By Ruthenium Red Does Not Suppress Hypothalamic Neuronal Thermosensitivity

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

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

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.

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.

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

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

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

vii

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

viii

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

9. Changes in Thermosensitivity Before and During Application of Ruthenium Red

in Low Slope Insensitive Neurons ...... 27

10. Changes in Thermosensitivity Before and During Application of Ruthenium Red

in Total Population of Neurons ...... 28

11. Differences in Firing Rate Thermosensitivity Responses to Ruthenium Red ...... 33

12. Differences in Resting Membrane Thermosensitivity Responses to Ruthenium

Red ...... 35

Chapter 1: Introduction

The preoptic-anterior hypothalamus (POAH) senses change in core temperature and evokes physiological and behavioral responses that regulate body temperature (17,

36). Early thermode studies in cats and dogs demonstrated that selective warming of the

POAH led to a suppression of ongoing shivering and caused an increase in heat loss responses (18). Conversely, selective cooling caused heat production responses such as shivering and non-shivering thermogenesis (18-20, 23). While most spontaneously firing

POAH neurons are considered to be temperature insensitive, about 20% are classified as warm sensitive (54-56). Warm sensitive neurons increase their firing rates during hypothalamic warming and decrease their firing rates during hypothalamic cooling (2,

37). In addition, there are morphological differences between POAH temperature insensitive neurons and warm sensitive neurons. The dendrites of temperature insensitive neurons are oriented in rostral and caudal directions, parallel to the third ventricle. The dendrites of warm sensitive neurons, however, are oriented in medial and lateral directions, toward ascending pathways, to synaptically receive ascending afferent projections from skin and spinal thermoreceptor pathways (4, 15).

The cellular mechanism by which POAH neurons detect temperature has not been firmly established. The recent identification of the transient receptor potential (TRP) superfamily of cation channels suggests a novel player in the transduction of thermal 1

information peripherally (7, 31, 34); however, the role of TRP channels within the central nervous system is still in question. Some studies suggest that warm-sensitivity in POAH neurons is due to activity of TRPV channels (28). Thermal sensitive cationic TRP

(TRPV1-4) channels contribute to the firing rate thermosensitivity of receptors found in the skin epithelium and dorsal root ganglion neurons. In addition, it is often assumed that

TRP channels are responsible for central hypothalamic thermosensitivity (1, 10, 16, 39).

When TRPV is activated by heat, TRPV produces an inward calcium/sodium current, which leads to depolarization, generating action potential activity. It has been hypothesized that this mechanism is responsible for the ability of hypothalamic neurons to detect temperature by increasing their firing rates with warming and decreasing their firing rates with cooling (27, 29, 31)

The transient receptor potential (TRP) superfamily of membrane proteins consists of seven subfamilies of six-transmembrane spanning proteins: TRPA, TRPC, TRPM,

TRPV, TRPN and TRPML (33, 42). Most TRP channels are permeable to Ca+2 and have varying degrees of calcium selectivity (47). Perhaps most relevant to thermosensation, the TRP-Vanilloid (TRPV) group of receptors distinguishes itself from most other members of the TRP channel superfamily due to its ability to be activated by heat. The members of the TRPV family (TRPV1-TRPV4) have varying temperature thresholds of activation, ranging from innocuous (TRPV4) 28-43°C to noxious (TRPV1-3) 34-52°C

(5, 24, 38). The TRPV4 channels are the most likely to be active at physiological temperatures within the CNS when compared to the more noxious heat gating properties

of TRPV1-3 (32). TRPV channels are currently known to be activated through phorbol ester derivatives (40) in addition to temperature change (10, 48). TRPV channels can be blocked by a low concentration (1uM) of ruthenium red. Ruthenium red is a widely utilized non-selective cationic TRPV channel inhibitor (13, 45, 46).

