Inappropriate synapses evoke monosynaptic EPSCs. (A) Schematic of the ex vivo spinal cord–hindlimb preparation utilized for whole-cell patch-clamp from TA motor neurons. Bipolar concentric needle electrodes were placed in the TA and Gs muscles to record EMG. Suction electrodes were placed in the CP and Tb nerves for stimulation en passant. The L4 and L5 ventral roots were cut and placed in suction electrodes. Intracellular electrode contained Neurobiotin. At birth, the TA muscle was injected with CTb-488, while the Gs muscle was injected with CAV2^CRE virus in PV::FlpO;Ai65-TdTomato mice. (B) Whole-cell responses from TA motor neurons following stimulation of the homonymous CP nerve (blue) or the antagonistic Tb nerve (red) in WT, C3^−/−, and CD47^−/− mice at P4–6. The EPSC response is the mean of five responses (evoked at 0.1 Hz). Dotted lines depict the duration of 3.5 ms, which was used as the criterion for monosynaptic duration. (C) EMG responses corresponding to the experimental groups as shown in B above. The TA (top traces) and Gs EMG responses (bottom traces) were acquired concurrently following stimulation of either the CP nerve (blue) or the Tb nerve (red). Superimposed traces shown are five responses evoked at 0.1 Hz. (D) Amplitude of EPSC evoked in TA motor neurons following CP (plain bars) or Tb (hatched bars) nerve stimulation in WT (blue), C3^−/− (green), and CD47^−/− (purple) mice (WT: n = 7 motor neurons; C3^−/−: n = 5; CD47^−/−: n = 7). One motor neuron per mouse was recorded. (E) Latency of EPSC for experimental groups shown in D. Note that in WT mice, only one TA motor neuron exhibited a small EPSC following Tb nerve stimulation. (F) Voltage responses in TA motor neurons following steps of current injection (in patch-clamp experiments) in WT, C3^−/−, and CD47^−/− mice (as shown in B). (G) Input resistance (left) and rheobase (right) of TA motor neurons in the three experimental groups. Significance: *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA multiple comparisons with Bonferroni’s test. (H) Confocal images of recorded, filled, and subsequently visualized TA motor neurons from the three experimental groups: PV::FlpO;Ai65 (WT), PV::FlpO;Ai65;C3^−/− (C3^−/−), and PV::FlpO;Ai65;CD47^−/− (CD47^−/−) mice. Images correspond to TA-recorded motor neurons for each mouse line from experiments shown in B, C, and F. Quadruple immunostaining against VGluT1 (red), DsRed (green), Neurobiotin (blue), and injected CTb488 (magenta). Images are Z-stack projections of single optical planes totaling 3.5 μm in the Z-axis. Inserts are single optical planes, corresponding to higher magnification of the recorded motor neuron soma (top; CTb); white arrow indicates inappropriate synapses on the soma of the TA motor neuron in C3^−/− and CD47^−/− mice only (bottom images). Scale bar: 20 μm.