Study of brain uptake and biodistribution of [11C]toluene in non-human primates and mice.

Inhalant abuse is a rapidly growing health problem particularly among adolescents. Yet we know little about the neural mechanisms underlying the abuse liability of inhalants, particularly when compared to other addictive drugs. Specifically, our understanding of the relationship between the regional brain phamacokinetics and features classically associated with drug reinforcement is lacking. Under the hypothesis that the abuse liability of toluene can be related to its pharmacokinetic properties and the pattern of regional brain uptake, we developed the methodology for radiolabeling and purifying [11C]toluene for use in PET studies. Here we report the regional brain distribution and kinetics of the widely abused solvent toluene in non-human primates and the whole body biodistribution in mice. To our knowledge, this is the first reported study of the in vivo brain pharmacokinetics of labeled toluene in non-human primates. Rapid uptake of radioactivity into striatal and frontal regions was followed by rapid clearance from the brain. Concurrent findings in rodents indicate similar radio-tracer kinetics, with excretion through kidneys and liver. Taken together, our data provides insight into pharmacokinetic features possibly associated with the abuse liability of toluene.

Effects of endogenous neurotransmitters on the in vivo binding of dopamine and 5-HT radiotracers in mice.

Several studies have indicated that the in vivo binding of D(2) receptor positron emission tomography radiotracers can, under some conditions, be influenced by competition with endogenous dopamine. The present study was undertaken to compare the extent to which the in vivo binding in mice of radiotracers to other amine neuroreceptors, namely D(1), 5-HT(2A) and 5-HT(1A) receptors, can also be modulated by neurotransmitter competition. For dopamine radiotracers we examined [3H]raclopride as a D(2) radiotracer and [3H]A69024 as a D(1) radiotracer. Striatal binding of both radiotracers was substantially reduced by administration of the dopamine releaser, amphetamine, although only at a high dose. [3H]raclopride was decreased more than [3H]A69024. Dopamine depletion with 4-hydroxybutyrate strongly increased [3H]raclopride binding but failed to increase [3H]A69024 binding. For 5-HT radiotracers we examined [3H]N-methylspiperone as a 5-HT(2A) radiotracer and [3H]WAY 100635 as a 5-HT(1A) radiotracer. Cortical binding of both radiotracers was unaffected by the 5-HT releaser, p-chloroamphetamine. [3H]WAY 100635 binding was additionally unaffected by 5-HT release with fenfluramine and by 5-HT depletion with p-chlorophenylalanine. In conclusion, of the four radiotracers examined, [3H]raclopride binding to D(2) receptors had greatest sensitivity to changes in endogenous neurotransmitter levels. [3H]A69024 binding to D(1) receptors was affected only by neurotransmitter increases. [3H]N-methylspiperone binding to 5-HT(2A) receptors and [3H]WAY 100635 binding to 5-HT(1A) receptors appeared insensitive to changes in neurotransmitter levels.

Effect of amphetamine-induced dopamine release on radiotracer binding to D1 and D2 receptors in rat brain striatal slices.

The in vivo binding of positron emission tomography (PET) and single photon emission computer tomography (SPECT) radiotracers to dopamine D2 receptors in the striatum can be influenced by competition with endogenous dopamine. The present study was undertaken to determine if a similar inhibition of radiotracer binding to dopamine receptors could be observed following pharmacologically-evoked dopamine release in rat brain striatal slices. Striatal slices were incubated in a large volume of oxygenated Krebs saline and exposed to amphetamine or methamphetamine to evoke dopamine release within the slice. Amphetamine and methamphetamine, at concentrations up to 30 microM, reduced [3H]raclopride binding in the slices by 77% and 86%, respectively, with 50% inhibition at 1.6 microM amphetamine or 3.0 microM methamphetamine. Neither drug produced a significant effect on binding of [3H]SCH 23390 in the slices. This suggests that dopamine was able to interfere with radiotracer binding to D2 but not D1 receptors. The dopamine uptake blockers, cocaine and methylphenidate, had relatively little effect by themselves on [3H]raclopride binding but, by inhibiting amphetamine-induced dopamine release, significantly reduced inhibition of [3H]raclopride binding by a low (3 microM) amphetamine concentration. At a higher (30 microM) amphetamine concentration the inhibition of [3H]raclopride binding was not antagonized by uptake blockers and data obtained from homogenate binding experiments indicated a direct displacement of [3H]raclopride binding by amphetamine at this concentration. In conclusion the data obtained in the present study demonstrate that the effects of amphetamine on striatal radiotracer accumulation observed in PET and SPECT can also be observed in brain slices in vitro and, at least at low amphetamine concentrations, are mediated by competition with released dopamine.

