REVIEW ARTICLE. Imaging the Dopamine System with In Vivo [ 11 C]raclopride Displacement Studies: Understanding the True Mechanism

Transcription

1 B Academy of Molecular Imaging 2005 Mol Imaging Biol (2005) 7:45Y52 Published Online: 30 December 2004 DOI: /s REVIEW ARTICLE Imaging the Dopamine System with In Vivo [ 11 C]raclopride Displacement Studies: Understanding the True Mechanism Nathalie Ginovart, PhD PET Centre, Centre for Addiction and Mental Health, University of Toronto, 250 College Street, M5T 1R8, Toronto, Ontario, Canada Abstract Measuring changes in dopamine (DA) levels in humans using radioligand-displacement studies and positron emission tomography (PET) has provided important empirical findings in diseases and normal neurophysiology. These studies are based on the assumption that DA exerts a competitive inhibition on D 2 -radioligand binding. However, the transfer of this hypothesis to a proven mechanism has not been fully achieved yet and an accumulating number of studies challenge it. In addition, new evidence suggests that DA exerts a noncompetitive inhibition on D 2 -radioligand binding under amphetamine conditions. This article reviews the theoretical basis for the DA competition hypothesis, the in vivo and in vitro evidences supporting a noncompetitive action of DA on D 2 -radioligand binding under amphetamine conditions, and discusses possible mechanisms underlying this noncompetitive interaction. Finally, we propose that such noncompetitive interactions may have important implications for how one interprets findings obtained from radioligand-displacement PET studies in neuropsychiatric diseases, especially in schizophrenia in which a dysregulation of the DA-promoted internalization of D 2 receptors was recently suggested. Key words: Amphetamine challenge, [ 11 C]raclopride, D 2 -receptors competition, Internalization, Positron emission tomography, Schizophrenia Background Arecent important development in neurosciences is the ability to assess changes in the levels of endogenous neurotransmitters in living humans using positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging. Using these techniques, the level of endogenous dopamine (DA) has been assessed in a number of pathological and physiological conditions, and this is having important scientific consequences. For example, the DA hypothesis of schizophrenia has experienced a renewal of interest from the observation that patients with schizophrenia show an abnormally high level and release of Correspondence to: Nathalie Ginovart, PhD; nathalie.ginovart@ camhpet.ca DA [1, 2]. The view that addiction involves an enhanced responsiveness of the striatal DA system has been challenged by the finding of a decreased release of striatal DA in cocaine abusers as compared to controls [3]. In addition, studies showing that behavioral tasks, such as playing video game or writing, and placebo response induce an increased release of striatal DA [4Y6] illustrated the ability of in vivo imaging to detect physiological changes in DA levels. While these techniques are being extensively used they do not provide a direct measurement of endogenous DA levels. In fact, changes in endogenous levels of DA are inferred from changes in the binding of [ 11 C]raclopride/[ 123 I]IBZM to the DA D 2 receptors. The standard observation is that drugs that increase the levels of endogenous DA decrease the binding of these radioligands to postsynaptic D 2 receptors, whereas the opposite effect is observed with drugs that decrease endogenous DA levels. Assessment of changes in endoge-

2 46 N. Ginovart: Imaging the Dopamine System with In Vivo [ 11 C]raclopride Displacement Studies nous DA levels is based on the hypothesis that the changes in D 2 -radioligand binding are due to competition with DA. However, the validation of this hypothesis has not been fully achieved yet and an accumulating number of studies even challenge it. This article reviews the theoretical basis for the DA competition hypothesis, the in vivo and in vitro evidences supporting a noncompetitive action of DA on D 2 - radioligand binding, and discusses possible mechanism(s) underlying this noncompetitive interaction. Endogenous DA Levels Affect Raclopride Binding From an Artifact to a Method In 1989, Seeman et al. [7] and Ross and Jackson [8] demonstrated that [ 3 H]raclopride, a ligand used to measure D 2 receptors, was sensitive to endogenous DA, whereas [ 3 H]NMSP, another ligand used to measure D 2 receptors, was not similarly vulnerable [7]. At first this sensitivity to endogenous DA was seen as a liability for [ 11 C]raclopride [9]. However, soon this liability was seen as the first opportunity to measure endogenous DA levels in humans by looking at variations in the level of binding of [ 11 C]raclopride. Since that first demonstration, numerous studies in humans and animals have confirmed that when levels of synaptic DA are increased (by the use of amphetamine, methylphenidate, GBR12909, cocaine, and fenfluramine) [10Y15], the level of binding of [ 11 C]raclopride as measured by PET and [ 123 I]IBZM as measured by SPECT is decreased. Conversely, when the levels of DA are decreased using reserpine and!-methyl-p-tyrosine [16, 17], the levels of [ 11 C]raclopride and [ 123 I]IBZM are increased. Thus, the fact that changes in DA are associated with changes in [ 11 C]raclopride is a very well replicated finding. The DA Competition The Initial Explanation for the BRaclopride Effect[ Competition with endogenous DA has been proposed as the underlying mechanism to explain the changes in [ 11 C]raclopride and [ 123 I]IBZM binding following manipulation changes in endogenous DA. One of the first requirements for the competition model is to show its dependence on endogenous dopaminevand this indeed is the case. Amphetamine is known to raise extracellular DA levels by inducing DA efflux from the intracytoplasmic stores to the synaptic space via reverse transport of the DA transporter [18, 19]. Manipulations that counteract the effect of amphetamine on DA release also counteract its raclopride effect. For example, Innis et al. [11] showed that depleting DA stores with reserpine abolished the effect of subsequent amphetamine administration on [ 123 I]IBZM binding. Villemagne et al. [20] showed that blocking the DA transporter with GBR12909 attenuates amphetamineinduced decrease in [ 11 C]raclopride binding. The model is further supported by some doseyresponse data. Studies of combined microdialysis/raclopride effect show that the administration of amphetamine dose-dependently increased DA concentration and decreased in vivo [ 123 I]IBZM [21] and [ 11 C]raclopride [22] binding in the striatum. A direct effect of endogenous DA on [ 11 C]raclopride is also suggested from experiments showing that unilateral stimulations of the nigrostriatal neurons, which