by Ruthenium Red Does Not Suppress Hypothalamic Neuronal Thermosen

57 Pages·2012·1.65 MB·English

Blockade of the Transient Receptor Potential Vanilloid (TRPV) by Ruthenium Red Does
Not Suppress Hypothalamic Neuronal Thermosensitivity

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in
the Graduate School of The Ohio State University

By
Nicholas Thompson Unger
Graduate Program in Biophysics

The Ohio State University
2012

Master's Examination Committee:
Dr. Jack A. Boulant, Advisor
Dr. Maqsood A. Chotani
Copyright by
Nicholas Thompson Unger
2012
Abstract

The thermoregulatory preoptic-anterior hypothalamus (POAH) contains both
temperature sensitive and insensitive neurons. The cellular mechanisms underlying
POAH neuronal thermosensitivity have not been firmly established. While controversial,
recent studies suggest that POAH neuronal thermosensitivity is caused by the vanilloid-
sensitive transient receptor potential (TRPV) family of TRP receptors. This hypothesis
was tested by determining the effect of ruthenium red, a known TRPV channel inhibitor,
on POAH neuron populations showing differing degrees of temperature sensitivity.
Whole-cell patch microelectrodes recorded the intracellular activity of POAH neurons in
rat hypothalamic tissue slices perfused with control artificial cerebral spinal fluid (aCSF)
and experimental media containing either 1, 10 or 100 μM ruthenium red. Each neuron
was characterized by its spontaneous firing rate at 36°C and its firing rate
thermosensitivity (impulses/sec/°C) during changes in tissue temperature. Ruthenium red
did not reduce the firing rate thermosensitivity nor the membrane potential
thermosensitivity of POAH neurons. In fact, some POAH neurons increased their
thermosensitivities during ruthenium red application. This supports our previous studies
indicating that TRP channels are not responsible for thermally induced changes in the
resting membrane potentials and firing rates of rostral hypothalamic neurons.

ii
Dedication

This document is dedicated to my lovely and patient wife. She has always
supported me throughout my academic endeavors.

iii
Acknowledgments

I would like to thank the many people for their help in this project. The most
important person to help me was Lorry Kaple. She was both a patient and wise teacher in
the subtleties of electrophysiological techniques. Yongjie Miao has been instrumental in
helping me both formulate and understand the various statistical methods chosen to
represent my data. I would like to sincerely thank Dr. Maqsood Chotani for his
encouragement for me to finish my thesis work and for his support throughout my
endeavor. Also, Dr. Selvi Jeyaraj was instrumental in helping me revise my thesis,
elevating it to a more professional level. I would like to personally acknowledge Dr.
Pamela Lucchesi for her dedication to the various students under her charge. She was a
key orchestrator in my return to graduate school. Lastly, I would like to thank Dr. Jack
Boulant, for allowing me the pleasure of working in his lab, teaching and training me in
the various intricacies of neurophysiology.

iv
Vita

May 2002 .......................................................Evangel Christian Academy High School
2006................................................................B.A. Biology, Capital University
2006 to 2008 .................................................Graduate Research Associate, Department
of Physiology and Cell Biology, The Ohio
State University
2009 to 2010 .................................................Research Assistant, Center for
Cardiovascular and Pulmonary Research,
Nationwide Children’s Hospital
2011 to Present ..............................................Graduate Research Associate, Department
of Physiology and Cell Biology, The Ohio
State University

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Publications

Unger NT, Kaple ML, Bishop GA, Boulant JA. 2007. Role of hyperpolarization-activated
currents in hypothalamic neuronal thermosensitivity. FASEB Journal. Vol. 6, no. 21:
A1313. (IF: 6.721)

Unger NT, Kaple ML, Boulant JA. 2008. TRPV blockade by ruthenium red does not
suppress thermosensitivity in hypothalamic neurons. FASEB Journal, no. 22. (IF: 6.721)

Jeyaraj SC, Unger NT, Chotani MA. Rap1 GTPases: an emerging role in the
cardiovasculature. Life Sci. 2011 Apr 11;88(15-16):645-52. Epub 2011 Feb 2. Review.

Fields of Study

Major Field: Biophysics

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Table of Contents

1. Abstract ................................................................................................................... ii
2. Dedication .............................................................................................................. iii
3. Acknowledgments.................................................................................................. iv
4. Vita .......................................................................................................................... v
5. Publications ............................................................................................................ vi
6. Fields of Study ....................................................................................................... vi
7. Table of Contents .................................................................................................. vii
8. List of Tables ....................................................................................................... viii
9. List of Figures ........................................................................................................ ix
10. Chapter 1: Introduction .......................................................................................... 1
11. Chapter 2: Methods ................................................................................................. 4
12. Chapter 3: Results ................................................................................................... 9
13. Chapter 4: Discussion ........................................................................................... 37
14. References ............................................................................................................. 41

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List of Tables

1. Characteristics of the Neuronal Population ............................................................ 9
2. Effect of Ruthenium Red on Firing Rate Thermosensitivity ................................ 23
3. Effect of Ruthenium Red on Firing Rate .............................................................. 24
4. Effect of Ruthenium Red on Resting Membrane Thermosensitivity.................... 30

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List of Figures

1. Examples of Three Different Types of POAH Neurons ......................................... 8
2. Increased Firing Thermosensitivity in Response to Varying Concentrations of
Ruthenium Red ..................................................................................................... 11
3. 10 μM Ruthenium Red Increases Firing Rate Thermosensitivity in a Warm
Sensitive Neuron ................................................................................................... 14
4. 100 μM Ruthenium Red Increases Firing Rate Thermosensitivity in a Warm
Sensitive Neuron ................................................................................................... 16
5. 1 μM Ruthenium Red Increases Firing Rate Thermosensitivity in an Moderate
Slope Temperature Insensitive Neuron ................................................................. 19
6. 10 μM Ruthenium Red Increases Firing Rate Thermosensitivity in an Moderate
Slope Temperature Insensitive Neuron ................................................................. 21
7. Changes in Thermosensitivity Before and During Application of Ruthenium Red
in Warm Sensitive Neurons .................................................................................. 25
8. Changes in Thermosensitivity Before and During Application of Ruthenium Red
in Moderate Slope Insensitive Neurons ................................................................ 26
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