The spinal cord has the capacity to coordinate motor activities such as locomotion. Following spinal transection, functional activity can be regained, to a degree, following motor training. To identify microcircuits involved in this recovery, we studied a population of mouse spinal interneurons known to receive direct afferent inputs and project to intermediate and ventral regions of the spinal cord. We demonstrate that while dI3 interneurons are not necessary for normal locomotor activity, locomotor circuits rhythmically inhibit them and dI3 interneurons can activate these circuits. Removing dI3 interneurons from spinal microcircuits by eliminating their synaptic transmission left locomotion more or less unchanged, but abolished functional recovery, indicating that dI3 interneurons are a necessary cellular substrate for motor system plasticity following transection. We suggest that dI3 interneurons compare inputs from locomotor circuits with sensory afferent inputs to compute sensory prediction errors that then modify locomotor circuits to effect motor recovery.

The properties of mammalian spinal interneurons that underlie rhythmic locomotor networks remain poorly described. Using postnatal transgenic mice in which expression of green fluorescent protein is driven by the promoter for the homeodomain transcription factor Hb9, as well as Hb9-lacZ knock-in mice, we describe a novel population of glutamatergic interneurons located adjacent to the ventral commissure from cervical to midlumbar spinal cord levels. Hb9+ interneurons exhibit strong postinhibitory rebound and demonstrate pronounced membrane potential oscillations in response to chemical stimuli that induce locomotor activity. These data provide a molecular and physiological delineation of a small population of ventral spinal interneurons that exhibit homogeneous electrophysiological features, the properties of which suggest that they are candidate locomotor rhythm-generating interneurons.

Mammalian motor programs are controlled by networks of spinal interneurons that set the rhythm and intensity of motor neuron firing. Motor neurons have long been known to receive prominent "C bouton" cholinergic inputs from spinal interneurons, but the source and function of these synaptic inputs have remained obscure. We show here that the transcription factor Pitx2 marks a small cluster of spinal cholinergic interneurons, V0(C) neurons, that represents the sole source of C bouton inputs to motor neurons. The activity of these cholinergic interneurons is tightly phase locked with motor neuron bursting during fictive locomotor activity, suggesting a role in the modulation of motor neuron firing frequency. Genetic inactivation of the output of these neurons impairs a locomotor task-dependent increase in motor neuron firing and muscle activation. Thus, V0(C) interneurons represent a defined class of spinal cholinergic interneurons with an intrinsic neuromodulatory role in the control of locomotor behavior.

Nerve injuries resulting in prolonged periods of denervation result in poor recovery of motor function. We have previously shown that embryonic stem cell-derived motoneurons transplanted at the time of transection into a peripheral nerve can functionally reinnervate muscle. For clinical relevance, we now focused on delaying transplantation to assess reinnervation after prolonged denervation.

Deletion of SCN9A encoding the voltage-gated sodium channel NaV1.7 in humans leads to profound pain insensitivity and anosmia. Conditional deletion of NaV1.7 in sensory neurons of mice also abolishes pain, suggesting that the locus of analgesia is the nociceptor. Here we demonstrate, using in vivo calcium imaging and extracellular recording, that NaV1.7 knockout mice have essentially normal nociceptor activity. However, synaptic transmission from nociceptor central terminals in the spinal cord is greatly reduced by an opioid-dependent mechanism. Analgesia is also reversed substantially by central but not peripheral application of opioid antagonists. In contrast, the lack of neurotransmitter release from olfactory sensory neurons is opioid independent. Male and female humans with NaV1.7-null mutations show naloxone-reversible analgesia. Thus, inhibition of neurotransmitter release is the principal mechanism of anosmia and analgesia in mouse and human Nav1.7-null mutants.

Layering of neural circuits facilitates the separation of neurons with high spatial sensitivity from those that play integrative temporal roles. Although anatomical layers are readily identifiable in the brain, layering is not structurally obvious in the spinal cord. But computational studies of motor behaviors have led to the concept of layered processing in the spinal cord. It has been postulated that spinal V3 interneurons (INs) play multiple roles in locomotion, leading us to investigate whether they form layered microcircuits. Using patch-clamp recordings in combination with holographic glutamate uncaging, we demonstrate focal, layered modules, in which ventromedial V3 INs form synapses with one another and with ventrolateral V3 INs, which in turn form synapses with ipsilateral motoneurons. Motoneurons, in turn, provide recurrent excitatory, glutamatergic input to V3 INs. Thus, ventral V3 interneurons form layered microcircuits that could function to ensure well-timed, spatially specific movements.

