B Academy of Molecular Imaging 2005 Mol Imaging Biol (2005) 7:45Y52 Published Online: 30 December 2004 DOI: 10.1007/s11307-005-0932-0

REVIEW ARTICLE Imaging the Dopamine System with In Vivo [11C]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 D2-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

D2-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 D2-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 D2 receptors was recently suggested.

11 Key words: Amphetamine challenge, [ C]raclopride, D2-receptors competition, Internalization, Positron emission tomography, Schizophrenia

Background DA [1, 2]. The view that addiction involves an enhanced responsiveness of the striatal DA system has been challenged recent important development in neurosciences is the by the finding of a decreased release of striatal DA in cocaine Aability to assess changes in the levels of endogenous abusers as compared to controls [3]. In addition, studies neurotransmitters in living humans using positron emission showing that behavioral tasks, such as playing video game tomography (PET) and single photon emission computed or writing, and placebo response induce an increased release tomography (SPECT) imaging. Using these techniques, the of striatal DA [4Y6] illustrated the ability of in vivo imaging level of endogenous dopamine (DA) has been assessed in a to detect physiological changes in DA levels. number of pathological and physiological conditions, and While these techniques are being extensively used they do this is having important scientific consequences. For not provide a direct measurement of endogenous DA levels. example, the DA hypothesis of schizophrenia has experienced In fact, changes in endogenous levels of DA are inferred from a renewal of interest from the observation that patients with changes in the binding of [11C]raclopride/[123I]IBZM to the schizophrenia show an abnormally high level and release of DA D2 receptors. The standard observation is that drugs that increase the levels of endogenous DA decrease the binding of these radioligands to postsynaptic D2 receptors, whereas Correspondence to: Nathalie Ginovart, PhD; e-mail: nathalie.ginovart@ the opposite effect is observed with drugs that decrease camhpet.ca endogenous DA levels. Assessment of changes in endoge- 46 N. Ginovart: Imaging the Dopamine System with In Vivo [11C]raclopride Displacement Studies