The role of thermal TRP channels beyond the peripheral nervous system has not been well established. Several investigators have shown histological staining of TRP within the hypothalamus (9, 31, 41) ; however, the intracellular presence of TRP within

POAH neuronal cell bodies has been contested (11, 49). Assuming that thermal TRP channels are playing a putative role in central thermosensitivity, it would be expected that blockade of these channels would likely lead to temperature regulation deficits. There is disagreement, however, among studies that have utilized various subtype specific TRP gene deletions in mice to determine their subsequent effects on thermal sensation. Using

TRPV1 null mice, some studies have reported thermoregulatory abnormalities (43,35), while other studies utilizing these TRPV1 null mice show no significant changes in body temperature or thermal preferences (8, 22). This same lack of thermoregulatory phenotype is evident in both TRPV3 and TRPV4 deficient mice (21, 30, 31). If thermal

TRP channels are present in POAH neurons then blockade of their currents should abolish or attenuate hypothalamic neuronal thermosensitivity. The present study tested this hypothesis by determining neuronal thermosensitivity before and after exposure to ruthenium red, which is a non-selective cationic TRPV channel blocker.

Chapter 2: Methods

Slice Preparation

Hypothalamic tissue slices were prepared in accordance with regulations set forth by the National Institute of Health and the Ohio State University Laboratory Animal Care and Use Committee. Male Sprague-Dawley rats (150g-320g) were decapitated by guillotine, and the brain was quickly removed. As described in previous papers (54-56), a hypothalamic tissue block was cut, mounted on a vibratome, and submerged in oxygenated perfusion medium. Using a vibratome, five to six 350 μm thick horizontal tissue slices were sectioned from the tissue block. As in previous studies, slices were placed in a recording chamber (26), and allowed to incubate for 2 hours before electrophysiological recordings were made. Artificial cerebrospinal fluid (aCSF), the 7.4 pH, 300 osmol control perfusion medium, contained (mM): 124 NaCl, 26 NaHCO3, 10 glucose, 5 KCl, 2.4 CaCl2, 1.3 MgSO4 and 1.24 KH3PO4. This medium was oxygenated with 95% O2–5% CO2. During the experiments, perfusions were switched between the control medium and a similar medium containing 1, 10 or 100 μM ruthenium red, in order to block TRPV channels (13, 45, 46). The temperature of the medium perfusing the recording chamber was constantly monitored by a thermocouple placed under the tissue slice. The temperature of the perfusion medium was maintained at 36°C-37°C by a thermoelectric Peltier assembly. This thermoelectric assembly also permitted the aCSF 4

and the tissue slice to be warmed and cooled periodically in order to determine the effect of temperature on neuronal electrophysiological activity.

Intracellular Electrophysiological Recordings

Each neuron’s membrane potential and action potential activity was measured using a 1-2 μm tip whole-cell patch microelectrode filled with a solution containing the following (in mM): 10 EGTA, 10 Hepes, 140 potassium gluconate, 1 MgCl2, 1 CaCl2,

2 ATP and 5 NaCl. This electrode solution had an osmolality of 295-300 mOsm and a pH of 7.2-7.3. As in previous studies (14), an AgCl ground electrode was placed in the outer bath, which was connected to the inner bath by a filter paper bridge. As previously determined experimentally (14) the liquid junction potential for this solution was 12mV.

This potential was subtracted from all values reported in the present study. Neuronal activity reported in this study was not subjected to any holding current.

Data Analysis

All currents and voltages were recorded by an Axopatch 200A amplifier (Axon

Instruments) and relayed by a Digidata 1440A for storage and analysis. The criteria used to determine if a neuron was suitable for use in this study consisted of the following:

1) a stable resting membrane potential (RMP) , defined as a change of no more than 3 mV during recordings at control temperatures of 36-37°C, and 2) action potential spike amplitudes of at least 55 mV. After a control recording at 36–37°C, neuronal firing rate thermosensitivity was determined by altering the tissue temperature, usually changing between 32°C and 39°C. Each neurons thermosensitivity was determine by its thermal coefficient (m), which is defined as the slope of the regression line produced when firing rate is plotted as a function of temperature (impulses s−1 °C−1). As in other studies (3, 25), a neuron was classified as warm sensitive if it had a positive thermal coefficient that was at least +0.7 impulses s−1 °C−1 (see Fig. 1A). All other neurons with spontaneous action potentials were classified as temperature insensitive. No cold sensitive neurons were recorded in this study. As in previous studies (14, 50), temperature-insensitive neurons were subdivided into two groups: moderate slope and low slope. Moderate slope temperature-insensitive neurons increased their firing rates slightly during increases in temperature; and their thermal coefficients were less than +0.7 impulses s−1 °C−1 but greater than +0.2 impulses s−1 °C−1 (see Fig. 1B). During temperature changes, the firing rates of low slope temperature-insensitive neurons were virtually unchanged, and the absolute values of their thermal coefficients were ≤ 0.2 impulses s−1 °C−1 (see Fig. 1C).

