No Evidence of Altered in Vivobenzodiazepine Receptor Binding in Schizophrenia

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No Evidence of Altered In Vivo Benzodiazepine Receptor Binding in Schizophrenia Anissa Abi-Dargham, M.D., Marc Laruelle, M.D., John Krystal, M.D., Cyril D’Souza, M.D., Sami Zoghbi, Ph.D., Ronald M. Baldwin, Ph.D., John Seibyl, M.D., Osama Mawlawi, Ph.D., Gabriel de Erasquin, M.D., Ph.D., Dennis Charney, M.D., and Robert B. Innis, M.D., Ph.D.

Deficits in gamma-amino-butyric acid (GABA) of this measurement was established in four healthy neurotransmitter systems have been implicated in the volunteers. No differences in regional BDZ VT were pathophysiology of schizophrenia for more than two observed between 16 male schizophrenic patients and 16 decades. Previous postmortem and in vivo studies of matched controls. No relationships were observed between benzodiazepine (BDZ) receptor density have reported BDZ VT and severity of psychotic symptoms in any of the alterations in several brain regions of schizophrenic regions examined. In conclusion, this study failed to patients. The goal of this study was to better characterize identify alterations of BDZ receptors density in possible alterations of the in vivo regional distribution schizophrenia. If this illness is associated with deficits in volume (VT) of BDZ receptors in schizophrenia, using the GABA transmission, these deficits do not substantially selective BDZ antagonist [123I]iomazenil and single photon involve BDZ receptor expression or regulation. emission computerized tomography (SPECT). Regional [Neuropsychopharmacology 20:650-661, 1999]

KEY WORDS: Schizophrenia; Benzodiazepine receptors; that disinhibition of dopamine function in schizophre- [123I]iomazenil; SPECT nia may stem from a deficit in inhibitory, GABA medi- ated systems (Roberts 1972; Stevens 1975; van Kammen Alterations of gamma-amino-butyric acid (GABA) sys- 1977). In addition, postmortem studies have reported tem have been implicated in the pathophysiology of alterations in regional concentrations of several mark- schizophrenia for more than two decades. The recogni- ers of the GABAergic system in schizophrenia. tion that GABA has an inhibitory effect on dopamine In accordance with the hypothesis of a GABA de- transmission prompted several authors to postulate ficit in schizophrenia, GABAergic cells and markers of GABAergic cells have usually been reported to be decreased in postmortem studies in patients with schizo- From the Departments of Psychiatry and Diagnostic Radiology (AA-D, ML, JK, CD’S, SZ, RMB, JS, GDE, DC, RBI), Yale University phrenia (Bird et al. 1977; Perry et al. 1979; Simpson et al. School of Medicine, New Haven, and VA Connecticut Healthcare 1989; Reynolds et al. 1990; Benes et al. 1991; Sherman et System, West Haven, Connecticut; and Department of Psychiatry al. 1991; Akbarian et al. 1995b; Beasley and Reynolds and Radiology (AA-D, ML, OM), College of Physicians and Sur- geons, Columbia University, New York, and New York State Psy- 1997). However, few of these observations have been chiatry Institute, New York, New York. replicated (Bennett et al. 1979; Cross et al. 1979; Akbar- Address correspondence to: Anissa Abi-Dargham, New York ian et al. 1995b; Woo et al. 1997). State Psychiatric Institute, Unit 42, 1051 Riverside Dr, New York, NY, 10032 As far as postsynaptic markers are concerned, in- 3 Received May 19, 1998; accepted October 1, 1998. creased binding of the GABAA receptor agonist [ H]mus-

NEUROPSYCHOPHARMACOLOGY 1999–VOL. 20, NO. 6 © 1999 American College of Neuropsychopharmacology Published by Elsevier Science Inc. 0893-133X/99/$–see front matter 655 Avenue of the Americas, New York, NY 10010 PII S0893-133X(98)00107-9

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cimol has been consistently reported in prefrontal cor- is insensitive to possible group differences in blood tex, caudate nucleus, hippocampus and cingulate flow and peripheral clearance which affect semi-quanti- cortex in schizophrenia (Hanada et al. 1987; Benes et al. tative methods following single bolus injection (Carson

