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We are interested in understanding the development of reciprocal inhibition, an important sensory-motor circuit that prevents co-contraction of antagonist muscles during locomotion. This effect is mediated by a subset of glycinergic neurons termed Ia inhibitory interneurons (IaINs). Transgenic mouse models are useful in helping to identify subsets of neurons in developing animals. In this thesis we first wanted to test the BAC-GlyT2-eGFP mice model by measuring GFP expression overlap in neurons with glycine in late-stage embryos (E17.5) and early postnatal animals (P0/P1). Our results suggest more than 98% of glycinergic neurons at these stages also express GFP in GlyT2-EGFP transgenic animals. Secondly, we quantitatively measured the number of IaINs at P0 and P1 using a combination of GFP expression in this transgenic model together with other immunohistochemical markers of IaINs. We found that IaINs account for 17% of glycinergic neurons found in lamina VII at this stage.

Peripheral nerve regeneration fails to restore complete normal function after surgical repair of severed nerves, and this failure has primarily been attributed to errors in connecting with peripheral targets. However, recent evidence suggests that central deficits remain even after peripheral target reinnervation is largely successful. It has long been established that regeneration fails to restore the stretch reflex despite observation that many of the neural components are intact. Regenerated Ia afferents are largely successful in reinnervating muscle spindles, are capable of encoding stretch, and elicit EPSPs in homonymous motoneurons, while regenerated motor pools are capable of responding to uninjured sources of excitatory input. We now know this areflexia is due in part to a retraction of Ia afferent collaterals from motor pools in lamina IX (Alvarez et al., 2011). However, Ia afferents project not only to homonymous motoneurons but also to heteronymous synergist motor pools, even ones that are injury-spared. Ia afferents also project to antagonist motor pools through an interposed inhibitory interneuron. Therefore stretch of a muscle is capable of producing reflex contraction of both itself and synergist muscles while producing reflex inhibition of antagonist muscles. The function of these heteronymous projections after regeneration, however, remains unknown. The goal of this thesis is to determine the limitations of recovery of spinal circuit function after peripheral nerve regeneration by direct examination of stretch-evoked reflexes among synergists and antagonists. Direct examination of the force response to stretch in vivo is extremely valuable as changes in behavior, the muscle response to stretch, after peripheral nerve regeneration must necessarily reflect changes in the underlying circuitry. We found that heteronymous stretch reflexes initiated by reinnervated muscle were dramatically decreased in both regenerated and injury-spared synergist motoneuronal pools. Additionally, both homonymous and heteronymous stretch reflexes were reduced in an injury-spared synergist after regeneration. These results give physiological evidence for retraction of regenerated Ia afferents from all synergist motor pools, and this retraction may extend to afferents that are injury-spared. Dysfunction also extends to antagonist stretch-evoked reflexes as we found a shift from net inhibition to net excitation of the injury-spared muscle due to reinnervated antagonist stretch. This shift is readily explained by the differential preservation of synapses located in lamina where interneurons mediating these responses are presumably located (Alvarez et al., 2011). Therefore functional deficits after peripheral nerve regeneration extend to heteronymous connections of Ia afferents with synergists and antagonists, both reinnervated and injury-spared. Taken together, these findings suggest that there is a profound discoordination of spinal reflexes and reorganization of spinal circuits after peripheral nerve regeneration.

Locomotor development is dependent on the maturation of spinal cord circuits controlling motor output, but little is known about the development of the spinal interneurons that control motoneuron activity. This study focused on the development of Renshaw cells (RCs) and Ia inhibitory interneurons (IaINs), which mediate recurrent and reciprocal inhibition, respectively, two basic inhibitory circuits for motorneuron control. Both interneurons originate from the same progenitor pool (p1) giving rise to ventral spinal embryonic interneurons denominated V1. V1-derived interneurons (V1-INs) establish local inhibitory connections with ipsilateral motoneurons and express the transcription factor engrailed-1. This characteristic permitted the generation of transgenic mice that were used in this study to genetically label V1 interneuron lineages from embryo to adult. Adult V1-derived Renshaw cells and IaINs share some similar properties, both being inhibitory and establishing ipsilateral connections; but differ in morphology, location in relation to motor pools, expression of calcium binding proteins (calbindin vs. parvabumin), synaptic connectivity and function. These differences are already present in neonates, therefore the purpose of this study was to determine possible embryonic differentiation mechanisms.Using 5‟-bromodeoxyuridine birth-dating we demonstrated that V1-INs can be divided into early and late born groups. The early group quickly upregulates calbindinivexpression and includes the Renshaw cells, which maintain calbindin expression through life. The second group includes many cells that postnatally upregulate parvalbumin, including IaINs. This later born group is characterized by upregulation of the transcription factor FoxP2 as they start to differentiate and is retained up to the first postnatal week in many V1-derived IaINs. In contrast, Renshaw cells express the transcription factor MafB that seems relatively specific to them within the V1-INs. Furthermore, Renshaw cells appear attracted to the ventral root exit region and follow a unique migratory route to become specifically placed at this location. In contrast, other V1 interneurons settle more medially and far from the ventral root exit region. MafB expression is upregulated in Renshaw cells only after they have reached their final position among motor axons. Therefore, the specific migration of Renshaw cells might be responsible for their final differentiation and unique relationship with motor axons in adult.