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Dopamine transporter knockdown by antisense oligonucleotides attenuates reinstatement of cocaine self-administration… Schachter, Michael Eli 2001

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1 DOPAMINE T R A N S P O R T E R KNOCKDOWN BY ANTISENSE OL IGONUCLEOTIDES ATTENUATES RE INSTATEMENT OF COCAINE SELF-ADMINISTRATION AFTER EXTINCTION by MICHAEL ELI S C H A C H T E R B.Sc, McGill University, 1998 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE D E G R E E OF MASTER OF SC IENCE in THE FACULTY OF GRADUATE STUDIES Graduate Program in Neurbscience We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISFf COLUMBIA June 15, 2001 © Michael Eli Schachter, 2001 f In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada 11 Abstract Abstract: Mice lacking the dopamine transporter (DAT) s e l f -administer cocaine (Rocha et al., 1998). However, i n DAT knockout mice a v a r i e t y of compensatory changes may occur, including increased basal dopamine le v e l s , decreased dopamine receptor number and behavioral hyperactivity. In the present cocaine self-administration study, we attempted to reduce these confounds by knocking down DAT expression i n adult Long-Evans rats using phosphorothioate antisense oligonucleotides targeting DAT mRNA. Animals were trained to self-administer a low-dose of cocaine (0.1 mg/infusion, FR2, TO30s) a f t e r which drug seeking was extinguished (50-110 hours) by substituting saline for cocaine. Next, half the rats were treated with DAT antisense (1 nmol/day for 6 days) b i l a t e r a l l y into the VTA using osmotic minipumps, and half the rats received a missense control sequence. On the s i x t h day of antisense/missense-administration, reinstatement of drug taking was assessed following one infusion of cocaine (0.1 mg/infusion) (priming) . Drug taking was also assessed without priming on two subsequent days. DAT antisense resulted i n a s i g n i f i c a n t (42%) reduction i n DAT binding i n the nucleus accumbens, assessed using [3H]WIN 35,428. Rats treated with DAT antisense responded s e l e c t i v e l y on the drug-appropriate lever during the f i r s t session following extin c t i o n . However discriminant responding was completely disrupted by the second session and on the f i n a l t r i a l , cocaine self-administration was s i g n i f i c a n t l y reduced. Drug taking i n the missense group was comparable to pre-extinction l e v e l s . These .data suggest that cocaine's i n t e r a c t i o n with DAT i s a necessary component of i t s r e i n f o r c i n g properties. i v Fig. 1 - "7.8" during cocaine self-administration TABLE OF CONTENTS A b s t r a c t i i T a b l e o f C o n t e n t s v L i s t o f T a b l e s v i i i L i s t o f F i g u r e s i x A c k n o w l e d g e m e n t s x SECTION 1 - INTRODUCTION 1 W i t h d r a w a l , D e p e n d e n c e , T o l e r a n c e a n d S e n s i t i z a t i o n .. 2 R e l a p s e 4 H e d o n i c H o m e o s t a t i c D y s r e g u l a t i o n 7 A n i m a l M o d e l s of A d d i c t i o n 8 Fixed-Ratios and Binges 9 Interpreting the Dose Response Curve 10 Progressive-Ratios and Second Order Schedules of Reinforcement 12 A n a t o m y of the D o p a m i n e S y s t e m : 14 P s y c h o s t i m u l a n t s a n d D o p a m i n e : 15 A l t e r n a t e E x p e r i m e n t a l S t ra teg ies : 2 0 Inducible Knockouts 2 0 Antisense Technology 2 2 P u r p o s e o f the P r e s e n t E x p e r i m e n t : 2 8 SECTION 2 - MATERIALS AND METHODS 29 S u b j e c t s 2 9 vi Food Training 29 Apparatus for operant training and intravenous self-administration 30 Surgery 30 Intravenous Catheter Surgery 31 Stereotaxic Surgery . 31 Minipump Implantation 32 Experimental Procedure 32 Antisense Oligonucleotides 34 Reinstatement 35 Autoradiography 35 SECTION 3 - RESULTS 36 Acquisition . . 36 Extinction 37 Reinstatement 38 DAT Knockdown 39 Histology 39 SECTION 4 - DISCUSSION 40 Interpreting the results 43 DAT, NET, and SERT 45 Future Directions • • • • 52 Conclusion 54 SECTION 5 - FIGURES 55 SECTION 6 - BIBLIOGRAPHY LIST OF TABLES Table 1. Acquisition of Cocaine Self-Administration ix LIST OF FIGURES Fig. 1 - Animal "7.8" During Self-Administration . . . . iv Fig. 2 - Intravenous self-administration of cocaine in Rhesus Monkeys 10 Fig. 3 - Phosphorotioate, End-modified, and Centrally-Modified mixed backbone oligonucleotides . . . 25 Fig. 4 - Schematic representation of the experimental procedure 34 Fig. 5 - Mean presses on the active lever during the first and final extinction sessions 55 Fig. 6 - Mean presses on the active lever during the First 90 minutes of first five extinction sessions 56 Fig. 7 - Mean presses on the active and inactive levers during the first and final extinction sessions .. 57 Fig. 8 - Acquisition and Drug-Induced Reinstatement Of Pressing on the Active Lever After Extinction 58 Fig. 9 - Ratio of active to inactive lever presses during Three reinstatement/reacquisition trials . . . . 59 Fig. 10 - Densitometric Analysis of [3]H-WIN Binding . . 60 Fig. 11 - VTA locations of oligonucleotide infusion canula 61 A C K N O W L E D G E M E N T S This thesis was made possible through the combined efforts of a large number of people. My supervisors, Drs. Tony Phillips and Jon Stoessl, provided a project, funding, guidance and tolerance. Thank you both. My committee assessed my proposal and helped make it better. Fred Lepiane and Rick Kornelson were invaluable for their technical support and assistance with the details when I was away in Baltimore. Jackalina Van Kampen's work with antisense in Dr. Stoessl's lab broke ground for me to follow, and she as well as Pornarin Taepavarapruk (for IVSA) taught me the relevant techniques. In the lab, Allisdair, John, Rachel and Soyon were always up for a talk or an outing to Koerner's - thanks guys. And, most importantly of course, I'd like to thank my family and friends for everything. Mom, dad, Theo, grandma Helen, Grandpa Hi (and now alphabetically) Benny, Coughs, Goobs, Ian, Jay, Joey, Moore, Rich, Scully, Ted, Tones, Yann and everyone else. Wherever I go, you guys are with me. 1 I n t r o d u c t i o n : Drug addiction, and cocaine abuse in particular, is a serious problem in North America with over five million cocaine users each year in the U.S. [C a i n e. 1998] Generally, illicit drug use accounts for $50 billion in criminal earnings as well as 35% of new HIV infections each year [MacCoun & Reuter, 1997] Massive governmental efforts at policing the use and dissemination of these drugs amount to more than twice the sum spent on biomedical research in America, yet supplies remain strong and the number of first-time users is increasing [Bloom, 1997] At present there are no reliable treatments for addiction. By definition, drug addiction is "a syndrome characterized by compulsive drug-seeking behavior that results in an impairment in social and psychological functions or damage to health" [ ^ , 1 9 9 4 1 . Despite negative outcomes drug use remains irrepressible by the addict. Thus, addiction is also marked by loss of control over formerly voluntary acts [Goldstein, 1994] Drug use disrupts the logic of self-preservation; by overriding all other motivators it becomes the single most important goal for the addict [Leschner, 2000] Subjectively, depending on the route of administration, small doses of cocaine (100 mg or less) produce a "rush" and subsequent feeling of euphoria. These effects are typically accompanied by increases in alertness, body temperature, heart rate, blood pressure and energy, dilation of pupils, constriction of blood vessels and suppression of appetite. Larger doses intensify and prolong the high, but are sometimes accompanied by unforeseen side effects, such as tremors, vertigo, muscle twitches, paranoia, and occasional fatalities. Deaths usually result from drug-induced seizures or cardiac arrest [National Institute on Drug Abuse Research Report Series, 1999] _ With chronic cocaine abuse, users often fall into a characteristic cycle of binge use, abstinence, withdrawal and relapse, or some variation thereof [Connors etai., 1996] other phenomenon, such as tolerance and sensitization also arise. 2 Withdrawal , Dependence, Tolerance, and Sensit izat ion: Wi thdrawal , or a "negative affective state", sometimes develops upon cessat ion of drug- intake or a signif icant decrease i n dose. Descr ibed as a "quasi rebound opposite i n direction to the in i t ia l d rug effects", the phenomenon is known to coincide wi th disappearance of d rug from the body [OBrien, 1995] Symptoms range from mi ld ly severe abdomina l c ramps and hypertension for opiates, to more serious concerns s u c h as seizures i n the case of a lcohol wi thdrawal [Goldstein, 1994] j n h u m a n cocaine abusers dysphor ic states fol lowing d rug binges occur in some, but not a l l users , cons ist ing of t ransient t i redness, anxiety a n d / o r depression for durat ions of no more than a few days after d rug use [O'Brien, 1995] A s renewed drug - tak ing can relieve these symptoms, w i thdrawal has been identif ied as one factor that may contr ibute to propagation of the addictive cycle. A n i m a l models of psychost imulant wi thdrawal have been developed, w h i c h provide a n objective (quantifiable) view of the phenomenon, lending support to the hypothesis that negative reinforcement 1 may be important i n mainta in ing the cycle of addict ion [Arroyo et at, i998]_ p o r example, rats al lowed to sel f -administer cocaine for long periods of time have been shown to develop elevations i n b ra in reward thresholds for in t racran ia l se l f -s t imulat ion (ICSS) [Markou and Koob, i99i]_ These elevations were proport ional to the amount of cocaine taken du r ing the preceding drug-session, and presumably the subsequent requirement for greater st imulat ion intensity to produce reward suff icient to warrant cont inued responding is indicative of a less responsive hedonic system (a state of withdrawal) . Fur ther evidence for the presence of negative d rug -associat ions i n an ima l models is provided by Ettenberg et al, who observed a character ist ic "approach-avoidance" behavior in rats that were approaching a box i n w h i c h they had received IV doses of cocaine [Ettenberg & Geist, 1993] 7/his un ique behavior was noted to be indist inguishable from that shown by other an ima ls approaching a box in w h i c h food avai labi l i ty was paired w i th foot-1 In negative reinforcement behavior that relieves a negative or undesirable stimulus is repeated. In contrast, positive reinforcement promotes behavior that results in a pleasurable or positive outcome. 3 shock IGeist & Ettenberg, 1997] T/hus, the an ima l is presented wi th a cost-benefit scenario i n w h i c h it wants food, but mus t weigh whether the positive experience of satiat ing hunger justifies a painful foot-shock. In both cases the anxiolyt ic agent diazepam reduces the approach-avoidance response i n a dose-dependent manner [Ettenberg et ai, 1999] Cocaine is also k n o w n to s t imulate the release of stress hormones corticosterone and A C T H [Moidow & Fischman, i987] } lending further support for a possible anxiogenic effect. Together, these data indicate that both positive and negative drug-effects exist, and may contr ibute to motivated drug-seeking behavior. This idea has been termed the Opponent-Process Theory of motivated behavior [Solomon & corbit, 1973]. This framework aside, phys ica l wi thdrawal from most drugs of abuse can now be treated us ing various therapeutic options, the most prominent of w h i c h is methadone treatment for opiate addicts. However, no treatment for wi thdrawal has proven to be a functional solut ion to addict ion. Therefore, addic t ion is no longer equated wi th phys ica l dependence. Dependence is characterized by the development of wi thdrawal symptoms a n d d rug tolerance 2 [First, 1994] i n fact, a l though dependence does occur i n some cases of addict ion, it is no longer considered sufficient, or necessary to describe addict ion. The concept of dependence does not include compulsive drug-seeking behavior, a core aspect of the new definition of addict ion, and its m a i n components -tolerance and wi thdrawal - disappear wi th in a few days P e r k e & Hyman, 2000] g y contrast, addic t ion is a s t r ikingly persistent condi t ion marked by long-term neura l changes. For example, sensit ization, a fourth phenomenon associated w i t h the addictive cycle, persists after phys ica l dependence has long-since subsided. Sensi t izat ion is defined as a leftward shift i n the dose-response curve [Aitman, 1996] Neurochemical correlates of sensit izat ion inc lude increased dopamine response i n the nucleus accumbens to a subsequent injection of a drug, even after prolonged abstinence [Chefer et al, 2000; Robinson & Badiani, 1998] 2 Tolerance is a progressive reduction of pharmacological response after repeated exposure to drug. 4 B e h a v i o r a l l y i t i s c h a r a c t e r i z e d b y t h e d e v e l o p m e n t o f p r o g r e s s i v e l y g r e a t e r r e s p o n s e s t o a c o n s t a n t d o s e o f d r u g . N o t a b l y , c o n s t a n t d r u g - a d m i n i s t r a t i o n a s o p p o s e d t o a n i n t e r m i t t e n t s c h e d u l e s e e m s to b r i n g a b o u t t o l e r a n c e i n s t e a d o f s e n s i t i z a t i o n . D a l i a et al. d e m o n s t r a t e d t h i s e f f e c t u s i n g a n i n d w e l l i n g o s m o t i c p u m p , w h i c h c o n t i n u a l l y d e l i v e r e d d r u g a t a s t e a d y r a t e t o p r o m o t e t h e e x p r e s s i o n o f t o l e r a n c e ID a l i a e t al> 1 9 9 8 1 o v e r s e n s i t i z a t i o n . C o c a i n e - s e n s i t i z e d r a t s w e r e e x p o s e d to c o n t i n u o u s d r u g a d m i n i s t r a t i o n f o r s e v e n d a y s (80 m g / k g p e r d a y ) a n d t h e n c h a l l e n g e d w i t h a n a c u t e c o c a i n e i n j e c t i o n (7 .5 m g / k g i .p . ) . T h e p r e v i o u s l y d e m o n s t r a t e d s e n s i t i z e d r e s p o n s e w a s a b s e n t . H o w e v e r , 1 0 d a y s a f t e r r e m o v a l o f t h e p u m p , w h e n t e s t e d a g a i n , t h e s e n s i t i z e d r e s p o n s e h a d r e t u r n e d . T h i s f i n d i n g s u g g e s t s t h a t s e n s i t i z a t i o n a n d t o l e r a n c e a r e i n d e p e n d e n t p r o c e s s e s . W h e n p r e s e n t c o n c u r r e n t l y , t o l e r a n c e a p p e a r s to b e p r e f e r e n t i a l l y e x p r e s s e d d e s p i t e a s h o r t e r f u n c t i o n a l l i f e s p a n Pai ia eta*., 1998] T h e n e u r a l c o r r e l a t e s o f b o t h p h e n o m e n a m a y o c c u r a t t h e s a m e t i m e , b u t t h e e f f e c t s o f t o l e r a n c e a r e b e h a v i o r a l l y e v i d e n t a t f i r s t , a n d l a t e r r e p l a c e d b y t h e m o r e l o n g - l a s t i n g e f f e c t s o f s e n s i t i z a t i o n . R e l a p s e : T h e f i n a l a s p e c t o f t h e a d d i c t i v e c y c l e , a n d t h e m o s t p r o b l e m a t i c f r o m a t r e a t m e n t p o i n t o f v i e w , i s t h e t e n d e n c y f o r i n d i v i d u a l s t o r e i n i t i a t e d r u g - t a k i n g a f t e r a p e r i o d o f a b s t i n e n c e . T h i s i s t e r m e d r e l a p s e . H u m a n s t u d i e s o f r e l a p s e h a v e p r i m a r i l y f o c u s e d o n t h e r o l e o f e n v i r o n m e n t a l c u e s i n c l u d i n g p e o p l e , p l a c e s , o r o b j e c t s p r e v i o u s l y p a i r e d w i t h p s y c h o s t i m u l a n t s s u c h a s c o c a i n e . T h e s e t y p e s o f s t i m u l i h a v e b e e n s h o w n to p r o d u c e c r a v i n g , a n d u l t i m a t e l y r e l a p s e , i n a b s t i n e n t h u m a n a d d i c t s l ™ * . 