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1 EFFECT OF NICOTINE ON BODY WEIGHT AND FOOD INTAKE DURING AND AFTER INTRAVENOUS ADMINISTRATION By IAN E. THOMPSON A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009
2 2009 Ian E. Thompson
3 To my parents for their support and to my advisor Dr. Neil E. Rowland for his guidance
4 TABLE OF CONTENTS page LIST OF FIGURES .........................................................................................................................5ABSTRACT ...................................................................................................................... ...............6 CHAP TER 1 INTRODUCTION .................................................................................................................. ..72 METHODS ..................................................................................................................... ........17Subjects ...................................................................................................................... .............17Intravenous Catheters ......................................................................................................... ....17Surgery ....................................................................................................................... .....17Maintenance ....................................................................................................................18Operant Chambers ..................................................................................................................18Nicotine ...................................................................................................................... .............19Procedure ..................................................................................................................... ...........20Baseline Establishment .................................................................................................... 20Nicotine Administration and Removal ............................................................................ 20Data Analysis ..........................................................................................................................213 RESULTS ..................................................................................................................... ..........23Body Weight ................................................................................................................... ........23Food Intake .............................................................................................................................23Meal Number ..........................................................................................................................25Meal Size ................................................................................................................................264 DISCUSSION .................................................................................................................. .......40REFERENCES .................................................................................................................... ..........48BIOGRAPHICAL SKETCH .........................................................................................................53
5 LIST OF FIGURES Figure page 3-1 Daily body weight (in grams) of rats receiving 2 different dos es of nicotine or vehicle. ...................................................................................................................... .........293-2 Daily change in body weight of rats r eceiving 2 different dos es of nicotine or vehicle. ...................................................................................................................... .........303-3 Total Food intake for rats receiving 2 different doses of nicotine or vehicle. ...................313-4 Dark phase food intake for rats receiving 2 different doses of nico tine or vehicle. .......... 323-5 Light phase food intake of rats receiving 2 different doses of nicotine or vehicle. ........... 333-6 Mean total meal number for rats receivi ng 2 different doses of nicotine or vehicle. ........ 343-7 Mean dark phase meal number for rats receiving 2 different do ses of nicotine or vehicle. ...................................................................................................................... .........353-8 Mean light phase meal number for rats receiving 2 different do ses of nicotine or vehicle. ...................................................................................................................... .........363-9 Mean 23-hr meal size for rats receiving 2 different doses of nicotine or vehicle. ............. 373-10 Mean dark phase meal size for rats receivi ng 2 different doses of nicotine or vehicle. .... 383-11 Mean light phase meal size for rats receivi ng 2 different doses of nicotine or vehicle. .... 39
6 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECT OF NICOTINE ON BODY WEIGHT AND FOOD INTAKE DURING AND AFTER INTRAVENOUS ADMINSTRATION By Ian E. Thompson August 2009 Chair: Neil E. Rowland Major: Psychology Several laboratory (rat) mode ls have been developed to investigate the relationship between chronic nicotine administ ration and food intake, and thes e suggest that nicotine may attenuate weight gain by suppres sing appetite and modulating ea ting behavior. However, many of these models lack certain ch aracteristics inherent to human nicotine administration, including the increased postcessation weight gain that is frequently seen in the human population. The present study tested a rat model that used noncontingent doses of nicotine given intravenously to analyze the effects of nicotine on body weight and pattern s of food intake. Rats were placed in operant chambers for 23 hours per day and given 30 noncontingent injections of nicotine at one of two doses (0.03mg/kg/injection or 0.06mg/kg/inj) and their patterns of food intake were compared to those of control rats given vehicle injections on a similar schedule. Both doses of nicotine caused an attenuation of body weight gain when compared to vehicle. These reductions in weight gain were due to ch anges in light phase food intake, including decreases in meal size and meal number. Following the te rmination of nicotine, rats that had received the drug began to increase in body weight at a rate that was higher than controls a nd this was due to increased food intake during this period. By the conclusion of the study, rats receiving nicotine had attained a body weight equal to that of the controls.
7 CHAPTER 1 INTRODUCTION Cigarette sm oking is one of the leading causes of mortality in the world, contributing to several different types of cancer, respiratory diseases and cardi ovascular pathologies (Ezzati et al. 2005). As more of the negative consequences of cigarette smoking are exposed, the rationale to quit smoking grows stronger and stronger. Despite advances in the development of pharmacological and behavioral aids to nicotine cessation (Reus and Smith 2008), success rates remain low (Hughes et al. 2008). This is mostly due to nicotine being an addictive substance, but other factors may contribute to the high relapse rates, an idea supported by the studies using denicotinized cigarettes (Rose et al. 2000). The ability of nicotin e to mitigate weight gain is a secondary result of smoking, but may have a signif icant impact on an individuals desire to quit smoking. The stigmas associated with obesit y, both social and professional, are well documented (Puhl and Brownell 2001) and smoki ng may represent a readily available, and dangerous, form of weight control. Additionally the subsequent weight gain associated with smoking cessation may discourage individuals from attempting to quit, or cause a relapse during abstinence. Many smokers, especially women, cite the weight gain associ ated with cessation of nicotine as the prime deterrent to quitting (Per kins et al. 1997) and are unwilling to quit if any weight gain might accompany smoking cessation (Pomerleau and Kurth 1996). While men tend to have fewer concerns regarding weight gain following smoking cessation, it is still an issue for a large proportion of male smokers (Pomerleau and Kurth 1996). Programs designed to help people quit smoking frequently deal with the behavior of smoking and ignore this potential accompanying problem, which may help explain th e high relapse rates of those attempting to quit (Perkins et al. 1997). While these belief s may be based on anecdotal or circumstantial evidence, they suggest an interaction betw een nicotine, food intake and body weight.
