Cannabis-sensitive dopaminergic markers in postmortem central nervous system: changes in schizophrenia

Cannabis-Sensitive Dopaminergic Markers in Postmortem Central Nervous System: Changes in Schizophrenia Brian Dean, Robyn Bradbury, and David Leon Copolov Background: This study investigated if changes in presynaptic markers on dopaminergic neurons (dopamine transporter [DAT], tyrosine hydroxylase [TH]) were present in the caudate from subjects with schizophrenia who had ⌬9(⫺)tetrahydrocannabinol (THC) in their blood at autopsy. These changes were posited because animal studies show that treatment with THC decreases dopamine uptake and TH in the striatum. Methods: Studies utilized caudate, obtained postmortem, from 14 schizophrenic and 14 control subjects. [3H]mazindol binding to caudate, measured using autoradiography, was taken as a measure of DAT; TH levels were estimated using an antihuman TH antibody and Western blotting. Results: There was decreased [3H]mazindol binding to DAT in the caudate from the schizophrenic subjects with no detectable blood THC levels (THC(⫺)) compared with THC(⫺) control subjects (mean ⫾ SEM: 240 ⫾ 19 vs. 296 ⫾ 14 fmol/mg estimated tissue equivalents, p ⫽ .01). There were no significant differences between levels of DAT in the caudate from schizophrenic and control subjects that had THC in their blood. Tyrosine hyroxylase was not different in any diagnostic cohort. Conclusions: Our data suggests that DAT is decreased in the caudate from THC(⫺) subjects with schizophrenia, a change that may be reversed by ingesting THC from cannabis. Biol Psychiatry 2003;53:585–592 © 2003 Society of Biological Psychiatry Key Words: Dopamine transporter, tyrosine hydroxylase, caudate-putamen, substantia nigra, schizophrenia, cannabis

From The Rebecca L. Cooper Research Laboratories, The Mental Health Research Institute of Victoria, Parkville, Victoria, Australia. Address reprint requests to Brian Dean, The Rebecca L. Cooper Research Laboratories, Mental Health Research Institute of Victoria, Locked Bag 11, Parkville, Victoria 3052, Australia. Received March 20, 2002; revised May 31, 2002; accepted July 14, 2002.

© 2003 Society of Biological Psychiatry

Introduction

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e have recently reported that the cannabinoid1 (CB1) receptor is increased in the frontal cortex of subjects with schizophrenia and in the caudate-putamen (CP) of subjects who had ⌬9(⫺)tetrahydrocannabinol (THC) detected in their blood at autopsy, whether or not they had schizophrenia (Dean et al 2001). The CB1 receptor is the site in the brain where both endogenous cannabinoids and the active component of cannabis, THC (Ameri 1999), are proposed to exert their effects. Thus our data, plus the finding that there are elevated levels of endogenous cannabinoids in the cerebral spinal fluids from subjects with schizophrenia (Leweke et al 1999), suggest a role for the cannabinoid system in the pathology of schizophrenia. Moreover, clinical data suggesting that cannabis use exacerbates existing psychoses (Hollister 1998) raises the possibility that inhalation of THC may modulate pathways in the brain that are central to psychoses. It is still not clear which systems in the brain become dysfunctional to induce the onset of psychoses; however, studies showing that dopamine receptor agonists cause or worsen psychoses, whereas dopamine2 receptor antagonists act as antipsychotic agents, have underpinned the long-standing dopamine hypothesis of schizophrenia (Meltzer and Stahl 1976). This hypothesis suggests that overactive dopaminergic systems in the central nervous system (CNS) are central to the genesis of psychoses. Based on the dopamine hypothesis of schizophrenia, it can be postulated that any effects of THC inhalation that increase the activity of the dopaminergic system in the CNS could cause or exacerbate psychoses. The difficulties in studying the effect of the inhalation of cannabis in human CNS have resulted in studies on the effects of THC in animals. One such study showed that THC both inhibits dopamine uptake and facilitates its release by rat striatal synaptosomes (Sakurai-Yamashita et al 1989). From these data, it was concluded that THC acts to stimulate nigrostriatal dopaminergic pathways in the rat. A more recent study has shown that anandamide, the endogenous ligand for cannabinoids receptors (Ameri 0006-3223/03/$30.00 doi:10.1016/S0006-3223(03)01545-7

