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Studies on dopaminergic and GABAergic markers in striatum reveals a decrease in the dopamine transporter in schizophrenia
Schizophrenia Research 52 (2001) 107±114
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Studies on dopaminergic and GABAergic markers in striatum reveals a decrease in the dopamine transporter in schizophrenia Brian Dean a,b,c,*, Tabasum Hussain a,c a
The Rebecca Cooper Research Laboratories, The Division of Molecular Schizophrenia, The Mental Health Research Institute, Parkville, Victoria, 3052, Australia b The University of Melbourne Department of Psychiatry, Parkville, 3052, Victoria, Australia c Department of Psychological Medicine, Monash University, Clayton, 3006, Victoria, Australia Received 20 February 2000; accepted 2 June 2000
Abstract Changes in the interaction between dopaminergic and GABAergic systems in the striatum have been suggested to be important in the pathology of schizophrenia. If that hypothesis is correct, these changes could produce inter-related changes in the dopaminergic and GABAergic systems in the striatum from schizophrenic subjects. To test this proposition we measured important markers on dopaminergic and GABAergic neurons in striatum obtained post-mortem from schizophrenic and nonschizophrenic subjects. There was a signi®cant decrease in the density of the dopamine transporter (mean ^ SEM: 230 ^ 31 vs. 334 ^ 22 fmol/mg ETE; P 0.01), but not nitric oxide synthase, dopamine D2-like, D1-like, D3 or GABAA receptors in subjects with schizophrenia. There were no inter-related changes in the dopaminergic or GABAergic markers. In the schizophrenic subjects, the density of dopamine D1-like receptors decreased with age and was positively correlated with the density of dopamine D2-like receptors. This study does not readily add weight to the hypothesis that changes in the interaction between dopamine and GABA in the striatum are important in the pathology of schizophrenia. However, our ®ndings could indicate that changes in the dopamine transporter within the striatum, either because of decreased transporter numbers per se or as a result of innervating neuronal loss, may be involved in the pathology of the illness. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Dopamine receptors; Dopamine transporter; GABAA receptor; Nitric oxide synthase; Schizophrenia; Striatum
1. Introduction The dopamine hypothesis of schizophrenia proposes that overactivity of dopaminergic neurons in the central nervous system (CNS) causes the psychoses associated with the illness (Meltzer, 1987). A lack of direct evidence supporting the dopamine (DA) hypothesis has led to the proposal that changes in the interactions * Corresponding author. Tel.: 1613-9388-1633; fax: 1613-93875061. E-mail address: [email protected] (B. Dean).
between the dopaminergic and the g-aminobutyric acid (GABA) systems of the brain may be a cause of some of the symptoms of the illness (Carlsson et al., 1997). This hypothesis is based on a body of experimental observations suggesting that the interplay between dopaminergic and GABAergic neurons is vital to maintaining functional homeostasis, particularly in the striatum (Carlsson et al., 1997). If the dopamine±GABA hypothesis of schizophrenia is correct, it could be that there are inter-related changes in important dopaminergic and GABAergic markers in the striatum from subjects with schizophrenia.
0920-9964/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0920- 996 4( 00) 00096- 7
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A number of studies have examined either dopaminergic or GABAergic markers in striatum obtained post-mortem from subjects with schizophrenia. Such studies have repeatedly shown an increase in the density of DA D2-like receptors in the striatum from subjects with schizophrenia (for review, see Kahn and Davidson, 1997). Increased GABAA receptors have also been reported in the striatum from schizophrenic subjects (Hanada et al., 1986). These changes could have resulted from inter-related changes in the dopaminergic and GABAergic systems in the striatum from subjects with schizophrenia, as would be predicted by the dopamine±GABA hypothesis. However, because the markers were not measured in tissue from the same individuals, the interactive nature of the changes cannot be assessed. To that end, we have measured important dopaminergic (DA D2-like receptors, DA D1-like receptors, D3 receptor and dopamine transporter [DAT]) and GABAergic (GABAA receptors and nitric oxide synthase [NOS]) markers in striatum so that we can determine if there are inter-related changes in these markers in schizophrenia. 2. Materials and methods 2.1. Materials [ 3H]7-OH-DPAT, [ 3H]SCH 23390, [ 3H]N G-nitroL-arginine HCl, [ 3H]Micro-scales and Hyper®lm- 3H w were obtained from Amersham Australia Pty Ltd., Sydney, Australia. [ 3H]mazindol, [ 3H]YM-09151-2 and [ 3H]muscimol were obtained from New England Nuclear via AMRAD Pharmacia Biotech, Melbourne, Australia. All other chemicals were obtained from Sigma Aldrich Pty Ltd., Castle Hill, New South Wales, Australia. 2.2. Tissue collection Permission to carry out this study was obtained from the Western Region Psychiatric Human Ethics Committee. Striatum was collected at autopsy from the left brain hemisphere of 13 subjects with a provisional diagnosis of schizophrenia and from the same hemisphere of 13 subjects with no history of psychiatric or neurological illness (controls). These subjects were matched for sex and were of a similar age to the
Table 1 Summary (mean ^ SEM) of demographic, pharmacological and tissue related data for schizophrenic and control subjects. PMI post-mortem intervals, DOI duration of illness, FRAD ®nal recorded antipsychotic drug dose
Sex (M/F) Age (yr) PMI (h) pH DOI (yr) FRAD (mg chlorpromazine equiv./day)
Schizophrenic
Control
10/3 47 ^ 5.2 45 ^ 5.1 6.05 ^ 0.07 45 ^ 5.1 436 ^ 120
10/3 47 ^ 4.9 47 ^ 3.4 6.38 ^ 0.04
schizophrenic subjects. The striatum was excised from frozen slice 5 of the left hemisphere, which had been sliced using a standardised protocol (Vonsattel et al., 1995). Post-mortem interval (PMI), in cases where death was witnessed, was the time between death and autopsy. When death was not witnessed, tissue was only taken from individuals that had been seen alive within 5 h of being found dead. In those cases PMI was taken as the interval between the donor being found dead plus half the interval between the subject last being seen alive and being found dead. In all cases, the cadavers were refrigerated within 5 h of being found and tissue was rapidly frozen to 2708C within 30 min of autopsy and stored until required. The pH of the brain tissue was measured as described previously (Kingsbury et al., 1995) (Table 1). 2.3. Diagnostic evaluation An extensive review of the case histories of the subjects with a provisional diagnosis of schizophrenia was carried out by a senior psychologist and psychiatrist using a structured instrument (Hill et al., 1996a,b). This allowed a diagnosis of schizophrenia to be made in accordance with the DSM-IV criteria (American Psychiatric Association, 1994). During the case history review the duration of illness (DOI) for each subject was calculated as the time from ®rst hospital admission to death. The ®nal recorded dose of antipsychotic drug was recorded and then converted to chlorpromazine equivalents (Foster,
