Stimulant-induced dopamine increases are markedly blunted in active cocaine abusers.

Dopamine signaling in nucleus accumbens is essential for cocaine reward. Interestingly, imaging studies have reported blunted dopamine increases in striatum (assessed as reduced binding of [(11)C]raclopride to D2/D3 receptors) in detoxified cocaine abusers. Here, we evaluate whether the blunted dopamine response reflected the effects of detoxification and the lack of cocaine-cues during stimulant exposure. For this purpose we studied 62 participants (43 non-detoxified cocaine abusers and 19 controls) using positron emission tomography and [(11)C]raclopride (radioligand sensitive to endogenous dopamine) to measure dopamine increases induced by intravenous methylphenidate and in 24 of the cocaine abusers, we also compared dopamine increases when methylphenidate was administered concomitantly with a cocaine cue-video versus a neutral-video. In controls, methylphenidate increased dopamine in dorsal (effect size 1.4; P<0.001) and ventral striatum (location of accumbens) (effect size 0.89; P<0.001), but in cocaine abusers methylphenidate's effects did not differ from placebo and were similar whether cocaine-cues were present or not. In cocaine abusers despite the markedly attenuated dopaminergic effects, the methylphenidate-induced changes in ventral striatum were associated with intense drug craving. Our findings are consistent with markedly reduced signaling through D2 receptors during intoxication in active cocaine abusers regardless of cues exposure, which might contribute to compulsive drug use.

A strategy for removing the bias in the graphical analysis method.

The graphical analysis method, which transforms multiple time measurements of plasma and tissue uptake data into a linear plot, is a useful tool for rapidly obtaining information about the binding of radioligands used in PET studies. The strength of the method is that it does not require a particular model structure. However, a bias is introduced in the case of noisy data resulting in the underestimation of the distribution volume (DV), the slope obtained from the graphical method. To remove the bias, a modification of the method developed by Feng et al. (1993), the generalized linear least squares (GLLS) method, which provides unbiased estimates for compartment models was used. The one compartment GLLS method has a relatively simple form, which was used to estimate the DV directly and as a smoothing technique for more general classes of model structures. In the latter case, the GLLS method was applied to the data in two parts, that is, one set of parameters was determined for times 0 to T1 and a second set from T1 to the end time. The curve generated from these two sets of parameters then was used as input to the graphical method. This has been tested using simulations of data similar to that of the PET ligand [11C]-d-threo-methylphenidate (MP, DV = 35 mL/mL) and 11C raclopride (RAC, DV = 1.92 mL/mL) and compared with two examples from image data with the same tracers. The noise model was based on counting statistics through the half-life of the isotope and the scanning time. Five hundred data sets at each noise level were analyzed. Results (DV) for the graphical analysis (DV(G)), the nonlinear least squares (NLS) method (DV(NLS)), the one-tissue compartment GLLS method (DV(F)), and the two part GLLS followed by graphical analysis (DV(FG)) were compared. DV(FG) was found to increase somewhat with increasing noise and in some data sets at high noise levels no estimate could be obtained. However, at intermediate levels it provided a good estimation of the true DV. This method was extended to use a reference tissue in place of the input function to generate the distribution volume ratio (DVR) to the reference region. A linearized form of the simplified reference tissue method of Lammertsma and Hume (1996) was used. The DVR generated directly from the model (DVR(FL)) was compared with DVR(FG) (determined from a "smoothed" uptake curve as for DV(FG)) using the graphical method.

Distribution volume ratios without blood sampling from graphical analysis of PET data.