Cannabinoid receptor-mediated inhibition of acetylcholine release from hippocampal and cortical synaptosomes.

In previous studies cannabinoid agonists have been found to inhibit and cannabinoid antagonists to enhance electrically-evoked [(3)H]-acetycholine (ACh) release in hippocampal slices. The present study was undertaken to determine if similar cannabinoid effects could be observed in synaptosomes. [(3)H]-ACh release was evoked by two methods, both sensitive to presynaptic receptor effects. The first involved the addition of 1.3 mM calcium following perfusion with calcium-free Krebs and the second the addition of 11 mM potassium following perfusion with normal Krebs. In hippocampal synaptosomes the 1.3 mM calcium-evoked release and the high potassium-evoked [(3)H]-ACh release were inhibited by the cannabinoid agonist, WIN 55212-2, by 59 and 39%, respectively, and with an EC(50) of approximately 1 nM. WIN 55212-2 produced a similar, although less potent, inhibition of [(3)H]-ACh release in cortical synaptosomes. No inhibitory effect of WIN 55212-2 on [(3)H]-ACh release in striatal synaptosomes was observed, supporting previous data collected in this area with brain slices. The cannabinoid antagonist, SR 141716A, produced a robust enhancement of 1.3 mM calcium-evoked [(3)H]-ACh release in hippocampal synaptosomes (EC(50)<0.3 nM) but had no effect on potassium-evoked release or on [(3)H]-ACh release in the cortex or striatum. In conclusion our data demonstrates the inhibitory effects of WIN 55212-2 observed on ACh release in brain slices can be observed in hippocampal and cortex synaptosomes, suggesting a direct action of these compounds on the synaptic terminals. The SR 141716A-induced enhancement of ACh release can similarly be observed in hippocampal synaptosomes and is probably due to an inverse agonist action at constitutively active receptors.

Sensitivity of binding of high-affinity dopamine receptor radioligands to increased synaptic dopamine.

PET and SPECT studies have documented that D2 radioligands of moderate affinity, but not radioligands of high affinity, are sensitive to pharmacological challenges that alter synaptic dopamine levels. The objective of this work was to determine whether the brain kinetics of high-affinity radioligands for dopamine D1 ([(3)H]SCH 23390) and D2 ([(123)I]epidepride) receptors were altered by a prolonged elevation of synaptic dopamine induced by the potent cocaine analog RTI-55. Mice were injected intravenously with radioligands either 30 min after or 4 h before intraperitoneal administration of RTI-55 (2 mg/kg). In separate experiments, the pharmacological effects of RTI-55 were assessed biochemically by measuring uptake of dopamine in synaptosomes prepared from RTI-treated mice and behaviorally by monitoring locomotor activity. Consistent with the expected elevation of synaptic dopamine, RTI-55 induced a long-lasting decrement in dopamine uptake measured ex vivo, and a prolonged increase in locomotor activity. RTI-55 injected prior to the radioligands induced a significant (P < 0.05) increase in striatal concentration of [(123)I]epidepride at 15 min, relative to saline-treated controls, but there were no differences between the two groups at later time-points. For [(3)H]SCH 23390, both initial striatal uptake and subsequent clearance were slightly increased by preadministration of RTI-55. Administration of RTI-55 4 h after the radioligands (i.e., when it was presumed that a state of near equilibrium binding of the radioligands had been reached), was associated with a significant reduction of striatal radioactivity for both radiotracers. Our results are consistent with increased competition between dopamine and radioligand for binding to both D1 and D2 receptors after treatment with RTI-55. We suggest that the magnitude of the competition is reduced by failure of the receptor binding of high-affinity radioligands to rapidly attain equilibrium.

