Postsynaptic activation of the PKA pathway enhances Ia EPSPs and restores synaptic structures. (A) Effect of iontophoretic injection of cAMPS-Sp on Ia EPSP amplitude. Afferent volleys (top traces) and EPSPs in TS MNs (bottom traces) evoked by electrical stimulation of the TS nerve recorded immediately before and a few minutes after the injection of the compound. (B) Effect of iontophoretic injection of cAMPS-Sp on paired-pulse ratios. Same MNs and same time points as in A. (C) Quantification of the increase in EPSP size. “Before” values were measured just before the injection; “after” values are averages over the whole duration of the recording after the injection. Each symbol represents one MN, and the stars next to the symbols represent the MNs shown in A. (C₁) In wtSOD1 animals (four mice), cAMPS-Sp increased the size of the EPSPs by 7 ± 8%; n = 7; paired t test, *, P = 0.04. (C₂) In mutSOD1 animals (two mice), the EPSP size increased by 14 ± 5% on average, n = 6; paired t test, **, P = 0.007. In mutant animals, the experiments were conducted in four MNs: Ia EPSPs coming from either of the two TS branches were tested in two MNs, whereas only one source of Ia excitation was tested in the remaining two MNs (see different symbols and line styles in C₂). The difference persists if we consider only EPSPs elicited by stimulation of the LG nerve in each MN. The EPSP size increased by 14 ± 6% on average, n = 4; paired t test, P = 0.02. (D) Quantification of the change in the paired-pulse ratio after cAMPS-Sp injection. Same organization as in C. In wtSOD1 animals (four mice), cAMPS-Sp did not significantly change the paired-pulse ratio (D₁, average difference 1 ± 2%; n = 6, paired t test, P = 0.23), while it caused an increase by 5 ± 5%, n = 6 in mutSOD1 animals (D₂, two mice, paired-pulse ratio before, 1.00 ± 0.06, n = 6; vs. after, 1.05 ± 0.08, n = 6; paired t test, *, P = 0.038). As before, the difference persists if we consider only EPSPs elicited by the LG nerve in each MN. cAMPS-Sp increased the paired-pulse ratio from 1.00 ± 0.07 to 1.05 ± 0.10, n = 4; paired t test, P = 0.048. (E) Experimental design for DREADD experiments. (F) MNs expressing D(Gs) and immunostained for VGluT1 and GluR4 under either vehicle (F₁), acute CNO (F₂), or chronic CNO treatment (F₃). The dotted line represents the approximate outline of the MNs. MNs are identified by VAChT staining. Scale bars: 20 µm (inset: 1 µm). (G) Same organization as F, but immunostained for VGluT1 and Homer1b. (H) Significant increase in GluR4 cluster area in VGlut1 synapses of D(Gs)⁺ MNs (magenta) compared with contralateral D(Gs)⁻ MNs (gray) upon acute (4.3 ± 2.6 µm², n = 43; vs. 2.3 ± 1.7 µm², n = 27; from three mice; two-way ANOVA followed by Tukey HSD, **, P = 0.001) and chronic (3.9 ± 2.5 µm², n = 144; vs. 2.3 ± 1.5 µm², n = 128; from four mice; Tukey HSD, **, P = 0.001) CNO treatment, but not in vehicle-treated mice (2.1 ± 1.6 µm², n = 119; vs. 2.3 ± 1.6 µm², n = 111; from three mice; Tukey HSD, P = 0.9). (I) Significant increase in Homer1b cluster area in D(Gs)⁺ MNs compared with contralateral D(Gs)⁻ MNs upon acute (2.4 ± 1.6 µm², n = 109; vs. 1.8 ± 1.2 µm^2,n = 83; two-way ANOVA followed by Tukey HSD, **, P = 0.0147) and chronic (2.4 ± 1.5, n = 88; vs. 1.6 ± 1.4 µm², n = 69; from four mice; Tukey HSD, **, P = 0.0019) CNO treatment, but not in vehicle-treated mice (1.4 ± 1.2 µm² in D(Gs)⁺ MNs, n = 51; vs. 1.3 ± 1.6 µm² in D(Gs)⁻ MNs, n = 117; from three mice; Tukey HSD, P = 0.9).