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Role of CRF2 Receptors in the Negative Mood State Associated with Acute Nicotine Withdrawal

Permanent Link: http://ufdc.ufl.edu/UFE0025044/00001

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Title: Role of CRF2 Receptors in the Negative Mood State Associated with Acute Nicotine Withdrawal
Physical Description: 1 online resource (38 p.)
Language: english
Creator: Kumar, Pranoo
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: acute, corticotropin, crf2, nicotine, withdrawal
Medicine -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Corticotropin releasing factor, also known as CRF, has been suggested to play a role in depression, anxiety disorders, drug addictions, and the the negative mood state associated with tobacco withdrawal. Nicotine is the main psychoactive component of tobacco smoke that leads to the tobacco addiction. Nicotine withdrawal leads to an increased release of CRF which can mediate a deficit in brain reward function and increased anxiety-like behavior. This experiment investigated the role of CRF2 receptors in the negative mood state associated with nicotine withdrawal. The rat intracranial self-stimulation (ICSS) procedure was used to assess the negative mood state associated with nicotine withdrawal. Nicotine withdrawal lead to elevated brain reward thresholds, which is indicative of a decrease sensitivity to rewarding electrical stimuli. The CRF2 receptor antagonist astressin- 2B did not prevent the elevations in brain reward threshold associated with nicotine withdrawal. This shows that the CRF2 receptors do not play a role in the negative mood state associated with nicotine withdrawal.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Pranoo Kumar.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Bruijnzeel, Adriaan Willem.

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Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0025044:00001

Permanent Link: http://ufdc.ufl.edu/UFE0025044/00001

Material Information

Title: Role of CRF2 Receptors in the Negative Mood State Associated with Acute Nicotine Withdrawal
Physical Description: 1 online resource (38 p.)
Language: english
Creator: Kumar, Pranoo
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: acute, corticotropin, crf2, nicotine, withdrawal
Medicine -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Corticotropin releasing factor, also known as CRF, has been suggested to play a role in depression, anxiety disorders, drug addictions, and the the negative mood state associated with tobacco withdrawal. Nicotine is the main psychoactive component of tobacco smoke that leads to the tobacco addiction. Nicotine withdrawal leads to an increased release of CRF which can mediate a deficit in brain reward function and increased anxiety-like behavior. This experiment investigated the role of CRF2 receptors in the negative mood state associated with nicotine withdrawal. The rat intracranial self-stimulation (ICSS) procedure was used to assess the negative mood state associated with nicotine withdrawal. Nicotine withdrawal lead to elevated brain reward thresholds, which is indicative of a decrease sensitivity to rewarding electrical stimuli. The CRF2 receptor antagonist astressin- 2B did not prevent the elevations in brain reward threshold associated with nicotine withdrawal. This shows that the CRF2 receptors do not play a role in the negative mood state associated with nicotine withdrawal.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Pranoo Kumar.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Bruijnzeel, Adriaan Willem.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0025044:00001


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1 ROLE OF CRF2 RECEPTORS IN THE NEGATIVE MOOD STATE ASSOCIATED WITH ACUTE NICOTINE WITHDRAWAL By PRANATI KUMAR A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009

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2 2009 Pranati Kumar

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3 To my parents, Pramodh and Pratima Kumar, for their continued support and love throughout this experience

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4 ACKNOWL EDGMENTS I would like to thank the members of my supervisory committee, Drs. Mark Gold, Adrie Bruijnzeel, and Gregory Schultz, for their continued mentoring and support throughout this process. They are all true inspirations and I thank them for their enco uragement. I would like to recognize Adrie Bruijnzeel once again and thank him for his personal commitment to guiding me throughout this process. I would also like to thank Joyce Conners for her true dedication and commitment to advising me through this Ma sters program and informing me about classes and seminars. I would also like to thank the members of my lab for their support and help throughout the experimental process. If it were not for these wonderful people, I would not have been able to achieve thi s goal.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES ................................................................................................................................ 6 LIST OF FIGURES .............................................................................................................................. 7 ABSTRACT .......................................................................................................................................... 8 CHAPTER 1 INTRODUCTION ......................................................................................................................... 9 Tobacco Use and Effects .............................................................................................................. 9 Corticotropin Releasing Factor .................................................................................................. 10 Aim of Investigation ................................................................................................................... 11 2 MATERIALS AND METHODS ............................................................................................... 15 Subjects ........................................................................................................................................ 15 Drugs ............................................................................................................................................ 15 Surgical Procedure of Cannula and Electrode ........................................................................... 16 Intracranial Self Stimulation Procedure (ICSS) ....................................................................... 16 Osmotic Minipump Surgery ....................................................................................................... 17 Experimental Procedure .............................................................................................................. 17 Statistics ....................................................................................................................................... 18 3 RESULTS .................................................................................................................................... 22 4 DISCUSSION .............................................................................................................................. 26 LIST OF REFERENCES ................................................................................................................... 34 BIOGRAPHICAL SKETCH ............................................................................................................. 38

