INVESTIGATE MECHANISMS UNDERLYING REINFORCING AND THERAPEUTIC EFFECTS OF STIMULANT DRUGS Dopamine (DA) D4 Receptor Knockout (KO) Model--The 7 repeat allele of the DA D4 receptor appears to be a genetic risk factor for Attention Deficit Hyperactivity Disorder (ADHD). The KO model was used to investigate the functional significance of the D4 gene in brain morphology, function, pharmacology and behavior that may be of relevance to understanding ADHD. The following summarizes our findings measuring the effects of acute administration of methylphenidate (MP) and amphetamine in DA D4R transgenic mice on locomotor activity and on conditioned place preference (CPP). MP is a stimulant used to treat ADHD. It is not clear what factors account for the variability in physiological and behavioral responses to MP. Polymorphisms in the D4R are associated with novelty-seeking behavior, a hallmark of ADHD. We used the KO model to study the behavioral effects of MP. Previous studies showed that these mice are hypersensitive to psychostimulants, display reduced spontaneous locomotor activity, and perform better on rotorod tests compared to their D4R+/+ counterparts. The behavioral effects of MP were tested in 36 adult male D4R+/+, D4R+/- and D4R-/- mice using both a locomotor open field activity assay and CPP; CPP testing is used to assess preference for environmental cues associated with MP. Preliminary results indicate significant differences in behavioral response to MP among the three mice strains. Specifically, the 12 D4R-/- mice showed no preference for the MP-paired environment, compared to the 12 D4R+/- and 12 D4R+/+ mice. These findings support the notion that D4R plays a critical role in the behavioral profile of MP. The KO model was also used to assess the effects of MP on regional brain glucose metabolism, an indicator of brain function. Volkow et al (1997) showed that cerebellar metabolism consistently increased (10%) after MP administration in all clinical subjects while regional activity in other areas varied. They also found a positive correlation between D2R and metabolic rate, suggesting a link between DA and metabolism due to lack of receptors in the cerebellum and varying levels in other regions. We utilized D4R-/- mice to study the brain metabolic effects of MP and, in particular, examine the contribution to D4R in brain metabolic response to MP by measuring the effects of MP on brain glucose metabolism in 36 adult male D4R+/+, D4R+/- and D4R-/- mice using micro PET and FDG. Each animal received a baseline scan and a MP challenge (10mg/kg ip; 5 min following MP mice were injected with FDG, allowed 30 min uptake, anesthetized and scanned). Preliminary results indicated differences in baseline activity between strains in several cortical areas. The most profound difference was noted in the cerebellum baseline vs MP challenge. Our findings support the notion that D4R plays a critical role in the brain metabolic response to MP and may further be used to predict individual response to drug treatment. Animal Model to Assess Behavioral Effects of MP--The visual discrimination task (VST) is a sustained attention task that signals the formation of a habit by taking into account hits, misses, false alarms, and correct rejections. Animals that are trained in VST are very reluctant to unlearn the behavior. Damos & Parker (1994) showed that high-false alarm rates might indicate recreational drug use in humans, Gouzoulis-Mayfrank et al (2000) found that ecstasy users do poorly when compared to non-users in attention memory-learning tasks, and Bandstra et al (2001) showed that cocaine had effects on sustained attention processing in children that had prenatal exposure to cocaine. MP has increased accuracy in visual sustained attention tasks. In a rat condition position responding discrimination task, MP had similar effects to d-amphetamine and cocaine, which increassed accuracy in rats during a sustained attention task. This study assessed the behavioral effects MP had on sustained attention accuracy using a VST and spontaneously hypertensive rats (SHR). SHR are an excellent rodent model for ADHD because they have behavioral and neurochemical characteristics similar to ADHD. Two-week old preadolescent male SHR (n=36) and Wistar rats (n=36) were tested on a food VST using four shaping cue-light intervals (1-s, 500 ms, 50 ms, and 25 ms) and after baseline was established, were exposed to 2 mg/kg MP; 1 mg/kg MP; or vehicle for 2 weeks. Finally, histomorphometric effects of MP were accessed using the Golgi-Cox staining method to demonstrate dendritic density and branching. NEUROBIOLOGY OF ADDICTIVE BEHAVIORS: Neurobiology of Compulsive Overeating and Obesity--Obesity is an epidemic affecting one-third of all American adults (NIH, 2003) and the CDC (2004) reports that about 15% of children and adolescents are overweight. In some instances, the phenomenology of obesity has many similarities to the compulsive behaviors observed in addiction. DA is involved in reinforcing the effects of drugs of abuse and food. The involvement of DA in pathological eating and obesity is poorly understood and has not been directly assessed. DA plays a major role in regulating food intake by modulating food reward via the mesolimbic circuitry of the brain and there is a lot of evidence to suggest that DA may be one of the target neurotransmitters linking the genetic and environmental factors that cause obesity. DA is involved in the regulation of food intake and Chen et al (2004) found that obese persons have decreased D2R availability in the striatum. In this study, we used 4-week old male Zucker obese (that have a leptin deficiency that makes them more prone to obesity) and lean rats, divided into unrestricted and restricted (20 g/day) diet groups. The objective of the study was to understand the developmental and diet effects on D2R levels in obese and lean Zucker rats. D2R levels were examined by both ARG ([3H] spiperone) and micro PET ([11]C-raclopride) at 4 and 16 weeks. Data was analyzed using a 1-way ANOVA at 4 and 16 weeks and consisted of the D2R levels within each group as well as between groups. MECHANISMS UNDERLYING VULNERABILITY TO DRUG ABUSE AND ADDICTION: Effects of Exposure to MP During Early Development in Subsequent Vulnerability to Drugs--MP is the most prescribed drug used in treating approximately 3 million American children diagnosed as ADHD and its use has become controversial. The debate over MP is growing due to concerns relating to its medicinal value and long-term effects. The question of whether chronic exposure to MP influences a child?s risk for drug abuse later in life remains unclear. The present study utilized a rodent chronic oral MP paradigm. Four-week old male Sprague Dawley rats were divided into three treatment groups: 2 mg/kg MP; 1mg/kg MP; and Vehicle (water). Rats were individually housed and had 24h free access to food and a drinking bottle containing one of the above. Body weight and locomotor activity in an open field chamber were monitored for all rats for 30 weeks. After 28 weeks, oral MP was replaced with water. At this point, all animals were microsurgically implanted with a jugular vein catheter and placed in operant test chambers for daily 1h sessions. In each session, rats had the opportunity to press an active lever that would result in an intravenous infusion of cocaine (1 mg/kg). Rats were maintained on this cocaine self-administration protocol for up to 60 days. Significant differences were observed in the number of cocaine infusions, lever presses and in the pattern of administration between the three groups. This study also examined the DA D2 receptor profile in these same rats before MP exposure and after 28 weeks of MP exposure using [11]C-raclopride and micro PET.
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