THE ROLE OF THE LATERAL HABENULA IN THE AFFECTIVE DIMENSION OF PAIN MICHELLE MARIE WHITE. Presented to the Faculty of the Graduate School of

Transcription

1 THE ROLE OF THE LATERAL HABENULA IN THE AFFECTIVE DIMENSION OF PAIN by MICHELLE MARIE WHITE Presented to the Faculty of the Graduate School of The University of Texas at Arlington in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE IN PSYCHOLOGY THE UNIVERSITY OF TEXAS AT ARLINGTON DECEMBER 2015

2 Copyright by Michelle Marie White 2015 All Rights Reserved ii

3 Acknowledgements I would like to thank everyone involved, including my committee, my parents, and my dog. October 26, 2015 iii

4 Abstract THE ROLE OF THE LATERAL HABENULA IN THE AFFECTIVE DIMENSION OF PAIN Michelle Marie White, MS The University of Texas at Arlington, 2015 Supervising Professor: Perry Fuchs The lateral habenula (LHb) functions as a hub that integrates information from cortical regions to aid in emotional decision-making, as well as helping to create response strategies under stress (Hikosaka, 2010; Proulx, Hikosaka, & Malinow, 2014; Thorton, Bradbury, & Davies, 1990; Evans & Thorton, 1984; Thorton & Evans, 1984; Thorton & Evans, 1982; Thorton, Evans, & Harris, 1985). In addition, the LHb functions in a descending pain modulatory circuit involving both the nucleus accumbens and periacqueductal gray (Shelton, Becerra, & Borsook, 2012). These two functions of the LHb indicate its importance in decision-making during heightened states of arousal, such as pain. The purpose of the current study was to assess neuronal activation in the LHb during a test of pain affect in addition to sensory testing. Specifically, the hypothesis was tested that cells of the LHb become activated during the Place Escape/Avoidance Paradigm, a test of pain affect in which animals must decide to avoid or withstand painful stimuli. While the assertion has been made that the LHb is involved in the affective component of pain, this claim has not been directly tested. To test the current hypothesis of the LHb s contribution to the affective dimension of pain, animals received injections of one of three solutions (saline, 0.5% or 2% carrageenan) to the left hindpaw, and then underwent either MPWT testing, PEAP testing, a combination of both, or simply no testing at all. For MPWT testing, animals iv

5 injected with 2% carrageenan had significantly lower threshold values compared to 0.5% carrageenan (p <.001) and controls (p <.001), which indicated that carrageenan was successful in inducing mechanical hypersensitivity. During the PEAP test, animals in pain were expected to first escape and avoid an area that they normally prefer, and indeed, behavioral results supported this notion. Animals in both inflammatory groups avoided stimulation and spent significantly more time in the light side of the chamber compared to controls for most of the duration of the test (both ps <.05). Additionally, cells of the LHb were examined to assess whether there were higher levels of neuronal activation as measured by c-fos protein expression. Results suggested that animals injected with 2% carrageenan had significantly higher levels of c- Fos expression in the LHb compared to both 0.5% carrageenan (p <.001) and saline controls (p <.001). To further examine the hypothesis that the LHb contributes to pain affect, the relationship between levels of c-fos expression and PEAP scores were examined. Interestingly, there was not a significant relationship between these two variables (p = n.s.). Also, there was also not a significant relationship between c-fos expression and MPWT scores (p = n.s.). This may support the idea that the LHb functions in sensory and affective processing, but to a much lesser extent than previously expected. This research holds importance because it offers further insight into the mechanisms underlying the pain experience. v

6 Table of Contents Acknowledgements...iii Abstract... iv List of Illustrations... viii List of Tables... ix Chapter 1 Introduction A Multidimensional View of Pain Pain Affect The Habenula and Processing of Aversive Stimuli The Habenula and Pain Affect Purpose... 5 Chapter 2 Preliminary Research Study One Subjects and Groups Procedures Immunohistochemistry (IHC) Results from Preliminary Study One Study Two Subjects and Groups Procedures Mechanical Paw Withdrawal Threshold (MPWT) Testing Results from Preliminary Study Two Chapter 3 Method Subjects and Groups Procedures vi

7 3.2.1 Mechanical Paw Withdrawal Threshold (MPWT) Testing Place Escape Avoidance Paradigm (PEAP) Testing Immunohistochemistry (IHC) Chapter 4 Results Behavioral Results MPWT Outcomes PEAP Outcomes Immunohistochemical (IHC) Results IHC Outcomes General Findings: Behavioral Results Mechanical Paw Withdrawal Threshold Place Escape/Avoidance Paradigm General Findings: IHC Results Immunohistochemistry Chapter 5 Discussion Study Summary Conclusion References Biographical Information vii

8 List of Illustrations Figure 2-1 Preliminary Study One c-fos Cell Counts... 9 Figure 2-2 Preliminary Study Two Threshold Values Figure 4-1 Left MPWT Outcomes Figure 4-2 Right MPWT Outcomes Figure 4-3 Approach-Avoidance Outcomes Figure 4-4 Immunohistochemical Outcomes by Condition Figure 4-5 Number of c-fos Positive Cells and MPWT Values Figure 4-6 Number of c-fos Positive Cells and Approach-Avoidance Outcomes viii