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1999), increases the release of dopamine in the striatum (Cadogan et al 1997) and mesolimbic system (Gessa et al 1998) of the CNS by activating the CB1 receptor. These data suggest that the CB1 receptor may have a physiologic role in regulating dopaminergic activity. Further evidence to support the role of THC as a generalized activator of the dopaminergic systems of the CNS has come from the study of tyrosine hydroxylase (TH), a rate-limiting enzyme in the synthesis of dopamine (Mallet 1996). A number of studies in rats have shown that exposure to THC increases both the activity and expression of TH (Bonnin et al 1996; Hernandez et al 1997). Both these findings suggest that THC would cause an increase in the synthesis of catecholamines, including dopamine, a hypothesis supported by the demonstration that THC increases the rate of conversion of radioactive tyrosine to radioactive dopamine (Rodriguez et al 1990). Therefore, if animal studies are reflective of the action of THC in human CNS, it would be predicted that inhalation of cannabis would act to increase dopamine synthesis and release while inhibiting the clearance of the neurotransmitter via the dopamine transporter (DAT). Such a series of outcomes would most likely increase levels of extra-neuronal dopamine, increasing the activity of dopaminergic pathways in the brain and hence, according to the dopamine hypothesis of schizophrenia, would either cause or exacerbate psychoses. To further our studies on the possible role of the cannabinoid system and/or its pharmacologic manipulation in psychoses, we have now examined the status of critical cannabis-sensitive dopaminergic markers in brains from subjects with schizophrenia and subjects who had inhaled cannabis near time of death. To parallel studies in animals, it would have been necessary to measure the activity of the dopamine uptake system and TH in human brain, but such measurements are affected by minor variations in tissue collection and processing (Yoshimoto et al 1993). Thus, we elected to use in situ radioligand binding to measure the density of DAT in the striatum and Western blots to measure the levels of TH in the caudate and substantia nigra (SN); both of these proteins have been shown to be stable postmortem (Dean and Hussain 2001; Torack and Morris 1992). Our studies have initially focused on the nigrostriatal areas because studies suggest that the dopaminergic pathways between these regions are affected by both THC (SakuraiYamashita et al 1989) and the pathology of schizophrenia (Risch 1996).

Methods and Materials Materials [3H]mazindol was obtained from NEN Life Sciences Products (Boston, MA). [3H]microscales and Hyperfilm ECL (enhanced

chemiluminescence) were obtained from Amersham Australia (Sydney, Australia). BAS-TR plates were obtained from Fuji Photo Film (Tokyo, Japan). Mouse monoclonal antihuman TH was obtained from Chemicon Australia (Boronia, Australia). Mazindol was a gift from Sandoz (Sydney, Australia), and desmethylimipramine (DMI) was from Research Biochemicals and obtained, like all other chemicals and the rabbit antimouse immunoglobulin G (IgG) antibody couple to horseradish peroxidase, from Sigma Aldrich (Castle Hill, New South Wales, Australia). Pierce Supersignal chemiluminescent kit was obtained from Pierce Biotechnology (Rockford, IL). All materials used in Western blots were obtained from Bio-Rad Laboratories (Regents Park, Australia).

