The formation of specific synaptic connections by local interneurons is critical for the processing of neuronal information. However, little is known about the factors that regulate interneuronal connectivity in the central nervous system. Our long-term goal is to understand the genetic mechanisms that control interneuronal circuit formation. The objective of the proposed experiments is to describe how a cell-intrinsic factor and its downstream effectors determine GABAergic interneuronal identity and circuit connectivity. We focus our analysis on an identified and molecularly characterized subclass of spinal GABAergic inhibitory interneurons that form direct axo-axonic contacts on sensory afferent terminals, thereby inhibiting them presynaptically. We will test the hypothesis that the transcription factor Ptf1a controls synaptic targeting and differentiation of a class of spinal GABAergic interneurons, and that a transcriptional target of Ptf1a, NrCAM, contributes with Contactin-5 and Caspr4 to an adhesive signaling complex that directs specific synaptic connectivity. We test our hypothesis with the following three aims: #1) Characterize distinct GABAergic interneuron subtypes based on the timing of Ptf1a expression in neuronal precursors; #2) Define the role of Ptf1a in directing connectivity of GABApre interneurons; and #3) Assess the role of the Ptf1a effector gene NrCAM and the potential NrCAM receptor complex Contactin-5/CASPR4 in specifying GABApre target selection. In the first aim, we use timed tamoxifen injections to label and characterize single Ptf1a-expressing interneurons. In the second aim, we use mouse genetics to assess whether Ptf1a is necessary and sufficient for the targeting and differentiation of GABApre synapses. In the third aim, we use mouse genetics to perturb cell adhesion signaling and we analyze the consequences of this both micro-anatomically and functionally, via a novel electrophysiological assay of presynaptic inhibition. Taken together, the proposed experiments will determine which aspects of spinal GABAergic interneuronal identity and connectivity are directed by Ptf1a, and will suggest a downstream molecular mechanism by which specific synaptic connectivity is conferred. Our proposed research is innovative both technically and conceptually. Technically, we will combine new mouse lines with novel in vivo molecular genetics and electrophysiological analyses to manipulate and functionally characterize spinal GABAergic circuits in an otherwise intact network in vivo. Conceptually, we will explore the necessity and sufficiency of an intrinsic transcription factor signal (Ptf1a) for determining specific GABAergic identity and connectivity. Our proposed work is significant in that we will demonstrate - for the first time - a transcriptionl mechanism mediating synaptic specificity of inhibitory central circuits in vivo, and a novel role for cell adhesion-based signaling in directing specific interneuronal connectivity. Our analysis will contribute to a basic scientific understanding of neuronal circuit formation and will provide foundation for regenerative therapies aimed at rebuilding GABAergic circuitry disrupted by human disease.
The proposed research is relevant to public health because it provides basic scientific knowledge about the development of neuronal circuitry in the central nervous system that will ultimately facilitate rational treatments for diverse disorders including spinal cord injury, neurodegenerative disease and schizophrenia. This work supports the NIH's goal to expand the knowledge base in medical and associated sciences, and ultimately supports the NINDS's mission to reduce the burden of neurological disease.