Locomotor activity and occupancy of brain cannabinoid CB1 receptors by the antagonist/inverse agonist AM281.

The goals of this study were to examine the relationship between intravenous doses of the cannabinoid CB1 receptor antagonist AM281 (N-(morpholin-4-yl)-5-(4-iodophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) and the degree of occupancy of this receptor, and to relate occupancy to the ability of this compound to antagonize the sedative effects of the cannabinoid receptor agonist WIN 55,212-2. Occupancy was determined by measuring the ability of intravenous doses of AM281 to inhibit in vivo binding of [(131)I]AM281 in brain areas, and locomotor activity was assessed by measuring the rate of beam crossings in a photocell apparatus. As previously documented, WIN 55,212-2 (1 mg/kg, i.v.) significantly reduced locomotor activity at early times after administration. Co-injection of AM281 (0.3 mg/kg i/v) and WIN 55, 212-2 restored the rate of beam crossings to that seen on injection of vehicle. In addition, AM281 (0.3 mg/kg i/v) approximately doubled locomotor activity between 60-120 min when injected alone. The IC(50) value for displacement of [(131)I]AM281 by AM281 was 0.45 mg/kg. These observations confirm earlier indications that AM281 is a CB1 receptor antagonist or inverse agonist and suggest the existence of an endogenous cannabinoid tone that moderates exploratory locomotor activity.

Potentiation of the action of anandamide on hippocampal slices by the fatty acid amide hydrolase inhibitor, palmitylsulphonyl fluoride (AM 374).

The electrically-evoked release of [3H]acetylcholine from hippocampal brain slices is inhibited by cannabinoid receptor agonists. The effect of palmitylsulphonyl fluoride (AM 374), a recently developed inhibitor of fatty acid amide hydrolase, in influencing the potency of exogenously added anandamide in this preparation was examined. Anandamide alone had relatively little effect on [3H]acetylcholine release. By contrast, in the presence of AM 374 (0.1 microM), anandamide produced a significant inhibition of [3H]acetylcholine release at all concentrations tested (0.1-10 microM). In addition to experiments with AM 374 the effects of N-(4-hydroxyphenyl)arachidonamide (AM 404), a putative anandamide uptake inhibitor, was also examined. However, AM 404 at concentrations up to 10 microM, was not found to significantly enhance the effect of anandamide on electrically-evoked [3H]acetylcholine release. These results indicate that AM 374 potently inhibits endogenous amidase activity and thus facilitates access of exogenous anandamide to cannabinoid receptors in the hippocampal tissue.

Dopamine-transporter occupancy after intravenous doses of cocaine and methylphenidate in mice and humans.

OBJECTIVES: Recent studies using positron emission tomography (PET) have established the relationship between an intravenous dose of cocaine and the percentage occupancy of the dopamine transporter in humans, and have documented the requirement of more than 50% occupancy for perception of the "high". The present experiments were conducted to examine dose-occupancy and dose-effect relationships in mice for cocaine and also for methylphenidate, a dopamine uptake blocker used in pediatric psychiatry. METHODS: Percentage occupancies of the dopamine transporter by cocaine and methylphenidate were estimated after intravenous injection in mice from the displacement of in vivo binding of [(3)H]cocaine from the striatum. Locomotor activity was measured in a photocell apparatus. RESULTS: The relationship between drug doses (milligrams of hydrochloride salt per kilogram body weight) and percentage occupancy of the dopamine transporter was indistinguishable for cocaine and methylphenidate, and corresponded to about 50% occupancy at 0.25 mg/kg and about 80% at 1 mg/kg. This was similar to the relationship between drug dose and transporter occupancy, previously measured in human and baboons using [(11)C]cocaine or [(11)C]d-threo-methylphenidate and PET. Methylphenidate increased locomotor activity in the mice substantially more than cocaine at the same dose and the same degree of dopamine-transporter receptor occupancy. CONCLUSIONS: The range of dopamine-transporter occupancy required for behavioral activation in the mice was thus similar to that previously reported for experience of a cocaine- or methylphenidate-induced "high" in human subjects. Our results are consistent with other studies in which both cocaine and methylphenidate were evaluated in animal behavioral assays and were found to have very similar psychopharmacological properties.