1992; Benes et al. 1996). However, GABAA receptor sub- et al. 1992). In addition, regions of interest (ROI) were units mRNA was unchanged in prefrontal cortex (Ak- identified following coregistration with MRI. This method barian et al. 1995a). Benzodiazepine receptors have yields several potential outcome measures, depending been reported to be unchanged in the hippocampus on the nature of the concentration parameter used in and prefrontal cortex (Reynolds and Stroud 1993; Benes the denominator of the distribution volume ratio. The et al. 1997; Pandey et al. 1997), decreased in hippocam- first goal of this investigation was to assess the repro- pus, anterior cingulate cortex, somatomotor cortex, cer- ducibility and reliability of each candidate outcome ebellum, globus pallidus (Squires et al. 1993), and in- measure in a testրretest study in four healthy subjects. creased in frontal, temporal and putamen (Kiuchi et al. The second goal was to compare BDZ receptor density 1989). The increase in [3H]muscimol binding to the between patients with schizophrenia and healthy con- ր GABA binding site of the GABAA BDZ receptor com- trols using the most reliable outcome measure. plex, in the face of unchanged BDZ receptor binding in some regions of schizophrenic brains, has been inter- preted as indicating an uncoupling of GABAA and BDZ MATERIALS AND METHODS receptors in schizophrenia (Benes et al. 1997). Thus, the Subjects overall picture is suggestive of a functional andրor structural decrease in GABAergic activity, possibly as- The study was performed according to protocols ap- sociated with a compensatory regulation of postsynap- proved by the Yale School of Medicine and VA Con- tic receptors. Still, many contradictory reports have necticut at West Haven Institutional Review Boards. been published, and most of these findings await inde- Patients were recruited from the VA Connecticut at pendent replication. West Haven. Study criteria for patients with schizo- In contrast to the abundant postmortem literature, phrenia were as follows: 1) diagnosis of schizophrenia very few in vivo studies of GABA transmission in according to Diagnostic and Statistical Manual (DSM- schizophrenia have been reported. Busato et al. (1997) IV); 2) no other DSM-IV axis I diagnosis; 3) no history of using SPECT and the BDZ receptor radiotracer [123I]iom- alcohol or substance abuse or dependence; 4) absence of azenil, reported no regional differences in the uptake of benzodiazepine administration for at least 21 days; 5) [123I]iomazenil between 15 patients with schizophrenia no concomitant or past severe medical conditions; 6) no and 12 healthy controls. In the schizophrenic group, se- pregnancy; 7) no metallic objects in the body; and 8) verity of positive symptoms was associated with de- ability to provide informed consent. Patient clinical creased [123I]iomazenil uptake in the left medial tempo- evaluation at intake included the Positive and Negative ral region, and severity of negative symptoms was Symptoms Scales (PANS) (Kay et al. 1987), a scale that correlated with decreased medial frontal [123I]iomazenil includes the Brief Psychiatric Rating Scale (BPRS) uptake. However, these correlations were not signifi- (Overall and Gorham 1962), and the AIMS (Munetz and cant after Bonferroni correction. Schröder et al. (1997) Benjamin 1988). Since BDZ receptor density is not con- investigated [123I]iomazenil binding in 20 patients with sistently affected by chronic antipsychotic administra- schizophrenia, but no control group was studied. They tion (Gavish et al. 1988; Giardino et al. 1991), neurolep- reported that BPRS scores were correlated with in- tic withdrawal was not required for this study. creased [123I]iomazenil binding in the medial prefrontal Study criteria for healthy controls were: 1) absence of cortex. Both studies were semi-quantitative and, as past or present neurological or psychiatric illnesses; 2) such, are subject to numerous potential artifacts. Yet, absence of concomitant or past severe medical condi- these reports suggested that BDZ receptors density tions; 3) no pregnancy; 4) no metallic objects in the might be associated with symptomatic features of body; and 5) informed consent. Healthy controls were schizophrenia. group matched to patients for age (Ϯ5 years), gender, In the present study, we used the constant infu- and race. Four healthy controls were scanned twice, at 1 sionրsustained equilibrium paradigm with SPECT and to 2 week intervals, to evaluate the reproducibility and [123I]iomazenil (Laruelle et al. 1993, 1994; Abi-Dargham reliability of the measurements. et al. 1994b; Lassen 1996) to examine possible alterations of BDZ receptor binding potential (BP) in 16 pa- Radiolableing tients with schizophrenia and 16 matched controls, and to study possible relationships of regional BDZ receptor Sodium [123I]iodide (no-carrier-added in 0.1 M NaOH) density with symptomatology. In contrast to the semi- was purchased from Nordion International, Vancouver, quantitative methods used in the above-described stud- B.C. [123I]Iomazenil is a high affinity antagonist of the ր ϭ Њ ies, the constant infusion sustained equilibrium method BDZ receptor (KD 0.35 nM at 22 C) (Beer et al. 1990;