8 The literature is rife with c ontradictions regarding nicotine s effect on food intake and body weight in humans with some studies indicating little or no associati on (Klesges et al. 1991) while others indicate that thei r interaction is quite a robust phenomenon (Eisenberg and Quinn 2006; Klesges et al. 1997). These studies face many methodological problems, notably the use of self-report of nicotin e abstinence, food intake and body wei ght, as well as the tendency to report abstinence at a single time point. Some studies have attempted to solve these pitfalls by using only individuals that met criteria for cont inued abstinence (Klesges et al. 1997) and have indicated that the amount of we ight gain following smoking cessati on that is routinely cited (~5 kg) may be higher than what is actually seen, especially in individuals with maintained abstinence. Obesity is also a social problem of increasing significance, and seve ral investigators have drawn a parallel between the behavior of those suffering from drug addic tion and excessive food intake leading to obesity. Each is characteri zed by repeated, compulsive intake despite the adverse consequences that are know n to be associated with that behavior. Not only do the two pathologies resemble one another behaviora lly, but both utilize similar neurobiological mechanisms such as the mesolimbic dopamine pathway and endogenous opioids (Knecht et al. 2008; Volkow and Wise 2005; Wise 1997). With this in mind, it is not surp rising that use of an addictive drug, such as nicotine, may interact or interfere with the normal regulation of food intake. While there are several possible lo cations within the brain that ma y be the site, or sites, of nicotines effect on food intake, most studies identify the lateral hypothalamus (LHA) as the most likely candidate; it has exte nsive connections to a multitude of cortical and subcortical structures, including the mesolimbic dopamine system, and is an important modulator of food
9 intake (Berthoud 2002; Hoebel et al. 1989). A st udy by Yang et al. (1999) found that infusions of nicotine into the LHA caused an immediate increase in dopamine followed by a gradual decrease back to baseline. Feeding was shown to elicit smaller, but significant increases in dopamine in the LHA; this might indicate that onc e dopamine reaches a threshold level, it signals the end of a meal. Additionally, long term periph eral infusions of nicotine caused an increase in the basal levels of dopamine when compared to controls (Miyata et al. 1999). If basal levels of dopamine are elevated, then the hypothetical thre shold level signaling the end of a meal would be attained more quickly and the meal would en d prematurely. Following removal of nicotine, dopamine was shown to decrease to below the leve ls of control which could explain the increase in food intake and weight gain that can be seen during nicotine cessation. In addition to dopamine, serotonin has also be en shown to have the ability to modulate food intake, most likely as an inhibitor of f ood intake (Leibowitz and Alexander 1998). One group found that serotonin levels increased gradua lly during food intake and remained elevated for a time following the meal, indicating a possible signal for inter-meal sa tiety (Schwartz et al. 1990; Schwartz et al. 1989). Additionally, anorectic compounds such as fenfluramine and fluoxetine have been shown to increase serotonin levels in the LHA (Hoebel et al. 1989). Infusions of nicotine into the LHA caused larger, lasting increases in serotonin levels that also coincided with a decrease in food intake during a period following the nicotine infusion (Yang et al. 1999). In separate study, several days of peripheral nicotine administration via mini-pump caused an elevation in baseline serotonin le vels (Miyata et al. 1999). Unlike dopamine, serotonins levels remained elevated for prolonged periods after a meal had concluded which points to a possible role as a signa l of a fed state. If this is the case, nicotines ability to
10 elevate the basal levels of serotonin in the L HA may cause early termination of meals and an increase of inter-meal intervals. Another food-associated neurotransmitter that has been studied extensively in the context of nicotines effects on food intake is neurope ptide Y. Neuropeptide Y (NPY) is a potent stimulator of food intake, and infusions of NPY into the hypothalamus can cause hyperphagia and obesity (Bishop et al. 2002; Stanley and Leib owitz 1985). NPY levels have also been found to increase in paraventricular nucleus (PVN) of the hypothalamus during food deprivation (Kalra et al. 1991). Because of this direct relationship betw een food intake and NPY, it was postulated that nicotine may decrease NPY levels in the brain. However, the results have been mixed. Chen et al. (2006) found th at NPY levels were reduced in the PVN of mice exposed to cigarette smoke for 5 days every week for 4 weeks. This occurred despite decreased leptin levels which are usually associated w ith increased NPY levels (Schwa rtz et al. 1996). Another study found decreased NPY levels in the PVN of rats in response to 12 days of nicotine administration by osmotic mini-pump although NPY messenger ribonucleic acid (mRNA) expression was significantly higher when compared to pair-fed c ontrols (Frankish et al. 1995). Furthermore, it was found that peripheral nicotine administration blunted the feedi ng response to injections of NPY into the PVN (Bishop et al. 2002). Howeve r, other studies have shown that prolonged cigarette smoke exposure has no effect on NPY levels in the PVN despite weight loss and reductions in circulating leptin (Chen et al. 2005) and another study found similar results when using intraperitoneal injections of nicotine (Kramer et al. 2007). One study has reported increases in brain NPY levels following nicotine administration (Li et al 2000b). In each of these studies, rats still exhibited nicoti ne-induced hypophagia, indi cating that NPY was ineffective in stimulating food intake in the nicotin e treated rats. It is possible that the prolonged
11 elevated NPY levels caused by chronic nicotine admi nistration resulted in either a desensitization or downregulation of NPY receptors in certain pa rts of the hypothalamus, attenuating its ability to stimulate feeding. Increased levels of NPY following nicotine administration were associated with decreases in NPY receptor binding in the anterior and posterior hypothalamus, and this deduction explain some of the inco nsistencies (Li et al. 2000a). Jo et al. (2002; 2005) put forth a novel hypothesis that may explain the discrepancy between results regarding nicotine and neuropep tide Y in which nicotine increases inhibitory tone, via -Aminobutyric acid (GABA), in the LHA. This increased GABA-ergic activity may suppress the effects of appetite-induced increas es neuropeptide Y signaling from the arcuate nucleus. This hypothesis may explain the decrea sed food intake that is concurrent with nicotines effects in the LHA and on NPY levels within the hypothalamus. Several animal models have been generated in order to examine the relationship between nicotine, food intake and body weight (Bellinger et al. 2003b; Grunberg et al. 1988b; Miyata et al. 2001) but these models lack certain characteris tics of nicotine use seen in humans that may have important implications for the effects of nicotine on food intake and body weight. The goal of this project is to test a rat model that looks at the interactio n between nicotine, food intake and body weight, and also possesses charact eristics of nicotine use in humans that have yet to be seen in previous animal models. The first animal models that tested the effects of prolonge d exposure to nicotine on food intake used implantable Alzet osmotic mini-pumps delivering a continuous dose of nicotine over an extended period of time (Bishop et al. 2002; 2004; Grunberg 1982; Grunberg et al. 1988a; Grunberg et al. 1988b; Miyata et al. 2001). By providing a stab le nicotine infusion throughout the day, mini-pumps create a steady-state serum ni cotine concentration. While this particular
12 route of administration has its adva ntages, it also has leaks. Nicotine administered peripherally is subject to first pass metabolism by the liver (~70%) so doses must be adjusted accordingly (Matta et al. 2007). The nicotine doses used ha ve tended to range from 2.5mg/kg/day (Grunberg 1982) to as high as 12 mg/kg/day (Grunberg et al. 1988a; Grunberg et al. 1988b); the approximate amount of nicotine reaching the bloodstream can be calculated by multiplying the dose by the fraction that is not meta bolized by the liver, but this is an estimate at best. LeSage et al. (2002) measured both venous and arterial nicotine levels followi ng continuous nicotine infusion from an osmotic mini-pump. They found that a dose of 1.0 mg/kg/day resulted in a stable venous nicotine level th at closely resembled the steady state venous concentration seen during human smoking (Benowitz 1997) while 2.1mg/kg/ day resulted in a stab le venous nicotine level that is similar to peak levels seen in human smokers following a cigarette (Gourlay and Benowitz 1997). Additionally, subcutaneous mini -pumps did not produce the arterial spike in nicotine concentration that is seen during smoking. Two separa te studies have noted that, following paced cigarette smoking in humans, the nico tine level in arterial blood is ~2.5 times as high as the levels seen in th e venous blood (Gourlay and Benowitz 1997; Rose et al. 1999). Osmotic mini-pumps can produce sustained arterial nicotine levels, but they cannot produce the spike seen in human smoking (LeSage et al. 2002). If nicotines ability to alter body weight and food intake relies on activating nicotinic receptors in the brain, transient excursions in nicotine concentration may be needed to elicit a sustained effect. The stable serum levels of nicotine produ ced by continuous infusion may desensitize nicotinic acetylcholine receptors (nAChRs) in a way that might not occur when using intermittent boluses of nicotine. Previous studies have indicated that prolonged exposure to nicotine can desensitize nAChRs on dopamine neurons (Pidoplichko et al. 1997). In another
