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. Author manuscript; available in PMC: 2022 May 9.
Published in final edited form as: Neuron. 2016 Jun 9;91(1):119–132. doi: 10.1016/j.neuron.2016.05.016

Figure 2. Oligodendroglial NMDA Receptor Mutants with Reduced GLUT1 Incorporation into Myelin.

Figure 2.

(A) Immunostaining of NR1 in optic nerve cross sections at age P80 from control and NR1 cKO mice (left panel). Higher magnifications (middle and right panels) reveal that NR1 (green) is absent from mutants and overlaps with MBP (red) in control mice. Scale bars, 20 μm (left) and 1 μm. See also Figure S2A.

(B) Quantitation of NR1 staining intensity from whole optic nerve sections (n = 3; p = 0.019, Student’s t test).

(C) Cre-mediated recombination of genomic DNA in individual optic nerves of each genotype as quantified by qPCR. Depicted is the relative abundance of the floxed NR1 allele after recombination (normalized to the abundance in NR1fl/fl littermate controls, defined as 100% at different ages). In NR1 mutant nerves, the abundance of NR1 flox copies was determined at ages P5 (98% ± 22% flox copies remaining; n = 5 versus 4, p = 0.939), age P10 (69% ± 7%; n = 4, p = 0.0119), age P16 (60% ± 4%; n = 4 versus 3, p = 0.0024), and P96 (38% ± 2%; n = 4 versus 9, p < 0.0001).

(D) Western blot analysis of NR1 expression in individual nerves from mutant and control mice at age P75. Quantification revealed a reduction by 61% ± 12% in NR1 cKO nerves (n = 3, p = 0.0079). GAPDH, loading control.

(E) By western blotting, GLUT1 is reduced in purified myelin of NR1 mutants (Sirt2, loading control).

(F) Quantification of E (n = 3; p = 0.008, Student’s t test).

(G) Localization of GLUT1 by immunogold labeling. In optic nerve cross sections, GLUT1 was detected in myelin sheaths, the outer tongue, and paranodal loops. Scale bar, 200 nm. Gold particles, red arrows.

(H) Reduced abundance of immunogold labeled GLUT1 in myelin of NR1 mutant optic nerves when compared to littermate controls (10–12 randomly taken images per animal, n = 3; p = 0.0025, Student’s t test).

(I) By immunogold labeling, MCT1 is associated with adaxonal (outer tongue) and abaxonal (inner tongue) myelinic channels, without difference between mutants and controls. Scale bar, 100 nm.