Serotonin 5-HT2 Receptors in Schizophrenia: A PET Study Using [18F]Setoperone in Neuroleptic-Naive Patients and Normal Subjects

Ralph Lewis, M.D., F.R.C.P.C., Shitij Kapur, M.D., Ph.D., F.R.C.P.C., Corey Jones, B.Sc., Jean DaSilva, Ph.D., Gregory M. Brown, M.D., Ph.D., F.R.C.P.C., Alan A. Wilson, Ph.D., Sylvain Houle, M.D., Ph.D., F.R.C.P.C., and Robert B. Zipursky, M.D., F.R.C.P.C.

Objective: Several postmortem studies have reported a decreased density of serotonin 5-HT2 receptors in the prefrontal cortex in schizophrenia. The purpose of this study was to investigate this in patients with schizophrenia by means of [18F]setoperone and positron emission tomography (PET) imaging. Method: Thirteen neuroleptic-free patients with schizophrenia, 10 of whom were also neuroleptic-naive, were compared with a group of 26 normal subjects in the same age range. The density of 5-HT2 receptors was as- sessed with the use of [18F]setoperone and PET in standardized cortical regions of inter- est. Results: Increasing age was associated with similar declines in 5-HT2 receptors in all cortical regions in the patient group and in the normal comparison group. After control for the effect of age, there was no statistically significant difference between the patients and the comparison subjects in 5-HT2 receptor density in any of the cortical regions. Conclu- sions: This study failed to find the decrease in 5-HT2 receptors reported in postmortem studies of schizophrenia. The study had the power to detect a decrease of 25% or more in 5-HT2 receptors, which was anticipated on the basis of the previous postmortem studies. Thus, a primary serotonergic abnormality in schizophrenia, if one exists, is either small or unlikely to be at the level of the 5-HT2 receptors. This finding does not rule out a therapeutic role for 5-HT2 antagonists in schizophrenia, but it does suggest that the therapeutic contri- bution is likely to be an indirect one. (Am J Psychiatry 1999; 156:72–78)

The serotonergic hypothesis of schizophrenia pre- to a greater focus on and investigation of the dopamine ceded the better-known dopaminergic hypothesis. Fol- system (5). With the success of clozapine as an “atypi- lowing Gaddum’s demonstration in 1953 that LSD, a cal” antipsychotic (6), interest in the role of serotonin hallucinogenic drug, had affinity for serotonin recep- in schizophrenia has been renewed. tors, several lines of evidence suggested a role for sero- The serotonergic hypothesis is best viewed as com- tonin in the pathophysiology of schizophrenia (1–4). plementary to the dopaminergic hypothesis, rather However, the development of effective antipsychotic than as an alternative to it, since these systems are an- drugs that were antagonists at dopamine receptors led atomically connected and functionally interactive (7, 8). Several authors have proposed that the atypical an- Presented in part at the annual meeting of the Society of Biologi- cal Psychiatry, San Diego, May 14–18, 1997. Received Dec. 2, tipsychotics derive their unique efficacy at least partly 1997; revision received May 28, 1998; accepted June 10, 1998. from their dual serotonin-dopamine antagonism, ex- From The Clarke Institute of Psychiatry, Department of Psychiatry, ploiting the neurochemical interaction between these University of Toronto. Address reprint requests to Dr. Kapur, PET two systems (2, 4, 7, 9, 10). Despite the recent interest Centre, The Clarke Institute of Psychiatry, 250 College St., Tor- onto, ON, Canada M5T 1R8; [email protected] (e-mail). in the role of serotonin 5-HT2 receptors, the nature of Supported by an award from the Ontario Mental Health Founda- the 5-HT2 abnormality in schizophrenia, if any, is not tion to Dr. Lewis and by a Clinician Scientist Award from the Medi- known. Until recently, the means to specifically mea- cal Research Council of Canada to Dr. Kapur. sure 5-HT2 receptor density in the brains of living pa- The authors thank Terry Bell, Kevin Cheung, Steven Dobbin, Doug Hussey, and Erin Toole for their technical assistance; Dr. tients with schizophrenia were unavailable. Therefore, Paul Roy for his assistance with patient recruitment; and Janssen- our current knowledge is derived mainly from studies Cilag France for providing the setoperone precursor. of 5-HT2 receptors in postmortem brain tissue and on

72 Am J Psychiatry 156:1, January 1999 LEWIS, KAPUR, JONES, ET AL.

TABLE 1. Postmortem Studies Comparing 5-HT2 Receptor Binding in Cortical Brain Tissue From Subjects With an Antemortem Diagnosis of Schizophrenia and From Nonschizophrenic Comparison Subjects

Study Year Ligand Finding on 5-HT2 Receptor Binding Bennett et al. (17) 1979 [3H]LSD Decreased in frontal cortex in schizophrenic subjects Whitaker et al. (18) 1981 [3H]LSD No difference in total sample; increased in frontal cortex in a subset of neu- roleptic-free schizophrenic subjects Reynolds et al. (19) 1983 [3H]Ketanserin No difference Mita et al. (20) 1986 [3H]Ketanserin Decreased in prefrontal cortex in schizophrenic subjects; also, no difference between neuroleptic-treated and -untreated subjects Arora and Meltzer (21) 1991 [3H]Spiperone Decreased in frontal cortex in schizophrenic subjects Laruelle et al. (22) 1993 [3H]Ketanserin Decreased in prefrontal cortex in chronic psychotic subjects; also, no differ- ence between neuroleptic-treated and -untreated subjects Joyce et al. (23) 1993 [3H]Ketanserin No difference in frontal cortex, but increased in subcortical limbic system and and [125I]LSD related cortical areas in schizophrenic subjects Dean et al. (24) 1996 [3H]Ketanserin No difference (with use of particulate membranes) Dean and Hayes (25) 1996 [3H]Ketanserin Decreased in prefrontal cortex (with use of autoradiography) Burnet et al. (26) 1996 [3H]Ketanserin Decreased in prefrontal cortex; also decreased mRNA in same area