9 List of Tables Table 3-1 Group Assignment and Subject Size per Group ix

10 Chapter 1 Introduction The encoding and processing of noxious stimuli by the nervous system is referred to as nociception (Loeser & Treede, 2008). It is the objective marker of tissue injury and its processing in higher cortical areas results in the subjective, perceptual experience that many interpret as pain (Rainville, 2002). Pain helps to protect an organism from events that induce harm or discomfort. Although simple to identify once felt, pain remains difficult to delineate due to its inherent emotional quality (Freire, Guimaraes, Leal, & Pereira, 2009). The International Association for the Study of Pain (IASP) defined pain as an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage (Merskey& Bogduk, 1994). Pain that persists past a period of normal healing or occurs despite the repair process being ceased is referred to as chronic pain (Merskey & Bogduk, 1994). Similar to other homeostatic drives, when pain crosses a certain threshold or reaches chronicity in duration, it loses its protective function and becomes maladaptive. In the United States alone, chronic pain affects more than 100 million adults and is the leading cause for physician consultation (Institute of Medicine, 2011; Turk & Dworkin, 2004). Financially, pain is expensive, costing the United States half a trillion dollars annually; this loss is measured in terms of health care usage, lost wages, and quality of life (Institute of Medicine, 2011). Pain is a far-reaching issue, and a better understanding of the mechanisms of pain processing is always present. 1.1 A Multidimensional View of Pain Historically, the view that pain was comprised solely of sensory processing dominated research until the 1960s (Gatchel, Peng, Peters, Fuchs, & Turk, 2007). Melzack and Wall s (1965) publication of gate control theory ushered in a modern era of pain research in which the leading paradigm was now to define pain in terms of both its physiological and psychological 1

11 properties. Sensation, emotion, and evaluation all took importance as components involved in the pain experience (Melzack & Casey, 1968). Indeed, subsequent research supported this notion, showing that pain can be influenced by past experience, levels of negative affect (anxiety, depression, among others), and expectation (Loeser & Melzack, 1999). 1.2 Pain Affect As Melzack and Casey (1968) stated, To consider only the sensory features of pain is to look at only part of the problem (p. 423). Pain affect describes how aversive or unpleasant pain is perceived and signifies its emotional quality (Craig, 2003; Melzack & Casey, 1968). It is important for understanding how an organism is motivated to end its pain by means of escape or avoidance (LaBuda & Fuchs, 2000). Craig (2003) has proposed that pain is categorically similar to other homeostatic drives, such as hunger and thirst, in that it demands a response. Pain affect is qualitative in nature, but can be captured quantitatively in the laboratory. For example, escape and avoidance behaviors are often used as proxy measures for pain affect. These behaviors occur in response to stimulation of painful areas, and indicate motivation to end pain (LaBuda & Fuchs, 2000). Preclinical models of pain avoidance, such as conditioned place avoidance (CPA) examine how an unconditioned stimulus (UCS), given during an amount of time in a specific area, induce avoidance behavior when animals are tested in the same area at a later date. The amount of avoidance behavior arising from the UCS is believed to reflect the negative affective state produced by the nociceptive stimulus (Johansen, Fields, & Manning, 2001). With the further development of affective pain tests, such as the Place Escape/Avoidance Paradigm (PEAP), the process of measuring pain affect has been streamlined to a single test session (LaBuda & Fuchs, 2000). The PEAP test uses a half-dark/half-light box that capitalizes on the inherent preference of rodents to be in dark areas. This preference can be manipulated in various ways to examine how aversive an animal perceives their pain. For example, an inflammatory pain condition can be induced in the left hindpaw of an experimentally naïve 2

12 animal. If this hindpaw is stimulated in the dark area of the PEAP chamber, the animal will typically modify its behavior, such that it escapes the stimulus and moves to the light half of the chamber. In this side of the chamber, the animal is then stimulated in the noninflamed right paw. With repeated stimulation over time, the animal learns to altogether avoid the dark side of the chamber and shifts its preference to the light side (LaBuda & Fuchs, 2000). The shift in behavior is indicative of the unpleasantness of pain. The PEAP test can therefore reliably disentangle sensory aspects of pain from affective aspects (Baastrup, Jensen, & Finnerup, 2011; McNabb, Uhelski, & Fuchs, 2012; Pedersen & Blackburn-Munro, 2006; Uhelski, Davis, & Fuchs, 2012). 1.3 The Habenula and Processing of Aversive Stimuli Neural substrates of the pain have been extensively studied and allow for insight into the pain experience. The lateral habenula (LHb) is an important region of interest that integrates input from cortical affective regions to aid in decision-making. It is located slightly above the thalamus and has been proposed to function in a descending pain modulatory circuit with both the nucleus accumbens and periacqueductal gray, among others (Shelton, Becerra, & Borsook, 2012). It contains two separate nuclei, including a medial and lateral nucleus, as well as a habenular commissure (Sutherland, 1980). The habenular nuclei link limbic forebrain structures to the midbrain (Sutherland, 1982; Wang & Aghajanian, 1977). The nuclear complex is involved in an array of functions, including homeostasis, sleep-wake cycles, pain and stress processing, and reproductive behaviors, with the participation of emotion being the common denominator among these (Andres, Von During, & Veh, 1999). The habenula contains a dense amount of mu-opioid receptors, and injection of morphine into this area produces analgesia (Atweh & Kuhar, 1977; Cohen & Melzack, 1985). Mahieux and Benabid (1987) demonstrated that electrical stimulation of the habenula during the tail-flick test led to analgesia, which was reversed by administration of naloxone. Similarly, stimulation of the habenula during the formalin test leads to a reduction of pain behaviors characteristic of 3

