Effects of trihexyphenidyl and L-dopa on brain muscarinic cholinergic receptor binding measured by positron emission tomography

Summary The effects of pharmacological intervention on brain muscarinic cholinergic receptor (mAChR) binding were assessed in seven patients with Parkinson's disease by positron emission tomography and carbon-11 labelled N-methyl-4-piperidyl benzilate ([¹¹C]NMPB). [¹¹C]NMPB was injected twice, approximately 2 hours apart, in each patient, to assess the effect of single doses of 4 mg of trihexyphenidyl (n=5) or 400 mg of L-dopa with 57 mg of benserazide (n=2) on the binding parameter of mAChRs (K₃). There was a mean 28% inhibition of K₃ values in the brain in the presence of trihexyphenidyl, which was assumed to reflect mAChR occupancy. No significant change in K₃ was observed in the presence of L-dopa. This study demonstrates the feasibility of measuring mAChR occupancy by an anticholinergic medication with PET.     

Imaging of cholinergic terminals using the radiotracer [18F](+)-4-fluorobenzyltrozamicol: In vitro binding studies and positron emission tomography studies in nonhuman primates

The goal of the present set of studies was to characterize the in vitro binding properties and in vivo tissue kinetics for the vesicular acetylcholine transporter (VAcChT) radiotracer, [¹⁸F](+)-4-fluorobenzyltrozamicol ([¹⁸F](+)-FBT). In vitro binding studies were conducted in order to determine the affinity of the (+)- and (−)- stereoisomers of FBT for the VAcChT as well as sigma (σ₂ and σ₂) receptors. (+)-FBT was found to have a high affinity (K_i = 0.22 nM) for the VAcChT and lower affinities for σ₁ (21.6 nM) and σ₂ (35.9 nM) receptors, whereas (−)-FBT had similar affinities for the VAcChT and σ₁ receptors (∼20 nM) and a lower affinity for σ₂ (110 nM) receptors. PET imaging studies were conducted in rhesus monkeys (n = 3) with [¹⁸F](+)-FBT. [¹⁸F](+)-FBT was found to have a high accumulation and slow rate of washout from the basal ganglia, which is consistent with the labeling of cholinergic interneurons in this brain region. [¹⁸F](+)-FBT also displayed reversible binding kinetics during the 3 h time course of PET and produced radiolabeled metabolites that did not cross the blood-brain barrier. The results from the current in vitro and in vivo studies indicate that [¹⁸F](+)-FBT is a promising ligand for studying cholinergic terminal density, with PET, via the VAcChT. Synapse 25:368–380, 1997. © 1997 Wiley-Liss, Inc.     

Receptor binding and selectivity of three 11C-labelled dopamine receptor antagonists in the brain of Rhesus monkeys studied with positron emission tomography

ABSTRACT— The regional distribution of 3 11C-labelled dopamine receptor antagonists, N-methyl spiperone, raclopride and clozapine, in the brain of Rhesus monkeys was studied by positron emission tomography (PET). The measured radioactivities in the striatal area were similar for the 3 antagonists, although the highest selectivity as compared to cerebellum was found for 11C-raclopride 60 min after administration. The selectivity of the radiotracers for the serotonin and D₂-dopamine receptors was evaluated after pretreatment of the monkeys with serotonin and dopamine receptor antagonists. 11C-N-methylspiperone and 11C-clozapine both bound to serotonin receptors in the frontal cortex and to D₂-dopamine receptors in the striatal area. Raclopride was selectively bound to the D₂-dopamine receptors. The radioactivities measured in the striatal area with cerebellum as reference were fitted to a 3-compartment model which made possible evaluation of receptor binding characteristics. The rate proportional to the association rate constant for the receptor, k_on and number of receptors, B_max, varied from 0.02–0.07 min^-1 between the studied radiolabelled drugs, whereas the apparent dissociation rate was highest for clozapine. This means that clozapine had the lowest affinity for the receptors in the striatum, assuming that the B_max values are identical. The observed difference in selective receptor binding and binding characteristics of the 3 tracers may have an influence both on the clinical efficacy and side effects of the studied dopamine receptor antagonists.     

