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Impact of Second Messenger Modulation on Activity-Dependent and Basal Properties of Excitatory Synapses Chun Yun Chang Washington University in St

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Impact of Second Messenger Modulation on Activity-Dependent and Basal Properties of Excitatory Synapses Chun Yun Chang Washington University in St Washington University in St. Louis Washington University Open Scholarship All Theses and Dissertations (ETDs) January 2010 Impact of Second Messenger Modulation on Activity-Dependent and Basal Properties of Excitatory Synapses Chun Yun Chang Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/etd Recommended Citation Chang, Chun Yun, "Impact of Second Messenger Modulation on Activity-Dependent and Basal Properties of Excitatory Synapses" (2010). All Theses and Dissertations (ETDs). 59. https://openscholarship.wustl.edu/etd/59 This Dissertation is brought to you for free and open access by Washington University Open Scholarship. It has been accepted for inclusion in All Theses and Dissertations (ETDs) by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. Washington University in St. Louis Division of Biology and Biomedical Science Developmental Biology Dissertation Examination Committee: Steven Mennerick Ph.D., Chairperson Aaron DiAntonio M.D. Ph.D. James Huettner Ph.D. Peter Lukasiewicz Ph.D. Lawrence Salkoff Ph.D. Robert S. Wilkinson Ph.D. Impact of second messenger modulation on activity-dependent and basal properties of excitatory synapses By Chun Yun Chang A dissertation presented to the Graduate School of Art and Science of Washington University in St. Louis in partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2010 Saint Louis, Missouri Abstract of the dissertation Impact of second messenger modulation on changes in activity-dependent and basal properties of excitatory synapses by Chun Yun Chang Doctor of Philosophy in Biology and biomedical Science (Program in Developmental Biology) Washington University in St. Louis, 2010 Associate Professor Steven J. Mennerick, Chairperson Cognitive processing in the central nervous system relies on accurate information propagation; neurotransmission is the fundamental mechanism underlying network information flow. Because network information is coded by the timing and the strength of neuronal activity, synaptic properties that translate neuronal activity into synaptic output profoundly determine the precision of information transfer. Synaptic properties are in turn shaped by changes in network activity to ensure appropriate synaptic output. Activity-dependent adjustment of synaptic properties is often initiated by second messenger signals. Understanding how second messengers sculpt synaptic properties and produce changes in synaptic output is key for elucidating the interplay between network activity and synaptic properties. We studied the effect of second messenger modification on activity-dependent and static properties of rat hippocampal excitatory synapses using electrophysiological and optical approaches. We focused on two second-messenger pathways that potentiate transmission: cAMP and diacyl glycerol (DAG) signals. In parallel, we also compared the effects of manipulating calcium influx, which is known to potentiate synaptic transmission through increasing release probability (Pr). During high frequency stimulation, we found that both cAMP and DAG signals potentiated phasic transmission, as previously characterized. In parallel with increasing phasic transmission, the modulators also enhanced high-frequency associated asynchronous transmission, which emerges late during stimulus trains ii and is relatively long-lasting. However, such parallel potentiation of phasic and asynchronous transmission was not seen in elevated calcium; high calcium preferentially promoted asynchronous transmission. With low frequency stimulation, we found that cAMP and high calcium enhanced synaptic output by potentiating synapses with basally high Pr. Conversely, DAG signals recruited neurotransmission from both high Pr and low Pr terminals, which include presynaptically quiescent synapses. Taken together, these results suggest that second messenger modulation of synapses differentially shapes the static properties of the synapses; second messengers also fine-tune activity-dependent synaptic responses differently from manipulating calcium influx. These results likely have physiological relevance to second messenger-dependent sculpting of temporal and spatial synaptic properties. iii Acknowledgment This work would not be possible without the help of many people. I am greatly indebted to them for their help, guidance, encouragement, and support. Thanks Steve for shepherding me in the meadow of science and for his role model as a mentor; Peter, Aaron, Jim and Bob for their guidance, encouragement and support in formulating this