Interactions between Brain-Derived Neurotrophic Factor and Endocannabinoids at Neocortical Inhibitory Synapses Liangfang Zhao, Ph.D. University of Connecticut, 2014 Endogenous cannabinoids (endocannabinoids; eCBs) play important roles in synaptic transmission. Known as retrograde messengers, eCBs are released on demand from postsynaptic sites and suppress presynaptic neurotransmitter release in several brain regions where the primary receptor, the type 1 cannabinoid receptor (CB1R) is expressed. Similarly, neurotrophins, particularly brain-derived neurotrophic factor (BDNF), act as potent modulators of synaptic transmission in many brain regions, most notably the neocortex and hippocampus. The effect of BDNF in synaptic transmission is mainly mediated by activation of tropomyosin receptor kinase B (trkB) receptors. There is growing evidence for cross-talk between BDNF and eCB signaling. Recently it has been shown that BDNF can induce eCB release at cortical inhibitory synapses, but little is known about the underlying signaling pathways or functional relevance of this interaction. In the present studies, we examined the intracellular signaling pathways that underlie BDNF-induced eCB release, as well as their interactions in regulating activity-dependent long-term depression at Liangfang Zhao – University of Connecticut, 2014 inhibitory synapses (iLTD). Using pharmacological approaches and whole-cell patch clamp recordings from layer 2/3 pyramidal neurons in mouse somatosensory cortical slices, we found that PLCγ underlies BDNF-induced eCB release, as it was blocked by PLC inhibitors. Other downstream signaling pathways of trkB, namely protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways, are not involved. This effect is also independent of mGluR activation. Furthermore, we found that theta burst stimulation (TBS) induces a form of eCB-mediated LTD at inhibitory synapses (eCB-iLTD) that is independent of mGluR activation but requires endogenous BDNF as well as downstream PLCγ signaling. Endocannabinoid- mediated iLTD can also be induced by combining a subthreshold concentration of exogenous BDNF with weak TBS that is insufficient to induce iLTD alone. Taken together, these results identified the signaling pathway underlying BDNF- induced eCB release, and identified a novel form of eCB-iLTD that requires endogenous BDNF signaling. Our studies may contribute to the understanding of functional interactions between BDNF and eCB signaling, as well as provide insights in understanding activity-dependent modulation of cortical circuits. Interactions between Brain-Derived Neurotrophic Factor and Endocannabinoids at Neocortical Inhibitory Synapses Liangfang Zhao B.S., Capital Normal University, Beijing, China, 2009 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2014 Copyright by Liangfang Zhao 2014 APPROVAL PAGE Doctor of Philosophy Dissertation Interactions between Brain-Derived Neurotrophic Factor and Endocannabinoids at Neocortical Inhibitory Synapses Presented by Liangfang Zhao, B.S. Major Advisor __________________________________________ Eric S. Levine, Ph. D. Associate Advisor ________________________________________ Richard E. Mains, Ph. D. Associate Advisor ________________________________________ Xin-Ming Ma, Ph. D. Associate Advisor ________________________________________ Douglas L. Oliver, Ph. D. University of Connecticut 2014 i Dedications To my beloved grandparents Qiang-Sheng Sun (1930 - ) and Kuang-Yi Liang (1927 - 2014), for everything they have given me. ii Acknowledgements This dissertation could not have been written without the help and guidance of my major advisor, Dr. Eric Levine, who has provided me tremendous inspiration, wisdom and challenge throughout my graduate career. I am forever grateful for his strict but supportive training in the purpose of teaching me to fish, as well as the intellectual freedom and friendship he has provided me. I would also like to thank my thesis advisory committee, Drs. Douglas Oliver, Richard Mains, and Xin-Ming Ma, for their persistent guidance and constructive criticism to make me a better scientist. I also deeply appreciate the past and current members of the Levine Lab: Dr. Lawrence Hsieh, Dr. Fouad Lemtiri-Chlieh, Tiwanna Robinson, James Fink, Kia Bolduc, Dr. Mason Yeh, for their support, companionship and scientific insights. I am also greatly indebted to all my lovely friends, in particular, Xi Bie, Yi- Zhou Zhu, Yi-He Ma, Qi-Yu Liang, Tong Chen, Zi-Yan Zhang, Ying-Hui Zhang, as well as my family, for giving me the emotional support and encouragement any-time when I needed them. Even if there are always significant time differences and distances, they always feel so close to me. Most of all I would like to thank my loving boyfriend Daniel Sheehy, for his undying love and support that are beyond the power of language during this academic endeavor. iii Table of Contents Chapter 1. Introduction, Background, and Rationale ...................................... 