13 study, constant infusions of 1 and 4 mg/kg/day resu lted in the elimination of an increase in extracellular dopamine in the nucleus accumbens th at can usually be elicited by a small bolus of nicotine in drug nave rats. This increase in extracellular dopamine in response to a bolus of nicotine can be seen in rats receiving .25mg/kg/day, indicating that regularly spaced injections or infusions may not have the same desensitization effect (Benwe ll et al. 1994). These problems of nicotine dose and route of ad ministration call into question some of the results seen in the studies which use subcutan eous mini-pumps. Early studies (Grunberg 1982; Grunberg et al. 1988a; Grunberg et al. 1988b) sh owed mitigation of weight gain following nicotine administration, but the data shown was a mean over the en tire administra tion period. It is unclear whether this represented an initial decreas e in weight that corrected itself, or if it was a truly sustained reduction in weight gain. More recent studies (Bishop et al. 2002; 2004) have shown that the attenuation of weight gain followi ng nicotine administration is significant, but disappears after ~6 days. After this initial period, rats gain weight at rates that are roughly equal to those seen in controls. These initial change s in weight are usually accompanied by a decrease in food intake. The animals that were subjected to nicotine did show some signs of rebound hyperphagia following removal of th e nicotine, which mirrors the phenomenon that is frequently cited as a major deterrent to smoking cessation (Pomerleau and Kurth 1996). Miyata et al. (2001) found that the differences in food intake between placebo-treated controls and rats given nicotine contributes to the difference in we ight but does not explain all of the difference. This decrease in food intake was primarily due to a severe decrease in meal size without concurrent increases in meal number, in dicating that the nicotine treated rats had no trouble initiating meals, but satiet y was achieved much quicker than in controls. These changes
14 disappeared within 5 days, despite continued nico tine administration, and thereafter, rats gained weight at rates equa l to controls. Intermittent intraperitoneal (i.p.) injections have been used recently in several studies (Bellinger et al. 2003b; Bellinger et al. 2005; Guan et al. 200 4; Wellman et al. 2005). Intermittent intraperitoneal injections more closely resemble the episodic nicotine intake seen in human smokers and rat self-administration models and as a result avoid some of the problems associated with osmotic mini-pumps, such as re ceptor desensitization and an inability to produce sufficient periodic spikes in arterial nicotine levels. Additionally, these studies are able to modulate the nicotine dose based on the daily body weights of the subjects, something that studies using osmotic mini-pumps cannot do. While the intention of mimick ing the periodic nature of human smoking is a good step, i.p. injections still face problems similar to thos e faced by subcutaneous mini-pumps. Peripheral injections are a slower form of drug administra tion than inhalation due to the need of the blood vessels in the peritoneal cavity to absorb the ag ent in contrast to inhalation or intravenous injection, where the time from administration for th e drug to reach the brain is in the order of a few seconds. The increased duration of absorption with i.p. injections means that the bolus is more drawn out and the increase in serum nicotine le vels is much more gradual. Also, as noted before, i.p. injections are also subject to first pass metabolism by the liver, which means that only 30% of nicotine administered will actually reach the bloodstream. While some studies (Bellinger et al. 2005; Guan et al. 2004; Wellman et al. 200 5) tried to account for this by adjusting their dose, there are no empirical data showing seru m levels of nicotine following intraperitoneal injection.
15 Additionally, these studies tended to use 4 equa lly spaced (once every 3-h) i.p. injections during the dark phase. While the half-life (t1/2) of nicotine is ~120 minutes in humans, it is much shorter in rats (~45 minutes); this means that nicotine levels probably never attain a stable level that is congruent with human smokers. This inab ility to adequately elevate serum nicotine levels results in effects seen in the studies using osmotic mini-pumps; attenuation of weight gain is seen upon nicotine administration but ceases in about 6-7 days. Food intake is suppressed during this period of decreased weight gain an d interestingly, it is also due mostly to a decrease in meal size without compensatory increases in meal number (Bellinger et al. 2003b; Bellinger et al. 2005). This converging evidence indicates that nicotine may influence the neural substrates that control food intake. The present project attempts to avoid limitations seen in previous st udies by utilizing an intravenous route of administra tion, which more accurately replic ates how human smokers selfadminister nicotine. Intravenous administrati on of nicotine is the mo st prevalent route of administration used in self-administration studies and rats will self administer a variety of doses of nicotine (Chaudhri et al. 2007; Donny et al. 1999; Donny et al. 1998; Shoaib et al. 1997), some of which result in nicotine serum levels similar to those seen in humans (LeSage et al. 2002). ODell et al. (2007) examined circadian rhythms of food and nicotine intake in a 23-hour nicotine intravenous self-administration protocol and noted not only decreased meal size but also decreased meal frequency during th e dark phase, both of which cont ributed to a decrease in food intake during nicotine administration. Unlike th e studies using mini-pumps or intraperitoneal injections, these effects on food intake were present even on th e last day of the 40 day study, which indicated that the transient effects seen in previous studies may have been due to the route
16 of administration. Meal analys is was reported for only the first and last day of nicotine administration, and did not extend to postcessation food intake. Models such as that used by ODell et al. ( 2007), which use self-administration concurrent with food intake monitoring, present a model with the most similarities to what is seen in humans, but still presents some problems; In mode ls such as this, initiation and maintenance of adequate levels of self-admin istration require some food restriction (D onny et al. 1998). Additionally, the number of rats that attain self-administration can be relatively variable from group to group, and the amount of nicotine that ge ts administered from rat to rat may cover a wide range. With these problems in mind, this project uses noncontingent doses of nicotine, equally spaced out during the rats active cycle. Previous studies (LeSage et al. 2002) have noted that rats receiving regularly spaced inj ections at a certain dose (0.03mg/kg/injection) achieve stable arterial and venous nicotine levels and also achieve th e arterial spike ratio that is seen in smokers (Gourlay and Benowitz 1997; Rose et al. 1999). Using a protocol such as this will mimic the method by which humans administer nicotine and allow standardization of the amount of nicotine each rat receives.
17 CHAPTER 2 METHODS Subjects Male Adult Sprague-Dawley Rats were purchased from Harlan (Indianapolis, IN). Rats were given 2 days to acclimate to the vivarium be fore the start of the experiment. Female rats were not used because previous work has been done suggesting that nicotine administration may interact with estrous cycle and alter food intake (Bellinger et al 2005; Miyata et al. 2001) When the rats were not being run in the operant chambers, they were housed in polycarbonate cages with Sani-Chips as bedding. They were also given ad libitum access to water; Purina (No. 5001) chow was provided during recovery from surgery. The vivarium was kept at a constant 22-26 C with 40-60% humidity. The Psychology Department vivarium is sta ffed and managed by Animal Care Services (ACS) at the University of Florida and has Association for Assessment and Accreditation of Laboratory Animal Care International (AAAL AC) accreditation. The ca re and use complies with all federal and local (Institutional Animal Care and Use Committee) standards. Operant chambers were in the same room as the animal housing and were maintain ed by the investigator Intravenous Catheters Surgery Rats were anesthetized using a m ixture is oflurane and oxygen; hindpaw pinching was used to periodically check that the ra t was unconscious. After the rat wa s sufficiently sedated, the hair was removed from the area (~1cm x 3 cm) where th e right forepaw meets the torso on the ventral side of the rat. A single incision was made in the skin and an indwelling, autoclaved catheter was placed in the right jugular vein via a sma ll incision and secured with silk string. The catheter consisted of a single 9cm length of t ubing (.58mm inside diameter (ID) x .94mm outside