Firing rate and resting membrane thermosensitivity comparisons between neuronal populations were made statistically using a two-tailed t test of equal variance, differences were considered significant if P < 0.05. Values reported in this study are shown as means

± SE

Figure 1. Examples of three different types of POAH neurons. Left: The experimental records of three types of neurons are shown. Firing rate is defined as impulses per second. Right: Regression line is plotted as a function of firing rate (FR) over temperature. The slope of the line m defines the thermosensitivity of the neuron measured as impulses s−1 °C−1. A) Low-slope insensitive neuron is shown as having a slope of

+0.16 impulses s−1 °C−1. B) Moderate-Slope insensitive neuron possessing a slope of

+0.53 impulses s−1 °C−1. C) Warm sensitive neuron having a slope of 1.49 impulses s−1

°C−1.

Figure 1. 8

Chapter 3: Results

Table 1. Characteristics of the Neuronal Population. Average firing rate and resting membrane potential thermosensitivity is shown for each neuronal type. Values are shown as means ± S.E.M, and in terms of the m firing rate thermal coefficient. Shown is the resting membrane potential thermal sensitivity which shows no statistical correlation to firing rate thermal sensitivity between neuronal subtypes. Firing rate thermal coefficient

(m) shown as impulses s−1 °C−1 , Resting membrane potential thermal coefficient in terms of Millivolts s−1 °C−1.

Thermosensitivity (TS)

Firing Rate Resting Membrane Classification T(imp/S/°C) (mV/S/°C) Number of Cells

Warm Sensitive 0.98 ± 0.18 0.21 ± 0.06 n =6

Moderate-Slope Insensitive 0.37 ± 0.03 0.22 ± 0.04 n =7

Low-Slope Insensitive -0.01 ±0.03 0.22 ± 0.04 n =8

All Neurons 0.39 ± 0.10 0.20 ± 0.03 n =21

General Overview of Neuronal Population

A total of 21 neurons were recorded in hypothalamic tissue slices from 20 rats and categorized according to their temperature sensitivity. Typically, only one neuron per rat was recorded. In addition, only one neuron per tissue slice was recorded. Of the recorded neurons, 81% (15/21) were categorized as temperature insensitive and 19% (6/21) were classified as warm sensitive. These ratios of warm sensitive neurons to temperature insensitive neurons are similar to previous studies (12, 15-16). Most neurons in this study were recorded in the POAH (67%); however, other areas included nearby hypothalamic nuclei (i.e. ventromedial hypothalamus and dorsomedial hypothalamus). There were no differences in neuronal responses between the different areas. Table 1 shows the three distinct categories of neuronal types. Warm sensitive neurons (n=6) had average firing rates of +0.98± 0.18 impulses s−1 °C−1. Moderate-slope neurons (n=7) had average firing rates of +0.37± 0.03 impulses s−1 °C−1, and Low-slope neurons (n=8) had average firing rates of -0.01± 0.03 impulses s−1 °C−1. There were no statistical differences in the resting membrane potential thermosensitivities between the different neuronal types. This supports our previous study (15) which indicates that thermally induced changes to resting membrane potential does not affect neuronal firing rate thermosensitivity.

Figure 2. Increased Firing Thermosensitivity in Response to Varying

Concentrations of Ruthenium Red. Regression graphs plot firing rate (FR, imp/sec) as a function of temperature. Neuronal thermosensitivity (m) is expressed in terms of impulses s−1 °C−1. A: Moderate-slope temperature insensitive neuron whose firing rate thermosensitivity increased during administration of 1 μM ruthenium red. B: Warm sensitive neuron whose firing rate thermosensitivity increased during administration of

10 μM ruthenium red. C: Warm-sensitive neuron whose firing rate thermosensitivity increased during administration of 100 μM ruthenium red. 11

Effect of Ruthenium Red on Hypothalamic Neurons

The firing rates of hypothalamic neurons were recorded before, during and after perfusion with experimental aCSF which contained varying concentration of the TRP channel blocker ruthenium red. The concentrations of ruthenium red used in the present study varied from low concentration (1 μM, n=7), medium concentration (10 μM, n=4) and high concentration (100 μM, n=10). There were no statistical differences between the varying concentrations of ruthenium red and their effect on neuronal thermosensitivity.

