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Voltage Dependence and Activation Mechanisms of The ii VOLTAGE DEPENDENCE AND ACTIVATION MECHANISMS OF THE NEUROMODULATORY INWARD CURRENT (IMI) IN THE STOMATOGASTRIC GANGLION OF C. BOREALIS By MICHAEL LESLIE GRAY A dissertation submitted to the Graduate School-New Brunswick Rutgers, The State University of New Jersey In partial fulfillment of the requirements For the degree of Doctor of Philosophy Graduate Program in Behavioral and Neural Sciences Written under the direction of Jorge P. Golowasch And approved by _____________________________________ _____________________________________ _____________________________________ _____________________________________ _____________________________________ Newark, New Jersey October 2015 iii © Copyright by Michael Gray All rights Reserved ii ABSTRACT OF THE DISSERTATION VOLTAGE DEPENDENCE AND ACTIVATION MECHANISMS OF THE NEUROMODULATORY INWARD CURRENT (IMI) IN THE STOMATOGASTRIC GANGLION OF C. BOREALIS By Michael Gray Dissertation Director: Jorge Golowasch The Neuromodulatory Inward Current (IMI) of the stomatogastric ganglion (STG) in Cancer borealis is an inward difference current elicited by neuromodulators whose current voltage relation (IV curve) has been shown to be crucial to transitioning the pyloric network of this system from non-oscillating to oscillating states. Here, models of the second messenger signaling of both this current’s activation and voltage dependence are constructed and tested. First, we examine how the proctolin receptor activates IMI with the hypothesis that it signals through an intermediate signaling component. This is tested by bath application and pressure injection of agonists and antagonists of second messenger systems and examining their effect on neuromodulator induced IMI. Consistent with this hypothesis, it is found that this proctolin-induced IMI is sensitive to modulators of G-protein function. Also consistent with this hypothesis, it is found that proctolin-induced IMI is sensitive to modulators of intracellular calcium concentration and calmodulin activated kinases. In contrast, there ii iii seems to be no effect to acute applications of pharmaceutical activators and inhibitors of cyclic nucleotides, Phospholipase C, and receptor tyrosine kinase signaling. Next, we examine the mechanism of IMI voltage dependence that has been shown to be dependent upon extracellular calcium level and calmodulin signaling which we show is necessary for IMI voltage dependence by finding that application of calmodulin antagonists in normal calcium reduces IMI voltage dependence. However, it is found that calmodulin activators do not rescue IMI voltage dependence in low calcium so we propose that a Calcium Sensitive Receptor (CaSR) maintains IMI voltage dependence by active detection of extracellular calcium. This hypothesis is supported by several lines of evidence. First, IMI slope was increased by the specific calcium sensitive receptor antagonist NPS-2143 suggesting CaSR involvement. Second, IMI slope conductance was increased by exposure to the βγ-subunit inhibitor, gallein, suggesting that voltage dependence can be modulated by the βγ-subunit of trimeric g-proteins. Third, W7 induced linearization of IMI was reduced by pre-incubation of preparations with endocytosis inhibitors, suggesting that downregulation of a receptor is mediating W7 induced changes in IMI voltage dependence. Together, these results support the hypothesis that IMI voltage dependence is mediated at least in part by a CaSR-like G- protein coupled receptor (GPCR). iii iv Table of Contents: Abstract………………………………………………………………………………………………………………………..…..…ii Table of Contents…………………………………………………………………………………………………………….….iv Table of Figures………………………………………………………………………………………………….…………….…vi Chapter 1: Introduction ........................................................................................................... 1 1.1 Introduction and Importance .................................................................................... 1 1.2 IMI and the Stomatogastric Nervous System (STNS): ................................................. 6 1.3 Receptors and Downstream Second Messenger Pathways of Proctolin and CCAP in Crustaceans and Invertebrates ...................................................................................... 18 1.4 Voltage Dependence Mechanisms of Currents Similar to IMI in Other Systems ...... 29 1.5 The Calcium Sensitive Receptor (CaSR) and the sodium Leak Current (NaLCN) ..... 37 1.6 Thesis Statement: .................................................................................................... 43 Chapter 2: Methods ............................................................................................................... 46 2.1 Introduction: ............................................................................................................ 46 2.2 General methods ..................................................................................................... 47 2.3 Measurement of IMI ................................................................................................. 53 2.4 Properties of IMI that Affect its Measurement. ........................................................ 57 2.5 Quantification of IMI ................................................................................................. 59 2.6 IMI Stability................................................................................................................ 67 2.7 Problems with Measurement in Low Calcium are Mitigated by Use of Bovine Serum Albumin (BSA). ............................................................................................................... 74 2.8 Discussion ................................................................................................................ 80 Chapter 3: IMI Activation ........................................................................................................ 82 3.1 Introduction ............................................................................................................. 82 3.2: G-protein Coupled Receptors are Necessary for Proctolin-induced IMI.................. 84 3.3 Role of Cyclic Nucleotides in Proctolin-induced IMI Activation. ............................... 97 3.4: Phospholipase C Signaling .................................................................................... 116 3.5 Other Phosphatases and Kinases. .......................................................................... 123 3.6: Calmodulin, and Calmodulin-Activated Proteins .................................................. 130 iv v 3.7: Discussion ............................................................................................................. 141 Appendix A: Effects for G-protein modulators ............................................................ 154 Appendix B: Effects for Cyclic Nucleotide Agents ........................................................ 169 Appendix C: Effects of PLC Pharmaceutical Agents ..................................................... 176 Appendix D: Effects of phosphatase and tyrosine kinase inhibitors ........................... 185 Appendix E: Effects of calmodulin (Cam) and Cam-activated inhibitors and activators.193 Chapter 4: IMI Voltage Dependence ..................................................................................... 203 4.1 Introduction ........................................................................................................... 203 4.2 Divalents Block Proctolin-induced IMI Uniformly and Do not Restore Proctolin-induced IMI Voltage Dependence. .............................................................................................. 205 4.3 Calmodulin inhibitors Reduce IMI voltage dependence ......................................... 208 4.4 Calmodulin Activators do Not Restore IMI Voltage Dependence in Low Calcium .. 222 4.5 Other Tested Agents that Had No Effect on Proctolin-Induced IMI Slope. ............. 231 As discussed in Chapter 3, we had accumulated substantial data for different second messenger pathways and wished to determine if there were interactions between these pathways and proctolin-induced IMI voltage dependence. As these experiments were originally designed to test activation and not voltage dependence, as discussed in Chapter 3, this data must be caveated as exploratory rather than confirmatory (Payne and Dyer, 1975; Wagenmakers et al., 2012). ............................................................................... 231 4.6 Calmodulin Activated Proteins. ............................................................................. 241 4.6 Calcium Sensing Receptor: A New Hypothesis to Explain Proctolin-induced IMI Voltage Dependence. ................................................................................................................ 248 Appendix F: Other Calcium/calmodulin activators that failed to restore proctolin-induced IMI voltage dependence in low calcium. ........................................................................... 270 Appendix G: Calmodulin Inhibitors .............................................................................. 272 Chapter 5: Discussion ........................................................................................................... 288 5.1. Activation .............................................................................................................. 288 5.2: Voltage dependence: ............................................................................................ 293
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