Fig. 6.

(A–O) Expression of the N-methyl-D-aspartate (NMDA) receptor 2A subunit (NR2A) in lumbar spinal cord of postnatal day (P) 8 wild-type (WT) and Smn ^–/–;SMN2 ^+/+;SMNΔ7 ^+/+ (SMNΔ7) mice chronically treated with either saline or 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). (A–L) Representative confocal micrographs of spinal cord cryostat sections from (A–C, G–I) saline- and (D–F, J–L) AICAR-treated (A–F) WT and (G–L) SMNΔ7 mice showing NR2A-immunoreactivity (green) in motor neurons (MNs), visualized with fluorescent Nissl staining (red). (M) Graph showing the quantification NR2A intensity in MN somata of mice subjected to the treatments; 75–85 MNs from 3–4 mice per experimental condition were analyzed. (N) Representative Western blots to determine NR2A expression in spinal cord extracts of mice after different treatments; β-actin was used as loading control. (O) Densitometry analysis of NR2A protein levels in Western blots; data were obtained from 3–4 mice per experimental condition and are expressed as the percentage of change in relation to the ratio of NR2A to β-actin of WT mice treated with saline. (P–S) Western blot assays, performed on (P) lumbar spinal cord and (R) skeletal muscle extracts of P8 WT and SMNΔ7 mice following saline or AICAR treatment, demonstrating no changes in survival of motor neuron (SMN) levels induced by the adenosine monophosphate-activated protein kinase agonist. β-actin and α-tubulin were used as loading controls; each sample corresponds to the spinal cord or muscle extract of one animal. (Q, S) Densitometry analysis of SMN protein levels in Western blots; data were obtained from 3 mice per experimental condition and are expressed as the percentage of change in relation to the ratio of SMN to β-actin or α-tubulin of saline-treated WT mice. Values in graphs are shown as mean ± SEM and were analyzed by using one-way analysis of variance (Bonferroni’s post-hoc test). Scale bar in (L) = 30 μm (applies to A–L)