nous DA levels is based on the hypothesis that the changes DA transporter with GBR12909 attenuates amphetamine- 11 in D2-radioligand binding are due to competition with DA. induced decrease in [ C]raclopride binding. The model is However, the validation of this hypothesis has not been further supported by some doseYresponse data. Studies of fully achieved yet and an accumulating number of studies combined microdialysis/raclopride effect show that the even challenge it. This article reviews the theoretical basis administration of amphetamine dose-dependently increased for the DA competition hypothesis, the in vivo and in vitro DA concentration and decreased in vivo [123I]IBZM [21] 11 evidences supporting a noncompetitive action of DA on D2- and [ C]raclopride [22] binding in the striatum. A direct radioligand binding, and discusses possible mechanism(s) effect of endogenous DA on [11C]raclopride is also underlying this noncompetitive interaction. suggested from experiments showing that unilateral stim- ulations of the nigrostriatal neurons, which induce only transient changes in striatal DA levels, induced only 11 Endogenous DA Levels Affect transient changes in [ C]raclopride binding [23]. The Raclopride Binding—From an Artifact exquisite temporal resolution of the radioligand binding to a Method response to the stimulation-induced changes in DA levels also suggests that changes in [11C]raclopride binding are In 1989, Seeman et al. [7] and Ross and Jackson [8] directly related to fluctuations in extracellular DA levels. demonstrated that [3H]raclopride, a ligand used to measure These studies confirm that the raclopride effect is mediated D2 receptors, was sensitive to endogenous DA, whereas by enhanced DA releaseVbut they do not tell us how 3 [ H]NMSP, another ligand used to measure D2 receptors, increase in DA release leads to a decrease in radioligand was not similarly vulnerable [7]. At first this sensitivity to binding. endogenous DA was seen as a liability for [11C]raclopride [9]. However, soon this liability was seen as the first opportunity to measure endogenous DA levels in humans by 11 Evidence in Favor of the DA looking at variations in the level of binding of [ C]raclo- Competition Model pride. Since that first demonstration, numerous studies in humans and animals have confirmed that when levels of One of the hallmarks of a true competition, i.e., when two synaptic DA are increased (by the use of amphetamine, agents are reversibly competing for the same receptor, is methylphenidate, GBR12909, cocaine, and fenfluramine) that it shows up as a change in the affinity of each [10Y15], the level of binding of [11C]raclopride as measured receptor, but does not result in a change in total receptor by PET and [123I]IBZM as measured by SPECT is number. The DA competition model thus predicts that decreased. Conversely, when the levels of DA are decreased changes in [11C]raclopride binding induced by changes in using reserpine and !-methyl-p-tyrosine [16, 17], the levels DA levels reflect a change in affinity (i.e., 1/KD) but not in 11 123 of [ C]raclopride and [ I]IBZM are increased. Thus, the receptor density (i.e., Bmax). This theory is confirmed by fact that changes in DA are associated with changes in in vivo PET data in nonhuman primates showing that [11C]raclopride is a very well replicated finding. depletion of endogenous DA using reserpine, which leads to a marked increase in [11C]raclopride binding, induces an increase in D2-receptor affinity with no evident change in ! The DA Competition—The Initial Bmax [17]. Similarly, depletion of striatal DA using - methyl-para-tyrosine in rodents has been reported to Explanation for the BRaclopride 123 Effect[ increase [ I]IBZM binding with no detectable effects on Bmax [16]. These studies thus showed that the effect of DA Competition with endogenous DA has been proposed depletion paradigms on D2-radioligand binding is due to as the underlying mechanism to explain the changes removal of endogenous DA rather than to a noncompetitive 11 123 in [ C]raclopride and [ I]IBZM binding following action (such as D2-receptor upregulation) and thus confirm manipulation changes in endogenous DA. One of the first a competitive action of endogenous DA at baseline requirements for the competition model is to show its conditions. dependence on endogenous dopamineVand this indeed is the case. Amphetamine is known to raise extracellular DA levels by inducing DA efflux from the intracytoplasmic Inconsistencies and Incompatibilities stores to the synaptic space via reverse transport of the DA with the DA Competition Model transporter [18, 19]. Manipulations that counteract the effect of amphetamine on DA release also counteract its raclo- Although the aforementioned data support the DA competi- pride effect. For example, Innis et al. [11] showed that tion model theory, a number of inconsistencies have, depleting DA stores with reserpine abolished the effect of however, emerged that questioned the validity of this subsequent amphetamine administration on [123I]IBZM model. These inconsistencies are summarized in the binding. Villemagne et al. [20] showed that blocking the following. N. Ginovart: Imaging the Dopamine System with In Vivo [11C]raclopride Displacement Studies 47