Measurement of density and affinity for dopamine D2 receptors by a single positron emission tomography scan with multiple injections of [11C]raclopride

Positron emission tomography (PET) with [¹¹C]raclopride has been used to investigate the density (B_max) and affinity (K_d) of dopamine D₂ receptors related to several neurological and psychiatric disorders. However, in assessing the B_max and K_d, multiple PET scans are necessary under variable specific activities of administered [¹¹C]raclopride, resulting in a long study period and unexpected physiological variations. In this paper, we have developed a method of multiple-injection graphical analysis (MI-GA) that provides the B_max and K_d values from a single PET scan with three sequential injections of [¹¹C]raclopride, and we validated the proposed method by performing numerous simulations and PET studies on monkeys. In the simulations, the three-injection protocol was designed according to prior knowledge of the receptor kinetics, and the errors of B_max and K_d estimated by MI-GA were analyzed. Simulations showed that our method could support the calculation of B_max and K_d, despite a slight overestimation compared with the true magnitudes. In monkey studies, we could calculate the B_max and K_d of diseased or normal striatum in a 150 mins scan with the three-injection protocol of [¹¹C]raclopride. Estimated B_max and K_d values of D₂ receptors in normal or partially dopamine-depleted striatum were comparable to the previously reported values.     

Striatal dopamine metabolism in living monkeys examined by positron emission tomography

Positron emission tomography, using the dopa analogue [18F]6-fluoro-L-dopa, has been used to depict the neostriatum in living monkeys. The amount of 18F that accumulated preferentially in the striatum could be augmented by a peripheral decarboxylase inhibitor. Striatal 18F could also be discharged with reserpine. This is the first time that the regional distribution of a neurotransmitter has been demonstrated in monkeys.     

Dopamine receptor properties in Parkinson''s disease and Huntington''s chorea evaluated by positron emission tomography using 11C-N-methyl-spiperone

Dopaminergic receptor properties in the striatum of patients with Parkinson's disease (PD) and Huntington's chorea (HD) were studied by positron emission tomography (PET), using 11C-N-methyl-spiperone as a dopamine D2 receptor ligand. The time-dependent regional radioactive uptake in the caudate nucleus and the putamen was measured and fitted to a 3-compartment pharmacokinetic model. The rate constant k3 for specific binding to the receptor compartment in the striatum was determined in relation to the binding in regions with a low density of specific binding sites, such as the cerebellum and the frontal cortex . k3, which is a measure of the receptor density, was reduced in one patient with HD but less affected in PD in comparison with healthy controls. The pattern of k3 values calculated from the 6 PD patients is discussed in relation to any side-to-side differences in dopamine receptor densities in hemiparkinsonism and to possible "hypersensitivity" of dopamine receptors in the early stage of the disease and down-regulation in more advanced disease.     

Muscarinic and dopaminergic receptor subtypes on striatal cholinergic interneurons

Unilateral stereotaxic injection of small amounts of the cholinotoxin, AF64A, caused minimal nonselective tissue damage and resulted in a significant loss of the presynaptic cholinergic markers [3H]hemicholinium-3 (45% reduction) and choline acetyltransferase (27% reduction). No significant change from control was observed in tyrosine hydroxylase or tryptophan hydroxylase activity; presynaptic neuronal markers for dopamine- and serotonin-containing neurons, respectively. The AF64A lesion resulted in a significant reduction of dopamine D2 receptors as evidenced by a decrease in [3H]sulpiride binding (42% reduction) and decrease of muscarinic non-M1 receptors as shown by a reduction in [3H]QNB binding in the presence of 100 nM pirenzepine (36% reduction). Saturation studies revealed that the change in [3H]sulpiride and [3H]QNB binding was due to a change in Bmax not Kd. Intrastriatal injection of AF64A failed to alter dopamine D1 or muscarinic M1 receptors labeled with [3H]SCH23390 and [3H]pirenzepine, respectively. In addition, no change in [3H]forskolin-labeled adenylate cyclase was observed. These results demonstrate that a subpopulation of muscarinic receptors (non-M1) are presynaptic on cholinergic interneurons (hence, autoreceptors), and a subpopulation of dopamine D2 receptors are postsynaptic on cholinergic interneurons. Furthermore, dopamine D1, muscarinic M1 and [3H]forskolin-labeled adenylate cyclase are not localized to striatal cholinergic interneurons.     