The distribution volume ratio (DVR), which is a linear function of receptor availability, is widely used as a model parameter in imaging studies. The DVR corresponds to the ratio of the DV of a receptor-containing region to a nonreceptor region and generally requires the measurement of an arterial input function. Here we propose a graphical method for determining the DVR that does not require blood sampling. This method uses data from a nonreceptor region with an average tissue-to-plasma efflux constant k2 to approximate the plasma integral. Data from positron emission tomography studies with [11C]raclopride (n = 20) and [11C]d-threo-methylphenidate ([11C]dMP) (n = 8) in which plasma data were taken and used to compare results from two graphical methods, one that uses plasma data and one that does not. k2 was 0.163 and 0.051 min-1 for [11C]raclopride and [11C]dMP, respectively. Results from both methods were very similar, and the average percentage difference between the methods was -0.11% for [11C]raclopride and 0.46% for [11C]dMP for DVR of basal ganglia (BG) to cerebellum (CB). Good agreement between the two methods was also achieved for DVR images created by both methods. This technique provides an alternative method of analysis not requiring blood sampling that gives equivalent results for the two ligands studied. It requires initial studies with blood sampling to determine the average kinetic constant and to test applicability. In some cases, it may be possible to neglect the k2 term if the BG/CB ratio becomes reasonably constant for a sufficiently long period of time over the course of the experiment.

Gamma vinyl-GABA differentially modulates NMDA antagonist-induced increases in mesocortical versus mesolimbic DA transmission.

To explore the role of endogenous GABA in NMDA antagonist induced dopamine (DA) release, we used in vivo microdialysis to study the effects of pretreatment with gamma-vinyl GABA (GVG) on phencyclidine (PCP)-induced DA release in terminal regions of midbrain DA neurons. GVG, an irreversible inhibitor of the GABA catabolizing enzyme GABA-AT, significantly reduced the DA response to PCP (7.0 mg/kg) in freely moving animals. Preferential increases in PCP-induced DA release in the PFC (four-fold those of NAcc) were dose-dependently inhibited by acute pretreatment with GVG at doses of 150 (51% inhibition), 300 (68% inhibition), and 500 (82% inhibition) mg/kg, whereas NAcc PCP-induced DA activity was unresponsive to 150 mg/kg and only partially inhibited by 300 and 500 mg/kg. Subchronic treatment with GVG did not enhance the inhibitory capacity of the GABAergic system. While GVG evidently modulates PCP-induced increases in mesocorticolimbic DA transmission, the character of this modulation is regionally specific, with cortical NMDA-antagonist induced increases appearing more sensitive to inhibition by endogenous GABA than subcortical areas.

Selective reduction of radiotracer trapping by deuterium substitution: comparison of carbon-11-L-deprenyl and carbon-11-deprenyl-D2 for MAO B mapping.

UNLABELLED: Recent human PET studies with the monoamine oxidase B (MAO B) tracer [11C]L-deprenyl show that the rapid rate of radiotracer trapping relative to transport reduces the sensitivity of the tracer in regions of high MAO B concentration. This study investigates the use of deuterium substituted L-deprenyl ([11C]L-deprenyl-D2) to reduce the rate of trapping in tissue and to improve sensitivity. METHODS: Five normal subjects (43-64 yr) were studied with [11C]L-deprenyl and [11C]L-deprenyl-D2 on the same day. Time-activity data from different brain regions and the arterial plasma were analyzed using a three-compartment model as well as graphical analysis for irreversible systems. RESULTS: For both tracers, maximum radioactivity accumulation occurred at about 5 min. For [11C]L-deprenyl, 11C concentration peaked at 5 min and remained constant throughout the study. With [11C]L-deprenyl-D2, peak 11C concentration also occurred at about 5 min but was followed by an initial washout. Carbon-11 concentration generally plateaued from 30 to 60 min. The plateau for [11C]L-deprenyl was higher than the plateau for [11C]L-deprenyl-D2. Data analysis by a three-compartment model and by graphical analysis showed that deuterium substitution: (a) does not affect plasma to tissue transport (K1); (b) reduces the rate of trapping of 11C in all brain regions; (c) facilitates the separation of model terms related to radiotracer delivery from radiotracer trapping in tissue; and (d) improves tracer sensitivity. CONCLUSION: This study demonstrates that deuterium substitution causes a significant reduction in the rate of trapping of labeled deprenyl, providing a direct link between radiotracer uptake and MAO B in the human brain and enhancing tracer sensitivity to changes in MAO B concentration.