1 1.1 Overview .............................................................................................. 1 1.2 The endogenous cannabinoid system ................................................. 2 1.2.1 Cannabinoid receptors ............................................................ 2 1.2.2 Endogenous ligands in the brain and their synthesis .............. 6 1.2.3 Endocannabinoid mobilization and metabolism ...................... 8 1.3 Endocannabinoid-mediated synaptic plasticity .................................. 12 1.3.1 Endocannabinoid-mediated short-term plasticity .................. 12 1.3.2 Endocannabinoid-mediated long-term plasticity .................... 14 1.4 The brain-derived neurotrophic factor (BDNF) system ...................... 18 1.4.1 BDNF, trkB receptor and BDNF-trkB signaling pathways .... 18 1.4.2 Distribution of trkB receptors ................................................. 20 1.4.3 Distribution and release of endogenous BDNF ..................... 21 1.5 Functional role of BDNF in synaptic plasticity .................................... 23 1.5.1 Acute effect of BDNF on synaptic transmission .................... 23 1.5.2 BDNF and long-term synaptic plasticity ................................ 26 1.6 Interactions between BDNF and endocannabinoid systems .............. 28 1.7 Rationale and hypothesis................................................................... 31 Chapter 2. Materials and Methods .................................................................. 35 2.1 Animal handling and slice preparation .............................................. 35 2.2 Electrophysiology .............................................................................. 36 2.3 Chemicals ......................................................................................... 37 2.4 Data analysis ..................................................................................... 38 Chapter 3. BDNF-endocannabinoid interactions at neocortical inhibitory synapses require phospholipase C signaling ................................................ 39 3.1 The effect of BDNF on IPSC requires postsynaptic phospholipase-C signaling ................................................................ 42 3.2 The effect of BDNF is independent of PKC, MAPK and PI3K signaling ............................................................................ 46 3.3 The effect of BDNF is independent of mGluR signaling .................... 49 iv 3.4 Summary and discussion .................................................................. 52 Chapter 4. Role for Endogenous BDNF in Endocannabinoid-mediated Long-Term Depression at Neocortical Inhibitory Synapses ................. 53 4.1 Strong theta frequency burst stimulation induces eCB-dependent iLTD ........................................................................ 55 4.2 iLTD in layer 2/3 of somatosensory cortex is independent of mGluR signaling ................................................................................ 57 4.3 iLTD in layer 2/3 of somatosensory cortex requires endogenous BDNF and phospholipase-C signaling .......................... 59 4.4 Exogenous BDNF facilitates iLTD induction ...................................... 61 4.5 Summary and discussion .................................................................. 65 Chapter 5. Discussion and conclusions ......................................................... 66 5.1 Summary and interpretation of findings ............................................ 66 5.2 Control experiments for pharmacological experiments ..................... 72 5.2.1 Controls for negative drug effects ......................................... 73 5.2.2 Controls for positive drug effects ........................................... 75 5.3 Limitations of the present studies ...................................................... 77 5.4 Functional impact of BDNF-eCB interactions on cortical synaptic plasticity ............................................................................................ 80 5.5 Conclusions and future directions for BDNF-eCB interactions .......... 83 References ........................................................................................................ 87 v Chapter 1 Introduction, Background, and Rationale This chapter is a modified version of a review article in preparation: Zhao L and Levine ES, It takes two to tango: Interactions between