On the relationship of first-episode psychosis to the amphetamine-sensitized state: a dopamine D2/3 receptor agonist radioligand study

Schizophrenia is characterized by increased behavioral and neurochemical responses to dopamine-releasing drugs. This prompted the hypothesis of psychosis as a state of “endogenous” sensitization of the dopamine system although the exact basis of dopaminergic disturbances and the possible role of prefrontal cortical regulation have remained uncertain. To show that patients with first-episode psychosis release more dopamine upon amphetamine-stimulation than healthy volunteers, and to reveal for the first time that prospective sensitization induced by repeated amphetamine exposure increases dopamine-release in stimulant-naïve healthy volunteers to levels observed in patients, we collected data on amphetamine-induced dopamine release using the dopamine D2/3 receptor agonist radioligand [11C]-(+)-PHNO and positron emission tomography. Healthy volunteers (n = 28, 14 female) underwent a baseline and then a post-amphetamine scan before and after a mildly sensitizing regimen of repeated oral amphetamine. Unmedicated patients with first-episode psychosis (n = 21; 6 female) underwent a single pair of baseline and then post-amphetamine scans. Furthermore, T1 weighted magnetic resonance imaging of the prefrontal cortex was performed. Patients with first-episode psychosis showed larger release of dopamine compared to healthy volunteers. After sensitization of healthy volunteers their dopamine release was significantly amplified and no longer different from that seen in patients. Healthy volunteers showed a negative correlation between prefrontal cortical volume and dopamine release. There was no such relationship after sensitization or in patients. Our data in patients with untreated first-episode psychosis confirm the “endogenous sensitization” hypothesis and support the notion of impaired prefrontal control of the dopamine system in schizophrenia.


Introduction
Several lines of evidence demonstrate increased subcortical dopamine (DA) transmission in psychotic patients with schizophrenia (SCZ). Positron emission tomography (PET) studies show increased dopamine synthesis capacity and heightened behavioral and neurochemical responses towards DA-releasing compounds [1][2][3][4][5][6] . The common mechanism of action of all antipsychotic drugs-reducing DA transmission at postsynaptic D 2/3 receptors-confirms the key role of DA signaling in psychosis 7 . While the pathophysiological basis of DA dysfunction in SCZ remains unknown, current versions of the DA theory of SCZ 8 posit that upstream pathogenic factors converge on subcortical DA pathways to mediate the expression and intensity of psychotic symptoms [9][10][11] . Sensitization denotes a process by which repeated exposure to a stimulus induces a progressive increase in responses to the very same stimulus 12,13 . When repeatedly administered, D-amphetamine (AMPH) induces behavioral sensitization and a progressive amplification in AMPH-induced DA release [14][15][16] . Since psychotic patients show elevated responses to AMPH without any prior drug exposure 1 , psychosis has been conceptualized as a state of "endogenous sensitization" 10,17,18 . The prefrontal cortex (PFC), origin of reciprocal regulatory connections to subcortical DA neurons 19 , has often been found to be structurally and functionally impaired in SCZ 20,21 . Earlier studies have shown particularly strong relationships between subcortical DA metabolism and the lefthemispheric dorso-lateral PFC (DLPFC) and inferior frontal gyrus (IFG) in SCZ and subjects at-risk mental state for SCZ 22,23 . In order to collect experimental evidence supporting this concept, we used PET and the DA D 2/3 receptor agonist radioligand (+)-4-propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho [1,2-b] [1,4]oxazin-9-ol ([ 11 C]-(+)-PHNO) 24 for measuring AMPH-induced changes in D 2/3 receptor binding, semi-quantitative index of DA release, in drug-naïve patients with first-episode psychosis (FEP). Healthy volunteers (HV) were studied before and after exposure to a mildly sensitizing regime of repeated AMPH administration. In order to identify upstream pathogenic mechanisms of psychotic hyperdopaminergia, we analyzed volumetric parameters in the PFC for their relationship to indices of subcortical DA release.

