Molecular Mechanisms Regulating Inhibitory Circuitry in the Spinal Cord

Establishing specific neuronal circuits is fundamental for the generation of coordinated muscle function. However, the signals underlying the specificity of connection between interneurons and their targets in the mammalian central nervous system (CNS) remain largely unknown to date. Our long-term research goal is to understand the rules of interneuron circuit wiring and the molecular mechanisms that control it. The objective of the proposed research is to describe how a combination of adhesive and neurotrophic signals determine GABAergic interneuron circuit connectivity. A class of GABAergic interneurons, termed GABApre, forms synaptic contacts with the terminals of proprioceptive sensory afferents, and thus directly controls proprioceptive sensory input through an inhibitory strategy known as presynaptic inhibition. We will test the hypothesis that the connectivity of a class of spinal GABAergic neurons varies between functionally-distinct sensory neurons and that this connectivity is mediated via (ii) differential expression of muscle-derived neurotrophin (NT)-3 and (ii) a matrix of IgSF adhesion proteins. The rationale underlying this proposal is that through understanding the mechanisms underlying GABAergic interneuron circuit formation, we will enhance our understanding of - and ultimately control over - inhibitory neuronal circuit development and function in vivo. We will test our hypothesis with the following three aims: #1) Determine the functional specificity of GABAergic interneuron circuitry; #2) Investigate the influence of muscle-derived NT-3 on GABApre synapse formation; and #3) Assess the role of Contactin-5 in GABApre-sensory synapse formation. In the first aim, we combine timed tamoxifen injections, mouse genetics and CTb labeling to analyze whether the specific connectivity of individual GABApre interneurons may be functionally relevant. In the second aim, we examine the expression of Gabrg1 in functionally distinct proprioceptive sensory neurons and assess consequences of changing NT-3 levels on both Gabrg1 expression and GABApre terminal number. In the third aim, we perturb cell adhesion signaling using mouse genetics and perform phenotypic analysis using molecular, micro-anatomic and functional assays, including an electrophysiological measure of presynaptic inhibition. We also use an in vitro binding assay to screen for new adhesion molecule candidates relevant for GABApre synaptic specificity in vivo. The research proposed in this application is innovative because it combines a molecularly-defined interneuronal circuit with a unique constellation of methodologies to integrate functional specificity with target-derived signals. The research will provide an understanding of functional diverse GABApre circuitry and also advance our understanding of how basic circuit paradigms may be adapted for diverse motor functions. The proposed work is significant because it will contribute to fundamental knowledge of the formation of circuit-level mechanisms and neural strategies used in the CNS; this in turn will advance our efforts to develop effective therapies to rebuild circuitry and muscle function after spinal cord injury or other neurological diseases.

Establishing specific neuronal circuits is fundamental for the generation of coordinated muscle function. However, the signals underlying the specificity of connection between interneurons and their targets in the mammalian central nervous system (CNS) remain largely unknown to date. Our long-term research goal is to understand the rules of interneuron circuit wiring and the molecular mechanisms that control it. The objective of the proposed research is to describe how a combination of adhesive and neurotrophic signals determine GABAergic interneuron circuit connectivity. A class of GABAergic interneurons, termed GABApre, forms synaptic contacts with the terminals of proprioceptive sensory afferents, and thus directly controls proprioceptive sensory input through an inhibitory strategy known as presynaptic inhibition. We will test the hypothesis that the connectivity of a class of spinal GABAergic neurons varies between functionally-distinct sensory neurons and that this connectivity is mediated via (ii) differential expression of muscle-derived neurotrophin (NT)-3 and (ii) a matrix of IgSF adhesion proteins. The rationale underlying this proposal is that through understanding the mechanisms underlying GABAergic interneuron circuit formation, we will enhance our understanding of — and ultimately control over — inhibitory neuronal circuit development and function in vivo. We will test our hypothesis with the following three aims: #1) Determine the functional specificity of GABAergic interneuron circuitry; #2) Investigate the influence of muscle-derived NT-3 on GABApre synapse formation; and #3) Assess the role of Contactin-5 in GABApre-sensory synapse formation. In the first aim, we combine timed tamoxifen injections, mouse genetics and CTb labeling to analyze whether the specific connectivity of individual GABApre interneurons may be functionally relevant. In the second aim, we examine the expression of Gabrg1 in functionally distinct proprioceptive sensory neurons and assess consequences of changing NT-3 levels on both Gabrg1 expression and GABApre terminal number. In the third aim, we perturb cell adhesion signaling using mouse genetics and perform phenotypic analysis using molecular, micro-anatomic and functional assays, including an electrophysiological measure of presynaptic inhibition. We also use an in vitro binding assay to screen for new adhesion molecule candidates relevant for GABApre synaptic specificity in vivo. The research proposed in this application is innovative because it combines a molecularly-defined interneuronal circuit with a unique constellation of methodologies to integrate functional specificity with target-derived signals. The research will provide an understanding of functional diverse GABApre circuitry and also advance our understanding of how basic circuit paradigms may be adapted for diverse motor functions. The proposed work is significant because it will contribute to fundamental knowledge of the formation of circuit-level mechanisms and neural strategies used in the CNS; this in turn will advance our efforts to develop effective therapies to rebuild circuitry and muscle function after spinal cord injury or other neurological diseases.