Fig 2: Developmental expression of nNOS in the neocortex. (A–E) nNos mRNA expression at different embryonic and postnatal stages. Black and red arrowheads in (A) and (B) point to expression in the superficial cortex and the ventral MGE, respectively. Arrowheads in (C') and (C”) point to cells expressing high levels of nNOS in the cortex and striatum, respectively. (F–J) nNOS protein expression at different embryonic and postnatal stages. Type I and type II cells are marked by black and red arrows, respectively in (I) and (J). Scale bars: A–J, 500 µm. C, C” and inserts in D, E, I, J, 50 µm. Insert in H, 20 µm.

Fig 3: Stimulation of MMP-9 protein expression and inflammatory markers via pMCAO. a The expression levels of both the active (86 kDa) and the inactive (92 kDa) forms of MMP-9 were determined at different times after surgery. b The ischaemic-induced phosphorylation of p38 and CaMKII as well as the expression levels of nNOS and GFAP were evaluated 8 days after surgery. The enhancing effects of pMCAO (light grey) were reduced by S1RA administration (dark grey). Each bar represents the means ± SEM of the data from three determinations performed using different gels and blots. Groups of 8–10 mice were used for each interval. Immunosignals (average optical density of the pixels within the object area/mm2; Quantity One Software, Bio-Rad, Madrid, Spain) were expressed as the change relative to the sham-operated group (attributed an arbitrary value of 1; white bars). Asterisk denotes the significant difference from the sham-operated mice, ? from the pMCAO group, all pairwise Holm-Sidak multiple comparison test following ANOVA, p < 0.05

Fig 4: A subset of cPPN nNOS neurons of runner mice lose ChAT transcripts and gain GAD1 transcripts.a Double staining of the cPPN in a non-runner control mouse for nNOS (green) and ChAT (red). Scale bar, 100 µm. b Co-localization of nNOS and ChAT in the cPPN. For a, b, n = 1068 cells from three non-runner control mice. c Stereological counts of DAB staining of nNOS in control and 1-week runner cPPNs. n = 6 animals per group. Mann–Whitney U test. NS, not significant. d Triple staining of ChAT and GAD1 transcripts and nNOS protein in the cPPN of a control and a 1-week runner mouse. Right panels are boxed regions in merged images at higher magnification. Scale bar, 20 µm. e Scatterplot of numbers of in situ stained ChAT puncta (y-axis) against numbers of in situ stained GAD1 puncta (x-axis). Each dot represents one neuron. The vertical line divides neurons that contain zero from those containing more GAD1 transcript puncta and the horizontal line divides neurons that contain less from those containing more than eight ChAT transcript puncta. f, g Y-axes are the mean number of ChAT (f) and GAD1 (g) fluorescent puncta in single nNOS+ neurons. For d–g, n = 4 animals per group; n = 123 cells for Ctrl and 137 cells for Run. Statistical significance *p < 0.05, ***p < 0.001 was assessed by two-sided Welch’s t-test. Data shown are mean ± SEM.

Fig 5: Schematic illustration of the analgesic mechanism of the dual-target pain killer ZL006-05. Chronic use of PSD-95-nNOS blocker develops analgesic tolerance due to NO reduction-induced GABAergic disinhibition, while ZL006-05 can avoid the analgesic tolerance by co-targeting α2-containing GABAARs and nNOS-PSD-95 interaction.