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

Olfactory sensory neurons in the olfactory epithelium are responsible for detecting the odors we smell and are constantly dying. However, in order for the sense of smell to be maintained, the olfactory system has the unique ability to generate new neurons. After an olfactory sensory neuron is born in the olfactory epithelium, it must extend an axon towards the olfactory bulb in the central nervous system. Within the olfactory bulb, these axons make specific synaptic contacts with the dendritic processes of mitral cells, which are the main projection neurons from the olfactory bulbs into higher cortical areas in the brain. In addition to regeneration due to normal turnover, the olfactory system is also capable of recovery after an injury. The olfactory system’s ability to recover is remarkable because it is capable of regeneration after a mild injury (a portion of olfactory epithelium is removed) or a severe injury (in which the entire olfactory epithelium is removed.) A well-established model for producing a severe type of injury in the olfactory epithelium is by inducing a chemical ablation by a single injection of the drug methimazole. A specific interest in the regenerative process after injury is reestablishment of synaptic connections. We hypothesized that expression of synaptic markers will allow for establishing a timeline of functional recovery of the olfactory system after injury. Our lab has studied three synaptic vesicle associated proteins, vesicular glutamate transporter -1 (VGlut-1), vesicular glutamate transporter-2 (VGlut-2), and synaptophysin, as well as one activity-regulated protein, tyrosine hydroxylase. These studies found specific temporal expression profiles at 2, 7 and 14 days post injury. Our initial data show that VGlut-1 and VGlut-2 are decreased after injury, indicative of a reduction in synaptic connectivity in both olfactory sensory neuron axons and in dendrites of mitral cell neurons. These changes in synaptic connectivity help in understanding functional connectivity after an injury and can further be used to correlate histological axonal tracing with behavioral studies.