1 9 " ; O B r i e n > 1 9 9 6 ; shiffman et ai, 1996]. S i m i l a r l y , a n i m a l m o d e l s o f r e l a p s e e m p l o y i n g c o n d i t i o n e d c u e s to m e a s u r e d r u g - s e e k i n g h a v e d e m o n s t r a t e d a s t r i k i n g l y p o w e r f u l a n d e n d u r i n g e f fec t . R a t s t r a i n e d to d i s c r i m i n a t e b e t w e e n a c o c a i n e - a s s o c i a t e d c u e ( ' S + ' - w h i t e n o i s e ) a n d a s a l i n e - a s s o c i a t e d c u e ('S"' - h o u s e l i g h t on ) i n s e r i a l d a i l y s e l f -a d m i n i s t r a t i o n s e s s i o n s , d e m o n s t r a t e d r e - i n i t i a t i o n o f d r u g - s e e k i n g b e h a v i o r i n 5 response to presentation of the S + , but not S", after ext inct ion l ~ W e i s s e t a l - 2 0 0 0 I . Th is re- ini t ia t ion was accompanied by elevations i n dopamine levels i n the nuc leus accumbens and amygdala. Drug-seeking persisted i n these an imals despite an average 16.5-day abstinence period and throughout eight re-acquis i t ion sessions i n the absence of the pr imary reward (cocaine). Moreover, a subsequent s tudy demonstrated that presentation of the discr iminat ive s t imulus (S+) cou ld reinstate drug-seeking after ext inct ion followed by an even longer, four month , abstinence period [CiccocioP Po et ai, 2001] j n th is c a s e presentat ion of the S + was also shown to induce expression of the c-/os gene i n the basolateral amygdala and media l prefrontal cortex. Reinstatement and c-fos gene expression were both attenuated by pre-treatment wi th a D i - r ecep to r antagonist . The fact that animals remain susceptible to cue- induced reinstatement of drug-seeking for long periods of time, wi thout benefit of further drug-reinforcement i n the in ter im, suggests that condi t ioned cues develop remarkably robust associations wi th drug. Fur thermore , i n cases where cocaine-adminis t ra t ion follows cue presentation it is believed that the d rug amplifies the cue's impact on subsequent drug- taking behavior [E veritt et ai, 1999] Th i s impl ies that identifying and addressing these types of drug-associated cues may be cri t ical ly important i n the prevention of relapse i n h u m a n addicts. In addi t ion to this evidence concerning the importance of condi t ioned cues, it is also k n o w n that relapse is more l ikely to occur du r ing periods of life stress i n h u m a n s [Shaham etal, 2000] However, the correlation between stress and relapse i n h u m a n studies does not, i n itself, provide evidence for a causa l l ink . Stewart et al. addressed this question us ing an an ima l model of relapse, w h i c h showed that exposure to stress along wi th re-exposure to d rug are the two most effective ways to cause relapse i n drug-addicted rats [Stewart, 2000] p o r example, 10-15 minutes of intermittent footshock stress has been shown to be as effective as re-exposure to drug i n reini t iat ing h igh levels of drug-seeking after ext inct ion and a 4-6 week period of abstinence [shaham, 1996] Fur ther 6 s t u d i e s h a v e b e e n a b l e t o g e n e r a l i z e t h i s f i n d i n g t o i n c l u d e r e s p o n d i n g f o r n i c o t i n e Puczek et at, i999] } a l c o h o l I L e e t al- 1 9 9 8 1 a n d e l e c t r i c a l b r a i n s t i m u l a t i o n to t h e t e g m e n t a l a r e a [Deutch and Howarth, 1962; shaiev et at, 2000]^ a u G f w h i c h c a n b e r e i n s t a t e d b y f o o t - s h o c k . I n t e r e s t i n g l y , f o o t s h o c k s t r e s s g i v e n o u t s i d e t h e se l f -a d m i n i s t r a t i o n e n v i r o n m e n t p r i o r t o p r e s e n t a t i o n w i t h s t a r t o f s e s s i o n c u e s w a s n o t e f f e c t i v e i n r e i n i t i a t i n g d r u g - s e e k i n g Puczek et ai, 1998] S i m i l a r l y , r e s t r a i n t s t r e s s u s i n g P l e x i g l a s r e s t r a i n e r s t o i m m o b i l i z e r a t s f o r 1 5 m i n u t e p e r i o d s p r i o r t o a c c e s s t o t h e d r u g s e l f - a d m i n i s t r a t i o n e n v i r o n m e n t p r o m o t e d o n l y w e a k r e i n i t i a t i o n o f d r u g - s e e k i n g . T h e s e e x c e p t i o n s m a y h a v e t o d o w i t h t h e p h y s i c a l s e p a r a t i o n b e t w e e n t h e s t r e s s a n d d r u g s e l f - a d m i n i s t r a t i o n e n v i r o n m e n t s , p r o m p t i n g s p e c u l a t i o n t h a t c o n t e x t u a l c u e s m a y f u n c t i o n a s o c c a s i o n s e t t e r s , w h i c h c o n t r i b u t e t o t h e a b i l i t y o f d i s c r e t e c u e s t o i n i t i a t e d r u g - s e e k i n g . H o w e v e r , f o r h u m a n a d d i c t s , t h e c o n t e x t o f d r u g a d m i n i s t r a t i o n i s m u c h m o r e v a r i e d , r e l y i n g o n m o r e m o b i l e s p e c i f i c c u e s t o t r i g g e r c r a v i n g o r d r u g - s e e k i n g . T h e r e f o r e , h u m a n s t r e s s o r s a r e b o t h m o r e v a r i a b l e a n d g e n e r a l i z e m o r e r e a d i l y t h a n t h o s e e m p l o y e d i n a n i m a l s t u d i e s . N o n e t h e l e s s , o t h e r s t r e s s o r s s u c h a s f o o d d e p r i v a t i o n a n d i n t r a c e r e b r o v e n t r i c u l a r i n j e c t i o n s o f t h e b r a i n p e p t i d e , c o r t i c o t r o p i n - r e l e a s i n g f a c t o r ( C R F ) , h a v e b e e n s h o w n t o b e e f f e c t i v e i n i n i t i a t i n g d r u g - s e e k i n g i n a n i m a l m o d e l s [shaham etai, 2000] T h e n e u r a l m e c h a n i s m s o f s t r e s s - i n d u c e d r e i n s t a t e m e n t o f d r u g - s e e k i n g f o r h e r o i n h a v e b e e n i n v e s t i g a t e d b y e x a m i n i n g t h e e f f ec t o f a mu o p i o i d r e c e p t o r a n t a g o n i s t , n a l t r e x o n e , a n d v a r i o u s d o p a m i n e r e c e p t o r a n t a g o n i s t s [Shaham and Stewart, 1996]. R e s u l t s s h o w e d t h a t mu o p i o i d r e c e p t o r b l o c k a d e d o e s n o t e f f ec t s t r e s s - i n d u c e d r e i n s t a t e m e n t , w h i l e o n l y c h r o n i c b l o c k a d e b y a m i x e d D A r e c e p t o r a n t a g o n i s t , f l u p e n t h i x o l , a t t e n u a t e d r e i n s t a t e m e n t . D l - l i k e a n d D 2 -l i k e r e c e p t o r a n t a g o n i s t s a l o n e d i d n o t i n h i b i t f o o t s h o c k - i n d u c e d r e l a p s e . S i n c e d r u g - i n d u c e d r e i n s t a t e m e n t m e c h a n i s m s a r e k n o w n t o b e m e d i a t e d b y t h e s e s p e c i f i c n e u r a l s y s t e m s , i t a p p e a r s t h a t s t r e s s - i n d u c e d r e i n s t a t e m e n t o p e r a t e s b y a s e p a r a t e o r p a r t i a l l y s e p a r a t e m e c h a n i s m . 7 O n e c a n d i d a t e for a s t r e s s - i n d u c e d re lapse m e c h a n i s m is the stress-re lated h o r m o n e , C o r t i c o t r o p i n Re leas ing F a c t o r . F o o t - s h o c k s p r i o r to d r u g acces s have b e e n s h o w n to release e n d o g e n o u s C R F a n d acute inject ions of the pept ide are k n o w n to reinstate h e r o i n seek ing [Shaham et at, 2000] j n a d d i t i o n , a -h e l i c a l C R F , a C R F receptor antagonis t , h a s b e e n s h o w n to a t tenuate f o o t s h o c k - i n d u c e d re ins ta tement [Shaham etal, 1997] T h u s , the C R F r e s p o n s e m a y be one m e c h a n i s m by w h i c h s tressful life events c o u l d p r o m o t e re lapse a n d there is s p e c u l a t i o n that C R F receptor antagoni s t s m i g h t s o m e d a y be u s e f u l i n m a i n t a i n i n g a b s t i n e n c e [Stewart, 2000] H e d o n i c H o m e o s t a t i c D y s r e g u l a t i o n : In l ight of these putat ive n e u r a l m e c h a n i s m s , a n d the e n d u r i n g t e n d e n c y to re lapse , a d d i c t i o n s h o u l d be seen as a c h r o n i c , r e c u r r i n g c o n d i t i o n , not u n l i k e o ther i l lnesses s u c h as diabetes or c h r o n i c h y p e r t e n s i o n , w h i c h resu l t f r o m long- las t ing , d r u g - i n d u c e d a l terat ions i n b r a i n f u n c t i o n [Lesnner, 1997] N e u r a l a d a p t a t i o n s s u c h as those re lat ing stress a n d v u l n e r a b i l i t y to re lapse o c c u r as the resu l t of repeated exposure to p s y c h o s t i m u l a n t s . Therefore , a d d i c t i o n c a n be seen as a b r e a k d o w n i n h e d o n i c - h o m e o s t a s i s [K o o b & L e M o a l> l ^ 7 \ . W h e r e a s a n i n d i v i d u a l n o r m a l l y m a i n t a i n s e q u i l i b r i u m a m o n g behav iors d irec ted t o w a r d p l e a s u r e - s e e k i n g a n d n o n - h e d o n i c c o n c e r n s , he or she often fails to do so w h e n add ic ted . K o o b h a s n a m e d this "hedonic homeos ta t i c dysregulat ion". In this f ramework , a l l b o d y sys tems , i n c l u d i n g the n e u r a l r e w a r d p r o c e s s i n g sys tem, f u n c t i o n w i t h i n a typ ica l range of opera t ing p a r a m e t e r s a r o u n d the homeosta t i c set-point pr ior to a d d i c t i o n . E x t e r n a l forces that shift b o d y sys tems away f r o m the homeos ta t i c set-point , a n d therefore t h r e a t e n stabi l i ty , are c o u n t e r e d w i th act ions i n oppos i t i on to the shift. T h i s proces s of a d a p t a t i o n is k n o w n as al lostasis . D r u g u s e alters n e u r a l p a r a m e t e r s w i t h i n the body's r e w a r d sys t em a n d thereby c h a n g e s the h o m e o s t a t i c set-point . Pro longed d r u g u s e c a n reset the se t -point to a pa tho log i ca l level, c u l m i n a t i n g i n a fai lure to self-regulate d r u g u s e (i.e. 8 addiction) [K o o b & L e M o a 1 ' 1 9 9 7 L Allostatic processes attempt to compensate for stress imposed by the acute effects of drug-use. For example, following two-weeks of daily one-hour cocaine self-administration sessions, rats demonstrated elevations in heart rate and core temperature at the time of day associated with drug sessions [Tomatzky & Miczek, 1999] i n addition, animals trained in one-hour cocaine self-administration sessions and later shifted to six-hours of access gradually escalated their total cocaine intake, whereas animals that remained limited to one-hour access sessions did not l^^a & Koob, 1999] Presumably, this escalation may signify a change in hedonic set-point, perhaps reflecting an altered dopaminergic "trigger point" l W i s e e t al> 1 9 9 5 1 , as a result of chronic and substantial drug administration. Alternatively, a myriad of proposed molecular, cellular, neurochemical, and synaptic changes may contribute to the clinical manifestation of the addictive cycle. One of the most useful tools in these continuing investigations has proven to be the intravenous self-administration paradigm in animal models of addiction. Animal Models of Addiction: Several species of animals, including sub-human primates, mice and rats have demonstrated the capacity to learn discriminating operant or instrumental behavior leading to i.v. infusions of various drugs [Brady, 1 9 9 i f Presumably, acquisition of these responses, which confers control over drug-administration, is motivated by the drugs' rewarding properties. Thus, animal behavior with respect to drugs of abuse can be seen to parallel that of humans insofar as both species have demonstrated incentive-motivated activities to obtaining drug. Moreover, the animal self-administration paradigm is insulated from complicating cultural factors that human studies cannot easily tease out. Hence, experimental manipulations that effect animal self-administration behavior may be seen as a more direct reflection of the relevant biological mechanisms, than corresponding human behavior. Using different schedules