platelet membranes (11, 12). The 5-HT system in affinity for the 5-HT2C receptor is lower still (Ki∼50– platelets is developmentally related to the 5-HT system 80 nM) (31). It has been shown to bind specifically to in the brain, but the exact relationship (and, hence, the 5-HT2 receptors in both in vitro and ex vivo prepara- correlation in disease states) is not completely under- tions. When radiolabeled with fluorine-18, setoperone stood (4). has several features that make it well-suited for PET Postmortem techniques have demonstrated the pres- studies of 5-HT2 receptors. It rapidly equilibrates ence of 5-HT2 receptors throughout the cortex and in across the blood-brain barrier, binds reversibly, and subcortical structures in normal human brains (13– has no metabolites that cross the blood-brain barrier 16). Postmortem investigations of cortical 5-HT2 re- (32). In PET studies, the cortical signal can be blocked ceptors in schizophrenia are summarized in table 1. by the competitive antagonist ketanserin and is unaf- A review of the literature on postmortem findings fected by sulpiride, a D2 blocker, suggesting that [18F]setoperone binds specifically to 5-HT receptors suggests a decrease in 5-HT2 receptors in schizophre- 2A nia, but this is not conclusive. While some studies sug- (27, 28, 30). Furthermore, the cerebellum is a region gest a decrease (17, 20–22, 25, 26), others do not con- almost devoid of 5-HT2 receptors in postmortem stud- firm this (18, 19, 23, 24). The reported decrease in ies (13, 15), and accordingly, the [18F]setoperone sig- nal from the cerebellum is not affected by 5-HT an- Bmax (the number of 5-HT2 receptors) is in the range of 2 25%–55 % (17, 20–22, 25, 26). While there is some tagonists. Since the cerebellum represents free ligand and nonspecific [18F]setoperone binding (30), using it inconsistency regarding studies of Bmax, there is una- as a reference region enables one to obtain semiquanti- nimity regarding the affinity (Kd) of these receptors: no tative estimates of B /K , indexes of 5-HT binding study has reported a significant alteration in the Kd of max d 2 the receptors in patients with schizophrenia (17–26). potential, without arterial puncture or kinetic model- The previous studies focused mainly on the prefrontal ing, making it suitable for routine clinical studies (33). cortex; even so, the different studies focused on differ- With the use of these methods, it has been shown that 18 ent subregions within it. Few studies have investigated [ F]setoperone is sensitive to the effects of age on 5- other cortical regions (Laruelle et al. [22] reported on HT2 receptors, shows robust effects of illnesses such as the occipital cortex, while Joyce et al. [23] reported on Alzheimer’s dementia (34), and is parametrically sensi- the limbic regions), even though 5-HT2 receptors are tive to the effects of antipsychotics that bind to 5-HT2 uniformly distributed throughout the cortex. Postmor- receptors (35). tem studies are often confounded by issues of inaccu- An alternative ligand for the study of 5-HT2 recep- rate diagnosis, different causes of death, different post- tors is [18F]altanserin. [18F]Altanserin’s specificity for mortem intervals before tissue processing for analysis, subcortical 5-HT2 receptors is slightly higher than that and the effects of concurrent illnesses and medications of [18F]setoperone, since setoperone has moderate af- at the time of death. Some of the studies attempted to finity for D2 receptors (36). However, because D2 re- address the question of the effect of exposure to neuro- ceptors are undetectable in the cortex, the effective leptics, with differing results (18, 20, 22, 25). To ad- specificity for these two ligands is equal for the study dress these shortcomings, we studied 5-HT2 receptors of cortical 5-HT2 receptors (37). Another radioligand in neuroleptic-free patients and a group of normal that is used is N1-([11C]methyl)-2-Br-LSD, which is comparison subjects, using [18F]setoperone and less selective than [18F]setoperone or [18F]altanserin positron emission tomography (PET) imaging (27–30). (37, 38). Setoperone binds to 5-HT2 receptors with high affin- At the outset we were interested in two issues. First, ity (Ki∼1 nM). It is relatively selective for the 5-HT2A on the basis of postmortem data, we hypothesized that 18 subtype, since its affinity for the dopamine D2 receptor the binding potential of [ F]setoperone for 5-HT2 re- is an order of magnitude lower (Ki∼10–25 nM), and its ceptors would be decreased in the prefrontal cortex in

