Francisco Alvarez (Advisor), Dan Halm (Committee Member), Barbara Hull (Committee Member), James Olson (Committee Member), Robert Putnam (Committee Member), Stephen Schneider (Committee Member)
Doctor of Philosophy (PhD)
Motoneurons are among the best studied neurons in the central nervous system. The motoneuron synapses have been well characterized in the periphery where they release acetylcholine at the neuromuscular junction. However excitatory amino acids also seem to be released from motoneuron terminals in the periphery, and centrally at their synapses contacting Renshaw cells. Although excitatory amino acids are suggested to be released from motoneuron synapses it is not known which excitatory amino acids (either aspartate or glutamate) are released, nor is the mechanism for their release known. To examine the presence and mechanism of release for aspartate and glutamate at motoneuron synapses on Renshaw cells, several immunocytochemistry experiments using both epifluorescence and electron microscopy techniques were used to determine if any of the known vesicular glutamate transporters (VGLUTs) or other transporters were present and to quantify the enrichment of aspartate and glutamate in these terminals. Moreover, immunofluorescent experiments using the Hb9::EGFP mouse model were done to confirm the specificity of VAChT immunolabeling for identifying motoneuron contacts on calbindin immunoreactive (-IR) Renshaw cells. The results from these experiments show that the known VGLUTs are not detectable at motoneuron contacts on Renshaw cells, and therefore aspartate and glutamate must be released via a VGLUT-independent mechanism. Immunofluorescent experiments with HB9::EGFP mice confirmed that VAChT is an appropriate marker for labeling motoneuron contacts on Renshaw cells. Electron microscopy experiments determined that both glutamate and aspartate are enriched in VAChT-IR contacts on Renshaw cells. Further immunofluorescent experiments looking for potential transporters for packaging excitatory amino acids into synaptic vesicles revealed that both SLC10A4 and SLC17A5 (members of the solute carrier protein family that also include the VGLUTs) were present in motoneurons. SLC10A4-IR was present in motoneuron terminals, whereas SLC17A5-IR was restricted to the motoneuron somata. In conclusion, aspartate and glutamate are enriched in motoneuron terminals contacting Renshaw cells. However, these synapses lack detectable VGLUT-IR. This suggests that there is another mechanism present for packaging aspartate and glutamate into vesicles. Future studies examining SLC10A4 are needed to determine if it is capable of transporting aspartate and glutamate into synaptic vesicles for release at motoneuron synapses.
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