18 diameter (OD), MicroRenathane MRE-037, Braintree Scientific, Inc, Braintree, MA) with 2 collars (~1cm in length; .64mm ID x 1.20mm OD), one placed 3.2 cm from one end and the other at the opposite end, flush with the catheter end. These collars were in place to maintain proper catheter placement in the vein and reinforce the catheters connection to the port, respectively. After insertion and security of the catheter within the vein, the free end was tunneled subcutaneously to an in cision made on the back of the rat where it was attached to a plastic port with surgical mesh attached (313000BM-15, Plastics One Inc., Roanoke, VA). The exterior of the port was fitted with a small (~1.5cm) piece of pl astic tubing (.51mm ID x 1.52mm OD; Norton Performance Plastics, Akron, OH) and cl osed with a pin when not in use. Both incisions were then sutured with a non-absorb able suture (Braunamid white, Aesculap Inc., Center Valley, PA). Immediately following surger y, rats were given 5mg/kg of an analgesic and anti-inflammatory medication (Ketorolac tromet hamine, Sigma Chemicals) and 10mg/kg of a broad spectrum antibiotic (enrofloxacin, Bayer HealthCare LLC, Shawnee Mission, KS). Maintenance For 3 days following surgery, catheters were fl ushed twice a day with a heparinized saline solution (0.17 m g/ml) and once a day with a co cktail consisting of a thrombolytic agent (streptokinase, Sigma Chemicals Co., 400 units) a nd an antibiotic (Timentin, 6 mg) in 0.2ml heparinized saline. After 3 days had passed, catheters were flushe d with heparinized saline daily and the thrombolytic/antibiotic cocktail every othe r day. This protocol had been proven to be effective in maintaining cat heter patency in a pilot study with a similar timeline. Operant Chambers 8 Operant cham bers (30.5 cm x 24.1cm x 21.0 cm, ENV-008CT, Med Associates, St. Albans, VT) were used throughout the experiment and were the main housing for the rats. Each chamber was located in its own soundproof enclosure and contained a single hole where ad
19 libitum access to water was provided via a sipper tube. Each chamber also possessed a single non-retractable lever and a 28V house light locate d 2-cm below the cage ceiling. This light was set on a 12hr;12hr dark/light cycle in order to mimic night /day cycles. Each rats port was connected to a piece of polyethylene tubing (P E60) protected by a steel spring which was hooked up to a syringe pump (PHM-100 set at 3.33RPM, Me d Associates) via a fluid swivel (Instech Laboratories, Inc. Plymouth Meeting, PA) to allow unlimited movement within the chamber. Vehicle and nicotine infusions were passed th rough a nitrocellulose filter (0.22m; Cameo #25ES; Osmonics) before injection. A hole was located at the center of the ceiling to allow passage of the polyethylene tubing out of the chamber. The lever was located on the wall opposite of the water tube; presses on this leve r caused a feeder (ENV-203M, Med Associcates) to release a single pellet ( 45mg, Purified Rodent Tablet, 5TUL, TestDiet, Richmond, IN; composition expressed as a %kilocalories: fat: 12.7, carbohydrate: 66.8, pr otein: 20.5) into a trough located next to the feeder. All rats were on an FR 1 reinforcement schedule for pellets, which provided complete nutrition. All apparatu s were interfaced to a computer through an input/output module (DIG-716, Med Associates) and total pellets and infusions were monitored and recorded by a software package (Med-PC Version IV). Chambers were cleaned daily; pans for Sani-Chips and water were refreshed daily and chambers were wiped down with a 70% ethanol solution every da y between sessions. Nicotine Nicotine hydrogen tartrate (S igma Chemicals) was dissolved in a sodium-phosphate buffer (pH~7.35) containing 17mg heparin per 100mls. Vehicle was the buffer-heparin solution without the nicotine. Doses of nicotine used were be 0.01, 0.03, 0.06mg/kg/injection, calculated as the free base. Rats were weighed each day an d the doses were recalculated as necessary by any changes in body weight.
20 Procedure Baseline Establishment Rats were allowed to acclim ate in their home cages for two days to both the vivarium and the TestDiet Precision pellets upon arrival. Following acclimation, rats were placed in the operant chambers for 23 hours/day and allowed to lever press for food on a fixed ratio-1 (FR1) schedule. The rats were taken out of their cages once a day for one hour to allow time for chamber maintenance an d cleaning. Baseline measures of body weight gain, total pellets consumed, meal size, meal number and meal length were established during this time. These parameters were separated into dark and light phases for further an alysis. A meal was defined as no fewer than four (4) pellets in a ten-minute bin with no more than a 10 minute (one bin) interpellet interval (Clifton 2000). While rats have the ability to press th e lever and receive food reward without eating previous pellets, preliminary studies have shown that rats do not leave uneaten pellets in the trough. Ba seline establishment was conducted for 8 days in order for the rats to develop stable food intake and meal patterns. Nicotine Administration and Removal After 7 days of collecting baseline infor ma tion, rats were implanted with intravenous catheters as detailed above. Ra ts were given two days to reco ver from surgery. Upon recovery rats were given 2 days in the chambers with ve hicle injections given on the same schedule as the nicotine injections via pump in order to ensure food intake pa rameters had not been altered by the surgery; no rats showed any significant deviat ions in food intake following surgery. While in the chambers, rats were given noncontingent, one second injections of nicotine every 30 minutes for the entire dark phase (12 hours) and for the la st 3 hours of the light phase for 12 days. This resulted in a total of 30 injections in a 23-hour period. The rats were divided into 2 drug-dose groups (n=6) and one control group (n=2) that got 30 injections of vehicle on the same schedule
21 as the nicotine injections. There were two groups receiving nicoti ne; one group received 0.06mg/kg.inj by the end of the drug period (Hi gh Dose) and one group r eceived 0.03mg/kg/inj by the end of the nicotine administration period (Low Dose). Both groups had their drug dose increased gradually over the first three days three days to avoid possible adverse effects. Groups were counterbalanced based on baseline total food intake. Food was available ad libitum via the lever a nd water was available at no cost. The body weight and meal parameters measured during baseline establishment were also measured throughout the 12 days of nicotine administration. After 12 days of nicotine administration, nicotine were replaced with vehicle and rats were placed in chambers for 14 days and allowed to lever press for food as indicated above. Body weight and meal parameters were still recorded. Because only 8 chambers were available, this study was run in 4 replications, with all 3 groups represented approximately equally in each replication. The data from each replication were then combined for analysis. Data Analysis In the experiment, raw data showed the number of pellets consumed in each 10 minute bin for the entire 23-hour (1380 minutes) session, resulting in 138 bins, as well as the total number of pellets consumed. Uneaten pellets we re counted and subtracted from the total; this amount was usually less than 5 per day. As men tioned above, individual meals were defined as no less than 4 pellets in a single 10 min bin with no more than one 10 minute interval between pellets. After meals were counted, they were split into light and dark phases for further analysis. Meal size was determined by dividing total pell ets by number of meals and this was also done for the light and dark phases. Body weights we re collected daily; additionally, weights were
22 subtracted from the previous days measuremen t to get a measure of daily bodyweight change. All meal parameters were averaged across each group. All measures were analyzed with SPSS statisti cal software (SPSS, Inc., Chicago, IL) using a two-way analysis of variance (ANOVA) with gr oups as and day as the independent variables; body weight, change in body weight, pellets cons umed (total, light, and dark), meal number (total, light, and dark) or meal si ze (total, light, and dark) were th e dependent variable. Tukeys post-hoc test for multiple comparisons was done on each individual day to look for daily differences between groups on any one measure. All data were divided into three time periods for analysis: baseline determinati on, nicotine administration, and ni cotine cessation. In all cases, p< .05 was considered significant. Graphs were generated using Sigma Plot (Systat Software, Inc., San Jose, CA).
