These findings support a view in which excitatory premotor neurons providing direct excitation to motor neurons are distinct from rhythm-generating excitatory neurons. Shox2 INs are clearly not the only rhythm-generating neurons in the locomotor network since rhythm remains in the absence of the Shox2 INs, although reduced in frequency. The molecular identity of other contributing interneurons is not known. Moreover, even within the Shox2+ non-V2a neurons, rhythm generation may be distributed among neurons

derived from several progenitor domains. The picture that emerges from our study is therefore that rhythm generation in the mammalian locomotor network seems to emerge from the combined action of multiple populations of molecularly defined neurons. Furthermore, our study shows that a single molecularly defined population may contribute Linsitinib Temozolomide clinical trial to several

aspects of the locomotor function. It is plausible that defining a finer-grained molecular code may help to clarify the identity of these functional subgroups. All experimental procedures followed the guidelines of the Animal Welfare Agency and were approved by the local Animal Care and Use Committees and competent veterinary authorities. For details of generation of the Shox2::Cre mouse line, see the Supplemental Experimental Procedures. The chx10::LNL::DTA mice were similar to those used in Crone et al. (2008). For conditional deletion of vGluT2, mice with loxP sites flanking exon 2 of the Slc17a6 gene, which encodes for vGluT2 were

used (see Talpalar et al., 2011; Supplemental Experimental Procedures). Rosa26-CAG-LSL-eNpHR3.0-EYFP-WPRE, ROSA26-YFP, Tau-GFP-nlsLacZ, and the Z/EG mice were obtained from Jackson Laboratory. Immunohistochemistry was performed using standard protocols with antibodies listed in the Supplemental Experimental Procedures. Combined in situ hybridization histochemistry/immunohistochemistry was performed on 12–20 μm cryostat sections, omitting the proteinase K step. vGluT2 full-length (GenBank AI841371) and exon 2 riboprobes were used. Midline crossing was evaluated by retrograde labeling with tetramethylrhodamine dextran (Supplemental Experimental Procedures). Spinal cords from mice aged 0–5 days (P0–5) were isolated. Transverse nearly slice preparations were used for connectivity and morphology and rhythmicity studies while dorsal-horn-removed preparations (Dougherty and Kiehn, 2010a) were used for studies of rhythmicity (Supplemental Experimental Procedures). All preparations were perfused with Ringer’s solution (111 NaCl, 3 KCl, 11 glucose, 25 NaHCO3, 1.3 MgSO4, 1.1 KH2PO4, 2.5 CaCl2, pH 7.4, and aerated with 95% O2/5% CO2) at a flow rate of 4–5 ml/min. Ventral root activity (signal band-pass filtered 100–1,000 Hz; gain 5–10,000) was recorded from ventral roots in L1 L2, L3, L4, or L5 with glass suction electrodes.