Materials and methods
All procedures of this study (Clinical Trial Registry: EUDRACT 2010-019586-29) were approved by the ethics committee of the Medical University of Vienna and pertinent federal regulatory authorities. After a test-retest phase ensuring reliability of local [ 11 C]-(+)-PHNO PET imaging procedures (six male HV), 42 HV and 29 antipsychotic-naïve (or minimally exposed) patients with FEP capable of providing informed consent were recruited between 2013 and 2017; HV were required to be of good health based on physical examination, history, ECG, and laboratory results. Exclusion criteria comprised any intake of drugs of abuse except nicotine, caffeine, and alcohol (occasional use only), five or more stimulant exposures lifetime, psychiatric disorders (evaluated with the DSM-IV based M.I.N.I. questionnaire 25 ), having a first-degree relative with schizophrenia or bipolar disorder or having any contraindications against receiving a PET, MRI or damphetamine; in addition, FEP patients were required to have a minimum Positive and Negative Symptom Scale (PANSS 26,27 ) score of 55 with >3 on at least two PANSS psychosis items or >4 on one psychosis item; no or minimal lifetime exposure to antipsychotics; no lifetime exposure to antipsychotic depot preparations; no antipsychotics within two weeks prior to scanning (for details see Supplemental Material). Diagnoses of FEP in SCZ according to DSM-IV were independently made by at least two experienced psychiatrists (N.M.; N.P-R., S.K., M. W.). Three patients were previously exposed to olanzapine, aripiprazole, or quetiapine. Antipsychotics had been discontinued at least two months prior to inclusion without attaining a predefined threshold of two treatment weeks or lifetime exposure up to 50 mg haloperidolequivalent. Three patients had a history of antidepressant treatment discontinued at least two months prior to inclusion. During the study, eleven patients required symptomatic treatment for psychomotor agitation or insomnia (lorazepam 1-10 mg or zolpidem 10 mg per day). Data sets (complete or partial) of 28 + 6 HV and 21 FEP patients entered final analysis (see Supplemental  Material and Supplementary Table 1 for full details).

Study setup and prospective sensitization
First, drug-naïve HV (HV UNSENS ) underwent an AMPH-free [ 11 C]-(+)-PHNO PET scan (baseline 1 , PET 1 ). On a separate day less than five 5 days apart, a second scan (PET 2 ) was performed 90-120 min after oral administration of 0.4 mg/kg body weight AMPH (Attentin ® , MEDICE Arzneimittel GmbH, Iserlohn, D), a time point at which subjective effects are peaking and blood levels are still rising (blood levels are highest after 3-4 h 28 ). The AMPH dose was chosen according to earlier studies showing that dosages between 0.3 and 0.5 mg/kg body weight induce reliable reductions in [ 11 C]-(+)-PHNO BP ND values 29,30 . The dose corresponds to a low to medium dose used for treating attention deficithyperactivity disorder in children. Administration of AMPH at this same dose was repeated two more times at intervals of two days. Two to four weeks thereafter, the now sensitized HV (HV SENS ) underwent another AMPHfree scan (baseline 2 , PET 3 ), and within five days, a fourth scan (PET 4 ) preceded by AMPH dose four (Fig. 1, lower panel).
Patients with FEP received one baseline scan (PET 1 ) and one AMPH-scan (PET 2 ; protocol as above). AMPH led to temporary increases in heart rate and blood pressure ( Supplementary Fig. 1). Occasionally, participants reported mild headache and insomnia the night after AMPH administration. Neither HV nor patients experienced any serious AMPH-related adverse events.

Behavioral and hormonal measurements
Subjective AMPH effects were recorded using the drug effects questionnaire 32 and the subjective states questionnaire (SSQ) 33 . The PANSS scale 26 was administered by certified raters for measuring baseline psychopathology in FEP patients, the brief psychiatric rating scale (BPRS 34 ) was used to measure AMPH-induced changes in psychopathology. Blood for serum AMPH levels was collected at the beginning of PET scans. Heart rate, blood pressure (systolic and diastolic were determined repeatedly before and during PET scans (see Supplemental Material).