Failure to Detect DA Competition with amphetamine on [11C]raclopride binding resulted from a Radioligands Other Than Benzamides dual mechanism: a decrease in the density of striatal D2 receptors and a decrease in their affinity, which is probably The DA competition model is consistent with data obtained due to competition. These results, which showed that the with benzamide radioligands (such as raclopride, IBZM, amphetamine-induced release of DA leads to a decrease in fallypride, FLB457), which are always affected by changes raclopride-labeled receptor rather than just a change in in endogenous DA [11Y13, 16, 17, 24Y26]. In contrast, the receptor affinity, directly refute the assumption of simple binding of butyrophenone compounds (such as spiperone, competition and suggested that noncompetitive interactions NMSP, pimozide) to D receptors [27Y29]aswellas 2 are involved in this effect. However, conflicting PET benzazepine compounds (such as SCH23390, NNC112, studies performed in primates recently showed that a single NNC756) to D receptors [30Y32] shows either no change 1 dose of methamphetamine (2 mg/kg; i.p.) induced an or even paradoxical increase in binding in the face of higher increase in [11C]raclopride KV with no significant change DA. Thus, it seems that this effect is peculiar to the D in B [39]. The reason for these discrepant results is not benzamides, rather than being a general principle governing max clear. However, it might be, at least partly, explained by all neurotransmitters and ligands. noticeable differences between the experimental protocols used in the two studiesVi.e., intraperitoneal vs. intravenous Temporal Discrepancy Between Changes in DA route of amphetamine administration, multiple radioligand Levels and in Radioligand Binding concentration assay requiring six to seven hours vs. three hours of postamphetamine data analysis; [11C]raclopride Another inconsistency in the validity of the DA competition bolus plus infusion paradigm vs. bolus paradigm, quantifi- model is the temporal discrepancies between changes in cation of [11C]raclopride BP values using the Logan vs. the radioligand binding as measured with PET and SPECT and transient equilibrium method, in Doudet’s vs. Ginovart changes in DA levels as measured with microdialysis. et al.’s study, respectively. 11 Indeed, the time course of changes in [ C]raclopride/ Thus, although the displacement of [11C]raclopride is a 123 [ I]IBZM binding far outlives the changes in DA levels, well-confirmed finding, the competition model is difficult calling into question the idea of direct competition. Micro- to reconcile with some of the aforementioned studies dialysis studies have shown that amphetamine elevates showing that noncompetitive interactions also play an striatal DA levels with peak values occurring within 20 important role in this phenomenon. In addition, there are minutes and that DA levels return to control values within two other problems with the competition model: Why is the 120 minutes [21, 33, 34]. Amphetamine-induced elevation displacement seen only with benzamides and not with other in DA levels is thus transient. This contrasts with the long- ligands? Why does the displacement outlast the levels of 11 123 lasting decrease in both [ C]raclopride and [ I]IBZM DA? binding observed in vivo. Indeed, [123I]IBZM binding has been shown to be still reduced at six hours following a single dose of amphetamine in nonhuman primates [21]. Proposed Theories to Explain the [11C]Raclopride binding is still reduced up to six hours fol- Failure of the DA Competition Model lowing a single dose of amphetamine in nonhuman primates The Lower Affinity of Raclopride Makes It More [35] and in humans [36]. Such an inconsistency suggests Sensitive Than [11C]NMSP that factors other than endogenous DA play a significant role in the alteration in radioligand binding observed fol- It has been proposed that the lower affinity of benzamides lowing DA manipulation. 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 Amphetamine Causes a Decrease in 11 V logic of receptor theory and laws of competition this is not [ C]raclopride Binding Site Number A Finding the case. In fact, Farde et al. [40] have shown that the Incompatible with the Competition Model vulnerability of a radiotracer to a competitive inhibitor is A recent ex vivo binding study performed on rat striatal not dependent on its affinity as long as binding quantifica- homogenates recently showed that increasing DA levels tion is performed at tracer dose. Therefore, differences in 3 affinity, by itself, are not a reasonable explanation for why with amphetamine causes a decrease in [ H]raclopride Bmax, rather than a change in apparent affinity, a finding that some radioligands show displacement and others do not. directly refutes the assumption of simple competition [37]. Because of the potential limitations of extrapolating in vitro Raclopride and Spiperone Bind to Different Sites binding data to the in vivo situation, we recently inves- of the D Receptor tigated whether amphetamine pretreatment also causes a 2 11 decrease in [ C]raclopride Bmax as assessed in vivo in cats Previous data in the literature suggest that benzamides and using PET [38]. This study showed that the effect of butyrophenones might bind to different binding on D2 48 N. Ginovart: Imaging the Dopamine System with In Vivo [11C]raclopride Displacement Studies