Large receptor reserve for cannabinoid actions in the central nervous system.

The receptor occupancy required to produce cannabinoid effects in the central nervous system was determined in both a neurochemical and a behavioral assay for cannabinoid actions. In the neurochemical experiments, performed on superfused rat hippocampal slices, electrically evoked [3H]acetylcholine release was inhibited by the cannabinoid agonist, WIN 55212 to 2 with an EC50 of 0.005 microM and maximum effect of 79%. In parallel experiments examining binding of the radiolabeled CB1 antagonist [131I]AM 281 (N-(morpholin-4-yl)-5-(4-[131I]iodophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) to living hippocampal slices, WIN 55212 to 2 inhibited [131I]AM 281 binding with an EC50 of 1.3 microM. From these two sets of data it was determined that 50% of maximal inhibition of [3H]acetylcholine release in hippocampal slices occurs at a receptor occupancy of only 0.13% and 95% of maximal inhibition at a receptor occupancy of 7.5%, suggesting the presence of a receptor reserve that is large compared with other G protein-coupled receptor systems in the central nervous system. In behavioral experiments, WIN 55212 to 2 inhibited spontaneous locomotor activity in mice with an ED50 of 0.3 mg/kg, i. v. In in vivo binding experiments using [131I]AM 281, WIN 55212 to 2 failed to produce significant inhibition of radiotracer binding in the mouse brains, except at very high doses (10 mg/kg or greater, i. v.). By contrast, the CB1 antagonist SR 141716A (10 mg/kg, i.p.), completely abolished specific [131I]AM 281 binding. These experiments suggest that behavioral effects of cannabinoids, like neurochemical effects, are produced at very low receptor occupancy.

Blockade of striatal dopamine transporters by intravenous methylphenidate is not sufficient to induce self-reports of "high".

The reinforcing effects of cocaine and methylphenidate have been linked to their ability to block dopamine transporters (DAT). Using positron emission tomography (PET), we previously showed that intravenous cocaine induced a significant level of DAT blockade, which was associated with the intensity for self-reports of "high" in cocaine abusers. In this study, we measured DAT occupancies after intravenous methylphenidate and assessed whether they also were associated with the "high". Occupation of DAT by intravenous MP was measured with PET using [11C]cocaine, as a DAT ligand, in eight normal control subjects tested with different methylphenidate doses. The ratio of the distribution volume of [11C]cocaine in striatum to that in cerebellum, which corresponds to Bmax/Kd + 1, was used as measure of DAT availability. In parallel, self-reports of "high" were measured. Methylphenidate produced a dose-dependent blockade of DAT with an estimated ED50 of 0.075 mg/kg. DAT occupancies were significantly correlated with the "high" (p <.03). However, four of the eight subjects, despite having significant levels of DAT blockade, did not perceive the "high". Methylphenidate is as effective as cocaine in blocking DAT in the human brain (cocaine ED50 = 0.13 mg/kg), and DAT blockade, as for cocaine, was also associated with the "high". However, the fact that there were subjects who despite significant DAT blockade did not experience the "high" suggests that DAT blockade, although necessary, is not sufficient to produce the "high".

In vitro and ex vivo autoradiographic studies of nicotinic acetylcholine receptors using [18F]fluoronochloroepibatidine in rodent and human brain.