induce only transient changes in striatal DA levels, induced only transient changes in [ 11 C]raclopride binding [23]. The exquisite temporal resolution of the radioligand binding response to the stimulation-induced changes in DA levels also suggests that changes in [ 11 C]raclopride binding are directly related to fluctuations in extracellular DA levels. These studies confirm that the raclopride effect is mediated by enhanced DA releasevbut they do not tell us how increase in DA release leads to a decrease in radioligand binding. Evidence in Favor of the DA Competition Model One of the hallmarks of a true competition, i.e., when two agents are reversibly competing for the same receptor, is that it shows up as a change in the affinity of each receptor, but does not result in a change in total receptor number. The DA competition model thus predicts that changes in [ 11 C]raclopride binding induced by changes in DA levels reflect a change in affinity (i.e., 1/K D ) but not in receptor density (i.e., B max ). This theory is confirmed by in vivo PET data in nonhuman primates showing that depletion of endogenous DA using reserpine, which leads to a marked increase in [ 11 C]raclopride binding, induces an increase in D 2 -receptor affinity with no evident change in B max [17]. Similarly, depletion of striatal DA using!- methyl-para-tyrosine in rodents has been reported to increase [ 123 I]IBZM binding with no detectable effects on B max [16]. These studies thus showed that the effect of DA depletion paradigms on D 2 -radioligand binding is due to removal of endogenous DA rather than to a noncompetitive action (such as D 2 -receptor upregulation) and thus confirm a competitive action of endogenous DA at baseline conditions. Inconsistencies and Incompatibilities with the DA Competition Model Although the aforementioned data support the DA competition model theory, a number of inconsistencies have, however, emerged that questioned the validity of this model. These inconsistencies are summarized in the following.

3 N. Ginovart: Imaging the Dopamine System with In Vivo [ 11 C]raclopride Displacement Studies 47 Failure to Detect DA Competition with Radioligands Other Than Benzamides The DA competition model is consistent with data obtained with benzamide radioligands (such as raclopride, IBZM, fallypride, FLB457), which are always affected by changes in endogenous DA [11Y13, 16, 17, 24Y26]. In contrast, the binding of butyrophenone compounds (such as spiperone, NMSP, pimozide) to D 2 receptors [27Y29] as well as benzazepine compounds (such as SCH23390, NNC112, NNC756) to D 1 receptors [30Y32] shows either no change or even paradoxical increase in binding in the face of higher DA. Thus, it seems that this effect is peculiar to the benzamides, rather than being a general principle governing all neurotransmitters and ligands. Temporal Discrepancy Between Changes in DA Levels and in Radioligand Binding Another inconsistency in the validity of the DA competition model is the temporal discrepancies between changes in radioligand binding as measured with PET and SPECT and changes in DA levels as measured with microdialysis. Indeed, the time course of changes in [ 11 C]raclopride/ [ 123 I]IBZM binding far outlives the changes in DA levels, calling into question the idea of direct competition. Microdialysis studies have shown that amphetamine elevates striatal DA levels with peak values occurring within 20 minutes and that DA levels return to control values within 120 minutes [21, 33, 34]. Amphetamine-induced elevation in DA levels is thus transient. This contrasts with the longlasting decrease in both [ 11 C]raclopride and [ 123 I]IBZM binding observed in vivo. Indeed, [ 123 I]IBZM binding has been shown to be still reduced at six hours following a single dose of amphetamine in nonhuman primates [21]. [ 11 C]Raclopride binding is still reduced up to six hours following a single dose of amphetamine in nonhuman primates [35] and in humans [36]. Such an inconsistency suggests that factors other than endogenous DA play a significant role in the alteration in radioligand binding observed following DA manipulation. Amphetamine Causes a Decrease in [ 11 C]raclopride Binding Site NumberVA Finding Incompatible with the Competition Model A recent ex vivo binding study performed on rat striatal homogenates recently showed that increasing DA levels with amphetamine causes a decrease in [ 3 H]raclopride B max, rather than a change in apparent affinity, a finding that directly refutes the assumption of simple competition [37]. Because of the potential limitations of extrapolating in vitro binding data to the in vivo situation, we recently investigated whether amphetamine pretreatment also causes a decrease in [ 11 C]raclopride B max as assessed in vivo in cats using PET [38]. This study showed that the effect of amphetamine on [ 11 C]raclopride binding resulted from a dual mechanism: a decrease in the density of striatal D 2 receptors and a decrease in their affinity, which is probably due to competition. These results, which showed that the amphetamine-induced release of DA leads to a decrease in raclopride-labeled receptor rather than just a change in receptor affinity, directly refute the assumption of simple competition and suggested that noncompetitive interactions are involved in this effect. However, conflicting PET studies performed in primates recently showed that a single dose of methamphetamine (2 mg/kg; i.p.) induced an increase in [ 11 C]raclopride K D V with no significant change in B max [39]. The reason for these discrepant results is not clear. However, it might be, at least partly, explained by noticeable differences between the experimental protocols used in the two studiesvi.e., intraperitoneal vs. intravenous route of amphetamine administration, multiple radioligand concentration assay requiring six to seven hours vs. three hours of postamphetamine data analysis; [ 11 C]raclopride bolus plus infusion paradigm vs. bolus paradigm, quantification of [ 11 C]raclopride BP values using the Logan vs. the transient equilibrium method, in Doudet s vs. Ginovart et al. s study, respectively. Thus, although the displacement of [ 11 C]raclopride is a well-confirmed finding, the competition model is difficult to reconcile with some of the aforementioned studies showing that noncompetitive interactions also play an important role in this phenomenon. In addition, there are two other problems with the competition model: Why is the displacement seen only with benzamides and not with other ligands? Why does the displacement outlast the levels of DA? Proposed Theories to Explain the Failure of the DA Competition Model The Lower Affinity of Raclopride Makes It More Sensitive Than [ 11 C]NMSP It has been proposed that the lower affinity of benzamides as compared to that of butyrophenones might explain their higher sensitivity to endogenous DA [7]. While on the surface this suggestion seems sensible, under the formal logic of receptor theory and laws of competition this is not the case. In fact, Farde et al. [40] have shown that the vulnerability of a radiotracer to a competitive inhibitor is not dependent on its affinity as long as binding quantification is performed at tracer dose. Therefore, differences in affinity, by itself, are not a reasonable explanation for why some radioligands show displacement and others do not. Raclopride and Spiperone Bind to Different Sites of the D 2 Receptor Previous data in the literature suggest that benzamides and butyrophenones might bind to different binding on D 2

