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.

Non-technical summary Our focus was on whether amyotrophic lateral sclerosis (ALS) might be precipitated by early developmental changes in large spinal motoneurons, which are vulnerable to early die-off in ALS. It has been shown that some electrical properties in motoneurons are profoundly altered soon after birth in mutant superoxide dismutase-1 (SOD1) mice, a standard animal model of ALS. These same properties undergo rapid developmental changes in normal mice during this time period. Our goal was to compare the development of motoneuron electrical properties in normal and SOD1 mice. Properties were measured from birth to 12 days of age, when the mouse is considered juvenile, but long before symptom onset. Most electrical properties in the SOD1 motoneurons showed an accelerated pace of maturation during this early developmental period compared with the normal motoneurons. If this trend persists, it could, along with other disease factors, hasten the onset of normal motoneuron degeneration due to ageing and result in the development of ALS.Abstract Spinal motoneurons are highly vulnerable in amyotrophic lateral sclerosis (ALS). Previous research using a standard animal model, the mutant superoxide dismutase-1 (SOD1) mouse, has revealed deficits in many cellular properties throughout its lifespan. The electrical properties underlying motoneuron excitability are some of the earliest to change; starting at 1 week postnatal, persistent inward currents (PICs) mediated by Na + are upregulated and electrical conductance, a measure of cell size, increases. However, during this period these properties and many others undergo large developmental changes which have not been fully analysed. Therefore, we undertook a systematic analysis of electrical properties in more than 100 normal and mutant SOD1 motoneurons from 0 to 12 days postnatal, the neonatal to juvenile period. We compared normal mice with the most severe SOD1 model, the G93A high-expressor line. We found that the Na + PIC and the conductance increased during development. However, mutant SOD1 motoneurons showed much greater increases than normal motoneurons; the mean Na + PIC in SOD1 motoneurons was double that of wild-type motoneurons. Additionally, in mutant SOD1 motoneurons the PIC mediated by Ca 2+ increased, spike width decreased and the time course of the after-spike after-hyperpolarization shortened. These changes were advances of the normal effects of maturation. Thus, our results show that the development of normal and mutant SOD1 motoneurons follows generally similar patterns, but that the rate of development is accelerated in the mutant SOD1 motoneurons. Statistical analysis of all measured properties indicates that approximately 55% of changes attributed to the G93A SOD1 mutation can be attributed to an increased rate of maturation.

The loss of motor neurons (MN) is a hallmark of the neuromuscular disease spinal muscular atrophy (SMA); however it is unclear whether this phenotype autonomously originates within the MN. To address this question, we developed an inducible mouse model of severe SMA that has reduced survival and motor function, motor unit pathology and hyperexcitable MNs. Using an Hb9-Cre allele, we increased Smn levels autonomously within MNs, and demonstrate that MN-rescue significantly improves all phenotypes and pathologies commonly described in SMA mice. MN-rescue also corrects the hyperexcitability in SMA motor neurons, and prevents sensory-motor synaptic stripping. Survival in MN-rescued SMA mice is extended by only 5 days, due in part to failed autonomic innervation of the heart. Collectively, this work demonstrates that the SMA phenotype autonomously originates in MNs and that sensory-motor synapse loss is a consequence, not a cause, of MN dysfunction.

Left-right alternation depends on activity in commissural interneurons (CINs) that have axons crossing in the midline. In this study, we investigate the CIN connectivity to local motor neurons using a newly developed preparation of the in vitro neonatal mouse spinal cord that allows us to identify all classes of CINs. Nineteen of 29 short-range CINs with axonal projections Ͻ1.5 segments (sCINs) directly excited, directly inhibited, or indirectly inhibited contralateral motor neurons in the quiescent spinal cord. Excitation was glutamatergic and inhibition was mixed glycinergic and/or GABAergic. Long-range CINs were also found to have input to local, contralateral motor neurons. Thirteen of 29 descending CINs had similar synaptic connectivity to contralateral motor neurons as the sCINs, including direct excitation and direct and indirect inhibition. Some (9 of 23) rostrally projecting ascending CINs, and a few (2 of 10) CINs with bifurcating axons that both ascend and descend, indirectly inhibited local, contralateral motor neurons. Rhythmic firing during locomotor-like activity was observed in a number of CINs with segmental synaptic effects on contralateral motor neurons. This study outlines the basic connectivity pattern of CINs in the mouse spinal cord on a segmental level. Our study suggests that, based on observed synaptic connectivity, both short-and long-range CINs are likely involved in segmental left-right coordination and that the CIN system is organized into a dual-inhibitory and single-excitatory system. These systems are organized in a way that they could provide appropriate coordination during locomotion.

The excitability of spinal motoneurons is both fundamental for motor behavior and essential in diagnosis of neural disorders. There are two mechanisms for altering this excitability. The classic mechanism is mediated by synaptic inputs that depolarize or hyperpolarize motoneurons by generating postsynaptic potentials. This "ionotropic" mechanism works via neurotransmitters that open ion channels in the cell membrane. In the second mechanism, neurotransmitters bind to receptors that activate intracellular signaling pathways. These pathways modulate the properties of the voltage-sensitive channels that determine the intrinsic input-output properties of motoneurons. This "neuromodulatory" mechanism usually does not directly activate motoneurons but instead dramatically alters the neuron's response to ionotropic inputs. We present extensive evidence that neuromodulatory inputs exert a much more powerful effect on motoneuron excitability than ionotropic inputs. The most potent neuromodulators are probably serotonin and norepinephrine, which are released by axons originating in the brainstem and can increase motoneuron excitability fivefold or more. Thus, the standard tests of motoneuron excitability (H-reflexes, tendon taps, tendon vibration and stretch reflexes) are strongly influenced by the level of neuromodulatory input to motoneurons. This insight is likely to be profoundly important for clinical diagnosis and treatment.