13 formalin-induced pain, including paw elevation, paw licking, and paw biting (Cohen & Melzack, 1986). The duration of stimulation is related to the duration of analgesia produced, such that the more frequently animals received stimulation, the longer the period of analgesia (Cohen & Melzack, 1986). Sex differences have also been demonstrated in stimulation-produced analgesia in regards to the habenula (Terenzi, Guimaraes, & Prado, 1990; Terenzi & Prado, 1990). In male rats, stimulation resulted in immediate analgesia that dissipated after fifteen minutes (Terenzi, Guimaraes, & Prado, 1990). In female rats, stimulation led to a slow developing analgesia that peaked at minutes and lasted for 3 hours and longer (Terenzi & Prado, 1990). These sex differences may be due to the participation of the habenular complex in hormonal control of the estrous cycle of female rats (Zouhar & DeGroot, 1963). Additional research has shown that the manner in which the habenula influences pain processing is complex and depends on a variety of factors (Cohen, Kimes, & London, 1991; Cohen & Melzack, 1985; Cohen & Melzack, 1993; Fuchs & Cox, 1992). The LHb has also been implicated in learning to avoid aversive stimuli under stressful conditions (Thorton & Bradbury, 1989). Thorton, Bradbury, and Davies (1990) showed that animals with lesions to the habenula not only demonstrated deficits in avoidance learning, but also tended to perseverate in tasks in which the animal was forced to operate under stress. Additionally, Thorton and Davies (1991) have noted that lesions to the habenula affected the ability of subjects to change response strategies when placed under stressful, demanding conditions. Animals with lesions to the habenula tended to adopt nonadaptive strategies when tested on operant schedules where a certain response pattern was required in order to gain maximum reinforcement (Evans & Thorton, 1984; Thorton & Evans, 1984). In forced swim tests, animals with habenular lesions showed a reduction in escape strategies and escape initiation, further demonstrating an inability to create adaptive strategies when under stress (Thorton & Evans, 1982; Thorton, Evans, & Harris, 1985). 4

14 1.4 The Habenula and Pain Affect The habenula has been implicated in emotional decision-making (Hikosaka, 2010). Undoubtedly, the ability to make emotional decisions under stress is a crucial skill in affective tests of pain in which animals must decide to avoid or escape painful stimuli or to withstand a painful stimulus at the expense of being in a preferred area. Thorton and Evans (1984) have noted the habenula as a site where motivational processes gain access to the motor system and where response strategies are organized. Accordingly, neurons in the LHb encode negative reward prediction, meaning they are excited by unexpected non-rewarding events and inhibited by unexpected rewarding events (Proulx, Hikosaka, & Malinow, 2014). Indeed, the habenula is an area organized to coordinate interactions between reward processing and pain modulation via transmission of dopamine and serotonin (Lee & Goto, 2011). In the PEAP test, a need exists for an animal to make a conscious decision as to whether it should avoid/escape the painful stimulus or withstand it. Presumably, when pain is constant, the absence of pain is rewarding. Therefore, it is possible that some dopamine neurotransmission underlies decision-making involved in the PEAP test. The LHb contains efferent connections to the ventral tegmental area (VTA), an area well known to be involved in motivation relating to rewarding stimuli (Christoph, Leonzio, & Wilcox, 1986). The LHb also densely projects to the midbrain dorsal and median raphe nuclei, which contain a high amount of serotonergic projections of the brain (Aghajanian & Wang, 1977). 1.5 Purpose The LHb functions as a hub that integrates information from cortical regions to aid in emotional decision-making, and aids in creating response strategies under stress (Hikosaka, 2010; Proulx, Hikosaka, & Malinow, 2014; Thorton, Bradbury, & Davies, 1990; Evans & Thorton, 1984; Thorton & Evans, 1984; Thorton & Evans, 1982; Thorton, Evans, & Harris, 1985). Additionally, the LHb functions in a descending pain modulatory circuit involving both the NAc and PAG. These two functions of the LHb indicate its importance in decision-making during 5

15 heightened states of arousal, such as pain. As such, the purpose of the current experiment was to measure neuronal activation in the LHb after a test of pain affect in comparison to more basic sensory testing. Specifically, the hypothesis was tested that cells of the LHb become activated during the PEAP test. While the assertion has been made that the LHb is involved in the affective component of pain, this claim has not been directly tested. Therefore, the aim of this research is to offer further insight into the neurobiological underpinnings at play when pain is present. 6

16 Chapter 2 Preliminary Research 2.1 Study One To establish the role of the LHb in pain processing, a pilot study was conducted in which animals received either injections of carrageenan, an inflammatory agent, or saline into the left hindpaw. Three hours later, at the peak of maximum behavioral hyperalgesia to the carrageenan injection (Hedo, Laird, & Lopez-Garcia, 1999), animals were then sacrificed and perfused in order to examine neuronal activity in the LHb via immunohistochemistry (IHC) techniques. The amount of neuronal activation was quantified via c-fos expression in the brain, which is a marker of activity in neurons that occurs in response to stimulation (Harris, 1998) Subjects and Groups Six female Sprague-Dawley rats, weighing approximately g, were randomly selected from the University of Texas at Arlington vivarium and given 24 hours to habituate to the laboratory colony room. Animals were placed on a 12:12 hour light/dark cycle with food and water available ad libitum. All research was conducted in the University of Texas at Arlington Animal Care Facility. The study had approval of the Institution s Animal Care and Use Committee and it was conducted in accordance with The Ethical Guidelines for Investigations of Experimental Pain in Conscious Animals (Zimmerman, 1983) Procedures Subjects were randomly assigned into one of two experimental groups, which included those who received injections of carrageenan (inflammatory condition) and those that received injections saline (control condition). Subjects received injections of either 0.05 ml of 1% carrageenan or saline at 9:00 AM in the morning. Three hours later, subjects were deeply anesthetized using chloral hydrate and perfused prior to brain extractions and IHC procedures. 7