4 48 N. Ginovart: Imaging the Dopamine System with In Vivo [ 11 C]raclopride Displacement Studies receptors. For example, the density of D 2 -receptor sites measured in vitro using [ 3 H]raclopride is about twofold higher than that measured using [ 3 H]spiperone in rat striata homogenates [41, 42]. Similarly, D 2 -receptor density values measured in the human striatum in vivo range from 30 to 40 nmol/ml when using [ 11 C]raclopride [40, 43] and from 15 to 25 nmol/ml when using [ 11 C]NMSP [44, 45]. These discrepancies between D 2 -receptor densities have led to the proposal that benzamides and butyrophenones do not bind to the same configuration of the D 2 receptor. Butyrophenones may bind primarily to the monomer form, whereas benzamides may bind to both the monomer and dimer forms of the receptor [41, 46]. Differences in the monomerydimer equilibrium could thus explain the differences in D 2 -receptor densities revealed by the two classes of compounds. Although this BmonomerYdimer theory[ itself is the subject of controversy, most people in the field, however, acknowledge the existence of a specific Bbutyrophenone binding site [47, 48].[ Along with this hypothesis, Hall et al. [49] showed that DA has a lower affinity for the NMSP binding sites than for the raclopride binding sites. A remote localization of the NMSP-butyrophenone binding sites may limit its access by endogenous DA and could explain the lack of effect of DA manipulations on in vitro [ 3 H]spiperone and in vivo [ 11 C]NMSP bindings. However, it cannot explain the other discrepancies (temporal discordance observed with [ 11 C]raclopride, lack of effect on D 1 ligands, paradoxical increases in [ 3 H]spiperone binding in some reports). While flaws in the competition model have been recognized before, no comprehensive model has emerged to replace it. While radioligand differences in affinity and/or binding site could play a role, they are unable to explain the discrepancies and paradoxes observed. Other Discrepancies Between Changes in DA Levels and in Radioligand Binding Previous studies have shown that ketamine, a noncompetitive N-methyl-D-aspartate antagonist that induces psychotic symptoms in humans, decreased the striatal binding of [ 11 C]raclopride in human subjects [50, 51]. A subsequent study in nonhuman primates showed that this ketamine-induced decrease in [ 11 C]raclopride binding was dose-dependent but induced no significant changes in extracellular DA concentration as measured with microdialysis [52]. In the same way, scopolamine (a muscarinic antagonist) decreased the binding of [ 11 C]raclopride in a dose-dependent manner but induced no significant changes in extracellular DA concentration as measured with microdialysis [53]. The same authors also demonstrated that administration of the indirect DA modulator benztropine (a muscarinic cholinergic antagonist) or ketanserine (a 5-HT 2 antagonist) also increased the striatal levels of DA and decreased [ 11 C]raclopride binding in a dose-dependent manner [22]. However, the relationships between the magnitude of radiotracer displacement and the magnitude of change in DA levels were different for direct dopamine enhancers (GBR12909 and methamphetamine) and for indirect dopamine modulators (benztropine and ketanserine). These experimental data thus support the view that, even for benzamides, the alternation of radiotracer binding as measured by in vivo imaging techniques is also affected by the synaptic concentration of DA. Noncompetitive Models Might be an Alternative to the Competition Model If competitive interaction of DA with D 2 receptors is ruled out, then three possible mechanisms can be evoked to explain the amphetamine-induced loss of [ 11 C]raclopride binding sites. First, DA and [ 11 C]raclopride may bind to two distinct states of the D 2 receptor (D 2 high and D 2 low) [54] for which [ 11 C]raclopride has equal affinity but for which DA has high and low affinity, respectively (two-state model). Tight DA binding to the D 2 -high sites would occlude these sites for the binding of [ 11 C]raclopride and would explain the apparent decrease in D 2 -receptor density obtained after amphetamine. This two-state model has already been proposed by Seeman et al. [7, 55] to explain the apparent fall in [ 3 H]raclopride B max obtained in vitro after incubation of striata membranes with exogenous DA. This two-state model, however, does not seem to apply to explain the decrease in [ 3 H]raclopride B max observed after amphetamine on rat striata membranes [37]. Indeed, the effect of amphetamine on [ 3 H]raclopride B max was not reversed by treatment of the striata membranes with Gpp(NH)p (5V-guanylylimidodiphosphate), which converts D 2 receptors from their high- to their low-agonist affinity state [56], suggesting that the two-state model was not a reasonable explanation for this effect. Second, DA may bind to an allosteric site on the receptor, which is not recognized by the radioligand but which can modulate its binding site (allosteric model). Dopamine binding to D 2 receptors may cause a conformational change in the receptor that would distort or even mask [ 11 C]raclopride binding sites. Although an allosteric modulation of D 2 receptors by endogenous DA cannot be excluded, it seems unlikely as most of the compounds that have been identified thus far as modulators are neither synthesized endogenously nor naturally encountered [57, 58]. Third, the massive release of DA triggered by amphetamine may cause an internalization of D 2 receptors (internalization model). A DA-promoted internalization of striatal D 2 receptors following amphetamine pretreatment has already been suggested in previous ex vivo studies in rodents [37, 59]. In these studies, subcellular fractionation experiments performed using [ 3 H]raclopride and/or [ 3 H]spiperone indicated that amphetamine resulted in a redistribution of D 2 receptors from the cell surface to intracellular compartments and that the loss of D 2 receptors from the plasma membrane was likely responsible for the decrease in [ 3 H]raclopride binding observed after amphetamine.