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6 LIST OF TABLE S Table page 2 1 Procedural list of experiment. ................................................................................................ 19

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7 LIST OF FIGURES Figure page 1 1 Brain Reward System involving nicotine intake. ................................................................. 12 1 2 Diagram of the effect of CRF1 and CRF2. ............................................................................ 13 1 3 Diagram of the eff ect of CRF1 and CRF2 antagonists. ........................................................ 13 1 4 Diagram of the brain and regions of importance in relation to dopamine release. ............ 14 2 1 Apparatus used during the cannula and electrode surgical procedure. ............................... 19 2 2 Using a flat skull and taken from bregma, coordinates for the electrode and cannula were identified. ....................................................................................................................... 20 2 3 Osmotic minipumps were used and filled with either saline or nicotine dissolved in saline. ...................................................................................................................................... 20 2 4 The ICSS procedure was performed in ope rant boxes within sound attenuated chambers ................................................................................................................................. 21 3 1 Effect of CRF2 receptor antagonist astressin 2B on brain reward threshold elevations associated with mecamylamine -precipitated nicotine with drawal ...................................... 24 3 2 Effect of CRF2 receptor antagonist astressin 2B on response latencies of rats chronically treated with saline or nicotine and acutely treated with mecamylamine ........ 24 3 3 Effect of CRF1 receptor antagonist R278995/CRA0450 on brain reward threshold elevations associated with mecamylamine -precipitated nicotine withdrawal .................... 25 3 4 Effect of CRF1 receptor antagonist R278995/CRA0450 on response latencies of rats chronically treated with saline or nicotine and acutely treated with mecamylamine ........ 25 4 1 Mode l of the blood-brain barrier including the capillaries and astrocytes ......................... 32 4 2 Different modes of transportation of drugs include intramuscular, transdermal, subcutaneous, and intravenous. ............................................................................................. 32 4 3 When a drug is taken orally, it must pass through the liver before being distributed to the appropriate region of the body ........................................................................................ 33

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8 A bstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree o f Master of Science ROLE OF CRF2 RECEPTORS IN THE NEGATIVE MOOD STATE ASSOCIATED WITH ACUTE NICOTINE WITHDRAWAL By Prana ti Kumar August 2009 Chair: Adrie Bruijnze e l Cochair: Mark Gold, Gregory Schultz Major: Medical Sciences Corticotropin releasing factor, also know n as CRF, has been suggested to play a role in depression, anxiety disorders, drug addictions, and the the negative mood state associated with tobacco withdrawal Nicotine is the ma in psychoactive component of tobacco smoke that leads to the tob acco addiction Nicotine withdrawal leads to an increased release of CRF which can mediate a deficit in brain reward function and increased anxiety -like behavior. This experiment investigated the role of CRF2 receptors in the negative mood state associated with nicotine withdrawal. The rat intracranial self -stimulation (ICSS) procedure was used to assess the negative mood state associated with nicotine withdrawal. Nicotine withdrawal lead to elevated brain reward thresholds, which is indicative of a decreas e sensitivity to rewarding electrical stimuli. The CRF2 receptor antagonist astressin 2B did not prevent the elevations in brain reward threshold associated with nicotine withdrawal This shows that the CRF2 receptors do not play a role in the negative m ood state associated with nicotine withdrawal.

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9 CHAPTER 1 INTRODUCTION Tobacco Use and Effects Tobacco use is one of the leading cause of preventable deaths in the United States, with annual rates continuing to grow worldwide (Center for Disease Control 2002). Since 1964, nearly 12 million Americans have died, 438,000 annually, or about 1 in every 5 death occurrences (Center for Disease Control 2002). This alone is more than HIV related, illegal drug use such as cocaine and heroin, alcohol use, motor vehicle injuries, suicide, and murder cases combined (Center for Disease Control 2002; McGinnis and Foege 1993). Nicotine is the major psychoactive ingredient of tobacco smoke and is thought to cause the addictive symptoms that lead to dependence and persi stence of smoking (Kenny and Markou 2001; Kenny and Markou 2005: Stolerman and Jarvis 1995). The acute effects of nicotine use include activation of the brain reward systems, such as the mesolimbic dopamine system (George et al. 2007; Koob and LeMoal 2005; Tiffany et al. 2004). The mesolimbic pathway, also known as the reward pathway, is one of the dopaminergic pathways in the brain in which the neurotransmitter dopamine is transported from one region of the brain to another. Dopamine is transported from the ventral tegmental area of the midbrain to the nucleus accumbens, amygdala, hippocampus, and the medial prefontral cortex, all of which make up the limbic system (Tisch et al. 2004). Feelings of relaxation, alleviation of stress and anxiety, energy i ncrease, and euphoric sensation occur as a result of this passage (Clarke 1987; Pomerleau and Pomerleau 1992). During abstinence however, negative emotional behavioral symptoms occur as a result of the neuradaptative changes that have caused dependency and continued use of tobacco (Epping Jordan et al. 1998; Hughes et al. 1994). This is known as nicotine withdrawal syndrome and is characterized by irritability, anxiety, sleep deprivation, increased appetite, cravings, restlessness,