17 2.1.3 Immunohistochemistry (IHC) Animals were deeply anesthetized with chloral hydrate and intracardially perfused with 10mM phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in PBS. Brains were then removed and stored overnight in 4% paraformaldehyde at 4 C. The next morning, brains were transferred to a 20% glycerol in 0.1 M PBS solution for cryoprotection. Coronal sections (40μm) were cut on a freezing microtome and processed for IHC. c-fos staining was revealed by use of the avidin-biotin peroxidase complex method. Peroxidase activity was visualized by reaction with diaminobenzidine (Vector Laboratories, Burlingame, CA). For this procedure, brain sections were first treated with 0.3% H2O2 to destroy endogenous peroxidases and then incubated for 1 hour in 0.3% Triton X-100 and 3% normal goat serum to minimize nonspecific labeling. Tissue sections were then incubated overnight at room temperature in 1% normal goat serum, 0.3% Triton X-100 and c-fos antibody (1:150; Santa Cruz, Biotechnology, Santa Cruz, CA). Sections were washed, placed for 1.5 hours in 1:200 dilution of biotinylated goatantirabbit immunoglobulin (DakoCytomation, Carpinteria, CA), washed, and placed for 1.5 hours in 1:200 dilution of avidin-biotin complex from the Elite kit (Vector Laboratories, Burlingame, CA). Peroxidase activity was visualized by reaction with diaminobenzidine (Vector Laboratories, Burlingame, CA). An experimenter, not blinded to the condition, used coded slides to manually quantify the numbers of c-fos positive cells in the LHb (approximately -2.8 mm posterior to bregma) using a brain atlas for guidance (Paxinos & Watson, 1998) Results from Preliminary Study One An independent t-test was conducted to compare means between the groups on the cell count in the LHb. The results from the current pilot study indicated that the LHb was significantly more activated in the presence of an inflammatory pain condition than with no pain condition. Subjects with injections of carrageenan had significantly higher activation in the LHb as compared to subjects who received injections of saline t(4) = 3.17, p =

18 60 Number of c-fos Positive Cells % Carrageenan Saline 0 Condition Figure 2-1 Preliminary Study One c-fos Cell Counts 2.2 Study Two Another small pilot study was conducted to assess how varying percentages of carrageenan affected mechanical pain thresholds Subjects and Groups Fifteen female Sprague-Dawley rats, weighing approximately g, were randomly selected from the University of Texas at Arlington vivarium and given 24 hours to habituate to the laboratory colony room. Animals were placed on a 12:12 hour light/dark cycle with food and water available ad libitum. All research was conducted in the University of Texas at Arlington Animal Care Facility. The study had approval of the Institution s Animal Care and 9

19 Use Committee, and it was conducted in accordance with The Ethical Guidelines for Investigations of Experimental Pain in Conscious Animals (Zimmerman, 1983) Procedures Subjects were randomly assigned into one of three experimental groups, which included those who received injections of 0.5% carrageenan, 1% carrageenan, or 2% carrageenan. Subjects received injections of 0.05 ml carrageenan or saline at 9:00 AM in the morning. Three hours later, subjects underwent mechanical paw withdrawal threshold (MPWT) testing to assess for the presence of tactile allodynia Mechanical Paw Withdrawal Threshold (MPWT) Testing MPWT was accomplished by stimulation with Von Frey monofilaments of varied force ranging from 3.85 to mn. Subjects were placed atop a wire mesh floor inside of Plexiglas chambers and allowed to habituate for 10 minutes. Stimuli were applied for one second to the plantar surface of each hindpaw. Tactile sensitivity for each hindpaw was measured using the up-and-down method (Dixon, 1980) with eight von Frey monofilaments (3.85, 5.68, 9.74, 18.39, 39.42, 77.3, 135.3, and mn). Responses, or lack thereof, determined the pattern of application of the stimuli. Responses included actions in reference to the stimulus such as licking, flicking, flinching that results in removal of the paw, and total withdrawal of the paw. Lack of a response resulted in the application of a higher force stimulus and presence of a response resulted in application of a lower force stimulus. MPWT was established using a formula which considers the pattern of responding, the force for the initial response, and force of the last stimulation. Higher MPWT scores reflected less sensitivity, whereas lower MPWT scores indicated greater sensitivity and discomfort. Three trials were conducted for each paw and the scores were averaged. The maximum, or rate at which there was no response or sensitivity, was mn. 10

20 2.2.4 Results from Preliminary Study Two A one-way analysis of variance (ANOVA) was calculated on subjects MPWT score. There were no statistically significant differences detected among groups, F(2,14) = 2.03, p = % Carrageenan 1% Carrageenan 2% Carrageenan Threshold Value (mn) Condition Figure 2-2 Preliminary Study Two Threshold Values 11

21 Chapter 3 Method The current study consisted of one experiment that included quantifying levels of c-fos expression after animals went through different testing conditions (no test, MPWT, PEAP, or both MWPT and PEAP). Groups received different percentages of carrageenan (0.5% or 2%) or saline in equivalent volume to examine the effects of noxious stimulation at varying levels of intensity on c-fos expression. 3.1 Subjects and Groups One hundred and thirty-eight female Sprague-Dawley rats, weighing approximately g, were randomly selected from the University of Texas at Arlington vivarium and given 24 hours to habituate to the laboratory colony room. Six animals were excluded due to unsuccessful perfusions. Animals were placed on a 12:12 hour light/dark cycle with food and water available ad libitum. All research was conducted in the University of Texas at Arlington Animal Care Facility and in accordance with The Ethical Guidelines for Investigations of Experimental Pain in Conscious Animals (Zimmerman, 1983). 3.2 Procedures Subjects were randomly assigned into one of four experimental groups. Within each of the four groups, there were three condition levels (saline, 0.5% carrageenan, or 2% carrageenan). Subjects received injections of 0.05 ml of 0.5% carrageenan (n = 44), 2% carrageenan (n = 47), or saline (n = 47) at 9:00 AM in the morning. Three hours later, animals were tested, with subjects in the different groups receiving a different testing protocol. In the first group (injection only), subjects simply received injections of either saline or one of the two doses of carrageenan. In the other three groups, subjects received injections of saline or carrageenan and then three hours later underwent either MPWT testing, PEAP testing, or a combination of both. A table of group assignment is provided in Table 3-1. Following testing, subjects were deeply anesthetized using chloral hydrate and perfused prior to brain extractions 12

