Neural Activity During Biting Prepares a Retractor Muscle for Force Generation During Swallowing in Aplysia
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PRE-MODULATION: NEURAL ACTIVITY DURING BITING PREPARES A RETRACTOR MUSCLE FOR FORCE GENERATION DURING SWALLOWING IN APLYSIA by HUI LU Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Advisor: Hillel J. Chiel, Ph. D. Department of Biology CASE WESTERN RESERVE UNIVERSITY August, 2014 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Hui Lu candidate for the degree of Doctor of Philosophy *. Committee Chair Robin Snyder Committee Member Hillel Chiel Committee Member Roy Ritzmann Committee Member Jessica Fox Committee Member Kenneth Gustafson Date of Defense April 1, 2014 *We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents Chapter 1 Introduction Mechanisms underlying multifunctionality ………………………… 2 Motivation and research rationale …………………………………... 6 Hypothesis and specific aims ……………………………………… 10 Experimental design ……………………………………………….. 11 Brief introduction to the structure of later chapters ……………….. 13 References …………………………………………………………. 15 Figures and Tables ………………………………………………… 21 Chapter 2 Selective extracellular stimulation of individual neurons in ganglia Summary …………………………………………………………... 25 Introduction …………………………………. ……………………. 26 Materials and Methods …………………………………………….. 28 Results ……………………………………………………………... 38 Discussion …………………………………………………………. 52 Acknowledgments …………………………………………………. 58 References …………………………………………………………. 60 Figures and Tables ………………………………………………… 64 Chapter 3 Extracellularly identifying motor neurons for a muscle motor pool in Aplysia californica Summary …………………………………………………………... 92 Introduction …………………………………. ……………………. 93 Materials and Methods …………………………………………….. 94 Representative Results …………………………………………….. 96 Discussion …………………………………………………………. 97 Acknowledgments ………………………………………………... 103 References ………………………………………………………... 104 Figures and Tables ……………………………………………….. 107 Chapter 4 Pre-modulation: Neural activity during biting prepares a retractor muscle for force generation during swallowing in Aplysia Summary …………………………………………………………. 115 Introduction ………………………………………………………. 117 Materials and Methods …………………………………………… 118 Results ……………………………………………………………. 129 Discussion …………………………………………………………140 Acknowledgments ………………………………………………... 146 References ………………………………………………………... 147 Figures and Tables ……………………………………………….. 151 Chapter 5 Summary and future work Results summary …………………………………………………. 164 Limitations of this study …………………………………………. 166 Implications of this study ………………………………………… 170 Future work ………………………………………………………. 173 References …………………………………………………………177 Appendix A ...…………………………………………………………………181 B …………………………………………………………………...187 C …………………………………………………………………...203 Bibliography …………………………………………………………… 206 List of Figures Chapter 1 Figure 1-1: A schematic diagram for extrinsic and intrinsic neuromodulation ………………………………………21 Figure 1-2: A schematic diagram for the anatomy of the buccal mass of Aplysia ……………………………………………...22 Figure 1-3: A schematic diagram for muscle innervation and intrinsic and extrinsic modulation of I1/I3 ……………………..23 Figure 1-4: Schematic diagrams for intrinsic modulatory effects from FMRFamide and SCP on the contraction force amplitudes of I1/I3 ………………………………………………...24 Chapter 2 Figure 2-1: Schematic geometry of stimulating and recording electrodes in the in vitro experiments using Aplysia buccal ganglia ………………………………………...64 Figure 2-2: The experimental setup for measurements of the resistivity of Aplysia saline …………………………...65 Figure 2-3: Morphology and equivalent electrical circuit of the Aplysia NEURON model ……………………………..66 Figure 2-4: The polarization of the axon hillock varies as the extracellular stimulating electrode is placed at different positions ……………………………………………….67 Figure 2-5: The polarization along the neuron by extracellular ganglionic stimulation ………………………………...69 Figure 2-6: Qualitative comparisons between experimental results and the NEURON model’s predictions: anodic currents activate a neuron by extracellular stimulation on the side of the soma opposite to the axon …………………….70 Figure 2-7: Qualitative comparisons between experimental results and the NEURON model’s predictions: cathodic currents inhibit the neuron by extracellular stimulation near the cell bodies ……………………………………………71 Figure 2-8: Threshold currents for both anodic activation and cathodic inhibition increase with the distance from the stimulating electrode to the soma …………………….72 Figure 2-9: Threshold currents of the same neuron were very close when the sheath was intact and after the sheath was removed ………………………………………………73 Figure 2-10: Anodic currents can selectively activate an individual neuron ………………………………………………..74 