Am J Psychiatry 156:1, January 1999 73 SEROTONIN RECEPTORS IN SCHIZOPHRENIA schizophrenic patients as compared with healthy com- Data Analysis munity volunteers. Second, we wanted to explore any Cortical regions of interest were manually traced onto the axial differences in 5-HT2 receptors in nonfrontal regions, PET images, with the use of Alice 3.0 image analysis software (Hay- since this issue has never been systematically addressed den Image Processing Group, Denver) implemented on a personal in any previous study. computer-based Windows NT platform, according to a set of conser- vative anatomical rules based on neuroanatomical landmarks and guided by a standard neuroanatomical atlas (42). The coregistered MRI slices were used as a visual reference to provide more precise METHOD anatomical definition in the drawing of the regions of interest on the PET images. Eight cortical regions of interest were defined: the left The study was approved by the Human Subjects Review Commit- and right prefrontal, temporal, parietal, and occipital cortices. A sin- tee of the University of Toronto. The subjects participated after re- gle region of interest was defined that encompassed both hemi- ceiving information regarding the study and providing written con- spheres of the cerebellum. The prefrontal cortical regions of interest sent. Thirteen patients (10 male and three female), whose mean age were defined on five contiguous PET slices for each hemisphere; the was 31.1 years (SD=6.9, range=21–43), were included in the study. temporal, parietal, and occipital regions of interest were defined on A 14th patient (male, aged 46 years) participated in the study, but his three contiguous slices in each hemisphere; and the cerebellar re- data were excluded from the analysis because of the poor quality of gions of interest were defined on two contiguous slices. These multi- his PET scan. All patients had a DSM-IV diagnosis of schizophrenia. ple slices were used in order to reduce partial volume effects. All of Patients were assessed by a chart review, discussion with the treating the slices within a given region of interest were combined for the physician, and a clinical interview in which the Structured Clinical analysis. All of the regions of interest were drawn by one of us Interview for DSM-IV Axis I Disorders (39) was used. The current (R.L.), who was blind to the identity of the subjects. symptomatic status of the patients was assessed with the Positive For each cortical region of interest, a time-activity curve was ob- and Negative Syndrome Scale (40). tained, reflecting the fluorine-18 activity in the region of interest. The patients were recruited from a university-affiliated psychiat- The ratio of fluorine-18 activity in the specific cortical region (S) to ric hospital receiving referrals from a large urban region. The pa- that in the cerebellum (C) (i.e., S/C–1) at 65–90 minutes was used as tients’ mean duration of illness, from the first onset of psychotic an index of 5-HT2 receptors. The cerebellum was used as a reference symptoms, was 4.7 years (SD=3.7 years, range=3 weeks to 10 years) region, serving as a measure of free and nonspecific binding of 18 at the time of their scans. Two patients had been ill for less than 6 [ F]setoperone. The time window of 65–90 minutes was chosen be- months at the time of their scans and were therefore provisionally di- cause the S/C–1 ratio was stable and showed no statistical change agnosed as having schizophreniform disorder. In both of these cases, during this window (33). Theoretically, it can be shown that under such conditions and assumptions, the S/C–1 ratio is linearly pro- the diagnosis was revised to schizophrenia in subsequent follow-up. 18 Ten patients were neuroleptic-naive, and three were neuroleptic-free. portional to the Bmax/Kd of [ F]setoperone for 5-HT2 receptors. In The neuroleptic-free patients had the following neuroleptic-free in- addition, Petit-Taboue et al. (43) have shown empirically that the tervals and prior cumulative exposure to neuroleptics: 7 weeks free ratio during this time window is highly correlated (r values=0.91– with 15 months’ exposure, 4 months free with 4 years’ exposure, 0.97) with the values of Bmax/Kd as obtained from compartmental and 10 months free with 1 month’s exposure. The previous expo- kinetic modeling. We call this semiquantitative index “5-HT2R” in sures were accompanied by poor compliance in all cases. Only one further discussions to distinguish it from fully quantitative values of patient had received a depot neuroleptic, and that was more than 3 Bmax or Kd. years before the study. Before this study we had established that this imaging protocol The normal comparison group consisted of 26 subjects (11 male can be repeated in the same individual with high test-retest reliability and 15 female) whose mean age was 31.3 years (SD=6.7, range=19– (intraclass correlation coefficients=0.96–0.98) (33). Also, we had 43). These subjects were healthy volunteers recruited from the com- standardized the technique of data analysis and established high in- trarater and interrater reliability (intraclass correlation coefficient, munity by advertisement. They were stratified over the age range of > > interest (eight to 10 subjects for each decade) to provide a reliable es- type III: intrarater r 0.98 for author R.L., and interrater r 0.95 for timate of age effects. They were screened with the Structured Clini- authors R.L., S.K., and C.J.) (33). cal Interview for DSM-III-R—Non-Patient Edition (41) to ensure Previous studies (30, 34, 44–46) had shown decreases in 5-HT2 that they had no history of psychiatric disorder. receptors with age, a finding that we confirmed. Therefore, it was planned that age would be a covariate in our analysis. We used two Neither patients nor comparison subjects were currently using statistical strategies. First, since we had an a priori hypothesis re- any psychotropic medications (with the exception of benzodiaz- garding a decrease in these receptors in the prefrontal cortex of pa- epines for the patients), alcohol, or street drugs. None of the subjects tients with schizophrenia, we compared the 5-HT R for the two had a history of alcohol or drug dependence, significant head injury, 2 groups by using an analysis of covariance (ANCOVA) with age as a major neurological disorder, or other serious medical illness. covariate to assess the main effect of diagnosis (i.e., schizophrenia) and to investigate any significant interactions between age and diag- Radiosynthesis and PET Imaging nosis. Second, to investigate the 5-HT2R in other brain regions, we undertook a multivariate analysis of covariance (MANCOVA) with [18F]Setoperone was synthesized by a method described previ- the four major brain regions (frontal, temporal, parietal, and occipi- ously (33). PET scans were acquired with the use of a GEMS tal) as dependent variables and age as a covariate. Statistical analyses PC2048-15B head-dedicated PET camera (GE Medical Systems, were done with SPSS version 7.0 for Windows (SPSS Inc., Chicago). Milwaukee). Subjects were fitted with a thermoplastic mask to limit head movement. A transmission scan was acquired with a germa- nium-68 rotating pin source to measure attenuation, prior to a bolus injection of [18F]setoperone (injected dose range=4.24–5.57 mCi; RESULTS specific activity range=360–6210 mCi/µmol). PET data were ac- quired over the next 90 minutes and reconstructed into 22 time frames (five 1-minute frames and then 17 5-minute frames), with 15 The values of 5-HT2R for the right and for the left 6.5-mm-thick axial slices, with the use of a Hanning filter of 5-mm regions were not statistically differentiable and were full width at half maximum. Magnetic resonance imaging (MRI) highly correlated for all of the cortical regions (Pear- scans were acquired separately with a GE Signa 1.5-T scanner, spin- son’s r=0.98–0.99); they were therefore averaged for echo sequence, in an axial orientation, yielding a whole-brain image further analysis. There was no significant effect of sex with 42 3-mm-thick slices. The MRI scans were coregistered with the PET scans by means of the surface-matching algorithm imple- in the group of normal comparison subjects (AN- mented in ANALYZE (CNS Software, Rochester, Minn.). COVA: F=1.64, df=1, 22, p=0.21). There were too few