22 and IHC procedures. Crucially, all subjects, regardless of group assignment, were sacrificed at the same time on a given testing day. Table 3-1 Group Assignment and Subject Size per Group Group Condition No Test MPWT PEAP MPWT + PEAP Saline % Carrageenan % Carrageenan Mechanical Paw Withdrawal Threshold (MPWT) Testing Refer to section Place Escape Avoidance Paradigm (PEAP) Testing PEAP testing occurred after placing subjects into a half-light/half-dark chamber (40.5 x 30.5 x 15.5 cm) atop raised wire mesh. During the 30-minute test, the plantar surfaces of the hindpaws were stimulated every 15 seconds with a suprathreshold Von Frey filament (476 mn of force). Specifically, stimulation was applied to the injected left hindpaw while in the dark side of the chamber and to the right/unaffected hindpaw when in the light side of the chamber. The wire mesh and chamber was cleaned between test subjects to minimize scent cues, and subjects were randomly assigned to placement of the light side of the chamber on either the right or left side of the mesh Immunohistochemistry (IHC) c-fos staining was revealed by use of the avidin-biotin peroxidase complex method. Peroxidase activity was visualized by reaction with diaminobenzidine (Vector Laboratories, Burlingame, CA). For this procedure, brain sections were first treated with 0.3% H2O2 to destroy endogenous peroxidases and then incubated for 1 hour in 0.3% Triton X-100 and 3% 13

23 normal donkey serum to minimize nonspecific labeling. Tissue sections were then incubated overnight at room temperature in 1% normal donkey serum, 0.3% Triton X-100 and c-fos antibody (1:1500; Santa Cruz, Biotechnology, Santa Cruz, CA). Sections were washed, placed for 1.5 hours in 1:200 dilution of biotinylated donkey-antirabbit immunoglobulin (DakoCytomation, Carpinteria, CA), washed, and placed for 1.5 hours in 1:200 dilution of avidinbiotin complex from the Elite kit (Vector Laboratories, Burlingame, CA). Peroxidase activity was visualized by reaction with diaminobenzidine (Vector Laboratories, Burlingame, CA). An experimenter, not blinded to the condition, used coded slides to manually quantify the numbers of c-fos positive cells in the LHb (approximately mm to mm posterior to bregma) using a brain atlas for guidance (Paxinos & Watson, 1998). 14

24 Chapter 4 Results 4.1 Behavioral Results For statistical analyses, SPSS Software Version 23.0 was used. Post hoc effects were not investigated when there was no interaction MPWT Outcomes Oneway ANOVAs were conducted for both left and right hindpaw scores to determine mechanical hypersensitivity and to assess efficacy of the experimental conditions. See Figure 4-1 and Figure 4-2 for scores by group. For the left hindpaw data, there was a statistically significant difference between groups, F(2,68) = , p <.001. Fisher s LSD post-hoc tests revealed that the 2% carrageenan group threshold values were significantly lower than both 0.5% carrageenan (p <.001) and saline (p <.001). Results for the right hindpaw data indicated that there were not statistically significant differences among groups, F(2,68) =.049, p = n.s. 15

25 500 Saline 0.5% Carrageenan 2% Carrageenan 400 Threshold Value (mn) Condition Figure 4-1 Left MPWT Outcomes 16

26 Saline 0.5% Carrageenan 2% Carrageenan Threshold Values (mn) Condition Figure 4-2 Right MPWT Outcomes PEAP Outcomes Mixed repeated measures ANOVAs were conducted with condition (saline, 0.5% carrageenan, or 2% carrageenan) as the between-subject variables and time as the withinsubject variable to assess preference percentages over the course of the test. Simple effects were not investigated when there was no interaction effect. Results of approach-avoidance behavior can be seen in Figure 4-3. For the approach-avoidance data, results indicated that there was a significant main effect of time, F(5,335) = 3.34, p =.006. There was also a significant interaction between time and condition, F(10,335) = 3.28, p <.001. Fisher s LSD post-hoc tests showed that both doses of carrageenan spent significantly more time in the light side of the chamber at the ten minute time bin, p =.005 for 0.5% carrageenan and p =.001 for 2% carrageenan; at the fifteen minute 17

27 time bin, p <.001 for 0.5% carrageenan and p <.001 for 2% carrageenan; at the twenty minute time bin, p =.002 for 0.5% carrageenan and p <.001 for 2% carrageenan; at the twenty-five minute time bin, p =.002 for 0.5% carrageenan and p <.001 for 2% carrageenan; and finally, at the thirty minute time bin, p <.001 for 0.5% carrageenan and p <.001 for 2% carrageenan. Percent of Time Spent in Light Side of Chamber Saline 0.5% Carrageenan 2% Carrageenan Time Figure 4-3 Approach-Avoidance Outcomes 4.2 Immunohistochemical (IHC) Results For statistical analyses, SPSS Software Version 23.0 was used. Post hoc effects were not investigated when there was no interaction. 18

28 4.2.1 IHC Outcomes The mean number of c-fos positive cells present in the LHb for the no-test group was compared using a univariate ANOVA. The no-test group was chosen for analysis in order to examine the direct effects of carrageenan on c-fos expression without the additional influence of behavioral testing procedures. Analysis of cell counts showed that there was not a statistically significant difference among groups F(2,28) = 2.84, p =.075. However, it was shown that animals injected with 2% carrageenan demonstrated significantly more c-fos positive cells in the LHb relative to saline controls (p =.038). The 0.5% carrageenan group, while higher in terms of c-fos expression, was only marginally statistically different from saline controls (p =.062). There was not a significant difference between the doses of carrageenan, p = n.s. See Figure 4-4 for results of IHC data. Number of c-fos Positive Cells Saline 0.5% Carrageenan 2% Carrageenan 0 Condition Figure 4-4 Immunohistochemical Outcomes by Condition 19

