Proprioceptive sensory neurons (pSNs) serve a key role in refining the output of the spinal motor system through the provision of feedback signals that convey the state of muscle activity to central and spinal motor neurons. Distinct pSN subtypes engage with select spinal circuits dedicated to specific musculoskeletal tasks (e.g. postural control, knee flexion, ankle extension etc). This precision in sensory-motor connectivity is presumed to rest in large part on the molecular distinctions between the various proprioceptor subtypes, yet surprisingly little is known of the way in which proprioceptor subtype identity is established. Challenging prevailing views, our recent studies suggest that certain aspects pSN subtype character are mediated by graded signaling by neurotrophin 3 (NT3) rather than by intrinsic transcriptional determinants. This idea is founded on several observations, most notably the finding that embryonic muscles exhibit muscle- by-muscle differences in NT3 expression levels at the time when pSNs establish their subtype identity. The hypothesis that variations in the strength of NT3 signaling direct pSN subtype character leads to two predictions. First, if graded NT3 signaling drives pSN subtype diversity, NT3 should elicit distinct molecular responses in pSNs that innervate muscle targets expressing different levels of NT3. Second, based on the notion that pSN identity is inherently linked to spinal connectivity patterns, changes in NT3 signaling levels should result in alterations in pSN connectivity patterns. The experiments in the proposal are designed to test these expectations. In agreement with our predictions, in preliminary studies, we identified several molecular markers that are differentiall expressed between pSN subsets that innervate NT3high -and those that innervate NT3low muscle targets. These molecular markers not only provide new insights into the various aspects of pSN subtype identity, but importantly, are powerful tools through which to assess the role of NT3 in regulating pSN diversity (Aim 1). In addition (Aim 2), we will take advantage of newly developed strategies - based on anterograde transsynaptic transfer of recombinant rabies virus - to construct an anatomical framework of the spinal connectivity patterns of defined NT3low and NT3high pSN subsets, and examine the role of NT3 signaling in establishing these patterns. Ultimately, these analysis'should lead to new insights in cardinal molecular and network features of pSN subtypes and may begin to reveal the organizational rules that underlie the formation of spinal sensory- motor circuits.
The immediate objective of the experiments described in this protocol is to gain better insight into the molecular identity of cardinal proprioceptor subtypes, and to define their target neurons in the spinal cord. The longer term significance of these studies is two-fold: First, there is an increasing interest in the development of therapeutic strategies that utilize proprioceptive spinal micro-circuits to reconfigure functional motor activiy following traumatic spinal cord injury (SCI). In addition, recent studies indicate that proprioceptr neuronal loss may in part underlie the motor neuronal death in Spinal Muscular Atrophy (SMA). Thus, a better understanding of the proprioceptive sensory neuronal subtypes, as well as the spinal circuits through which they exert influence on motor output, is critical in the advancement of new treatment methods that alleviate the severely debilitating consequences of SCI and/or motor neuron disease.