Tissue Collection Following ethical approval from the North-Western Health Care Human Research and Ethics Committees, CP and SN were collected at autopsy from the left brain hemisphere of 14 subjects with a provisional diagnosis of schizophrenia suggested in a police report of death to the coroner and from 14 subjects with no known history of psychiatric illness (control subjects) (Table 1). All tissue was collected at the Victorian Institute of Forensic Medicine, following approved procedures and after consultation with the next of kin, where a forensic neuropathologist carried out a neuropathological examination. The control subjects were matched for gender and were of a similar age to the schizophrenic subjects. To minimize variation in experimental parameters, all tissue blocks were prepared by standardized procedures. Thus, blocks of CP were taken from a rostral region so as to include the nucleus accumbens and exclude the globus pallidus (pars lateralis). Substantia nigra tissue was from the “rostral” component of the structure at the level of the subthalamic nucleus in a coronal section. We did not delineate between the pars compacta (ventral) and the pars reticulata (dorsal) components of the nucleus; hence both cell types were within SN homogenates. The blocked tissue was rapidly frozen to ⫺70°C and stored at this temperature until required. In cases where death did not involve suicide, tissue was collected from subjects whose death was witnessed, and the postmortem interval (PMI) was the time from death to autopsy. In cases of suicide, tissue was only taken from individuals seen alive within 5 hours of being found dead. In such cases, the PMI was the interval between the donor being found dead and autopsy plus half the time between when they were last seen alive and found dead. In all cases, the cadavers were refrigerated within 5 hours of being found. The provisional diagnosis of schizophrenia was confirmed by a senior psychologist and senior psychiatrist after an extensive case history review using the Diagnostic Instrument for Brain Studies (Hill et al 1996), a structured instrument for the collection of clinical, pharmacologic, and other relevant information from case histories. In this study, the diagnosis of schizophrenia was made according to DSM-IV criteria (American Psychiatric Association 1994). In addition to the DSM-IV diagnosis of schizophrenia, there were two schizophrenic subjects who would fulfill the DSM-IV criteria for cannabis abuse at death (subjects 4 and 5) and two subjects who would have fulfilled those criteria during their lifetime (2 and 3) but not at death. Two of the

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Table 1. Demographic Data from, and Radioligand Data for, Schizophrenic and Control Subjects from Whom Brain Tissue Was Obtained Postmortem for the Study of CB1 Receptors

ID Schizophrenic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Analyses THC(⫹) THC(⫺) Control 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Analyses THC(⫹) THC(⫺)

[3H]mazindol binding (mol/ mg ETE)

Sex

Age (y)

PMI (hr)

pH

DOI (y)

FRD

THC

Cause of death

M M F M M M M M F M M M M F

25 22 35 41 45 38 36 42 27 44 48 55 23 36

49 37 15 31 68 40 38 47 41 32 30 25 43 45

6.38 6.07 6.26 6.20 6.48 5.52 6.04 6.26 5.85 6.28 6.62 6.10 6.40 6.28

2 3 7 11 12 15 12 22 10 23 24 33 6 4

200 450 300 500 300 160 200 1000 750 600 1250 400 1750 160

DET DET 75 10 15

Suicide: overdose of mixed drugs Pericarditis Coronary arterial thrombosis Suicide: combined drug toxicity Suicide: hanging Mediastinitis Suicide: overdose amitryptiline Coronary arterial atheroma Suicide: asphyxia Ischemic heart disease Bronchopneumonia Coronary arterial atheroma Suicide: hanging Suicide: CO poisoning

Mean SEM Mean SEM

34 4.4 39 3.3

40 8.9 38 2.5

6.27 .07 6.15 .10

7.0 2.0 16.6 3.2

M M M M F F M M M M M M M M

30 26 25 29 21 32 25 42 30 38 50 23 43 21

27 24 50 15 58 56 36 26 24 46 69 36 45 51

5.86 6.42 6.48 6.46 6.03 6.16 6.15 6.32 6.46 6.42 6.43 6.13 6.25 6.58

Mean SEM Mean SEM

38 1.2 33 3.3

29 7.5 45 4.6

6.31 .14 6.29 .06

350 55 697 184 23 30 85 14

Coronary arterial atheroma Electrocution Exsanguination Congestive cardiac failure Myocarditis Coronary arterial atheroma Right ventricular hypertrophy Coronary arterial atheroma Electrocution Trauma and asphyxia Ischemic heart disease Asthma Drowning Exsanguination