Image analysis Analysis in regions of interest
Frame-wise motion correction and co-registration of attenuation-corrected average PET images to T1weighted MRIs was performed using AFNI software. PET images of two FEP patients who did not undergo MR imaging (see above) were co-registered to normalized [ 11 C]-(+)-PHNO template images (one each for nointervention and AMPH scans) created by averaging spatially normalized PET images (early and late low-contrast PET    frames omitted) of 15 HV. Individually optimized bilateral regions of interest (ROIs) were obtained for the caudate nucleus (CAU), putamen (PUT), ventral striatum (VST), and cortical cerebellum (CER) using the automated image analysis software ROMI 35 as described elsewhere 30 . Since automated algorithms provided no satisfactory ROI delineation in globus pallidus (GP) and substantia nigra/ ventral tegmental area (SNVTA), GP and SN/VTA were delineated manually by a single rater on individual PD MR images (fused with PET images for aiding delineation of SNVTA). ROI delineation was evaluated independently by a second rater visually controlling for anatomical fit and assessing outliers and time drift in ROI sizes. Decaycorrected time-activity curves (TACs) were extracted from the dynamic sequence. The simplified reference tissue model (SRTM2) 36,37 implemented in PMOD software (Version 3.6; PMOD Technologies Ltd, Zurich, Switzerland) was used to derive binding potential (BP ND ) values in each ROI. Cerebellar cortex (CER) avoiding midline structures served as reference region since it is virtually devoid of DA D 2/3 receptors in humans [38][39][40] .

Parametric analysis
Voxel-wise BP ND maps were calculated using the PMOD 3.6 SRTM basis function implementation 41 . TACs previously derived in CER (low-binding) and VST (highbinding) were used to optimize iterative model fitting procedures. Effects of AMPH and AMPH-sensitization were analyzed using the AFNI programs 3dttest++ and 3dLME.

Volumetric analyses
T1-weighted MR images were processed using Freesurfer 6.0 software (http://surfer.nmr.mgh.harvard.edu/). Cortical gray voxels were allocated to a set of regions predefined in the Destrieux atlas 42 using a Bayesian algorithm. Skull-stripping, gross accuracy of delineation, and surface modeling were quality-controlled by visual inspection of processed images.

Statistical analysis
The sample size of this study was planned according to data collected with antagonist radioligands in sensitization and patients with schizophrenia [3][4][5]15 . Analysis of differences in DA release (ΔBP ND ) between HV before and after sensitization was carried out using ΔBP ND as dependent variable and group (FEP vs. HV UN-SENS, and FEP vs. HVSENS , respectively) as fixed variable; ROI was entered as random variable. For analyzing receptor binding, BP ND was entered as dependent variable, subject status (HV UNSENS , HV SENS , FEP) as fixed between-group factor, scan condition (AMPH yes/no) as fixed repeated factor, and ROI as fixed or random repeated factor. MLM analyses testing the effects of covariates (PFC volume parameters) were carried out analogously. All main effects and all relevant interactions were entered into the respective models. The subject-identifier variable was entered as random factor where appropriate. Two-tailed t-tests (paired where appropriate) were used for post-hoc tests after assuring normal distribution. Correlations were calculated using Pearson product moment (r) or, if appropriate, Spearman rank sum (rho) correlation coefficients. Results confirming the main a-priori study hypotheses (Figs. 1, 2) were not corrected for multiple comparisons. More exploratory results (Figs. [3][4][5] were carried out using ROI-based and parametric methods (voxel-wise maps for PET images, vertex-wise analysis of MR images) for independent confirmation. Results of the ROI-based analysis on the relationship between PANSS items and BP ND values ( Fig. 3; 30 PANSS items, 5 ROIs, two hemispheres and on/off AMPH) and the relationship between regional cortical volumes as implemented in Freesurfer 6.0 software 42 and ΔBP ND ( Fig. 4; 33 cortical regions, 5 ROIs, two hemispheres; three groups) were Bonferroni corrected, resulting in adjusted significance levels of p corr1 = 0.00008 and p corr2 = 0.00005, respectively.