of drug availability, pharmacological manipulations, and various experimental designs, investigators have been able 9 to ascribe functions to specific brain areas, identify important neurotransmitters, recognize mechanisms of drug action and develop models of different aspects of the addictive cycle including tolerance, sensitization, relapse and withdrawal. In addition, self-administration experiments are arguably the most useful predictor of abuse potential for newly developed drugs before they are offered to humans [Jonanson, 1990]. Fixed-Ratios and Binges Fixed-ratio schedules of i.v. cocaine reinforcement have been used extensively to study parameters of drug-taking in various animals. For example, a fixed-ratio 1 (FR1) schedule requires the animal to make one operant response (often a lever-press or nose-poke) to obtain an i.v. infusion of drug. Similarly, reward acquisition in a FR2 schedule is contingent upon successful completion of two responses before receiving the reward. Fixed ratio schedules of reinforcement have helped investigators characterize patterns of drug administration that are common among animals. Over extended cocaine self-administration sessions (24 hours to 5 days), or "binges", which might model human behavior more closely than paradigms involving regularly scheduled, short training sessions, a typical pattern of responding can be observed: this involves an initial "loading" phase (0-1 hr) wherein blood and brain levels of drug quickly increase, followed by "maintenance" of behavior (1-22 hrs) characterized by a relatively constant inter-response interval (IRI), and finally a "dysregulation" phase (s >22 hrs) in which there is increased IRI variability [Tomatzky & Miczek, 20oo]3, Notably, maintenance and dysregulation phases appear to involve relatively constant drug-in take. Accordingly, the important distinction seems to be primarily one of increasing IRI variability in the dysregulated phase. Moreover, during dysregulation there are usually one or more periods of "abstinence" in which animals voluntarily refrain from drug 3 The time course shown here is that reported by Tomatzky & Miczek. Notably, these time parameters seem to be dose-dependent. Based on preliminary data from a study currently underway, increasing the dose appears to promote earlier dysregulation. 10 intake for more than one hour (as defined by Tornatzky & Miczek), or two hOUrS (Di CianO et til, 1998), followed by relapse ITomatzky and Miczek, 2000; Bozarth and Wise, 1985]. Interpreting the Dose Response Curve During loading and maintenance phases, data suggest that instrumental responses for cocaine (and other psychostimulants) may be described by a biphasic inverted U-shaped function composed of an ascending and descending limb (Fig. 2 ! F i s h e r . Raskin & Uhlenhuth, 1987]) _ Consequently, when the dose of drug used in a "typical" self-administration experiment is increased the rate of responding decreases (the "interesting range" of doses - those used in many self-administration studies, which maintain stable responding on fixed ratio schedules - are generally on the descending portion of the curve t w i s e ' 1 9 8 71). Likewise, when such "descending limb" doses are decreased self-administration hastens tKoob- 1 9 9 9 L By contrast, doses on the ascending arm generally do not maintain stable responding under fixed-ratio conditions. For instance, there may be a period of rapid responding followed by no responding, or some other variation thereof ^ f i s t - 1 9 8 7 L However, a fraction of animals do discriminate between responses leading to infusions of low doses of cocaine and responses with no consequences (see results section). Notably, this situation for low doses is markedly different from the consistent finding that non-reinforcing substances such as saline do not support any discriminative responding. Speculatively, the inverse relationship between dose and response rate on the descending arm of the curve may belie an underlying "satiety effect" for which larger doses satiate the animal's desire for drug for a correspondingly longer 11 period of time. Thus, higher doses require fewer responses to maintain satiety than lower doses do. This hypothesis agrees with data from varying dose self-administration paradigms in which larger doses are followed by longer inter-response intervals [Yokel & Pickens, 1974] Low doses (those on the ascending limb of the biphasic dose-response function) do not appear to satiate, because responding is erratic and unreliable [wise> 1 9 8 7 1 . The absence of data showing consistent and graded responding proportional to increasing dose for the ascending portion of the curve has led to the suggestion that the dose-response curve for drug self-administration is more accurately represented as a monophasic decreasing function in which the lowest stably-administered4 dose supports the highest response rate l W i s e > 1987] That is, there is no observable ascending limb on which doses maintain stable responding, as there is for the descending arm. This view notwithstanding, responding for doses typically ascribed to the ascending arm is qualitatively and quantitatively different than that seen for non-reinforcing substances. Non-reinforcers do not evoke instrumental behavior leading to intake. In addition, since animals will discriminate between active and inactive levers for low doses of cocaine, despite unstable IRIs, it may be argued that these doses provide reward without the relatively immediate satiety associated with higher doses. Whereas an animal may be rapidly satiated by a high dose of drug, which quickly brings blood and brain levels past a given "satiety threshold", it may require several, or many, infusions of a lower dose to pass this threshold. This framework might explain the bursts of responding followed by no responding, which is sometimes observed with low doses. Interestingly, this same pattern - burst/abstinence -is observable in some animals in the "dysregulation phase" of continuous self-administration [Tomatzky and Miczek, 2000] _ This similarity may reflect the buildup of tolerance after continuous administration of drug, which can be viewed as functionally akin to administration of a progressively lower dose over time. Thus, the animal may begin to treat a physically constant "descending limb" 4 Stably-administered in this context refers to a situation in which there is relatively low IRI variability; that is, the time between responses is consistent. 12 d o s e , a s o n e t h a t p r o g r e s s i v e l y b e c o m e s f u n c t i o n a l l y s m a l l e r , r e s u l t i n g i n i r r e g u l a r a n d e s c a l a t i n g i n t a k e o v e r t i m e . T h i s f r a m e w o r k t a k e s a s i m p l i c i t , t h e p r e v a i l i n g n o t i o n t h a t a n i m a l s c a n m o n i t o r p s y c h o s t i m u l a n t l e v e l s a n d t i t r a t e t h e i r i n t a k e a c c o r d i n g l y . A l t h o u g h t o t a l i n t a k e o f b o t h c o c a i n e a n d a m p h e t a m i n e i s g r e a t e r w h e n t h e d o s e i s h i g h e r l B r a d y- 1 9 9 1 ; D i C i a n o e t a l - 1 9 9 5 1 , c o c a i n e e l e v a t e s e x t r a c e l l u l a r d o p a m i n e i n t h e n u c l e u s a c c u m b e n s i n a d o s e - d e p e n d e n t m a n n e r , w h e r e a s a m p h e t a m i n e - i n d u c e d e l e v a t i o n s a r e n o t d o s e - d e p e n d e n t IDi C i a n o e t a l - 1 9 9 5 1 . T h i s d o s e - d e p e n d e n t r e l a t i o n s h i p o f t h e d o p a m i n e r e s p o n s e w i t h c o c a i n e s u g g e s t s t h a t e x t r a c e l l u l a r d o p a m i n e l e v e l s a c t a s a g a u g e t h a t m a y i n d i c a t e w h e n a n o p e r a n t r e s p o n s e l e a d i n g to f u r t h e r c o c a i n e a d m i n i s t r a t i o n i s d e s i r a b l e . B y c o n t r a s t , d i f f e r e n t d o s e s o f s e l f - a d m i n i s t e r e d a m p h e t a m i n e p r o d u c e i n i t i a l e l e v a t i o n s i n b r a i n a n d b o d y l e v e l s o f t h e d r u g , f o l l o w e d b y m a i n t e n a n c e o f a c o n s t a n t s u p r a - t h r e s h o l d l e v e l t h e r e a f t e r . T h u s , a m p h e t a m i n e i n t a k e m a y r e f l e c t b r a i n a n d b o d y l e v e l s o f t h e d r u g r a t h e r t h a n d o p a m i n e l e v e l s W^ei and Pickens, 1974] E l e c t r o c h e m i c a l s t u d i e s w i t h c o c a i n e a l s o s h o w a f a s t e r r i s e t i m e i n e x t r a c e l l u l a r d o p a m i n e t h a n f o r d - a m p h e t a m i n e , a n d i t i s r e g a r d e d a s t h e m o r e r e w a r d i n g o f t h e t w o [Yanagita, 1973] T h i s f i n d i n g h a s l e d t o s p e c u l a t i o n t h a t t h e s t e e p e r c o c a i n e - i n d u c e d i n c r e a s e i n n u c l e u s a c c u m b e n s d o p a m i n e o v e r t i m e i s m o r e r e w a r d i n g t h a n t h e c o m p a r a b l y s l o w e r , b u t m o r e , e n d u r i n g d o p a m i n e r g i c r e s p o n s e t o a m p h e t a m i n e . I n c o n t r a s t to f i x e d r a t i o s c h e d u l e s o f r e i n f o r c e m e n t , w h i c h p r o v i d e i n f o r m a t i o n a b o u t w h e t h e r a d r u g i s r e i n f o r c i n g a n d t h e p a r a m e t e r s o f d r u g -t a k i n g , p r o g r e s s i v e r a t i o s c h e d u l e s , p i o n e e r e d b y H o d o s i n 1 9 6 1 to s t u d y l i q u i d r e i n f o r c e m e n t i n r a t s [Hodos, i96i] ; a r e t h o u g h t to p r o v i d e a m e a s u r e o f t h e r e w a r d i n g v a l u e o f d r u g s Pepoortere et at, 1993]. Progressive-Ratios and Second Order Schedules of Reinforcement I n a p r o g r e s s i v e r a t i o s c h e d u l e t h e n u m b e r o f r e s p o n s e s r e q u i r e d to o b t a i n a r e w a r d i s i n c r e a s e d a f t e r e a c h s u c c e s s i v e r e w a r d u n t i l t h e a n i m a l f a i l s t o m a k e t h e r e q u i s i t e n u m b e r o f r e s p o n s e s . T h e h i g h e s t r a t i o t h a t i s 13 s u c c e s s f u l l y c o m p l e t e d , o r t h e n u m b e r o f r e i n f o r c e r s o b t a i n e d , i s t e r m e d t h e " b r e a k p o i n t " . T h e b r e a k p o i n t r e f l e c t s t h e d e g r e e t o w h i c h a n a n i m a l i s w i l l i n g t o w o r k f o r r e w a r d , a n d i s t h u s c o n s i d e r e d a m e a s u r e o f t h e r e w a r d i n g v a l u e o f s u b s t a n c e s . F o r c o c a i n e , t h e b r e a k p o i n t h a s b e e n s h o w n t o i n c r e a s e a s a f u n c t i o n o f d o s e [Roberts et ai, 1989; Depoortere et ai, 1993]^ w h i c h i s c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t h i g h e r d o s e s a r e g e n e r a l l y m o r e r e w a r d i n g . W h i l e f i x e d a n d p r o g r e s s i v e r a t i o s c h e d u l e s o f r e i n f o r c e m e n t p r o v i d e i n f o r m a t i o n a b o u t d r u g - t a k i n g b e h a v i o r , s e v e r a l a u t h o r s h a v e a r g u e d t h a t t h e s e p a r a d i g m s d o n o t c l e a r l y d i s c r i m i n a t e b e t w e e n d r u g - d r i v e n a n d d r u g -s e e k i n g b e h a v i o r 1 ^ ° ? ° e t al- 1 9 9 8 & Markou et ai, 1993], p o r i n s t a n c e , i n p r o g r e s s i v e r a t i o s t u d i e s , t h e n o t i o n t h a t c o c a i n e ' s m o t o r i c e f f e c t s m i g h t l e a d t o h i g h e r b r e a k p o i n t s a t h i g h e r d o s e s i s r e a s o n a b l e . F o l l o w i n g t h e f i r s t i n f u s i o n i n a d r u g - t a k i n g s e s s i o n w i t h e x p e r i e n c e d a n i m a l s , w h i c h i s o f t e n g i v e n n o n -c o n t i n g e n t l y b y t h e e x p e r i m e n t e r i n t h e s e s t u d i e s , a s s e s s m e n t o f t h e a n i m a l ' s m o t i v a t i o n t o o b t a i n d r u g w h i l e i n a d r u g - f r e e s t a t e , i s i m p o s s i b l e . O p e r a n t b e h a v i o r n o l o n g e r r e f l e c t s o n l y t h e a n i m a l ' s d e s i r e t o o b t a i n d r u g , b u t a l s o t h e a s s o c i a t e d c h a n g e i n r a t e o f b e h a v i o r . T o a d d r e s s t h i s i s s u e , s e c o n d o r d e r s c h e d u l e s o f r e i n f o r c e m e n t h a v e b e e n d e v e l o p e d , w h i c h , l i k e p r o g r e s s i v e r a t i o s c h e d u l e s , a r e d e s i g n e d t o p r o v i d e a m e a s u r e o f t h e r e i n f o r c i n g p r o p e r t i e s o f d r u g s . T h e s e m a k e u s e o f t h e f a c t t h a t p r e s e n t a t i o n o f a c o n d i t i o n e d s t i m u l u s ( C S ) h a s b e e n s h o w n t o m a i n t a i n h i g h r e s p o n s e r a t e s o v e r p r o l o n g e d p e r i o d s o f t i m e w i t h o u t e x p o s u r e t o t h e d r u g i t s e l f 1 ^ ^ ° e t al- 1 9 9 8 & wwteiaw et ai, 1996]. R a t s h a v e b e e n s h o w n t o i n i t i a t e o p e r a n t r e s p o n d i n g f o r c o c a i n e i n r e s p o n s e t o p r e s e n t a t i o n o f a C S p r e v i o u s l y p a i r e d w i t h t h e d r u g . A r r o y o et al. u s e d t h i s f i n d i n g t o a s s e s s c o c a i n e - s e e k i n g b e h a v i o r w i t h a s e c o n d o r d e r s c h e d u l e o f r e i n f o r c e m e n t . R a t s w e r e t r a i n e d w i t h a n i n i t i a l 1 5 m i n u t e f i x e d i n t e r v a l i n w h i c h d r u g w a s u n a v a i l a b l e . N o n e t h e l e s s , t h e C S w a s p r e s e n t e d i n a s s o c i a t i o n w i t h e v e r y t e n t h o p e r a n t r e s p o n s e . A f t e r f i f t e e n m i n u t e s , a c o c a i n e i n f u s i o n c o u l d b e e a r n e d b y r e s p o n d i n g t e n t i m e s o n a n a c t i v e l e v e r l A r r °yo et ai, 1998] R e s u l t s i n d i c a t e d t h a t t h e r a t e o f r e s p o n d i n g d u r i n g t h e i n i t i a l 1 5 m i n u t e