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10 and impatience (Gross an d Stitzer 1989). Relapse occurs soon thereafter in humans because of the inability to overcome these symptoms (Kenny and Markou 2001; Kenny and Markou 2005). Cessation of nicotine shows similar results in animals including anxiety and stress and a defici t in brain reward function (Costall et al. 1989; Costall et al. 1990; Markou and Koob 1991; Schulteis et al. 1994; Schulteis et al. 1995; Stinus et al. 1990). Dopamine secretion is known to be the reason for other addictive substances such as cocaine, op iates, and alcohol (Fu et al. 2000; Nisell et al. 1995; Pontieri et al. 1992). Nicotine increases the rate of firing signals of dopamine to the ventral tegmental area and as a result stimulates nucleus accumbens (Nacc) dopamine release (Fu et al. 2000; Nis sell et al. 1996; Fisher et al. 1998; Nissell et al. 1994; Schilstrom et al. 1998a,b). In addition to the Nacc, the extended amygdala, which includes the central nucleus of the amygdala or CeA, is another important site of drug abuse induction (Funk and K oob 2007). It is in this region that the stress response is activated during the withdrawal syndrome to which the CRF system is activated (Bruijnzeel and Gold 2005). Corticotropin Releasing Factor Corticotropin releasing factor (CRF) is thought to play a significant role in the development of addiction during tobacco use as well as withdrawal symptoms during abstinence (Lu et al. 2000). CRF, a 41 amino acid peptide derived from a 191 preprohormone, is recognized as a hypothalamic factor that stimulates corticotropin (Adrenocorticotropin hormone or ACTH) from the anterior lobe of the pituitary gland when coupled with corticotropes containing CRF receptors (Vale et al. 1981; Cummings et al. 1983). This factor is distributed throughout the central nervous system (CNS), with a high concentration in the paraventricular nucleus of the hypothalamus and areas of the extended amygdala (Lu et al. 2000; Funk and Koob 2007; Vale et al. 1981; Bloom et al. 1982; Dunn and Berridge 1990). There are two types of

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11 cortic otropin releasing factor receptor subtypes, CRF1 and CRF2, which are thought to stimulate stress response and a negative emotional system when coupled with CRF (George et al. 2007; Takahashi 2001). CRF1 and CRF2 receptors are Gs coupled receptors (Koob 1 999). CRF1 receptors are expressed in the medial septum, pituitary, cortex, cerebellum, hindbrain, and olfactory bulb while the CRF2 receptors are found in the lateral septum, ventralmedial hypothalamus, and choriod plexus (Koob 1999). CRF and Ucn (urocor tin) play significant roles in the central nervous system as they activate arousal and have the potential to produce seizures (Koob 1999). Urocortin structurally related to CRF and is a ligand to CRF2 recptors. CRF and Ucn bind with high affinity to CRF1 receptors while only Ucn is able to bind to CRF2 receptors successfully (Koob 1999). In relation to the seizures and negative neural, behavioral and physical responses, various CRF antagonists are able to abolish these effects (Koob 1999; Curtis et al. 1 994; Vale et al. 1981). Recently, specific experiments have been performed using CRF receptor antagonists and agonists to alleviate the symptoms brought on by cessation of drug abuse (Bruijnzeel et al. 2006). They do this by blocking the receptor of CRF (Lu et al. 2000; Urban et al. 2006). Aim of Investigation The aim of this experiment was to investigate the role of CRF2 receptors in the negative mood state associated with acute nicotine withdrawal. It was hypothesized that the CRF2 receptors play a me diating role in the deficit in brain reward associated with and negative mood state associated with acute nicotine withdrawal.

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12 Nicotine intake Activates Nicotinic acetylcholine receptor (nAChR) Activates Mesolimbic pathway dopm ine system Stimulates Dopamine release from the ventral tegmental area of the brain to 2 regions 1. Nucleus accumbens 2. Central nucleus of the amygdala Stimulates Activates Euphoric feelings occur because of this passage This region activates a stress response during the withdrawal period of nicotine intake Activates Corticotropin releasing factor CRF1 CRF2 Figure 1 1. Brain Reward System involving nicotine intake.

