STUDIES ON THE DEVELOPMENT OF PET RADIOTRACERS FOR MEASURING EPIGENETIC MARKS IN VIVO. Histone deacetylases (HDACs) are enzymes involved in epigenetic modifications that shift the balance toward chromatin condensation and silencing of gene expression. Here, we evaluate the utility of 6-((18)Ffluoroacetamido)-1-hexanoicanilide ((18)FFAHA) for positron emission tomography (PET) imaging of HDAC activity in the baboon brain. For this purpose, we assessed its in vivo biodistribution, sensitivity to HDAC inhibition, metabolic stability and the distribution of the putative metabolite (18)Ffluoroacetate ((18)FFAC). METHODS: (18)FFAHA and its metabolite (18)FFAC were prepared, and their in vivo biodistribution and pharmacokinetics were determined in baboons. (18)FFAHA metabolism and its sensitivity to HDAC inhibition using suberanilohydroxamic acid (SAHA) were assessed in arterial plasma and by in vitro incubation studies. The chemical form of F-18 in rodent brain was assessed by ex vivo studies. Distribution volumes for (18)FFAHA in the brain were derived. RESULTS: (18)FFAHA was rapidly metabolized to (18)FFAC, and both labeled compounds entered the brain. (18)FFAHA exhibited regional differences in brain uptake and kinetics. In contrast, (18)FFAC showed little variation in regional brain uptake and kinetics. A kinetic analysis that takes into account the uptake of peripherally produced (18)FFAC indicated that SAHA inhibited binding of (18)FFAHA in the baboon brain dose-dependently. In vitro studies demonstrated SAHA-sensitive metabolism of (18)FFAHA to (18)FFAC within the cell and diffusion of (18)FFAC out of the cell. All radioactivity in brain homogenate from rodents was (18)FFAC at 7 min postinjection of (18)FFAHA. CONCLUSION: The rapid metabolism of (18)FFAHA to (18)FFAC in the periphery complicates the quantitative analysis of HDAC in the brain. However, dose-dependent blocking studies with SAHA and kinetic modeling indicated that a specific interaction of (18)FFAHA in the brain was observed. Validating the nature of this interaction as HDAC specific will require additional studies. STUDIES ON THE ROLE OF NMDA PARTICAL AGONIST ON THE EXTINCTION OF DRUG-INDUCED CONDITIONING Recently, it was shown that D-cycloserine (DCS, a NMDA partial agonist) facilitated extinction of fear as well as cocaine conditioned place preference (CPP) in rats. METHODS: The present study examined the effects of DCS (15 mg/kg i.p. and 30 mg/kg i.p.) on extinction and renewal of cocaine-induced CPP in C57bL/c mice. In parallel, we examined the effects of DCS on locomotor activity. RESULTS: Extinction to cocaine CPP was significantly faster with DCS than with vehicle treatment (three versus six sessions, respectively). After extinction was achieved, mice were retested for CPP 1 and 2 weeks later. All animals maintained extinction to CPP 1 week later, but at 2 weeks, the vehicle and the 15 mg/kg DCS-treated animals maintained the extinction, but the 30 mg/kg DCS-treated mice had renewed CPP. During induction of cocaine CPP, mice displayed enhanced locomotor activity following treatment with cocaine, as expected, based on previous literature. During extinction, there were no differences in locomotor activity between the vehicle and the 15 mg/kg DCS-treated mice, whereas the 30 mg/kg DCS-treated animal showed significant locomotor activity inhibition. These results corroborate in mice the previously reported acceleration of extinction to cocaine-induced CPP by DCS in rats. However, we also show that the higher DCS doses facilitated CPP reestablishment after extinction. Thus, while DCS could be beneficial in accelerating the extinction to conditioned responses in addiction it could, at higher doses, increase the risk of relapse. Thus, studies evaluating the beneficial therapeutic effects of DCS should assess not only the short-term effects but also the potential of longer lasting undesirable effects. STUDIES ON THE ROLE OF DOPAMINE D4 RECEPTORS IN THE PHARMACOLOGICAL EFFECTS OF STIMULANT DRUGS Methylphenidate (MP) and amphetamine (AMPH) are the most frequently prescribed medications for the treatment of attention-deficit/hyperactivity disorder (ADHD). Both drugs are believed to derive their therapeutic benefit by virtue of their dopamine (DA)-enhancing effects, yet an explanation for the observation that some patients with ADHD respond well to one medication but not to the other remains elusive. The dopaminergic effects of MP and AMPH are also thought to underlie their reinforcing properties and ultimately their abuse. Polymorphisms in the human gene that codes for the DA D4 receptor (D4R) have been repeatedly associated with ADHD and may correlate with the therapeutic as well as the reinforcing effects of responses to these psychostimulant medications. Conditioned place preference (CPP) for MP, AMPH and cocaine were evaluated in wild-type (WT) mice and their genetically engineered littermates, congenic on the C57Bl/6J background, that completely lack D4Rs (knockout or KO). In addition, the locomotor activity in these mice during the conditioning phase of CPP was tested in the CPP chambers. D4 receptor KO and WT mice showed CPP and increased locomotor activity in response to each of the three psychostimulants tested. D4R differentially modulates the CPP responses to MP, AMPH and cocaine. While the D4R genotype affected CPP responses to MP (high dose only) and AMPH (low dose only) it had no effects on cocaine. Inasmuch as CPP is considered an indicator of sensitivity to reinforcing responses to drugs these data suggest a significant but limited role of D4Rs in modulating conditioning responses to MP and AMPH. In the locomotor test, D4 receptor KO mice displayed attenuated increases in AMPH-induced locomotor activity whereas responses to cocaine and MP did not differ. These results suggest distinct mechanisms for D4 receptor modulation of the reinforcing (perhaps via attenuating dopaminergic signaling) and locomotor properties of these stimulant drugs. Thus, individuals with D4 receptor polymorphisms might show enhanced reinforcing responses to MP and AMPH and attenuated locomotor response to AMPH. STUDIES ON THE INTERACTIONS BETWEEN LEPTIN AND DOPAMINE SIGNALING AND THEIR ROLE IN OBESITY The dopamine (DA) D(2) receptor (D2R) agonist bromocriptine (BC) decreases body fat in animal and human models and increases lean muscle mass, improves glucose intolerance and insulin resistance, and reduces triglycerides and free fatty acids. We have previously shown a negative correlation between D2R and body weight in obese individuals and in rodents, and that chronic food restriction increases D2R binding in genetically obese rats. The purpose of this study was to assess whether the antiobesity and metabolic effects of BC are related to changes in midbrain DA and D2R activity by measuring D2R and DA transporter (DAT) binding in a genetic (leptin-receptor-deficient) and environmental (diet-induced) rodent obesity model. Obese (fa/fa) (leptin-receptor-deficient), lean (FA/FA) Zucker rats and rats with diet-induced obesity (DIO) were treated with 10 mg/kg BC for 4 weeks. Body weight, food intake, locomotor activity and blood glucose levels were measured along with D2R- and DAT-binding levels using in vitro receptor autoradiography. RESULTS: BC decreased food intake and body fat and increased locomotor activity in both the (fa/fa) and DIO rats. Furthermore, BC increased D2R binding in (fa/fa) but not in DIO rats. Finally, BC increased DAT binding in DIO rats but not in the (fa/fa) rats. These observations are all consistent with the existence of unique leptin-DA interactions and the hypothesis that there is hyposensitivity of the DA system in obesity.
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