5 N. Ginovart: Imaging the Dopamine System with In Vivo [ 11 C]raclopride Displacement Studies 49 Although a two-state model or an allosteric modulation of D 2 receptors by DA cannot be excluded, the noncompetitive interaction evidenced between endogenous DA and [ 11 C]raclopride for binding on D 2 receptors is likely related to a DA-promoted internalization of the receptors after amphetamine. Agonist-Induced Internalization of D 2 Receptors: An Alternative to the Simple Competition Model Laruelle [60] was the first to propose the internalization model as a potential alternative to the competition model theory. A DA-promoted internalization of D 2 receptors following amphetamine could account for the flaws previously noticed in the competition model theory. Especially, it might explain the temporal persistence of the raclopride effect after amphetamine as well as why it is observed only with raclopride-like and not with spiperone-like radioligands. Receptor Internalization: A Regulatory Process of Cell Functional Activity Agonists not only bind and stimulate receptors, they also initiate a shift in their position from cell surface to intracellular compartments, a process that is termed internalization of receptors. This rapid internalization process is associated with the appearance of receptors in intracellular vesicles and with their concomitant disappearance from the cell surface membrane. Internalized receptors are conveyed to the endosomes, from where some may be recycled back to the cell surface, whereas others are transported to the lysosomes and degraded. This internalization regulates the number of receptors at the cell surface, thereby modulating the responsiveness of the cell to further stimulation. Receptor internalization has now been observed for a variety of neuronal receptors [61Y68]. Raclopride and Spiperone Show Differential Responses to Internalization Agonist-promoted internalization of D 2 receptors is now a well-established phenomenon that has been shown to occur in vitro in cells expressing D 2 receptors [69Y71]. Barton et al. [72] were the first to propose that agonist-mediated D 2 - receptor internalization differently affects radioligand binding depending on the ligand lipophilicity. These authors showed that preexposure of retinoblastoma cells to DA resulted in decreased binding of the hydrophilic benzamide [ 3 H]iodosulpiride with no change in the binding of the lipophilic butyrophenone [ 3 H]NMSP. They suggested that [ 3 H]iodosulpiride only detects receptors present on the cell surface, whereas [ 3 H]NMSP detects both the receptors present on the cell surface and those sequestered. Since this pioneering study, several studies have shown that DAinduced internalization of D 2 receptors can be measured in vitro by measuring the loss of [ 3 H]sulpiride binding from the cell surface [70, 73]. Evidence also exists that such a differential response of [ 3 H]raclopride and [ 3 H]spiperone to D 2 -receptor internalization occurs in vivo. Indeed, while the binding of both radioligands decreased at the cell surface following amphetamine administration, only [ 3 H]spiperone binding showed an increased accumulation in the intracellular endosomal compartment [37, 59]. These data thus suggest that although [ 3 H]raclopride and [ 3 H]spiperone both bind to D 2 receptors, [ 3 H]raclopride does not have access to a subpopulation of D 2 receptors that are located in intracellular compartments but that are still accessible to [ 3 H]spiperone. This finding compares with a previous study already showing that part of the D 2 -binding sites found in striata homogenates is exclusively bound by NMSP and can only be partially accessed by raclopride [49]. Taken together, these data suggest that the subset of D 2 -binding sites exclusively bound by NMSP and not accessed by raclopride could correspond to internalized receptors, and the differential ability of spiperone-like and raclopride-like ligands to access these receptors might explain their different response to internalization. Agonist-Induced Internalization Explains Current Paradoxes The internalization hypothesis can account for the two major problems of the competition model. First, by delinking [ 11 C]raclopride-binding decrease from online DA competition, it can explain the temporal persistence of the [ 11 C]raclopride effect that has been observed after amphetamine. As DA internalizes receptors, they become unavailable to [ 11 C]raclopride not because of competition, but because of shift in their compartmental localization. Thus, even if DA levels came back to baseline, [ 11 C]raclopride levels will not come back to baseline until the receptors are recycled to the surface. Thus, it is the receptor dynamics and not DA competition which explains the prolonged decrease in [ 11 C]raclopride. The model can also account for the raclopride/spiperone differences. Indeed, agonist-mediated D 2 -receptor internalization could account for both the decrease in benzamide binding and the lack of changes in butyrophenone binding reported following amphetamine. The internalization model predicts that released DA promotes a shift (i.e., internalization) of the D 2 receptors from the cell membrane to intracellular compartments. Two important differences in the physicochemical properties of two compounds could account for their different abilities to access or to bind internalized receptors. First, raclopride and spiperone greatly differ in their lipophilicity, raclopride being less lipophilic than spiperone and NMSP [74Y76]. The low lipophilicity of raclopride-like ligands would limit their access to internalized receptors. Dopamine-promoted internalization of D 2 receptors would not affect spiperonelike ligand binding since these ligands are lipophilic enough