23 CHAPTER 3 RESULTS Body Weight There was no significant group difference in body weights (expressed in gram s) or daily change in body weight (expressed in gram s per day) during the baseline period ( p >.05). There was a significant group effect of treatment on body weight (Figure 3-1) during the nicotine administration period, with the High Dose (HD) and Low Dose (LD) nicotine groups weighing significantly less than the vehicle (VEH) group [F(2,400) = 32.809, p < .05]. Using the Tukeys post-hoc test, body weights of the VEH group (Mean SEM; 325.09.93) were found to be significantly higher than the HD ( 308.91.00) and LD (303.72.97) groups during the drug infusion period ( p <.001). In order to determine whether the significantly decreased weight in rats receiving nico tine was due to a constant attenuation of weight gain or an initial decrease, data were calculated as a f unction of daily body weight change (Figure 3-2). An ANOVA of the daily body weight change during the drug infusi on period found a significant group effect [F(2, 385) = 5.330, p <.01]. Post-hoc tests revealed a si gnificant difference between the VEH group (2.12.45) and both the HD (0.23.48) and LD (0.32.47) nicotine groups ( p <.02), but no difference between the two nicotine groups. Upon cessation of nicotine, the rats in the two groups receiving nicotine (HD: 2.58.46; LD: 3.49.47) began to gain weight at a rate th at exceeded those seen in the control rats (2.02.43), although this difference wa s not significant. By the end of the study, the nicotine rats had achieved body weights equal to that of the rats in the control group. Food Intake There was no significant difference in total food intake between any of the groups during the baseline testing period (expressed in number of pellets per day; HD: 463.60.87; LD:
24 435.85.19; VEH: 441.57.19). During the infusion pe riod, there was a si gnificant difference in food intake between groups [F(2, 403) = 8.443, p <.001]. As shown in Figure 3-3, total food intake decreased in all groups during the infusion period, but rats receiving nicotine showed a much larger decrease than rats in the control gr oup. This is reflected in the post-hoc test which revealed that the VEH group (385.27.34) had much higher total food intakes than both nicotine groups ( p <.002) ; there was no significant differe nce between the two groups receiving nicotine (HD: 347.93.67; LD: 347.99.47), indicati ng there was no effect of dose. Rats receiving nicotine reached their nadir of food intake during the last three days of the drug treatment regimen, while the control group began to increase their food intake midway thought the infusion regimen. During the nicotine cessa tion period, there was no significant difference in total food intake between any of the groups (HD: 415.79.18; LD: 413.64.47; VEH: 418.03.69). Food intake during the dark pha se (expressed in number of pellets per day; Figure 3-4) between any of the groups during the baseline testing peri od (HD: 301.85.89; LD: 280.86.10; VEH: 274.68.14) or drug infusi on period (HD: 245.85.49; LD: 235.13.35; VEH: 241.93.21). During the nico tine cessation period, there was a significant group effect on dark phase food intake [F(2, 402) = 4.310, p <.02]. A post-hoc test revealed that the LD group (284.79.47) had a much higher dark ph ase food intake than the control group (255.42.70) during the nicotine cessation period. While the di fference between the control group and HD group was not significant, the HD group (270.53.21) had a higher dark phase food intake than the control group throughout most of the nicotine cessation period. Rats in the HD and LD nicotine groups reached their peak dark phase food intakes 7-10 and 4-6 days after nicotine cessation, respectively. Both nicoti ne groups food dark phase intakes decreased
25 towards the end of the cessation period and approached the dark phase food intake of the control group. There was no significant difference in the light phase food intake (expressed in number of pellets per day; Figure 3-5) during the baseline period (HD: 154.55.76; LD: 152.68.15; VEH: 165.25.19), there was a significant difference during the nicotine infusion period [F(2,403) = 39.473, p < .001). Post-hoc tests revealed si gnificant differences between the VEH group (145.31.77) and both the HD (98.78.94) and the LD group (111.46 3.84). This difference was relatively stable throughout the nicotine infusion pe riod and there was no significant difference between the two nicotine groups during the infusion period. During the nicotine cessation period, a significant group effect was seen [F(2, 401) = 10.914, p <.001]. Post-hoc tests found differences betw een the VEH group (152.09.73) and the LD (126.07.15) group, and HD (141.53.00) and LD groups. The light phase food intakes of the two nicotine groups reached the level of th e VEH group by the end of the experiment. Meal Number There was no significant difference in total daily meal number (Figure 3-6) between any of the groups (HD: 10.79.27; LD: 10.66.25; VE H: 10.68.25) during the baseline testing period or the nicotine in fusion period (HD: 11.14.23; LD: 10.65.22; VEH: 11.16.22). There was a difference in meal number betw een the VEH group and the two nicotine groups during the last 3 days of drug infusion and the first three days following removal of nicotine. There was no significant difference in the num ber of meals taken in each group during the nicotine cessation period (HD: 12.55.26; LD: 12.18.26; VEH: 12.66.24), although all groups experienced an increase in total meal number during the last phase of the study. There was no significant difference in dark phase meal number (Figure 3-7) between the VEH group (6.03.17) and either nicotine gr oup (HD: 5.97.19; LD: 5.70.17) during the
26 baseline testing period. Ther e was a significant group effect on meals during the dark phase during the drug infusion period [F(2,370) = 33.916, p < .001]. Tukeys posthoc test revealed significant differences between the HD group (6.99.16) and both the LD group (6.35.16) and VEH group (5.96.16). There was no significan t difference in dark phase meal number between any of the groups (HD: 6.93.16; LD: 6.82.17; VEH: 6.64.15) during the period in which nicotine was removed. There was no significant difference in the number of light phase meals (Fig. 3-8) during the baseline period (HD: 4.76.19; LD: 4.93.17; VEH: 4.70.17). There was a significant group effect of light phase meals [F(2,403)= 44.27, p <.001] during the drug infusion period. Post-hoc tests revealed that the VEH group ( 5.20.13) had a significantly higher number of meals during the light phase when compared to the HD (4.15.13) and LD groups (4.30.13). Upon removal of the nicotine, there was a signi ficant group effect on light phase meal number [F(2, 398) = 4.85, p < .02]; post-hoc tests revealed a si gnificant difference between the VEH group (6.02.15) and the LD group (5.36.16). Th ere was no significant difference between the HD group (5.62.15) and the LD group or the VEH group. Meal Size The baseline testing period di splayed no significant differenc es (Figure 3-9) between any of the groups regarding total m eal size (e xpressed in pellets per meal; HD: 45.78.74; LD: 43.71.59; VEH: 42.22.60), although the meal size wa s much larger in all groups during the baseline period when compared to the drug infusion period and the post te sting period. During the infusion period, there was a significant group effect on total meal size [F (2, 403) = 6.989, p <.001]. Post-hoc tests reveal ed a significant difference between VEH group (35.531.75) and the HD group (31.47.79); the LD group (33.48.77) also had a smaller meal size compared
27 to the control group but the difference was not si gnificant. There was no effect of group on total meal size (HD: 33.71.78; LD: 34.65.81; VEH: 33.72.74). There was a significant group effect in dark pha se meal size (expressed in pellets per meal; Figure 3-10) during the baseline testing period [F(2, 261) =3.066, p <.05]; both nicotine groups (HD: 55.27.06; LD: 55.92.80) had larger meal sizes than the VEH group (47.01.80) during the baseline period. During the drug in fusion period, there was a significant of group on dark phase meal size [F(2, 403) = 6.14, p <.005]. The HD group had significantly decreased dark phase meal size (36.18 1.18) when compared to th e VEH group (41.80.12); the LD group (38.33.14) had smaller dark phase meal sizes during the drug infusion period as well but this difference was not significant. Upon the rem oval of nicotine, there was a significant group effect on dark phase meal size [F(2, 398) = 3.32, p<.05]. The LD group (43.94.19) had significantly higher dark phase meal sizes when compared to the HD group (39.88.15). However, the HD group had larger dark phase m eal sizes for the majority of the nicotine cessation period, and it was only toward the end of the experiment th at they showed smaller meal sizes than the VEH group (40.72.08). There was no significant difference in light phase meal size during the baseline testing period (HD: 34.38.59; LD: 32.84 1.44; VEH: 35.83.45). During the drug infusion period, there was a significant group eff ect [F(2, 403) = 11.62, p< .001] on li ght phase meal size; posthoc tests found significant differences be tween the HD group (23.89 0.78) and both the LD group (26.83.76) and the VEH group (29.08.74). The LD group had increased light phase meal size compared to the VEH group during one 3day interval but had much smaller meal sizes during every other interval. Du ring the nicotine cessation period, there was no significant effect
28 of group on the size of meals during the light phase (HD: 25.800.68; LD: 24.84.70; VEH: 26.95.64).