f i x e d 14 interval in which cocaine was not available increased sharply as the end of the interval approached and the animal presumably began to anticipate availability of drug. During the second interval (after earning one infusion of cocaine), the animals earned the presentation of the CS significantly faster than in the first interval, suggesting the presence of a rate-altering effect of drug on behavior. Omission of the CS for three consecutive sessions progressively increased the latency to complete the first ten responses, and re-introduction of the CS restored responding to pre-omission levels. Therefore, this study using second order scheduling to disconfound the rate-altering behavioral effects of cocaine and the drug's reinforcing properties, suggests that conditioned stimuli significantly enhance animal's motivation to perform instrumental behavior leading to intravenous cocaine-administration. While these and other studies have helped to characterize and identify parameters of the addictive cycle, investigations into the mechanisms of acute psychostimulant action have also relied heavily on IVSA methodology. Most drugs of abuse have effects on the mesolimbic dopamine system, which has served as a point of departure to identify some of the neural correlates of the reward processing system. Thus, a brief outline of the anatomy of the dopamine system follows, before a review of dopamine's involvement in psychostimulant addiction. Anatomy of the Dopamine System: Dopaminergic projections originate in the midbrain from cell bodies in the Substantia Nigra Pars Compacta (SNc) and Ventral Tegmental Area (VTA). In particular, projections from the VTA to the nucleus accumbens (nAcc), an area of the ventral striatum, are thought to be important in the brain's reward processing circuitry. In general, these efferents have three main targets: the neo-striatum (caudate and putamen), the limbic cortex (medial prefrontal, cingulate, and entorhinal regions) and lower limbic areas including the nAcc in the ventral striatum. VTA projections to the limbic cortex and lower limbic 15 reg ions are referred to as mesocortical a n d mesolimbic dopamine systems, respect ively . Moreover , cel l bodies i n the V T A project p r e d o m i n a n t l y to the v e n t r a l s t r i a t u m (the c lass ic r e w a r d pathway) a n d those i n the S N c project m a i n l y to d o r s a l s t r i a t u m ( impl icated i n movement) . A l o n g the l ength of these project ions varicos i t ies c o n t a i n i n g d o p a m i n e r g i c s y n a p t i c vesic les exist, w h i c h , for m e s o l i m b i c axons , s y n a p s e w i t h dendr i t e s of m e d i u m s p i n y G A B A e r g i c n e u r o n s i n the n A c c . D o p a m i n e r g i c i n p u t to the n A c c serves to m o d u l a t e i n f o r m a t i o n p r o c e s s i n g t h r o u g h o u t the ex tended a m y g d a l a a n d a larger set of n u c l e i collectively k n o w n as the b a s a l gangl ia . A n a t o m i c a l l y , the n A c c cons i s t s of two d i s t inc t regions , the she l l a n d the core, w h i c h differ i n subt le ways . B o t h she l l a n d core receive projec t ions f r o m V T A , b u t the shel l , l y ing med ia l l y a n d ventra l ly to the core, is d i s t i n g u i s h e d by efferent project ions to the lateral , h y p o t h a l a m u s a n d b e d n u c l e u s of the s tr ia t e r m i n a l i s (BNST) [E v e r i t t e t al- 1 9 " 1 . Ce l l s of the core are a l m o s t i n d i s t i n g u i s h a b l e f r o m cells of the c a u d a t e a n d p u t a m e n (the m a i n n u c l e i of the d o r s a l s tr ia tum) . B y contras t , the she l l c a n be seen as a m i x t u r e of s tr ia ta l n e u r o n s a n d those charac ter i s t i c of the a m y g d a l a [Heimer & wiison, 1975] T h u s , it h a s b e e n a r g u e d that the she l l represents a t rans i t i ona l zone f r o m s t r i a t u m to the ex tended a m y g d a l a . T h e n u c l e u s a c c u m b e n s a n d a m y g d a l a share fur ther s imi lar i ty i n that b o t h receive afferent c o n n e c t i o n s f r o m V T A . Moreover , the c e n t r a l n u c l e u s of the a m y g d a l a (CeA) h a s dec idedly s tr iata l charac ter i s t i c s , sugges t ing that it m a y f u n c t i o n as a spec ia l ized reg ion of s t r i a t u m i n some cases [ E v e r i t t etai, 1999] T h e s t r i a t u m also receives g lutamaterg ic i n p u t f r o m Neocortex , H i p p o c a m p u s a n d A m y g d a l a . Neocort ica l i n p u t s c o n n e c t p r e d o m i n a n t l y w i th d o r s a l par t s of s t r i a t u m whereas the h i p p o c a m p u s a n d a m y g d a l a project m o s t l y to v e n t r a l areas [McGeorge & Fauii, 1989]. P s y c h o s t i m u l a n t s a n d D o p a m i n e : W i t h th is a n a t o m i c a l f r a m e w o r k i n m i n d , several c o n v e r g i n g l ines of ev idence present a c o m p e l l i n g a r g u m e n t s u p p o r t i n g the h y p o t h e s i s that d o p a m i n e r g i c s igna l ing i n the N A c c p lays a c e n t r a l role i n the re in forc ing 16 effects of c o c a i n e a n d a m p h e t a m i n e [ C a i n e . 1 9 9 8 1 . F o r e x a m p l e , c o c a i n e i s k n o w n to b i n d d i r e c t l y to p l a s m a m e m b r a n e d o p a m i n e t r a n s p o r t e r (DAT) p r o t e i n s lmtz e t ai, 1990] T h i s b l o c k s r e u p t a k e of d o p a m i n e i n t o p r e s y n a p t i c t e r m i n a l s of the m e s o l i m b i c d o p a m i n e s y s t e m , a n d t h e r e b y i n c r e a s e s e x t r a c e l l u l a r l eve l s of d o p a m i n e i n the n u c l e u s a c c u m b e n s l D i C i a n 0 e t al- 1 9 9 5 1 . I n a d d i t i o n , t r e a t m e n t w i t h d o p a m i n e - r e c e p t o r a n t a g o n i s t s h a s b e e n s h o w n to s i g n i f i c a n t l y w e a k e n s e l f - a d m i n i s t r a t i o n of c o c a i n e b y a n i m a l s l M e l l ° & Negus, 1996] a n d se lec t ive l e s i o n i n g o f d o p a m i n e r g i c n e u r o n s i s k n o w n to a t t e n u a t e r e s p o n d i n g for c o c a i n e [Roberts et ai, 1980] B y c o n t r a s t , d o p a m i n e r ecep to r a g o n i s t s a re s e l f - a d m i n i s t e r e d w h e n s u b s t i t u t e d for c o c a i n e i n d r u g - e x p e r i e n c e d a n i m a l s [Bergman, 1989]- a n d n o t a b l y , s t u d i e s w i t h spec i f ic D i - a n d D2-iike a g o n i s t s sugges t a p a r t i c u l a r l y i m p o r t a n t ro le for the D2 r e cep to r i n m e d i a t i n g the r e w a r d i n g p r o p e r t i e s o f c o c a i n e [Caine et ai, 1999] D o p a m i n e r e u p t a k e i n h i b i t o r s o t h e r t h a n c o c a i n e are a l so self-a d m i n i s t e r e d (an effect t h a t co r r e l a t e s w i t h t h e i r a f f in i ty for the D A T ) , a n d m i c e t h a t a re g e n e t i c a l l y m o d i f i e d to over e x p r e s s the D A T p r o t e i n h a v e b e e n r e p o r t e d to s h o w e leva ted c o c a i n e - c o n d i t i o n e d p l a c e p r e f e r e n c e 5 f S o r a e t a'-> 1 9 9 8 ~ unpublished observations]. i n h u m a n s , (PET) i m a g i n g s t u d i e s h a v e e s t a b l i s h e d a r e l a t i o n s h i p b e t w e e n d o p a m i n e t r a n s p o r t e r o c c u p a n c y b y c o c a i n e , a n d the sub jec t ive effects of the d r u g No^ow e t a l - 1 9 9 7 1 . L i k e w i s e , a s t u d y b y R o m a c h et al, s h o w e d the D 1 / D 5 r e c e p t o r - a n t a g o n i s t , e c o p i p a m ( S C H 3 9 1 6 6 ) dose -d e p e n d e n t l y b l o c k e d the sub jec t ive e u p h o r i c a n d s t i m u l a n t effects, b u t n o t the p h y s i o l o g i c a l i n c r e a s e i n h e a r t ra te a s s o c i a t e d w i t h i n t r a v e n o u s c o c a i n e u s e i n d e p e n d e n t h u m a n u s e r s t K o o b ' 1 9 9 9 & Romach et ai, 1999] T h u s , D 1 / D 5 r e c e p t o r a c t i v a t i o n a s s o c i a t e d w i t h c o c a i n e - i n d u c e d d o p a m i n e e l e v a t i o n m a y a l s o be a n e s s e n t i a l a s p e c t of the r e w a r d i n g p r o p e r t i e s of the d r u g . A n o t h e r s t u d y b y P i l l a et al, d e m o n s t r a t e d t h a t the p a r t i a l D 3 r ecep to r a g o n i s t (a s u b s t r a t e w i t h h i g h a f f in i ty b u t l o w i n t r i n s i c a c t i v i t y a t i t s receptor) , B P 8 9 7 , w e a k e n e d r e s p o n d i n g for a d r u g - a s s o c i a t e d c u e d u r i n g a 15 m i n u t e p e r i o d before c o c a i n e b e c a m e 5 The Conditioned Place Preference (CPP) paradigm assesses the degree to which animals demonstrate preference for locations that have been previously paired with drug. It is regarded as a way to measure the rewarding value of drugs. 17 available to drug-experienced rats I r a i a e t a l - 1999] This agent had no effect on drug-taking behavior after the first dose of the primary reinforcer was given. Taken together, these data clearly show that dopamine contributes to the reinforcing effects of cocaine. Both Di-uke and D2-iike receptors may participate in mediating reward, and conditioned drug-craving may employ a D3 receptor mechanism. Of additional importance, is the fact that cocaine is known to bind to other monoamine transporters. The D A T protein is one member of the larger Na + /CT-dependent family of plasma membrane transporters, which also includes the serotonin and norepinephrine transporters (SERT and NET) [Masson etal, 1999). Cocaine and other drugs that block the D A T also block S E R T and N E T proteins. In fact, cocaine has a higher affinity for the S E R T than for the D A T [white, 1998] Moreover, in-vitro data from recombinant cell lines transfected with h u m a n h D A T , h N E T and h S E R T show that the h N E T has a higher affinity for dopamine than the h D A T IGiros e t al- 1 9 9 4 ; Eshieman et ai, 1999]. This observation highlights the fact that all three transporters have a certain affinity for all three monoamine neurotransmitters, and each is named not according to their affinity for ligands, but for the type of neuron on which it is predominantly expressed. Hence, the D A T is mostly expressed on dopamine-releasing neurons. Evidence from N E T and D A T knockout mice provide a further example. Dopamine uptake by frontal cortex synaptosomes was shown to be approximately equal in D A T knockout and wild-type animals, but 5 5 % lower in N E T knockout tissue. Thus , the N E T , not the DAT, seems to be the main instrument of dopamine uptake in the frontal cortex I M ° r ° n etal, 2000] These data notwithstanding, the connection between cocaine's reinforcing properties and its interaction with other monoamine transporter proteins in various brain regions remains unclear. One recent study involving D A T knockout mice supports a greater role for serotonin than previously thought [R o c h a e t a l ' 1 9 9 8 L Despite the complete absence of D A T proteins in these animals (the putative chief substrate for cocaine's action) and failure of cocaine 18 to induce a dopamine response in dorso/ventral striatum [Rocha eta/., i998]; mutant mice were able to acquire self-administration behavior for cocaine. This surprising finding provides evidence for the view that some mechanism other than a cocaine-mediated increase in extracellular dopamine might be responsible for the drug's reinforcing properties. The authors go on to suggest that cocaine-induced elevations in serotonin, rather than dopamine, may be the basis for self-administration in these animals. This conclusion is based on the fact that the cocaine congener [ 1 2 5 I]RTI-55 bound to the Serotonin Transporter (SERT) in slices taken from mutant brains, and immediate-early gene activation (c-fos) was induced by cocaine, in serotonergic brain areas of the mutant mice. Further confirmation that D A T knockout mice experience reward from cocaine comes from evidence that they exhibit conditioned place-preference for locations associated with drug-administration I S o r a e t al< 1 9 9 8 ] . These data suggest that an interaction between D A T and cocaine may not be crucial to the reinforcing or rewarding properties to be realized in these animals. Although Rocha et al.'s findings raise some interesting questions; interpretation of the data is not entirely straightforward. Knockout mice are subject to a wide range of compensatory changes, which occur in animals that develop without the ability to quickly terminate dopaminergic transmission. DAT-/- mice are profoundly hyperactive, they show a 7 5 % reduction in dopamine release compared to WT mice in response to electrical stimulation, have a 5-fold higher concentration of extracellular dopamine in the striatum, less than 5 % the normal neuronal dopamine stores, and a decreased number of dopamine receptors [Jones et at, 1998] j n addition, D A T knockout mice have recently been shown to exhibit conditioned place preference for environments paired with the selective S E R T and N E T blockers, Fluoxetine and Nisoxetine [Haii et ai., 2000] wild-type and heterozygous controls, which express half-normal levels of D A T protein, showed no such preference. These data suggest that the previously demonstrated cocaine self-administration in D A T knockout mice [Rocha etai, 1998] m a y have been maintained through actions at the S E R T a n d / o r 19 NET proteins. Notably, this explanation cannot be generalized to wild-type animals because they do not show place-preference for selective SERT and NET blockers. The clear implication from these data is that the relative contributions of NET and/or SERT blockade to cocaine's reinforcing properties are much greater in mutant animals. Perhaps in the absence of regulated dopamine signaling, norepinephrine and serotonin acquire certain dopaminergic