29 To further explore the relationship between neuronal activation in the LHb and the behavioral results (MPWT and PEAP scores), linear regression analyses were conducted. Analyses indicated that there was not a significant linear relationship existed between the degree of mechanical hypersensitivity and neuronal activation in the LHb, F(1,43) =.000, p = n.s., R 2 =.000. Results can be seen in Figure 4-5. Moreover, there was not a significant linear relationship between averaged preference data from PEAP scores and neuronal activation in the LHb, F(1,42) =.203, p = n.s., R 2 =.005. The results from this regression analysis are shown in Figure 4-6. Number of c-fos Positive Cells % Carrageenan 2% Carrageenan Regression Line Mechanical Paw Withdrawal Threshold Value (mn) Figure 4-5 Number of c-fos Positive Cells and MPWT Values 20

30 Number of c-fos Positive Cells % Carrageenan 2% Carrageenan Regression Line Percent of Time Spent in Light Side of Chamber Figure 4-6 Number of c-fos Positive Cells and Approach-Avoidance Outcomes Lastly, to examine whether c-fos levels differed depending upon group assignment (no test, MPWT, PEAP, or MPWT and PEAP), a mixed ANOVA was conducted. Results indicated that there was not a statistically significant difference among groups, F(3,122) = 1.79, p = n.s. 4.3 General Findings: Behavioral Results Mechanical Paw Withdrawal Threshold For the current study, MPWT scores acquired were in line with expectations, meaning that carrageenan injections induced inflammation. In fact, the results indicated a simple, stepwise pattern in which increases in inflammatory intensity led to decreases in threshold values. Also, there were significant statistical differences among groups, which demonstrated that carrageenan was efficacious in inducing allodynia, at least among animals that underwent 21

31 MPWT testing. Therefore, a concern for lack of allodynia was not an issue moving forward with statistical analyses Place Escape/Avoidance Paradigm As expected, results from the PEAP tests revealed that animals injected with either dose of carrageenan (0.5% and 2%) significantly avoided stimulation more to the inflamed hindpaw compared to control animals. Interestingly, although MPWT scores were significantly different among each condition, PEAP scores did not reflect this same trend. 4.4 General Findings: IHC Results Immunohistochemistry In accordance with previous literature (Harris, 1998), there were heightened levels of c- Fos expression resulting from both doses of carrageenan, and the 2% carrageenan dose was statistically different from saline controls. In addition, there was not a significant relationship demonstrated between MPWT values and levels of c-fos expression. Curiously, there was not a significant relationship shown between PEAP scores and levels of c-fos expression, which was not expected. In addition to these findings, c-fos expression was examined based upon group assignment (no test, MPWT, PEAP, or MPWT and PEAP). In contrast to expectations, c- Fos expression did not differ significantly among the test conditions. 22

32 Chapter 5 Discussion Pain is the subjective, psychological experience that accompanies physical injury to the body. Although helpful when felt briefly, it can be debilitating if chronic. Merksey and Bogduk (1994) have defined chronic pain is defined as pain that is present beyond the normal healing process, and exemplifies aberrant processing in the central nervous system. Unfortunately, over 100 million people in the United States develop chronic pain at some point in their lives (Institute of Medicine, 2011). Pain may also engender a host of negative emotions such as depression, anxiety, and anger (Gatchel et al., 2007). Financially, pain costs the United States half a trillion dollars annually, as measured in terms of health care usage, lost wages, and quality of life (Institute of Medicine, 2011). Moreover, individuals suffering from chronic pain will likely have to take time off from work, resulting in a loss of income that further adds to any existing negative emotions. Indisputably, chronic pain is a major stressor that leads to a steep decline in one s quality of life (Gatchel et al., 2007). Much is currently known about chronic pain; still, with the problem of suffering remaining unsolved, additional research is always needed. Pain affect, as a construct, is an experimental proxy for suffering, and therefore its study in a preclinical setting can ultimately be beneficial for clinical populations. As mentioned earlier, pain affect describes how unpleasant pain is perceived and signifies its emotional quality (Craig, 2003; Melzack & Casey, 1968). It is important for understanding how an organism is motivated to end its pain by means of escape or avoidance, and, in this way, pain is intuitively similar to other homeostatic mechanisms in that it demands a response when it crosses a certain threshold (Craig, 2003; LaBuda & Fuchs, 2000). With this in mind, the aim of this research was to assess neuronal activation in an area previously reported to be a part of the pain experience, the lateral habenula. The LHb is an epithalamic structure, tiny in comparison to other players in pain processing. Nonetheless, it is 23