29 b a s e l i n e 1 b a s e l i n e 2 b a s e l i n e 3 b a s e l i n e 4 b a s e l i n e 5 b a s e l i n e 6 b a s e l i n e 7 b a s e l i n e 8 b a s e l i n e 9 d r u g 1 d r u g 2 d r u g 3 d r u g 4 d r u g 5 d r u g 6 d r u g 7 d r u g 8 d r u g 9 d r u g 1 0 d r u g 1 1 d r u g 1 2 p o s t d a y 1 p o s t d a y 2 p o s t d a y 3 p o s t d a y 4 p o s t d a y 5 p o s t d a y 6 p o s t d a y 7 p o s t d a y 8 p o s t d a y 9 p o s t d a y 1 0 p o s t d a y 1 1 p o s t d a y 1 2 p o s t d a y 1 3 p o s t d a y 1 4 p o s t d a y 1 5Body Weight (g) 0 240 260 280 300 320 340 360 380 400 High Dose (0.06mg/kg/inj) Low Dose (0.03mg/kg/inj) Vehicle Nicotine# # # # Figure 3-1. Daily body weight (in grams; Mean SEM) of rats receiving 2 different doses of nicotine or vehicle (*: significant differe nce between nicotine (High dose (HD) & Low Dose (LD)) groups and vehicle group (V EH); **: significant difference between LD group and HD group; @: significant di fference between HD and VEH groups; #: significant difference betw een LD and VEH group; p < .05).
30 b a s e l i n e 1 9 D r u g 1 3 D r u g 4 6 D r u g 7 9 D r u g 1 0 1 2 P o s t d a y 1 3 P o s t d a y 4 6 P o s t d a y 7 1 0 P o s t d a y 1 1 1 5Daily change in body weight (g per day) -4 -2 0 2 4 6 8 High Dose (0.06mg/kg/inj) Low Dose (0.03mg/kg/inj) Vehicle Nicotine Figure 3-2. Daily change in body weight (in grams per day; Mean SEM) of rats receiving 2 different doses of nicotine or vehicle (*: significant di fference between nicotine (HD & LD) groups and VEH; **: significant difference between LD group and HD group; @: significant difference between HD a nd VEH groups; #: significant difference between LD and VEH group; p < .05).
31 b a s e l i n e 1 9 D r u g 1 3 D r u g 4 6 D r u g 7 9 D r u g 1 0 1 2 P o s t d a y 1 3 P o s t d a y 4 6 P o s t d a y 7 1 0 P o s t d a y 1 1 1 5Number of pellets per day 0 300 350 400 450 500 High Dose (0.06mg/kg/inj) Low Dose (0.03mg/kg/inj) Vehicle Nicotine* Figure 3-3. Total Food intake (in pellets per da y; Mean SEM) for rats receiving 2 different doses of nicotine or vehicl e (*: significant difference between nicotine (HD & LD) groups and VEH; **: signifi cant difference between LD group and HD group; @: significant difference between HD and VEH gr oups; #: significant difference between LD and VEH group; p < .05).
32 # b a s e l i n e 1 9 D r u g 1 3 D r u g 4 6 D r u g 7 9 D r u g 1 0 1 2 P o s t d a y 1 3 P o s t d a y 4 6 P o s t d a y 7 1 0 P o s t d a y 1 1 1 5Number of pellets duri ng dark phase per day 0 180 200 220 240 260 280 300 320 340 High Dose (0.06mg/kg/inj) Low Dose (0.03mg/kg/inj) Vehicle Nicotine Figure 3-4. Dark phase food intake (in pellets per day; Mean SEM) for rats receiving 2 different doses of nicotine or vehicle (*: significant di fference between nicotine (HD & LD) groups and VEH; **: significant difference between LD group and HD group; @: significant difference between HD a nd VEH groups; #: significant difference between LD and VEH group; p < .05).
33 b a s e l i n e 1 9 D r u g 1 3 D r u g 4 6 D r u g 7 9 D r u g 1 0 1 2 P o s t d a y 1 3 P o s t d a y 4 6 P o s t d a y 7 1 0 P o s t d a y 1 1 1 5Number of Pellets duri ng light phase per day 0 60 80 100 120 140 160 180 200 High Dose (0.06mg/kg/inj) Low Dose (0.03mg/kg/inj) Vehicle Nicotine#@* Figure 3-5. Light phase food inta ke (in pellets per day; Mean SEM) of rats receiving 2 different doses of nicotine or vehicle (*: significant di fference between nicotine (HD & LD) groups and VEH; **: significant difference between LD group and HD group; @: significant difference between HD a nd VEH groups; #: significant difference between LD and VEH group; p < .05).
34 b a s e l i n e 1 9 D r u g 1 3 D r u g 4 6 D r u g 7 9 D r u g 1 0 1 2 P o s t d a y 1 3 P o s t d a y 4 6 P o s t d a y 7 1 0 P o s t d a y 1 1 1 5Number of total meals per day 0 10 12 14 16 High Dose (0.06mg/kg/inj) Low Dose (0.03mg/kg/inj) Vehicle Nicotine Figure 3-6. Mean total meal nu mber (Mean SEM) for rats receiving 2 different doses of nicotine or vehicle (*: significant differe nce between nicotine (HD & LD) groups and VEH; **: significant difference between LD group and HD group; @: significant difference between HD and VEH groups; #: significant difference between LD and VEH group; p < .05).
35 b a s e l i n e 1 9 D r u g 1 3 D r u g 4 6 D r u g 7 9 D r u g 1 0 1 2 P o s t d a y 1 3 P o s t d a y 4 6 P o s t d a y 7 1 0 P o s t d a y 1 1 1 5Number of meals in dark phase per day 0 4 6 8 10 High Dose (0.06mg/kg/inj) Low Dose (0.03mg/kg/inj) Vehicle Nicotine**@ @ @ Figure 3-7. Mean dark phase meal (Mean SEM) number for rats receiving 2 different doses of nicotine or vehicle (*: significant differe nce between nicotine (HD & LD) groups and VEH; **: significant difference between LD group and HD group; @: significant difference between HD and VEH groups; #: significant difference between LD and VEH group; p < .05).
36 b a s e l i n e 1 9 D r u g 1 3 D r u g 4 6 D r u g 7 9 D r u g 1 0 1 2 P o s t d a y 1 3 P o s t d a y 4 6 P o s t d a y 7 1 0 P o s t d a y 1 1 1 5Number of meals during light phase per day 0 4 6 8 High Dose (0.06mg/kg/inj) Low Dose (0.03mg/kg/inj) Vehicle Nicotine* *@* Figure 3-8. Mean light phase meal number (Mean SEM) for rats receiving 2 different doses of nicotine or vehicle (*: significant differe nce between nicotine (HD & LD) groups and VEH; **: significant difference between LD group and HD group; @: significant difference between HD and VEH groups; #: significant difference between LD and VEH group; p < .05).
37 b a s e l i n e 1 9 D r u g 1 3 D r u g 4 6 D r u g 7 9 D r u g 1 0 1 2 P o s t d a y 1 3 P o s t d a y 4 6 P o s t d a y 7 1 0 P o s t d a y 1 1 1 5Number of pellets per meal 0 30 40 50 High Dose (0.06mg/kg/inj) Low Dose (0.03mg/kg/inj) Vehicle Nicotine Figure 3-9. Mean 23-hr meal si ze (in pellets per meal; Mean SEM) for rats receiving 2 different doses of nicotine or vehicle (*: significant di fference between nicotine (HD & LD) groups and VEH; **: significant difference between LD group and HD group; @: significant difference between HD a nd VEH groups; #: significant difference between LD and VEH group; p < .05).