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13 Nicotine withdrawal CRF1 CRF2 Negative Mood States Figure 1 2. Diagram of the effect of CRF1 and CRF2. Nicotine Withdrawal CRF1 antagonist CRF2 antagonist Attenuation of Negative Mood States Figure 1 3. Diagram of the effect of CRF1 and CRF2 antagonists.

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14 Figure Figure 1 4. Diagram of the brain and regions of importance in relation to dopamine release.

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15 CHAPTER 2 MATERIALS AND METHOD S Subjects The animals used in this experiment were Male Wistar rats (Charles River, Raleigh, NC) weighing between 200 and 300g. These subjects were placed in cages, grouphoused, and given water and ad libitum fo od Their conditions were temperature and humidity controlled as well. Testing occurred between 9 AM and 12 PM to ensure stable results everyday. They were also maintained on a 12 hour light dark cycle, where the lights went off at 6 PM, within the r oom they were placed in. Animal care was given with the highest and utmost care and treated in accordance with the National Institute of Health guidelines. Animal facilities and experimental protocols were in accordance with the Association for the Asses sment and Accreditation of Laboratory Animal care and approved by the University of Florida Institutional Animal Care and Use Committee. Drugs Nicotine and mecamylamine were purchased from Sigma (Sigma -Aldrich, St. Louis, MO, USA) and dissolved in salin e CRF2 receptor antagonist, cyclo(3134)[Dphe11,His12,Nle17,C MeLeu13,39, Glu31,Lys34]Ac Sau(8 40) or astressin 2B, was dissolved in distilled water and chilled in ic e until used. CRF1 receptor antagonist used, 1 [8 (2,4 -dichlorophenyl) -2 -methylquinolin 4 yl] 1,2,3,6 tetrahydropyridine 4 -carboxamide benzenesulfonate (R278995/CRA0450) was dissolved in distilled water and chilled in ice approximately one hour before bei ng used. The CRF1 receptor antagonist R278995/CRA0450 is selective for the CRF1 receptor subtype while astressin 2B is selective for the CRF2 receptor subtype

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16 Surgical Procedure of Cannula and Electrode Approximately 5 7 days after arrival of the rats, cannulas and electrodes were placed surgically into the brain. They were anesthetized by inhalation of 1 3% isoflurane in oxygen and placed in a stereotaxic frame. An 11 mm in length electrode was placed in the medial forebrain and an 11 mm in len gth cannula was placed above the left lateral ventricle. The coordinates for each placement were taken using a flat skull. The coordinates for cannula placement were the following: anterior posterior 0.9mm, medial lateral + 1.4 mm, dorsal ventral 3.0 m m. The electrodes were implanted using the following coordinates: anterior posterior 0.5mm, medial lateral + 1.7mm, and dorsal ventral 8.3mm. The incisor bar was adjusted up to 5.0 mm above the interaural line before the electrode was placed. Four ind entations were made in the skull to accommodate the screws being placed. Dental cement was used to secure the screws, electrodes, and cannulas. Each animal was given an injection of 0.5 ml flunixin, an anti inflammatory, immediately after surgery as well as the next day, and allowed 7 days for recovery. Steel gauge stylets were made and inserted into the cannulas to prevent infection between tests. Caps placed on the electrodes served to prevent the rats from damaging them. Intracranial Self Stimulatio n Procedure (ICSS) Animals were tested and trained using this procedure. They were placed in operant boxes, which were located in sound attenuated chambers. Each chamber contained a metal response wheel on the side of the wall. Constant current stimula tors delivered brain stimulation to the rats. This procedure was divided into three stages, CTW1, CTW2, and CTW3. The first stage, CTW1, allowed the rats to respond freely on a fixed 1 ratio schedule and as the wheel was spun, the rats received 0.1 ms s quare pulses at 100Hz and at a current intensity beginning at 180 A, 500 ms in duration. It was adjusted according to the animals reaction from thereon. Once stability was reached on this stage, the animal was moved to the second stage, CTW2. Here, the

