Year: 2016
Type: dissertation
Abstract: The goals of this study were to define the nicotinic acetylcholine receptor (nAChR) subtypes expressed in the rat habenula.This was done using three main techniques: single and sequential immunoprecipitation using subunit-specific antibodies, single-point binding with α-conotoxin mII, and saturation binding with [ 3 H]-epibatidine and competition binding with nicotine, cytisine, AT-1001, and sazetidine-A.Single immunoprecipitation experiments revealed that every subunit probed was expressed in the habenula in significant quantities.The α5 subunit was expressed in approximately 26% of nAChRs, the α4 subunit was expressed in approximately 40% of receptors, α3 was expressed in approximately 32% of receptors, β2 was expressed in approximately 48% of receptors, and β4 was expressed in approximately 51% of nAChRs in the habenula.Sequential immunoprecipitations demonstrated that every subunit expressed was significantly associated with every other subunit with one notable exception: the α4 and α5 subunits were not discernably associated in the habenula.Additionally, we found that approximately 20% of nAChRs in the habenula contain both the β2 and β4 subunits.Finally, through binding assays with [ 3 H]-epibatidine and α-conotoxin MII, we determined that there are no detectable α6-containing nAChRs in the habenula.Saturation binding assays determined that nAChRs in the habenula have a Kd for [ 3 H]epibatidine of 78 pM.Additionally, we found that approximately 20% of nAChRs have a high affinity for Sazetidine-A (a ligand with a high specificity for β2-containing nAChRs) and approximately 70% of nAChRs in the rat habenula had a high affinity for AT-1001 (a ligand with a high specificity for β4-containing nAChRs).From this data, and the data from the immunoprecipitation reactions, we hypothesized that the β2β4* nAChRs in the habenula indicate binding properties much like those of β4-containing nAChRs.Finally, nAChR subunits in the olfactory bulb and olfactory tubercle were probed by single and sequential immunoprecipitation.In the olfactory bulb, the α5 subunit was expressed in approximately 28% of nAChRs, the α4 subunit was expressed in approximately 48% of receptors, α3 was expressed in approximately 41% of receptors, β2 was expressed in approximately 71% of receptors, and β4 was expressed in approximately 33% of nAChRs.v Acknowledgements First and foremost, I would like to thank my mentor, Dr. Ken Kellar, for taking me on as a student and undertaking the task of transforming me from a bright-eyed, bushy-tailed graduate student fresh out of college to a scientist.Over the past five years, Ken has given me the freedom to explore my project in my own way, allowed me to make mistakes, and hone my creative scientific thinking, all while being there to constantly guide me and give me focus if I was floundering.He has expressed confidence in my abilities when my own confidence was weak, and catalyzed my intellectual growth in a way I did not even realize was possible when I joined the department.I would also like to sincerely thank Dr. Robert Yasuda for being an exemplary committee member, mentor, scientist, and friend.Bob has taken me under his wing and helped me through everything from questions about experiments and methods to my existential crises regarding my career, and I would not have had the level of success I experienced without his guidance.I would also like to thank my other committee members: Dr. Barry Wolfe, for constantly having an open door and being willing to talk about whatever science I wanted, Dr. Dan Pak, for his insights into the molecular portions of my projects and constructive critiques, and Dr. Mark Burns, for providing neuroscience-centric perspective on my work.I would also like to thank the entire department of Pharmacology; in particular, Dr. Ken Dretchen, who has been a wonderful mentor and makes the entire department truly feel like
Author: Rashmi Venkatesh
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2562162714