Wright State University CORE Scholar Browse all Theses and Dissertations Theses and Dissertations 2009 Characterization of Excitatory Amino Acid Neurotransmitters at Motoneuron Synapses Contacting Renshaw Cells Dannette Shanon Richards Wright State University Follow this and additional works at: https://corescholar.libraries.wright.edu/etd_all Part of the Biomedical Engineering and Bioengineering Commons Repository Citation Richards, Dannette Shanon, "Characterization of Excitatory Amino Acid Neurotransmitters at Motoneuron Synapses Contacting Renshaw Cells" (2009). Browse all Theses and Dissertations. 971. https://corescholar.libraries.wright.edu/etd_all/971 This Dissertation is brought to you for free and open access by the Theses and Dissertations at CORE Scholar. It has been accepted for inclusion in Browse all Theses and Dissertations by an authorized administrator of CORE Scholar. For more information, please contact [email protected]. CHARACTERIZATION OF EXCITATORY AMINO ACID NEUROTRANSMITTERS AT MOTONEURON SYNAPSES CONTACTING RENSHAW CELLS A dissertation submitted in partial fulfillment of the requirements of the degree of Doctor of Philosophy By DANNETTE SHANON RICHARDS M.S., Wright State University, 2001 B.S., Saint Mary-of-the-Woods College, 1997 __________________________________________________ 2009 Wright State University WRIGHT STATE UNIVERSITY SCHOOL OF GRADUATE STUDIES November 10, 2009 I HEREBY RECOMMEND THAT THE DISSERTATION PREPARED UNDER MY SUPERVISION BY Dannette Shanon Richards ENTITLED Characterization of excitatory amino acid neurotransmitters at motoneuron synapses contacting Renshaw cells BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DECREE OF Doctor of Philosophy. ____________________________________ Francisco J. Alvarez, Ph.D. Dissertation Director ____________________________________ Gerald, M. Alter, Ph.D. Director, Biomedical Sciences Ph.D. Program ____________________________________ Joseph F. Thomas, Jr., Ph.D. Dean, School of Graduate Studies Committee on Final Examination ____________________________________ Dan Halm, Ph.D. ____________________________________ Barbara Hull, Ph.D. ____________________________________ James Olson, Ph.D. ____________________________________ Robert Putnam, Ph.D. ____________________________________ Stephen Schneider, Ph.D. ABSTRACT Richards, Dannette Shanon. Ph.D., Biomedical Sciences Ph.D. Program, Wright State University, 2009. Characterization of excitatory amino acid neurotransmitters at motoneuron synapses contacting Renshaw cells. 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 iii 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. iv Table of Contents Page I. Introduction………………………………………………………………………… 1 II. Background………………………………………………………………………. 4 Renshaw cell physiology…………………………….……………………… 4 Renshaw cell anatomy……………………………………………………… 5 Renshaw cell function………………………………………………………... 5 Renshaw cell synaptic pharmacology………………………………………… 9 Pharmacology of motoneuron synapses on Renshaw cells…………………… 10 Other inputs on Renshaw cells……………………………………..………… 11 Motor axon synapses………………...……………………………………… 13 Glutamate and aspartate: neurotransmitters in the CNS and developmental actions………………………………………………………………………… 15 Vesicular glutamate transporters or VGLUTs……………………………….. 17 Other possible vesicular excitatory amino acid transporters…………………. 20 III. Specific aims…………………………………………………………………….. 21 IV. Aim 1: Characterization of vesicular glutamate transporters (VGLUT) presence at central motoneuron synapses………………………………………………………… 23 Rationale…………………………………………………………………… 23 Materials and methods…………………………………………….………… 25 Animals, fixation, sectioning……………………………………………. 25 Multiple color immunofluorescence for VGLUT 1, 2 or 3 and VAChT on calbindin-IR Renshaw cells………………………………………… 26 Motor axon retrograde labeling and combination with immunohistochemistry…………………………………………… 27 v Tyramide amplification…………………………………………… 28 Microscopy and image analysis…………………………………… 29 Analysis of VGLUTs and VAChT colocalization………………… 29 Analysis of retrogradely labeled synaptic varicosities……………… 30 Results……………………………………………………………………… 32 Distribution of different VGLUT isoforms in the Renshaw cell region 32 VAChT-IR boutons in the Renshaw cell region lack VGLUT immunoreactivities………………………………………………… 33 Retrogradely labeled synaptic varicosities contain VAChT but not VGLUTs…………………………………………………………… 36 Discussion…………………………………………………………………… 53 V. Aim 2: Characterization of the Hb9::EGFP mouse model for labeling motoneuron synapses contacting Renshaw cells………………………………………………… 58 Rationale…………………………………………………………………… 58 Materials and methods……………………………………………………… 61 Animals……………………………………………………………… 61 Fixation and sectioning……………………………………………… 61 Neurolucida analyses of EGFP expressing neurons in Hb9::EGFP animals……………………………………………………………….. 62 Confocal analysis of Hb9::EGFP axons in the Renshaw cell region… 63 Results……………………………………………………………………… 65 Motoneurons are the only spinal neurons that co-express EGFP and cholinergic markers (ChAT, VAChT)……………………………… 65 Motor axon synapses on Renshaw cells co-express VAChT and EGFP, but EGFP is down-regulated with development…………………… 67 Discussion………………………………………………………………… 81 vi VI. Aim 3: Electron microscopy analyses of aspartate and glutamate enrichment in motoneuron synapses on Renshaw cells……………………… 84 Rationale…………………………………………………………………… 84 Materials and methods………………………………………………………. 86 Antibody specificity………………………………………………… 86 Pre-embedding electron microscopy immunolabeling for calbindin and VAChT……………………………………………………………… 87 Cryosubstitution and Lowicryl embedding………………………… 89 Colloidal gold post-embedding with aspartate and glutamate antibodies…………………………………………………………… 89 Electron microscopy image capturing and analysis………………… 90 Statistical comparisons……………………………………………… 91 Results……………………………………………………………………… 92 Specificity of polyclonal aspartate and glutamate antibodies……….. 92 VAChT-IR and calbindin-IR structures in the ventral horn Renshaw cell area…………………………………………………… 93 Aspartate immunoreactivitiy………………………………………… 95 Glutamate immunoreactivity………………………………………… 99 Discussion………………………………………………………………….. 123 Antibody specificity………………………………………………… 123 Glutamate and aspartate in motor axon synaptic boutons………… 123 Co-release of excitatory amino acids with acetylcholine………… 124 Functional significance…………………………………………… 126 VII. Aim 4: Characterization of the presence of members of the solute carrier (SLC) protein family in the spinal cord ventral horn……………………………………… 131 vii Rationale…………………………………………………………………… 131 Materials and methods……………………………………………………… 135 Confocal analysis of SLC10A4 and SLC10A5 contacts on Renshaw cells………………………………………………………………… 135 Results……………………………………………………………………… 136 SLC10A4-IR in the spinal cord…………………………………… 136 SLC17A5 in the spinal cord………………………………………… 136 Discussion………………………………………………………………… 143 SLC10A4: a putative aspartate/glutamate transporter?...................... 143 SLC15A5…………………………………………………………… 146 VIII. General conclusions and discussion………………………………………… 148 IX. Significance……………………………………………………………………... 150 X. References……………………………………………………………………..