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Exploring Local Neuronal Circuitry with Controlled Iontophoresis

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Exploring Local Neuronal Circuitry with Controlled Iontophoresis EXPLORING LOCAL NEURONAL CIRCUITRY WITH CONTROLLED IONTOPHORESIS Anna Marie Belle A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry Chapel Hill 2013 Approved by: Mark Wightman James Jorgenson Royce Murray Regina Carelli Dorothy Erie © 2013 Anna Marie Belle ALL RIGHTS RESERVED ii ABSTRACT Anna Marie Belle: EXPLORING LOCAL NEURONAL CIRCUITRY WITH CONTROLLED IONTOPHORESIS (Under the direction of R. Mark Wightman) Understanding how neurochemical messengers propagate neuronal signals and ensure delivery of nutrients to fuel this process is the first step to proper treatment and prevention of disorders involving neurological dysregulation. Here, the technique of controlled iontophoresis is coupled to electrochemical detection of dopamine, serotonin, and molecular oxygen (O2) and concurrent monitoring of cell firing to begin to probe the mechanics of local neural circuits. Iontophoresis is a drug delivery technique where application of current causes charged molecules to migrate through a glass capillary. In controlled iontophoresis these capillaries are coupled to a carbon-fiber microelectrode and ejections are monitored electrochemically. The ability to control and modify iontophoretic ejections in real-time makes controlled iontophoresis a significant advancement over previous iterations of iontophoresis as explained and demonstrated herein. Use of this technique first established that we can in fact selectively modulate electrically evoked dopamine release in the striatum of anesthetized rats with local application of an autoreceptor antagonist. Then, the technique was used to examine functional hyperemia, the link between cerebral blood flow and neurochemical release, by monitoring local changes in O2. Direct glutamate application, known to cause vasodilation, increased local O2 concentration linking these O2 changes to blood flow. Additionally, with controlled iontophoresis the relative concentrations of glutamate applied could be compared. This led to the discovery that application of high concentrations of glutamate induced ionic iii oscillations that are attributed to calcium. Next, the role of serotonin in functional hyperemia is examined in two regions with different serotonergic topography. Unlike glutamate, serotonin application is shown have a much more complex role in functional hyperemia, inducing increases, decreases, and no change in O2. Finally, in order to really understand neuronal signaling, it must be given a context. This work concludes with collection of concurrent electrochemical/electrophysiological data while controlled iontophoresis is used to selectively modulate dopamine release and cell firing in freely-moving animals engaged in behavioral tasks. Preliminary application of this technique has confirmed the existence of subpopulations of medium spiny neurons in the nucleus accumbens and shown that each of these subpopulations plays a unique role in intracranial self-stimulation. iv ACKNOWLEDGEMENTS The work presented herein was directly and indirectly influenced and its author nurtured by many people over the years. First, to my advisor, Mark Wightman, your guidance, support, and enthusiasm for learning has shaped me into the scientist I am now and for that I am truly grateful. You have provided a chemistry laboratory full of endless opportunities limited only by my own creativity and need for sleep. To Dr. Natalie Rios Herr, thank you for teaching me the ways of the Wightman Lab as a young first year and for your own work on controlled iontophoresis, without which this dissertation would not be possible. To Dr. Catarina Owesson-White, thank you for confronting the seemingly impossible project of iontophoresis in freely-moving animals with me and giving me a Zumba buddy. Our efforts are presented in Chapters 5 and 6. To Kevin Wood, you were a fantastic undergraduate researcher and thank you for collecting the serotonin data in Chapter 4. To Preethi Gowrishankar, thank you so much for all your help over the past 3 years, without your help there is no way I could have collected the data in Chapters 3 and 4. You continually went above and beyond as an undergraduate student! Finally, to former and current Wightmanites thank you so much for all of the encouragement, help, friendship and fun both during and after work. There are also several people who have fostered my love of science in general and chemistry in particular over the years. To my extended family, thank you for all of the gifts and holiday projects, it’s amazing the influence that Childcraft Encyclopedias and a crystal radio can have on a child. I will always remain gratefully to my high school chemistry v teacher, Marilyn Theisen. While there’s a chance that I would still love chemistry without her instruction, I am not willing to risk it. Thanks also to Dr. Kevin Chambliss and Dr. Jason Belden, for their support and mentorship during my undergraduate research. Finally, I will be forever gratefully to Kevin Chambliss, not only for mentorship over the years but for suggesting I look into ‘Chapel Hill’ for graduate school. There have been a number of folks near and far who have helped me through this experience as well. To Natalie, Kristi and Kristen, thanks for the free therapy and laughs over the years. And, to Michael, my friend-for-life, thanks for making this whole experience a bit more fun. Finally, thanks to my family for all of your support over the years. Justin thanks for being my first collaborator and the best brother ever. My gratitude and love also goes to my parents Joseph and Deborah Belle who are mentioned last simply because they should be mentioned in every paragraph above! vi TABLE OF CONTENTS List of Tables ...................................................................................................................... xiii List of Figures.....................................................................................................................xiii i List of Abbreviations and Symbols ..................................................................................... xvv Chapter 1. Monitoring and modulating dopamine release and unit activity in real-time ........................................................................................................................... 1 Introduction ....................................................................................................................... 1 Dopamine Neurotransmission ........................................................................................... 2 Evolution of a Waveform ................................................................................................... 5 Seeing The Brain Communicate: A combination of measurement procedures ....................................................................................................................... 8 Combining electrochemistry with electrophysiology ....................................................... 9 Controlled iontophoresis ...............................................................................................14 Conclusions .....................................................................................................................17 Dissertation Overview ......................................................................................................18 References ......................................................................................................................19 2. Iontophoresis for quantitative modulation of the D2 autoreceptor ....................................27 Introduction ......................................................................................................................27 Drug receptor theory ....................................................................................................27 Drug delivery methods .................................................................................................29 Iontophoresis ...............................................................................................................31 General overview .........................................................................................................37 vii Materials & Methods ........................................................................................................39 Chemicals ....................................................................................................................39 Construction of iontophoresis probes ...........................................................................39 Electrochemistry ...........................................................................................................42 Iontophoresis ejections .................................................................................................42 Anesthetized rat surgery ..............................................................................................42 Calibration procedure ...................................................................................................43 Capillary electrophoresis ..............................................................................................43 In vivo protocol .............................................................................................................44 Construction of dose response curves .........................................................................45
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