Elaborate behaviours are produced by tightly controlled flexor-extensor motor neuron activation patterns. Motor neurons are regulated by a network of interneurons within the spinal cord, but the computational processes involved in motor control are not fully understood. The neuroanatomical arrangement of motor and premotor neurons into topographic patterns related to their controlled muscles is thought to facilitate how information is processed by spinal circuits. Rabies retrograde monosynaptic tracing has been used to label premotor interneurons innervating specific motor neuron pools, with previous studies reporting topographic mediolateral positional biases in flexor and extensor premotor interneurons. To more precisely define how premotor interneurons contacting specific motor pools are organized, we used multiple complementary viral-tracing approaches in mice to minimize systematic biases associated with each method. Contrary to expectations, we found that premotor interneurons contacting motor pools controlling flexion and extension of the ankle are highly intermingled rather than segregated into specific domains like motor neurons. Thus, premotor spinal neurons controlling different muscles process motor instructions in the absence of clear spatial patterns among the flexor-extensor circuit components.

The spinal cord contains neural circuits that can produce the rhythm and pattern of locomotor activity. It has previously been postulated that a population of glutamatergic neurons, termed Hb9 interneurons, contributes to locomotor rhythmogenesis. These neurons were identified by their expression of the homeobox gene, Hb9, which is also expressed in motor neurons. We developed a mouse line in which Cre recombinase activity is inducible in neurons expressing Hb9. We then used this line to eliminate vesicular glutamate transporter 2 from Hb9 interneurons, and found that there were no deficits in treadmill locomotion. We conclude that glutamatergic neurotransmission by Hb9 interneurons is not required for locomotor behaviour. The role of these neurons in neural circuits remains elusive.

Mammalian motor systems adapt to the demands of their environment. For example, muscle fibre types change in response to increased load or endurance demands. However, for adaptations to be effective, motoneurons must adapt such that their properties match those of the innervated muscle fibres. We used a rat model of chronic functional overload to assess adaptations to both motoneuron size and a key modulatory synapse responsible for amplification of motor output, C-boutons. Overload of extensor digitorum longus (EDL) muscles was induced by removal of their synergists, tibialis anterior muscles. Following 21 days survival, EDL muscles showed an increase in fatigue resistance and a decrease in force output, indicating a shift to a slower phenotype. These changes were reflected by a decrease in motoneuron size. However, C-bouton complexes remained largely unaffected by overload. The C-boutons themselves, quantified by expression of vesicular acetylcholine transporter, were similar in size and density in the control and overload conditions. Expression of the post-synaptic voltage-gated potassium channel (KV 2.1) was also unchanged. Small conductance calcium-activated potassium channels (SK3) were expressed in most EDL motoneurons, despite this being an almost exclusively fast motor pool. Overload induced a decrease in the proportion of SK3+ cells, however, there was no change in density or size of clusters. We propose that reductions in motoneuron size may promote early recruitment of EDL motoneurons, but that C-bouton plasticity is not necessary to increase the force output required in response to muscle overload.

Many mammals are born with immature motor systems that develop through a critical period of postnatal development. In rodents, postnatal maturation of movement occurs from rostral to caudal, correlating with maturation of descending supraspinal and local spinal circuits. We asked whether development of fundamental electrophysiological properties of spinal motoneurons follows the same rostro-caudal sequence. We show that in both regions, repetitive firing parameters increase and excitability decreases with development; however, these characteristics mature earlier in cervical motoneurons. We suggest that in addition to autonomous mechanisms, motoneuron development depends on activity resulting from their circuit milieu.

Axon guidance receptors such as deleted in colorectal cancer (DCC) contribute to the normal formation of neural circuits, and their mutations can be associated with neural defects. In humans, heterozygous mutations in DCC have been linked to congenital mirror movements, which are involuntary movements on one side of the body that mirror voluntary movements of the opposite side. In mice, obvious hopping phenotypes have been reported for bi-allelic Dcc mutations, while heterozygous mutants have not been closely examined. We hypothesized that a detailed characterization of Dcc heterozygous mice may reveal impaired corticospinal and spinal functions. Anterograde tracing of the Dcc+/- motor cortex revealed a normally projecting corticospinal tract, intracortical microstimulation (ICMS) evoked normal contralateral motor responses, and behavioral tests showed normal skilled forelimb coordination. Gait analyses also showed a normal locomotor pattern and rhythm in adult Dcc+/- mice during treadmill locomotion, except for a decreased occurrence of out-of-phase walk and an increased duty cycle of the stance phase at slow walking speed. Neonatal isolated Dcc+/- spinal cords had normal left-right and flexor-extensor coupling, along with normal locomotor pattern and rhythm, except for an increase in the flexor-related motoneuronal output. Although Dcc+/- mice do not exhibit any obvious bilateral impairments like those in humans, they exhibit subtle motor deficits during neonatal and adult locomotion.