A fluorine-18-labeled analog of the potent nicotinic agonist epibatidine is a candidate radioligand for positron emission tomographic (PET) studies of nicotinic acetylcholine receptors (nAcChR). Following intravenous administration of [18F]exo-2-(2'-fluoro-5'-pyridinyl)-7-azabicyclo[2.2.1]heptane (NFEP), high uptake in thalamus was visualized in sections of mouse and rat brain by autoradiography using a phosphor imaging device. Binding of [18F]NFEP to rat thalamic homogenate was consistent with a single class of binding site with a Kd value of 71 pM. In vitro autoradiography of thaw-mounted sections of human thalamus revealed a heterogeneous pattern of binding; Bmax values for ventrolateral nucleus, insular cortex and dorsomedial nucleus, and internal capsule were 20, 8, and 3 pmol/cc of tissue, respectively. However, similar Kd values close to 50 pM were calculated for all regions. These studies support the suitability of [18F]NFEP as a radioligand for PET studies of nAcChR in the living human brain.

Evaluation of the importance of rebinding to receptors in slowing the approach to equilibrium of high-affinity PET and SPECT radiotracers.

The importance of rebinding to receptors in influencing the kinetics of in vivo binding of PET and SPECT radiotracers was evaluated by examining the binding of a high-affinity D1 receptor radiotracer, [3H]SCH 23390, in tissue homogenates, living brain slices, and in vivo. In rat striatal homogenates, [3H]SCH 23390 binding reached equilibrium with a half-time of 6 min. By contrast, in striatal brain slices incubated in [3H] SCH 23390, the radioactivity levels in the slice increased in a linear fashion over the 4-h incubation, with no indication of an approach to equilibrium at the termination of the experiment. In in vivo experiments, [3H]SCH 23390 was given as a slow intravenous infusion to mice, using a paradigm that kept the plasma concentration at a constant level. Under these conditions, striatal [3H] SCH 23390 levels increased in a linear fashion over the 4-h infusion period, similar to what was observed in the brain slices, and as in the slices there was no indication of approach to a steady state. However, when given instead as a single-bolus intravenous dose, the striatal [3H]SCH 23390 levels reached a peak only 15 min after injection. Calculations based on the slice experiments, in which the blood-brain barrier is absent, suggested that the rate-limiting step accounting for the failure of [3H]SCH 23390 levels to reach equilibrium was its hindered diffusion as a result of repeated rebinding to receptors. This phenomenon may also be important in vivo and should be considered as a factor in determining the time-course of binding of radiotracers in PET and SPECT experiments where either the receptor density or radiotracer affinity is high.

Imaging the brain marijuana receptor: development of a radioligand that binds to cannabinoid CB1 receptors in vivo.

The major active ingredient of marijuana, (-)-delta9-tetrahydrocannabinol, exerts its psychoactive effects via binding to cannabinoid CB1 receptors, which are widely distributed in the brain. Radionuclide imaging of CB1 receptors in living human subjects would help explore the presently unknown physiological roles of this receptor system, as well as the neurochemical consequences of marijuana dependence. Currently available cannabinoid receptor radioligands are exceedingly lipophilic and unsuitable for in vivo use. We report the development of a novel radioligand, [123I]AM281[N-(morpholin-4-yl)-5-(4-[123I]iodophenyl)-1-(2,4 -dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide], that is structurally related to the CB1-selective antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide]. Baboon single photon emission computed tomography studies, mouse brain dissection studies, and ex vivo autoradiography in rat brain demonstrated rapid passage of [123I]AM281 into the brain after intravenous injection, appropriate regional brain specificity of binding, and reduction of binding after treatment with SR141716A. AM281 has an affinity in the low nanomolar range for cerebellar binding sites labeled with [3H]SR141716A in vitro, and binding of [123I]AM281 is inhibited by several structurally distinct cannabinoid receptor ligands. We conclude that [123I]AM281 has appropriate properties for in vivo studies of cannabinoid CB1 receptors and is suitable for imaging these receptors in the living human brain.