Plasma input function determination for PET using a commercial laboratory robot.

A commercial laboratory robot system (Zymate PyTechnology II Laboratory Automation System) was interfaced to standard and custom laboratory equipment and programmed to perform rapid radiochemical assays necessary for plasma input function determination in quantitative PET studies in humans and baboons. A Zymark XP robot arm was used to carry out two assays: (1) the determination of total plasma radioactivity concentrations in a series of small-volume whole blood samples and (2) the determination of unchanged (parent) radiotracer in plasma using only solid phase extraction methods. Steady state robotic throughput for determination of total plasma radioactivity in whole blood samples (0.350 mL) is 14.3 samples/h, which includes automated centrifugation, pipetting, weighing and radioactivity counting. Robotic throughput for the assay of parent radiotracer in plasma is 4-6 samples/h depending on the radiotracer. Percents of total radioactivities present as parent radiotracers at 60 min, postinjection of 25 +/- 5.0 (N = 25), 26 +/- 6.8 (N = 68), 13 +/- 4.4 (N = 30), 32 +/- 7.2 (N = 18), 16 +/- 4.9 (N = 20), were obtained for carbon-11 labeled benztropine, raclopride, methylphenidate, SR 46349B (trans, 4-[(3Z)3-(2-dimethylamino-ethyl) oxyimino-3 (2-fluorophenyl)propen-1-yl]phenol), and cocaine respectively in baboon plasma and 84 +/- 6.4 (N = 9), 18 +/- 11 (N = 10), 74 +/- 5.7 (N = 118) and 16 +/- 3.7 (N = 18) for carbon-11 labeled benztropine, deprenyl, raclopride, and methylphenidate respectively in human plasma. The automated system has been used for more than 4 years for all plasma analyses for 7 different C-11 labeled compounds used routinely in our laboratory. The robotic radiotracer assay runs unattended and includes automated cleanup procedures that eliminates all human contact with plasma-contaminated containers.

Remote processing, delivery and injection of H2[15O] produced from a N2/H2 gas target using a simple and compact apparatus.

We report here a simple apparatus for remote trapping and processing of H2[15O] produced from the N2/H2 target. The system performs a three step operation for H2[15O] delivery at the PET imaging facility which includes the following: (i) collecting the radiotracer in sterile water; (ii) adjusting preparation pH through removal of radiolytically produced ammonia, while at the same time adjusting solution isotonicity; and (iii) delivery of the radiotracer preparation to the injection syringe in a sterile and pyrogen-free form suitable for human studies. The processing apparatus is simple, can be remotely operated and fits inside a Capintec Dose Monitoring Chamber for direct measurement of accumulated radioactivity. Using this system, 300 mCi of H2[15O] (15 microA of 8 MeV D+ on target) is transferred from target through 120 m x 3.18 mm o.d. Impolene tubing to yield 100 mCi of H2[15O] which is isotonic, neutral and suitable for human studies. A remote hydraulically driven system for i.v. injection of the H2[15O] is also described. The device allows for direct measurement of syringe dose while filling, and for easy, as well as safe transport of the injection syringe assembly to the patient's bedside via a shielded delivery cart. This cart houses a hydraulic piston that allows the physician to "manually" inject the radiotracer without directly handling the syringe.

Therapeutic doses of amphetamine or methylphenidate differentially increase synaptic and extracellular dopamine.