DA release in FEP and sensitization
The concept of "endogenous sensitization" in psychosis 17 predicts that the difference in AMPH-induced DA release between FEP patients and HV should disappear or substantially diminish after HV are sensitized to AMPH. Analysis of AMPH-induced changes in [ 11 C]-(+)-PHNO binding (ΔBP ND ) showed significant differences between unsensitized HV and FEP (HV UNSENS vs. FEP: F (1;184.04) = 11.6, p = .0008: Fig. 1a). After HV were sensitized, groups were no longer significantly different (HV SENS vs. FEP: F (1;175.6)) = 0.99, p = .32; Fig. 1a, Supplementary Table 2). Including alcohol and nicotine consumption and sex as covariates into the model did not relevantly alter results (HV UNSENS vs. FEP: F (1;139.9) = 11.6, p = 0.0008, see Supplemental Material for details). In good agreement with the ROI-based analysis, parametric ΔBP ND maps showed the most robust effects of sensitization in the VST (Fig. 1b). Indicating behavioral sensitization in HV SENS , repeated AMPH-administration induced progressive enhancement of subjective AMPH effects up to levels observed in FEP (Fig. 1c, Supplementary Fig. 2).
The same model, when applied to FEP and HV after sensitization, did no longer show a significant two-way interaction between group and condition (F (1;275.8) = 0.6, p = 0.42). However, the group*ROI (F (4;123) = 11.2, p = 9.4 × 10 −8 ), and a group*condition* ROI interactions remained significant, (F (8;123.1) = 5.5, p = 5 × 10 −5 ; Fig. 2a, b). At least in part, this is due to the fact that irrespective of AMPH pretreatment, D 2/3 receptor binding was consistently lower in FEP than in HVs in neo-striatal ROIs (CAU, PUT, VST) but higher in GP (and trend-wise also in SNVTA; Fig. 2b; Supplementary Table 3). Baseline D 2/3 receptor binding and sensitization showed a significant positive correlation for all regions in HV (see Supplementary Fig. 3). An analysis of functional subdivisions of the basal ganglia as previously published did not reflect our findings (Supplementary Fig. 4).
Indices of DA release were positively correlated with behavioral AMPH effects (see Supplemental Material). It is known that benzodiazepines might interact with dopamine neurotransmission 43 . Thus we compared DA release between patients receiving benzodiazepines and those who did not require sedation, we found no significant difference (16 available datasets for calculating DA release, 7 not receiving lorazepam, 9 receiving lorazepam; two-sided t-test p > 0.05 for all ROIs). striatum; GP, globus pallidus; SNVTA, substantia nigra/ventral-tegmental area) in healthy volunteers before (HV UNSENS ; sky-blue) and after AMPH sensitization (HV SENS ; deep-blue) and in patients with first-episode psychosis (FEP; red). X-axis break between PET2 and PET3 in HV indicates a scan-free interval (2-4 weeks) during prospective AMPH sensitization. All direct AMPH effects (PET1 vs. PET2 HV UNSENS and FEP, PET3 vs. PET4 HV sens ) were significant (paired t-tests p = 0.03-2.5 × 10 −5 ; not marked). Note that AMPH sensitization led to a significant increase in D 2/3 receptor binding from PET1 to PET3 in VST. b Alternative representation of data shown in a highlighting systematic differences in D 2/3 receptor binding across conditions between HV and patients with FEP. D 2/3 binding in FEP is lower than in HV in D 2 receptor-rich neo-striatal regions (CAU, PUT, VST), while it is elevated in D 3 receptor-rich regions of the paleo-striatum (GP and SNVTA). Error bars represent 95% confidence intervals. *p < 0.05, **p < 0.01, ***p < 0.001, post-hoc two-tailed t-test.  Table 4). In contrast to earlier studies 3-6,44-46 but in line with a recent study using the D 2/3 receptor agonist radioligand [ 11 C]N-propyl-apomorphine ([ 11 C] NPA 47 ), we did not observe significant relationships between positive symptoms and DA release. However, there were significant relationships between [ 11 C]-(+)-PHNO BP ND values in the AMPH condition and negative symptoms. Correlations were strongest in PUT and driven mainly by the PANSS item N2 ("emotional withdrawal"; right PUT: rho = 0.93, p = 7.7 × 10 −8 ; Fig.  3c, d; Supplementary Fig. 5). Typical behavioral effects of  AMPH in healthy volunteers included increased production of speech, extraversion, alertness, and enhanced energy.

