CHOLINERGIC INTERNEURON MEDIATED ACTIVATION OF G- PROTEIN COUPLED RECEPTORS IN THE DORSAL STRIATUM by APHRODITI A. MAMALIGAS Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Neurosciences CASE WESTERN RESERVE UNIVERSITY August, 2018 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Aphroditi A. Mamaligas Candidate for the degree of Doctor of Philosophy.* Thesis advisor: Christopher Ford, PhD Committee Chair: David Friel, PhD Committee Member: Lynn Landmesser, PhD Committee Member: Evan Deneris, PhD Date of Defense: May 23, 2018 *We also certify that written approval has been obtained for any proprietary material contained therein. 2 TABLE OF CONTENTS List of figures..........................................................................................................6 Acknowledgements................................................................................................8 List of abbreviations................................................................................................9 Abstract................................................................................................................12 Chapter 1..........................................................................................14 Introduction.............................................................................................15 Striatal microcircuitry.................................................................................16 Striatal cholinergic interneuron activity and role in disease........................19 ChI anatomy and distribution across the striatum.......................................21 Striatal ACh release and signaling.............................................................22 Modulation of striatal inputs and interneurons.................................23 Cholinergic regulation of MSN muscarinic receptors.......................27 GPCR synapses and volume transmission in the central nervous system.29 Rationale...................................................................................................31 Figures and figure legends........................................................................33 3 Chapter 2..........................................................................................35 Nicotinic and opioid receptor regulation of striatal dopamine D2- receptor mediated transmission............................................................35 Abstract.....................................................................................................36 Introduction...............................................................................................37 Experimental procedures...........................................................................39 Results......................................................................................................42 Discussion.................................................................................................49 Figures and figure legends........................................................................55 Chapter 3..........................................................................................69 Spontaneous synaptic activation of muscarinic receptors by striatal cholinergic interneuron firing.................................................................69 Abstract.....................................................................................................70 Introduction...............................................................................................71 Experimental procedures...........................................................................73 Results......................................................................................................78 Discussion.................................................................................................94 Figures and figure legends......................................................................100 4 Chapter 4........................................................................................130 Cortical and thalamic inputs evoke cholinergic transmission in the striatum..................................................................................................130 Abstract...................................................................................................131 Introduction.............................................................................................132 Experimental procedures.........................................................................134 Results....................................................................................................138 Discussion...............................................................................................150 Figures and figure legends......................................................................153 Chapter 5........................................................................................170 Discussion...............................................................................................171 Synaptic point-to-point transmission at M4 synapses..............................173 Unlaminated striatal structure and implications for ChI connectivity.........174 Future directions......................................................................................175 References.....................................................................................181 5 LIST OF FIGURES • Chapter 1 o Figure 1.1……………………………………………………………….33 • Chapter 2 o Figure 2.1…………………………………………….…………………55 o Figure 2.2……………………………………………………………….58 o Figure 2.3……………………………………………………………….61 o Figure 2.4……………………………………………………………….63 o Figure 2.5……………………………………………………………….65 o Figure 2.6……………………………………………………………….67 • Chapter 3 o Figure 3.1…………………………………………………………….100 o Figure 3.2…………………………………………………………….103 o Figure 3.3…………………………………………………………….105 o Figure 3.4…………………………………………………………….108 o Figure 3.5…………………………………………………………….110 o Figure 3.6…………………………………………………………….113 o Figure 3.7…………………………………………………………….115 o Figure 3.8…………………………………………………………….117 o Figure 3.9…………………………………………………………….119 6 o Figure 3.10……………………………………………………………121 o Figure 3.11……………………………………………………………123 o Figure 3.12……………………………………………………………125 o Figure 3.13……………………………………………………………127 • Chapter 4 o Figure 4.1……………………………………………………………..153 o Figure 4.2……………………………………………………………..156 o Figure 4.3……………………………………………………………..159 o Figure 4.4……………………………………………………………..161 o Figure 4.5……………………………………………………………..164 o Figure 4.6……………………………………………………………..167 7 ACKNOWLEDGEMENTS I would like to thank my mentor Chris Ford and the members of the Ford lab for their help, support, and contributions to my development as a scientist. I thank my thesis committee for their critiques, questions, and helpful suggestions at committee meetings. I thank Suhanti Banerjee, Kathy Lobur, and Michael Grybko for their assistance with animal husbandry. I thank Yuan Cai and Michael Grybko for assistance with recording experiments. I thank Sarah Zych for help with fluorescence imaging. I thank Ben Strowbridge for custom code used for the 2-photon microscope. I thank the CWRU Department of Neurosciences and the Department of Physiology and Biophysics, as well as the University of Colorado Denver Department of Pharmacology for their support and feedback during my graduate training. Finally, my thesis work was supported by NINDS R01 NS95809 and NIDA R01 DA35821 (to CPF). 8 LIST OF ABBREVIATIONS AAV – adeno associated virus ACh – acetylcholine AChE – acetylcholinesterase aCSF – artificial cerebrospinal fluid AP – action potential ChAT – choline acetyltransferase ChI – cholinergic interneuron ChR2 – channelrhodopsin-2 CNS – central nervous system CV – coefficient of variation DAT – dopamine transporter dMSN – direct-pathway medium spiny neuron EPSC – excitatory postsynaptic current EPSP – excitatory postsynaptic potential FSI – fast spiking interneuron GIRK2 (Kir 3.2) – G-protein coupled inwardly rectifying potassium channel GPCR – G-protein coupled receptor GPe – globus pallidus (external) GPi – globus pallidus (internal) HCN – hyperpolarization-activated cyclic nucleotide-gated channel iMSN – indirect-pathway medium spiny neuron 9 IPSC – inhibitory postsynaptic current IP3 – inositol trisphosphate Kir 2 – inward rectifier potassium channel 2 KOR – kappa opioid receptor LTD – long term depression LTP – long term potentiation LTSI – low threshold spiking interneuron LRRK2 – leucine-rich repeat kinase 2 mAChR – muscarinic acetylcholine receptor MOR – mu opioid receptor MSN – medium spiny neuron NAc – nucleus accumbens nAChR – nicotinic acetylcholine receptor PD – Parkinson’s disease Pf – parafascicular nucleus of the thalamus PIP2 – phosphatidylinositol 4,5-bisphosphate PKA – protein kinase A PKC – protein kinase C PLC – phospholipase C PPR – paired pulse ratio Pr – probability of release sEPSC – spontaneous excitatory postsynaptic current 10 sIPSC – spontaneous inhibitory postsynaptic current SK – small conductance Ca2+-activated potassium channel SNc – substantia nigra pars compacta SNr – substantia nigra pars reticulata TAN – tonically active neuron TTX - tetrodotoxin uIPSC – unitary inhibitory postsynaptic current VAChT – vesicular acetylcholine transporter VTA – ventral tegmental area 2-AG – 2-arachinonoylglycerol 11 Cholinergic interneuron mediated activation of G-protein coupled receptors in the dorsal striatum