Ketamine increases the striatal N-[C]methylspiperone binding in vivo: positron emission tomography study using conscious rhesus monkey

A system for positron emission tomography study of conscious monkeys was newly developed. By use of this system in combination with a microdialysis technique, the effect of ketamine on the binding and release of dopamine was investigated. The administration of ketamine (5 mg/kg) caused sedation accompanied by psychotic symptoms such as nystagmus and stereotyped movements of extremities. During this psychotomimetic period produced by ketamine, a significant increase in the accumulation of the dopamine D₂ receptor ligand N-[¹¹C]methylspiperone was observed in the striatum compared with the level in the conscious state, while no significant change was observed in the frontal cortex and cerebellum. In contrast to the use of ketamine as the anesthetic, pentobarbital (25 mg/kg), which produced deeper anesthesia but no psychotic symptoms, caused a decrease in the accumulation of N-[¹¹C]methylspiperone in the striatum. Kinetic analysis, conducted by a graphical method, revealed that the value of the association constant (K3) for N-[¹¹C]methylspiperone binding in the striatum was increased to approximately 130% by ketamine and decreased to approximately 70% by pentobarbital compared with the control values. Furthermore, the release of dopamine from the striatum measured by microdialysis was not affected by ketamine anesthesia. These results indicate that ketamine facilitates striatal dopaminergic neurotransmission through increasing the binding activity of dopamine D₂ receptors in the striatum, and suggest that these changes may be related to the psychotomimetic behavioral symptoms of this drug.     

Age-related changes in the striatal dopaminergic system in the living brain: a multiparametric PET study in conscious monkeys

In the present study, age-related changes in the striatal dopaminergic system were examined in the living brains of conscious young (6.2 +/- 1.5 years old) and aged (20.2 +/- 2.6 years old) monkeys (Macaca mulatta) using positron emission tomography (PET). L-[beta-(11)C]DOPA and [(11)C]beta-CFT were applied to determine dopamine presynaptic functions such as synthesis rate and transporter (DAT) availability, respectively. Striatal dopamine D(1)- (D(1)R) and D(2)-like receptor (D(2)R) binding were measured with [(11)C]SCH23390 and [(11)C]raclopride, respectively. Although the markers of presynaptic terminals showed parallel age-related declines, the reduction of dopamine synthesis rate measured with L-[beta-(11)C]DOPA was slightly smaller than that of DAT determined with [(11)C]beta-CFT. The binding of [(11)C]raclopride to D(2)R in vivo was significantly reduced with aging, while that of [(11)C]SCH23390 to D(1)R showed no such marked age-related reduction. When the DAT inhibitor GBR12909 (0.5 and 5 mg/kg) was administered, DAT availability, dopamine synthesis, and D(2)R binding were significantly decreased in a dose-dependent manner in both age groups; however, the degrees of the decreases in these parameters were significantly higher in young rather than in aged animals. Dopamine concentration in the striatal extracellular fluid (ECF), as measured by microdialysis, was increased by administration of GBR12909 in a dose-dependent manner and the degree of the increase in dopamine level decreased with age. These results demonstrate that age-related changes of dopamine neuronal functions were not limited to the resting condition but were also seen in the functional responses to the neurotransmitter modulation.     