74 Am J Psychiatry 156:1, January 1999 LEWIS, KAPUR, JONES, ET AL.

a women in the patient group (N=3) to permit a valid FIGURE 1. 5-HT2R in the Prefrontal Cortex of Patients With Schizophrenia and of Normal Comparison Subjects comparison of the effect of sex on 5-HT2R in the pa- tients. To rule out any bias due to too few female pa- tients, we analyzed the results in the male subgroup only (i.e., the 11 male comparison subjects and the 10 male patients). The results in the male subgroup were no different from those for the complete study group; therefore, we report here a pooled analysis of male and female subjects, comparison subjects as well as patients. There was no significant difference in activity of [18F]setoperone in the cerebellum (i.e., the denomina- tor “C” in the ratio S/C–1, used to derive the 5-HT2R) between patients and comparison subjects (F=0.60, df=1, 35, p=0.45), indicating that there was no signifi- cant difference between the groups in free ligand and a 18 nonspecific binding; a difference here could have con- The binding potential of [ F]setoperone to 5-HT2 receptors. founded the interpretation of 5-HT2R and therefore was important to evaluate. Furthermore, there was no mean=1.93); parietal region—patients’ mean=1.66 significant difference between the groups in the vol- (SD=0.49; age-adjusted mean=1.65), and comparison umes of any of the regions of interest, suggesting that a subjects’ mean=1.80 (SD=0.63; age-adjusted mean= difference in determination of the regions of interest 1.80); occipital region—patients’ mean=1.75 (SD= was not confounding the results. 0.41; age-adjusted mean=1.75), and comparison sub- As expected, for the study group as a whole (patients jects’ mean=1.92 (SD=0.59; age-adjusted mean=1.92). and comparison subjects), there was a substantial de- We found no significant relationship between Posi- crease in 5-HT R with age (Pearson’s r=0.80; F=67.42, 2 tive and Negative Syndrome Scale total score, positive df=1, 37, p<0.001) (figure 1). With age as a covariate, or negative subscale scores, and 5-HT R for any of the we found no significant effect of diagnosis on 5-HT R 2 2 regions of interest, suggesting that differences in 5- (i.e., no significant difference in 5-HT R between pa- 2 HT R are not associated with profile of symptoms. Fi- tients and comparison subjects) in the prefrontal re- 2 nally, three of our patients had received neuroleptics, gion. The mean prefrontal 5-HT R was=1.66 (SD= 2 while the other 10 were neuroleptic-naive. Repeating 0.50; age-adjusted mean=1.65) for the patient group the MANCOVA in the neuroleptic-naive group, we and 1.85 (SD=0.66; age-adjusted mean=1.85) for the still found no significant effect of illness (Pillai’s trace comparison group (ANCOVA: F=1.45, df=1, 35, p= statistic=0.06, and Wilks’s lambda=0.94; F=0.45, df= 0.24) (figure 1). While age had a significant effect on 5- 4, 29, p=0.78). HT2R in both patients and comparison subjects, the age-associated decline was not significantly different between the two groups (ANCOVA: F=0.76, df=1, 35, p=0.39). DISCUSSION The second important question was the effect of schizophrenia on 5-HT2 receptors in all of the brain re- This study constitutes the first systematic effort to gions. Since this was an exploratory question, and the assess 5-HT2 receptors in schizophrenic patients with regions are highly interrelated, the appropriate method the use of neuroreceptor imaging. With [18F]setoper- to address this question is a multivariate analysis. A one and PET, the study did not find a significant de- MANCOVA showed an effect of age but no effect of crease in 5-HT2 receptors in patients with schizophre- illness. With the four cortical regions as dependent nia. These findings are at odds with some of the variables and age as a covariate, there was no effect previous postmortem studies. We now discuss the lim- of illness (Pillai’s trace statistic=0.08, and Wilks’s itations of postmortem studies, the limitations of our lambda=0.92; F=0.69, df=4, 32, p=0.60). Age itself own methods, and the implications of these findings had a very significant multivariate effect (Pillai’s trace for understanding the role of 5-HT in the pathophysi- statistic=0.68, and Wilks’s lambda=0.32; F=16.69, df= ology and therapeutics of schizophrenia. 4, 32, p<0.001), but there was no interaction between Studying neuroreceptors in living patients offers sev- the effects of age and illness (Pillai’s trace statistic= eral advantages over postmortem studies. In vitro post- 0.06, and Wilks’s lambda=0.94; F=0.54, df=4, 32, p= mortem studies face confounding variables that may 0.71). Quantitatively, the 5-HT2R results were highly explain some of the inconsistencies in findings between correlated across the brain (Pearson’s r values=0.95– studies (4, 23, 25, 26). Technical methods vary be- 0.98), and the values in the different brain regions tween laboratories. Postmortem changes in the brain were as follows: temporal region—patients’ mean= may affect neuroreceptors, depending on the time 1.79 (SD=0.52; age-adjusted mean=1.78), and com- elapsed between death and preparation of the speci- parison subjects’ mean=1.92 (SD=0.68; age-adjusted men. Clinical data about the subjects are usually re-