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1989) and, wherever available, toxicology reports were obtained. 2.4. Measurement of radioligand binding using autoradiography 2.4.1. Autoradiography The binding of each radioligand, at a single concentration (,3 £ Kd), to sections of striatum was measured. Thus, this study uses single point saturation analysis to gain a good estimate of the density of radioligand binding sites. 5 £ 20 mm frozen tissue sections (2208C) were cut from striatum from each subject for each of the six radioligands and mounted on gelatin coated slides. After exposure to radioligand in the absence (total binding: three sections) or presence [non-speci®c binding (NSB): two sections] of non-radioactive drugs, all sections were washed twice in ice-cold assay buffer, dipped into ice-cold distilled water and then dried thoroughly. Tissue sections along with [ 3H]Micro-scales were then apposed to Hyper®lm- 3H w until an image of appropriate optical density was obtained. The density of binding sites and the speci®c activity of the radioligand utilised dictated exposure time. Resulting images were analysed with reference to the images from the [ 3H]Micro-scales using a MCID image analysis system. Results were obtained as dpm/mg estimated wet weight tissue equivalents (ETE) and converted to fmol/mg ETE. 2.4.2. Measurement of dopaminergic markers The binding of [ 3H]YM-09151-2 (4 nM) to the D2, 3 and 4 receptors was measured as described in earlier studies (Murray et al., 1995). Speci®c binding was taken as the difference in radioligand binding in the absence and presence of (1)-butaclamol (10 25 M) after incubating at room temperature for 60 min in 50 mM Tris buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2 at pH 7.4. Speci®c binding of [ 3H]7-OH-DPAT (2 nM) to the D3 receptor was taken as the difference in radioligand binding in the absence or presence of 10 26 M haloperidol after incubating in 50 mM Hepes (pH 7.5) containing 1 mM EDTA for 60 min at room temperature (Herroelen et al., 1994). For [ 3H]7-OH-DPAT binding, tissue sections were washed twice for 5 min at room temperature in buffer and dried thoroughly
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prior to the measurement of radioligand binding. The speci®c binding of [ 3H]SCH 23390 (3 nM) to the D1-like receptors was taken as the difference in binding of the radioligand in the absence and presence of 10 26 M cis-¯upenthixol after incubating in 50 mM Tris buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2 at pH 7.4 for 60 min at room temperature (Pimoule et al., 1985). Finally, the speci®c binding of [ 3H]mazindol (15 nM) to DAT was taken as the difference in binding of radioligand in the presence of 0.3 mM desmethylimipramine (DMI) minus binding in the presence of DMI and mazindol (10 26 M) after incubating in 50 mM Tris buffer containing 300 mM NaCl, 5 mM KCl for 60 min at 48C (Javitch et al., 1983). 2.4.3. Measurement of GABAergic markers The density of [ 3H]muscimol binding to the GABAA receptor was measured as described previously (Dean et al., 1999). Sections were washed three times in ice-cold assay buffer [50 mM Tris citrate (pH 7.1)] at 48C for 5 min and air-dried using a stream of cool air at room temperature. The speci®c binding of [ 3H]muscimol (90 nM) was measured as the difference in radioligand binding in the absence or presence of 10 26 M SR-95531 after incubating in assay buffer for 60 min at 48C. The density of the NOS was measured as described previously (Doyle and Slater, 1995). Sections were washed at room temperature for 90 min in assay buffer (50 mM Tris±HCl containing 3 mM CaCl2 and 0.025% Triton X-100). The tissue sections were then dried thoroughly and the density of NOS measured as the difference in binding of [ 3H]N Gnitro-L-arginine HCl (20 nM) in the absence or presence of Nv-nitro-L-arginine (10 26 M) after incubating for 120 min at room temperature. 2.4.4. Statistical analysis All statistical analysis was carried out using the Minitab Statistical Software Release II. Initially all data sets were analysed individually and shown to have a binomial distribution. Statistical differences were then identi®ed by comparing age, PMI, DOI and the binding of each radioligand in the schizophrenic subjects to that in the control groups using Student's t test. Possible relationships between age, DOI, PMI, pH and ®nal recorded drug dose
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Fig. 1. The density (mean ^ SEM) of dopamine D1-like (D1), dopamine D2-like (D2), dopamine D3 (D3) and GABAA receptors (GABA) as well as nitric oxide synthase (NOS) and the dopamine transporter (DAT) in striatum from schizophrenic and control subjects.
(dependent variables) with the binding of each radioligand were assessed using Pearson's productmoment correlation coef®cients calculated using an assumed straight line ®t.
(tri¯uoperazine/¯upenthixol n 1, ¯uphenazine/ chlorpromazine n 1, ¯uphenazine/chlorpromazine n 1, ¯upenthixol/chlorpromazine n 1). One subject had received clozapine. Signi®cantly, for eight subjects plasma levels of antipsychotic drugs were available with six subjects having plasma levels below the sensitivity of the assay (detection limit 5 mg/l) and the other two subjects recording plasma levels of thioridazine (0.2 mg/l) and pimozide (0.06 mg/l). In addition, four of the 13 subjects were recorded as receiving 2 mg benztropine/day in their case histories. Brain pH was signi®cantly lower for the schizophrenic subjects (P , 0.001; Table 1), whilst there was no signi®cant difference in the mean age or PMI between the groups. In the control subjects, there were no signi®cant correlations between any
3. Results Initially, the binding of all radioligands to the caudate and putamen was analysed separately. In no case did the binding of any radioligand differ between these regions within tissue from an individual donor. Hence, in this study the binding of each radioligand was taken as an integrated measure across both regions to give a single measure of binding for the striatum. There was a signi®cant decrease in the density of [ 3H]mazindol binding to striatum from schizophrenic subjects (Fig. 1). In the tissue from the schizophrenic subjects there were trends towards increases in the density of [ 3H]muscimol (P 0.09) and [ 3H]N Gnitro-L-arginine HCl (P 0.07) binding, whilst the binding [ 3H]YM-09151-2, [ 3H]7-OH-DPAT or [ 3H]SCH 23390 did not differ signi®cantly. There was a number of confounding factors in this study. All the schizophrenic subjects had been recorded as receiving antipsychotic drugs close to death in their case histories. In the main subjects had received typical antipsychotic drugs either singly (¯uphenazine n 3, tri¯uoperaxine n 2, haloperidol n 2, pimozide n 1) or in combination
Fig. 2. The correlation between the density of dopamine D1-like receptors in the striatum with donor age in schizophrenic and control subjects.
