Then, we used sural nerve stimulation in neonatal and adult preparations in dI3OFF and control mice to assess this putative disynaptic pathway. Stimulation of the sural nerve in in vitro preparations (P1–P3; Figure 6C) led to L5 DR volleys of longer delay (1–2.5 ms, n = 5; Figure 6D) than those obtained with tibial nerve stimulation, which was consistent with slower

TGF-beta pathway conduction velocities in cutaneous afferents compared to muscle afferents. The thresholds for eliciting these responses were similar for the two nerves, (2–4 μA), demonstrating that, although we could not be specific about the fiber type stimulated, we were using the lowest possible currents to evoke responses. Next, we assessed disynaptic reflex responses. The latencies of ventral root reflexes in response to Osimertinib cell line sural

nerve stimulation were 4–5 ms (n = 3) longer than their latencies in response to tibial nerve stimulation (Figure 6E), which was reflective of the fact that tibial nerve stimulation elicits monosynaptic, Ia afferent-evoked reflexes and suggests that the reflex evoked by sural nerve stimulation involves one to two additional synapses (Figure 6B). The stimulation thresholds for eliciting short-latency reflexes by sural nerve stimulation ranged from 1.5–2 T (n = 5), where T is defined as the smallest stimulation strength at which a DR volley was seen. This suggests that the short-latency response from sural nerve stimulation is mediated by cutaneous afferents, possibly

ones with low thresholds. In dI3OFF mice, DR volleys in response to sural nerve stimulation were similar to those in control mice (Figure S5A), but the mean–normalized, short-latency ventral root response was significantly smaller (p < 0.05; Figure 6F) in dI3OFF mice (1.1 ± 0.3, mean ± pooled SD, n = 4) in comparison to control mice (3.2 ± of 0.8, n = 7). The short-latency reflexes were present in six of seven control animals, as opposed to zero of four dI3OFF animals (p < 0.05, chi-square test), indicating that dI3 INs mediate a short-latency response, which is most likely a disynaptic cutaneous to motor reflex in neonatal mice. To determine whether dI3 INs mediate this reflex in awake adult mice (Figure 6G), we ensured that monosynaptic reflexes were not affected in dI3OFF mice. Single-pulse tibial nerve stimulation (Figure 6Hi) produced both a direct M-wave and an H-reflex response (latency in the range of 2–3 ms; Figure 6Hii). Both the M-wave and H-reflex were observable in control and in dI3OFF animals, and the ratios of H-reflex to M-wave, calculated at 2 T, were similar (p = 0.2) in controls (0.19 ± 0.06, n = 3) and mutants (0.30 ± 0.16, n = 4), which was indicative of normal Ia afferent reflexes and motoneuron activity in dI3OFF mice.