[11C]β-CIT-FE,a radioligand for quantitation of the dopamine transporter in the living brain using positron emission tomography

The cocaine analogue β-CIT-FE (N-(2-fluoroethyl)-2β-carbomethoxy-3β-(4-iodophenyl)nortropane) was labeled with ¹¹C for positron emission tomography (PET) studies of the dopamine transporter. After intravenous administration to a cynomolgus monkey, [¹¹C]β-CIT-FE accumulated in the striatum with a striatum-to-cerebellum ratio of about 9 after 60 min. Pseudoequilibrium of specific [¹¹C]β-CIT-FE binding in the striatum was obtained within 30–50 min. The radioactivity ratios of the thalamus to the cerebellum and the neocortex to the cerebellum were about 2 and 1.5, respectively. In displacement and pretreatment experiments, radioactivity in the striatum but not in the cerebellum was reduced after injection of β-CIT or the dopamine transporter inhibitor GBR 12909, indicating that striatal radioactivity following injection of [¹¹C]β-CIT-FE represents reversible binding to dopamine transporter sites. After displacement or pretreatment with cocaine there was a marked effect not only in the striatum but also in the thalamus and neocortex. [¹¹C]β-CIT-FE has potential as a useful PET radioligand for quantitation of the dopamine transporter in the primate brain in vivo. © 1996 Wiley-Liss, Inc.     

Effect of 6R- -erythro-5,6,7,8-tetrahydrobiopterin and infusion of -tyrosine on the in vivo l-[β-C]DOPA disposition in the monkey brain

The effect of 6R- -erythro-5,6,7,8-tetrahydrobiopterin (6R-BH₄) and -tyrosine infusion on [¹¹C]dopamine synthesis was analyzed in the striatum of Rhesus monkey using positron emission tomography (PET). The rate for decarboxylation from -[β-¹¹C]DOPA to [¹¹C]dopamine was calculated using a graphical method with cerebellum as a reference region. Although the peripheral administration of 6R-BH₄ at low dose (2 mg/kg) did not provide a significant increase in the rate of dopamine biosynthesis, a high dose of 6R-BH₄ (20 mg/kg) induced an elevation of the rate. This 6R-BH₄-induced elevation of the dopamine synthesis rate was further dose-dependently enhanced by the continuous infusion of -tyrosine (0.2 and 1.0 μmol/min/kg). -Tyrosine infusion with a rate of 1.0 μmol/min/kg caused an enhancement of the rate even during low dose administration of 6R-BH₄ (2 mg/kg). -Tyrosine infusion alone did not induce any elevation of the dopamine biosynthesis rate. The analysis of plasma indicated that the metabolic ratios of -[β-¹¹C]DOPA to each metabolite were not affected by 6R-BH₄ and/or -tyrosine infusion. The results suggest that the low dose loading of tyrosine facilitates the activity of 6R-BH₄ on the presynaptic dopamine biosynthesis, and also that the combined effects can be monitored by PET using -[β-¹¹C]DOPA as a biochemical probe.     

Protective effects of minocycline on the reduction of dopamine transporters in the striatum after administration of methamphetamine: a positron emission tomography study in conscious monkeys.

BACKGROUND: Positron emission tomography (PET) studies of methamphetamine (METH) abusers suggest that psychotic symptoms of METH abusers may be attributable to the reduction of dopamine transporters (DAT) in the human brain. However, there are currently no particular pharmacological treatments for the wide range of symptoms associated with METH abuse. METHODS: Using a PET study in conscious monkeys, we investigated whether the second generation antibiotic minocycline could protect against the reduction of DAT in monkeys treated with METH (2 mg/kg x 3, 3-hour intervals). RESULTS: Pretreatment and subsequent administration of minocycline significantly attenuated the reduction of DAT in the striatum of monkeys treated with METH. Furthermore, posttreatment and subsequent administration of minocycline also significantly attenuated the reduction of DAT. In contrast, repeated administration of minocycline alone did not alter the density of DAT in the striatum of monkeys treated with METH. CONCLUSIONS: Our findings suggest that minocycline protects against METH-induced neurotoxicity in the monkey brain. Therefore, minocycline is likely to be a promising therapeutic agent for the treatment of several symptoms associated with METH use in humans.     