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17 animals received a non contingent stimulus to which they had a window of time to respond in order to receive a second contingent response. If they did not respond within this time frame, the initial response was considered irrelevant and not counted. As a result, the response was not reinforced and a delay, also called a time-out, began until the next non contingent stimulus was given. The inter -trial interval, or ITI, started at 3s to a penalty time of 1s, followed by 5s: 2s, and finally 10s: 5s. The animal was promoted to the next level once they reached an accuracy level of about 90 to 95%. CTW3, the third and final stage, began the discrete trial current threshold procedure testing. Each testing session, which lasted about 30 minutes, involved four alternating series of ascending and descending current intensities. Each testing measured brain reward thresholds and response latencies. The midpoint between the stimulus response intensities and failed response intensities represented the brain re ward threshold. Response latencies were described as the interval of time between the non -contingent stimulus and a positive response, or the reaction time in relation to the animals behavior. Osmotic Minipump Surgery The use of an osmotic minipump is an alternative to repetitive injections of a certain drug or substance over a prolonged period of time. Pumps (Alzet model 2ML4) contained either saline or nicotine (9 mg/kg/day of nicotine tartrate salt solution) and the rats were divided based on simila r current threshold levels. The rats underwent anesthesia and the pumps were placed subcutaneously. The recovery period was quick as they resumed testing the day after surgery. Experimental Procedure The experiment tested the effect of CRF2 antagonist on precipitated nicotine withdrawal. After the rats were established having stable baseline brain reward thresholds on the ICSS procedure, they were prepared with 28 day osmotic minipumps containing either saline (n=8) or nicotine dissolved in saline (n =8). The rate of flow was 9mg/kg/day of nicotine tartrate salt.

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18 Measurements of brain reward thresholds and response latencies, like stated earlier, were taken between 9AM and 12PM, and nAChR mecamylamine injections (3mg / kg) were given about 6 days afte r implantation of the pumps to develop dependence of nicotine and precipitate withdrawal. The CRF2 antagonist astressin 2B (1 20 was given 15 minutes prior to mecamylamine treatment and rats were placed in the ICSS chambers 5 minutes after mecamylamine injections. Mecamylamine injections were given every 72 hours to ensure full reestablishment of nicotine dependence. Th e second experiment was identical to the first, except for the use of a CRF1 antagonist R278995/CRA0450 (1 20 and was administered once again to rats with nicotine (n=14) and saline (n=12). Statistics Brain reward thresholds and response latenc ies were expressed as percentages of the pre test day values. Percentages were analyzed using a mixed two way analysis of variance (ANOVA), with the CRF2 antagonist dose as the within-subjects factor and the pump content (saline or nicotine) and the betw een subjects factor. A significance factor of 0.05 and 0.01 was used to indicate the elevations in brain reward thresholds of nicotine treated rats to those of saline treated and the effect of mecamylamine treatment. Statistically significant results wer e followed by post hoc analysis using Newman-Keuls test.

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19 Table 2 1. Procedural list of experiment. 1 Electrode and cannula insertion surgery 2 ICSS procedure with stable baseline brain reward thresholds 3 Osmotic minipump surgery containing saline o r nicotine dissolved in saline 4 Measurement of brain reward thresholds 5 nAChR (nicotinic acetylcholine receptor) mecamylamine injections given 6 days after impantation 6 CRF antagonist (1 or 2) given 15 minutes prior to mecamylamine treatment and rats were placed in the ICSS chambers 5 minutes after mecamylamine injections 7 Mecamylamine injections given every 72 hours to ensure full re establishment of nicotine dependency Figure 2 1 Apparatus used during the cannula and electrode surgical procedure. Rat skull placed here between the earbars.

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20 Figure 2 2 Using a flat skull and taken from bregma, coordinates for the electrode and cannula were identified. Figure 2 3 Osmotic minipumps were used and filled with either saline or nicotine dissolved in saline.

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21 Figure 2 4 The ICSS procedure was performed in operant boxes within soundattenuated chambers. The rats were connected to the box by placement of the wire inside the box to the electrode located on the brain. Sound attenuated chamber Operant box Metal response wheel

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22 CHAPTER 3 RESULTS Results from the effect of astressin 2B on acute nicotine withdrawal showed that mecamylamine elevated brain reward thresholds of nicotine induced rats however showed no effect on the saline rats (Figure 3 1; Dose: F1,13=25.67, P<0.0002). During pretrea tment with astressin 2b, the brain reward thresholds experienced no significant effect in either group (Figure 3 1; Dose: F4,52=0.304, not significant; Dose x Treatment interaction: F4,52=0.149, not significant). There was a slight effect of mecamylamine initially on the response latencies of nicotine induced rats compared to saline treated rats (Figure 3 2; Dose: F1,13=21.583, P<0.0004). However, according to Figure 3 2, astressin 2B had no effect on the response latencies of either group (Figure 3 2; D ose: F4,52=1.079, not significant; Dose x Treatment interaction: F4,52=0.394, not significant). Results from the effect of R278995/CRA0450 on acute nicotine withdrawal showed that mecamylamine elevated brain reward thresholds of nicotine induced rats howe ver showed no effect on the saline rats (Figure 3 3 ; Dose: F1,24= 28.491, P<0.0001). However, when pretreated with the CRF1 antagonist, the brain reward thresholds were lower to that of mecamylamine induced elevation levels in nicotine treated rats but not saline treated rats (Dose x Treatment interaction: F4,96=3.162, P<0.017) According to Figure 3 3, after performing a statistical analysis test, the Newman Keuls post hoc test, it was verified that at 20 g of R278995/CRA0450, there was no elevation of brain reward thresholds compared to that of saline treated rats (P<0.01). At 10 g, lower brain reward thresholds compared to rats treated with nicotine and acutely treated with mecamylamine and vehicle (0 g R278995/CRA0450) were initially detected (P <0.05). Figure 34 indicates the baseline latencies and while mecamylamine did increase the response latencies for nicotine induced rats, it had little effect