38 ** b a s e lin e 1 9 D r u g 1 3 D r u g 4 6 D r u g 7 9 D r u g 1 0 1 2 P o s td a y 1 3 P o s td a y 4 6 P o s td a y 7 1 0 P o s td a y 1 1 1 5Number of pellets per meal 0 35 40 45 50 55 60 65 High Dose (0.06mg/kg/inj) Low Dose (0.03mg/kg/inj) Vehicle Nicotine Figure 3-10. Mean dark phase meal size (in pellets per meal; Mean SEM) for rats receiving 2 different doses of nicotine or vehicle (*: significant di fference between nicotine (HD & LD) groups and VEH; **: significant difference between LD group and HD group; @: significant difference between HD a nd VEH groups; #: significant difference between LD and VEH group; p < .05).
39 b a s e l i n e 1 9 D r u g 1 3 D r u g 4 6 D r u g 7 9 D r u g 1 0 1 2 P o s t d a y 1 3 P o s t d a y 4 6 P o s t d a y 7 1 0 P o s t d a y 1 1 1 5Number of pellets per meal 0 20 25 30 35 40 High Dose (0.06mg/kg/inj) Low Dose (0.03mg/kg/inj) Vehicle Nicotine@ @** Figure 3-11. Mean light phase meal size (in pellets per meal; Mean SEM) for rats receiving 2 different doses of nicotine or vehicle (*: significant di fference between nicotine (HD & LD) groups and VEH; **: significant difference between LD group and HD group; @: significant difference between HD a nd VEH groups; #: significant difference between LD and VEH group; p < .05).
40 CHAPTER 4 DISCUSSION This project sought to develop an in travenous model of intermittent nicotine administration to test the hypotheses that nico tine causes a decrease in food inta ke and a concurrent attenuation in body weight gain, and that removal of the nico tine will result in an incr ease in body weight at a rate that will exceed the rate of body weight gain seen in control rats. Rats receiving nicotine in this study exhibite d a complex change in food intake parameters both during and after nicotine administration. Rats in both nicotine groups exhibited a significant reduction in body weight gain during nicotine administ ration when compared to the group receiving vehicle infusions, although there appeared to be no si gnificant dose effect between the two nicotine groups. While this change in rate of body weight gain took ~3 days to manifest itself, it persisted throughout the rest of the drug infusion period. This reduction in weight gain can be mostly attributed to a ~40 pe llet per day decrease in food intake, an effect which persisted throughout the nicotine administrati on period. Previous st udies (Bellinger et al. 2003a; Bellinger et al. 2003b; Wellman et al. 2005) have found initial decreases in food intake that eventually disappeared after a couple days. This difference may be due to the differences in the route of administration, as one study that examined food intake in rats on a 23-hour intravenous self-administration pr otocol exhibited decr eased total food intake throughout the self administration period (O'Dell et al. 2007). This decrease in tota l food intake was primarily due to a decrease food intake during th e light phase, although a divergen ce in dark phase food intake started to appear during the end of the nicotine administration period and it is possible that a significant difference may have a ppeared had nicotine administrati on continued. This decrease in light phase food intake is in contrast to pr eviously published reports which had found that the decrease in food intake was primarily due to changes in dark phase food intake, without any
41 significant changes to light phase food intake. Th e decrease in light phase food intake did not begin until ~3 days into the drug infusion pe riod and coincided with the attenuation of body weight gain, indicating th at it was principally responsible for the change in rate of body weight gain. The decrease in total food intake that was s een in the two groups receiving nicotine was due to a significant decrease in meal size and the lack of a compensatory increase in total meal number. An analysis of the parameters of light phase food intake indicated that the decrease in light phase food intake was cause d by significant decreases in bot h meal size and meal number, both of which persisted throughout the entire dr ug infusion period. Dark phase food intake remained relatively unchanged throughout the nico tine administration period. However, a closer look reveals that intermittent nicotine infusions accompanie d decreases in meal size and increases in meal number. These increases in meal number were able to compensate for the smaller meals and resulted in a dark phase food inta ke that was equal to that of the control group. This change in dark phase meal structure has be en shown in previous studies (Bellinger et al. 2003b; Bellinger et al. 2005; Well man et al. 2005), although the compensatory changes seen in those studies occurred after se veral days of nicotine administ ration. The rats in this study exhibited an almost immediate increase in meal nu mber to account for the decrease in meal size during the dark phase. These changes were not sufficient enough to make up the caloric deficit that was accrued during the light phase and was only able to compensate for the decreased meal size in the dark phase. Overall, a decrease in m eal size, particularly in the light phase, was the prime source of the decrease in food intake seen in the rats receiving nicotine. Following the removal of nicotine, rats in th e two nicotine groups experienced an increase in the rate of body weight gain that was highe r than that of the c ontrol group, although not
42 significantly so. This change resulted in the ra ts receiving nicotine r eaching a body weight equal to that of the control rats by the end of the study. This increase in body weight was accompanied by an increase in food intake relative to the f ood intake seen in the nicotine groups during the drug infusion period. Their food intake during the post-nicotine period was not significantly higher than the food intake of the rats in the vehicle group, but all three groups did experience an increase in food intake during the post-drug period. Despite both groups having similar amounts of total food intake during the nicotine cessation peri od, the rats that had received nicotine gained much more weight. Interestingly, the lig ht phase food intake of the two nicotine groups were significantly lower than that of the c ontrol group during the majority of the post-drug period, although they eventually reached the level of the control group by the end of the experiment. The dark phase food intake of both nicotine groups increased to levels well above that of the control group, which accounts for the defi cit in light phase food intake that remained. The dark phase food intake began to decline toward the end of the experimental period, so it is unknown whether the rats that had received nicotine would have exceeded the body weight of the control group. This decline may have been du e to the normalization of the light phase food intake, but further study is needed to determin e if the body weights would remain equal or if significant differences would appear following a longer peri od of nicotine cessation. Although all three groups experienced an increase in meal number during the post-drug period, the increased total food intake seen in the ni cotine groups was due to an increase in total meal size while the control group in creased their total food intake vi a increases in meal number. While not significant, the nicotine groups ate larger meals during th e post drug period when compared to the control group. This change in total meal size was caused by an increase during the dark phase; the light phase meal size retu rned to levels simila r to the control group upon
43 removal of the nicotine. Total meal number also gradually attained the levels of the control group due to the number of light phase meals increasing steadily over the nicotine cessation period. Dark phase meal number, which was elev ated in a possible compensatory behavior to make up for the smaller dark phase meals, decreased during the cessati on period and was not significantly different th en the control group. These results reinforce the idea that there is an inverse re lationship between nicotine and body weight. Similar to previously published reports, nicotine caused a substantial blunting of body weight gain by decreasing food intake. Th ese changes in food intake were brought on by significant decreases in meal size and meal number, especially during the light phase. This is in contrast to other studies (Bel linger et al. 2003a; Bellinger et al. 2003b; Bellinger et al. 2005; Miyata et al. 2001; Wellman et al. 2005) which witnessed decreased food intake during the dark phase without any change to light phase food inta ke; this decrease in dark phase food intake was due to decreases in meal size without any change in meal number. After 5-7 days, rats in those studies frequently engaged in a compensatory increase in meal number to account for the decrease in meal size. After this compensation took place, rats gain ed weight at levels that were roughly equal to that of the control groups. Rats in the present study also experienced decreased dark phase meal size, but the increase in meal number occurred simultaneously and dark phase food intake remained unchanged. These differences could be due to the route of administration used in this study and previous stud ies. Past work in this area has used either constant infusion via osmotic mini-pumps or relatively large intrap eritoneal injections; it is difficult to determine how much nicotine is reaching the bloodstream using these methods, and by extension, how much is reaching the sites of acti on. It is possible that nicotine administered subcutaneously or intraperitoneally does not elevate blood nicotine levels enough to a ffect light phase food intake.