Fig. 2 shows examples of different neurons recorded during exposure to the three different ruthenium red concentrations. Figures 3-7 are other examples of various neurons exposed to ruthenium red.

Figure 2, shows regression graphs of neurons whose firing rates increased during administration of 1 μM, 10 μM or 100 μM ruthenium red. Included are a moderate-slope insensitive neuron (Fig. 2a), and two warm sensitive neurons (Fig. 2b-c).

In these examples ruthenium red administration regardless of concentration increased firing rate thermosensitivity. In two of these examples (Fig. 2a-b), control conditions were obtained after ruthenium red washed out of the cell and basal levels of thermosensitivity continued. The washout of ruthenium red did not occur in one cell

(Fig. 2c) due to the extremely high concentration of the drug and baseline levels of thermosensitivity were not able to be resumed. 12

Figure 3. 10 μM Ruthenium Red Increases Firing Rate Thermosensitivity in a

Warm Sensitive Neuron. Top: Experimental record of firing rate, tissue slice temperature, resting membrane potential over time. Bottom: Regression plots (m) of firing rate (FR) and resting membrane potential (RMP) as a function of temperature.

Administration of 10 μM of ruthenium red increased the firing rate thermosensitivity but had no major effect on the resting membrane thermosensitivity. Bottom: Regression plots showing neuronal thermosensitivity before (A), during (B) and after (C) TRPV blockade.

Figure 3. 14

Figure 4. 100 μM Ruthenium Red Increases Firing Rate Thermosensitivity in a

Administration of 100 μM of ruthenium red increased the firing rate thermosensitivity but had no major effect on the resting membrane thermosensitivity. Bottom: Regression plots showing neuronal thermosensitivity before (A), during (B) and after (C) TRPV blockade

Figure 4. 16

Figures 3 and 4 show the firing rate responses to temperature changes before (A) during (B) and after (C) exposure to different concentrations of ruthenium red. The warm sensitive neuron in Fig. 3 had a firing rate thermosensitivity of +0.85 impulses s−1 °C which increased to +1.13 of impulses s−1 °C with application of 10 μM of ruthenium red, which was able to be washed out with control media. In Fig. 4, the warm sensitive neuron was found to have a neuronal firing rate thermosensitivity of +1.56 impulses s−1 °C which was increased to +1.76 impulses s−1 °C with administration of 100 μM of ruthenium red, however; washout of the higher concentration of ruthenium red was unable to be obtained.

Figure 5. 1 μM Ruthenium Red Increases Firing Rate Thermosensitivity in an

Moderate Slope Temperature Insensitive Neuron. Top: Experimental record of firing rate, tissue slice temperature, resting membrane potential over time. Bottom: Regression plots (m) of firing rate (FR) and resting membrane potential (RMP) as a function of temperature. Administration of 1 μM of ruthenium red increased the firing rate thermosensitivity but had no major effect on the resting membrane thermosensitivity.

Bottom: Regression plots showing neuronal thermosensitivity before (A), during (B) and after (C) TRPV blockade.

Figure 5. 19

Figure 6. 10 μM Ruthenium Red Increases Firing Rate Thermosensitivity in an

Moderate Slope Temperature Insensitive Neuron. Top: Experimental record of firing rate, tissue slice temperature, resting membrane potential over time. Bottom: Regression plots (m) of firing rate (FR) and resting membrane potential (RMP) as a function of temperature. Administration of 10 μM of ruthenium red increased firing rate thermosensitivity but had no major effect on the resting membrane thermosensitivity.

Bottom: Regression plots showing neuronal thermosensitivity before (A), during (B) and after (C) TRPV blockade.

Figure 6. 21

Figures 5 and 6, show the firing rate records of two different moderate slope insensitive neurons. Both neurons displayed an increase in firing rate thermosensitivity upon administration of ruthenium red. Again, this increase in firing rate thermosensitivity was independent of the dosage of ruthenium red administered. The moderate slope neuron in Fig. 5 had a firing rate thermosensitivity of +0.41 impulses s−1 °C which increased to +0.53 impulses s−1 °C with application of 1 μM of ruthenium red.