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experimental variables and donor age, tissue PMI and tissue pH. By contrast, in the tissue from the schizophrenic subjects, there was a signi®cant decrease in [ 3H]SCH 23390 binding with age (Fig. 2) and a direct correlation between [ 3H]YM-09152-2 and [ 3H]SCH 23390 binding (schizophrenics r 0.73, P , 0.04; controls r 20.32, P 0.29). No other signi®cant correlations were identi®ed. There was no signi®cant difference in the density of any radioligand binding to striatum from schizophrenic subjects who had or had not received benztropine. 4. Discussion This study has shown a decrease in the density of DAT in the striatum from subjects with schizophrenia. This ®nding is in agreement with another study using [ 3H]mazindol which also reported decreased DAT in the striatum from schizophrenic subjects (Joyce et al., 1988). A study using [ 3H]-CFT also reported a trend to a decrease in DAT (232%) in the caudate putamen from subjects with schizophrenia that did not reach signi®cance (Knable et al., 1994). This was a study using tissue from seven schizophrenic and eight control subjects, and it would therefore seem likely that inclusion of more samples would have made the means in DAT density signi®cantly different. Studies using methodological conditions that would make the radioligand used less selective for DAT {i.e. [ 3H]GBR 12935 (Czudek and Reynolds, 1989; Pearce et al., 1990) and [ 3H]cocaine (Pimoule et al., 1985)} failed to show a decrease in DAT in striatum in schizophrenia. Thus, on balance, current data would suggest that there is a decrease in DAT in the striatum from subjects with schizophrenia. One confounding issue in this study is that all subjects had been treated with antipsychotic drugs prior to death, and therefore the decrease in DAT could be a drug rather than a pathological related phenomenon. Against the argument that the change in DAT is an effect of antipsychotic drugs is the observation that treating animals with antipsychotic drugs does not alter the density of DAT (Rivest et al., 1995; Scheffel et al., 1996; Ase et al., 1999). Thus, the decrease in DAT in the striatum from subjects with schizophrenia does not appear to be due to antipsychotic treatment, and therefore could be involved in
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the pathology of the illness. However, it is still not clear whether the decrease in DAT is due to a loss of this molecule per se or is related to a decrease in innervating dopaminergic neurons. Techniques able to quantify the density of DAT on individual neurons will need to be used to address this important question. This study has shown trends towards increases in the density of GABAA receptors and NOS in striatum from subjects with schizophrenia. Power analyses suggest that the measurement of GABAA receptors in cohorts of 50 individuals and NOS in cohorts of 60 individuals could result in these differences becoming signi®cant. Therefore, this study cannot completely exclude that there are small differences in GABAA receptors or NOS in striatum from subjects with schizophrenia. An increase in GABAA receptors in the striatum has been reported in schizophrenia (Hanada et al., 1986). However, that study used Triton solubilised membranes, a process that was shown to increase [ 3H]muscimol binding above that seen in the non-solubilised membrane. Our study used tissue sections containing non-solubilised membranes and therefore the different outcomes in the two studies could be due to differences in methodology. We are not aware of any other studies comparing levels of NOS in striatum from schizophrenic and control subjects. NOS has been reported as not changed in the frontal cortex (Dean et al., 1999) and unchanged (Doyle and Slater, 1995) or increased (Karson et al., 1996) in the cerebellum. In addition, the total number of NOS containing paraventricular neurons was reported as decreased in schizophrenia (Bernstein et al., 1998). Thus, current data would suggest that there might be regionally speci®c changes in NOS in schizophrenia that do not occur in the striatum. This, and most other studies (Pimoule et al., 1985; Seeman et al., 1987; Reynolds and Czudek, 1988; Knable et al., 1994), have reported no change in the density of the DA D1-like receptors in striatum from subjects with schizophrenia. One study has reported a decrease in these receptors in the striatum from schizophrenic subjects (Hess et al., 1987). Thus most evidence would suggest that DA D1-like receptors are not altered in the striatum from subjects with schizophrenia. This study did not show a signi®cant difference in
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[ H]YM-09152-2 binding to tissue from subjects with schizophrenia. Under the conditions used in this study, [ 3H]YM-09152-2 could bind to both DA D2like receptors (Seeman et al., 1993) and s receptors (Helmeste et al., 1996). To determine the relative contribution of DA D2-like and s receptors to [ 3H]YM-09152-2 binding in our study, we measured the ability of the s receptor antagonist SKF 10047 (Rogers et al., 1989) to displace [ 3H]YM-09152-2 from human striatum from three subjects with no psychiatric history. These experiments showed that SKF 10047 displaced approximately 10% of [ 3H]YM-09152-2 binding and (1)-butaclamol did not increase the displacement of [ 3H]YM-09152-2. Together these data suggest that 90% of [ 3H]YM09152-2 binding we measured would be DA D2-like receptors and that the density of these receptors is not altered in schizophrenia. The demonstration that DA D2 receptor binding is not altered in the striatum from subjects with schizophrenia differs from the ®nding in other studies which, in the main, report the density of these receptors to be increased (for review, see Kahn and Davidson, 1997). However, our ®nding is in line with another study using post-mortem tissue (Reynolds et al., 