functions; or perhaps other monoamine transporters compensate by regulating dopamine. Speculation aside, these data highlight the point that DAT-knockout and wildtype animals represent fundamentally different, and incomparable, biological systems. Therefore, an inference based on the knockout model about the contribution of DAT blockade to cocaine's reinforcing properties in wild-type animals is not supported. This view notwithstanding, the fact that serotonin and norepinephrine appear to be rewarding in knockout animals, is not unimportant. Presumably, compensatory changes in DAT knockout animals render noradrenergic and serotonergic systems, more important in reward processing, which implies that these systems may have a role, albeit a different or diminished one, in non-mutant animals as well. Further characterization of the importance of compensatory changes in DAT knockout animals may help to clarify this issue. Also worth mentioning is the fact that extracellular dopamine is elevated during self-administration of cocaine in both wild-type and DAT"/- mice [ J o n e s e t ai, 1996] j n wild-type mice cocaine dose-dependently elevates dopamine in the striatum by up to 1000% above baseline. Similarly, DAT - /- animals maintain 5-fold higher extracellular dopamine levels than wild-type controls, which are not altered by cocaine administration. Thus, since it is known that serotonin receptor agonists by themselves are not self-administered in wild-type animals [white, 1998]^ t l i e possibility exists that the interplay between cocaine-induced elevations of serotonin levels in DAT -/- mice and constitutively elevated dopamine levels may act synergistically to produce drug-reward. This hypothesis is further supported by the previously mentioned fact that DAT knockout mice develop conditioned place preference to environments paired 20 with the selective S E R T blocker, Fluoxetine IHaU e t al- 2 0 0 0 l Indirect support for this proposal also comes from studies of the drug, Mazindol, which is not abused by humans or self-administered by animals. Mazindol strongly inhibits dopamine and norepinephrine transport, but only slightly inhibits serotonin transport l S o r a e t a l - 1 9 9 8 L This divergence from cocaine's serotonergic effects might conceivably play a part in Mazindol's diminished abuse potential, and indirectly supports the idea that cocaine's effect on the S E R T contributes to its rewarding properties. Alternate Experimental Strategies: The use of D A T knockout experiments to investigate mechanisms of cocaine reward is, in one way, better than pharmacological strategies. Knockout methodology avoids complexities inherent in interpreting receptor agonists/antagonist studies because these agents may have multiple, and difficult to assess, actions on several receptors. Furthermore, this strategy-addresses the drug's interaction with its primary substrate (the DAT) rather than attempting to tease out the effects of dopamine on specific receptors, which may have overlapping roles. Thus , by attempting to remove cocaine's regulation of dopamine levels, the investigation is free to question its' other effects. That said, there are two possible ways to address the problems inherent in the D A T knockout model, wherein the gene is absent for the entire lifetime of an organism. The first is to engineer an inducible knockout [Mayford et at, 1996; Chen et ai, i998] ? a n c i the second to employ antisense technology to produce a transient "knockdown" of the gene in adult animals. Inducible Knockouts Recent advances in molecular biology and genetic engineering have made it possible to control gene expression in vivo both regionally and temporally. One of the first studies to successfully employ this strategy was one by Mayford 21 et al. in which expression of a calcium-calmodulin-dependent kinase II (CaMKII)-encoding transgene was put under the control of the tet-O promoter [Mayford et ai, 1996]. A second transgene encoding tTA (a tetracycline-inhibitable transcription factor), under the control of the CaMKII promoter, was also added to the genome. This system provided region-specific expression of the CaMKII transgene, which could be turned off using the tetracycline analogue, doxycycline. A further study tested a modified tetracycline-regulated system using the Neuron-Specific Enolase (NSE) promoter to direct high levels of Luciferase, delta FosB and C R E B expression in specific brain areas [C h e n e t al> 1998] j n ^ i s study, three different strains of mutant mice demonstrated transgene expression in striatum, cerebellum and striatum, and cortex respectively, which was completely shut down using low doses of doxycycline in drinking water within 3 weeks. The authors concluded that the precise site of insertion of the NSE- tTA transgene into the genome determined which brain regions express the desired, transactivated gene product. Collectively, these studies show that regulated, tissue-specific gene expression is now a feasible and promising strategy for investigating gene function. However, with respect to the immediate use of this strategy to explore the role of the dopamine transporter in cocaine reward, it should be noted that an appropriate system does not yet exist. Moreover, the case for D A T differs from those of CaMKII, or transcription factors, delta FosB and C R E B in at least one important way. The principle manipulation of interest with D A T would be an inducible knockout of the endogenous gene. By contrast, inducible systems to date, have concentrated on issues relating to expression of transgenes, which are expressed in addition to their endogenous counterparts. This situation is not appropriate for investigations of D A T because endogenous D A T expression is constitutive in certain brain areas (notably ventral striatal terminals of dopaminergic midbrain neurons), whereas CaMKII, delta FosB and C R E B are not expressed at high levels under controlled conditions. Thus , inducing expression of a transgenic D A T would provide hyper-expression, not expression where there was none before. Hence, despite a promising theoretical 22 prospect, the considerable task of engineering an appropriately inducible system for D A T investigations has yet to be accomplished. 23 Antisense Techno logy Antisense technology specific to D A T is currently available. Like inducible systems, this approach studies a phenotype that has benefited from a full complement of functional genes during critical stages of development. This helps to avoid the confounding compensatory changes that complicate interpretation of knockout experiments. Antisense technology makes use of short strands of synthetic nucleic acid, complementary to the so called "sense" strand of m R N A encoding specific genes, to hybridize to the mRNA and knock down production of the gene product Persidis, 1999; Myers & Dean, 2ooo]. Three putative mechanisms have been proposed to explain antisense function. RNase H , a degradative enzyme in the nucleus with a mandate to destroy double-stranded nucleic acid, may cleave m R N A at the duplex site [R°ush, 1997] Antisense may also sterically block ribosomes from translating the encoded protein and if targeted to the appropriate section of mRNA, it may disrupt splicing. [Myers & Dean, 2000]. Thus , two of the three mechanisms occur in the nucleus, although mechanisms of nuclear entry by oligos are poorly characterized and not always observed even in cases where putative antisense effects are shown [stein, 1999] Nonetheless, use of antisense to inhibit protein synthesis is, in theory, relatively efficient compared to traditional methods. Under normal circumstances a strand of m R N A exits the nucleus after being transcribed from a gene, and is repeatedly translated into protein by ribosomes. Consequently, many copies of a particular protein are produced from a single mRNA strand. Therefore, when drugs target proteins, as most common therapeutic or experimental pharmacological agents do, they must eliminate many fold more effectors than a drug that targets the mRNA transript from which the protein originates. That is one advantage of antisense technology. A second benefit is the exquisite specificity that it provides. Most drugs that attack gene products demonstrate some cross-reactivity to other proteins with similar structure, charge characteristics, or amino acid motifs, which can cause side effects. Antisense, by contrast, targets the gene in question alone, limiting non-specific effects 24 [Agrawai, 1997] Finally, with respect to experimental investigation of gene function, knockout experiments in which the gene in question is removed from the DNA code of germ cells have been the gold standard in the past. This allows researchers to infer information about a gene by examining the phenotype that occurs when the gene is absent. However, the primary phenotype expressed in these paradigms reflects the gene's absence when its first critical requirement occurs during development, and as such may not always be representative of the gene's function in a normal adult organism [Driver et ai, 1999] g y contrast, antisense methodology produces a secondary phenotype reflecting absence of the gene's full service in a mature animal that has benefited from normal gene function during development. Early studies with antisense in test tube cell cultures were promising, as specific oligos were shown to be able to diminish production of various gene products, but it was later demonstrated that the short strands were not stable in vivo. They were vulnerable to enzymatic degradation by endo- and exonucleases l " E d e r e t al- 1 9 9 1 1 . Thus , the first generation of modified antisense molecules, called phosphorothioates, were engineered which employed sulphur atoms substituted for non-bridging oxygen atoms of phosphate groups within the nucleic acid backbone (fig. 3a) [Agrawai &KandimaUa,2000]. This afforded a greater resistance to degradation by enzymes [R°usn, 1997] However, further studies showed that these first generation drugs were causing non-sequence-specific effects [Stein' 1 9 9 9 1 , and in some cases fatal side-effects such as hyperactivation of the body's complement system of immunity. It seems the polar nature of the sulphur modifications allowed the nucleotide sequences to stick to molecules on the cell surface such as heparin-binding proteins [Guvakova, i995] > producing some of the observed problems. Thus , a second generation of oligos called mixed backbone oligonucleotides, which come in end-modified (the type being employed in our current study to knock down D A T expression) and centrally-modified forms, have been developed with 25 Fig. 3: (a) Phosphorothioate (b) End-modified mixed-backbone (c) Centrally-modified mixed-oligodeoxynucleotides oligonucleotides backbone oligonucleotides diminished anionic charges that have been shown to survive longer, and produce fewer side effects (see examples of modified mixed backbone oligonucleotides, Fig. 3b and c). These combine the most functional features of PS-oligonucleotides (i.e. resistance to degradation, aqueous solubility, cell entry etc.) with reduced polyanionic-induced side effects and increased bonding to m R N A (one consequence of phosphorothioate modifications is reduced affinity for m R N A - mixed backbone construction ameliorates this problem) [Agrawai & Kandimalla, 2000] _ In addition to appropriate backbone construction, the precise antisense sequence used to block translation of a given mRNA transcript must be very 26 carefully chosen (often through educated trial and error) to make sure non-sequence specific effects are not confused with a bona fide antisense mechanism. For instance, it has been shown that the presence of CpG motifs can, in some cases, cause an immunostimulatory response including induction of cytokines such as IL-12, IL-6, IFN-Y, TNF-a and chemokines [Kiinman, et ai, 199.6; zhao, et ai, 1997] Furthermore, oligonucleotide sequences may hairpin forming duplex sections and in some cases may contain a cis-transcription recognition sequence that can sequence-specifically bind to transcription factors to interfere with transcription. This phenomenon is called a decoy mechanism and results in global inhibition of transcription that may augment inhibition of the specific gene in question. Therefore, it is sometimes difficult to tell when true antisense activity is behind observed results [Agrawai & Kandimaiia, 2000] Another problem lies in strands containing four guanine bases in sequence (GGGG) and self-complementary or palindromic sequences, which can form problematic secondary structures. Oligo length is also a concern. Ideally oligos should be 15-20 base pairs; long enough to differentiate among target sequences that differ by as little as one base, but short enough to minimize the probability of non-sequence specific effects, which increases in parallel with length [Myers & Dean, 20001 At the outset of antisense technology the primary focus was therapeutic, a tool to combat disease, as highlighted by the recent licensing of Vitravene for the treatment of cytomegalovirus (CMV) retinitis in AIDS patients by the US FDA in 1998 [Crooke, 1998]. This highlights the maturing utility of this technology. More recently it has been applied to study the contribution of specific genes to behavior, also with increasing success. In both contexts, it is of utmost importance to include proper controls as well as direct measures of antisense efficacy via evaluation of mRNA or protein levels, if not both. Proper control paradigms should compare the active sequence with scrambled sequences of the same length, containing identical bases in pseudo-random order (making sure to avoid problem sequences as 27 outlined above), or a series of mismatched sequences with decreasing potency as