Examination of the effect of the cannabinoid receptor agonist, CP 55,940, on electrically evoked transmitter release from rat brain slices.

In the present study we examined the effect of the cannabinoid receptor agonist, [[1 a,2-(R)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyc lohexyl]-phenol; CP 55,940] on [14C]acetylcholine and [3H]norepinephrine release from hippocampal slices and on [14C]acetylcholine release from striatal slices. CP 55,940 potently inhibited electrically evoked [14C]acetylcholine release from hippocampal slices, with an EC50 of 0.02 microM and a maximal inhibition of 61% at 1 microM. The inhibition of acetylcholine release by CP 55,940 was partially antagonized (60%) by the cannabinoid receptor antagonist, [[N-piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide hydrochloride; SR 141716A]. Alone, SR 141716A significantly enhanced stimulated [14C]acetylcholine release. In contrast to the effects of CP 55,940 on [14C]acetylcholine release, electrically evoked [3H]norepinephrine release from hippocampal slices and [14C]acetylcholine release from striatal slices were both unaffected by this compound. Similarly, hippocampal [3H]norepinephrine release and striatal [14C]acetylcholine release were not affected by SR 141716A. In conclusion, the results of this study extend our previous data indicating that cannabinoid receptors modulate acetylcholine release in the hippocampus. The effects of cannabinoid receptor activation on [3H]acetylcholine release in the hippocampus does not appear to extend to [3H]norepinephrine release from this region or to acetylcholine release from the striatum.

Binding of the non-classical cannabinoid CP 55,940, and the diarylpyrazole AM251 to rodent brain cannabinoid receptors.

The binding of [123I]AM251 (a radioiodinated analog of the cannabinoid CB1 receptor antagonist SR141716A) was compared to that of [3H]CP 55,940 in mouse and rat brain preparations. Scatchard analysis of the binding of [123I]AM251 and [3H]CP 55,940 to membranes prepared from mouse cerebellum, striatum and hippocampus yielded similar Bmax values (15-41 pmol/g wet wt tissue). Kd values were lower for [123I]AM251 (0.23-0.62 nM) than for [3H]CP 55,940 (1.3-4 nM). CP 55,940 and SR141716A increased dissociation of [123I]AM251 from binding sites in mouse cerebellar homogenates to a similar extent. The structurally dissimilar cannabinoid receptor ligands THC, methanandamide, WIN 55, 212-2, CP 55,940 and SR141716A were each able to fully compete with binding of both [123I]AM251 and [3H]CP 55,940 in mouse cerebellum. In vitro autoradiography demonstrated that the distribution of binding sites for [123I]AM251 in rat brain was very similar to published distributions of binding sites for [3H]CP 55,940. Together, these observations suggest that AM251 binds to the same site (the cannabinoid CB1 receptor) in rodent brains as CP 55,940. However, the binding site domains which interact with AM251 and CP 55,940 may not be identical, since IC50 values for cannabinoid receptor ligands depended on whether [123I]AM251 or [3H]CP 55,940 was used as radioligand.

The effect of intravenous administration of delta-9-tetrahydrocannabinol on the activity of A10 dopamine neurons recorded in vivo in anesthetized rats.

In this pilot study, we examined the effect of the intravenous administration of delta-9-tetrahydrocannabinol (THC) on the activity of spontaneously active A10 dopamine (DA) neurons of anesthetized, male albino rats. This was accomplished using the technique of in vivo extracellular single-unit recording. The administration of THC (0.05-1.6 mg/kg i.v.) did not significantly alter either the basal firing rate or the firing pattern of spontaneously active A10 DA neurons compared to vehicle controls. Our findings suggest that, unlike a number of drugs of abuse, THC does not alter the activity of A10 DA neurons and that the previously reported THC-induced increase in brain DA levels is not due to its action on firing rate or pattern in A10 DA neurons.