652 A. Abi-Dargham et al. NEUROPSYCHOPHARMACOLOGY 1999–VOL. 20, NO. 6

Laruelle et al. 1994). [123I]Iomazenil was prepared by io- these subjects, the attenuation ellipse and the ROIs dodestannylation of its tributylstannyl precursor as were directly drawn on the SPECT images, as previ- previously described (Zoghbi et al. 1992; Zea-Ponce et ously described for the study of the testրretest repro- al. 1993) in average yield 63.3 Ϯ 10.7% and radiochemi- ducibility of BDZ distribution volume measurements cal purity 97.6 Ϯ 2.0% (with these and subsequent data by kinetic analysis of single bolus [123I]iomazenil injec- expressed as mean Ϯ S.D.; n ϭ 19). Sterility was con- tion experiments (Abi-Dargham et al. 1995). firmed by lack of growth in two media: fluid thioglycol- For the clinical study, attenuation correction and ROI late at 35ЊC and soybean-casein digest at 25ЊC for two identification were performed after coregistration to the weeks. Apyrogenicity was confirmed by the LAL test MRI. The aligned four SPECT acquisitions were summed (Endosafe, Charleston, NC). on a slice-by-slice basis, and the summed SPECT data set was transferred to the program ANALYZE (Mayo Foundation, Rochester, Minnesota). MRI T2 weighted Data Acquisition images were coregistered to the summed SPECT image All subjects received potassium iodide (SSKI solution using a contour-fitting algorithm, after manual tracings 0.6 g) in the 24 h period prior to scan. Four fiducial of the contour of the brain on both SPECT and MRI im- ␮ 99m markers filled with 10 Ci of [ Tc]NaTcO4 were at- ages (Jiang and Robb 1992). The coregistered MRI was tached on both sides of the subject’s head at the level of then used to draw the attenuation correction ellipse the cantho-meatal line to identify this plane during im- around the skull on each slice. Ellipses were transferred age analysis. Subjects received a priming bolus of to the corresponding SPECT slices, and attenuation cor- [123I]iomazenil (3.83 Ϯ 0.43 mCi for controls and 3.85 Ϯ rection was performed assuming uniform attenuation 0.41 mCi for patients), followed by a continuous infu- equal to that of water, corrected for higher scatter in the sion at a constant rate (1.01 Ϯ 0.11 mCiրh and 1.02 Ϯ center of the object (attenuation coefficient ␮ ϭ 0.12 0.11 mCiրh, respectively) using a computer-controlled cmϪ1). The attenuation corrected SPECT was then pump (IMED pump, Jemini PC-1, San Diego, CA). Du- coregistered to the original MRI. Final SPECT voxel vol- ration of infusion was 7 h (total injected dose of 10.68 Ϯ ume was 0.94 ϫ 0.94 ϫ 3 mm ϭ 2.64 mm3. This proce- 1.26 mCi and 10.44 Ϯ 1.16 mCi for controls and patients, dure was followed in order to: 1) perform the attenuation respectively). SPECT scanning was initiated at 6 h into correction before any significant rotation or reslicing of the infusion. Four consecutive 10 min frames were ac- the SPECT data; and 2) orient the SPECT slices of all quired with the Ceraspect camera (Digital Scintigraph- subjects into the AC-PC line for data analysis. ROIs ics, Waltham, MA), with a resolution in water of 12 mm were drawn on each MRI plane by the same rater, in the three planes (Zubal et al. 1990). Three venous blindly to the diagnosis, according to anatomical crite- blood samples were collected in the middle of the scan- ria derived from the Duvernoy atlas (Duvernoy 1991). ning session. MRI scans were acquired on the GE 1.5 Sampled ROIs included right and left frontal, cingulate, Tesla Signa super-conducting magnet. A sagittal scout temporal, parietal, and occipital cortices, right and left scan was used to define the anterior-posterior commis- hippocampus, amygdala, parahippocampal gyrus, thal- sure (AC-PC). Axial slices in the AC-PC plane were ac- amus, striatum, and cerebellum. A region was also quired using a double spin echo protocol with TE’s of drawn on the midbrain white matter, which is the re- 30 ms and 80 ms, TR 4000 ms, matrix 256 ϫ 192, 1 NEX, gion with the lowest density of BDZ receptors in the 3 mm thickness, no gap (pixel size 0.94 ϫ 0.94). The T2 brain. Cortical ROIs included both gray and white mat- weighted images (TE 80 ms) were used in this analysis. ter. These ROIs were then transferred to the corresponding SPECT planes. Average cpmրpixel ROI activi- ties were decay corrected and expresed in ␮Ciրcc using Image Analysis an average calibration factor derived from 123I distrib- SPECT images were reconstructed using a Butterworth uted source phantom studies performed at regular in- filter (cutoff ϭ 1 cm, power factor ϭ 10) and displayed tervals throughout the duration of the studies. Because on a 64 ϫ 64 ϫ 32 matrix (pixel size ϭ 3.33 ϫ 3.33 mm, reduced [3H]nipecotic acid binding to GABA transport- slice thickness ϭ 3.32 mm, voxel volume ϭ 36.76 mm3) ers has been reported to be more prominent in the left using the Ceraspect software. The four acquisitions than the right temporal regions, right and left regions were individually inspected for evidence of head move- were analyzed separately. A laterality index was calcu- ment, and reoriented, if needed, to the first acquisition, lated as (L Ϫ R)ր(L ϩ R). This index varies from Ϫ1 (all using the fiducial markers as landmarks. Occipital to the right) to ϩ1 (all to the left). counts were measured on each acquisition, and the time activity curve was analyzed to check the quality of Plasma Analysis the equilibrium (a regional change of less than 10%րh was chosen as equilibrium criteria). Blood samples were analyzed as previously described No MRIs were acquired for the testրretest subjects. In (Zoghbi et al. 1992). A 200 ␮L aliquot of the plasma was

