Longitudinal extension of primary afferents is regulated by spingosine 1-phosphate receptors and tyrosine kinase receptor B in the embryonic spinal cord via a brain derived neurotrophic factor related mechanism

Primary sensory afferent outgrowth within the developing longitudinal pathway of the spinal cord is important for intrasegmental and intersegmental communication that underlies coordination and development of reflexes and contributes to sensory perception. The endogenous mechanisms that regulate primary sensory afferent extension are the primary focus of this dissertation. This dissertation tested the hypothesis that primary sensory afferent extension in the longitudinal pathway is regulated by sphingosine 1-phosphate type 1 receptor (S1P1R) and tyrosine kinase receptor B (TrkB) through a brain derived neurotrophic factor (BDNF) related mechanism. To test this hypothesis we used embryonic day five (E5) chicken embryos, as this is the developmental time point when sensory afferents are growing along the longitudinal axis of the spinal cord but have not yet turned ventrally to make connections with the grey matter of the spinal cord. Chicken embryos were removed from their in ovo environment to allow for labeling of primary afferent neurons in the thoracic 3/4 (T3/4) dorsal root ganglia (DRG). Tissue was then put into culture with or without various pharmacological agents and subsequently assayed for length of growth of the labeled primary afferent axons along the longitudinal axis of the spinal cord. Results showed both BDNF and fingolimod-p, an S1P1R agonist known to increase BDNF mRNA and protein production/secretion in cortical neurons, increased primary axon extension along the longitudinal pathway. Further, fingolimod-p increased BDNF mRNA production in DRG in this system. Conversely, inhibition of BDNF or S1PRs attenuated primary afferent axon extension along the longitudinal pathway. We found BDNF signaling to be required for fingolimod-p’s effects as addition of αBDNF attenuated the effects of fingolimod-p on axon outgrowth. TrkB, the high affinity receptor for BDNF, is expressed in chicken DRG during embryonic development. We hypothesized that TrkB activation by BDNF regulates DRG axon extension in the longitudinal pathway through the PLC-γ signaling pathway. We found inhibition of TrkB and/or PLC-γ signaling pathway attenuated DRG axon extension with or without BDNF stimulation. Additional pathways associated with TrkB activation: mitogen activated kinase (MAPK) and phosphoinositide 3-kinase (PI3K) appeared to either have no effect on DRG axon extension or were involved in DRG axon extension through a mechanism that is not related to TrkB. Collectively, these studies suggest an endogenous mechanism for the regulation of DRG axon outgrowth within the longitudinal pathway. With this mechanism, DRG axon outgrowth may be enhanced or attenuated following manipulation of S1P1R, BDNF and/or TrkB. Further, these findings suggest an action through BDNF on CNS axons as a potential therapeutic effect of fingolimod-p, a treatment for relapsing remitting forms of Multiple Sclerosis.