receptors. For example, the density of D2-receptor sites magnitude of radiotracer displacement and the magnitude measured in vitro using [3H]raclopride is about twofold of change in DA levels were different for direct dopamine higher than that measured using [3H]spiperone in rat striata enhancers (GBR12909 and methamphetamine) and for homogenates [41, 42]. Similarly, D2-receptor density values indirect dopamine modulators (benztropine and ketanser- measured in the human striatum in vivo range from 30 to 40 ine). These experimental data thus support the view that, nmol/ml when using [11C]raclopride [40, 43] and from 15 to even for benzamides, the alternation of radiotracer binding 25 nmol/ml when using [11C]NMSP [44, 45]. These as measured by in vivo imaging techniques is also affected discrepancies between D2-receptor densities have led to by the synaptic concentration of DA. the proposal that benzamides and butyrophenones do not bind to the same configuration of the D2 receptor. Noncompetitive Models Might be an Butyrophenones may bind primarily to the monomer form, Alternative to the Competition Model whereas benzamides may bind to both the monomer and dimer forms of the receptor [41, 46]. Differences in the If competitive interaction of DA with D2 receptors is ruled monomerYdimer equilibrium could thus explain the differ- out, then three possible mechanisms can be evoked to 11 ences in D2-receptor densities revealed by the two classes of explain the amphetamine-induced loss of [ C]raclopride compounds. Although this BmonomerYdimer theory[ itself binding sites. First, DA and [11C]raclopride may bind to two is the subject of controversy, most people in the field, distinct states of the D2 receptor (D2 high and D2 low) [54] however, acknowledge the existence of a specific Bbuty- for which [11C]raclopride has equal affinity but for which rophenone binding site [47, 48].[ Along with this hypoth- DA has high and low affinity, respectively (two-state esis, Hall et al. [49] showed that DA has a lower affinity for model). Tight DA binding to the D2-high sites would the NMSP binding sites than for the raclopride binding occlude these sites for the binding of [11C]raclopride and sites. A remote localization of the NMSP-butyrophenone would explain the apparent decrease in D2-receptor density binding sites may limit its access by endogenous DA and obtained after amphetamine. This two-state model has could explain the lack of effect of DA manipulations on in already been proposed by Seeman et al. [7, 55] to explain 3 11 3 vitro [ H]spiperone and in vivo [ C]NMSP bindings. the apparent fall in [ H]raclopride Bmax obtained in vitro However, it cannot explain the other discrepancies (tempo- after incubation of striata membranes with exogenous DA. ral discordance observed with [11C]raclopride, lack of effect This two-state model, however, does not seem to apply to 3 3 on D1 ligands, paradoxical increases in [ H]spiperone explain the decrease in [ H]raclopride Bmax observed after binding in some reports). amphetamine on rat striata membranes [37]. Indeed, the 3 While flaws in the competition model have been effect of amphetamine on [ H]raclopride Bmax was not recognized before, no comprehensive model has emerged reversed by treatment of the striata membranes with to replace it. While radioligand differences in affinity and/or Gpp(NH)p (5V-guanylylimidodiphosphate), which converts binding site could play a role, they are unable to explain the D2 receptors from their high- to their low-agonist affinity discrepancies and paradoxes observed. state [56], suggesting that the two-state model was not a reasonable explanation for this effect. Second, DA may bind Other Discrepancies Between Changes in DA to an allosteric site on the receptor, which is not recognized Levels and in Radioligand Binding by the radioligand but which can modulate its binding site (allosteric model). Dopamine binding to D2 receptors may Previous studies have shown that ketamine, a noncompeti- cause a conformational change in the receptor that would 11 tive N-methyl-D-aspartate antagonist that induces psychotic distort or even mask [ C]raclopride binding sites. Although symptoms in humans, decreased the striatal binding of an allosteric modulation of D2 receptors by endogenous DA [11C]raclopride in human subjects [50, 51]. A subsequent cannot be excluded, it seems unlikely as most of the com- study in nonhuman primates showed that this ket- pounds that have been identified thus far as modulators are amine-induced decrease in [11C]raclopride binding was neither synthesized endogenously nor naturally encountered dose-dependent but induced no significant changes in [57, 58]. Third, the massive release of DA triggered by extracellular DA concentration as measured with micro- amphetamine may cause an internalization of D2 receptors dialysis [52]. In the same way, scopolamine (a muscarinic (internalization model). A DA-promoted internalization of 11 antagonist) decreased the binding of [ C]raclopride in a striatal D2 receptors following amphetamine pretreatment dose-dependent manner but induced no significant changes has already been suggested in previous ex vivo studies in in extracellular DA concentration as measured with micro- rodents [37, 59]. In these studies, subcellular fractionation dialysis [53]. The same authors also demonstrated that experiments performed using [3H]raclopride and/or [3H]spi- administration of the indirect DA modulator benztropine (a perone indicated that amphetamine resulted in a redistrib- 2 muscarinic cholinergic antagonist) or ketanserine (a 5-HT ution of D2 receptors from the cell surface to intracellular antagonist) also increased the striatal levels of DA and compartments and that the loss of D2 receptors from the decreased [11C]raclopride binding in a dose-dependent plasma membrane was likely responsible for the decrease in manner [22]. However, the relationships between the [3H]raclopride binding observed after amphetamine. N. Ginovart: Imaging the Dopamine System with In Vivo [11C]raclopride Displacement Studies 49

