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Stop Animal Exploitation NOW!
S. A. E. N.
"Exposing the truth to wipe out animal experimentation"

National Institute on Drug Abuse

1 Z01 DA000405-11 NB
Development Of New Approaches To Neuroimaging with PET and SPECT

Lead Investigator: Alane S. Kimes, PhD (NB, NIDA)
PI/SOR: Elliot A Stein, PhD (NB, NIDA)
Lab staff: Svetlana Chefer, PhD (NB, NIDA)
Carlo Contoreggi, MD (NB, NIDA)
Donald B Vaupel, Ph.D. (NB, NIDA)
Betty Jo Salmeron, MD (NB, NIDA)
Alexey Mulkin (NB, NIDA)
NIH Collaborators: John Matochik (DNB, NIAAA)
Simona Spinelli, PhD (NN, NIAAA)
Extramural Collaborators: Edward F. Domino, MD (Pharmacology, University of Michigan)
Andrew G Horti, Ph.D. (Radiology, Johns Hopkins Universtiy)
 from October 01, 2006 to September 30, 2007
Human subject research: Human Tissues, Fluids, Cells, etc.
Human Subjects
Total Staff Years:2.8
Keywords:radioligands, positron emission tomography, magnetic resonance imaging, PET, SPECT nicotinic receptors, cannabinoid receptors
Goals and Objectives:PET imaging and quantification of receptors is a state-of-the-art methodology that allows multiple studies to be performed on human volunteers or research animals. The major focus of this project involved the development of methods to image nicotinic acetylcholine receptors (nAChRs), the ligand gated ion channels at which nicotine acts. These receptors play an important role in nicotine abuse and understanding its neurobiology may be central to developing more rational approaches to treatments for smoking cessation. The interaction of nicotine with high affinity nAChRs in the brain (predominantly the alpha4beta2 subtype) is thought to mediate the rewarding and addictive properties of tobacco smoke. The literature shows that assays of high affinity nAChRs in human postmortem brain tissue revealed that smokers have higher densities than non-smokers or exsmokers in several brain regions. We applied PET imaging to show that this effect can be observed in living smokers. PET imaging of the serotonin and dopamine receptors was used to elucidate effects of perinatal stress and a model of Parkinson’s Disease, respectively in non-human primates.
Summary:Part of this project determined if differences in nAChR density between smokers and non-smokers could be shown in vivo with PET and to determine the neuroanatomical extent of the difference. We used dynamic PET imaging with 2F-18F-A-85380 (2FA) to measure total volume of distribution (VT) in non-smokers and heavy smokers. Values for VT obtained by several modeling methods using a metabolite-corrected arterial input function for 2FA yielded similar results. The thalamus (TH), midbrain (MB), pons (P), cerebellum (CB), frontal cortex (FC), putamen (PUT) and corpus callosum (CC) were sampled. VT was significantly higher in smokers than in nonsmokers in CB, FC, MB, P and PUT. PET imaging of nAChRs suggests that it can be used to study the role of nicotine-induced upregulation of nAChRs in smoking behaviors and in smoking cessation. Quantifying nAChRs in this study required arterial blood sampling and dynamic scanning as the 2FA was administered as a bolus injection. A second part of this project evaluated the less invasive bolus plus constant infusion (B/I) paradigm for quantifying nAChRs. Volunteers underwent a bolus injection study and a study in which the 2FA was administered by B/I to evaluate the feasibility of using shorter scan times and data from venous blood. VT values from the B/I studies were very similar to those calculated from bolus studies. Test/retest showed a high reproducibility of VT measurements. We conclude that B/I methodology will be useful for clinical and research studies of human brain nAChRs. We developed a one-step solid-phase extraction (SPE) method for measuring the concentration of unmetabolized 2FA, which allowed many samples to be processed in a short period of time. SPE effectively separated 2FA from radioactive metabolites typically observed in blood plasma after 2FA administration to humans, yielding nearly identical values to those obtained with HPLC, and showed good reproducibility within and between runs. These results suggest that SPE is the method of choice for the determination of the plasma 2FA concentration when measurement of individual metabolites is not required.
Male squirrel monkeys underwent quantitative studies of nAChRs with 2FA. Non-displaceable volumes of distribution (VDnd) were determined following blockade of 2FA specific binding by nicotine infusion. Binding potential (BP*) values, estimated using Cb and muscle as reference regions, were compared for reproducibility of measurements. Administration of 2 mg/kg/day nicotine via osmotic pump nearly completely saturated specific binding to nAChRs and led to a very small changes in VT in CB and muscle (-9 4% and 0 6%, respectively), suggesting limited specific binding of 2FA in these areas. VT measured in muscle in 15 monkeys was reasonably constant but VDnd in studied brain regions exceeded VT in muscles by a factor of 1.3. Applying this factor and using muscle as a reference region, BP* values calculated for studied brain regions were in a good agreement with those obtained using CB as a reference region, suggesting that nAChRs can be accurately quantified using 2FA and muscle as a reference region.