Following this, the thermosensitivity decreased during the final washout with control media. In Fig. 6, the moderate slope insensitive neuron was found to have a neuronal firing rate thermosensitivity of +0.36 impulses s−1 °C which increased to +0.41 impulses s−1 °C with administration of 10 μM of ruthenium red, however; baseline thermosensitivity did not resume during washout with control media.

Table 2. Effect of Ruthenium Red on Neuronal Firing Rate Thermosensitivity. TS =

Thermosensitivity. N= Number of cells. FR = Neuronal firing rate. P value is significant if P < 0.05.

Thermosensitivity (TS)

Control FR TS Drug FR TS Classification (imp/S/°C) (imp/S/°C) Number of Cells p value

Warm Sensitive 0.98±0. 18 1.11 ± 0.22 n =6 p=0.57

Moderate-Slope 0.37± 0.03 0.45 ± 0.04 n =7 *p=.04 Insensitive

Low-Slope Insensitive -0.01 ±0.03 -0.07 ± 0.15 n =8 p =0.47

All Neurons 0.39 ± 0.10 0.45 ± 0.13 n =21 p= 0.21

Table 3. Effect of Ruthenium Red on Neuronal Firing Rate. TS = Thermosensitivity.

N= Number of cells. FR = Neuronal firing rate. P value is significant if P < 0.05

Firing Rate (FR)

Control FR Drug FR Classification (imp/S) (imp/S) Number of Cells p value

Warm Sensitive 19.1± 4.2 21.7 ± 5.0 n =4 p=0.20

Moderate-Slope 7.4 ± 1.4 6.9 ± 1.4 n =7 p=0.06 Insensitive

Low-Slope Insensitive 3.1 ± 1.3 3.8 ± 1.7 n =8 p =0.29

All Neurons 7.0 ± 1.5 7.5 ± 1.7 n =19 p= 0.19

Figure 7. Changes in Thermosensitivity Before and During Application of

Ruthenium Red in Warm Sensitive Neurons. Top: firing rate. Bottom: resting membrane thermosensitivity. Error bars represent SE. There was no significant change in thermosensitivity both in firing rate and in resting membrane thermosensitivity in warm sensitive neurons.

Figure 8. Changes in Thermosensitivity Before and During Application of

Ruthenium Red in Moderate Slope Insensitive Neurons. Top: firing rate.

Bottom: resting membrane thermosensitivity. Error bars represent SE. Ruthenium red significantly increased both firing rate thermosensitivity and resting membrane thermosensitivity of moderate slope insensitive neurons (P< 0.05).

Figure 9. Changes in Thermosensitivity Before and During Application of

Ruthenium Red in Low Slope Insensitive Neurons. Top: firing rate. Bottom: resting membrane thermosensitivity. Error bars represent SE. There was no significant change in thermosensitivity both in firing rate and in resting membrane thermosensitivity in low slope insensitive neurons. 27

Firing Rate Thermosensitivity 0.8

-1 0.6

°C 

-1 0.4

s 

imp 0.2

0.0

Control

Ruthenium Red

Figure 10. Changes in Thermosensitivity Before and During Application of

Ruthenium Red in Total Population of Neurons. Top: firing rate. Bottom: resting membrane thermosensitivity. Error bars represent SE. There was no significant change in thermosensitivity both in firing rate and in resting membrane thermosensitivity in total, unstratified population of neurons 28

Table 2 summarizes the effects of ruthenium red on neuronal firing rate thermosensitivity. Warm sensitive neurons (Fig. 7, top) displayed an overall average firing rate thermosensitivity of 0.98 ± 0.18 impulses s−1 °C which did not significantly change with administration of ruthenium red. Moderate slope insensitive neurons (Fig. 8, top) had an average firing rate thermosensitivity of 0.37 ± 0.03 impulses s−1 °C which significantly increased to 0.45 ± 0.04 impulses s−1 °C (p< 0.05) during ruthenium red.