Am J Psychiatry 156:1, January 1999 75 SEROTONIN RECEPTORS IN SCHIZOPHRENIA constructed retrospectively, and it is difficult to control scribed changes. While such approaches have been for clinical variables, especially exposure to neurolep- used for comparisons of regional blood flow and are in tics before death, which could affect 5-HT2 receptor principle possible for receptor studies, they have not binding. None of the postmortem studies included yet been standardized for these purposes. Examining neuroleptic-naive subjects, and the type and degree of this issue by using a voxel-by-voxel analysis remains a cumulative exposure varied greatly. Finally, there are project for the future. no true “normal” control subjects in postmortem stud- Finally, there is no logical way to “prove” the null ies, since all subjects have died through some disease hypothesis. The best one can do is to state the certainty process or trauma, factors which could affect 5-HT2 with which one can reject a null hypothesis. The post- receptor assessment. The combination of these factors mortem studies showed changes in receptor density of may explain the inconsistency in the literature on post- approximately 25%–55%. On the basis of these num- mortem studies and may also explain why our findings bers, we estimated that a study of 12 patients would are at variance with the postmortem data. give us sufficient power to rule out a 25% difference. Our study is not without its limitations. We used an A post hoc power analysis confirmed our initial as- index of 5-HT2 receptors that reflects the ratio of re- sumption. Since there was no difference in the effect of ceptor density to receptor affinity (i.e., Bmax/Kd) but age in the two study groups, and age-corrected vari- cannot provide independent measures of either Bmax or ance was similar across the two groups, a post hoc Kd. A separate measurement of Bmax would be theoret- analysis suggests that with 13 patients we had a more ically desirable. However, it should be pointed out that than 80% power to detect a 25% decrease at a signifi- none of the previous postmortem studies has suggested cance level of p<0.05 (55). Could a decrease smaller any change in Kd in schizophrenia. Thus, it is very un- than 25% exist? Our data do in fact show a nonsignif- likely that Kd could have obscured a change in Bmax. icant trend in that direction. The patients had a numer- Furthermore, a change in Kd would hide a change in ical decrease in the range of 10%, but such a change Bmax only if both Bmax and Kd changed simultaneously, did not reach significance in our study. In fact, if one in the same numerical direction, by exactly the same wanted to design a study to detect a 10% change with extent—a rather unlikely scenario. reasonable certainty (i.e., greater than 80% power to It is important to note that while our data suggest no detect a change at p<0.05), one would need 60–70 pa- change in receptor number, they do not address recep- tients, a considerable task for any single research site. tor function. The study does not tell us to what extent Thus, while we can rule out a decrease of 25% or 5-HT2 receptors have the same or different functional greater with reasonable certainty, a smaller change properties in the two groups. It is of interest that stud- may exist. ies using pharmacological challenges which probe the The results from this study have significant implica- 5-HT system (e.g., the 5-HT releasing agent and re- tions, since it is the first systematic PET study of 5-HT2 uptake inhibitor fenfluramine or the 5-HT agonist m- receptor density in untreated patients with schizophre- chlorophenylpiperazine) have pointed to abnormal 5- nia. The serotonergic hypothesis of schizophrenia HT function in schizophrenia (4, 47–52) or at least in holds that 5-HT is involved in the neurochemistry of that subgroup of patients who benefit from treatment psychosis. Serotonergic agents such as LSD can induce with 5-HT2 antagonist neuroleptics such as clozapine hallucinations. The literature reviewed in this article, (48, 53, 54). However, the results of these studies are based on postmortem studies, suggests that decreased not consistent. Furthermore, an alteration in the neu- numbers of 5-HT2 receptors may be the basis of a se- roendocrine response to a 5-HT2 challenge does not rotonergic abnormality in schizophrenia. If the nega- localize the deficit at the level of the 5-HT2 receptor, tive results from the present study in living patients are for it can equally plausibly represent postreceptor or correct, then this suggests that a serotonergic abnor- primary endocrine system alterations. mality, if it exists, is not at the level of 5-HT2 receptors. Our method of region of interest analysis also has its Targeting the 5-HT2 receptor has been an important limitations. While we analyzed more brain regions and factor in the design of the new generation of atypical more brain volumes than any of the previous postmor- antipsychotics, all of which possess 5-HT2 antagonist tem studies, we did not analyze the entire cortex. By properties. If there is no abnormality of 5-HT2 recep- choosing five slices for the prefrontal cortex and three tor density in schizophrenia and yet 5-HT2 antagonism for the temporal cortex, and so on, we are reporting on is held to confer some unique therapeutic efficacy, the representative regions. Such an analysis lumps together efficacy of 5-HT2 antagonist drugs must be produced smaller regions within the region of interest, and it is by some indirect mechanism. Such a discrepancy be- conceivable that if there are very localized changes in tween lack of receptor involvement in disease etiology schizophrenia (i.e., if the 5-HT2 decreases are limited and a value of that receptor in therapeutics is not new to a very circumscribed region, for example, just Brod- to schizophrenia at all. While it has been difficult to mann’s area 9), we may have missed them. However, confirm an alteration in dopamine D2 receptors in there is little uniformity in the postmortem findings schizophrenia, there is no denying a role of these recep- that could have focused our analysis further. A voxel- tors in therapeutics (56). Such could be the case for 5- by-voxel analysis of the entire brain space would have HT2 receptors. In fact, it has been postulated that 5- been a more sensitive approach for exploring circum- HT2 antagonists may exert their effects through a