To expand the capability of microPET to quantify nAChRs in the rat brain, we measured BP* in anesthetized rats that were imaged repeatedly over six months Using a B/I paradigm, 2FA was administered intravenously over 8 to 9 h. Steady state conditions developed within 5 h. A 2-h nicotine infusion initiated 2 h before the end of scanning displaced specifically bound 2FA. BP* averages for TH, forebrain, and CB were consistent with nAChR distribution in rat brain measured in vitro. Studies of nAChR occupancy determined that 0.29 nmol/kg/h nicotine occupied 50% of the nAChRs.
A novel radioligand F-18 NIDA131 for imaging extrathalamic nAChRs was characterized in vitro and in vivo. The Kd and T1/2 of dissociation of NIDA522131 measured in vitro were 4.9 pM and 81 min, respectively. The in vivo patterns of radioactivity distribution for F-18NIDA522131 and 2FA were similar and matched the distribution of nAChRs. F-18NIDA522131 exhibited better in vivo binding properties than 2FA, and accumulated in monkey brain to a substantially greater extent. VT and VDnd were substantially greater than those of 2FA. The toxicity of NIDA522131 in mice was comparable to 2FA and was consistent with a 2300 fold higher affinity for alpha4beta2* nAChRs than for alpha3beta4* nAChRs. These results suggest that F-18 NIDA131 is promising for studying extrathalamic nAChR in humans.
Nicotine may function as a gateway drug to illicit drug use. Nicotine produces cross sensitization to opioids in rats in a conditioned place preference (CPP) paradigm. We utilized CPP to test the hypothesis that nicotine produces behavioral cross sensitization to stimulants and demonstrated that nicotine pretreatment enhances the rewarding effects of amphetamine for at least 3 to 5 days following the cessation of nicotine, with this effect dissipating within 19 days. The underlying mechanism involves alpha4beta2 nAChRs as a competitive alpha4beta2 antagonist effectively blocks development of nicotine-induced cross sensitization. An alpha7 nicotinic antagonist also blocked cross-sensitization at doses that do not block nicotine self-administration in rats. This study clearly demonstrated that nicotine produces cross-sensitization to the rewarding effects of both psychostimulants measured with CPP.
We compared D2 dopamine receptor (D2DR) occupancy by dopamine (DA) in the MPTP-unilaterally lesioned (a model of Parkinson's disease) and contralateral PUT of four pig-tailed macaques. PET and in vitro binding techniques were used to measure BP* and D2DR density (Bmax), respectively. There were relatively higher values of BP* and Bmax and less amphetamine-induced decreases in C-11raclopride binding in the lesioned compared with the contralateral PUT in each animal. The percent differences between the measurements were similar for BP* and Bmax values. As BP* is a measure of unoccupied D2DRs, these findings suggest that endogenous DA occupies a similar fraction of receptors in the lesioned PUT and contralateral PUT. Therefore, the DA occupies a greater number of D2DRs in the lesioned than in the contralateral PUT, possibly because despite a loss in available DA, there is an increase in the ratio of D2DR in the low-affinity to those in the high affinity state on the lesioned side.
Childhood trauma is associated with increased risk for developing depression, anxiety, posttraumatic stress and substance abuse disorders. Rearing infant macaques with same-aged peers in the absence of adults, an established model of early adversity, induces high levels of anxiety-like behavior in monkeys. Chronic early life stress affects expression and function of 5-HT1A receptors (5HTR), which may increase vulnerability to mood and anxiety disorders. In addition, human data shows sex differences in 5HTR. Based on the hypothesis that peer-reared (PR) macaques would exhibit differences in 5HTR density (measured with 18FFPWAY and PET) relative to those raised by their mothers (MR), we evaluated these effects in male MR, female MR, male PR and female PR rhesus monkeys, at 24-29 months (corresponding to 6-7 human years). The distribution of 18FFPWAY was consistent with the known pattern of 5HTR density (highest accumulation in hippocampus and cingulate cortex, intermediate in the prefrontal cortex and the lowest in the CB) and showed a significant rearing X sex interaction. In females, BP values were elevated in the dorso-medial prefrontal cortex in the PR compared to the MR group, but in males, values were lower in medial cingulate cortex in the PR group than in the MR group, suggesting that early stress affects the number of available 5HTR differently in females and males.
Publications generated by this research:
  1. Chefer S, Kimes AS, Matochik J, Horti A, Kurian V, Shumway DA, Domino EF, London ED, Mukhin A (2007) Estimation of D2-like receptor occupancy by dopamine in the putamen of hemiparkinsonian monkeys. Neuropsychopharmacology, in press.
  2. Kimes AS, Chefer S, Matochik J, Contoreggi C, Vaupel DB, Stein EA, Mukhin A (2007) Quantification of nicotinic acetylcholine receptors in the human brain with PET: Bolus plus infusion administration of 2-18FF-A85380. Neuroimage, in press.
  3. LeFoll B, Chefer SI, Kimes AS, Shumway D, Goldberg SR, Stein EA, Mukhim AG (2007) Validation of an extra-cerebral reference region approach for quantification of brain nicotinic acetylcholine receptors in squirrel monkeys with PET and 2-[18F]FA. J Nucl Med 48:1492-1500. PubMed
  4. Shumway DA, Pavlova O, Mukhin A (2007) A simplified method for the measurement of nonmetabolized 2-18FF-A-85380 in blood plasma using solid-phase extraction. Nucl Med Biol 34:221-228.
  5. Vaupel DB, Stein EA, Mukhin AG (2006) Quantification of alpha4beta2 nicotinic receptors in the rat brain with microPET(R) and 2-(18)FF-A-85380. Neuroimage, in press (e-pub ahead of print). PubMed

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