thesis work; Larry Salkoff for the encouragement when I decided to take the transition step; Krista for the insightful inputs on the initial and the following projects; Larry Eisenman for helping and sharing the programming resources; Bill for unselfishly sharing the equipments; Xiaoping for helping the imaging experiments; John for sharing the experimental reagents; Amanda and Ann for maintaining the cultures and doing many behind-the-scene works; Pato and Mariangela for sharing after-work recreation hours; Devon, Gerry and Jenny for helping many areas in the lab; Chuck for the encouragement and for sharing his exploration in many scientific territories as well as his deep thought about professional career; Yuki and Kaz for Tokyo banana. Grateful to those who were, are, and will be walking with me in this journey. Grateful to the One. iv Table of content Abstract ………………………………..………………………………..……………………… ii Acknowledgment ………………………………..………………………………..…………… iv Table of content ………………………………..………………………………..…………….. v List of figures ………………………………..………………………………..………………… vii Chapter 1. Synaptic properties and transmitter release 1.1 Introduction ………………………………………………………………………... 1 1.2 Hippocampal formation and synaptic plasticity ……………………………….. 1 1.3 The synapse and its properties …………………………………………………. 3 1.3.1 Presynaptic compartments and vesicles in hippocampal excitatory Synapses ………………………………………………………………….. 3 1.3.2 Postsynaptic compartment and glutamate receptors …………………. 7 1.3.3 Calcium-triggered transmitter release ………………………………….. 9 1.3.4 The physiological advantages of chemical synapses ………………… 13 1.4 Probability of vesicle release (Pr) ………………………………………………. 14 1.4.1 Calcium and Pr ………………………………..………………………….. 15 1.4.2 Second messenger modulations and Pr ……………………………….. 16 1.4.3 Pr and functional connections …………………………………………… 19 1.5 Summary of objectives ………………………………..…………………………. 20 1.6 References ………………………………..………………………………………. 22 Chapter 2. Dynamic modulation of phasic and asynchronous release in hippocampal excitatory synapses. 2.1 Abstract …………………………………..……………………………………….. 35 2.2 Introduction ………………………………..……………………………………… 36 2.3 Material and Methods ………………………………..………………………….. 37 2.4 Results ………………………………..…………………………………………… 39 2.5 Discussion ………………………………..……………………………………….. 51 2.6 References ………………………………..……………………………………… 55 2.7 Figures ………………………………..…………………………………………… 61 Chapter 3. Rapid activation of presynaptic dormant synapses by phorbol esters. 3.1 Abstract ………………………………..………………………………………….. 72 3.2 Introduction ………………………………..……………………………………… 73 3.3 Material and Methods ………………………………..………………………….. 74 3.4 Results ………………………………..…………………………………………… 77 v 3.5 Discussion ………………………………..………………………………………. 90 3.6 References ………………………………..……………………………………… 95 3.7 Figures ………………………………..…………………………………………… 102 Chapter 4. Summary and future direction 4.1 Asynchronous transmission and beyond ……………………………………… 114 4.2 Presynaptic silent synapses – why they are silent, and why they break the silence ………………………………..………………………………………. 119 4.3 Conclusion ………………………………..………………………………………. 122 4.4 References .………………………………..……………………………………… 123 Appendix A. Dynamic modulation of phasic and asynchronous glutamate release in hippocampal synapses. By Chun Yun Chang and Steven Mennerick B A specific role for Ca2+-dependent adenylyl cyclases in recovery from adaptive presynaptic silencing. By Krista L. Moulder, Xiaoping Jiang, ChunYun Chang, Amanda A. Taylor, Ann M. Benz, Alana C. Conti, Louis J. Muglia, and Steven Mennerick vi List of figures Chapter 2 Figure 1. Repetitive stimulation at 20 Hz resulted in complementary alterations in phasic and asynchronous release. ……………………………………. 61 Figure 2. EGTA-AM preferentially eliminated the asynchronous component. ….. 63 2+ Figure 3. Asynchronous release was sensitive to [Ca ]o. ……………………….. 64 Figure 4. Asynchronous release was not dependent on specific presynaptic Ca2+ channels. …………………………………………………………………… 65 Figure 5. Asynchronous release was insensitive to blocking vesicle supply from newly recycled vesicles. ………………………………………………….. 76 Figure 6. Pharmacological manipulation of Pr potentiated EPSCs but did not affect the proportion of asynchronous release. …………………………. 68 Figure 7. PKC inhibition prevented increases in asynchronous release in PDBu. 70 Chapter 3 Figure 1. Modulators differentially potentiate NMDAR-mediated EPSCs after MK-801 block of synaptic NMDARs. ……………………………………. 102 Figure 2. Modulators equally potentiate AMPAR- and NMDAR-mediated EPSCs. 104 Figure 3. Acute PDBu application does not increase surface NMDARs. ……….. 105 Figure 4. PKC inhibition does not prevent INMDAR recovery by PDBu. ……………. 106 Figure 5. Pr differs at active versus dormant synapses after PDBu potentiation. 107 Figure 6. The initial INMDAR and PDBu-recovered INMDAR have similar NR2B contribution. …………………………………………………………………. 109 Figure 7. PDBu
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