Although a two-state model or an allosteric modulation induced internalization of D2 receptors can be measured in 3 of D2 receptors by DA cannot be excluded, the non- vitro by measuring the loss of [ H]sulpiride binding from competitive interaction evidenced between endogenous the cell surface [70, 73]. Evidence also exists that such a 11 3 3 DA and [ C]raclopride for binding on D2 receptors is differential response of [ H]raclopride and [ H]spiperone to likely related to a DA-promoted internalization of the D2-receptor internalization occurs in vivo. Indeed, while the receptors after amphetamine. binding of both radioligands decreased at the cell surface following amphetamine administration, only [3H]spiperone binding showed an increased accumulation in the intra- Agonist-Induced Internalization of D2 Receptors: An Alternative to the cellular endosomal compartment [37, 59]. These data thus suggest that although [3H]raclopride and [3H]spiperone both Simple Competition Model 3 bind to D2 receptors, [ H]raclopride does not have access to Laruelle [60] was the first to propose the internalization a subpopulation of D2 receptors that are located in intra- model as a potential alternative to the competition model cellular compartments but that are still accessible to 3 theory. A DA-promoted internalization of D2 receptors [ H]spiperone. This finding compares with a previous study following amphetamine could account for the flaws pre- already showing that part of the D2-binding sites found in viously noticed in the competition model theory. striata homogenates is exclusively bound by NMSP and can Especially, it might explain the temporal persistence of only be partially accessed by raclopride [49]. Taken the raclopride effect after amphetamine as well as why it is together, these data suggest that the subset of D2-binding observed only with raclopride-like and not with sites exclusively bound by NMSP and not accessed by spiperone-like radioligands. raclopride could correspond to internalized receptors, and the differential ability of spiperone-like and raclopride-like Receptor Internalization: A Regulatory Process of ligands to access these receptors might explain their differ- Cell Functional Activity ent response to internalization.

Agonists not only bind and stimulate receptors, they also Agonist-Induced Internalization Explains initiate a shift in their position from cell surface to Current Paradoxes intracellular compartments, a process that is termed inter- nalization of receptors. This rapid internalization process is The internalization hypothesis can account for the two major associated with the appearance of receptors in intracellular problems of the competition model. First, by delinking vesicles and with their concomitant disappearance from the [11C]raclopride-binding decrease from online DA competi- cell surface membrane. Internalized receptors are conveyed tion, it can explain the temporal persistence of the to the endosomes, from where some may be recycled back [11C]raclopride effect that has been observed after amphet- to the cell surface, whereas others are transported to the amine. As DA internalizes receptors, they become unavail- lysosomes and degraded. This internalization regulates the able to [11C]raclopride not because of competition, but number of receptors at the cell surface, thereby modulating because of shift in their compartmental localization. Thus, the responsiveness of the cell to further stimulation. even if DA levels came back to baseline, [11C]raclopride Receptor internalization has now been observed for a levels will not come back to baseline until the receptors are variety of neuronal receptors [61Y68]. recycled to the surface. Thus, it is the receptor dynamics and not DA competition which explains the prolonged 11 Raclopride and Spiperone Show Differential decrease in [ C]raclopride. The model can also account for Responses to Internalization the raclopride/spiperone differences. Indeed, agonist-medi- ated D2-receptor internalization could account for both the Agonist-promoted internalization of D2 receptors is now a decrease in benzamide binding and the lack of changes in well-established phenomenon that has been shown to occur butyrophenone binding reported following amphetamine. in vitro in cells expressing D2 receptors [69Y71]. Barton et The internalization model predicts that released DA al. [72] were the first to propose that agonist-mediated D2- promotes a shift (i.e., internalization) of the D2 receptors receptor internalization differently affects radioligand bind- from the cell membrane to intracellular compartments. Two ing depending on the ligand lipophilicity. These authors important differences in the physicochemical properties of showed that preexposure of retinoblastoma cells to DA two compounds could account for their different abilities to resulted in decreased binding of the hydrophilic benzamide access or to bind internalized receptors. First, raclopride and [3H]iodosulpiride with no change in the binding of the spiperone greatly differ in their lipophilicity, raclopride lipophilic butyrophenone [3H]NMSP. They suggested that being less lipophilic than spiperone and NMSP [74Y76]. [3H]iodosulpiride only detects receptors present on the cell The low lipophilicity of raclopride-like ligands would limit surface, whereas [3H]NMSP detects both the receptors their access to internalized receptors. Dopamine-promoted present on the cell surface and those sequestered. Since internalization of D2 receptors would not affect spiperone- this pioneering study, several studies have shown that DA- like ligand binding since these ligands are lipophilic enough 50 N. Ginovart: Imaging the Dopamine System with In Vivo [11C]raclopride Displacement Studies