44 O"dell et al. (2007) used a 23hour model of nicotine self-a dministration, which used an intravenous route of administrati on, and looked at food intake on the first and last days of the 40 day nicotine administration period. They also found significantly decreased light phase food intake, although this seemed to be due solely to decreased meal numbe r. Additionally, they found substantial decreases in dark phase food in take that was caused by decreases in both meal size and meal number. This difference could be due to the self-administration protocol which required pressing a lever for nicotine; the pres ent study used non contingent injections which meant that the rat did not have to interrupt eat ing to respond for food and vice versa. It should also be noted that while the number of meals, meal size a nd food intake in the present experiment were similar to prev ious studies, differences in th e criteria for determining the various meal parameters may have resulted in some of the differences. Overall, this study indicates that route of admini stration may have a significant e ffect on the impact of nicotine administration on food intake and that the magnitude of this impact may be greater if a route of administration is used that more accurately emul ates the method that is used by human smokers. The major discrepancy between this study and previous reports (Bel linger et al. 2003a; Bellinger et al. 2003b; Bellinger et al. 2005; Miyata et al. 2001; Wellman et al. 2005) is the reduced food intake during the light phase. In past studies, total f ood intake was suppressed during nicotine administration and th is was solely due to changes in dark phase meal parameters. While there was alteration of dark phase food in take in the present study, light phase food intake was also significantly altered, indicating that ni cotine had some effect on the neural mechanisms involved in food intake during that time of the day, despite little nicotine being administered at that time. Gries et al. (1996) looked at the circadian rhythms of ni cotine metabolism and found that it was at its peak during the active period and reached a nadir sometime during the inactive
45 period. Hepatic blood flow is a prime determinant of drug clearance and is influenced heavily by circadian rhythms as well; it reaches its peak dur ing the active periods, which causes an increase in drug clearance during this time. Additionall y, blood flow to the liver increases following meals, which are more frequent during the activ e periods. Therefore, decreased food intake during the inactive perio d, coupled with a lower basal level of hepatic blood flow, results in a decreased rate of nicotine clearance. This may result in nicotine levels remaining elevated for longer periods during the inactive periods comp ared to the active period despite no nicotine administration during this time. This effect is no t seen in other studies wh ich use different routes of administration because plasma levels of nicotine may not be high enough at the end of the dark phase to exert any action during a pr olonged period without ni cotine administration. Osmotic mini-pumps should have a similar effect on light phase food intake, due to 24-hour infusions of nicotine, but the studies that have used that particular route of administration for extended periods of time (Bishop et al. 2002; 2004; Grunberg 1982; Gr unberg et al. 1988a; Grunberg et al. 1988b; Miya ta et al. 2001) have not included an analysis for dark/light phase meal patterns in the study. Another possible reason behind the changes in diurnal food intake is due to nicotines effects on the brains internal clock, the supe rchiasmatic nucleus (SCN). The SCN is the primary pacemaker of the circadian rhythms in the brain and uses external cues such as light. The SCN also has connections with the lateral hy pothalamus (LHA) in order to regulate the daily rhythms of food intake; the light phase causes inhibition of the LHA and suppresses feeding, although this is not the only means by which the circadian rhythms of food intake are determined. It is possible that nicotine may somehow modify the circadia n rhythms via nicotinic receptors on the SCN (O'Hara et al. 1998). Tr achsel et al. (1995) found that nicotine causes
46 phase-advances in the circadian rhythms and other reports indicate that nicotine alters the circadian rhythms of dopamine and serotonin levels within the striatum, causing the levels of both dopamine and serotonin to be increased during the light phase when compared to controls (Pietil et al. 1995). The striatum has proj ections to and from the hypothalamus, and any alterations in the circadian rhyt hm of monoamine levels within the striatum may affect food intake patterns. While nicotine most likely exerts its primar y influence on body weight via food intake, there may be metabolic changes due to nicotine ad ministration. Rats in the two nicotine groups gained ~45g compared with a ~30g body weight gain by controls over the same period despite similar food intakes. Acute changes in metabolic rate have been found in humans receiving intravenous nicotine (Perkins et al. 1989). Bishop et al. (200 4) found that peripheral nicotine administration in rats increased the respiratory quotient, a meas ure of metabolic activity, after two days of exposure although this effect disappeared over time. Also, removal of the nicotine caused a short term decrease although this was not accompanied by any increase in body weight when compared to controls. However, anothe r measure of metabolic activity that was taken, energy expenditure, indicated no significant effect of nicotine. Nicotine has also been found to increase thermogenesis in brown adipose tissue in rats although there were no changes seen in body weight (Lupien and Bray 1988). Uncoupling proteins (UCPs), which are involved in cellular respiration, are thought to be involved in regulati ng thermogenesis in brown adipose tissue, but although it is unclear how they ar e involved. One group has found both increases (Chen et al. 2005) and decreases (Chen et al. 2006) in UCP levels within brown adipose tissue following prolonged exposure to cigarette smoke While changes in UCP messenger ribonucleic acid (mRNA) levels or other mechanisms i nvolved in regulating metabolic activity are
47 significant, it has yet to be definitively shown that nicotine changes metabolic activity. Studies that have hypothesized a link between nicotine and changes in metabolic activity have relied on physiological markers such as respiratory quot ient (Bishop et al. 2004) or UCP mRNA levels (Chen et al. 2005; 2006), or have be en inferred based on behavioral indicators such as increased locomotor activity (Benwell et al. 1994). Studies that incorporate both methods as well as show congruence between them would be a more c onvincing argument for nicotines effects on metabolic rate. Subsequent experiments with this model should focus on more accurately defining the neurobiological mechanisms surrounding the nicotine-induced changes in food intake, specifically, neurochemical changes within the h ypothalamus. Also, it would be interesting to determine if there are differences regardi ng macronutrient selecti on during the nicotine administration or cessation period as some studies have suggested (Gr unberg et al. 1988b). Studies using longer periods of both nicotine ad ministration and cessation may also give a more accurate picture of the eff ects of chronic nicotine on f ood intake and body weight.
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53 BIOGRAPHICAL SKETCH Ian E. Thompson graduated m agna cum laude fr om the University of Buffalo in 2007 with a Bachelor of Science Degree in biomedical sciences and psychology. He became interested in behavioral neuroscience following several classes in topics rela ted to drug addiction and began working in a lab that used behavioral neuroscience to study bioacoustics as an undergraduate. As he was completing his undergraduate degree, he began looking for a lab that studied the addictive properties of drugs such as nicotine using intravenous self-administration models. In the winter of 2007, he was accepted into the University of Floridas Behavioral Neuroscience Program in the Department of Psychology as a College of Liberal Arts and Sciences Alumni Fellow under Dr. Neil E. Rowland. His fields of study included research into gustatory secondary reinforcers and their ro les in nicotine administration in addition to the work for his masters thesis. He defended his masters thes is in the summer of 2009; it was concerned with testing the effects of nicotine on food intake and body weight using a different route of administration than previous studies. He r eceived his Master of Science Degree from the University of Florida in the summer of 2009. Afte r graduation from the University of Florida in the summer of 2009, he will begin prep aring to apply for medical school.