Methylphenidate (MP) and amphetamine (AMP) are first-line treatments for attention-deficit hyperactivity disorder. Although both drugs have similar therapeutic potencies, the stimulatory effect of AMP on extracellular dopamine (ECF DA) is greater than that of MP. We compared extracellular effects directly against synaptic changes. ECF DA was assessed by microdialysis in freely moving rodents and synaptic dopamine (DA) was measured using PET and [11C]-raclopride displacement in rodents and baboons. Microdialysis data demonstrated that MP (5.0 mg/kg, i.p.) increased ECF DA 360% +/- 31% in striatum, which was significantly less than that by AMP (2.5 mg/kg, i.p.; 1398% +/- 272%). This fourfold difference was not reflected by changes in synaptic DA. In fact, rodent PET studies showed no difference in striatal [11C]-raclopride binding induced by AMP (2.5 mg/kg, i.p.; 25% +/- 4% reduction) compared with that by MP (5.0 mg/kg, i.p.; 21% +/- 4% reduction). Primate PET experiments also showed no differences between AMP (0.5 mg/kg, i.v.; 24% +/- 4% reduction) and MP (1.0 mg/kg, i.v.; 25% +/- 7% reduction) induced changes in [11C]-raclopride binding potential. The similar potencies of MP and AMP to alter synaptic DA, despite their different potencies in raising ECF DA, could reflect their different molecular mechanisms.

Removal of the 2.2.2 cryptand (Kryptofix 2.2.2) from 18FDG by cation exchange.

The 2.2.2 cryptand [4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo-(8.8.8)-hexacosane] was trapped efficiently on Dowex AG50W-X8 (100-200 mesh) cation exchange resin. The concentration of the 2.2.2 cryptand in water with 0.1-2% methanol, or in 1 N HCl, was decreased by a factor of > 4000 using 1 mL (1.7 mequiv.) of AG50W-X8 and a flow rate of approx. 2 mL/min. K+, Cs+, Ag+ and Ba2+ forms were about equal in their ability to remove the 2.2.2 cryptand. A disposable cartridge containing 1 mL of hydrogen form resin was inserted into our automated 18FDG system so that the hydrolysate (in 2 cm3 of 1 N HCl) would pass through the cartridge before final purification. No cryptand was detected in the final product as determined by TLC with idoplantinate visualization. The detection limit was 2.5 micrograms/mL. Less than 3% of the total starting radioactivity was retained by the cation column. Quality assurance tests including apyrogenicity, sterility, radiochemical purity, carbohydrate composition and pH were not compromised by the incorporation of the cryptand removal cartridge.

A universal water target loading system with direct in-target liquid level sensing.

A reliable remote water target loading system has been designed and fabricated around the operation of a single pneumatic syringe dispenser that accesses one of two water reservoirs, filling one or more targets depending on the system configuration. For multiple radionuclide configuration, reservoirs are filled with 18O-enriched water for [18F]fluoride production, and natural abundance water for [13N]nitrate and [13N]nitrite production. When actuated, the system withdraws a calibrated volume of water from a selected reservoir, and automatically dispenses that charge through a short transfer line to the appropriate target. A second actuation then forces the dispenser to purge itself of residual water by dispensing a charge of helium gas through the same line thus completing the transfer of water to the target volume. Direct liquid level sensing inside the target, utilizing the specific resistance response of water, signals the operator that the target is loaded. For single radionuclide configuration, one or both reservoirs can be used with the same type of water, while dispensing is directed to a single target.

Modular automation in PET tracer manufacturing: application of an autosynthesizer to the production of 2-deoxy-2-[18F]fluoro-D-glucose.

A compact autosynthesizer was developed and used successfully for the production of 2-deoxy-2-[18F]fluoro-D-glucose [18FDG] from gaseous acetyl hypo[18F]fluorite. The autosynthesizer performs a sequence of general purpose synthesis procedures named Synthesis Unit Operations (SUO's). Each SUO is controlled through execution of a digital control algorithm with a BASIC language subroutine. This automatic synthesis system is based on two industry standard microcomputer architectures, the IBM PC and STD Bus, and it becomes a component of an evolving distributed microprocessor network of task-dedicated subsystems suitable for automated manufacturing of several useful radiotracers. The yield of 18FDG product using the autosynthesizer and remote manually controlled purification procedures is approximately 20% EOB. Radiochemical purity of this product as measured by thin layer chromatography was 96-99%. Chemical purity of the product was measured to be approximately 96%. 2-Deoxy-2-fluoro-D-mannose impurity from this method was determined to be approximately 4%.

