SUMMARY The reciprocal activation of flexor and extensor muscles constitutes the fundamental mechanism that tetrapod vertebrates use for locomotion and limb-driven reflex behaviors. This aspect of motor coordination is controlled by inhibitory neurons in the spinal cord; however, the identity of the spinal interneurons that serve this function is not known. Here we show that the production of an alternating flexor-extensor motor rhythm depends on the composite activities of two classes of ventrally-located inhibitory neurons, V1 and V2b interneurons (INs). Abrogating V1 and V2b IN-derived neurotransmission in the isolated spinal cord results in a synchronous pattern of L2 flexor-related and L5 extensor-related locomotor activity. Mice lacking V1 and V2b inhibition are unable to articulate their limb joints and display marked deficits in limb-driven reflex movements. Taken together, these findings identify V1- and V2b-derived neurons as the core interneuronal components of the limb central pattern generator (CPG) that coordinate flexor-extensor motor activity.

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The diversity of premotor interneurons in the mammalian spinal cord is generated from a few phylogenetically conserved embryonic classes of interneurons (V0, V1, V2, V3). Their mechanisms of diversification remain unresolved, although these are clearly important to understand motor circuit assembly in the spinal cord. Some Ia inhibitory interneurons (IaINs) and all Renshaw cells (RCs) derive from embryonic V1 interneurons; however, in adult they display distinct functional properties and synaptic inputs, for example proprioceptive inputs preferentially target IaINs, while motor axons target RCs. Previously, we found that both inputs converge on RCs in neonates, raising the possibility that proprioceptive (VGLUT1-positive) and motor axon synapses (VAChT-positive) initially target several different V1 interneurons populations and then become selected or deselected postnatally. Alternatively, specific inputs might precisely connect only with predefined groups of V1 interneurons. To test these hypotheses we analyzed synaptic development on V1-derived IaINs and compared them to RCs of the same age and spinal cord levels. V1-interneurons were labeled using genetically encoded lineage markers in mice. The results show that although neonatal V1-derived IaINs and RCs are competent to receive proprioceptive synapses, these synapses preferentially target the proximal somato-dendritic regions of IaINs and postnatally proliferate on IaINs, but not on RCs. In contrast, cholinergic synapses on RCs are specifically derived from motor axons, while on IaINs they originate from Pitx2 V0c interneurons. Thus, motor, proprioceptive, and even some interneuron inputs are biased toward specific subtypes of V1-interneurons. Postnatal strengthening of these inputs is later superimposed on this initial preferential targeting.

Renshaw cells provide a convenient model to study spinal circuit development during the emergence of motor behaviors with the goal of capturing principles of interneuron specification and circuit construction. This work is facilitated by a long history of research that generated essential knowledge about the characteristics that define Renshaw cells and the recurrent inhibitory circuit they form with motoneurons. In this review, we summarize recent data on the specification of Renshaw cells and their connections. A major insight from these studies is that the basic Renshaw cell phenotype is specified before circuit assembly, a result of their early neurogenesis and migration. Connectivity is later added, constrained by their placement in the spinal cord. Finally, different rates of synapse proliferation alter the relative weights of different inputs on postnatal Renshaw cells. Based on this work some general principles on the integration of spinal interneurons in developing motor circuits are derived.