Effects of alkylating agents on dopamine D(3) receptors in rat brain: selective protection by dopamine

Dopamine D₃ receptors are structurally highly homologous to other D₂-like dopamine receptors, but differ from them pharmacologically. D₃ receptors are notably resistant to alkylation by 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), which readily alkylates D₂ receptors. We compared EEDQ with N-(p-isothiocyanatophenethyl)spiperone (NIPS), a selective D₂-like receptor alkylating agent, for effects on D₃ and D₂ receptors in rat brain using autoradiographic analysis. Neither agent occluded D₃ receptors in vivo at doses that produced substantial blockade of D₂ receptors, even after catecholamine-depleting pretreatments. In vitro, however, D₃ receptors were readily alkylated by both NIPS (IC₅₀=40 nM) and EEDQ (IC₅₀=12 μM). These effects on D₃ sites were blocked by nM concentrations of dopamine, whereas μM concentrations were required to protect D₂ receptors from the alkylating agents. The findings are consistent with the view that alkylation of D₃ receptors in vivo is prevented by its high affinity for even minor concentrations of endogenous dopamine.     

Positron emission tomography (PET) in the study of dopamine receptors in the primate brain: evaluation of a kinetic model using 11C-N-methyl-spiperone

The regional kinetics of 11C-N-methyl-spiperone (11C-NMSP) are described in the monkey brain under tracer conditions and after displacement and protection experiments. The primary aim of the study was to investigate different methodological problems associated with use of 11C-NMSP in the quantitation of receptor properties before applying the method in clinical research. Special emphasis was placed on the evaluation of different mathematical approximations of a general compartment model. Different mathematical procedures are described and the results indicate that the approximations performed are reasonable. The simplest method for obtaining a reliable measurement of the rate constant k3 (which is approximately equal to proportional to the number of receptors) and k4 (the receptor dissociation rate constant) uses the radioactivity in the cerebellum as a measure of the tissue concentration of ligand equilibrating with the receptor compartment.     

A positron emission tomography study of the effects of treatment with valproate on brain 5-HT2A receptors in acute mania

Objective:  To examine the effects of treatment with valproate on brain 5-HT2A receptors in acute manic patients using positron emission tomography (PET) and [¹⁸F]-setoperone.
Methods:  Patients with DSM-IV bipolar I disorder–manic episode were recruited. Patients were drug free or drug naïve at the time of baseline PET scan. All patients were treated with valproate and one patient received lithium in addition to valproate for 3–5 weeks following which they had a post-treatment PET scan. The effect of treatment on brain 5-HT2A receptor binding was determined using statistical parametric mapping (SPM) and region of interest (ROI) analyses. Of the 12 manic patients recruited, seven patients had both baseline and post-treatment PET scans.
Results:  All seven patients improved with treatment and were in remission at the time of the second PET scan. Both SPM and ROI analyses showed that treatment with mood stabilizers had no significant effect on brain 5-HT2A receptor binding in manic patients.
Conclusion:  This study suggests that changes in brain 5-HT2A receptors are not involved in the antimanic effects of mood stabilizers however, we cannot exclude the possibility of 5-HT2A receptor involvement in down-stream signaling pathways.     

Amphetamine pretreatment induces a change in both D2-Receptor density and apparent affinity: a [11C]raclopride positron emission tomography study in cats.