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23 on saline rats ( Figure 3 4; Dose: F1,24=16.381, P<0.0005). There was no effect on response latencies throughout pretreatment of the CRF1 antagonist on nicotine as well as saline treated rats (Figure 3 4; Dose: F4,96=0.396, not significant; Dose x Treatment interaction: F4,96=1.217, not significant).

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24 Figure 3 1. Effect of CRF2 receptor antagonist astressin 2B on brain reward threshold elevations associated with mecamylamine -precipitated nicotine withdrawal. Rats were divided into two groups, saline or nicotine treated. Brain reward thresholds are expressed as a percentage of pre-test day valu es. Figure 3 2. Effect of CRF2 receptor antagonist astressin 2B on response latencies of rats chronically treated with saline or nicotine and acutely treated with mecamylamine. Response latencies are expressed as a percentage of pre -test day values.

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25 Figure 3 3. Effect of CRF1 receptor antagonist R278995/CRA0450 on brain reward threshold elevations associated with mecamylamine -precipitated nicotine withdrawal. Rats were divided into two groups, saline or nicotine treated. Brain reward thresholds are expressed as a percentage of pre -test day values. Asterisks (* P<0.05, ** P<0.01) indicate elevations in brain reward thresholds compared to those of the corresponding saline treated control group. Plus signs (+ P<0.05, ++ P<0.01) indicate lower brain reward thresholds compared to those of rats chronically treated with nicotine and acutely treated with mecamylamine and vehicle (0 g of R278995/CRA0450). Figure 3 4. Effect of CRF1 receptor antagonist R278995/CRA0450 on response latencies of rats c hronically treated with saline or nicotine and acutely treated with mecamylamine. Response latencies are expressed as a percentage of pre -test day values.

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26 CHAPTER 4 DISCUSSION Findings from these experiments indicate that the neuronal nicotinic acetylcho line receptor (nAChR) antagonist mecamylamine elevated the brain reward thresholds of the nicotine -treated rats and did not affect the brain reward thresholds of the control rats. Mecamylamine did not affect the reponse latencies of the nicotine or saline -treated animals. The CRF1 receptor antagonist R278995/CRA0450 prevented the elevations in brain reward thresholds associated with acute nicotine withdrawal and did not affect the brain reward thresholds of the control rats. The CRF1 receptor antagonist did not affect the response latencies. The CRF2 receptor antagonist astressin 2B had no effect on the brain reward thresholds or the response latency. While many studies have shown similar results investigating the role of CRF1 receptors associated with addic tive substances, the role of CRF2 receptors has produced rather conflicting results. Neuronal nicotinic acetylcholine receptors play a significant role in the neurobiology of nicotinic withdrawal syndrome. Evidence shows that nicotine acts at this receptor site and is the active component in tobacco smoke associated with the development and maintenance of tobacco addiction (Kenny and Markou, 2001). Nicotinic receptors are expressed in various areas of the body including the following: mature skeletal musc le, autonomic ganglia, and within the central nervous system. We will focus on the CNS as it relates to the behavioral actions due to nicotine (Kenny and Markou, 2001). Nicotine, by action at nAChR, has been shown to stimulate the release of most neurotra nsmitters throughout areas of brain. The nAChR antagonist mecamylamine has been proven in previous studies to block physiological effects of nicotine and improving abstinence rates in smoking cessation studies. Even more interesting is that a low dose is required to achieve these effects. Mecamylamine was one of the first medications studied