The diversity of premotor interneurons in the mammalian spinal cord is generated from a few phylogenetically conserved embryonic classes of interneurons (V0, V1, V2, V3). Their mechanisms of diversification remain unresolved, although these are clearly important to understand motor circuit assembly in the spinal cord. Some Ia inhibitory interneurons (IaINs) and all Renshaw cells (RCs) derive from embryonic V1 interneurons; however, in adult they display distinct functional properties and synaptic inputs, for example proprioceptive inputs preferentially target IaINs, while motor axons target RCs. Previously, we found that both inputs converge on RCs in neonates, raising the possibility that proprioceptive (VGLUT1-positive) and motor axon synapses (VAChT-positive) initially target several different V1 interneurons populations and then become selected or deselected postnatally. Alternatively, specific inputs might precisely connect only with predefined groups of V1 interneurons. To test these hypotheses we analyzed synaptic development on V1-derived IaINs and compared them to RCs of the same age and spinal cord levels. V1-interneurons were labeled using genetically encoded lineage markers in mice. The results show that although neonatal V1-derived IaINs and RCs are competent to receive proprioceptive synapses, these synapses preferentially target the proximal somato-dendritic regions of IaINs and postnatally proliferate on IaINs, but not on RCs. In contrast, cholinergic synapses on RCs are specifically derived from motor axons, while on IaINs they originate from Pitx2 V0c interneurons. Thus, motor, proprioceptive, and even some interneuron inputs are biased toward specific subtypes of V1-interneurons. Postnatal strengthening of these inputs is later superimposed on this initial preferential targeting. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe development of spinal cord local interneuronal circuits is currently poorly understood. Recent work has defined a relatively small number of cardinal interneurons in embryo that generates the much larger diversity of adult interneurons (Goulding, 2009). In the ventral horn just four embryonic subgroups (V0, V1, V2, V3) generate most adult premotor interneurons by diversification mechanisms that are currently largely unknown. Each group displays common properties that generally include origins from one type of progenitor cell (p0, p1, p2, p3), the direction taken by the primary axon, their cell migration and final location in the ventral horn with relation to motor pools, and their neurotransmitter phenotype. For example, V1 interneurons have common origins in the p1 progenitor area and are characterized by expression of the engrailed-1 (En-1) transcription factor. They migrate ventrolaterally, positioning themselves adjacent to motor pools, extend ascending axons that target ipsilateral motoneurons, and have an inhibitory phenotype (Matise and Joyner, 1997; Ericsson et al., 1997;Saueressig et al., 1999;Sapir et al., 2004;Alvarez et a...

We investigated factors controlling the development of connections between muscle spindle afferents, spinal motor neurons and inhibitory Renshaw cells. Several mutants were examined to establish the role of muscle spindles, muscle spindle-derived NT3 and excess NT3 in determining the specificity and strength of these connections. The findings suggest that although spindlederived factors are not necessary for the initial formation and specificity of the synapses, spindlederived NT3 seems necessary for strengthening homonymous connections between Ia afferents and motor neurons during the second postnatal week. We also found evidence for functional monosynaptic connections between sensory afferents and neonatal Renshaw cells although the density of these synapses decreases at P15. We conclude that muscle spindle synapses are weakened on Renshaw cells while they are strengthened on motor neurons. Interestingly, the loss of sensory synapses on Renshaw cells was reversed in mice over-expresssing NT3 in the periphery, suggesting that different levels of NT3 are required for functional maintenance and strengthening of spindle afferent inputs on motor neurons and Renshaw cells.

Motor function in mammalian species depends on the maturation of spinal circuits formed by a large variety of interneurons that regulate motoneuron firing and motor output. Interneuron activity is in turn modulated by the organization of their synaptic inputs, but the principles governing the development of specific synaptic architectures unique to each premotor interneuron are unknown. For example, Renshaw cells receive, at least in the neonate, convergent inputs from sensory afferents (likely Ia) and motor axons raising the question of whether they interact during Renshaw cell development. In other well-studied neurons, like Purkinje cells, heterosynaptic competition between inputs from different sources shapes synaptic organization. To examine the possibility that sensory afferents modulate synaptic maturation on developing Renshaw cells, we used three animal models in which afferent inputs in the ventral horn are dramatically reduced (Er81(−/−) knockout), weakened (Egr3(−/−) knockout) or strengthened (mlcNT3(+/−) transgenic). We demonstrate that increasing the strength of sensory inputs on Renshaw cells prevents their de-selection and reduces motor axon synaptic density and, in contrast, absent or diminished sensory afferent inputs correlate with increased densities of motor axons synapses. No effects were observed on other glutamatergic inputs. We conclude that the early strength of Ia synapses influences their maintenance or weakening during later development and that heterosynaptic influences from sensory synapses during early development regulates the density and organization of motor inputs on mature Renshaw cells.