1981) and several studies using positron emission tomography (Farde et al., 1990; Martinot et al., 1991), which have reported DA D2 receptors are not changed in schizophrenia. It is possible to hypothesise that the absence of an increase in the density of DA D2 receptors in this study is due to the presence of residual antipsychotic drugs from subjects with schizophrenia, which leads to a falsely low estimation of the receptor density. However, it has been suggested previously that the increase in DA D2 receptors in post-mortem tissue was due to treatment with antipsychotic drugs prior to death (Mackay et al., 1980) and has been measured in tissue from subjects treated up to death with antipsychotic drugs (Kahn and Davidson, 1997). Notably, in this study, 75% of individuals who had toxicology reports had no detectable levels of antipsychotic drugs in a blood sample taken at autopsy, indicating that a signi®cant number of individuals in this cohort of schizophrenic subjects may not have taken the drugs as prescribed in their history. Hence, this study could be interpreted as a study of tissue from subjects who had become `drug-free' some time before death. If
that is the case, antipsychotic drug treatment prior to death may no longer be a confounding factor, and hence no increase in DA D2 receptors was detected. The demonstration of an absence of residual antipsychotic drugs in the tissue of subjects with schizophrenia that do not have elevated DA D2 receptors, data that is not available on the cohort in this study, would be necessary to con®rm the latter hypothesis. The observation that DA D2 receptors were not altered in the striatum from the schizophrenic subjects is supported in part by our observation that there was no change in the binding of [ 3H]7OH-DPAT to the DA D3 receptor in schizophrenia. Our ®ndings on the DA D3 receptor agree with a previous report which showed this receptor was not altered in striatum from subjects with schizophrenia (Gurevich et al., 1997). This study has shown a signi®cant decrease in the density of DA D1 receptors with age in schizophrenia but not in the control subjects, a ®nding that has been reported previously (Hess et al., 1987). It has been suggested that there is a decrease in DA D1 receptor density early in life, that ends in the fourth decade (Cortes et al., 1989). Our data would support the hypotheses that subjects with schizophrenia continue to lose DA D1 receptors beyond the fourth decade of life, due to an ongoing degenerative process that may be involved in the pathology of schizophrenia. One of the objectives of this study was to determine if there are inter-related changes in dopaminergic and GABAergic neurons in the striatum from subjects with schizophrenia. There was a positive correlation between the density of DA D2 and DA D1 receptors in subjects with schizophrenia. In other studies examining DA D1 and DA D2-like receptors in striatum from schizophrenic and control subjects, no comment was made on the relationship between the density of these two families of receptors (Seeman et al., 1987; Joyce et al., 1988; Knable et al., 1994). It is therefore not possible to place our ®nding on the relationship between dopamine receptors in schizophrenia in relation to previous studies. By contrast, this study has failed to show inter-related changes in neurochemical markers on dopaminergic and GABAergic neurons in the striatum from subjects with schizophrenia. Thus, the study does not add weight to the hypothesis that concomitant changes in these neurotransmitter systems are involved in the pathology of schizophrenia (Carlsson et al., 1997), but may indicate the
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interaction between the two families of dopamine receptors is altered. In summary, this study adds to others (Joyce et al., 1988; Knable et al., 1994) which suggest there is a decrease in the density of DAT in the striatum from subjects with schizophrenia. Functional neuroimaging studies have shown that subjects with schizophrenia release increased concentrations of dopamine in response to a standardised amphetamine challenge (Laruelle et al., 1996; Breier et al., 1997). Together, these data from neuroimaging and post-mortem studies could indicate that the pathology of schizophrenia involves abnormalities in the uptake and release of dopamine. The consequences of altered dopaminergic function in the striatum, as suggested by a change in DAT, that appears to be present in both the caudate and putamen, is not yet clear. However, from current knowledge of neuroanatomy it could be predicted that changes in dopaminergic function in the putamen could primarily be associated with disturbances of motor function, whereas changes in the caudate would more likely have an effect on cortical functions that include eye movements, memory and the ability to change behavioural set (CoÏte and Crutcher, 1991). This raises the possibility that changes in the putamen could be related to extrapyramidal side effects resulting from treatment with antipsychotic drugs (Casey, 1996). By contrast, changes in the caudate could be involved with functional abnormalities of cortical±striatal function which have been proposed to be involved in the pathology of schizophrenia (Pantelis et al., 1997). Acknowledgements Brian Dean is the NARSAD Fellow at the Mental Health Research Institute of Victoria. Tabasum Hussain was the recipient of a National Health and Medical Research Dora Lush Scholarship (957473). This work has been supported in part by grants-in-aid from the State Government of Victoria, the National Alliance for Research on Schizophrenia and Depression, Rebecca L. Cooper Medical Research Foundation and The Woods Family Trust. The authors would like to thank Mr. Geoffrey Pavey for his excellent technical assistance and Ms. Christine Hill and Professor Nicholas Keks for their assistance with post-mortem diagnosis.