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27 for smoking cessation and prevents health related problems associated with tobacco smoking (Shytle et al., 2002). Increased CRF release is associated with precipita ted nicotine withdrawal and leads to increased anxiety like symptoms such as irritability and depression. These effects can be prevented be blocked by treatment with the CRF1 receptor antagonists. Increased CRF release has also been associated with alcoho l addiction as well as cocaine and other drug addictions. During alcohol abstinence, anxietylike behaviors, mood disturbances, and depression, quickly occur (Valdez et al., 2003). During this study, the underlying mechanism causing these changes was th ought to be a result of an increase in CRF activity (Valdez et al., 2003). However, with the use of a CRF receptor antagonist D-Phe -CRF(12 41) the effects of ethanol were attenuated (Valdez et al., 2003; Macey et al. 2000). Similar results were found i n another study using different CRF1 antagonists including anatalarmin, MJL 1 1092, and R121919 (Funk et al., 2007; Zorrilla et al., 2002; Marinelli et al., 2007). This article concluded that CRF1 receptors also played a significant role in mediating exc essive ethanol consumption in dependent animals however not in nondependent animals (Funk et al., 2007). In association with cocaine addiction, studies have concluded that the CRF system plays a significant role in the behavioral changes during abstinence (Lu et al., 2001; Erb and Stuart, 1999; Specio et al., 2007). The use of CRF1 antagonists such as -helical CRF, D Phe CRF(1241) significantly attenuated the affects and results concluded that the CRF1 receptor type mediated stress induced behavior as sociated with withdrawal (Lu et al., 2001; Erb and Stuart, 1999; Specio et al., 2007). Overall, previous results have shown that an increase in CRF in the central region contributes to the deficit in brain reward thresholds associated with cessation of al cohol, opiate, or tobacco use. Our results

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28 concluded that the CRF1 receptor type with the CRF system plays a significant role mediating behavioral changes. Conflicting results have been shown when investigating the role of the CRF2 receptor type. The CRF2 receptor is not as widespread as the CRF1 receptor subtype with expression mainly in the dorsal raphe, lateral septum, ventral hypothalamus, and extended amygdala (Van Pett et al., 2000; Funk and Koob, 2007). The anxietylike behaviors are more comp lex as some studies have shown CRF2 receptor to have shown anxiolytic like effects and others anxiogenic like behavior (Funk and Koob, 2007; Pellymounter et al., 2002, 2004; Rosbrough et al., 2003, 2004; Ho et al., 2001). A specific example of this was w ith the use of a CRF2 antagonist antisauvagine 30 and the anxiogenic like effects it produced in certain brain sites in rats and anxiolytic like effects in mice (Bakshi et al., 2002; Takahashi et al., 2001). It is thought that the opposing roles may be a reflection of the role of CRF2 receptors in various regions of the brain (Funk and Koob. 2007; Liu et al., 2004). In ethanol exposed experiments, such as presented by Funk and Koob (2007), exposure to urocortin 3 (Ucn 3), a highly selective CRF2 agonist in the CeA decreased ethanol administration and anxiety like behaviors in dependent rats. This allows one to believe that it may have been caused by activation of CRF2 receptors. In nondependent rats however, the administration of Ucn 3 increased ethanol consumption but only observed at the highest dose, similar to benzodiazepine diazepam (Funk and Koob, 2007; Schmitt et al., 2002). Some studies have also been performed testing the effect of CRF2 receptor activation on anxiety and have concluded that i t may also be dependent on the stress level of the animal (Henry et al., 2006). The mechanism, brain dose, brain location, and testing environment are all possible factors contributing to the role of CRF2 receptors in association to stress and are importan t for further pharmacological studies.

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29 It is important for future studies to address the importance of CRF2 receptors and antagonists associated with reducing anxiety and negative mood states. Even though our present study showed no significant effect of astressin 2B, it may have been due to placement of administration within the brain or the dosage given. However, the CRF1 receptor antagonist produced significant affects in attenuating the negative behavior leading us to believe that CRF1 receptors pla y a significant role in the mediating effects of nicotine. Further studies could be performed with this receptor subtype to understand the factors associated with the longlasting nature of dependence on this substance as well as ethanol and cocaine. D ifferent methods of prevention of relapse include: nicotine gum, dermal nicotine patches, electronic cigarettes, or nasal sprays. A nicotine vaccine is also currently being studied with both animal and human subjects called NicVAX (Heading 2003). This is an investigational vaccine designed to facilitate during smoking cessation and prevention of relapse. NicVAX is designed to stimulate production of antibodies within the immune system that bind to nicotine and thus, prevent crossing the blood brain barrier and into the brain (Heading 2003). Because of this action, the brain would not produce euphoric like feelings in response to the nicotine. It is important therefore to understand the bloodbrain barrier model as on average it takes only about 7 sec onds for a substance to reach the brain when inhaled. The bloodbrain barrier is an anatomicphysiologic feature of the brain that is composed of walls of capillaries in the central nervous system along with glial cells (astrocytes) and composed of epit helial cells sealed together in continuous tight junctions (Mosbys Medical Dictionary 2009). This barrier separates the blood from the parenchyma of the central nervous system (Mosbys Medical Dictionary 2009). It is selective as it prevents many substa nces, such as drugs, disease causing organisms, or other chemical compounds, from entering the central nervous system (Mosbys