76 Am J Psychiatry 156:1, January 1999 LEWIS, KAPUR, JONES, ET AL. modulation of the dopaminergic system (7). Thus, 9. Meltzer HY: The role of serotonin in schizophrenia and the while our data argue against a significant decrease in place of serotonin-dopamine antagonist antipsychotics. J Clin Psychopharmacol 1995; 15:2S–3S 5-HT2 receptors in patients with schizophrenia, they 10. Carpenter WT Jr: Serotonin-dopamine antagonists and treat- do not rule out the role of 5-HT2 receptors in the ther- ment of negative symptoms. J Clin Psychopharmacol 1995; apeutics of schizophrenia. 15:30S–35S Our study also shows a dramatic effect of age on 5- 11. Arora RC, Meltzer HY: Serotonin2 receptor binding in blood platelets of schizophrenic patients. Psychiatry Res 1993; 47: HT2 receptors. In fact, the 5-HT2 receptor index de- 111–119 creased by about 2%–3% per year in our subjects. 12. Pandey SC, Sharma RP, Janicak PG, Marks RC, Davis JM, Such a decline has been demonstrated by others previ- Pandey GN: Platelet serotonin-2 receptors in schizophrenia: ously (30, 34, 44–46). This decline has potentially im- effects of illness and neuroleptic treatment. Psychiatry Res portant consequences for medication dosage. All cur- 1993; 48:57–68 13. Schotte A, Maloteaux JM, Laduron PM: Characterization and rent atypical antipsychotics block 5-HT2 receptors. It regional distribution of serotonin S2 receptors in human brain. is conceivable that as one ages, the receptor reserve de- Brain Res 1983; 276:231–235 clines, and older patients may be more sensitive to the 14. Luabeya MK, Maloteaux JM, Laduron PM: Regional and cor- tical laminar distributions of serotonin S2, benzodiazepine, effects of 5-HT2 receptor blockers. Most clinical trials are focused on adult populations in their 30s or so; muscarinic, and dopamine D2 receptors in human brain. J Neurochem 1984; 43:1068–1071 thus, there are few systematic data in older age groups. 15. Pazos A, Probst A, Palacios JM: Serotonin receptors in the Our finding should alert clinicians to the possibility human brain, IV: autoradiographic mapping of serotonin-2 re- that the elderly may be particularly sensitive to these ceptors. Neuroscience 1987; 21:123–139 medications. 16. Radja F, LaPorte A-M, Daval GV, Verg D, Gozlan H, Hamon M: Autoradiography of serotonin receptor subtypes in the central In summary, we failed to find a decrease in 5-HT2 re- nervous system. Neurochem Int 1991; 18:1–15 ceptors in the prefrontal cortex or other brain regions 17. Bennett JP, Enna SJ, Bylund DB, Gillin JC, Wyatt RJ, Snyder in schizophrenia. Our results are in contrast to those of SH: Neurotransmitter receptors in frontal cortex of schizo- the majority of postmortem studies, which show a de- phrenics. Arch Gen Psychiatry 1979; 36:927–934 crease of 25%–55%. It is not logically possible to 18. Whitaker PM, Crow TJ, Ferrier IN: Tritiated LSD binding in frontal cortex in schizophrenia. Arch Gen Psychiatry 1981; 38: “prove” that there is no difference, but our data sug- 278–280 gest that if there is any decrease in 5-HT2 receptors at 19. Reynolds GP, Rossor MN, Iversen LL: Preliminary studies of all, the magnitude of decrease is small and may be lo- human cortical 5-HT2 receptors and their involvement in calized to a small brain region. The results do not rule schizophrenia and neuroleptic drug action. J Neural Transm Suppl 1983; 18:273–277 out the involvement of 5-HT2 receptors in the thera- 20. Mita T, Hanada S, Nishino N, Kuno T, Nakai H, Yamadori T, peutics of schizophrenia but suggest that