Caudate

Tyrosine hydroxylase (optical density) Caudate

SN

248 280 273 278 304 222 273 306 191 184 341 146 194 254

.12 .14 .08 .08 .13 .13 .10 .11 .03 .03 .14 .09 .36 .13

.12 .05 .10 .11 .16 .11 .12 .13 .13 .10 .10 .13 .02 .03

277 8.9 240 19

.11 .01 .12 .02

.11 .02 .10 .01

326 306 303 280 214 240 361 334 288 329 294 321 289 286

.10 .11 .12 .15 .07 .09 .10 .13 .08 .11 .09 .06 .11 .10

.14 .13 .13 .13 .02 .16 .12 .09 .11 .09 .08 .10 .10 .10

304 9.4 296 14

.12 .01 .09 .006

.13 .002 .10 .01

CB1, cannabinoid1; PMI, postmortem interval; DOI, duration of illness; FRD, final recorded antipsychotic drug dose (chlorpromazine equivalents per day); THC, blood ⌬9(⫺) tetrahydrocannabinol levels (ng/mL: DET, detected but below sensitivity of assay); ETE, estimated tissue equivalent; SN, substantia nigra; M, male; F, female.

schizophrenic subjects (6 and 10) would fulfill the DSM-IV criteria for alcohol abuse. There was no evidence to suggest that any of the schizophrenic subjects would fulfill the DSM-IV criteria for the abuse of other substances, and there was no evidence that any of the control subjects would have fulfilled any of the DSM-IV criteria for the abuse of any substance. Duration of illness (DOI) was calculated as the time from first hospital admission to death. In addition, information on the type and amount of antipsychotic drugs prescribed close to death was obtained from the case history, and the final recorded dose (FRD)

of antipsychotic drug was converted to chlorpromazine equivalents (Soulsby 1999). Toxicology reports were examined to identify which subjects had detectable levels of THC in blood taken at autopsy, and case histories were carefully examined for any suggestion of cannabis use. Because of the many confounding variables that influence the levels of THC and its metabolites in blood, the presence of these compounds in blood of subjects from whom CNS was collected can only be interpreted as indicating that cannabis had been inhaled, most likely within 2 weeks before autopsy (Solowij 1998).

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water, and then thoroughly dried. The washed sections were then fixed overnight in paraformaldehyde fumes in a desiccator. The fixed sections and a set of [3H]microscales were then apposed against a BAS-TR2025 imaging plate for 7 days before the plate was scanned in the BAS 5000 phosphoimager. The density of the resulting phosphoimages was then compared with the density of the [3H]microscales using Analytical Imaging Station (AIS) image analysis software (Imaging Research, St. Catharines, Canada) to obtain results as dpm/mg estimated tissue equivalents (ETE). These results were then transformed to fmol radioligand bound/mg ETE (fmol/mg ETE).

Measurement of Tyrosine Hydroxylase

Figure 1. (A) Typical high-resolution phosphoimages showing [3H]mazindol binding to human caudate-putamen in the absence (total binding) and presence (NSB) of 1 ␮ mazindol. (B) The specific (total binding ⫺ NSB) binding of [3H]mazindol to the caudate from schizophrenic and control subjects who had THC(⫹) or had not THC(⫺) inhaled cannabis close to death. ETE, estimated tissue equivalents; THC, ⌬9(⫺)tetrahydrocannabinol. NSB, non-specific binding.

Measurement of [3H]mazindol Binding For this study, 5 ⫻ 20 ␮m frozen tissue sections (⫺20°C) were cut from the CP from each subject so that a portion of the section was missing an area of tissue that was included in the material excised for a Western blot (Figure 1). The specific binding of [3H]mazindol (15 nmol/L) to DAT was taken as the difference in binding of radioligand in the presence of .3 ␮mol/L DMI minus binding in the presence of DMI and mazindol (1 ␮mol/L) after incubating in 50 mmol/L Tris buffer containing 300 mmol/L NaCl and 5 mmol/L KCl for 60 min at 4°C (Javitch et al 1983). The presence of [3H]mazindol at greater than three times its Kd for binding means this study used single saturation analysis to estimate the density of binding. In such studies, specific binding is a good estimate of the density of radioligand binding sites in the tissue (Rodbard 1981). After incubating with radioligand, all sections were washed twice in ice-cold assay buffer, dipped into ice-cold distilled