Model for estimating dopamine transporter occupancy and subsequent increases in synaptic dopamine using positron emission tomography and carbon-11-labeled cocaine.

Although increases in dopamine secondary to the inhibition of the dopamine transporter appear to underlie the reinforcing properties of cocaine, there is presently no model that relates the elevation of synaptic dopamine to the transporter occupancy by cocaine. We propose such a model based on positron emission tomographic (PET) measurements of the brain concentration of cocaine and the assumption of rapid equilibrium between free cocaine and cocaine bound to the dopamine transporter. A euphorigenic dose of cocaine (about 40 mg) is predicted to occupy 80-90% of the transporters, while a perceptible dose (about 5 mg) occupies about 40% of the transporters. If reuptake of dopamine is reduced in proportion to the fraction of transporters occupied by cocaine, our model indicates that synaptic dopamine rises supra-linearly with occupancy, so that 5 and 40 mg doses of cocaine give about 2- and 10-fold increases, respectively. A consequence is that a given dose of cocaine produces a similar degree of elevation of dopamine regardless of the prior level of occupation of the transporters by cocaine. This prediction is supported by recent PET/neuropsychological studies in our laboratory where dopamine transporter occupancy was measured after giving methylphenidate intravenously to volunteers; similarly intense "highs" were reported whether the initial occupancy was zero or 75-85%. It could also explain why attempts to block the psychostimulant-induced "high" by pretreating subjects with drugs that block the dopamine transporter have been unsuccessful, and why the use of methylphenidate to treat cocaine addicts led to increased cocaine consumption.

Effect of the cannabinoid receptor SPECT agent, AM 281, on hippocampal acetylcholine release from rat brain slices.

The SPECT ligand AM 281, a less lipophilic analog of the cannabinoid receptor antagonist SR 141716A, robustly potentiated electrically-evoked release of acetylcholine from superfused hippocampal slices and prevented the inhibition of acetylcholine release by the cannabimimetic drug WIN 55212-2. These results, similar to earlier observations with SR 141716A, indicate that AM 281 is either a cannabinoid receptor antagonist or inverse agonist. Despite showing lower affinity than SR 141716A in hippocampal membrane binding experiments, AM 281 had slightly greater potency than SR 141716A in the hippocampal slice experiments, perhaps because of reduced drug absorption to slice membranes and to the apparatus.

123I-labeled AM251: a radioiodinated ligand which binds in vivo to mouse brain cannabinoid CB1 receptors.

We have investigated the binding of 123I-labeled N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methy l-1 H-pyrazole-3-carboxamide (AM251), an analog of the cannabinoid receptor antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1 H-pyrazole-3-carboxamide] in the mouse brain. Following intravenous injection, the peak whole-brain uptake of about 1% of the administered activity occurred at about 2 h. By 8 h radioactivity in brain had declined to about half its peak value. High-performance liquid chromatographic analysis showed that > 70% of radioactivity extracted from brain at 2 h was still present as [123I]AM251. Co-injection of SR141716A inhibited the in vivo brain binding of [123I]AM251 dose dependently. At 2 mg/kg, the highest dose that could be tested, inhibition was 50% at 2 h post-administration. The ED50 value calculated assuming that 2 mg/kg gave near-maximal inhibition was about 0.1 mg/kg. In contrast to the brain, radioactivity in other major organs (blood, liver, kidney, heart and lung) was little affected by SR141716A. The regional binding of [123I]AM251 in the brain was consistent with the published distribution of cannabinoid receptors in rat brain, in that the order was hippocampus, striatum > cerebellum > brain stem. delta 9-Tetrahydrocannabinol co-administered intravenously at 10 mg/kg, a dose which induced catalepsy and decreased locomotor activity, decreased the 2 h brain uptake of [123I]AM251 by 10%, but this was not significant (P = 0.08). In in vitro binding assays with mouse hippocampal membranes, tetrahydrocannabinol inhibited binding of [123I]AM251 with an IC50 value of about 700 nM, compared with about 0.2 nM for SR141716A.