Low-slope insensitive neurons (Fig 9, top) had an average firing rate thermosensitivity of

-0.01 ± 0.03 impulses s−1 °C which again did not change significantly with the application of ruthenium red. Table 3 also demonstrates that there is no significant effect of ruthenium red on neuronal firing rate. There was no significant effect of ruthenium red in the total population of neurons (Fig. 10, top).

Table 4. Effect of Ruthenium Red on Neuronal Resting Membrane

Thermosensitivity. TS = Thermosensitivity. N = Number of cells. RMP = Resting

Membrane Potential. P value is significant if P < 0.05.

Resting Membrane Potential Thermosensitivity (RMP TS)

Control RMP Drug RMP TS Classification TS (mV/S/°C) (mV/S/°C) Number of Cells p value

Warm Sensitive 0.21± 0.06 0.18 ± 0.05 n =6 p=0.58

Moderate-Slope 0.22 ± 0.04 Insensitive 0.32 ± 0.02 n =7 *p=0.04

Low-Slope Insensitive 0.22 ± 0.04 0.25 ± 0.07 n =8 p=0.24

All Neurons 0.20 ± 0.03 0.24 ± 0.03 n =21 p= 0.10

In table 4, the effects of ruthenium red on resting membrane potential thermosensitivity are summarized. Warm sensitive neurons (Fig. 7, bottom) had a resting membrane potential thermosensitivity of 0.21 ± 0.06 mV s−1 °C which did not significantly change with the administration of ruthenium red. Moderate slope (Fig 8, bottom) insensitive neurons possessed a resting membrane thermosensitivity of 0.22 ±

0.04 mV s−1 °C which significantly changed during ruthenium red to 0.32 ± 0.02 mV s−1 °C (P< 0.05). Low slope insensitive neurons (Fig. 10, bottom) displayed a resting membrane thermosensitivity of 0.22 ± 0.04 mV s−1 °C which did not significantly change upon perfusion with ruthenium red. When the effect of ruthenium red on resting membrane thermosensitivity was examined in the entire neuronal population (Fig. 10, bottom), there was no significant change.

Figure 11. Differences in Firing Rate Thermosensitivity Responses to Ruthenium

Red. This graph is plotted as a function of firing rate thermosensitivity before administration of ruthenium red (x-axis) vs. thermosensitivity during ruthenium red (y- axis). Red = Warm sensitive. Yellow = Moderate Slope Insensitive. Grey = Low Slope

Insensitive. Points lying on the line represent neurons which did not change thermosensitivity upon exposure to ruthenium red. All points above the line represent an increased response; all points below the line represent a diminished thermosensitivity.

Figure 11.

Figure 12. Differences in Resting Membrane Thermosensitivity Responses to

Ruthenium Red. This graph is plotted as a function of resting membrane thermosensitivity before administration of ruthenium red (x-axis) vs. thermosensitivity during ruthenium red (y-axis). Red = Warm sensitive. Yellow = Moderate Slope

Insensitive. Grey = Low Slope Insensitive. Points lying on the line represent neurons which did not change thermosensitivity upon exposure to ruthenium red. All points above the line represent an increased response; all points below the line represent a diminished thermosensitivity.

Figure 12 35

Figure 11 shows each neuron’s firing rate before and after ruthenium red and figure 12 shows each neurons resting membrane potential thermosensitivity before and after ruthenium red. Each data point represents a single neuron recorded and characterized. Data points that are above the diagonal line signify that the thermosensitivity of the neuron increased, data points that lie below the diagonal line represent decreased neuronal thermosensitivity. The total population of neurons tended to increase firing rate thermosensitivity (Fig. 11) upon administration of ruthenium red, although this increase was only a significant within the moderate slope insensitive neuron population (see Fig 8, top). The total population of neurons did not appear to have changed in any particular pattern in regards to resting membrane thermosensitivity (Fig.

12) with application of ruthenium red. It is important to point out that the moderate slope insensitive neurons displayed a significant increase in resting membrane thermosensitivity (Fig. 8, bottom) and firing rate thermosensitivity (Fig. 8, top) with ruthenium red application.