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counted to measure the plasma activity concentration ational equation used to yield a practical measure of ␮ ր (CPACT, Ci mL). Extraction (ethyl acetate) was fol- binding potential (BPOP) (Equation 4) lowed by reverse phase HPLC to measure the metabo- CROI – CREF lite-corrected total plasma activity (parent compound BPOP = ------(4) ␮ ր CREF plasma concentration, CPPAR, Ci ml). Plasma protein binding was measured by ultrafiltration through Cen- Measurement of activity in a region devoid of recep- 123 trifree membrane filters (Amicon Division, W.R. Grace tors (CREF) is required for this method. For [ I]iomaze- & Co., Danvers, MA) (Gandelman et al. 1994). One ali- nil, the white matter activity has been used as measure quot from a pooled sample of plasma obtained from of CREF (Busatto et al. 1997). However, close to 50% of different healthy volunteers was processed as a stan- white matter “nonspecific” activity is activity scattered dard with each experiment to control for day to day from adjacent cortical areas, as demonstrated by the fact variability in the free fraction assay. The plasma free that 50% of [123I]iomazenil white matter activity is “dis- fraction (f1) measured in the subject sample was cor- placeable” by flumazenil (Abi-Dargham et al. 1994b). rected for inter-assay variability using the plasma free The absence of a measurable CREF has two consequences fraction measured in the standard (f1 std) and the aver- for the calculation of BP: 1) CROI is used instead of B in age of the standard measurement over the course of the the numerator, which is adequate since the nonspecific Ϯ ϫ ր study (f1 ave, 0.39 0.03) according to [(f1 f1 ave) f1 std]. component is negligible (Abi-Dargham et al. 1994b); The parent compound plasma concentration (CPPAR) and 2) the concentration parameter, i.e., the denomina- 123 was multiplied by f1 to yield the free plasma [ I]ioma- tor, must be derived from a plasma measurement. zenil concentration (CPFREE). Plasma clearance of total Since passive diffusion is the mechanism of transport activity, total parent [123I]iomazenil and free [123I]ioma- of [123I]iomazenil across the blood-brain barrier, free zenil (Lրh) were calculated as the ratio of the rate of in- [123I]iomazenil concentration in plasma and brain water fusion (␮Ciրh) to the corresponding plasma concentra- are equal under the steady-state conditions achieved ␮ ր tions (CPACT, CPPAR, and CPFREE, respectively, Ci L). during constant infusion (Kawai et al. 1991; Laruelle et al. 1994). Therefore, free [123I]iomazenil concentration in brain water can be estimated by the free plasma Outcome Measures 123 [ I]iomazenil concentration (CPFREE). Since the numer- At equilibrium, the ligand bound to the receptors (B) is ator includes both specific binding and nondisplaceable related to receptor parameters (maximal density of components (nonspecific binding and free), we use the available receptors, Bmax, and equilibrium dissociation term total distribution volume (VT) rather than BP to constant, KD) and to the free tracer concentration (Free) characterize the BDZ receptors availability (Equation 5) by the Michaelis-Menten equation (Equation 1) CROI × V = ------(5) Bmax Free T CP B = ------(1) FREE KD + Free While CPFREE is theoretically the most appropriate When experiments are carried out at tracer doses, denominator for the distribution volume ratio (Equa- Free is negligible compared to KD, and Equation 1 can tion 5), the measurement of CPFREE is prone to experi- be simplified as follows (Equation 2) mental errors. Other candidate denominators are avail- B × Free able, which might prove more robust (Table 1). The B = ------max (2) KD simplest outcome measure (VT INF) involves correcting the measured regional activity by the hourly infused The binding potential (BP) quantifies receptor avail- ϭ ր ability at tracer doses, and corresponds to the product dose (HID) (VT INF CROI HID). To take into account be- ր tween individual differences in the rate of peripheral of receptor density and affinity (Bmax KD). Rearranging Equation 2 shows that BP is equal to the BoundրFree ra- clearance of the infused activity, regional activity can be expressed relatively to the total plasma activity at equi- tio at equilibrium (Equation 3) ϭ ր librium (VT ACT CROI CPACT). This outcome measure re- B B BP ==------max- (3) quires a simple measurement of plasma activity. To ac- Free KD count for between individual differences in the In vivo, B is usually derived by subtracting the activ- metabolism of iomazenil, regional activity can be ex- ity in a region of reference (CREF) from the activity in a pressed relatively to the total (free and bound to plasma 123 region of interest (CROI). CREF is assumed to provide an proteins) plasma concentration of the parent [ I]ioma- estimate of the free and nonspecific binding in the re- zenil (CPPAR), which requires determination by HPLC of Ϫ ϭ gion of interest, so that CROI CREF B. The measure- the parent fraction in plasma extracts. This outcome Ј Ј ϭ ր ment of the free requires plasma analysis. When this measure has been denoted VT (VT CROI CPPAR) (Abi- measurement is not performed, CREF can be used as the Dargham et al. 1994a). Finally, to correct for possible denominator (i.e., concentration parameter) in the oper- differences in plasma protein binding of the radiotracer, 654 A. Abi-Dargham et al. 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the measurement of plasma free fraction (fl) must be measurement and can vary between 0 (no reliability) ϭ ր obtained (VT CROI CPFREE). The choice between these and 1 (maximum reliability, achieved in case of identity outcome measures can not be made without knowledge between test and retest, i.e., MSWS ϭ 0). Group differ- of the reproducibility of these measurements, because ences in outcome measures were assessed with a two- of the conflicts between potential biases and experimen- tailed unpaired t test, using p Ͻ .05 as significance level, tal errors. The more measurements are incorporated following an F test to evaluate the assumption of equal into the outcome measure, the greatest the protection variance. The relationship between continuous vari- against biases but the greatest the vulnerability to prop- ables was assessed with Pearson product correlation co- agation of experimental errors. Testրretest studies allow efficient, followed by ANOVA. to determine the reproducibility of each outcome mea- In addition to the region of interest analysis de- sure, as well as their reliability, i.e. their ability to detect scribed above, we also performed a pixel-by-pixel sta- between subject differences. We elected to evaluate the tistical analysis using statistical parametric mapping variability and reliability of these outcome measures by (SPM 96) (Friston et al. 1995). The advantage of SPM scanning four healthy subjects twice. The results of the compared to the ROI method is that SPM does not testրretest studies determined the outcome measure make any prediction regarding the size or location of an used in the clinical investigation. area of significant change. Therefore, this method is able to detect areas of change (statistical excursions) limited to small parts of the regions selected by the ROI Statistical Analysis analysis, as well as areas of change overlapping two re- All values are expressed as mean Ϯ SD. The variability gions. The disadvantage of SPM is the inevitable blur- of one measurement between test and retest was com- ring of the images resulting from the spatial normaliza- puted as the absolute value of the difference between tion procedure, as well as the intrinsic exploratory test and retest, expressed in percentage of the mean nature of its results. SPECT images were transformed value of both measurements. Differences in variability into BDZ VT maps by dividing the activity concentra- between different outcome measures were investigated tion in each pixel by the steady-state [123I]iomazenil by a repeated measure ANOVA with regions as cofac- plasma concentration. MRI scans were mapped into the tor. The reliability of the measurements was assessed Talairach space (Talairach and Tournoux 1988) using a relatively to the between subjects variance by the intra- 12 parameter affine transformation which was then ap- ␳ class correlation coefficient, , calculated as (Kirk 1982) plied to the subject’s BDZ VT map, in a concentration conservative manner. The atlas-fitted V maps were MSBS– MSWS T ------smoothed with a Gaussian kernel to minimize the effect MSBS+ () n– 1 MSWS of intersubject anatomical variability. Since the VT map where MSBS is the mean sum of square between sub- consists of absolute numbers which are directly compa- jects and MSWS is the mean sum of square within sub- rable between subjects, we performed the SPM analysis jects. This coefficient estimates the reliability of the without global normalization. The statistical z map

Table 1. Definition of Potential Outcome Measures

Factors Taken Into Account (Yes/No) Outcome Measure Activity Iomazenil Binding to Notation Equation Equation clearance metabolism plasma proteins

CROI V Hourly infused dose------No No No T INF HIR

CROI Yes No No VT ACT Total plasma activity ------CPACT

CROI Yes Yes No V Ј Total plasma ------T CP [123I]iomazenil activity PAR CROI Yes Yes Yes V Free plasma ------T CP [123I]iomazenil activity FREE

123 ␮ Abbreviations: CROI, [ I]iomazenil concentration in ROI at equilibrium ( Ci/g of tissue); HIR, hourly infused ␮ 123 dose ( Ci/g body weight); CPACT, plasma activity concentration (parent and metabolized [ I]iomazenil, bound ␮ ␮ and free) ( Ci/ml of plasma); CPPAR, plasma concentration of parent compound (bound and free) ( Ci/ml of 123 ␮ plasma); CPFREE, plasma concentration of free [ I]iomazenil ( Ci/ml of plasma); VT, total distribution volume. NEUROPSYCHOPHARMACOLOGY 1999–VOL. 20, NO. 6 Benzodiazepine Receptors in Schizophrenia 655

threshold was set at 2.53 (p ϭ .005). A more traditional Table 3. Variability and Reliability of