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J.F., Syrota, A., 1991. The estimated density of D2 striatal receptors in schizophrenia. A study with positron emission tomography and 76Br-bromolisuride. Br. J. Psychiatry 158, 346±350. Meltzer, H.Y., 1987. Biological studies in schizophrenia. Schizophr. Bull. 13, 77±107. Murray, A.M., Hyde, T.M., Knable, M.B., Herman, M.M., Bigelow, L.B., Carter, J.M., Weinberger, D.R., Kleinman, J.E., 1995. Distribution of putative D4 dopamine receptors in post-mortem striatum from patients with schizophrenia. J. Neurosci. 15, 2186±2191. Pantelis, C., Barnes, T.R.E., Nelson, H.E., Tanner, S., Weatherley, L., Owen, A.M., Robbins, T.W., 1997. Frontal±striatal cognitive de®cits in patients with chronic schizophrenia. Brain 120, 1823±1843. Pearce, R.K.B., Seeman, P., Jellinger, K., Tourtellotte, W.W., 1990. Dopamine uptake sites and dopamine receptors in Parkinson's disease and schizophrenia. Eur. Neurol. 30 (Suppl. 1), 9±14. Pimoule, C., Schoemaker, H., Reynolds, G.P., Langer, S.Z., 1985. [ 3H]SCH 23390 labeled D1 dopamine receptors are unchanged in schizophrenia and Parkinson's disease. Eur. J. Pharmacol. 114, 235±237. Reynolds, G.P., Czudek, C., 1988. Status of the dopaminergic system in post-mortem brain in schizophrenia. Psychopharmacol. Bull. 3, 345±347. Reynolds, G.P., Riederer, P., Jellinger, K., Gabriel, E., 1981. Dopamine receptors and schizophrenia: the neuroleptic drug problem. Neuropharmacology 20, 1319±1320. Rivest, R., Falardeau, P., Di Paolo, T., 1995. Brain dopamine transporter: gender differences and effect of chronic haloperidol. Brain Res. 692, 269±272. Rogers, C., Cecyre, D., Lemaire, S., 1989. Presence of sigma and phencyclidine (PCP)-like receptors in membrane preparations of bovine adrenal medulla. Biochem. Pharmacol. 38, 2467± 2472. Scheffel, U., Steinert, C., Kim, S.E., Ehlers, M.D., Boja, J.W., Kuhar, M.J., 1996. Effect of dopaminergic drugs on the in vivo binding of [ 3H]WIN 35,428 to central dopamine transporters. Synapse 23, 61±69. Seeman, P., Bzowej, N.H., Guan, H.-C., Bergeron, C., Reynolds, G.P., Bird, E.D., Riederer, P., Jellinger, K., Toutellotte, W.W., 1987. Human brain D1 and D2 dopamine receptors in schizophrenia, Alzheimer's, Parkinson's and Huntington's diseases. Neuropsychopharmacology 1, 5±15. Seeman, P., Guan, H.-C., Van Tol, H.H.M., 1993. Dopamine D4 receptors elevated in schizophrenia. Nature 365, 441±445. Vonsattel, J.P., Aizawa, H., Ge, P., DiFiglia, M., McKee, A.C., MacDonald, M., Gusella, J.F., Landwehrmeyer, G.B., Bird, E.D., Richardson Jr., E.P., Whyte, E.T.H., 1995. An improved approach to prepare human brains for research. J. Neuropathol. Exp. Neurol. 54, 42±56.
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Studies on dopaminergic and GABAergic markers in striatum reveals a decrease in the dopamine transporter in schizophrenia Brian Dean a,b,c,*, Tabasum Hussain a,c a
The Rebecca Cooper Research Laboratories, The Division of Molecular Schizophrenia, The Mental Health Research Institute, Parkville, Victoria, 3052, Australia b The University of Melbourne Department of Psychiatry, Parkville, 3052, Victoria, Australia c Department of Psychological Medicine, Monash University, Clayton, 3006, Victoria, Australia Received 20 February 2000; accepted 2 June 2000
Abstract Changes in the interaction between dopaminergic and GABAergic systems in the striatum have been suggested to be important in the pathology of schizophrenia. If that hypothesis is correct, these changes could produce inter-related changes in the dopaminergic and GABAergic systems in the striatum from schizophrenic subjects. To test this proposition we measured important markers on dopaminergic and GABAergic neurons in striatum obtained post-mortem from schizophrenic and nonschizophrenic subjects. There was a signi®cant decrease in the density of the dopamine transporter (mean ^ SEM: 230 ^ 31 vs. 334 ^ 22 fmol/mg ETE; P 0.01), but not nitric oxide synthase, dopamine D2-like, D1-like, D3 or GABAA receptors in subjects with schizophrenia. There were no inter-related changes in the dopaminergic or GABAergic markers. In the schizophrenic subjects, the density of dopamine D1-like receptors decreased with age and was positively correlated with the density of dopamine D2-like receptors. This study does not readily add weight to the hypothesis that changes in the interaction between dopamine and GABA in the striatum are important in the pathology of schizophrenia. However, our ®ndings could indicate that changes in the dopamine transporter within the striatum, either because of decreased transporter numbers per se or as a result of innervating neuronal loss, may be involved in the pathology of the illness. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Dopamine receptors; Dopamine transporter; GABAA receptor; Nitric oxide synthase; Schizophrenia; Striatum
1. Introduction The dopamine hypothesis of schizophrenia proposes that overactivity of dopaminergic neurons in the central nervous system (CNS) causes the psychoses associated with the illness (Meltzer, 1987). A lack of direct evidence supporting the dopamine (DA) hypothesis has led to the proposal that changes in the interactions * Corresponding author. Tel.: 1613-9388-1633; fax: 1613-93875061. E-mail address: [email protected] (B. Dean).
between the dopaminergic and the g-aminobutyric acid (GABA) systems of the brain may be a cause of some of the symptoms of the illness (Carlsson et al., 1997). This hypothesis is based on a body of experimental observations suggesting that the interplay between dopaminergic and GABAergic neurons is vital to maintaining functional homeostasis, particularly in the striatum (Carlsson et al., 1997). If the dopamine±GABA hypothesis of schizophrenia is correct, it could be that there are inter-related changes in important dopaminergic and GABAergic markers in the striatum from subjects with schizophrenia.