PFC volume and subcortical DA release
Dysfunctional PFC-DA interactions are prime candidates for "upstream" pathogenic mechanisms underlying subcortical hyperdopaminergia in patients with SCZ. Furthermore, brain circuits involved in cognitive functions encompassing the PFC are often found to be compromised in patients with SCZ 48 . Thus we derived volumetric parameters from MR images and, in a first step, analyzed the relationship between indices of DA release and overall PFC volume. Consistent with inhibition of DA release by the PFC 19,22 , we observed an inverse relationship between PFC volume and DA release in AMPH naïve HV UNSENS . After AMPH sensitization, the association was lost or even reversed and more resembled the pattern observed in FEP patients. An anatomically unbiased vertex-wise analysis of the relationship between sensitization of DA release in SNVTA (ΔBP ND-SENS − ΔBP ND-UNSENS ) and whole-cortex volumetric parameters identified a left-hemispheric cluster spanning the boundaries of middle and inferior frontal gyrus as main driver of the whole-PFC signal (Fig. 4a, b). In a next step, we analyzed correlations between DA release and volumes of individual PFC sub-regions as implemented into Freesurfer 6.0 software according to anatomical and functional priors 42 . In perfect agreement with the vertexwise analysis, we observed the by far strongest correlations in three anatomically adjacent left-hemispheric regions: Brodmann areas (BA) 46 (dorsolateral prefrontal cortex; DLPFC), and BA 44 and BA 45, or pars opercularis and triangularis of the left inferior frontal gyrus (IFG), together forming "Broca's area" 49 .
Plotting the correlations between volume of these regions (henceforth referred to as DLPFC/IFG) and DA release in the five subcortical ROIs (CAU, PUT, VST, GP, and SNVTA in rostro-caudal anatomical order) revealed a remarkable "herring bone"-like pattern (Fig. 5). This pattern is shaped by negative regression lines-indicating DLPFC/IFG-inhibition of DA release in HV UNSENS -and positive regression lines observed after the same subjects had undergone AMPH sensitization (HV UNSENS vs. HV SENS vs. FEP: F (2;278.9)) = 10.14, p = .00005). We did not observe any significant relationships between subcortical DA release and cortical volumes in FEP (Fig. 5).
To account for possible changes in brain volume induced by AMPH sensitization, we derived volumetric parameters in an independent group of AMPH naïve, sex and age matched HVs. There were no significant differences between our group of HV SENS and the independent HV group ( Supplementary Fig. 6). Although of restricted power, these results argue against gross volumetric effects arising from our sensitization procedure.