Tissue for the analysis of TH was collected from the caudate (see Figure 1) and SN from the subjects used in the study of [3H]mazindol binding. The tissue from both regions was homogenized with five strokes of a Teflon-glass homogenizer into 20 mmol/L Tris-HCl, pH 7.4, containing .2 mmol/L ethylene glycol bis-(2 aminoethyl)-N,N,N⬘,N⬘ tetraacetic acid and .1 mmol/L ethylenediaminetetraacetic acid with the protease inhibitors phenylmethylsulfonylfluoride (1 mmol/L), leupeptin (10 ␮g/mL), benzamidine (1 mmol/L), bacitracin (1 mg/mL), pepstatin (10 ␮g/mL), and chymostatin (10 ␮g/mL) being added on the day of use. The brain homogenates were stored on ice until the concentration of protein in each sample was determined using the Bio-Rad protein assay and then stored at ⫺70°C. On the day of assay, homogenates were thawed and denatured by heating at 95°C for 4 min after being diluted in an equal volume of electrophoresis buffer (.5 mol/L Tris-HCl, pH 6.8, containing 20% glycerol, 10% sodium dodecyl sulfate (SDS), 10% 2-␤-mercaptoethanol, and .05% bromophenol blue). Homogenates (10 ␮g/protein per well) were then loaded onto 4% SDS stacking gels and separated on 10% SDS minigels. The proteins were then transferred from the minigel to nitrocellulose membrane (Bio-Rad: Trans-Blot Transfer Medium) in Towbin transfer buffer (25 mmol/L Tris, pH 8.3, containing 192 mmol/L glycine and 20% methanol) overnight in a Bio-Rad Mini Transblot electrophoretic transfer cell. The proteins on each nitrocellulose membrane were stained with .2% Ponceau S Red in 3% trichloroacetic acid to ensure transfer had occurred. The nitrocellulose membranes containing the separated proteins were placed in Tween Tris-buffered saline (TTBS: 100 mmol/L Tris, pH 7.5, containing .9% sodium chloride, 5% blotting grade nonfat milk, and .1% Tween 20) for 1 hour at room temperature with agitation. The proteins on the nitrocellulose membrane were then exposed to a mouse monoclonal antihuman TH diluted 1:1000 in blocking buffer for 1 hour at room temperature with agitation. The solution containing the antihuman TH was then removed, and the nitrocellulose membrane washed 6⫻ for 5 min with TTBS for 30 min. The nitrocellulose membrane was then exposed to a rabbit antimouse IgG antibody couple to horseradish peroxidase diluted 1:2000 in blocking buffer for 1 hour at room temperature with agitation. The nitrocellulose membranes were then washed as occurred after the incubation with the antihuman TH antibody. The washed nitrocellulose membranes were transferred to a clean tray and exposed to the substrate solution from the Pierce Super-

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signal chemiluminescent kit for 5 min at room temperature with agitation. Following the removal of excess substrate solution, the nitrocellulose film was placed between two pieces of transparent film. In a darkroom, a piece of enhanced chemiluminescence (ECL) film was opposed to the nitrocellulose membrane in the pieces of transparent film for between 15 and 45 sec, depending on the strength of the chemiluminescent signal. The ECL film was then developed in Kodak D-19 x-ray developer and fixed with x-ray fixer. Once the ECL film was dry, it was imaged using an analog camera and the optical density (OD) of the image measured using a Microcomputer Imaging Device (MCID) image analysis system (Imaging Research, St. Catharines, Canada). To control for blot-toblot variation, the ECL film was exposed so that the OD of a standard membrane preparation run on every gel was effectively constant (inter-assay variation ⬍ 5%).

Statistical Analysis All statistical analyses were carried out using GraphPad Prism. Each data set was analyzed using the Kolmogorov-Smirnov test to determine if the distribution of the data was parametric. Data on [3H]mazindol binding and TH within region were compared using a two-way analysis of variance (ANOVA) with a post hoc Bonferonni correction, using diagnosis and drug status as variables. Student’s t tests were used to determine if there were any significant differences in the FRD or DOI between the THC(⫹) and THC(⫺) subjects with schizophrenia. Correlations between radioligand binding and TH levels with demographic data and confounding factors were expressed as a Pearson Product– Moment assuming a straight-line best fit.