0920-9964/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0920- 996 4( 00) 00096- 7
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A number of studies have examined either dopaminergic or GABAergic markers in striatum obtained post-mortem from subjects with schizophrenia. Such studies have repeatedly shown an increase in the density of DA D2-like receptors in the striatum from subjects with schizophrenia (for review, see Kahn and Davidson, 1997). Increased GABAA receptors have also been reported in the striatum from schizophrenic subjects (Hanada et al., 1986). These changes could have resulted from inter-related changes in the dopaminergic and GABAergic systems in the striatum from subjects with schizophrenia, as would be predicted by the dopamine±GABA hypothesis. However, because the markers were not measured in tissue from the same individuals, the interactive nature of the changes cannot be assessed. To that end, we have measured important dopaminergic (DA D2-like receptors, DA D1-like receptors, D3 receptor and dopamine transporter [DAT]) and GABAergic (GABAA receptors and nitric oxide synthase [NOS]) markers in striatum so that we can determine if there are inter-related changes in these markers in schizophrenia. 2. Materials and methods 2.1. Materials [ 3H]7-OH-DPAT, [ 3H]SCH 23390, [ 3H]N G-nitroL-arginine HCl, [ 3H]Micro-scales and Hyper®lm- 3H w were obtained from Amersham Australia Pty Ltd., Sydney, Australia. [ 3H]mazindol, [ 3H]YM-09151-2 and [ 3H]muscimol were obtained from New England Nuclear via AMRAD Pharmacia Biotech, Melbourne, Australia. All other chemicals were obtained from Sigma Aldrich Pty Ltd., Castle Hill, New South Wales, Australia. 2.2. Tissue collection Permission to carry out this study was obtained from the Western Region Psychiatric Human Ethics Committee. Striatum was collected at autopsy from the left brain hemisphere of 13 subjects with a provisional diagnosis of schizophrenia and from the same hemisphere of 13 subjects with no history of psychiatric or neurological illness (controls). These subjects were matched for sex and were of a similar age to the
Table 1 Summary (mean ^ SEM) of demographic, pharmacological and tissue related data for schizophrenic and control subjects. PMI post-mortem intervals, DOI duration of illness, FRAD ®nal recorded antipsychotic drug dose
Sex (M/F) Age (yr) PMI (h) pH DOI (yr) FRAD (mg chlorpromazine equiv./day)
Schizophrenic
Control
10/3 47 ^ 5.2 45 ^ 5.1 6.05 ^ 0.07 45 ^ 5.1 436 ^ 120
10/3 47 ^ 4.9 47 ^ 3.4 6.38 ^ 0.04
schizophrenic subjects. The striatum was excised from frozen slice 5 of the left hemisphere, which had been sliced using a standardised protocol (Vonsattel et al., 1995). Post-mortem interval (PMI), in cases where death was witnessed, was the time between death and autopsy. When death was not witnessed, tissue was only taken from individuals that had been seen alive within 5 h of being found dead. In those cases PMI was taken as the interval between the donor being found dead plus half the interval between the subject last being seen alive and being found dead. In all cases, the cadavers were refrigerated within 5 h of being found and tissue was rapidly frozen to 2708C within 30 min of autopsy and stored until required. The pH of the brain tissue was measured as described previously (Kingsbury et al., 1995) (Table 1). 2.3. Diagnostic evaluation An extensive review of the case histories of the subjects with a provisional diagnosis of schizophrenia was carried out by a senior psychologist and psychiatrist using a structured instrument (Hill et al., 1996a,b). This allowed a diagnosis of schizophrenia to be made in accordance with the DSM-IV criteria (American Psychiatric Association, 1994). During the case history review the duration of illness (DOI) for each subject was calculated as the time from ®rst hospital admission to death. The ®nal recorded dose of antipsychotic drug was recorded and then converted to chlorpromazine equivalents (Foster,
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1989) and, wherever available, toxicology reports were obtained. 2.4. Measurement of radioligand binding using autoradiography 2.4.1. Autoradiography The binding of each radioligand, at a single concentration (,3 £ Kd), to sections of striatum was measured. Thus, this study uses single point saturation analysis to gain a good estimate of the density of radioligand binding sites. 5 £ 20 mm frozen tissue sections (2208C) were cut from striatum from each subject for each of the six radioligands and mounted on gelatin coated slides. After exposure to radioligand in the absence (total binding: three sections) or presence [non-speci®c binding (NSB): two sections] of non-radioactive drugs, all sections were washed twice in ice-cold assay buffer, dipped into ice-cold distilled water and then dried thoroughly. Tissue sections along with [ 3H]Micro-scales were then apposed to Hyper®lm- 3H w until an image of appropriate optical density was obtained. The density of binding sites and the speci®c activity of the radioligand utilised dictated exposure time. Resulting images were analysed with reference to the images from the [ 3H]Micro-scales using a MCID image analysis system. Results were obtained as dpm/mg estimated wet weight tissue equivalents (ETE) and converted to fmol/mg ETE. 2.4.2. Measurement of dopaminergic markers The binding of [ 3H]YM-09151-2 (4 nM) to the D2, 3 and 4 receptors was measured as described in earlier studies (Murray et al., 1995). Speci®c binding was taken as the difference in radioligand binding in the absence and presence of (1)-butaclamol (10 25 M) after incubating at room temperature for 60 min in 50 mM Tris buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2 at pH 7.4. Speci®c binding of [ 3H]7-OH-DPAT (2 nM) to the D3 receptor was taken as the difference in radioligand binding in the absence or presence of 10 26 M haloperidol after incubating in 50 mM Hepes (pH 7.5) containing 1 mM EDTA for 60 min at room temperature (Herroelen et al., 1994). For [ 3H]7-OH-DPAT binding, tissue sections were washed twice for 5 min at room temperature in buffer and dried thoroughly
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prior to the measurement of radioligand binding. The speci®c binding of [ 3H]SCH 23390 (3 nM) to the D1-like receptors was taken as the difference in binding of the radioligand in the absence and presence of 10 26 M cis-¯upenthixol after incubating in 50 mM Tris buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2 at pH 7.4 for 60 min at room temperature (Pimoule et al., 1985). Finally, the speci®c binding of [ 3H]mazindol (15 nM) to DAT was taken as the difference in binding of radioligand in the presence of 0.3 mM desmethylimipramine (DMI) minus binding in the presence of DMI and mazindol (10 26 M) after incubating in 50 mM Tris buffer containing 300 mM NaCl, 5 mM KCl for 60 min at 48C (Javitch et al., 1983). 2.4.3. Measurement of GABAergic markers The density of [ 3H]muscimol binding to the GABAA receptor was measured as described previously (Dean et al., 1999). Sections were washed three times in ice-cold assay buffer [50 mM Tris citrate (pH 7.1)] at 48C for 5 min and air-dried using a stream of cool air at room temperature. The speci®c binding of [ 3H]muscimol (90 nM) was measured as the difference in radioligand binding in the absence or presence of 10 26 M SR-95531 after incubating in assay buffer for 60 min at 48C. The density of the NOS was measured as described previously (Doyle and Slater, 1995). Sections were washed at room temperature for 90 min in assay buffer (50 mM Tris±HCl containing 3 mM CaCl2 and 0.025% Triton X-100). The tissue sections were then dried thoroughly and the density of NOS measured as the difference in binding of [ 3H]N Gnitro-L-arginine HCl (20 nM) in the absence or presence of Nv-nitro-L-arginine (10 26 M) after incubating for 120 min at room temperature. 2.4.4. Statistical analysis All statistical analysis was carried out using the Minitab Statistical Software Release II. Initially all data sets were analysed individually and shown to have a binomial distribution. Statistical differences were then identi®ed by comparing age, PMI, DOI and the binding of each radioligand in the schizophrenic subjects to that in the control groups using Student's t test. Possible relationships between age, DOI, PMI, pH and ®nal recorded drug dose
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Fig. 1. The density (mean ^ SEM) of dopamine D1-like (D1), dopamine D2-like (D2), dopamine D3 (D3) and GABAA receptors (GABA) as well as nitric oxide synthase (NOS) and the dopamine transporter (DAT) in striatum from schizophrenic and control subjects.
