Figure 7J shows such an example
with recordings from an L5 MN in an induced Vglut2-KO animal in which the cell was voltage clamped to −30 mV to enhance the amplitude of the DR L3 evoked compound IPSC (trace 1). Stimulation of L5 DR evoked a short latency EPSC (trace 2). When the DR L3 stimulation was preceded by DR L5 stimulation, there was a small but clear reduction of the DR L3-evoked compound IPSC (trace 2+1). Similar findings were seen in two L5 MNs from wild-type mice and two other L5 MNs from induced this website Vglut2-KO mice with an average reduction of the IPSC by 34% ± 14%. Together, these experiments show that the reciprocal RC and Ia-IN pathways are present and functional in wild-type and Vglut2-KO E18.5 mice. Having confirmed that the RC and rIa-IN pathways are present in mice, including the Vglut2-KOs, we directly tested whether components of this network could be the source of the rhythm and flexor-extensor alternation. Given that RCs inhibit Ia-INs, the rhythm should be blocked in Vglut2-KOs by stimulating the ventral root if reciprocal connectivity between flexor- and extensor-related Ia-INs is the Compound C mouse source of the rhythm and of the alternating activity between flexor and extensors (Figure 8A; Hultborn et al., 1976 and Jordan, 1983). Prolonged stimulation
of the VR (with the same stimulation strength as needed to inhibit the reciprocal Ia pathway; Figure 7) blocked, or strongly attenuated, the ongoing rhythm and the PIK-5 rhythmic oscillations occurring in MNs of Vglut2-KO mice (Figures 8B and 8G; n = 5). This effect was seen when stimulating either L3 or L4 ventral roots (at the same stimulation strength needed to block the reflex) while recording from L2 and L5 (Figure 7G). This effect was blocked by nicotinic receptor blockers (Figure 8C; n = 3). A similar effect was seen in induced Vglut2-KO mice (Figures 8D
and 8G; n = 5). In contrast, prolonged stimulation of the ventral roots sped up the rhythm in control mice (Figures 8E and 8F; n = 10), similar to what has been shown before for the disinhibited rhythm in wild-type mice (Bonnot et al., 2009). Short (1–1.5 s) trains of stimulation of the L4 VRs in Vglut2-KO mice delivered late in the L2 bursting phase caused phase-advance of the next L2 burst with permanent phase shifts of the rhythm (Figure 8H; n = 8 trials in two animals). Similar duration trains delivered in the early phase of the L2 bursts had no effects (n = 12 trials in two animals). Such resetting of the rhythm in a phase-dependent way is indicative of synaptic interaction with the rhythm-generating network (Hultborn et al., 1998). To test whether RC rhythmic activity was needed for network activity in Vglut2-KO mice, we tested the effect of blocking or reducing the MN-to-RC drive with nicotinic blockers on drug-evoked rhythmic activity.