to cross the cell membrane and access these newly certainly true for most clinical PET studies performed using internalized receptors. Second, the sensitivity of raclopride this paradigm. However, it should be kept in mind that and spiperone binding to pH conditions is also very different. pathological states in which a dysregulation of receptor The affinity of substituted benzamides such as raclopride for trafficking exists may show either an exaggerated or an 11 D2 receptors is highly reduced at high pH conditions, attenuated [ C]raclopride response to amphetamine. For whereas that of butyrophenones such as spiperone is more instance, recent data [86, 87] support the hypothesis for a moderately affected by such environmental conditions dysregulation of the agonist-induced internalization process [77, 78]. For instance, reduction of pH from eight to six that modulates D2-receptor signaling in the brain of has been shown to have little effect on [3H]spiperone binding schizophrenic and bipolar patients. If, as suggested by these to bovine caudate nucleus membranes, whereas the same pH studies, D2-receptor internalization is indeed reduced in reduction led a substantial reduction in [3H]sulpiride binding schizophrenics when compared to normal controls, the [79]. The different pH dependencies of butyrophenones and higher amphetamine/[11C]raclopride effect observed in substituted benzamides for binding D2 receptors might these patients may mean that their drug-induced release of explain their different abilities to bind internalized receptors. DA is even greater than that previously postulated on the Indeed, upon internalization, receptors are delivered to basis of the DA competition theory alone. Conversely, endosomes that are acidified by ATP-dependent proton diseases in which D2-receptor internalization is increased pumps [80, 81]. One of the major functions of endosomes may show an exaggerated [11C]raclopride response to acidification is to dissociate ligands from their receptors, amphetamine when compared to normal controls despite a after which many receptors are recycled to the cell surface similar effect of the drug on DA outflow. Understanding the while the ligands are degraded [82, 83]. Several ligands true mechanism underlying the abnormally high amphet- dissociate from their receptors with only slight acidification amine/[11C]raclopride effect in schizophrenia may thus (pH approx. 6.8) of the endosomal compartment, whereas uncover a novel mechanism underlying the excessive some other ligands require more acidic conditions (pH G 6.0) stimulation of D2 receptors postulated in this disorder. to be released from their receptors [83Y85]. It thus appears Indeed, an alteration of D2-receptor internalization would be that subtle differences in endosomal pH can have a dif- predicted to result in overactive D2-receptor signaling under ferential impact on endosomal ligandYreceptor dissociation stimulation, as predicted by the DA hypothesis of schizo- depending on the ligand. The high sensitivity of raclopride- phrenia, and could occur without any detectable change in like ligand to high pH conditions might thus preclude their the baseline levels of D2 receptors. If impairment of D2- binding to internalized receptors, whereas the relative receptor trafficking rather than just impairment of DA insensitivity of butyrophenones to such conditions would release is proven to be involved in this effect in schizo- preserve their binding to receptor even in the high pH phrenics, it may open new leads for the development of endosomal conditions. therapeutic approaches targeted at modifying D2-receptor trafficking in this disorder.

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