Discussion
This study is the first to directly compare AMPHinduced DA release in patients with FEP to DA release in HV before and after prospective AMPH sensitization. Our data confirm the main hypothesis of this work: subcortical DA transmission in patients with FEP is in a state of "endogenous sensitization" towards AMPH. They thus replicate and extend the results of earlier studies that have used D 2/3 receptor antagonist-radioligands for studying DA release in SCZ 3-6 and the effects of AMPH sensitization in HV 15 . In contrast to these studies, we found no relationship between DA release and change in positive symptoms of SCZ. However, we observed a significant correlation between [ 11 C]-(+)-PHNO BP ND values after AMPH in the putamen and negative symptoms, in particular emotional withdrawal. Although the interpretation of this finding is not straightforward, the finding was highly significant and is somewhat supported another [ 11 C]-(+)-PHNO study 50 that found a negative relationship between stress-induced DA release in SNVTA and negative symptoms in individuals with clinical high risk for schizophrenia (and at trend level, also patients with FEP). Together, these results corroborate the notion that reduced DA transmission is a relevant element in the pathogenesis of negative schizophrenic symptoms.
Our results are at odds with those of a recent study using the D 2/3 receptor agonist radioligand [ 11 C]NPA 47 . In contrast to our results and those obtained with D 2/3 antagonist radioligands 3-6 , the study by Frankle et al. study did not find enhanced AMPH-induced DA release in schizophrenia. Besides differences in clinical variables (as for example a smaller proportion of medication-naïve patients in the Frankle et al. study), the discrepancy could also reflect different properties of the two radioligands. Since the method used in both studies does not allow for reliably distinguishing between the absolute number of receptors and receptor affinity (including changes in apparent affinity induced by competition with endogenous DA 51 ), it remains open how these parameters exactly relate to each other in dopaminergic dysfunction of SCZ 52 .
Relationships between volumetric and functional data suggest that AMPH-induced DA release in HV at first exposure to the drug is under inhibitory control of the left-hemispheric DLPFC/IFG. At the same time, AMPH sensitization increased DA release to a greater extent in subjects with larger DLPFC/IFG volumes. This relationship was particularly pronounced in GP and SNVTA (Fig.  4), where the [ 11 C]-(+)-PHNO signal is predominantly reflecting binding to DA D 3 rather than D 2 receptor subtypes [53][54][55] . Since it is unlikely, according to our data obtained in an independent HV sample, that AMPH sensitization had induced major volumetric changes, the "herring bone" pattern most likely reflects a true functional shift in control of subcortical DA release by the DLPFC/IFG from inhibition in HV UNSENS towards facilitation in HV SENS . This interpretation is supported by rodent data showing that expression of the sensitized state is critically dependent on the integrity of PFC neuronal tissue and its interaction with subcortical D 3 receptors 56 .
In our study, prospective sensitization of HV led to an increase in DA release to the magnitude observed in FEP. DLPFC/IFG volumes, while showing strong correlations to DA release in HV UNSENS , did not show any significant relationship to DA release in patients with FEP (Fig. 4). While we cannot rule out that this is due to lack of power, the very same region was found to show abnormal cortical folding in SCZ patients with a mean illness duration of two decades and extensive exposure to antipsychotics 57 . Thus, our data support the long-standing conjecture that the hyperdopaminergic state in SCZ is directly related to a dysfunction in top-down control of subcortical DA transmission by the PFC 22,58 , and they are consistent with the notion that antipsychotics act by rectifying the consequences of upstream pathogenic factors, which persist despite antipsychotic treatment.
Antipsychotic-sensitive hyperlocomotion is a standard measure for sensitization in rodents and a widely accepted animal model of SCZ. Although not formally quantified in this study, the most evident behavioral effect of AMPH in our participants was an increase in quantitative speech production. Thus, while expressing on a motor level in rodents, enhanced DA transmission in human AMPHsensitive brain circuits seems to affect primarily neuronal functions involved in the processing of language and speech, making speech being the human equivalent to rodent hyperlocomotion. Alterations in cytoarchitecture introduced into the rodent PFC by disrupting the cytoskeleton of dendritic spines increase subcortical DA levels and induce hyper-locomotion via direct projections between the PFC and SNVTA 59 . Similar alterations in the DLPFC/IFG of patients with SCZ may cause dysfunctions in hierarchical control within DLPFC/IFG-SNVTA-striato-thalamic loops and may be involved in mediating the autonomous (i.e., not subject to will) production of language in auditory verbal hallucinations of patients with SCZ. Simultaneously activated by certain grammatical constructs 60 , altered interactions between the DLPFC/IFG and the evolutionarily ancient SNVTA may also be involved in generating the fascinating and inherently grammatical symptom-complex of schizophrenic "ego disturbances".
As prospective AMPH sensitization in HV successfully mimics the endophenotype of increased DA release observed in "psychotic sensitization", our data prompt the question why the endophenotype associates with psychotic symptoms in SCZ but is occurring-unless reexposed to AMPH-without any relevant behavioral effects in HVs. Symptom-provocation studies 61 and ecological evidence strongly suggest this to be a matter of dose and intensity of exposure: While an escalation of the AMPH dose induces transient psychotic symptoms in psychiatrically healthy subjects 61 , subjects with stimulantuse disorders indeed exhibit disproportionally high rates of psychotic disorders 62 . Interestingly, subjects abusing methamphetamine or cocaine exhibit increased [ 11 C]-(+)-PHNO binding in the GP 63,64 . What seems to differentiate AMPH-induced from "endogenous psychotic" sensitization in our data-higher [ 11 C]-(+)-PHNO BP ND values in GP-may just be a matter of dose and length of exposure to AMPH.
An inherent limitation of our method is that it does not discriminate the effects of extracellular DA levels from changes in maximal D 2/3 receptor binding capacity. In addition, although more sensitive towards fluctuations in extracellular DA, the differences between HVs and FEP patients observed with the agonist radioligand [ 11 C]-(+)-PHNO are by no means larger than those observed with antagonist radioligands 1-6 . However, this could in part also be a consequence of increased D 2/3 receptor occupancy due to higher baseline extracellular DA levels in patients with FEP (Fig. 3b). Another limitation might be the fact that some patients required benzodiazepines due to agitation or anxiety. Although there was no significant difference between medicated and unmedicated patients we cannot rule out the possibility that this might be a confounding factor. Furthermore, although sex did not play a role when entered as a covariate, the imbalance between male and female patients in the FEP sample has to be mentioned as a limitation. Lastly, our design has the limitation that we did not expose, mainly for ethical reasons, our FEP patients to a sensitizing regime of repeated AMPH. However, rarely cited earlier work by Strakowski et al. 65 has shown that FEP patients fail to show a progressive enhancement in the response to repeated AMPH. This suggests that FEP patients, at least at the level of behavior, are already maximally sensitized. Our data support and extend this finding to a neurochemical level. In summary, we feel confident that our work provides the first direct experimental proof for the hypothesis that the pathogenic substrate underlying psychosis in SCZ is indeed a state of "endogenous sensitization" in subcortical DA systems.