the number of mismatches increases. However, a homology search should also be done for the control sequences to ensure that they are not inadvertently knocking down an unrelated protein, with possible confounding effects on the present experiment. It may also be wise to compare inhibition of the targeted protein with a similar, non-targeted, protein in order to rule out non-specific effects - with this approach, care must be taken to ensure that pharmacological interactions between the target protein and the control are not responsible for altered levels of the control. In measuring the response to antisense, one should expect dose-dependent decreases in mRNA or protein to occur. Yet, if RNase-independent antisense mechanisms are at play, they might only be observed at the level of the protein and differential half-life qualities of various proteins can have an impact on the length of antisense administration required. Therefore, a thorough investigation is required to verify that observed effects are due to sequence-specific antisense-mRNA interactions. Hence, reliance on phenotypic measures, such as inhibition of cellular proliferation or behavioral changes to claim true antisense mechanisms can at best be deemed an educated inference rather than scientific fact, and at worst be completely false [Myers & Dean, 2000] In the context of psychostimulant drug studies, and dopamine research in general, one can see immediate applications for the use of antisense methodology. Effective antisense sequences currently exist for specific dopamine receptor Subtypes DI [Van Kampen & Stoessl, 2000] ( Q2 [Rajakumar et al, 1997]> Q3 [Tepper et ai, i997]> a n d D5 i F i l i P e t a l - 2 0 0 ° ] , which, in contrast to traditional pharmacological manipulations, are capable of influencing these receptors with unparalleled specificity. These sequences will undoubtedly be useful for further elucidating the differential effects of psychostimulant-induced increases of dopamine at particular dopamine receptors. Additionally, the development of a SERT antisense sequence might also provide interesting insight in a cocaine IVSA experimental paradigm. Thus, excitement grows, as the formerly 28 impossible step of assessing these transporter manipulations singly and in tandem, within the same animal, is no longer out of the realm of possibility. Purpose of the present experiment: The purpose of this thesis was to employ an antisense sequence complementary to the initiation site of the DAT molecule to assess the effect of DAT knockdown on reinstatement of cocaine self-administration behavior after extinction. Animals were trained to lever press for a low dose of cocaine. Given the fact that antisense treatment does not produce full knockdown of the targeted protein, a low dose of cocaine was employed to maximize the sensitivity of the assay (see discussion). Following acquisition of cocaine self-administration, animals were thoroughly extinguished and treated with either antisense or missense oligonucleotides. DAT antisense resulted in a significant reduction in DAT binding in the nucleus accumbens, assessed using [3H]WIN 35,428. On the sixth day of antisense/missense-administration, reinstatement of drug taking was assessed following one infusion of cocaine (priming). Drug taking was also assessed without priming on two subsequent days. 29 MATERIALS AND METHODS All experiments were conducted in accordance with the standards of the Canadian Council on Animal Care. Subjects Animals (n = ~70) were screened for self-administration for the low dose of drug. Only those demonstrating preference for the active lever and reliable self-administration completed the full experiment. Due to the low dose of cocaine employed in this experiment, approximately one in five animals tested acquired self-administration to criterion. Those that retained catheter patency completed the experiment. Thus, approximately one in seven animals completed the full experiment. These subjects were 10 male Long-Evans rats (320-450g at the time of surgery; Charles River, Saint-Constant, Qubec), which were individually housed after surgery with free access to food and water. Colony temperature was controlled to 21°C with 40% humidity. Rats were maintained on a twelve hour light-dark cycle (0600h-1800h; lights on at 0600h). Test sessions were done during the light phase, with the exception of 10 hr extinction sessions, which began at 900 h for the first five animals and 1200h thereafter. Food Training To facilitate acquisition of drug self-administration animals were first trained on an FR2 schedule to bar press for 45 mg food pellets (BioServ). 30 Training continued daily unti l the criterion was reached (150 pellets in a 1 hr session - data not shown). During food training a 'session on' houselight went on at the beginning of the session, which signified food availability. Rats were maintained at approximately 85% of their baseline body weight for the duration of food training. This was done by allowing 1-2 hours of food access per day. Free-feeding conditions were restored upon completion of training. Apparatus for operant training and intravenous self-administration Al l training was conducted in transparent plexiglass operant chambers (32cm X 32cm X 41 cm). Two stainless steel operant levers, a 28V D.C. 170 mA light bulb (Spectro), and a pellet dispenser were fixed to the back wall of the chamber. The floor was lined with paper towels and covered with a metal grid. The testing chamber was placed within a light-proof and sound-attenuating black wooden chamber. Silastic tubing shielded by a metal spring extended from the i.v. catheter of the animal to a mercury-filled commutator and liquid swivel (Instech, Inc., Plymouth Meeting, Pennsylvania) mounted on the Plexiglas box. It continued out of the wooden box to a Harvard apparatus pump (Sage Apparatus, pump model 341, Orion Research Lab, Cambridge, Massachusetts). Lights, operant lever, pellet dispenser (with food hopper), and pump were regulated by a computer control system (MANX; Gilbert & Rice, 1979). 31 Surgery Surgical procedures occurred once animals had returned to baseline weight (that measured immediately before food deprivation began to facilitate food training). Catheterization and guide canulation procedures were performed first, and osmotic minipumps were implanted during a second surgery after drug training sessions and extinction. Intravenous Catheter Surgery: A l l instruments were autoclaved for 30 min before the first surgery of the day. In between surgeries on the same day instruments were scrubbed with hibitane antiseptic and soaked in 70% isopropyl for at least ten minutes. Rats were anaesthetized with xylazine (9 m k / k g i.p.; Rompun) and ketamine hydrochloride (100 mg/kg i.p.; MTC Pharmaceuticals) and supplemented with ketamine (-20 mg) when needed. Al l rats were implanted with a single jugular catheter aimed at the right vena cava. The catheter was then tied and glued to the vein to prevent movement and the free end was threaded subcutaneously to the top of the head. After stereotaxic procedures, it was mounted to the skull using dental acrylic cement. Al l rats were given Baytril (0.1 mL, s.e, 50 mg/mL) prior to surgery and twice daily for two days after surgery. Stereotaxic Surgery: At the completion of catheterization, rats were immediately placed in a stereotaxic device for the bilateral implantation of one 11 mm guide cannula into the VTA (-4.8 mm posterior to Bregma, +/- 1.0 mm lateral to the midline and -6.4 mm ventral to the skull's surface at that point); 32 (Paxinos & Watson, 1986). Guide cannulae and the free end of the i.v. catheter were then cemented to jeweler's screws placed in the four skull quadrants surrounding Bregma with dental acrylic cement. 12 mm wire obturators were placed in the guide cannula to prevent blockage and infection. Minipump Implantation: Following acquisition of intravenous self-administration behavior for cocaine, and subsequent extinction, animals were anaesthetized using halothane. A small incision was then made immediately posterior to the existing dental acrylic headcap, and the skin was pulled loose with forceps. Notably, silastic tubing of the i.v. catheter was present subcutaneously posterior to the headcap. This necessitated that careful incisions be made to prevent cutting the hidden tubing. In later surgeries, the position of the silastic tubing was modified to exit slightly to the right of the headcap thereby minimizing the possibility of damage during minipump surgery. A curved hemostat was then used to blunt dissect a subcutaneous pocket at the base of the neck. Osmotic minipumps (ALZET model 2001) were attached to 13 mm infusion cannulae (25 guage) by 50 PE polyethylene tubing and placed into the pocket beneath the skin. Infusion cannulae were inserted into the guide cannula, which terminated 2 mm above the VTA. Infusion cannulae were fixed in position with dental acrylic cement. Experimental Procedure Rats were trained to self-administer cocaine (0.1 mg/infusion) on a Fixed Ratio 2 Time Out 30 s (FR2 TO 30) schedule of reinforcement (see Fig. 4). Each 33 training session was 1.5 hours in duration. Illumination of the house light signaled the beginning of a session ("drug available") and each infusion was accompanied by a discrete CS consisting of five brief (0.5 sec) flashes of the houselight over five seconds. During the time out period after an infusion, the house light remained off, and lever pressing had no programmed consequences. Training sessions had no priming infusion and no maximum number of infusions. Animals were trained for a minimum of 6 sessions. Rats that made 25 or more active lever responses in each of the last three sessions, demonstrated a 2:1 response ratio (activeinactive) in at least one of last three days, and more active presses than inactive in all of last three days, were considered to have met the training criteria. At this point they graduated to 10 hr extinction sessions, in which all lever pressing resulted in saline infusions instead of cocaine. To emphasize the change to extinction, the first five infusions (roughly the volume contained in the i.v. line) of the first extinction session contained cocaine. Every infusion thereafter offered only saline and subsequent extinction sessions were run with saline alone. Infusions earned during extinction sessions were paired with the discrete CS (flashing light) and were similar to drug infusions in all respects. Animals were then randomly assigned to one of two groups (antisense Or missense control) for implantation of osmotic minipumps. 34 Drug Prime V Acquisition Extinction Antisense/Missense (6 Days) Day 1 Day 2 Day 3 REINSTATEMENT Normal D A T Expression Reduced D A T Expression TIME Fig. 4 - Schematic representation of the experimental procedure. Antisense Oligodeoxynucleotides Two 17 mer Oligodeoxynucleotide sequences were synthesized (Nucleic Acid and Protein Synthesis laboratory, UBC) with phosphorothioate modification at bases 1-3 and 15-17. The antisense sequence was complementary to the initiation site of the rat dopamine transporter (5-AGA-TTC-AGT-GGA-TCC- AT-3), while the control missense sequence contained the same bases in pseudo-random order (5'-AGC-ATT-GAA-CAA-GCC-AT-3') . The missense control sequence did not have any homology with known sequences based on a DBLAST database search. Oligodeoxynucleotides were dissolved in sterile saline and treatment occurred through osmotic minipumps, which administered lnmol /day (1 ul/hr) continuously for seven days. 35 Reinstatement Six days following minipump surgery, animals were placed back into operant chambers for a 1.5 hour reacquisition session, and given one priming infusion of cocaine (0.1 mg/infusion in 0.1 mL volume) accompanied by presentation of the CS. This procedure was repeated on days 7 and 8 of minipump function without the priming infusion. After the third reacquisition session animals were sacrificed by decapitation. Brains were flash frozen with dry ice and isopentane at -20°C, and stored at -80°C until autoradiography was performed. Autoradiography Immediately following the final reinstatement trial, animals were decapitated, their brains removed and flash frozen using dry ice with isopentane, and stored at -80°C until sectioning (10 um). Sections underwent 15-30 min of preincubation in 150 m M NaCI prior to incubation with 50 m M Tris-HCl buffer (pH 7.4) containing 300 mM NaCI, 5 m M KC1, and 10 n M 3 H-WIN 35-428 (NEN, Boston, MA, USA) for 40 min in humidified boxes. Nomifensine (10 \im, Research Biochemicals) was added to one of each pair of adjacent slides to rule out non-specific binding. A l l procedures were carried out at 4°C. Following incubation, sections were doubly washed for 60 seconds using the incubation buffer and then dried under cool air. Slides and tritium standards were then apposed to tritium-sensitive Hyperfilm- 3H (Amersham, Toronto, Canada) for 4 weeks and developed with D19 developer (Kodak, Rochester, NY, USA). 36 RESULTS Acquisition Animals met criteria for acquisition of self-administration behavior in an average of 9.9 ± 1 . 