Results Because of the collection procedure described, there were no significant differences in the age, PMI, or brain pH between the four cohorts of schizophrenic or control subjects, whether they did (THC(⫹)) or did not (THC(⫺)) have THC in their blood at autopsy (Table 1). All data sets were normally distributed, allowing parametric analysis. No significant difference in the density of [3H]mazindol binding could be detected across the CP within donor. Thus, for consistency with the data from Western blotting, we report only data from the caudate. Two-way ANOVA revealed that there was significant variation in the density of [3H]mazindol binding to the caudate with diagnosis [F(1,25) ⫽ 5.0, p ⬍ .05; Table 1] but not with drug status (THC(⫹) vs. THC(⫺)) [F(1,25) ⫽ 1.5, p ⫽ .23]. There was also no significant interaction between the two variables [F(1,25) ⫽ .6, p ⫽ .44]. Post hoc Bonferonni analysis revealed that the source of significant variation in [3H]mazindol binding with diagnosis was because of a decrease in [3H]mazindol binding to caudate from the THC(⫺) schizophrenic subjects when compared with the THC(⫺) control subjects (Table 1; Figure 1).

Figure 2. Typical Western blot showing tyrosine hydroxylase in homogenate from the caudate of a schizophrenic (A) and control (B) subject as well as the standard membrane preparation (C) run on every gel to accommodate gel-to-gel variation. MW, molecular weight.

There was no significant variance in TH levels with diagnosis [Caudate: F(1,25) ⫽ .25, p ⫽ .62; SN: F(1,25) ⫽ .42, p ⫽ .52; Table 1] or drug status [Caudate; F(1,25) ⫽ .05, p ⫽ .83; SN: F(1,25) ⫽ 2.3, p ⫽ .14), and there was no interaction between the two variables (Caudate: F(1,25) ⫽ .60, p ⫽ .45; SN: F(1,25) ⫽ .59, p ⫽ .45) (Table 1, Figure 2). There were no significant differences in the mean FRD (All: p ⫽ .46; SN: p ⫽ .47) or DOI (All: p ⫽ .11; SN: p ⫽ .12) between the THC(⫹) and THC(⫺) schizophrenic subjects (Table 1). There were no significant correlations between [3H]mazindol binding or TH in either brain region with age, PMI, brain pH, DOI, FRD, or blood drug levels at death.

Discussion This study has shown that there is a decrease in [3H]mazindol binding in the caudate from THC(⫺) schizophrenic subjects compared with that in the THC(⫺) control subjects, which was not apparent between the same diagnostic cohorts who had inhaled cannabis close to death. Under the conditions used in this study, [3H]mazindol would predominantly bind to the DAT (Javitch et al 1983). Thus these data support earlier findings that there is a decrease in DAT in the CP from subjects with schizophrenia (Dean and Hussain 2001; Joyce et al 1988). In the

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same cohorts of subjects, we showed that levels of TH were not different in either the caudate or SN from schizophrenic and control subjects whether or not they had THC in their blood at autopsy. In the case of both DAT and TH it is important to note that the design of this study means it is not controlled or randomized, and this limits the overall interpretation of these data; however, the study is relevant as it is the first to attempt to obtain data on the effect of cannabis ingestion on what are thought to be cannabis-sensitive markers on dopaminergic neurons in postmortem human brain from subjects with schizophrenia and how such changes may be related to psychotic illness. The hypothesis tested in this study was that cannabis inhalation would effect critical dopaminergic markers in the brain, leading to overactive dopaminergic pathways and hence causing or worsening psychoses. In the rat, one response to THC was a decrease in dopamine uptake by striatal synaptosomes (Sakurai-Yamashita et al 1989). The DAT is now recognized as the most critical component of the dopamine uptake mechanism of neurons (Amara 1993), and the level of dopamine uptake is related to the level of the DAT (Schoemaker et al 1985). If these data were applicable in humans, it would be predicted that levels of DAT would be decreased in subjects who had recently inhaled cannabis. Our data showing that neither THC(⫹) schizophrenic nor THC(⫹) control subjects had decreased levels of DAT in the caudate, which does not support the hypothesis that cannabis inhalation causes changes in DAT; however, we cannot exclude the possibility that changes in the activity of DAT can occur without changes in the levels of DAT protein. In addition, the intake of THC in humans is by inhalation, whereas studies in animals either apply THC directly to isolated brain preparations or are given via injection. Therefore, it remains possible that differences in administration routes, or in the actions of pure THC versus THC inhaled from cannabis, could account for the apparent differing effects of THC on DAT in animal and human CNS. The recent availability of suitable ligands to measure DAT using positron emission tomography and single photon emission computed tomography have resulted in a number of studies in schizophrenia. Two of these studies have reported no differences in levels of DAT in the striatum of subjects with schizophrenia (Laruelle et al 2000; Lavalaye et al 2001). By contrast, one study has reported an absence of a right–left asymmetry in DAT in subjects with schizophrenia (Laakso et al 2000), and another suggests there may be a loss of DAT in the striatum from a subset of subjects with chronic schizophrenia (Laakso et al 2000, 2001). Our study only examined DAT levels in one hemisphere, and therefore we cannot comment on asymmetry; however, we did not observe any relationship between levels of DAT and DOI,