any such ben- Mizoi Y, Tanaka C: Decreased serotonin S2 and increased do- efits are obtained through indirect mechanisms. It will pamine D2 receptors in chronic schizophrenics. Biol Psychia- be important to replicate these results with other try 1986; 21:1407–1414 ligands and with greater statistical power. Future re- 21. Arora RC, Meltzer HY: Serotonin2 (5-HT2) receptor binding in the frontal cortex of schizophrenic patients. J Neural Transm search will also need to be directed at other 5-HT re- Gen Sect 1991; 85:19–29 ceptor subtypes (in particular the 5-HT1A subtype) and 22. Laruelle M, Abi-Dargham A, Casanova MF, Toti R, Wein- at other levels (e.g., intracellular second messengers) berger DR, Kleinman JE: Selective abnormalities of prefrontal within the serotonergic pathways. serotonergic receptors in schizophrenia: a postmortem study. Arch Gen Psychiatry 1993; 50:810–818 REFERENCES 23. Joyce JN, Shane A, Lexow N, Winokur A, Casanova MF, Kleinman JE: Serotonin uptake sites and serotonin receptors are altered in the limbic system of schizophrenics. Neuropsy- 1. Breier A: Serotonin, schizophrenia and antipsychotic drug ac- chopharmacology 1993; 8:315–336 tion. Schizophr Res 1995; 14:187–202 24. Dean B, Hayes W, Opeskin K, Naylor L, Pavey G, Hill C, Keks 2. Hattunen M: The evolution of the serotonin-dopamine antago- N, Copolov DL: Serotonin2 receptors and the serotonin trans- nist concept. J Clin Psychopharmacol 1995; 15:4S–10S porter in the schizophrenic brain. Brain Res Behav Brain Res 3. Iqbal N, van Praag HM: The role of serotonin in schizophre- 1996; 73:169–175 nia. Eur Neuropsychopharmacol 1995; 5:11–23 25. Dean B, Hayes W: Decreased frontal cortical serotonin2A re- 4. Bleich A, Brown S-L, van Praag HM: A serotonergic theory of ceptors in schizophrenia. Schizophr Res 1996; 21:133–139 schizophrenia, in The Role of Serotonin in Psychiatric Disor- 26. Burnet PW, Eastwood SL, Harrison PJ: 5-HT1A and 5-HT2A ders. Edited by Brown S-L, van Praag HM. New York, Brun- receptor mRNAs and binding site densities are differentially ner/Mazel, 1991, pp 183–214 altered in schizophrenia. Neuropsychopharmacology 1996; 5. Seeman P, Lee T, Chau-Wong M, Wong K: Antipsychotic drug 15:442–455 doses and neuroleptic/dopamine receptors. Nature 1976; 27. Blin J, Pappata S, Kiyosawa M, Crouzel C, Baron JC: [18F]Se- 261:717–719 toperone: a new high-affinity ligand for positron emission to- 6. Kane J, Honigfeld G, Singer J, Meltzer H: Clozapine for the mography study of the serotonin-2 receptors in baboon brain treatment-resistant schizophrenic: a double-blind comparison in vivo. Eur J Pharmacol 1988; 147:73–82 with chlorpromazine. Arch Gen Psychiatry 1988; 45:789–796 28. Crouzel C, Venet M, Irie T, Sanz G, Boullais C: Labeling of a 7. Kapur S, Remington G: Serotonin-dopamine interaction and serotoninergic ligand with [18F]: [18F]setoperone. J Labelled its relevance to schizophrenia. Am J Psychiatry 1996; 153: Compounds and Radiopharmaceuticals 1988; 25:403–414 466–476 29. Maziere B, Crouzel C, Venet M, Stulzaft O, Sanz G, Ottaviani 8. Ohuoha DC, Hyde TM, Kleinman JE: The role of serotonin in M, Sejourne C, Pascal O, Bisserbe JC: Synthesis, affinity and schizophrenia: an overview of the nomenclature, distribution specificity of 18F-setoperone, a potential ligand for in-vivo im- and alterations of serotonin receptors in the central nervous aging of cortical serotonin receptors. Int J Radiation Applica- system. Psychopharmacology (Berl) 1993; 112:S5–S15 tions and Instrumentation, part B 1988; 15:463–468

