Optical characterization of dopamine release in the globus pallidus and striatum Jozsef Meszaros Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2017 c 2017 Jozsef Meszaros All rights reserved Abstract Optical characterization of dopamine release in the globus pallidus and striatum Jozsef Meszaros The measurement of dopamine neurotransmission in the brain has evolved alongside techniques for measuring neuronal activity. This evolution has progressed from coarse phys- ical inspection (using lesions or dialysis approaches) to measuring electrical signatures of the phenomena of interest. Most recently, an optical revolution has taken hold within the neurosciences. We present an optical dopamine measurement technique as a companion to burgeoning neural activity monitors such as GCaMP. The electrical consequences of indi- vidual glutamate molecules impinging upon a postsynaptic membrane can be captured in an electrophysiological trace. On the other hand, dopamine has no consistent, measureable postsynaptic effects and therefore cannot easily be measured electrophysiologically. Re- searchers have instead used the electrochemical features of dopamine to measure samples of it in physical space. This approach, termed cyclic voltammetry, has generated nearly all of the existing knowledge about the precise characteristics of dopamine release. While a reliable method for measuring dopamine release from heavily innervated areas, cyclic voltammetry lacks the resolving power to establish release from dopamine terminals in other areas of interest. One such area is the external globus pallidus (GPe), the focus of this work. Re- search performed nearly thirty years prior to this thesis established the presence of sparsely distributed dopamine varicosities within the GPe. Here, we leverage FFN102, a newly de- veloped optical method used as a proxy for dopamine release, to measure dopamine release in this area. Based on previous literature showing that dopamine varicosities were present in the GPe and that dopamine receptors exist on principal cells in the area, we hypothesized that these dopamine varicosities were capable of releasing FFN102. Moreover, previous work had shown that anatomically, the most prominent dopaminergic innervation to the GPe came from the substantia nigra. By validating the FFN102 method in the GPe, we showed that a substance is released which likely reflects dopamine vesicle release. Moreover, the use of two dopamine depletion mouse models allowed us to conclude that FFN102 is released exclusively from dopamine terminals. Finally, we advanced the understanding of dopamine release in the area by examining whether pharmacological manipulation could alter the amount of FFN102 released from dopamine terminals. Table of Contents List of Figures iii Acknowledgements v Preface vii 1 Introduction 1 1.1 Detecting dopamine: from anatomy to dynamics . 1 1.1.1 Visualizing dopamine . 2 1.1.2 Electrochemical detection of dopamine oxidation . 5 1.1.3 Direct capture of dopamine . 9 1.1.4 Fluorescent false neurotransmitters . 11 1.1.5 Calibration . 15 1.2 Dopamine within the brain . 18 1.2.1 Dopamine release in areas of high and low innervation . 18 1.2.2 Determinants of dopamine release . 23 2 Using Fluorescent False Neurotransmitters to Characterize Exocytosis from Dopamine Synaptic Vesicles within the GPe 27 2.1 Significance of developing a novel method for measuring dopamine vesicle release . 28 2.2 Materials and Methods . 29 2.2.1 6-OHDA lesion . 30 i List of Figures 1.1 The condensation reaction of Falck and Hilarp (I and II), with the final protein-catalyzed dehydration product discovered by Corrodi (III). Adapted from Corrodi and Jonsson(1967).........................3 1.2 Chambers used to vaporize glyoxylic acid (left) and treat slices (right). .4 1.3 A basic circuit for in vivo electrochemistry. .6 1.4 Electrochemistry of dopamine. .7 1.5 A theoretical voltammogram. .8 1.6 Electrochemistry of two catecholamines. .8 1.7 Innervation of muscle fiber endplates by two nerves (red and green) as shown using a fluorescent endocytic dye. 12 1.8 Demonstration of destaining in frog pectoris motor nerve terminals after elec- trical stimulation. 13 1.9 FFN102 molecule. 14 1.10 Correlating CV currents with gCamp activity. 17 1.11 Falck-Hilarp method used to visualize dopamine within the external globus pallidus . 19 1.12 Signal processing techniques to enhance CV signals . 20 1.13 Inferred dopamine release per pulse in vivo ................... 22 1.14 Dopamine release in the striatum and nucleus accumbens differ in their fre- quency dependence . 23 1.15 Cholinergic modulation of dopamine release, with and without nicotine . 25 iii 1.16 Depiction of long lasting dopamine D2 receptor inhibition . 26 2.1 Electrical stimulation of GPe-containing brain slices evokes FFN102 release . 43 2.2 FFN102 is necessary for recording electrically-evoked fluorescence transients 44 2.3 Colocalization between FFN and a marker for DAT cells . 