SPM analysis using ANCOVA to normalize global VT Distribution Volumes was also performed. Measure Variability (%) Reliability (␳) Ϯ Ϯ VTINF 14 1 .52 .16 Ϯ a Ϯ VTACT 10 3 .53 .20 Ϯ Ϯ RESULTS VTЈ 13 1 .66 .06 Ϯ Ϯ VT 15 1 .70 .05 TestրRetest Study ϭ Abbreviations: VTINF distribution volume relative to hourly infused ϭ The testրretest variability and reliability of the plasma dose; VTACT distribution volume relative to total plasma activity at measurements are provided in Table 2. The variability equilibrium. a Significantly different from three other outcome measures, ANOVA was lower than 10% for all measures. Clearance of the (p ϭ .004), followed by Fisher PLSD. parent compound was the measurement with lowest Clinical Study variability, 4 Ϯ 3%, and highest reliability 0.93. Correc- tion for free fraction resulted in a slight degradation of The study included sixteen patients and sixteen con- both reproducibility and reliability. However, correct- trols. Both groups were matched on gender (all males),

ing fl for between assay variability improved the repro- age and ethnicity (Table 4). Eleven patients were taking ducibility of the measurement (from 6 Ϯ 4% to 3 Ϯ 3%). neuroleptic drugs at the time of the scan, and five pa- For the distribution volumes, four regions were ex- tients were neuroleptic-free: three of them had been amined (frontal, temporal, cingulate and occipital). As previously treated and were off neuroleptics for more no statistically significant differences were observed in than 21 days, and two patients were drug naive. The av- reproducibility indices between these regions, values erage duration of prior neuroleptic exposure was 13 Ϯ derived from the four regions were averaged for the 10 years. Four patients had noticeable tardive dyskine- purpose of comparing the outcome measures (Table 3). sia (AIMS scores Ͼ 6). Significant differences in reproducibility were observed Table 5 summarizes plasma measurements in both ϭ between outcome measures (ANOVA, p .004): VT ACT groups. No differences were observed between groups had a significantly better reproducibility (10 Ϯ 3%), in total activity clearance. However, [123I]iomazenil me- Ϯ Ј Ϯ compared to VT INF (14 1%), VT (13 1%), and VT tabolism was slower in controls than in patients (per- Ϯ Ј Ϯ Ϯ (15 1%). The difference in variability between VT cent parent: 14 7% versus 12 3% in controls and pa- ϭ ϭ and VT was not significant (p .38). VT showed the tients, respectively, p .077). As a result, clearance of highest reliability (0.70 Ϯ 0.05), although between meth- the parent compound was slower in controls than in pa- ods differences in reliability were not significant (p ϭ tients (116 Ϯ 23 Lրh versus 142 Ϯ36 36 Lրh in controls ϭ .18). Because VT was the theoretically preferred out- and patients, respectively, p .023). The free fraction come measure (Table 1) and the variable with the great- was similar in both groups, indicating similar binding est power to detect inter-individual differences (i.e., to plasma proteins. The difference in rate of metabolism highest reliability, Table 3), this outcome measure was resulted in a slower clearance of the free compound in selected for the clinical study, despite the fact that its re- controls compared to patients (370 Ϯ 86 Lրh versus 441 Ϯ ր ϭ producibility was lower than VT ACT. 115 L h, p .058). In other terms, for a similar rate of infusion, the plasma concentration of [123I]iomazenil tended to be lower in the schizophrenic patients than in the controls. Table 2. Variability and Reliability of Equilibrium criteria were satisfied for every subject. Plasma Measurements The distribution of BDZ VT was in accordance with the known distribution of BDZ receptors in the human ␳ Measure Variability (%) Reliability ( ) brain (Braestrup et al. 1977; Luabeya et al. 1984). High- Clearance total activity 7 Ϯ 4 .52 Fraction parent by HPLC 6 ϩ 2 .93 Clearance parent 4 ϩ 3 .93 a Ϯ Table 4. Sample Demographics f1 uncorrected 6 4 .54 f correctedb 3 Ϯ 3 .57 1 Characteristics Controls Schizophrenics Clearance free (corrected) 8 Ϯ 6 .78 n1616 Reproducibility parameters derived from repeated experiments in Ϯ Ϯ four healthy subjects. Age (years) 45 10 44 10 af uncorrected: plasma free fraction measured with ultracentrifuga- Gender All males All males 1 a tion of the subject’s sample. Ethnicity (AA/C/H) 5/10/1 7/8/1 b Ϯ Ϯ f1 corrected: plasma free fraction measured with ultracentrifugation Weight (kg) 84 13 82 12 of the subject’s sample, corrected for between assay variability using in- ternal standards. a AA, Afro-Americans; C, Caucasians; H, Hispanics. 656 A. Abi-Dargham et al. NEUROPSYCHOPHARMACOLOGY 1999–VOL. 20, NO. 6