Am J Psychiatry 156:1, January 1999 77 SEROTONIN RECEPTORS IN SCHIZOPHRENIA

30. Blin J, Sette G, Fiorelli M, Bletry O, Elghozi JL, Crouzel C, 43. Petit-Taboue MC, Landeau B, Osmont A, Tillet I, Barre L, Baron JC: A method for the in vivo investigation of the seroto- Baron JC: Estimation of neocortical serotonin-2 receptor bind- nergic 5-HT2 receptors in the human cerebral cortex using ing potential by single-dose fluorine-18-setoperone kinetic positron emission tomography and 18F-labeled setoperone. J PET data analysis. J Nucl Med 1996; 37:95–104 Neurochem 1990; 54:1744–1754 44. Wong DF, Wagner HN Jr, Dannals RF, Links JM, Frost JJ, 31. Seeman P: Receptor Tables: Drug Dissociation Constants for Ravert HT, Wilson AA, Rosenbaum AE, Gjedde A, Douglass Neuroreceptors and Transporters, vol 2. Toronto, SZ Re- KH: Effects of age on dopamine and serotonin receptors mea- search, 1993 sured by positron tomography in the living human brain. Sci- 32. Blin J, Crouzel C: Blood-cerebrospinal fluid and blood-brain ence 1984; 226:1393–1396 barriers imaged by 18F-labeled metabolites of 18F-setoper- 45. Iyo M, Yamasaki T: The detection of age-related decrease of one studied in humans using positron emission tomography. J dopamine D1, D2 and serotonin 5-HT2 receptors in living hu- Neurochem 1992; 58:2303–2310 man brain. Prog Neuropsychopharmacol Biol Psychiatry 33. Kapur S, Jones C, DaSilva J, Wilson A, Houle S: Reliability of 1993; 17:415–421 a simple non-invasive method for the evaluation of 5-HT2 re- 46. Wang GJ, Volkow ND, Logan J, Fowler JS, Schlyer D, ceptors using [18F]-setoperone PET imaging. Nucl Med Com- MacGregor RR, Hitzemann RJ, Gur RC, Wolf AP: Evaluation mun 1997; 18:395–399 of age-related changes in serotonin 5-HT2 and dopamine D2 34. Blin J, Baron JC, Dubois B, Crouzel C, Fiorelli M, Attar-Levy D, receptor availability in healthy human subjects. Life Sci 1995; Pillon B, Fournier D, Vidailhet M, Agid Y: Loss of brain 5-HT2 56:PL249–PL253 receptors in Alzheimer’s disease: in vivo assessment with 47. Iqbal N, Asnis GM, Wetzler S, Kahn RS, Kay SR, van Praag positron emission tomography and [18F]setoperone. Brain HM: The MCPP challenge test in schizophrenia: hormonal 1993; 116:497–510 and behavioral responses. Biol Psychiatry 1991; 30:770–778 35. Kapur S, Zipursky R, Remington G, Jones C, McKay G, Houle 48. Kahn RS, Davidson M: Serotonin receptor responsivity in S: PET evidence that loxapine is an equipotent blocker of 5- schizophrenia. Int Clin Psychopharmacol 1993; 8:47–51 HT2 and D2 receptors: implications for the treatment of schizo- 49. Krystal JH, Seibyl JP, Price LH, Woods SW, Heninger GR, phrenia. Am J Psychiatry 1997; 154:1525–1529 Aghajanian GK, Charney DS: m-Chlorophenylpiperazine ef- 36. Sadzot B, Lemaire C, Maquet P, Salmon E, Plenevaux A, De- fects in neuroleptic-free schizophrenic patients: evidence im- gueldre C, Hermanne JP, Guillaume M, Cantineau R, Comar plicating serotonergic systems in the positive symptoms of D, Franck G: Serotonin 5HT(2) receptor imaging in the human schizophrenia. Arch Gen Psychiatry 1993; 50:624–635 brain using positron emission tomography and a new radioli- 50. Abel KM, O’Keane V, Murray RM: Enhancement of the prolac- gand, [F-18]altanserin: results in young normal controls. J tin response to d-fenfluramine in drug-naive schizophrenic pa- Cereb Blood Flow Metab 1995; 15:787–797 tients. Br J Psychiatry 1996; 168:57–60 37. Crouzel C, Guillaume M, Barre L, Lemaire C, Pike VW: 51. Abel KM, O’Keane V, Murray RM, Cleare AJ: Serotonergic Ligands and tracers for PET studies of the 5-HT system—cur- function and negative and depressive symptomatology in rent status. Int J Radiation Applications and Instrumentation, schizophrenia and major depression. Psychoneuroendocri- part B 1992; 19:857–870 nology 1997; 22:539–548 38. Wong DF, Lever JR, Hartig PR, Dannals RF, Villemagne V, 52. Lindenmayer JP, Adityanjee, Vital-Herne M, Bark N, Gro- Hoffman BJ, Wilson AA, Ravert HT, Links JM, Scheffel U, chowski S, Moynihan N: Heterogeneity of serotonergic re- Wagner HN: Localization of serotonin 5-HT2 receptors in liv- sponse in treatment-refractory schizophrenia patients. Biol ing human brain by positron emission tomography using N1- Psychiatry 1997; 42:6–12 ([11C]-methyl)-2-Br-LSD. Synapse 1987; 1:393–398 53. Kahn RS, Davidson M, Siever L, Gabriel S, Apter S, Davis KL: 39. First MB, Spitzer RL, Gibbon M, Williams JBW: Structured Serotonin function and treatment response to clozapine in Clinical Interview for DSM-IV Axis I Disorders (SCID), Clini- schizophrenic patients. Am J Psychiatry 1993; 150:1337– cian Version. Washington, DC, American Psychiatric Press, 1342 1995 54. Curtis VA, Wright P, Reveley A, Kerwin R, Lucey JV: Effect of 40. Opler LA, Kay SR, Lindenmayer JP, Fiszbein A: Structured clozapine on d-fenfluramine-evoked neuroendocrine re- Clinical Interview for the Positive and Negative Syndrome sponses in schizophrenia and its relationship to clinical im- Scale. Toronto, Multi-Health Systems, 1992 provement. Br J Psychiatry 1995; 166:642–646 41. Spitzer RL, Williams JBW, Gibbon M, First MB: Structured 55. Cohen J: Statistical Power Analysis for the Behavioral Sci- Clinical Interview for DSM-III-R—Non-Patient Edition (SCID- ences. Hillsdale, NJ, Lawrence Erlbaum Associates, 1988 NP, Version 1.0). Washington, DC, American Psychiatric 56. Andreasen NC, Carson R, Diksic M, Evans A, Farde L, Press, 1990 Gjedde A, Hakim A, Lal S, Nair N, Sedvall G, Tune L, Wong 42. Talairach J, Tournoux P: Co-Planar Stereotaxic Atlas of the DF: Workshop on schizophrenia, PET, and dopamine D2 re- Human Brain: Three-Dimensional Proportional System. Stutt- ceptors in the human neostriatum. Schizophr Bull 1988; 14: gart, Germany, Georg Thieme, 1988 471–484

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