BACKGROUND: Measuring changes in dopamine (DA) levels in humans using radioligand-displacement studies and positron emission tomography (PET) has provided important empirical findings in disease and normal neurophysiology. These studies are based on the assumption that DA exerts a competitive inhibition on radioligand binding. To test this, we used PET and a Scatchard approach to investigate whether the decrease in [11C]raclopride binding following amphetamine results from competitive or noncompetitive interactions with DA. METHODS: Scatchard analyses of [11C]raclopride/PET data were used to quantify changes in apparent D2-receptor density (Bmax) and radioligand apparent affinity (K'D) at baseline and after amphetamine pretreatment (2 mg/kg; intravenous) in cats. RESULTS: Amphetamine induced a 46% decrease in [11C]raclopride binding in the striatum of five cats. Scatchard analyses revealed that this decrease in binding was due to a 28% decrease in Bmax and a concomitant 35% increase in K'D. CONCLUSIONS: Competition with DA is an insufficient explanation for the decrease in [11C]raclopride binding observed after amphetamine. Noncompetitive interactions, likely representing D2-receptor internalization, also play an important role in this phenomenon. This finding may have important implications for the interpretation of amphetamine-raclopride PET studies in schizophrenia because dysregulation of the agonist-induced internalization of D2 receptors was recently suggested in this disorder.     

Effect of reserpine-induced depletion of synaptic dopamine on [11C]Raclopride binding to D2-dopamine receptors in the monkey brain

Positron emission tomography was used to examine the in vivo binding of [¹¹C]raclopride to D₂-dopamine (DA) receptors in the striatum of two Cynomolgus monkeys after a single dose of reserpine (1 mg/kg, i.v.). A Scatchard procedure was repeated five times to follow D₂ receptor density and apparent affinity for 7 weeks after reserpine. Reserpine-induced depletion of DA lead to a marked increase in [¹¹C]raclopride binding, which was still detectable 20 days after treatment. Scatchard analyses indicated that the measured increase in [¹¹C]raclopride binding reflected an increase in receptor affinity but no evident change in receptor density (B_max). Thus, the increase in [¹¹C]raclopride binding after reserpine should correspond to a reduced competition with endogenous DA for binding to D₂ receptors. The results were used to estimate the DA-induced D₂ occupancy to be about 40% at physiological conditions. Synapse 25:321–325, 1997. © 1997 Wiley-Liss, Inc.     

Genetic control of dopamine and serotonin receptors in brain regions of inbred mice

In this report the genetic determinants of dopamine and serotonin receptors are investigated. We have used two types of radioreceptor binding assays to identify and quantify these neurotransmitter receptors in various brain regions of inbred mice. In the first method dopamine and serotonin sites are quantified using [3H]spiperone in the presence of appropriate blanking agents. These results are compared with those obtained by the use of [3H]domperidone and [3H]mianserin to label D2 and S2 sites, respectively. Both methods yield nearly identical results. Strain differences in D2 sites are found in the striatum, olfactory tubercle and pituitary. The density of dopaminergic sites is uncorrelated in the 3 brain regions in all mouse strains studied, suggesting that genetic determination of receptor density is independently regulated in each region. Similar observations have been made for S2 receptors in the striatum, hypothalamus, olfactory tubercle and frontal cortex. Analysis of D3 and D2 binding sites in recombinant inbred lines suggests that each site may be determined monogenically.     

Estimation of regional cerebral utilization of [11C]-L-3, 4-dihydroxy-phenylalanine (DOPA) in the primate by positron emission tomography

The intracerebral kinetics of [11C]-labelled L-3,4-dihydroxyphenylalanine, L-DOPA, was investigated in rhesus monkeys by positron emission tomography (PET). Through the labelling of the L-DOPA molecule in different positions and observation of a series of pharmacological challenges it was possible to establish that the kinetic conversion of the radiotracer in the striatum represents the process of decarboxylation to [11C]-labelled dopamine. The rate constant for this process can be estimated using a two-compartment model. The use of [11C]-L-DOPA and PET will thus provide a possibility for in vivo studies of blood-brain barrier transport of the amino acid as well as for the estimation of the ability for brain tissue to decarboxylate the tracer by the action of aromatic L-amino acid decarboxylase.