6 50 N. Ginovart: Imaging the Dopamine System with In Vivo [ 11 C]raclopride Displacement Studies to cross the cell membrane and access these newly internalized receptors. Second, the sensitivity of raclopride and spiperone binding to ph conditions is also very different. The affinity of substituted benzamides such as raclopride for D 2 receptors is highly reduced at high ph conditions, whereas that of butyrophenones such as spiperone is more moderately affected by such environmental conditions [77, 78]. For instance, reduction of ph from eight to six has been shown to have little effect on [ 3 H]spiperone binding to bovine caudate nucleus membranes, whereas the same ph reduction led a substantial reduction in [ 3 H]sulpiride binding [79]. The different ph dependencies of butyrophenones and substituted benzamides for binding D 2 receptors might explain their different abilities to bind internalized receptors. Indeed, upon internalization, receptors are delivered to endosomes that are acidified by ATP-dependent proton pumps [80, 81]. One of the major functions of endosomes acidification is to dissociate ligands from their receptors, after which many receptors are recycled to the cell surface while the ligands are degraded [82, 83]. Several ligands dissociate from their receptors with only slight acidification (ph approx. 6.8) of the endosomal compartment, whereas some other ligands require more acidic conditions (ph G 6.0) to be released from their receptors [83Y85]. It thus appears that subtle differences in endosomal ph can have a differential impact on endosomal ligandyreceptor dissociation depending on the ligand. The high sensitivity of raclopridelike ligand to high ph conditions might thus preclude their binding to internalized receptors, whereas the relative insensitivity of butyrophenones to such conditions would preserve their binding to receptor even in the high ph endosomal conditions. Conclusion For nearly a decade, the results from experiments showing decreased [ 11 C]raclopride binding after amphetamine have been interpreted as representing increased competition with DA and thus increased DA release. However, new evidence suggests that noncompetitive interactions are equally important explanations for this effect. The interaction between endogenous DA levels and [ 11 C]raclopride for binding on D 2 receptors thus appears to be more complex as it is generally believed. While this interaction appears to be purely competitive at baseline conditions, the massive release of DA induced by amphetamine triggers a cascade of cellular events that ultimately lead to both competitive and noncompetitive interactions. Although the precise mechanism leading to noncompetitive interactions remains to be fully elucidated, recent data suggest it might be related to a DA-promoted internalization of D 2 receptors after amphetamine. As DA is indubitably the trigger of the mechanism ultimately leading to the drug-induced change in [ 11 C]raclopride binding, one may consider that it does not matter much whether this mechanism actually reflects DA competition, D 2 -receptor internalization, or both. This is certainly true for most clinical PET studies performed using this paradigm. However, it should be kept in mind that pathological states in which a dysregulation of receptor trafficking exists may show either an exaggerated or an attenuated [ 11 C]raclopride response to amphetamine. For instance, recent data [86, 87] support the hypothesis for a dysregulation of the agonist-induced internalization process that modulates D 2 -receptor signaling in the brain of schizophrenic and bipolar patients. If, as suggested by these studies, D 2 -receptor internalization is indeed reduced in schizophrenics when compared to normal controls, the higher amphetamine/[ 11 C]raclopride effect observed in these patients may mean that their drug-induced release of DA is even greater than that previously postulated on the basis of the DA competition theory alone. Conversely, diseases in which D 2 -receptor internalization is increased may show an exaggerated [ 11 C]raclopride response to amphetamine when compared to normal controls despite a similar effect of the drug on DA outflow. Understanding the true mechanism underlying the abnormally high amphetamine/[ 11 C]raclopride effect in schizophrenia may thus uncover a novel mechanism underlying the excessive stimulation of D 2 receptors postulated in this disorder. Indeed, an alteration of D 2 -receptor internalization would be predicted to result in overactive D 2 -receptor signaling under stimulation, as predicted by the DA hypothesis of schizophrenia, and could occur without any detectable change in the baseline levels of D 2 receptors. If impairment of D 2 - receptor trafficking rather than just impairment of DA release is proven to be involved in this effect in schizophrenics, it may open new leads for the development of therapeutic approaches targeted at modifying D 2 -receptor trafficking in this disorder. Acknowledgment. The author thanks Shitij Kapur, MD, PhD, for fruitful discussions during the preparation of