Figure 2-11: Cathodic currents can selectively inhibit an individual neuron ………………………………………………..76 Figure 2-12: The spatial specificity for anodic activation predicted by the multiple-cell NEURON model …………………...77 Figure 2-13: The spatial specificity for cathodic inhibition predicted by the multiple-cell NEURON model ………………..78 Figure 2-14: The spatial specificity for a group of neurons with different sizes and geometric configurations predicted by the NEURON model and the analytical model ………79 Figure 2-15: The temporal specificity for anodic activation of an individual neuron demonstrated experimentally ……..81 Figure 2-16: The temporal specificity for cathodic inhibition of an individual neuron demonstrated experimentally ……..83 Figure 2-17: Comparisons of the membrane polarization along a simulated neuron with different soma and axon diameters ……………………………………………..85 Figure 2-18: Comparisons of the membrane polarization along the simulated vertebrate neuron with different neuronal structures ……………………………………………..87 Table 2-1: The geometric parameters of the NEURON model for an Aplysia buccal neuron ………………………………..89 Table 2-2: The electrical parameters of the NEURON model for an Aplysia buccal neuron …………………………………90 Table 2-3: The electrical parameters of the NEURON model for an Aplysia buccal neuron …………………………………91 Chapter 3 Figure 3-1: Schematic of overall setup and the dish for the force studies ………………………………………………..107 Figure 3-2: Schematic of the buccal ganglia and electrodes setup …………………………………………………108 Figure 3-3: A picture and schematic of the neuron map for extracellular identification of the I1/I3 motor neurons in the Aplysia buccal ganglion ………………………….109 Figure 3-4: Identifying and characterizing the I1/I3 motor neuron B3 ……………………………………………………111 Figure 3-5: Identifying and characterizing the I1/I3 motor neuron B43 …………………………………………………..112 Figure 3-6: The optimized diagnostic tree for identifying some of the I1/I3 motor neurons using extracellular soma and nerve recordings ……………………………………………113 Figure 3-7: Comparison of success rates of neuron identification during force experiments using either the extracellular technique or the intracellular technique ……………..114 Chapter 4 Figure 4-1: Characterizing the BN2 motor programs during the retraction phase of biting and swallowing …………...151 Figure 4-2: B6, B9 and B3 are more active in swallowing than in biting …………………………………………………153 Figure 4-3: The activity of B6, B9 and B3 is correlated with the overall retraction force in swallowing ……………….155 Figure 4-4: The force/frequency relationships of B6, B9, and B3 in vitro ………………………………………………….157 Figure 4-5: The I1/I3 muscle forces evoked by B6, B9 and B3 at their physiological activity levels are small compared to the overall retraction force exerted during swallowing responses …………………………………………….158 Figure 4-6: B6, B9 and B3 generate no force in biting even after self- modulation, but pre-modulate I1/I3 and prepare it to generate larger forces during the initial swallow ……159 Figure 4-7: Intrinsic modulation from B6, B9 and B3 in swallowing …………………………………………...160 Figure 4-8: The nonlinear summation of the I1/I3 muscle forces evoked by B6, B9 and B3 ……………………………161 Figure 4-9: The I1/I3 muscle forces generated during ingestive-like patterns ………………………………………………162 Figure 4-10: Summary schematics for results ……………………..163 Acknowledgements I would like thank to my advisor Dr. Hillel Chiel for his unwavering faith, wise advice, and enormous patience and support. I would like to thank the other members of my committee, Dr. Roy Ritzmann, Dr. Jessica Fox and Dr. Kenneth Gustafson for their helpful comments. I would also like to thank my lab colleagues: Jeffrey McManus, Miranda Cullins, Cindy Chestek, Kendrick Shaw, Catherine Kehl and Jeff Gill for their help during different stages of my Ph.D. work. I would also like to express my special appreciation to my husband, Qing Ran, and parents, who supported me and encouraged me to strive towards my goal. This work was supported by NIH grant NS047073, EB004018 and T32 GM007250 as well as NSF grant DMS1010434 and IIS1065489. PRE-MODULATION: NEURAL ACTIVITY DURING BITING PREPARES A RETRACTOR MUSCLE FOR FORCE GENERATION DURING SWALLOWING IN APLYSIA Abstract by HUI LU The ability to rapidly modify behaviors to meet changing environmental demands is essential to both vertebrates and invertebrates. This flexibility can be achieved by coordinated neuromodulation at different levels (Katz, 1995), which changes the excitability of motor neurons (Trimmer 1994; Schotland et al., 1995) and the responsiveness of a muscle to the same neural activation (Cropper et al., 1994; Brezina et al., 1994). Although neuromodulation has been extensively studied in many systems, less is known about the role