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and therefore our data would not support the hypothesis that decreases in DAT are related to the chronic form of schizophrenia. Overall, neuroimaging data does not suggest that there is a decrease in DAT in schizophrenia; however, it is important to note that our data suggest that changes in DAT may not be apparent in subjects who have ingested THC close to death or, in the case of neuroimaging, close to testing. It is not clear whether the existing neuroimaging studies have taken cannabis use into account; however, given the relatively high incidence of cannabis use in schizophrenia (Johns 2001), controlling for this confounding factor could be critical in identifying the changes in DAT we report in this study. There are a number of studies that show that THC increases the activity, levels, and expression of TH in the rat brain (Bonnin et al 1996; Hernandez et al 1997; Rodriguez et al 1990). By contrast, we have found that TH is not increased in either the caudate or SN from THC(⫹) subjects. This does not suggest that TH changes acutely after the inhalation of cannabis in humans. Again, differences in results because of drug purity and/or routes of administration cannot be excluded as an explanation of results in animals and humans; however, our current data would not support the hypothesis that the inhalation of THC from cannabis is associated with short-term changes in TH that would be consistent with an activation of the nigrostriatal dopaminergic pathways and subsequent onset or worsening of psychoses. One confounding factor in this study is that tissue was obtained from subjects that have received antipsychotic drugs during their lifetime. This raises the possibility that the changes we have observed in DAT are due to antipsychotic drug effects and not associated with the pathology of the schizophrenia. Against this argument is the repeated finding that treating rats with antipsychotic drugs does not affect the levels of DAT (Allard et al 1990; Ase et al 1999; Reader et al 1998; Tarazi et al 2000). In addition, in this study both THC(⫹) and THC(⫺) subjects with schizophrenia had received antipsychotic drugs, and therefore the specificity of the change in DAT to the THC(⫺) schizophrenic subjects argues against this being simply an antipsychotic drug effect. In conclusion, this study suggests that there is a decrease in DAT in the caudate from THC(⫺) subjects with schizophrenia. From current knowledge of DAT it would be expected that the density of DAT would, at times, prove rate limiting to neuronal dopamine uptake (Horn 1990) resulting in an increase in extracellular dopamine. An increase in extracellular dopamine should cause an overactivation of the dopaminergic system in the brain. Thus, the decrease in DAT we have measured in caudate from subjects with schizophrenia could be providing direct

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support to the dopamine hypothesis of schizophrenia (Meltzer and Stahl 1976), which proposes that overactive dopaminergic systems are central to the pathology of the illness. Our data would also support the argument that absence of change in DAT in the THC(⫹) subjects with schizophrenia means that the ingestion of THC from cannabis may act to reverse changes in DAT associated with the pathology of the disorder. Brian Dean was a National Alliance for Research on Schizophrenia and Depression Young Investigator. This work was supported in part by grants-in-aid from the State Government of Victoria “Turning the Tide” Initiative and the National Health and Medical Research Council (Project Grant: 114253). We thank Mr. Geoffrey Pavey for his assistance in preparing the tissue for this study and Ms. Christine Hill and Professor Nicholas Keks for the assistance with postmortem diagnosis.

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