33 a crucial relay station that conveys afferents to more frontal areas and sends efferents back down in a descending pain modulatory circuit (Shelton, Becerra & Borsook, 2012). The habenula consists of two nuclei which serve to link limbic forebrain structures to the midbrain (Sutherland, 1982; Wang & Aghajanian, 1977). This structure is involved in an array of functions, including homeostasis, pain, and stress processing, among others (Andres, Von During, & Veh, 1999). The LHb, in particular, functions to participate in the interaction of pain and behavior; namely, how pain serves to motivate an animal to avoid sources of painful stimulation (Shelton, Becerra, & Borsook, 2012). In addition to its function in pain processing, the LHb assists in creating adaptive strategies when an organism is under stress (Thorton & Evans, 1982; Thorton, Evans, & Harris, 1985). For example, Thorton and Davies (1991) have noted that lesions to the habenula affected the ability of subjects to change response strategies when placed under demanding conditions, such that animals will perseverate even when the behavior is not advantageous to avoiding stress. Furthermore, research has shown that neurons in the LHb encode negative reward prediction, meaning they are excited by unexpected non-rewarding events and inhibited by unexpected rewarding events (Proulx, Hikosaka, & Malinow, 2014). Indeed, the habenula is an area organized to coordinate interactions between reward processing and pain modulation via transmission of dopamine and serotonin (Lee & Goto, 2011). With this in mind, it logically follows that the LHb may process information related to the affective component of pain. 5.1 Study Summary To test the current hypothesis of the LHb s contribution to the affective dimension of pain, animals received injections of one of three solutions (saline, 0.5% or 2% carrageenan) to the left hindpaw, and then underwent either MPWT testing, PEAP testing, a combination of both, or simply no testing at all. For MPWT testing, animals injected with 2% carrageenan had significantly lower threshold values compared to 0.5% carrageenan (p <.001) and controls (p <.001), indicating that the experimental conditions were effective. During the PEAP test, animals 24

34 in pain were expected to first escape and avoid an area that they normally prefer, and indeed, behavioral results supported this notion. Animals in both inflammatory groups did avoid stimulation and spent significantly more time in the light side of the chamber compared to controls for most of the duration of the test (both ps <.05). Additionally, cells of the LHb were examined to assess whether there were higher levels of neuronal activation as measured by c-fos protein expression. The no-test group was chosen for analysis in order to examine the direct effects of carrageenan on c-fos expression without the additional influence of behavioral testing procedures. Analysis of cell counts showed that there was not a statistically significant difference among groups (p =.075). However, it was shown that animals injected with 2% carrageenan demonstrated significantly more c-fos positive cells in the LHb relative to saline controls (p =.038). The 0.5% carrageenan group, while higher in terms of c-fos expression, was only marginally different from saline controls (p =.062). There was not a significant difference between the doses of carrageenan (p = n.s.). To further examine the hypothesis that the LHb contributes to pain affect, the relationship between levels of c-fos expression and PEAP scores were examined. There was not a significant relationship between these two variables (p = n.s.). Interestingly, there was not a significant relationship between c-fos expression and MPWT scores (p = n.s.). Although not significant, the relationship between PEAP scores and c-fos levels was stronger, and therefore this could support the idea that the LHb may participate (albeit in a small manner) in the affective component of pain processing. 5.2 Conclusion The complexity of pain can be owed in large part to its multidimensional nature. Historically, pain has been tested in a preclinical setting through simple reflexive measures, but with the advent of the new model of pain developed by Melzack and Casey (1968), researchers began to tease apart the sensory, affective, and cognitive aspects that comprise the pain experience. 25

35 A survey of supraspinal mechanisms is vital for understanding pain s various dimensions. For example, heightened or depressed activity in dopaminergic connections between various centers can influence emotional decision-making, the creation of response strategies under stress, as well as avoidance behaviors. Based upon results from this research, the LHb, while equipped with the hardware necessary to modulate pain affect, was not shown to play a major role as supported by data from the IHC techniques used herein. An examination of the neural substrates at play is a small first step that must be taken towards the ultimate goal of alleviating the problem of chronic pain. 26

36 References Andres, K.H., Von During, M., & Veh, R.W. (1999). Subnuclear organization of the rat habenular complexes. The Journal of Comparative Neurology, 407, Atweh, S.F., & Kuhar, M.J. (1977). Autoradiographic localization of opiate receptors in rat brain. II. The brain stem. Brain Research, 129, Baastrup, C., Jensen, T.S., & Finnerup, N.B. (2011). Pregabalin attenuates place escape/avoidance behavior in a rat model of spinal cord injury. Brain Research, 1370, Borsook, D., Edwards, R., Elman, I., Becerra, L., & Levine, J. (2013). Pain and analgesia: The value of salience circuits. Progress in Neurobiology, 104, Christoph, G.R., Leonzio, R.J., & Wilcox, K.S. (1986). Stimulation of the lateral habenula inhibits dopamine-containing neurons in the substantia nigra and ventral tegmental area of the rat. The Journal of Neuroscience, 6(3), Cohen, S.R., Kimes, A.S., & London, E.D. (1991). Morphine decreases cerebral glucose utilization in limbic and forebrain regions while pain has no effect. Neuropharmacology, 30, Cohen, S.R., & Melzack, R. (1985). Morphine injected into the habenula and dorsal postero thalamus produces analgesia in the formalin test. Brain Research, 359, Cohen, S.R., & Melzack, R. (1986). Habenular stimulation produces analgesia in the formalin test. Neuroscience Letters, 70, Craig, A.D. (2003). A new view of pain as a homeostatic emotion. Trends in Neurosciences, 26(6), Dixon, W. J. (1980). Efficient analysis of experimental observations. Annual Review of Pharmacological Toxicology, 20,

37 Evans, J.A.C., & Thorton, E.W. (1984). Impaired acquisition of DRL operant responding following lesion of the habenular nucleus. Physiological Psychology, 12, Fernandez, E., & Turk, D.C. (1992). Sensory and affective components of pain: separation and synthesis. Psychological Bulletin, 112(2), Freire, M.A.M., Guimaraes, J.S., Leal, W.G., & Pereira, A. (2009). Pain modulation by nitric oxide in the spinal cord. Frontiers in Neuroscience, 3(2), Fuchs, P.N., & Cox, V.C. (1992). Habenula lesions attenuate lateral hypothalamic analgesia in the formalin test. NeuroReport, 4, Gatchel, R. J., Peng, Y. B., Peters, M. L., Fuchs, P. N., & Turk, D. C. (2007). The biopsychosocial approach to chronic pain: Scientific advances and future directions. Psychological bulletin, 133(4), Hayes, D.J., & Northoff, G. (2012). Common brain activations for painful and non-painful aversive stimuli. BMC Neuroscience, 13(60), Hedo, G., Laird, J.M., Lopez-Garcia, J.A. (1999). Time-course of spinal sensitization following carrageenan-induced inflammation in the young rat: a comparative electrophysiological and behavioural study in vitro and in vivo. Neuroscience, 92(1), Hikosaka, O. (2010). The habenula: From stress evasion to value-based decisionmaking. Nature Reviews Neuroscience, 11, Institute of Medicine. (2011). Relieving pain in America: A blueprint for transforming prevention, care, education, and research. Washington D.C.: The National Academies Press. 28

