The initiation and coordination of activity in limb muscles are the main functions of neural circuits that control locomotion. Commissural neurons connect locomotor circuits on the two sides of the spinal cord, and represent the known neural substrate for left-right coordination. Here we demonstrate that a group of ipsilateral interneurons, V2a interneurons, plays an essential role in the control of left-right alternation. In the absence of V2a interneurons, the spinal cord fails to exhibit consistent left-right alternation. Locomotor burst activity shows increased variability, but flexor-extensor coordination is unaffected. Anatomical tracing studies reveal a direct excitatory input of V2a interneurons onto commissural interneurons, including a set of molecularly defined V0 neurons that drive left-right alternation. Our findings imply that the neural substrate for left-right coordination consists of at least two components; commissural neurons and a class of ipsilateral interneurons that activate commissural pathways.

Local circuits in the spinal cord that generate locomotion are termed central pattern generators (CPGs). These provide coordinated bilateral control over the normal limb alternation that underlies walking. The molecules that organize the mammalian CPG are unknown. Isolated spinal cords from mice lacking either the EphA4 receptor or its ligand ephrinB3 have lost left-right limb alternation and instead exhibit synchrony. We identified EphA4-positive neurons as an excitatory component of the locomotor CPG. Our study shows that dramatic locomotor changes can occur as a consequence of local genetic rewiring and identifies genes required for the development of normal locomotor behavior.

Motor neurons (MNs) are the principal neurons in the mammalian spinal cord whose activities cause muscles to contract. In addition to their peripheral axons, MNs have central collaterals that contact inhibitory Renshaw cells and other MNs. Since its original discovery >60 years ago, it has been a general notion that acetylcholine is the only transmitter released from MN synapses both peripherally and centrally. Here, we show, using a multidisciplinary approach, that mammalian spinal MNs, in addition to acetylcholine, corelease glutamate to excite Renshaw cells and other MNs but not to excite muscles. Our study demonstrates that glutamate can be released as a functional neurotransmitter from mammalian MNs.synaptic transmission ͉ spinal cord M otor neurons (MNs) are the output neurons from the central nervous system. Their activity directly leads to muscle contraction. By the 1940s, it was generally accepted that MNs release acetylcholine (ACh) at the neuromuscular junction (1). Shortly thereafter, it was shown that ACh also is released from MNs' central axonal branches contacting Renshaw cells (RCs) (2). As was found at the neuromuscular junction, this transmission was shown to be nicotinic (3, 4). The collaterals contacting other MNs (5) are also thought to be mediated by ACh, although this has not been shown directly (6). Since these initial discoveries and after many later investigations, it has been a general dogma that mammalian MNs contain and release one neurotransmitter, ACh, both centrally and peripherally. It has been suggested recently, based on anatomical data, that MNs might contain glutamate as a neurotransmitter (7,8). There has been, however, no direct electrophysiological evidence to support this. Here, we examine this question directly by investigating the transmission in central and peripheral MN synapses (Fig. 1a). Materials and MethodsRecordings from RCs and MNs. All procedures followed Swedish federal guidelines for animal care. Postnatal heterozygote glutamic acid decarboxylase (GAD) 67-GFP mice [postnatal day (P) 0 to P4] were anaesthetized with isoflurane and eviscerated, and spinal cords were removed with ventral laminectomy, as described in ref. 9. The spinal cord was placed in a recording chamber perfused with oxygenated Ringer's solution (128 mM NaCl͞4.69 mM KCl͞25 mM NaHCO 3 ͞1.18 mM KH 2 PO 4 ͞1.25 mM MgSO 4 ͞2.5 mM CaCl 2 ͞22 mM glucose aerated with 5% CO 2 in O 2 ) at room temperature. Whole-cell tight-seal recording of RCs and MNs were performed with patch electrodes pulled from thick-walled borosilicate glass (o.d. of 1.5 mm, i.d. of 1.0 mm; Harvard Instruments) to a final resistance of 5-8 M⍀. The electrode tips were filled with 138 mM K-gluconate, 10 mM Hepes, 0.0001 mM CaCl 2 , 5 mM ATP-Mg, and 0.3 mM GTP-Li. After filling of the tip, the electrodes were back-filled with the same solution, and to label the recorded cell, Alexa Fluor dye (0.15-0.20%; Molecular Probes) or neurobiotin (1-2%) was diluted into the electrode solution. Cells were filled during recording. Signals wer...

The ventral spinal cord consists of interneuron groups arising from distinct, genetically defined, progenitor domains along the dorsoventral axis. Many of these interneuron groups settle in the ventral spinal cord which, in mammals, contains the central pattern generator for locomotion. In order to better understand the locomotor networks, we have used different transgenic mice for anatomical characterization of one of these interneuron groups, called V2 interneurons. Neurons in this group are either V2a interneurons marked by the postmitotic expression of the transcription factor Chx10, or V2b interneurons which express the transcription factors Gata2 and Gata3. We found that all V2a and most V2b interneurons were ipsilaterally projecting in embryos as well as in newborns. V2a interneurons were for the most part glutamatergic while V2b interneurons were mainly GABAergic or glycinergic. Furthermore, we demonstrated that a large proportion of V2 interneurons expressed the axon guidance molecule EphA4, a molecule previously shown to be important for correct organization of locomotor networks. We also showed that V2 interneurons and motor neurons alone did not account for all EphA4-expressing neurons in the spinal cord. Together, these findings enable a better interpretation of neural networks underlying locomotion, and open up the search for as yet unknown components of the mammalian central pattern generator.