This dependence on NMDA receptor activation for induction of locomotor-like activity suggests that the burst-like properties that NMDA receptor activation can evoke in spinal neurons (Hochman et al., 1994 and Ziskind-Conhaim et al., 2008) is a requirement

for rhythm generation in the Vglut2-KO mice. The rhythm generation, therefore, seems to be a consequence of the interplay between cellular rhythmogenic properties and reciprocal inhibitory coupling between groups of inhibitory neurons, similar to what has been observed in many invertebrate motor networks (Marder and Calabrese, 1996) and in the mammalian cortex (Bartos et al., 2007). The fact that slow low-amplitude oscillations were seen in individual root recordings after blocking inhibition suggests that even MNs may display NMDA-induced oscillations, similarly to what was previously FK228 purchase described Z-VAD-FMK mw (Tresch and Kiehn, 2000). Notably, the frequency of the rhythm in Vglut2-KO mice was restricted to the lower part of the frequency spectrum (<0.4 Hz) reported for locomotor-like activity in the neonatal mouse (Talpalar and Kiehn, 2010), and for any given drug concentrations the frequency always remained lower in the Vglut2-KO compared to the control littermates. These observations suggest that, although the rIa-IN networks can generate a rhythm, this is not the normal state in

an intact locomotor network. Rather, the rIa-INs may be driven into rhythmicity by upstream flexor- and extensor-related excitatory CPG neurons (Kiehn, 2011 and McCrea and Rybak, 2008). These rhythmogenic excitatory networks may be connected via inhibitory neurons that are different from the rIa-INs (see Kiehn, 2011). We anticipate that under normal circumstances these excitatory circuits produce and pace the rhythm. However, when the Vglut2-dependent neurotransmission is removed, the rhythm generation can be shifted to the Ia inhibitory networks.

These latter networks could only be brought to bursting when stimulated with drugs and could not be accessed by the neural locomotor initiating signals. In this sense, the rhythmogenic capability of Ia inhibitory networks in the Vglut2-KO mice is a consequence of the removal of the excitatory network components. Our experiments, therefore, stress these the need for a careful and intervening analysis in order to understand the significance of changes in network structure when mouse mutants are investigated in the in vitro conditions and when locomotor-like activity is induced by drugs. The details of generating the Vglut2 knockout mice are reported elsewhere (Supplemental Experimental Procedures; Hnasko et al., 2010). The generation and specificity of the BAC-Vglut2-Chr2-YFP mouse is described in Hägglund et al. (2010). ROSA26-Cre-ER™ mice were obtained from Jackson Laboratory. The procedure for inducing Cre is described in the Supplemental Experimental Procedures.