Ion chromatographic analysis of high specific activity 18FDG preparations and detection of the chemical impurity 2-deoxy-2-chloro-D-glucose.

Because of the widespread use of 2-deoxy-2-[18F]fluoro-D-glucose (FDG) prepared by the "Julich" method or its variants it was decided necessary to determine the major chemical impurities present in the final product. An analytical system for quantifying FDG was developed using pulsed amperometry after separation by high-performance anion exchange chromatography. With this system a heretofore unidentified impurity, 2-deoxy-2-chloro-D-glucose (C1DG, ca 20-2000 micrograms; typically < 100 micrograms), was found in our preparation and in those from other laboratories using the "Julich" method. C1DG arises from Cl- ion displacement during the labeling procedure where Cl- ion comes from several sources, and Cl- ion displacement from the HCl used in the hydrolysis step. FDG mass was present in the same preparations at a level of ca 1-40 micrograms. Other major chemical constituents were glucose (ca 1-6 mg) and mannose (ca 10-18 micrograms). Glycerol, arising from sterilizing filters, was also detected in most preparations. Although C1DG is a chemical impurity which has not been detected previously in nca FDG preparations, its biochemical and pharmacological properties are similar to FDG and 2-deoxy-D-glucose. Thus it is unlikely that the presence of small quantities of C1DG found in typical FDG preparations (ca 100 micrograms) would have adverse pharmacological or toxicological consequences that would limit continued application of this radiopharmaceutical in basic and clinical studies.

Positron emission tomography (PET) studies of dopaminergic/cholinergic interactions in the baboon brain.

Interactions between the dopaminergic D2 receptor system and the muscarinic cholinergic system in the corpus striatum of adult female baboons (Papio anubis) were examined using positron emission tomography (PET) combined with [18F]N-methylspiroperidol [( 18F]NMSP) (to probe D2 receptor availability) and [N-11C-methyl]benztropine (to probe muscarinic cholinergic receptor availability). Pretreatment with benztropine, a long-lasting anticholinergic drug, bilaterally reduced the incorporation of radioactivity in the corpus striatum but did not alter that observed in the cerebellum or the rate of metabolism of [18F]NMSP in plasma. Pretreatment with unlabelled NMSP, a potent dopaminergic antagonist, reduced the incorporation of [N-11C-methyl]benztropine in all brain regions, with the greatest effect being in the corpus striatum greater than cortex greater than thalamus greater than cerebellum, but did not alter the rate of metabolism of the labelled benztropine in the plasma. These reductions in the incorporation of either [18F]NMSP or [N-11C-methyl]benztropine exceeded the normal variation in tracer incorporation in repeated studies in the same animal. This study demonstrates that PET can be used as a tool for investigating interactions between neurochemically different yet functionally linked neurotransmitters systems in vivo and provides insight into the consequences of multiple pharmacologic administration.

GABAergic attenuation of cocaine-induced dopamine release and locomotor activity.

GABA modulates dopamine concentrations in the nucleus accumbens and corpus striatum. Using in vivo microdialysis techniques we examined this modulatory role and the extent to which three different GABAergic drugs can attenuate cocaine's ability to increase extracellular dopamine concentrations and gross locomotor activity. Ethanol, lorazepam (Ativan), and gamma-vinyl GABA (GVG) significantly and dose-dependently attenuated cocaine-induced dopamine release in the corpus striatum of freely moving animals. Unlike ethanol or lorazepam, however, GVG is not a sedative hypnotic in the doses used, and hence the strategy of selectively increasing GABAergic activity by suicide inhibition of the catabolic enzyme, GABA-transaminase, offers the unique advantage of attenuating cocaine-induced dopamine release without the apparent side effects typically associated with sedative hypnotics.