38 Johansen, J.P., Fields, H.L., & Manning, B.H. (2001). The affective component of pain in rodents: Direct evidence for a contribution of the anterior cingulate cortex. Proceedings of the National Academy of Sciences, 98(14), LaBuda, C. J., & Fuchs, P. N. (2000). A behavioral test paradigm to measure the aversive quality of inflammatory and neuropathic pain in rats. Experimental neurology, 163(2), Lee, A.L., & Goto, Y. (2011). Neurodevelopmental disruption of cortico-striatal function caused by degeneration of habenula neurons. PLoS ONE, 6(4), Lee, E.H.Y., & Huang, S.L. (1988). Role of lateral habenula in the regulation of exploratory behavior and its relationship to stress in rats. Behavioural Brain Research, 30, Loeser, J.D., & Melzack, R. (1999). Pain: An overview. The Lancet, 353(9164), Loeser, J.D., & Treede, R.D. (2008). The Kyoto protocol of IASP basic pain terminology. Pain, 137, Mahieux, G., & Benabid, A.L. (1987). Naloxone-reversible analgesia induced by electrical stimulation of the habenula in the rat. Brain Research, 406, McNabb, C.T., Uhelski, M.L., & Fuchs, P.N. (2012). A direct comparison of affective pain processing underlying two traditional pain modalities in rodents. Neuroscience Letters, 507, Melzack, R., & Casey, K. L. (1968). Sensory motivational and central control determinants of pain: A new conceptual model. Kenshalo DR, ed. The Skin Senses. Springfield: CC Thomas (pp ). 29

39 Merskey, H., Bogduk, N. (Eds.). (1994). Classifications of chronic pain: Descriptions of chronic pain syndromes and definitions of pain terms. (2 nd ed.). Seattle, WA: IASP Press. Mouraux, A., Diukova, A., Lee, M.C., Wise, R.G., & Iannetti, G.D. (2011). A multisensory investigation of the functional significance of the pain matrix. Neuroimage, 54(3), Paxinos, G., & Watson, C. (1998). The Rat Brain, 4th ed. Academic Press, San Diego. Pederson, L.H., & Blackburn-Munro, G. (2006). Pharmacological characterization of place escape/avoidance behavior in the rat chornic constriction injury model of neuropathic pain. Psychopharamcology, 185, Proulx, C.D., Hikosaka, O., & Malinow, R. (2014). Reward processing by the lateral habenula in normal and depressive behaviors. Nature Neuroscience, 17(9), 1 7. Rainville, P. (2002). Brain mechanisms of pain affect and pain modulation. Current Opinion in Neurobiology, 12(2), Shelton, L., Becerra, L., & Borsook, D. (2012). Unmasking the mysteries of the habenula in pain and analgesia. Progress in Neurobiology, 96, Sutherland, R.J. (1982). The dorsal diencephalic conduction system: A review of the anatomy and functions of the habenular complex. Neuroscience & Biobehavioral Reviews, 6, Terenzi, M.S., Guimaraes, F.S., & Prado, W.A. (1990). Antinociception induced by stimulation of the habenular complex of the rat. Brain Research, 524, Terenzi, M.S., & Prado, W.A. (1990). Antinociception elicited by electrical or chemical stimulation of the rat habenular complex and its sensitivity to systemic antagonists. Brain Research, 535,

40 Thorton, E.W., & Bradbury, G.E. (1989). Effort and stress influence the effect of lesion of the habenula complex in one-way active avoidance learning. Physiology & Behavior, 45, Thorton, E.W., Bradbury, G.E., & Davies, C. (1990). Increased immobility in an automated forced swimming test following lesion of the habenula in rats: Absence of evidence for a contribution from motor impairment. Behavioral Neuroscience, 104, Thorton, E.W., & Davies, C. (1991). A water-maze discrimination learning deficit in the rat following lesion of the habenula. Physiology & Behavior, 49, Thorton, E.W., & Evans, J.A.C. (1982). The role of habenular nuclei in the selection of behavioral strategies. Physiological Psychology, 10(3), Thorton, E.W., & Evans, J.A.C. (1984). The effects of lesions of the habenula nucleus on lever press behaviour during a tandem operant schedule with contrasting response requirements. Behavioural Brain Research, 12, Thorton, E.W., Evans, J.A.C., & Harris, C. (1985). Attenuated response to nomifensine in rats during a swim test following lesion of the habenula complex. Psychopharmacology, 87, Turk, D.C., & Dworkin, R.H. (2004). What should be the core outcomes in chronic pain clinical trials? Arthritis Research & Therapy, 6(4), Uhelski, M.L., Davis, M.A., & Fuchs, P.N. (2012). Pain affect in the absence of pain sensation: Evidence of asomaesthesia after somatosensory cortex lesions in the rat. Pain, 153, Vale-Martinez, A., Marti-Nicolovius, M., Guillazo-Blanch, G., Coll-Andreu, M., & Morgado- Bernal, I. (1997). Effects of habenular lesions upon two-way active avoidance conditioning in rats. Neurobiology of Learning and Memory, 68,