46 2.4 Calcium dependence of evoked peaks . 48 2.5 FFN102 transients from dopamine depleted mice . 49 2.6 FFN transients in striatum and GPe . 51 2.7 Pharmacological modulation of FFN transients in the GPe . 53 iv Acknowledgments Thank you to my mentors David Sulzer and Christoph Kellendonk. Thank you Dave for always challenging the conventional understanding, for emphasizing my talents and enduring my shortcomings, for listening, and for putting in the long hours needed to sustain the laboratory (and my PhD). Thank you Christoph for your engagement, for your respect, and for the opportunity to work among the group of pleasant and successful scientists that you have assembled in your laboratory. Both of you have a breadth of knowledge that is expansive and non-overlapping. I hope that this collaboration has been a source of learning and an opportunity for building a relationship that lives long beyond this dissertation. Thank you to the members of my thesis committee, Ai Yamamoto and Ethan Bromberg- Martin, whose diversity of interests inspired me and who each served as models of inquisitive, thoughtful scientists. I hope I can adopt your approaches to respectful and attentive ques- tioning. Additionally, I am grateful to you Ai for being so open in your conversations about life as a scientist. Ethan, your willingness to meet and discuss approaches to analyzing data kept me engaged and interested. I was always enthused when I found out I was doing something the way you would have! And Nic Tritsch, thank you for your excitement about my topic and for leading a very stimulating and enjoyable dissertation examination. Thank you to my many, many intellectual influences. The teachers who showed me that thinking is not a chore, but a reward: Mr. Morgan (math), Mr. Ayotte (chemistry), and Ms. Farrell (physics). The professors who gave me the opportunities for extensive trial and error learning without shame or embarrassment: Dr. Hamilton (astronomy, UMD), Dr. Losert (physics, UMD), and Dr. Balice-Gordon (neuroscience, UPenn, now Pfizer). To Misha Isaak, formerly my legal writing instructor, currently serving as Deputy General Counsel to the Governor of Oregon, whose incisive and occasionally mordant critiques of my v work prepared me for the evaluations I would receive in graduate school. To Sue Osthoff, who gave me the space to explore many unconventional and half-baked ideas about how neurobiology could fit within the context of law and society. Through her example, Sue taught me about integrity, fairness, dialogue, and living authentically { knowledge sufficient to fill out many PhD theses. To previous graduate students, Josh Merel, David Pfau, and others, who I often turned to for advice and always looked to for inspiration. Thank you to my students. For better or worse, I have had the opportunity to stand before rooms of dozens of attentive, capable individuals and perform teaching as I under- stood it. This is of course an evolving understanding, and so my gratitude goes out to the students who tolerated my mistakes, adapted to occasional changes of course, and showed me what real effort looks like. Thank you especially to my friends, for never-ending opportunities to develop, exchange, learn, nurture and be nurtured, listen, give and receive, and all of the many things that comprise living. Pia and sisters, Chris, Conor, James, Laura, Luke, Sadie. This only exists because of you all! vi Preface "[W]hen a muscle contracts, when a gland secretes, or a nerve ending is excited, the cause in each case may be due to the liberation of some chemical substance, not necessarily set free in the circulation as in the case of secretin, but more likely liberated at the spot upon which it is to act." | Walter Ernest Dixon 1907 The Greek physician Hippocrates (c. 460 c. 370 BC) is broadly credited with the theory that bodily fluids could determine an individual's activity, disposition, and behavior (Sykiotis et al., 2005). Yet, the import of this theory, and any rigorous testing of it, had to wait until the 18th century. The experiments of Galvani, who used electricity to induce movement in a frog leg, reified the notion of 'animal electricity'. In 1917, George Howard Parker and Anne P. Van Heusen made the curious observation that the catfish amiurus nebulosus would move away from electrically charged rods in their vicinity, even when the rods were not in contact with the water (Parker and Van Heusen, 1917). As physiologists came to regard this exotic force as something of biological relevance, they struggled to understand how a nerve cell could convey its "action-current" to neigh- boring cells (Lillie, 1919). The idea that chemicals could bridge certain spaces came first for the neuromuscular junction, a conjecture of the German physician Dubois-Reymond, made in 1877 (Eccles, 1976). Without much evidence, T.R. Elliott, a colleague of Sidney Ringer, speculated in 1904, that sympathetic nerve endings might act by releasing adrenaline (Elliott, 1914).