, 2010). Here, we show glutamatergic synaptic Pfizer Licensed Compound Library connections that likely underestimate the number of connections that exist. We next examined whether Shox2 INs provide direct excitation to commissural interneurons (CINs). CINs serve an essential role in coordinating motor activity on the left and right sides the body (Grillner, 2006 and Kiehn, 2006). CINs are rhythmically active during locomotion in rodents (Butt et al., 2002b, Butt and Kiehn, 2003 and Quinlan

and Kiehn, 2007) and may be driven by excitatory neuronal activity during locomotion (Butt et al., 2002a). We therefore looked for connections between Shox2 INs and identified CINs in a transverse spinal slice preparation (Figure 7E). CINs were recorded in whole-cell mode while spikes were elicited in Shox2 INs by application of short (10 ms) kainate (100 mM) puffs delivered from a microelectrode placed in juxtaposition to individual Shox2 INs (Jonas et al., 1998; Figure S3), permitting stimulation of up to four Shox2 INs, in turn, for each recorded CIN. Of 26 recorded CINs, four received short latency EPSPs (mean amplitude: 1.5 mV) in response to kainate applications to a Shox2 IN (Figures 7F and 7G). Thus, a subset of

Shox2 INs projects directly to commissural neurons located in the same segments. In summary, the electrophysiological connectivity studies demonstrate connections between Shox2 INs and neurons projecting ipsilaterally buy KU-57788 and contralaterally in the ventral spinal cord. Studies of spinal networks have long implicated excitatory interneurons in the generation of locomotor rhythm, but their identity and precise contribution to CPG circuitry has remained ambiguous. This study of mouse locomotor networks reveals that a subset of lumbar spinal iEINs defined by expression of the homeodomain transcription factor Shox2, in the absence of Chx10, has a role in rhythm generation. Our findings provide insight

into the molecular identity of iEINs involved in mammalian locomotor control. The homeodomain 3-mercaptopyruvate sulfurtransferase protein Shox2 marks a discrete subset of ventrally positioned glutamatergic neurons with ipsilateral axons and targets. In postnatal spinal cord, we find that ∼75% of all Shox2 INs coexpress Chx10 and thus derive largely or exclusively from the p2 progenitor domain (Ericson et al., 1997). Previous studies have shown that p2 domain progenitors give rise to excitatory V2a (Chx10+; Ericson et al., 1997 and Peng et al., 2007), and inhibitory V2b/c (Gata3+/Sox1+; Panayi et al., 2010) neurons. Our study defines an additional p2-derived excitatory IN set, termed V2d INs and thus subdivides p2-derived excitatory INs into two populations that differ in the status of Shox2 expression. A small contingent of Shox2 INs represents excitatory neurons belonging to the Isl1+ dI3 and Lbx1+ dI5 populations.