Table 5. Plasma Measurements in Healthy Controls and No effect of age was observed on regional BDZ VT in Patients with Schizophrenia controls or in schizophrenics, and there was no age * diagnosis interaction. No regional BDZ V differences Plasma Parameter Controls Schizophrenics p T were observed between the patients off (n ϭ 5) or on ϭ Clearance total activity neuroleptics (n 11). No regional BDZ VT differences (L/h) 15.7 Ϯ 1.8 16.7 Ϯ 3.0 .247 were observed between patients with (n ϭ 4) or without Ϯ Ϯ Fraction parent (%) 14 7 12 3 .077 (n ϭ 12) significant tardive dyskinesias. Correlations Clearance parent [123I]iomazenil (L/h) 116 Ϯ 23 142 Ϯ 36 .023 were investigated between regional BDZ VT and posi- Ϯ Ϯ f1 normalized 0.32 0.05 0.32 0.05 .697 tive and negative symptoms severity. BDZ VT in several Clearance free regions was positively correlated with severity of posi- [123I]iomazenil (L/h) 370 Ϯ 86 441 ϩ 115 .058 tive symptoms: left amygdala, p ϭ .018; right hippo- ϭ ϭ Values are Mean Ϯ SD; p is the probability value of between group dif- campus, p .0076; right striatum, p .033; right thala- ϭ ferences by two tailed unpaired t-test. mus, p .0331. No regional BDZ VT was correlated with negative symptoms. Since a total of 40 correlations est binding was observed in neocortical areas, parahip- were performed (10 regions * 2 sides * 2 clinical clus- pocampal gyrus, and cerebellum, and lower values in ters), none of the above mentioned correlations sur- amygdala, hippocampus, striatum and thalamus. The vived a Bonferroni correction for significance. To at- tempt to replicate the observation of Schröder et al., we variance of regional BDZ VT was not significantly different between the groups. tested the correlation between BPRS scores and pre- We observed no between group differences in re- frontal BDZ VT: these measured were not correlated ϭ (p .63 and .33, for left and right prefrontal VT, respec- gional BDZ VT between controls and schizophrenics in any of the regions examined (Table 6). Figure 1 shows tively). the distributions of BDZ VT in left and right prefrontal region in both groups. In controls, laterality indices were significant for a right dominance (i.e., significantly DISCUSSION lower than zero, one sample t-test) in the temporal cortex (p ϭ .036), occipital cortex (p ϭ .011), and striatum This study failed to demonstrate any alterations in the (p ϭ .032). In schizophrenics, no region had a significant density and regional distribution of BDZ receptors in lateralization index. However, no significant between patients with schizophrenia. A preliminary report of groups differences were observed in the laterality indices. this study suggested an increased BDZ receptor avail- Results of the SPM analysis were in accordance with ability in several regions of schizophrenic subjects the region of interest analysis: no significant statistical compared to controls (n ϭ 7 per group), a difference excursions were detected by comparing the BDZ VT that reached significance in the occipital cortex (Abi- maps between patients and controls, either on the nor- Dargham et al. 1994a). However, in this extended sam- malized or non normalized analyses. ple, no significant differences between schizophrenics

Table 6. Regional BDZ Distribution Volumes (VTFcor, ml/g) in Schizophrenic Patients and Controls

Controls Schizophrenics Significance

Region Left Side Right Side AI Left Side Right Side AI p(1) p(2) p(3)

Cortical regions Frontal cortex 116 Ϯ 22 119 Ϯ 22 Ϫ0.02 Ϯ 0.03 112 Ϯ 21 111 ϩ 24 0.00 Ϯ 0.04 .59 .32 .12 Temporal cortex 108 Ϯ 23 111 Ϯ 20 Ϫ0.02 Ϯ 0.03* 105 Ϯ 20 108 Ϯ 19 Ϫ0.01 Ϯ 0.05 .77 .66 .72 Parietal cortex 97 Ϯ 19 99 Ϯ 25 0.00 Ϯ 0.06 98 Ϯ 19 99 Ϯ 19 0.00 Ϯ 0.03 .83 .99 .97 Occipital cortex 112 Ϯ 21 119 Ϯ 24 Ϫ0.03 Ϯ 0.04* 111 Ϯ 22 115 Ϯ 21 Ϫ0.02 Ϯ 0.04 .85 .68 .70 Limbic Regions Cingulate cortex 112 Ϯ 26 107 Ϯ 18 0.01 Ϯ 0.12 112 Ϯ 26 108 Ϯ 30 0.03 Ϯ 0.03 .98 .95 .78 Amygdala 57 Ϯ 21 61 Ϯ 16 Ϫ0.04 Ϯ 0.10 60 Ϯ 14 63 Ϯ 17 Ϫ0.02 Ϯ 0.12 .64 .67 .56 Hippocampus 62 Ϯ 20 65 Ϯ 18 Ϫ0.03 Ϯ 0.08 58 Ϯ 14 65 Ϯ 16 Ϫ0.06 Ϯ 0.10 .56 .48 .97 Parahippocampal gyrus 91 Ϯ 30 100 Ϯ 24 Ϫ0.05 Ϯ 0.12 91 Ϯ 20 96 Ϯ 27 Ϫ0.02 Ϯ 0.11 .94 .66 .45 Striatum 57 Ϯ 11 58 Ϯ 15 Ϫ0.04 Ϯ 0.07* 54 Ϯ 13 56 Ϯ 10 Ϫ0.02 Ϯ 0.08 .47 .77 .34 Thalamus 61 Ϯ 14 62 Ϯ 15 0.01 Ϯ 0.06 56 Ϯ 14 57 Ϯ 13 Ϫ0.02 Ϯ 0.06 .27 .35 .75 Cerebellum 121 Ϯ 20 120 Ϯ 21 .81

Values are Mean ϩ SD; AI is the asymmetry index, calculated as (L Ϫ R)/(L ϩ R); p is the probability value of between group difference by two tailed unpaired t test: p(1) is the probability of a regional difference comparing right regions; p(2) is the probability of a regional difference comparing left regions; p(3) is the probability of a difference in the asymmetry index. NEUROPSYCHOPHARMACOLOGY 1999–VOL. 20, NO. 6 Benzodiazepine Receptors in Schizophrenia 657

123 Figure 1. Scatter plot of benzodiazepine receptors distribution volumes (VT) measured with [ I]iomazenil SPECT in left and right prefrontal cortex (PFC) in 16 patients with schizophrenia (SCH) and 16 controls (CTR). No significant between- groups differences were detected.

