMNs (yellow arrowheads) at P30 (scale bar, 50 μm).

(I) Quantification of Kir4.1 signal intensity around individual MMP-9+ or MMP-9− MN (n = 4 mice, >100 MN counts/animal, boxplot, Mann-Whitney test).

(J) Kir4.1 loss in AS from VGLUT1 KO animals at P26. VGLUT1 KO mice were crossed with EAAT2-td-Tomato reporter for AS visualization.

(K) Quantification of Kir4.1 immunofluorescence intensity per AS (EAAT2-td-tomato+) (n = 2 mice, >50 AS counts/animal, boxplot, Mann-Whitney test; scale bar, 40 μm, insert: 20 μm). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Edges of boxplots denote interquartile range (25th–75th percentile) with whiskers denoting 1.5 times the interquartile range and the black line denoting the median value.