this manuscript. References 1. Breier A, Su TP, Saunders R, et al. (1997) Schizophrenia is associated with elevated amphetamine-induced synaptic dopamine concentrations: Evidence from a novel positron emission tomography method. Proc Natl Acad Sci U S A 94:2569Y Laruelle M, Abi-Dargham A, van Dyck CH, et al. (1996) Single photon emission computerized tomography imaging of amphetamineinduced dopamine release in drug-free schizophrenic subjects. Proc Natl Acad Sci U S A 93:9235Y Volkow ND, Wang GJ, Fowler JS, et al. (1997) Decreased striatal dopaminergic responsiveness in detoxified cocaine-dependent subjects. Nature 386:830Y de la Fuente-Fernandez R, Ruth TJ, Sossi V, et al. (2001) Expectation and dopamine release: Mechanism of the placebo effect in Parkinson s disease. Science 293:1164Y Koepp MJ, Gunn RN, Lawrence AD, et al. (1998) Evidence for striatal dopamine release during a video game. Nature 393:266Y Schommartz B, Larisch R, Vosberg H, Muller-Gartner HM (2000) Striatal dopamine release in reading and writing measured with [ 123 I]iodobenzamide and single photon emission computed tomography in right handed human subjects. Neurosci Lett 292:37Y40 7. Seeman P, Guan HC, Niznik HB (1989) Endogenous dopamine lowers the dopamine D 2 receptor density as measured by [ 3 H]raclopride: Implications for positron emission tomography of the human brain. Synapse 3:96Y97

7 N. Ginovart: Imaging the Dopamine System with In Vivo [ 11 C]raclopride Displacement Studies Ross SB, Jackson DM (1989) Kinetic properties of the accumulation of 3 H-raclopride in the mouse brain in vivo. Naunyn-Schmiedeberg s Arch Pharmacol 340:6Y12 9. Volkow ND, Wang GJ, Fowler JS, et al. (1994) Imaging endogenous dopamine competition with [ 11 C]raclopride in the human brain. Synapse 16:255Y Farde L, Nordstrom AL, Wiesel FA, et al. (1992) Positron emission tomographic analysis of central D 1 and D 2 dopamine receptor occupancy in patients treated with classical neuroleptics and clozapine. Relation to extrapyramidal side effects. Arch Gen Psychiatry 49: 538Y Innis RB, Malison RT, al-tikriti M, et al. (1992) Amphetaminestimulated dopamine release competes in vivo for [ 123 I]IBZM binding to the D 2 receptor in nonhuman primates. Synapse 10:177Y Volkow ND, Wang GJ, Fowler JS, et al. (1994) Imaging endogenous dopamine competition with [ 11 C]raclopride in the human brain. Synapse 16:255Y Laruelle M, Abi-Dargham A, van Dyck CH, et al. (1995) SPECT imaging of striatal dopamine release after amphetamine challenge. J Nucl Med 36:1182Y Smith GS, Dewey SL, Brodie JD, et al. (1997) Serotonergic modulation of dopamine measured with [ 11 C]raclopride and PET in normal human subjects. Am J Psychiatry 154:490Y Tsukada H, Nishiyama S, Kakiuchi T, et al. (1999) Isoflurane anesthesia enhances the inhibitory effects of cocaine and GBR12909 on dopamine transporter: PET studies in combination with microdialysis in the monkey brain. Brain Res 849:85Y Laruelle M, D Souza CD, Baldwin RM, et al. (1997) Imaging D 2 receptor occupancy by endogenous dopamine in humans. Neuropsychopharmacology 17:162Y Ginovart N, Farde L, Halldin C, Swahn CG (1997) Effect of reserpineinduced depletion of synaptic dopamine on [ 11 C]raclopride binding to D 2 -dopamine receptors in the monkey brain. Synapse 25:321Y Fischer JF, Cho AK (1979) Chemical release of dopamine from striatal homogenates: Evidence for an exchange diffusion model. J Pharmacol Exp Ther 208:203Y Sulzer D, Chen TK, Lau YY, et al. (1995) Amphetamine redistributes dopamine from synaptic vesicles to the cytosol and promotes reverse transport. J Neurosci 15:4102Y Villemagne VL, Rothman RB, Yokoi F, et al. (1999) Doses of GBR12909 that suppress cocaine self-administration in non-human primates substantially occupy dopamine transporters as measured by [ 11 C] WIN35,428 PET scans. Synapse 32:44Y Laruelle M, Iyer RN, al-tikriti MS, et al. (1997) Microdialysis and SPECT measurements of amphetamine-induced dopamine release in nonhuman primates. Synapse 25:1Y Tsukada H, Nishiyama S, Kakiuchi T, et al. (1999) Is synaptic dopamine concentration the exclusive factor which alters the in vivo binding of [ 11 C]raclopride?: PET studies combined with microdialysis in conscious monkeys. Brain Res 841:160Y Ginovart N, Hassoun W, Le Cavorsin M, et al. (2002) Effects of amphetamine and evoked dopamine release on [ 11 C]raclopride binding in anesthetized cats. Neuropsychopharmacology 27:72Y Mukherjee J, Yang ZY, Lew R, et al. (1997) Evaluation of D- amphetamine effects on the binding of dopamine D 2 -receptor radioligand, 18 F-fallypride in nonhuman primates using positron emission tomography. Synapse 27:1Y Chou YH, Halldin C, Farde L (2000) Effect of amphetamine on extrastriatal D 2 dopamine receptor binding in the primate brain: A PET study. Synapse 38:138Y Mach RH, Nader MA, Ehrenkaufer RL, et al. (1997) Use of positron emission tomography to study the dynamics of psychostimulantinduced dopamine release. Pharmacol Biochem Behav 57:477Y Onoe H, Inoue O, Suzuki K, et al. (1994) Ketamine increases the striatal N-[ 11 C]methylspiperone binding in vivo: Positron emission tomography study using conscious rhesus monkey. Brain Res 663: 191Y Kobayashi K, Inoue O, Watanabe Y, et al. (1995) Difference in response of D 2 receptor binding between 11 CYN-methylspiperone and 11 C-raclopride against anesthetics in rhesus monkey brain. J Neural Transm Gen Sect 100:147Y Hartvig P, Torstenson R, Tedroff J, et al. (1997) Amphetamine