3 sessions. By group, antisense-treated animals (n=5) took 7.8 +/- 1.1 sessions to meet criteria; missense-treated controls (n=5) required 12.0+/- 1.6 sessions (Table 1). A student's t-test Table 1. Acquisition of Cocaine Self-Administration Mean Number of Mean Number of Active Lever Acquisition Sessions Presses for Final 3 Trials Antisense 7.8 +/- 1.07 52.6 +/- 14.47 Missense 12.0 +/- 1.58 67.6 +/- 21.4 statistical analysis allows the null-hypothesis to be accepted (p=0.0589). The small p-value for this analysis is close to statistical significance, raising the possibility that animals allocated to the two experimental contingencies may have had unequal drug-experience. However, over the last three days of training both groups consumed equal quantities of drug. Two-way repeated measures ANOVA failed to find a significant effect of group (p=0.58), time (p=0.45), or interaction between group and time (p=0.61) over the last three days of acquisition training (3 data points; each of last three days). Moreover, 37 animals in both groups displayed preference for the drug-appropriate lever that was not statistically different as assessed by the relative ratios of active to inactive lever presses (unpaired student's t-test, p=0.126). Extinction Active lever pressing decreased significantly (59%) from an average of 87.4 +/- 13.4 total lever presses on the first day of extinction to 35.7 +/- 9.9 on the last day (unpaired student's t-test, p=0.005). Most importantly, prolonged extinction training also altered the pattern of lever pressing within individual sessions (Fig. 5). The first extinction session was characterized by an initial burst of lever pressing (31.6 +/- 6.5 mean presses in the first hour) followed by a lul l and second burst (9.0 +/- 2.2 mean presses in the ninth hour, which was the peak of the second burst). During the final extinction session, a smaller initial burst occurred (13.9 +/- 3.2 mean presses in the first hour) but the second burst was absent. Active lever presses in the first 90 minutes of the initial extinction session (approximating the "burst") was 38.800 +/- 6.719. This compares to 62.8 +/- 14.7 presses during the last 90-minute drug-available training session. Pressing during the next, and subsequent sessions dropped to very low levels (fig. 6). In addition, preference for the drug-appropriate lever during the initial hour of the first extinction session (left panel Fig. 7) was completely lost by the beginning of the final session (right panel Fig. 7). These data show that prolonged extinction trials were effective in eliminating the 38 association between specific operant responses on the drug-appropriate lever and cocaine reward. Reinstatement Following extinction half of the rats (n=5) were treated with DAT antisense (1 nmol/day for 6 days) bilaterally into the VTA, and half of the rats received a missense control sequence. On the sixth day of antisense/missense-administration, reinstatement of drug taking was assessed after one infusion of cocaine (0.1 mg/infusion) (priming). Drug taking was also assessed without priming on two subsequent days. Antisense-treatment resulted in a significant (95%) reduction on the third reinstatement trial, but not the first two, compared to the average of the last three drug-available training trials (Fig. 8) (paired t-test, t=3.4, p=0.03). Drug taking in the missense group was comparable to pre-extinction levels. With reference to discriminative behavior, rats treated with DAT antisense responded selectively on the drug-appropriate lever during the first session following extinction, but discriminant responding was completely disrupted by the second session (fig. 9). Over three days of reinstatement trials, the ratios of active to inactive lever presses for antisense-treated animals were 5.4, 1.6, and 0.92 respectively. Figure 9 presents these data in log-transformed form. Though not statistically significant, the ratios suggest a trend toward progressive loss of preference for the drug-appropriate lever. In contrast, ratios 39 for missense-treated animals over the same trials were 5.1, 7.7, and 29.7, consistent with the opposite trend toward increasing preference for the drug-associated lever in this group. DAT Knockdown Densitometric values for each animal were measured separately and pooled for analysis. Student's t-test analysis (p=0.026) revealed that 3 H-WIN 35-428 binding was reduced by 42% in the nucleus accumbens of antisense-treated animals compared to missense-treated controls (Fig. 10). Histology The placement of infusion canula in the Ventral Tegmental Area (VTA) is shown in Fig. 11. Canula placements from antisense-treated animals are marked with circle symbols and those from animals treated with missense are shown by diamond symbols. Al l infusion canula were well within an estimated diffusion diameter of 1 mm from VTA cells. 40 DISCUSSION The present study was undertaken to develop a highly sensitive and specific behavioral assay to assess the importance of cocaine's interaction with the dopamine transporter for its rewarding properties. The experiment monitored drug-induced reinstatement of cocaine self-administration after extinction. Notably, three separate "reinstatement" trials were given on three consecutive days in association with the final phase of antisense treatment. A single infusion of cocaine was used to prime reinstatement on the first day only. Thus, the final two trials may be more aptly termed "reacquisition", rather than reinstatement, which traditionally refers to a single test session. During the interval between extinction and reinstatement DAT expression was reduced using antisense oligonucleotides to block translation of the DAT protein. Thus, cocaine self-administration was established and extinguished during a period of normal DAT expression; but drug-induced reinstatement was uniquely tested in the same animals while DAT expression was significantly reduced. Under these experimental conditions, animals with reduced DAT expression at VTA dopamine terminals demonstrated two important differences from missense-treated controls. They showed a progressive reduction in contingent drug-infusions during reinstatement/reacquisition and a progressive loss of preference for the drug-appropriate lever over time. The unique design of this experiment is based on four complementary factors: 1) Use of DAT knockdown with antisense sought to limit 41 neuroadaptations arising from knockout methodology, while retaining the basic objective of removing the putative primary substrate of cocaine's pharmacological action (the DAT); 2) Animals were pre-screened to exclude those that did not show preference for the drug-associated lever. This allowed lever-preference to serve as a measure of the discriminant stimulus properties of the drug 6 ; 3) Following acquisition training, animals were thoroughly extinguished (50-110 hours) to ensure that reinstatement behavior was the result of priming and subsequent drug-taking, rather than residual drug-seeking from the previous drug experience; and 4) The protocol required animals to learn to self-administer a very low dose of cocaine. This point requires elaboration. The dose of cocaine employed for training and reinstatement (0.1 mg/infusion or -0.25 mg/kg/infusion for a 400g rat) was a slightly supra-threshold dose, shown previously to be just high enough to support self-administration. Traditionally, a significantly higher training dose (0.25 mg/infusion or ~0.7 mg/kg/infusion in Phillips' lab) has been used to establish self-administration. A pilot study using 0.075 mg/infusion showed that animals would not respond for this dose [data n o t shown] Thus, our observations confirm that 0.1 mg/infusion was indeed very close to threshold, representing a low, ascending-arm position on the "inverted U " dose-response curve. Despite a difficulty in obtaining stable responding in a significant proportion of experimental animals (after extensive trials approximately 1 in 5 animals were 6 Demonstration of preference for operant responses leading to certain stimuli is considered to be indicative of a rewarding stimulus. Accordingly, loss of preference can be seen as indicative of loss of reward. 42 found to reliably self-administer), our "low dose" was chosen to maximize the behavioral sensitivity of the experiment. This reasoning relies on the assumption that cocaine's interaction with the DAT is a necessary part of its reinforcing properties. If this assumption is true, then it is reasonable to conclude that a slightly supra-threshold dose for self-administration would block a minimum number of DAT sites to alter dopamine levels sufficiently to provide salient reinforcement for the animal. The number of DAT sites blocked is a function of two variables: the total number of DAT sites, and the concentration of drug in the synapse [personal communication with Jonathan Katz]_ Therefore, if DAT antisense treatment is successful in decreasing the number of DAT sites, a slightly supra-threshold dose, can no longer block enough sites to provide a dopamine response sufficient to contribute to the drug's reinforcing properties. For example, consider a cocaine dose, "x", that is sufficient to block 50% of available DAT binding sites. If antisense treatment is then successful in producing a 50% knockdown in DAT expression (our manipulation produced a 42% reduction), the same dose, "x", will block 50% of the remaining sites. Thus, only 25% of the original number of binding sites would be blocked in this example. 43 Interpreting the results -Rats treated with DAT antisense and primed to reinstate drug-taking immediately regained selective responding on the drug-appropriate lever during the first session following extinction. However, discriminant responding was completely disrupted by the second session and on the final trial, cocaine self-administration had virtually ceased. Drug-taking and preference for the drug-appropriate lever in the missense group was comparable to pre-extinction levels. The finding that DAT antisense treatment decreased drug-taking for a dose on the ascending arm of the dose-response curve is consistent with the conclusion that reducing expression of the dopamine transporter resulted in diminished cocaine reward. In addition, the observation that priming reinstated preference for the active lever, which was gradually diminished over the course of three 90-minute sessions suggests that the initial drug experience after antisense treatment may have been comparable to the memory of the discriminant stimulus properties of cocaine acquired during previous training. The discriminant stimulus properties of cocaine may include SERT- and NET-mediated effects in addition to the rewarding properties resulting from simultaneous effects on the DAT. As such, these discriminant properties of cocaine may have been sufficient to prime drug-seeking behavior during the first reinstatement session, but could not sustain continued drug-seeking in the second and third sessions. This observation is very similar to the previous finding that serotonin IB receptor agonists transiently maintained cocaine's 44 discriminant stimulus properties if substituted for the drug during self-administration Parsons et at, 1998] Addition of serotonin IB receptor agonists to saline vehicle dose-dependently reduced extinction bursts associated with substituting saline for cocaine, and, maintained self-administration for up to 90 minutes in experienced animals. However, activation of serotonin IB receptors, like cocaine administration in DAT knockdown rats, was apparently insufficient to maintain motivated operant responding. This similarity notwithstanding, serotonin IB receptor activation is only one consequence of cocaine-mediated serotonin elevation. There are 14 currently identified serotonin receptors, some with several isoforms. Moreover, it is very difficult to determine whether the discriminant stimulus properties observed of serotonin IB receptor agonists are specific to the agent, or conditioned to other pharmacological effects of cocaine. Caveats aside, the initial maintenance, and subsequent loss of preference for the active lever by the second trial represents a clear transition. Conceptually, this may be viewed as a search strategy employed in response to diminished reward; when the previous source was exhausted, addicted rats responded on the inactive lever as a potentially new supply. Thus, we conclude that non-DAT mediated cocaine effects have discriminant, but not rewarding, properties. 