(dependent variables) with the binding of each radioligand were assessed using Pearson's productmoment correlation coef®cients calculated using an assumed straight line ®t.
(tri¯uoperazine/¯upenthixol n 1, ¯uphenazine/ chlorpromazine n 1, ¯uphenazine/chlorpromazine n 1, ¯upenthixol/chlorpromazine n 1). One subject had received clozapine. Signi®cantly, for eight subjects plasma levels of antipsychotic drugs were available with six subjects having plasma levels below the sensitivity of the assay (detection limit 5 mg/l) and the other two subjects recording plasma levels of thioridazine (0.2 mg/l) and pimozide (0.06 mg/l). In addition, four of the 13 subjects were recorded as receiving 2 mg benztropine/day in their case histories. Brain pH was signi®cantly lower for the schizophrenic subjects (P , 0.001; Table 1), whilst there was no signi®cant difference in the mean age or PMI between the groups. In the control subjects, there were no signi®cant correlations between any
3. Results Initially, the binding of all radioligands to the caudate and putamen was analysed separately. In no case did the binding of any radioligand differ between these regions within tissue from an individual donor. Hence, in this study the binding of each radioligand was taken as an integrated measure across both regions to give a single measure of binding for the striatum. There was a signi®cant decrease in the density of [ 3H]mazindol binding to striatum from schizophrenic subjects (Fig. 1). In the tissue from the schizophrenic subjects there were trends towards increases in the density of [ 3H]muscimol (P 0.09) and [ 3H]N Gnitro-L-arginine HCl (P 0.07) binding, whilst the binding [ 3H]YM-09151-2, [ 3H]7-OH-DPAT or [ 3H]SCH 23390 did not differ signi®cantly. There was a number of confounding factors in this study. All the schizophrenic subjects had been recorded as receiving antipsychotic drugs close to death in their case histories. In the main subjects had received typical antipsychotic drugs either singly (¯uphenazine n 3, tri¯uoperaxine n 2, haloperidol n 2, pimozide n 1) or in combination
Fig. 2. The correlation between the density of dopamine D1-like receptors in the striatum with donor age in schizophrenic and control subjects.
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experimental variables and donor age, tissue PMI and tissue pH. By contrast, in the tissue from the schizophrenic subjects, there was a signi®cant decrease in [ 3H]SCH 23390 binding with age (Fig. 2) and a direct correlation between [ 3H]YM-09152-2 and [ 3H]SCH 23390 binding (schizophrenics r 0.73, P , 0.04; controls r 20.32, P 0.29). No other signi®cant correlations were identi®ed. There was no signi®cant difference in the density of any radioligand binding to striatum from schizophrenic subjects who had or had not received benztropine. 4. Discussion This study has shown a decrease in the density of DAT in the striatum from subjects with schizophrenia. This ®nding is in agreement with another study using [ 3H]mazindol which also reported decreased DAT in the striatum from schizophrenic subjects (Joyce et al., 1988). A study using [ 3H]-CFT also reported a trend to a decrease in DAT (232%) in the caudate putamen from subjects with schizophrenia that did not reach signi®cance (Knable et al., 1994). This was a study using tissue from seven schizophrenic and eight control subjects, and it would therefore seem likely that inclusion of more samples would have made the means in DAT density signi®cantly different. Studies using methodological conditions that would make the radioligand used less selective for DAT {i.e. [ 3H]GBR 12935 (Czudek and Reynolds, 1989; Pearce et al., 1990) and [ 3H]cocaine (Pimoule et al., 1985)} failed to show a decrease in DAT in striatum in schizophrenia. Thus, on balance, current data would suggest that there is a decrease in DAT in the striatum from subjects with schizophrenia. One confounding issue in this study is that all subjects had been treated with antipsychotic drugs prior to death, and therefore the decrease in DAT could be a drug rather than a pathological related phenomenon. Against the argument that the change in DAT is an effect of antipsychotic drugs is the observation that treating animals with antipsychotic drugs does not alter the density of DAT (Rivest et al., 1995; Scheffel et al., 1996; Ase et al., 1999). Thus, the decrease in DAT in the striatum from subjects with schizophrenia does not appear to be due to antipsychotic treatment, and therefore could be involved in
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the pathology of the illness. However, it is still not clear whether the decrease in DAT is due to a loss of this molecule per se or is related to a decrease in innervating dopaminergic neurons. Techniques able to quantify the density of DAT on individual neurons will need to be used to address this important question. This study has shown trends towards increases in the density of GABAA receptors and NOS in striatum from subjects with schizophrenia. Power analyses suggest that the measurement of GABAA receptors in cohorts of 50 individuals and NOS in cohorts of 60 individuals could result in these differences becoming signi®cant. Therefore, this study cannot completely exclude that there are small differences in GABAA receptors or NOS in striatum from subjects with schizophrenia. An increase in GABAA receptors in the striatum has been reported in schizophrenia (Hanada et al., 1986). However, that study used Triton solubilised membranes, a process that was shown to increase [ 3H]muscimol binding above that seen in the non-solubilised membrane. Our study used tissue sections containing non-solubilised membranes and therefore the different outcomes in the two studies could be due to differences in methodology. We are not aware of any other studies comparing levels of NOS in striatum from schizophrenic and control subjects. NOS has been reported as not changed in the frontal cortex (Dean et al., 1999) and unchanged (Doyle and Slater, 1995) or increased (Karson et al., 1996) in the cerebellum. In addition, the total number of NOS containing paraventricular neurons was reported as decreased in schizophrenia (Bernstein et al., 1998). Thus, current data would suggest that there might be regionally speci®c changes in NOS in schizophrenia that do not occur in the striatum. This, and most other studies (Pimoule et al., 1985; Seeman et al., 1987; Reynolds and Czudek, 1988; Knable et al., 1994), have reported no change in the density of the DA D1-like receptors in striatum from subjects with schizophrenia. One study has reported a decrease in these receptors in the striatum from schizophrenic subjects (Hess et al., 1987). Thus most evidence would suggest that DA D1-like receptors are not altered in the striatum from subjects with schizophrenia. This study did not show a signi®cant difference in