and controls were observable. Furthermore, unlike pre- method: 145 min of scanning time (versus 40 min for vious in vivo imaging studies, we were unable to detect the equilibrium method) and 18 arterial samples (ver- any correlations between clinical symptoms and re- sus 3 venous samples for the equilibrium method). The gional BDZ receptors density. slight gain in reproducibility of the kinetic method may This study was performed with the constant infu- not justify the considerable increase in labor, cost, and sionրequilibrium analysis technique (Abi-Dargham et patient discomfort associated with this method. How- al. 1994b). As previously discussed, this method is eas- ever, this observation suggests that acquiring data for ier to implement than the kinetic analysis of single bo- longer period of time and measuring more blood sam- lus experiments, as it does not require arterial samples could improve the reproducibility of the equilib- pling, multiple plasma measurements and prolonged rium method. imaging time. Compared to semi-quantitative “ratio” In this study, the reproducibility of fl measurements methods following single bolus injection, the constant were excellent, as it was in the kinetic study (3 Ϯ 3% infusion method is more accurate, since the results are and 6 Ϯ 5%, respectively), yet the reliability was poor not influenced by possible between groups differences (0.54 in this study versus 0.46 in the kinetic study). This in peripheral clearance or regional blood flow. result confirms the previous observation that fl mea- While the reproducibility and accuracy of the method surements, however accurate, are of little significance had been previously determined in baboons (Laruelle compared to the small between-subjects variability ob- et al. 1994), we had not yet examined the performance served in these testրretest studies. However, subjects in- of this method in humans. We previously reported the cluded in these testրretested studies were young and reproducibility and reliability of measurement of re- healthy, and this conclusion may not apply to compari- gional BDZ distribution volumes with [123I]iomazenil in son of more heterogeneous groups. Therefore, in this humans derived by kinetic analysis of single bolus ex- clinical study, we decided to use VT as outcome mea- periments (6 pairs) (Abi-Dargham et al. 1995). In the sure, because: 1) the use of VT corrects for between sub- present study, the reproducibility and reliability of out- jects differences in the free fraction; and 2) the cost of come measures determined with the constant infusion this correction in terms of reproducibilityրreliability is were slightly inferior to the kinetic study. The variability not apparent, since no significant differences were ob- Ϯ Ϯ ր (reliability) of VT was 13 1% (0.66), compared to 10 served in the test retest study between the reproducibil- Ј 2% (0.81) in the kinetic study, and the variability (reli- ity and reliability of VT and VT. Ϯ Ϯ ability) of VT was 15 1% (0.70), compared to 13 2% The importance of including the plasma free fraction (0.61) in the kinetic study. The advantage in reproduc- in the computation of the distribution volumes is illus- ibility and reliability of the single bolusրkinetic method trated by the following observation. In this study, we ր over the constant infusion equilibrium method is not observed a trend toward decreasing fl values with age surprising, since more data are acquired for the kinetic in the schizophrenic group (decrease of 0.02% per de- 658 A. Abi-Dargham et al. NEUROPSYCHOPHARMACOLOGY 1999–VOL. 20, NO. 6

cade, p ϭ .14), but not in the control group. In the SPECT limits the accuracy of the measurement in small schizophrenic group, we observed a parallel trend to- structures, such as the hippocampus and amygdala. At Ј ward a decrease in VT values with age, and this “age” best, the resolution of SPECT is comparable to the reso- Ј effect on VT reached significance in the frontal, cingu- lution of homogenate binding studies. Careful review late and cerebellar regions. Since BDZ receptor number of postmortem homogenate binding literature does not is not affected by age in healthy subjects (Abi-Dargham provide consistent evidence for alteration of BDZ re- et al. 1994b), an age effect on BDZ receptors in schizo- ceptors in schizophrenia. Four postmortem investiga- phrenia would have been a potentially interesting obser- tions have examined the binding of the BDZ agonist vation. In addition, one postmortem study had described [3H]flunitrazepam in homogenate membranes of schizo- such a correlation in prefrontal regions of schizophrenic phrenic brains. Changes reported by Squires et al. brains (Kiuchi et al. 1989). However, in this particular (1993) (a 30–60% decrease in [3H]flunitrazepam binding sample of schizophrenic patients, less free [123I]iomazenil in homogenated membranes of hippocampus and cin- was available for binding to BDZ receptors in older gulate cortex) should be detectable with SPECT, if subjects compared to younger subjects. After correction present in this sample. The significance of the Squires of the volume of distribution by the free fraction (i.e. us- study is hampered by the small number of subjects (n ϭ Ј ing VT instead of VT ), the “age effect” disappeared. The 4 per groups). In a larger sample (13 schizophrenics and important point is that the absence of correction for the 10 controls),Kiuchi et al. (1989) reported a 25% increase free fraction might have induced artifactual results. in most of the regions examined (which included fron- A similar observation illustrates the potential pitfalls tal, temporal, hippocampus and putamen). In the me- of not measuring peripheral metabolism of [123I]iomaze- dial frontal cortex, there was no overlap between the nil. In this study, schizophrenics showed a faster me- schizophrenic and control values. Again, an increase of tabolism than controls, which resulted in both lower this magnitude should be detectable with SPECT, and plasma values and lower total [123I]iomazenil regional the reason for the discrepancy between this postmor- uptake. Expressing the BDZ distribution volume rela- tem and the in vivo studies reported here and else- tive to the total activity (metabolites and parent), i.e. where is not obvious. The results of the present investi- using VT ACT as outcome measure, would have resulted gation agree with the postmortem studies of Reynolds in observing significantly lower “BDZ receptors” in and Stroud (1993) which included 15 schizophrenics and schizophrenic patients in frontal, thalamic and cerebel- 13 controls, and failed to show group differences in 3 lar regions. Worse, expressing the regional uptake rela- [ H]flunitrazepam Bmax and KD in the hippocampus, and tive to the injected dose (VT INF) would have resulted in of Pandey et al. (1997) who showed no changes in 3 observing significantly lower BDZ distribution vol- [ H]flumazenil KD or Bmax in the prefrontal cortex of umes in frontal, temporal, occipital, cingulate, striatal, schizophrenic patients. Altogether, as one study showed thalamic and cerebellar regions. These points illustrate an increase in density (Kiuchi et al. 1989), one showed a the potential pitfalls of semi-quantitative approaches to decrease (Squires et al. 1993), and three, in addition to in vivo neuroreceptor imaging. this one, showed no change (Reynolds and Stroud 1993; In agreement with the study of Busatto et al. (1997), Busatto et al., 1997; Pandey et al. 1997) it is reasonable we found no differences in any regions in BDZ recep- to propose that no substantial abnormalities in BDZ tors between schizophrenics and controls. Bussato et al. density are present in schizophrenic patients. Our re- (1997) reported several relationships between regional sults are also consistent with the observation that BDZ density and severity of positive and negative symp- mRNA expression of the main subunits of the adult ր toms. The authors acknowledged that these correlations GABAA BDZ receptor complex is unaltered in prefron- were exploratory at best, and that they did not survive tal cortex in schizophrenia (Akbarian et al. 1995a). Ob- Bonferroni corrections. The fact that these correlations viously, these investigations cannot rule out potential were not observed in the present study, which included alterations limited to specific layers of the cortex or sub- a similar number of subjects, and an improved quanti- fields of the hippocampus, such as recently described tative method (equilibrium conditions, plasma mea- (Benes et al. 1997). surement, and MRI based ROI analysis) confirmed that Interestingly, Pandey et al. (1997) reported down- these correlations were indeed statistical artifacts. The regulation of BDZ receptors in the frontal cortex of same comment applies to the correlation between BPRS schizophrenic patients who died while taking neuro- scores and frontal [123I]iomazenil uptake reported by leptic drugs compared to schizophrenic patients who Schröder et al. (1997). Together, these studies indicate were off drugs at time of death. In the present study, we that in vivo BDZ receptor density, measured with SPECT failed to replicate these results, since no regional differ- 123 [ I]iomazenil, is not affected in schizophrenia, and is ences in BDZ VT were observed between patients on or not related to symptomatology as previously claimed. off neuroleptics. Thus our results do not support alter- Evidently, this conclusion is only valid for the level ation of BDZ receptors density associated with long- of resolution obtained in vivo. The spatial resolution of term neuroleptic exposure, a conclusion consistent with NEUROPSYCHOPHARMACOLOGY 1999–VOL. 20, NO. 6 Benzodiazepine Receptors in Schizophrenia 659

