Fig 2: Loss of Nxph4 reduced Golgi-granule inhibitory synapse number.(A) Gephyrin and vGAT staining in the cerebellar granular layer indicates Golgi-granule inhibitory synapses. (B) Quantification of puncta co-expressing gephyrin and vGAT as an indicator of inhibitory synapse number. (C) vGlut1 and PSD-95 staining in the cerebellar granular layer indicates mossy fiber-granule cell excitatory synapses. (D) Quantification of puncta co-expressing vGlut1 and PSD-95 as an indicator of excitatory synapse number. Data are presented as mean ± SEM. n.s., not significant; *p<0.05; by t test (B) or Mann-Whitney U test (D).

Fig 3: (+)-Borneol is a positive allosteric modulator of α2- and α3-containing GABAARs without BZDs-associated behavioral effects. (A) The hypothesis: chronic use of PSD-95-nNOS inhibitor causes analgesic tolerance through the disinhibition of pain neural circuit at spinal level, and α2- and α3-containing GABAARs agonist reverses this process. (B) Left, chemical structure of (+)-borneol. Right, locomotor activities of the mice treated by (+)-borneol at supramaximal doses (45 mg/kg, i.v.) or diazepam (0.5 mg/kg, i.v.) in the open field test. Locomotor activity was expressed as distance travelled during 5 min after drug injection (F (3,36) = 37.33, ***p < 0.001, vs vehicle, ANOVA, n = 10). (C) Representative image showing patch-clamp from HEK293 cells transiently expressing GABAARs (images under 40 × magnification). (D) Dose/response plots of GABA-evoked currents in the HEK293 cells transiently expressing α1β2γ2, α2β3γ2 or α3β3γ2 GABAARs. (E-F) Representative recordings of GABA-evoked currents in the HEK293 cells transiently expressing α1β2γ2, α2β3γ2 or α3β3γ2 GABAARs (left) and dose/response plots (right) of potentiation of GABA-evoked currents by diazepam (E) or (+)-borneol (F), n =8. (G-H) Representative recordings of GABA-evoked currents in putative excitatory neurons (left) of brain cortex (G) and spinal dorsal horn (H) and dose/response plots (right) of potentiation of GABA-evoked currents by diazepam or (+)-borneol, n =12. All data were shown as mean ± SEM.

Fig 4: Ethanol treatment increases the level of PSD-95 found in the striatum 24 h after exposure. (A–F). Immunofluorescence images taken at 400× magnification; images left to right of DAPI, PSD-95, and FoxP1 staining. DAPI labels nuclei, and FoxP1 is a marker for MSNs. (A–C) depict ethanol-treated animals, while D–F depict saline-treated animals (Scale bar = 50 µm). (G–H) depict examples of PSD-95 immunofluorescence images used for counting puncta. (I). Quantification of PSD-95 puncta in immunofluorescence images (461.46 ± 58.38 saline, 984.17 ± 10.0 ethanol, *: p = 0.010).

Fig 5: Electron microscopic images of synapses and the protein expression of PSD95 and synaptophysin in the hippocampus. The presynaptic vesicles were decreased, and the PSD length was significantly shorter in Wnt10a-/- mice compared with WT mice (A). Arrows: presynaptic vesicles; yellow bars: PDS. Scale bars = 1 µm. The protein expressions of PSD95 and synaptophysin were significantly lower in Wnt10a-/- mice than in WT mice (B). Data are expressed as mean ± SEM; n = 5/group; * p < 0.05, ** p < 0.01, and *** p < 0.001.