effects on dopamine release and synthesis rate studied in the Rhesus monkey brain by positron emission tomography. J Neural Transm 104:329Y Abi-Dargham A, Simpson N, Kegeles L, et al. (1999) PET studies of binding competition between endogenous dopamine and the D 1 radiotracer [ 11 C]NNC 756. Synapse 32:93Y Chou YH, Karlsson P, Halldin C, et al. (1999) A PET study of D 1 -like dopamine receptor ligand binding during altered endogenous dopamine levels in the primate brain. Psychopharmacology 146:220Y Gifford AN, Park MH, Kash TL, et al. (2000) Effect of amphetamineinduced dopamine release on radiotracer binding to D 1 and D 2 receptors in rat brain striatal slices. Naunyn-Schmiedeberg s Arch Pharmacol 362:413Y Sharp T, Zetterstrom T, Ljungberg T, Ungerstedt U (1987) A direct comparison of amphetamine-induced behaviours and regional brain dopamine release in the rat using intracerebral dialysis. Brain Res 401:322Y Butcher SP, Fairbrother IS, Kelly JS, Arbuthnott GW (1988) Amphetamine-induced dopamine release in the rat striatum: An in vivo microdialysis study. J Neurochem 50:346Y Carson RE, Channing MA, Vuong B, et al. (2001) Amphetamineinduced dopamine release: Duration of action assessed with [ 11 C]raclopride in anesthetized monkeys. In: Gjedde A (ed) Physiological imaging of the brain with PET. New York: Springer-Verlag, pp 205Y Cardenas L, Houle S, Kapur S, Busto UE (2004) Oral D-amphetamine causes prolonged displacement of [ 11 C]raclopride as measured by PET. Synapse 51:27Y Sun W, Ginovart N, Ko F, et al. (2003) In vivo evidence for dopaminemediated internalization of D 2 -receptors after amphetamine: Differential findings with [ 3 H]raclopride versus [ 3 H]spiperone. Mol Pharmacol 63:456Y Ginovart N, Wilson AA, Houle S, Kapur S (2004) Amphetamine pretreatment induces a change in both D 2 -receptor density and apparent affinity: A [ 11 C]raclopride positron emission tomography study in cats. Biol Psychiatry 55:1188Y Doudet D, Holden JE (2003) Raclopride studies of dopamine release: Dependence on presynaptic integrity. Biol Psychiatry 54:1193Y Farde L, Wiesel FA, Stone-Elander S, et al. (1990) D 2 dopamine receptors in neurolepticynaive schizophrenic patients. A positron emission tomography study with [ 11 C]raclopride. Arch Gen Psychiatry 47:213Y Seeman P, Guan HC, Civelli O, et al. (1992) The cloned dopamine D 2 receptor reveals different densities for dopamine receptor antagonist ligands. Implications for human brain positron emission tomography. Eur J Pharmacol 227:139Y Terai M, Hidaka K, Nakamura Y (1989) Comparison of [ 3 H]YM with [ 3 H]spiperone and [ 3 H]raclopride for dopamine D 2 receptor binding to rat striatum. Eur J Pharmacol 173:177Y Farde L, Hall H, Pauli S, Halldin C (1995) Variability in D 2 -dopamine receptor density and affinity: A PET study with [ 11 C]raclopride in man. Synapse 20:200Y Wong DF, Wagner HN Jr, Tune LE, et al. (1986) Positron emission tomography reveals elevated D 2 dopamine receptors in drug-naive schizophrenics. Science 234:1558Y Nordstrom AL, Farde L, Eriksson L, Halldin C (1995) No elevated D 2 dopamine receptors in neurolepticynaive schizophrenic patients revealed by positron emission tomography and [ 11 C]N-methylspiperone. Psychiatry Res 61:67Y Zawarynski P, Tallerico T, Seeman P, et al. (1998) Dopamine D 2 receptor dimers in human and rat brain. FEBS Lett 441:383Y Vile JM, D Souza UM, Strange PG (1995) [ 3 H]Nemonapride and [ 3 H]spiperone label equivalent numbers of D 2 and D 3 dopamine receptors in a range of tissues and under different conditions. J Neurochem 64:940Y Malmberg A, Jerning E, Mohell N (1996) Critical reevaluation of spiperone and benzamide binding to dopamine D 2 receptors: Evidence for identical binding sites. Eur J Pharmacol 303:123Y Hall H, Wedel I, Halldin C, et al. (1990) Comparison of the in vitro receptor binding properties of N-[ 3 H]methylspiperone and [ 3 H]raclopride to rat and human brain membranes. J Neurochem 55:2048Y Breier A, Adler CM, Weisenfeld N, et al. (1998) Effects of NMDA antagonism on striatal dopamine release in healthy subjects: Application of a novel PET approach. Synapse 29:142Y Smith GS, Schloesser R, Brodie JD, et al. (1998) Glutamate modulation of dopamine measured in vivo with positron emission tomography (PET) and 11 C-raclopride in normal human subjects. Neuropsychopharmacology 18:18Y25

8 52 N. Ginovart: Imaging the Dopamine System with In Vivo [ 11 C]raclopride Displacement Studies 52. Tsukada H, Harada N, Nishiyama S, et al. (2000) Ketamine decreased striatal [ 11 C]raclopride binding with no alterations in static dopamine concentrations in the striatal extracellular fluid in the monkey brain: Multiparametric PET studies combined with microdialysis analysis. Synapse 37:95Y Tsukada H, Harada N, Nishiyama S, et al. (2000) Cholinergic neuronal modulation alters dopamine D 2 receptor availability in vivo by regulating receptor affinity induced by facilitated synaptic dopamine turnover: Positron emission tomography studies with microdialysis in the conscious monkey brain. J Neurosci 20:7067Y Sibley DR, De Lean A, Creese I (1982) Anterior pituitary dopamine receptors. Demonstration of interconvertible high and low affinity states of the D 2 -dopamine receptor. J Biol Chem 257:6351Y Seeman P, Niznik HB, Guan HC, et al. (1989) Link between D 1 and D 2 dopamine receptors is reduced in schizophrenia and Huntington diseased brain. Proc Natl Acad Sci U S A 86:10156Y Grigoriadis