preclinical studies (Gavish et al. 1988; Giardino et al. fer PB, Innis RB (1995): Reproducibility of SPECT mea- 1991). surement of benzodiazepine receptors in human brain The lack of change in BDZ receptors is at variance with iodine-123-iomazenil. J Nucl Med 36:167–175 with the observation of increased [3H]muscimol bind- Abi-Dargham A, Krystal J, Laruelle M, Zoghbi SS, D’Souza ing to GABA receptors in schizophrenia described C, Brenner L, Sernyak M, Baldwin RM, Zubal G, Hoffer A PB, Charney DS, Innis RB (1994a): SPECT imaging of with homogenate binding in the left prefrontal cortex benzodiazepine receptors in schizophrenics and healthy (increase of about 25%, number of schizophrenic cases, subjects. Soc Neurosci Abs 20 (part 1):24 19, Hanada et al. 1987) and with autoradiography in Abi-Dargham A, Laruelle M, Seibyl J, Rattner Z, Baldwin ϭ several layers of anterior cingulate (n 6; Benes et al. RM, Zoghbi SS, Zea-Ponce Y, Bremner JD, Hyde TM, 1992), and hippocampus subfields (n ϭ 8; Benes et al. Charney DS, Hoffer PB, Innis RB (1994b): SPECT 1996). Observing little differences in BDZ binding on measurement of benzodiazepine receptors in human 123 the same brain regions where marked differences in brain with [ I]iomazenil: Kinetic and equilibrium par- adigms. J Nucl Med 35:228–238 [3H]muscimol binding had been measured, Benes et al. (1997) proposed that schizophrenia might be associated Akbarian S, Huntsman MM, Kim JJ, Tafazzoli A, Potkin SG, Bunney W, Jr, Jones EG (1995a): GABAA receptor sub- with an uncoupling of GABAA and BDZ receptors. ր unit gene expression in human prefrontal cortex: Com- Since the pattern of expression of GABAA BDZ recep- parison of schizophrenics and controls. Cereb Cortex tors subunits mRNA is unchanged in schizophrenia 5:550–560 (Akbarian et al. 1995a), the mechanism underlying this Akbarian S, Kim JJ, Potkin SG, Hagman JO, Tafazzoli A, putative uncoupling remains to be elucidated. The de- Bunney W, Jr, Jones EG (1995b): Gene expression for velopment of an in vivo GABAA radiotracer would be glutamic acid decarboxylase is reduced without loss of useful to explore this issue further. neurons in prefrontal cortex of schizophrenics. Arch Gen Psychiatry 52:258–266 In conclusion, this study failed to detect any significant abnormalities of BDZ receptors density in a sample Beasley CL, Reynolds GP (1997): Parvalbumin-immunoreac- of 16 patients with schizophrenia compared to 16 tive neurons are reduced in the prefrontal cortex of schizophrenics. Schizophr Res 24:349–355 healthy controls, and contradicts previous reports of an association between BDZ receptor density and symp- Beer H-F, Blauenstein PA, Hasler PH, Delaloye B, Riccabona G, Bangerl I, Hunkeler W, Bonetti EP, Pieri L, Richards tomatic features of the illness. If a deficit in GABA func- JG, Schubiger PA (1990): In vitro and in vivo evaluation tion is implicated in the pathophysiology of schizo- of iodine-123 Ro 16–0154: A new imaging agent for phrenia, this deficit might not involve a substantial SPECT investigations of benzodiazepine receptors. J alteration in the expression of BDZ receptors. In vivo Nucl Med 31:1007–1014 ր neuroreceptor studies of the GABA site on the GABAA Benes FM, Khan Y, Vincent SL, Wickramasinghe R (1996): BDZ complex or magnetic resonance spectroscopy Differences in the subregional and cellular distribution studies of GABA levels are needed to provide addi- of GABAA receptor binding in the hippocampal formation of schizophrenic brain. 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