Glutamatergic reticulospinal neurons in the gigantocellular reticular nucleus (GRN) of the medullary reticular formation can function as command neurons, transmitting motor commands to spinal cord circuits to instruct movement. Recent advances in our understanding of this neuron-dense region have been facilitated by the discovery of expression of the transcriptional regulator, Chx10, in excitatory reticulospinal neurons. Here, we address the capacity of local circuitry in the GRN to contribute to reticulospinal output. We define two subpopulations of Chx10-expressing neurons in this region, based on distinct electrophysiological properties and soma size (small and large), and show that these populations correspond to local interneurons and reticulospinal neurons, respectively. Using focal release of caged glutamate combined with patch clamp recordings, we demonstrated that Chx10 neurons form microcircuits in which the Chx10 local interneurons project to and facilitate the firing of Chx10 reticulospinal neurons. We discuss the implications of these microcircuits in terms of movement selection.NEW & NOTEWORTHY Reticulospinal neurons in the medullary reticular formation integrate inputs from higher regions to effectively instruct spinal motor circuits. Using photoactivation of neurons in brainstem slices, we studied connectivity of reticular formation neurons that express the transcriptional regulator, Chx10. We show that a subpopulation of these neurons functions as local interneurons that affect descending commands. The results shed light on the internal organization and microcircuit formation of reticular formation neurons.

Motoneurons (MNs) control muscle contractions, and their recruitment by premotor circuits is tuned to produce accurate motor behaviours. To understand how these circuits coordinate movement across and between joints, it is necessary to understand whether spinal neurons pre-synaptic to motor pools have divergent projections to more than one MN population. Here, we used modified rabies virus tracing in mice to investigate premotor interneurons projecting to synergist flexor or extensor MNs, as well as those projecting to antagonist pairs of muscles controlling the ankle joint. We show that similar proportions of premotor neurons diverge to synergist and antagonist motor pools. Divergent premotor neurons were seen throughout the spinal cord, with decreasing numbers but increasing proportion with distance from the hindlimb enlargement. In the cervical cord, divergent long descending propriospinal neurons were found in contralateral lamina VIII, had large somata, were neither glycinergic, nor cholinergic, and projected to both lumbar and cervical MNs. We conclude that distributed spinal premotor neurons coordinate activity across multiple motor pools and that there are spinal neurons mediating co-contraction of antagonist muscles.

Dystonia, a neurological disorder defined by abnormal postures and disorganized movements, is considered to be a neural circuit disorder with dysfunction arising within and between multiple brain regions. Given that spinal neural circuits constitute the final pathway for motor control, we sought to determine their contribution to this movement disorder. Focusing on the most common inherited form of dystonia in humans, DYT1-TOR1A, we generated a conditional knockout of the torsin family 1 member A (Tor1a) gene in the mouse spinal cord and dorsal root ganglia (DRG). We found that these mice recapitulated the phenotype of the human condition, developing early-onset generalized torsional dystonia. Motor signs emerged early in the mouse hindlimbs before spreading caudo-rostrally to affect the pelvis, trunk, and forelimbs throughout postnatal maturation. Physiologically, these mice bore the hallmark features of dystonia, including spontaneous contractions at rest and excessive and disorganized contractions, including cocontractions of antagonist muscle groups, during voluntary movements. Spontaneous activity, disorganized motor output, and impaired monosynaptic reflexes, all signs of human dystonia, were recorded from isolated mouse spinal cords from these conditional knockout mice. All components of the monosynaptic reflex arc were affected, including motor neurons. Given that confining the Tor1a conditional knockout to DRG did not lead to early-onset dystonia, we conclude that the pathophysiological substrate of this mouse model of dystonia lies in spinal neural circuits. Together, these data provide new insights into our current understanding of dystonia pathophysiology.