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[ H]YM-09152-2 binding to tissue from subjects with schizophrenia. Under the conditions used in this study, [ 3H]YM-09152-2 could bind to both DA D2like receptors (Seeman et al., 1993) and s receptors (Helmeste et al., 1996). To determine the relative contribution of DA D2-like and s receptors to [ 3H]YM-09152-2 binding in our study, we measured the ability of the s receptor antagonist SKF 10047 (Rogers et al., 1989) to displace [ 3H]YM-09152-2 from human striatum from three subjects with no psychiatric history. These experiments showed that SKF 10047 displaced approximately 10% of [ 3H]YM-09152-2 binding and (1)-butaclamol did not increase the displacement of [ 3H]YM-09152-2. Together these data suggest that 90% of [ 3H]YM09152-2 binding we measured would be DA D2-like receptors and that the density of these receptors is not altered in schizophrenia. The demonstration that DA D2 receptor binding is not altered in the striatum from subjects with schizophrenia differs from the ®nding in other studies which, in the main, report the density of these receptors to be increased (for review, see Kahn and Davidson, 1997). However, our ®nding is in line with another study using post-mortem tissue (Reynolds et al., 1981) and several studies using positron emission tomography (Farde et al., 1990; Martinot et al., 1991), which have reported DA D2 receptors are not changed in schizophrenia. It is possible to hypothesise that the absence of an increase in the density of DA D2 receptors in this study is due to the presence of residual antipsychotic drugs from subjects with schizophrenia, which leads to a falsely low estimation of the receptor density. However, it has been suggested previously that the increase in DA D2 receptors in post-mortem tissue was due to treatment with antipsychotic drugs prior to death (Mackay et al., 1980) and has been measured in tissue from subjects treated up to death with antipsychotic drugs (Kahn and Davidson, 1997). Notably, in this study, 75% of individuals who had toxicology reports had no detectable levels of antipsychotic drugs in a blood sample taken at autopsy, indicating that a signi®cant number of individuals in this cohort of schizophrenic subjects may not have taken the drugs as prescribed in their history. Hence, this study could be interpreted as a study of tissue from subjects who had become `drug-free' some time before death. If
that is the case, antipsychotic drug treatment prior to death may no longer be a confounding factor, and hence no increase in DA D2 receptors was detected. The demonstration of an absence of residual antipsychotic drugs in the tissue of subjects with schizophrenia that do not have elevated DA D2 receptors, data that is not available on the cohort in this study, would be necessary to con®rm the latter hypothesis. The observation that DA D2 receptors were not altered in the striatum from the schizophrenic subjects is supported in part by our observation that there was no change in the binding of [ 3H]7OH-DPAT to the DA D3 receptor in schizophrenia. Our ®ndings on the DA D3 receptor agree with a previous report which showed this receptor was not altered in striatum from subjects with schizophrenia (Gurevich et al., 1997). This study has shown a signi®cant decrease in the density of DA D1 receptors with age in schizophrenia but not in the control subjects, a ®nding that has been reported previously (Hess et al., 1987). It has been suggested that there is a decrease in DA D1 receptor density early in life, that ends in the fourth decade (Cortes et al., 1989). Our data would support the hypotheses that subjects with schizophrenia continue to lose DA D1 receptors beyond the fourth decade of life, due to an ongoing degenerative process that may be involved in the pathology of schizophrenia. One of the objectives of this study was to determine if there are inter-related changes in dopaminergic and GABAergic neurons in the striatum from subjects with schizophrenia. There was a positive correlation between the density of DA D2 and DA D1 receptors in subjects with schizophrenia. In other studies examining DA D1 and DA D2-like receptors in striatum from schizophrenic and control subjects, no comment was made on the relationship between the density of these two families of receptors (Seeman et al., 1987; Joyce et al., 1988; Knable et al., 1994). It is therefore not possible to place our ®nding on the relationship between dopamine receptors in schizophrenia in relation to previous studies. By contrast, this study has failed to show inter-related changes in neurochemical markers on dopaminergic and GABAergic neurons in the striatum from subjects with schizophrenia. Thus, the study does not add weight to the hypothesis that concomitant changes in these neurotransmitter systems are involved in the pathology of schizophrenia (Carlsson et al., 1997), but may indicate the
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interaction between the two families of dopamine receptors is altered. In summary, this study adds to others (Joyce et al., 1988; Knable et al., 1994) which suggest there is a decrease in the density of DAT in the striatum from subjects with schizophrenia. Functional neuroimaging studies have shown that subjects with schizophrenia release increased concentrations of dopamine in response to a standardised amphetamine challenge (Laruelle et al., 1996; Breier et al., 1997). Together, these data from neuroimaging and post-mortem studies could indicate that the pathology of schizophrenia involves abnormalities in the uptake and release of dopamine. The consequences of altered dopaminergic function in the striatum, as suggested by a change in DAT, that appears to be present in both the caudate and putamen, is not yet clear. However, from current knowledge of neuroanatomy it could be predicted that changes in dopaminergic function in the putamen could primarily be associated with disturbances of motor function, whereas changes in the caudate would more likely have an effect on cortical functions that include eye movements, memory and the ability to change behavioural set (CoÏte and Crutcher, 1991). This raises the possibility that changes in the putamen could be related to extrapyramidal side effects resulting from treatment with antipsychotic drugs (Casey, 1996). By contrast, changes in the caudate could be involved with functional abnormalities of cortical±striatal function which have been proposed to be involved in the pathology of schizophrenia (Pantelis et al., 1997). Acknowledgements Brian Dean is the NARSAD Fellow at the Mental Health Research Institute of Victoria. Tabasum Hussain was the recipient of a National Health and Medical Research Dora Lush Scholarship (957473). This work has been supported in part by grants-in-aid from the State Government of Victoria, the National Alliance for Research on Schizophrenia and Depression, Rebecca L. Cooper Medical Research Foundation and The Woods Family Trust. The authors would like to thank Mr. Geoffrey Pavey for his excellent technical assistance and Ms. Christine Hill and Professor Nicholas Keks for their assistance with post-mortem diagnosis.
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