D, Seeman P (1985) Complete conversion of brain D 2 dopamine receptors from the high- to the low-affinity state for dopamine agonists, using sodium ions and guanine nucleotide. J Neurochem 44:1925Y Christopoulos A, Kenakin T (2002) G protein-coupled receptor allosterism and complexing. Pharmacol Rev 54:323Y Rees S, Morrow D, Kenakin T (2002) GPCR drug discovery through the exploitation of allosteric drug binding sites. Recept Channels 8:261Y Chugani DC, Ackermann RF, Phelps ME (1988) In vivo [ 3 H]spiperone binding: Evidence for accumulation in corpus striatum by agonistmediated receptor internalization. J Cereb Blood Flow Metab 8:291Y Laruelle M (2000) Imaging synaptic neurotransmission with in vivo binding competition techniques: A critical review. J Cereb Blood Flow Metab 20:423Y Chuang DM, Kinnier WJ, Farber L, Costa E (1980) A biochemical study of receptor internalization during beta-adrenergic receptor desensitization in frog erythrocytes. Mol Pharmacol 18:348Y Chuang DM (1982) Internalization of beta-adrenergic receptor binding sites: Involvements of lysosomal enzymes. Biochem Biophys Res Commun 105:1466Y Berry SA, Shah MC, Khan N, Roth BL (1996) Rapid agonist-induced internalization of the 5-hydroxytryptamine 2A receptor occurs via the endosome pathway in vitro. Mol Pharmacol 50:306Y Bhatnagar A, Willins DL, Gray JA, et al. (2001) The dynamindependent, arrestin-independent internalization of 5-hydroxytryptamine 2A (5-HT 2A ) serotonin receptors reveals differential sorting of arrestins and 5-HT 2A receptors during endocytosis. J Biol Chem 276:8269Y Mantyh PW, Allen CJ, Ghilardi JR, et al. (1995) Rapid endocytosis of a G protein-coupled receptor: Substance P evoked internalization of its receptor in the rat striatum in vivo. Proc Natl Acad Sci U S A 92:2622Y Garland AM, Grady EF, Payan DG, et al. (1994) Agonist-induced internalization of the substance P (NK1) receptor expressed in epithelial cells. Biochem J 303:177Y Sternini C, Spann M, Anton B, et al. (1996) Agonist-selective endocytosis of mu opioid receptor by neurons in vivo. Proc Natl Acad Sci U S A 93:9241Y Sternini C, Brecha NC, Minnis J, et al. (2000) Role of agonistdependent receptor internalization in the regulation of mu opioid receptors. Neuroscience 98:233Y Ng GY, Varghese G, Chung HT, et al. (1997) Resistance of the dopamine D 2 L receptor to desensitization accompanies the upregulation of receptors on to the surface of Sf9 cells. Endocrinology 138:4199Y Ito K, Haga T, Lameh J, Sadee W (1999) Sequestration of dopamine D 2 receptors depends on coexpression of G-protein-coupled receptor kinases 2 or 5. Eur J Biochem 260:112Y Kim KM, Valenzano KJ, Robinson SR, et al. (2001) Differential regulation of the dopamine D 2 and D 3 receptors by G protein-coupled receptor kinases and {beta}-arrestins. J Biol Chem 25: Barton AC, Black LE, Sibley DR (1991) Agonist-induced desensitization of D 2 dopamine receptors in human Y-79 retinoblastoma cells. Mol Pharmacol 39:650Y Itokawa M, Toru M, Ito K, et al. (1996) Sequestration of the short and long isoforms of dopamine D 2 receptors expressed in Chinese hamster ovary cells. Mol Pharmacol 49:560Y Leysen JE, Gommeren W, Laduron PM (1978) Spiperone: A ligand of choice for neuroleptic receptors: 1. Kinetics and characteristics of in vitro binding. Biochem Pharmacol 27:307Y Kohler C, Hall H, Ogren SO, Gawell L (1985) Specific in vitro and in vivo binding of 3 H-raclopride. A potent substituted benzamide drug with high affinity for dopamine D 2 -receptors in the rat brain. Biochem Pharmacol 34:2251Y Lyon RA, Titeler M, Frost JJ, et al. (1986) 3 H-3-N-methylspiperone labels D 2 dopamine receptors in basal ganglia and S 2 serotonin receptors in cerebral cortex. J Neurosci 6:2941Y Williamson RA, Strange PG (1990) Evidence for the importance of a carboxyl group in the binding of ligands to the D 2 dopamine receptor. J Neurochem 55:1357Y Neve KA (1991) Regulation of dopamine D 2 receptors by sodium and ph. Mol Pharmacol 39:570Y Presland JP, Strange PG (1991) ph dependence of sulpiride binding to D 2 dopamine receptors in bovine brain. Biochem Pharmacol 41:R9YR Al-Awqati Q (1986) Proton-translocating ATPases. Annu Rev Cell Biol 2:179Y Tycko B, Maxfield FR (1982) Rapid acidification of endocytic vesicles containing alpha 2-macroglobulin. Cell 28:643Y Davis CG, Goldstein JL, Sudhof TC, et al. (1987) Acid-dependent ligand dissociation and recycling of LDL receptor mediated by growth factor homology region. Nature 326:760Y DiPaola M, Maxfield FR (1984) Conformational changes in the receptors for epidermal growth factor and asialoglycoproteins induced by the mildly acidic ph found in endocytic vesicles. J Biol Chem 259:9163Y Yamashiro DJ, Borden LA, Maxfield FR (1989) Kinetics of alpha 2- macroglobulin endocytosis and degradation in mutant and wild-type Chinese hamster ovary cells. J Cell Physiol 139:377Y Borden LA, Einstein R, Gabel CA, Maxfield FR (1990) Acidificationdependent dissociation of endocytosed insulin precedes that of endocytosed proteins bearing the mannose 6-phosphate recognition marker. J Biol Chem 265:8497Y Koh PO, Undie AS, Kabbani N, et al. (2003) Up-regulation of neuronal calcium sensor-1 (NCS-1) in the prefrontal cortex of schizophrenic and bipolar patients. Proc Natl Acad Sci U S A 100:313Y Bergson C, Levenson R, Goldman-Rakic PS, Lidow MS (2003) Dopamine receptor-interacting proteins: The Ca 2+ connection in dopamine signaling. Trends Pharmacol Sci 24:486Y492