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30 Medical Dictionary 2009). While lipid -soluble substances such as alcohol, caffeine, and nicotine pass rather quickly and easily in the brain cells, most antibiotics and ions have difficulty in entering or enter slowly (Millodot: Dictionary of Optometry and Visual Science 2009). This is where NicVAX plays a significant role as the nicotine molecules bind to antibodies produced and cannot enter the barrier because they are too large. Another common way of administration of a drug is orally. With all administrations, oral, intramuscular, intravenous, and transdermal, there are four basic stages of the medicine life cycle including : absorption, distribution, metabolism, and excretion (National Institute of General Medical Sciences 2006). Medicines taken orally are transported by a special blood vessel from the digestive tract to the liver, where are large amount may be destroyed du e to metabolic enzymes (National Institute of General Medical Sciences 2006). During the distribution phase, side effects may occur (National Institute of General Medical Sciences 2006). Drugs destined for the central nervous system must pass through the blood brain barrier, as talked about before, and once the drug is distributed throughout the body and its respective areas, it is then broken down, or metabolized (National Institute of General Medical Sciences 2006). Once the enzymes are finished workin g with the medicine, the resulting inactive drug is excreted via the urine or feces (National Institute of General Medical Sciences 2006). An example of an oral drug used to treat nicotine addiction is Zyban, also known as Bupropion (Wu et al., 2006). It is an antidepressant that blocks the nicotine receptors in the brain and is generally different from other nicotinic treatment therapies because it does not introduce more nicotine into your body (Wu et al., 2006). Human experimentation has been used wit h this substance as well. The present study, along with others studied, has made a significant impact on this field. What is so interesting about the nicotine withdrawal syndrome is the complexity of the subject.

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31 This syndrome is comprised of multiple be havioral deficits and underlying neurobiological substrates. Further studies are required to identify the reasoning behind the mediating effects within specific regions of the brain. Studies with CRF1 receptor knockout mice have shown anxiogenic effects of CRF to be mediated by the CRF1 receptor subtype (Heinrichs et al., 1997; Smith et al., 1998). CRF2 knockout mice have shown varying evidence however while one study showed increased activity, another decreased activity, and a third no activity at all (Kishimoto et al., 2000; Bale et al., 2000; Coste et al., 2000). The general idea of a knockout mouse is where one or more genes have been turned off through a gene knockout. By turning this gene off, one is able to infer its function from any difference s in normal behavior or condition. This, as well as other animal model experiments, will eventually lead to strategies in treating nicotine dependency and treatment therapies. Overall we see that the blockade of the CRF1 receptor prevents the deficit in brain reward function associated with nicotine withdrawal. Studies further confirm that CRF2 receptors do not play a role in nicotine withdrawal. The use of a CRF1 receptor antagonist however may be beneficial to pharmacological therapies as it has played a significant role in the prevention of negative moods states associated with the cessation of smoking.

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32 Figure 4 1. Model of the blood-brain barrier including the capillaries and astrocytes. ( Mosby's Medical Dictionary, 8th edition. 2009, Elsev ier) Figure 4 2. Different modes of transportation of drugs include intramuscular, transdermal, subcutaneous, and intravenous. (National Institute of General Medical Sciences 2006).

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33 Figure 4 3. When a drug is taken orally, it must pass through the liver before being distributed to the appropriate region of the body. Other modes may bypass the liver, entering the bloodstream immediiately. (National Institute of General Medical Sciences 2006).

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38 BIOGRAPHICAL SKETCH Pranati Pramodh Kumar, also know n a s Pranoo, was bo rn in Hydrabad, India. She moved to the United States with her mother and father in 1987, where they resided for about 10 years in Texas. At the age of 10, her family moved to Jacksonville, Florida and she graduat ed from Paxon School for Advanced Studies in 2003 with her International Baccalaureate diploma. She earned her B.S. in Integrative Biology from the University of Florida in 2007. From here, she decided to continue her studies at the University of Florida after being accepted into the Master in Medical Sciences Program through the College of Medicine. Upon completion of the M.S. program, Pranati will continue her studies in the field of medicine and attend medical school. With the education and knowledge attained from this program as well as her future studies, she hopes to eventually become a pediatrician and travel around the world focusing on various global health issues. Her father and mother, Pramodh and Pratima Kumar, have an amazing and loving su pport throughout this process.