45 DAT, NET and S E R T -The present data appear to reaffirm the role of the DAT as cocaine's primary substrate, serving to transduce cocaine reward in the brain. However, a recent study showed that KO mice lacking the dopamine transporter exhibited an unexpected propensity to self-administer cocaine Poena et ai, 1998] This called into question the notion that cocaine's interaction with DAT, and consequent dopamine elevations in the nucleus accumbens, are of primary importance to the rewarding properties of the drug. Hence, cocaine's pharmacological actions at all three monoamine transporter proteins, DAT, NET and SERT P»tz etai, i990] j have come under renewed scrutiny. For example, although the SERT, unlike the NET, does not regulate dopamine at physiological concentrations [Hoffman et at, i99i] ; cocaine-mediated elevations in serotonin almost certainly play a role in the drug's effects. In fact, the role of serotonin in reward may be one of modulating dopamine release. Stimulation of 5-HT1B receptors has been shown to stimulate dopamine release in the nucleus accumbens and caudate/putamen [ ° u a n e t al> 1989] Moreover, as previously mentioned, 5-HT1B receptor agonists have some of the discriminative stimulus properties of cocaine Parsons et at, 1998] Mechanistically, activation of 5-HT1B receptors has been proposed to reduce GABAergic inhibition of VTA dopamine projections to striatum, resulting in enhanced dopamine release. Furthermore, anecdotal evidence from drugs like Mazindol, which strongly binds to the NET and DAT, but not the SERT, implies that cocaine's interaction with the SERT is a likely contributor to a drug's abuse 46 potential. Mazindol differs from cocaine by virtue of a weaker interaction with the SERT, and it is not consistently abused by humans or self-administered reliably by animals [ C a s t a n o n e t a / ' 2 0 0 ° ] . In addition, the cocaine-like ligand, [ 1 2 5I]RTI-55 binds to SERT from DAT knockout animals that self-administer cocaine [ R ° c h a et ai, 1998.] Moreover, although DAT knockout animals (DAT-/-) demonstrate conditioned place-preference (CPP) for cocaine, the additional removal of both SERT alleles on a D A T - / - genetic background (a double knockout: "DAT-/- ,SERT-/ -") completely abolishes this behavior [Soraeta/.,2001]_ These data suggest that regulation of serotonin is one factor in determining the rewarding properties of cocaine that may have putative modulatory impact on dopamine levels. However, the precise role and relative importance of serotonin and SERT remains unclear. Knockout models have attempted to address this question. Removal of only one SERT allele in DAT - / -animals has no effect on cocaine-CPP. By contrast, removal of a single DAT allele on a S E R T - / - genetic background (DAT+ / - ,SERT- / - ) is sufficient to prevent cocaine-CPP. One interpretation of this finding, championed by Sora et al, 2001, claims that cocaine's interaction with DAT is more essential to its rewarding properties than the interaction with SERT. However, if this view is to be based on the above findings, it is informative to note that knocking out one allele of the DAT or SERT gene results in different reductions in the corresponding transporter expression. Genetic knockout of one DAT allele on a S E R T - / - background produced a significant (66%) reduction of DAT expression 47 in striatum and frontal cortex compared to wildtype littermates, whereas the SERT+ / - ,DAT- / - genotype showed a non-significant (44%) reduction in SERT binding. Thus, it may be that loss of cocaine-CPP by removing one DAT allele in a SERT KO, and failure to prevent the effect by removing one SERT allele in DAT KOs, may reflect the overall success in reducing expression, rather than the relative importance of the transporter. In any case, while emphasis of these subtleties will not promote consensus on the issue, it does highlight the inherent difficulty of interpreting results from knockout animals with complex neural adaptations (see Introduction section, "Psychostimulants and Dopamine" - p . 19). Despite a bias in the literature toward investigations focused foremost on DAT followed by SERT, there is also evidence that the NET plays a significant role in cocaine's regulation of dopamine, and hence reward. Yamamoto and Novotney have shown that a fraction of cocaine-dependent inhibition of dopamine uptake in wildtype rats occurs by blocking the norepinephrine transporter [Yamamoto & Novotney, 1998]; infusions of the selective NET blocker desmethylimipramine (DMI) into the medial prefrontal cortex and nucleus accumbens shell of rats caused an increase in extracellular dopamine in these regions. Notably, there is increasing evidence that drugs of abuse elevate dopamine [Reviewed by Heimer et al, 1997] a n d stimulate locomotor activity [Personal communication with Satoshi Ikemoto - paper submitted for publication] preferentially in the shell of the nucleus accumbens versus the core, and failure to recognize this may have confused the literature somewhat by virtue of non-specific probe placement for 48 microdialysis. A second study corroborates Yamamoto and Novotney's findings, using nisoxetine, a selective NET blocker, to show a 20% reduction in dopamine uptake in nucleus accumbens synaptosomes from wild-type mice [Moron etal., 20oo]_ Thus, the NET may contribute to regulation of dopamine levels in the nucleus accumbens and medial prefrontal cortex. The NET story continues in DAT KO mice. Although Rocha et al.'s initial study lRocha et ai., 1998] suggested that "dorso/ventral striatal" dialysate dopamine levels in DAT KO mice are unresponsive to cocaine challenge, a new study has modified this view [Carboni et at, 2001] Cocaine- and amphetamine-induced elevations of dialysate dopamine have now been demonstrated in the medial portion of the nucleus accumbens in DAT KO mice. Recall, the nucleus accumbens shell lies medially and ventrally to the core. Reboxetine, a selective-NET blocker, administered systemically by i.p. injection, increased nucleus accumbens dopamine in DAT KOs to the same extent as cocaine, but not in wild-type animals 7 . Areas of the DAT KO striatum (i.e. dorsal, or caudate/putamen) where there is known to be negligible NET expression in wildtypes, did not show dopaminergic reactivity to cocaine, amphetamine, or reboxetine. These data support the conclusion that cocaine-mediated inhibition of dopamine uptake in the nucleus accumbens shell contributes to the phenomenon of self-administration in DAT KO mice. However, the important 7 The fact that Carboni et al. did not observe NET-blockade induced dopamine elevations in wildtype animals appears to disagree with Yamamoto & Novotny's and Moron et al.'s suggested NET-dependent regulation of dopamine in wildtype animals. This discordance may be explained by the fact that three different NET-selective blockers were used (reboxetine, DMI, and nisoxetine), each with a unique N E T affinity and method of administration (respectively, systemic i.p. injection, through the dialysis probe, and applied to synaptosomal pellets). 49 question of how cocaine elevates nucleus accumbens dopamine in DAT KO animals has no definitive answer. One possibility involves a direct effect of cocaine on the NET. Although DAT KO animals do not exhibit significantly altered expression of NET or SERT when compared to wildtype littermates [ S o r a e t ai, 2001]^  systemic administration of reboxetine elevates dopamine in DAT KOs, but not wildtype animals. Thus, subtle compensatory neuroadaptations may confer greater control over dopamine clearance to the norepinephrine transporter in DAT KO mice. In this framework, NET blockade by cocaine may provide the basis for the increased dopamine levels observed by Carboni et al. in the nucleus accumbens shell region of DAT KO mice. This is also one possible explanation for cocaine self-administration in these animals. However, two additional ideas have bearing on the issue of compensatory changes to NET in this context. First, it is possible that dopamine elevation by reboxetine in DAT KO animals reflects greater access to dopamine by NET, in the absence of DAT, rather than compensatory change. Afterall, NET expression in DAT KO animals is not significantly different from wildtype littermates ISora e t a i < 2001 j j n wildtype animals, DAT may efficiently clear small dopamine elevations produced by reboxetine, rendering its effect on dopamine undetectable. Second, Moron et al. have shown that dopamine uptake from DAT KO nucleus accumbens synaptosomes is unresponsive to even very high doses of cocaine. This seems incongruous with Yamamoto and Novotney's demonstration that local infusions of the NET-specific blocker, DMI, elevate 50 dopamine in the nucleus accumbens of wild-type rats. Conflicting, though they seem, these data may be reconciled by the hypothesis that the NET protein of DAT KO animals is somehow fundamentally different than those of wildtypes [personal communication with Bruce Hope]. Also important is the observation that cocaine does inhibit dopamine uptake by DAT KO synaptosomes originating from the medial prefrontal cortex, where NET expression is known to be high. Hence, might there be more than one isoform of the NET protein with different affinities for cocaine and perhaps dopamine? Moreover, could altered regional expression of these isoforms constitute a compensatory change in the DAT KO model? Although, to date, there has been no evidence for NET proteins with differing conformation or primary structure, further studies may bear this hypothesis out. First, an examination of the respective NET proteins from DAT KOs and wildtype animals would be interesting. Second, it would be informative to measure dopamine in DAT KO animals using local infusions of nisoxetine through nucleus accumbens dialysis probes. Nisoxetine is more selective for NET than reboxetine, and local administration would eliminate possible confounds of systemic noradrenergic receptor activation. This experiment would provide definitive evidence regarding the ability of NET to regulate dopamine in vivo in DAT KO animals. A third question involves the relative contribution of cocaine-mediated NET blockade to nucleus accumbens dopamine levels in wild-type animals. Yamamoto and Novotney showed that DMI can elevate dopamine, but does cocaine-mediated NET blockade in the 51 nucleus accumbens lead to elevations of dopamine as well as norepinephrine? Measuring dopamine uptake by nucleus accumbens synaptosomes from wildtype animals in the presence of cocaine and a selective DAT-blocker such as GBR 12909, might answer this question. If the presence of cocaine as well as GBR provided additional inhibition of dopamine uptake relative to GBR alone, cocaine-mediated NET blockade would be the most likely cause. 52 Future Directions Interpretation of results from the present thesis will be aided by future experiments aimed at further characterization of the DAT antisense knockdown model for the study of cocaine reward. Several approaches are currently planned, or underway. From a behavioral standpoint there are several experiments of immediate interest. Both acquisition of cocaine self-administration and maintenance of stable responding are potential targets of investigation. First, in an acquisition paradigm, one might predict that animals beginning training during maximal DAT knockdown would fail to acquire self-administration behavior during initial trials. Once DAT levels had recovered to normal, self-administration would likely also recover. Secondly, for animals stably administering a descending-arm cocaine dose, DAT knockdown might function like a competitive inhibitor, which typically hastens responding. A third behavioral assay, conditioned place-preference, could be useful to confirm that DAT knockdown reduces cocaine reward. Neurochemical investigations will also prove informative. Accordingly, a no-net flux microdialysis procedure will be used to evaluate whether baseline dopamine levels are altered by DAT antisense administration and the dopamine response to multiple dose cocaine challenge will also be measured. In addition, 53 the timecourse of antisense knockdown will be investigated using synaptosomal [3]H-dopamine uptake assays. 54 Conclusion -In summary, this study provides further evidence that cocaine's interaction with the DAT is an essential factor of the drug's reinforcing properties. Additionally, the ability of a priming dose of cocaine to reinstate selective responding in DAT antisense- treated animals on the drug-appropriate lever during the first reinstatement trial, but not the final two sessions, suggests a role for NET and/or SERT in cocaine reward. Thus, important questions remain to be answered regarding the extent to which NET and SERT regulate other monoamines, where this regulation occurs, what role(s) norepinephrine and serotonin may play in cocaine-reward, and the precise functional role of dopamine in reward. 55 40 -i First Extinction Session Final Extinction Session (hrs) Fig. 5 - Mean (± SEM) active lever presses in one-hour bins on the first and final extinction session, respectively. Repeated measures A N O V A revealed a statistically significant session effect (p=.005). Animals pressed the active lever an average of 87.4 +/- 13.4 times during the first extinction trial vs 35.7 +/- 9.9 during the last session. 56 50 - i Fig. 6 - Mean (±SEM) presses on the active lever during the first 90 minutes of extinction sessions 1-5. Repeated measures A N O V A revealed a significant sess ion effect (p=0.0003). 57 7 0 1 Fig. 7 - Mean presses ( ±SEM) per hour during the first (left panel) and final (right panel) extinction session, respectively. The first five infusions of the first extinction session contained cocaine (the volume of the line) after which active lever presses resulted in saline infusions. The first two data points (corresponding to "drug" on the x-ordinate, left panel) represent mean responses on active and inactive levers during the final 1.5-hour drug-available training session. 58 FIG. 8 Acquisition and Drug-Induced Reinstatement of Active Lever Pressing After Extinction Two-way analysis of variance (ANOVA) of mean active lever presses showed no overall effect of group (F=1.632; DF=1, p= 0 .2372) , but a significant effect of trials (F=2.608; DF=5 , p=.0392) and a group by trials effect (F=3.65; DF=5, p=0.0082). Post-hoc tests revealed lever presses on the active lever were greater for the missense-treated group on day 3 (asterisk) of reinstatement (unpaired t-test, p=0.0233). Negative numbers on x-ordinate indicate drug-available training sessions on days before extinction; likewise, positive numbers indicate days post extinction (i.e. reinstatement sessions) . Reinstatement Trials Fig 9 - Mean (±SEM) log ratio of active to inactive lever presses during the final two drug-available training sessions and 3 reinstatement trials. The first two data points corresponding to '-2' and on the x-ordinate indicate the log lever pressing ratio on days before extinction. Stippled lines show 1:1 ratio level indicating no preference. Repeated measures ANOVA analysis reveals a trend toward statistical significance: antisense effect F[l,8]=4.3, p=0.072, session effect F[8,32]=2.9, p=0.039, antisense X session effect F[8,32]=1.2, p=0.321. 60 2-1 1.5 H SG E b = fi •3 B S z NM E w u < H 0.5 H Missense Antisense Fig. 10 - Densitometric measurement of autoradiographs representing nucleus accumbens [ 3 ] H WIN-35, 428 binding following bilateral infusion of ant isense or missense ol igonucleotides into the V T A . Ant isense treatment (1 nmol/day, 7 days) resulted in a significant decrease in [3] H WIN-35, 428 binding compared to the missense control group. 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