Chapter 4: Discussion

The ionic mechanism of hypothalamic neuronal thermosensitivity is still under much debate. Earlier studies suggest that hypothalamic neuronal temperature sensitivity could be due to the inward depolarizing cationic current driven by TRPV channels. (1, 10,

17, 43). This would result in a thermal sensitive resting membrane depolarization and a subsequent temperature related increase in firing rate. In addition, the TRPV3 and

TRPV4 members of the TRPV family appear to be active over a physiological temperature range (5, 24, 38). Immunohistochemical studies show positive labeling of

TRPV4 within the POAH (9, 31, 41); however, other studies suggest that the neurons are not positively stained (11, 49). TRPV channels also possess subtype specific sensitization and desensitization characteristics (1); however, upon repeated thermal stimulation of temperature sensitive hypothalamic neurons, there are no differences in firing rate thermosensitivity (3). If TRPV channels are responsible for hypothalamic thermosensitivity, warm sensitive neurons should display an increased resting membrane thermosensitivity upon repeated warming (31). Previous studies, however, have found no significant correlation between resting membrane thermosensitivity and firing rate thermosensitivity (56),

The present study also found no significant difference between resting membrane thermosensitivity and firing rate thermosensitivity (Table 1). Although only 21 neurons were recorded in this study, statistical significance was still attained (Table 2, Fig 8).

Most importantly, the present study found that blockade of TRPV channels, did not decrease neuronal thermosensitivity. In addition to this study, another study found that ruthenium red administered intracerebrally had no detrimental effect on body temperature regulation (44). Moreover, double knockout TRPV3/TRPV4 mice, also showed no temperature regulatory deficits in a recent study (21). Together, these studies indicate that

TRP is not the primary determinant of thermosensitivity within the hypothalamus. It is possible that other types of ionic mechanism, such as inactivation of the potassium-A ( IA

) current or activation of hyperpolarization-cyclic nucleotide gated (HCN) channels may be responsible for neuronal thermosensitivity in the preoptic hypothalamus (54, 56, 15).

In the present study, ruthenium red often affected an increase in firing rate thermosensitivity (Fig 3-7). Ruthenium red at concentrations of 5uM has been indicated to partially inhibit TASK channels (12). TASK channels are expressed throughout the

POAH (49),(6). TASK channels are highly temperature sensitive and act to hyperpolarize the neuronal membrane. This would decrease firing rate thermosensitivity.

A previous study suggested that temperature insensitive neurons employ these potassium channels to remain insensitive to temperature (12). Neuronal modeling studies have also indicated that an increased TASK current will decrease neuronal firing rate

thermosensitivity (54). This suggests that warm sensitive neurons have low amounts of

TASK current, relative to temperature insensitive neurons, and would not be affected by ruthenium red inhibition of TASK (see Fig. 7). Moreover, temperature insensitive neurons, which are suggested to contain more TASK, would be more severely affected by ruthenium red blockade of TASK. Figure 7, warm sensitive neurons are not affected by ruthenium red blockade; however, Figure 8, moderate slope insensitive neurons become significantly more temperature sensitive with ruthenium red blockade. This supports the hypothesis of the previous study (12) that TASK channels cause hypothalamic neurons to remain temperature insensitive, and that TASK channels are differentially expressed in the temperature sensitive and insensitive neurons (12, 54).

Further studies must be completed to determine the precise cause of increased firing rate thermosensitivity with application of ruthenium red. As previously mentioned, ruthenium red blockade of TASK channels is a highly likely mechanism for increased firing rate thermosensitivity. TASK channels play an essential role in preventing a neuron from reaching its action potential threshold by modulating the depolarizing prepotential

(DPP) thereby lengthening the interspike interval of the neuron (14, 54). If ruthenium red is inhibiting TASK channel activity in the POAH, then there should be a lengthening of the interspike interval and flattening of the DPP. This could also be compared against known TASK channel blockers such as tetraethyl-ammonium (TEA) to see if similar neuronal thermosensitivity responses can be induced.

The present intracellular study is the first to examine the effects of TRP blockade on hypothalamic neuronal thermosensitivity. The most important finding of this study is that the blockade of TRPV channels by ruthenium red does not inhibit temperature sensitive neurons, nor does it decrease their thermosensitivies (see figs.). Therefore, TRP channels are not responsible for neuronal temperature sensitivity. Furthermore, during blockade, many neurons displayed an increase in temperature sensitivity, particularly the moderate slope temperature insensitive neurons (Fig 8). As described above, this increased thermosensitivity is likely due to inhibition of TASK channels by ruthenium red.

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