A change in the density of [3H]flumazenil, but not [3H]muscimol binding, in Brodmann’s Area 9 from subjects with bipolar disorder

Journal of Affective Disorders 66 (2001) 147–158www.elsevier.com/ locate / jad

Research report3 3A change in the density of [ H]flumazenil, but not [ H]muscimol

binding, in Brodmann’s Area 9 from subjects with bipolar disorder

a,b,c,d , a a,b a*Brian Dean , Geoffrey Pavey , Mark McLeod , Kenneth Opeskin ,a,d a,c,dNicholas Keks , David Copolov

aThe Rebecca L. Cooper Research Laboratories, The Mental Health Research Institute of Victoria, The University of Melbourne,Parkville, Victoria, Australia

bDepartment of Pathology, The Mental Health Research Institute of Victoria, Parkville, Victoria, AustraliacDepartment of Psychiatry, The Mental Health Research Institute of Victoria, Parkville, Victoria, Australia

dMonash Department of Psychological Medicine, The Alfred, Prahran, Victoria, Australia

Received 1 April 2000; received in revised form 24 July 2000; accepted 4 August 2000

Abstract

Background: This study examines the hypothesis that there are changes in cortical serotonergic, GABAergic andglutamatergic systems in bipolar disorder and schizophrenia. Methods: In situ radioligand binding and autoradiography wereused to measure neurochemical markers in Brodmann’s Area (BA) 9 from control subjects and subjects with bipolar disorderor schizophrenia (n 5 8 per group). Results: Compared to tissue from schizophrenic (mean6S.E.M, 385644 fmol /mg ETE)

3and control (383644 fmol /mg ETE) subjects, there was an increase in the density of [ H]flumazenil binding to thebenzodiazepine binding site on the GABA receptor in subjects with bipolar disorder (451617 fmol /mg ETE; P , 0.05).A

3There was no difference in the density of [ H]muscimol binding to the GABA receptor or in the density of the serotoninA 1A

receptor, serotonin receptor, ionotropic glutamate receptors or the serotonin transporter between the three cohorts. There2A

was an age-related decrease in NMDA receptor density in control subjects that was absent in schizophrenia and bipolar3disorder. An age-related increase in [ H]flumazenil binding in schizophrenia was absent in control and bipolar disorder

subjects. Limitations: This study involved a relatively small number of individuals. Conclusions: An increase in the3

g2-receptor sub-unit in the GABA receptor has been shown to increase benzodiazepine but not [ H]muscimol binding, thisA

is the mismatch in binding we have shown in BA 9 from subjects with bipolar disorder. Thus, a change in the assembly ofreceptor subunits into GABA receptors may be involved in the neuropathology of bipolar disorder. There may also beA

differences in age-related changes in cortical receptor density between bipolar disorder and schizophrenia. 2001 ElsevierScience B.V. All rights reserved.

Keywords: Schizophrenia; Bipolar disorder; GABA; GABA receptor; Glutamate; SerotoninA

*Corresponding author. Tel.: 1 61-3-9388-1633; fax: 1 61-3-9387-5061.E-mail address: [email protected] (B. Dean).

0165-0327/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0165-0327( 00 )00294-9

148 B. Dean et al. / Journal of Affective Disorders 66 (2001) 147 –158

1. Background serotonergic and GABAergic function had affectedmarkers on glutamatergic cells, we also measured

The neuropathological basis of bipolar disorder levels of ionotropic glutamate receptors in frontalremains to be elucidated. There is a growing body of cortex from the same subjects.evidence to suggest that some of the symptoms ofbipolar disorder result from abnormalities in thefunctioning of the frontal lobe (Joseph, 1999). How- 2. Methodsever, such data have yet to be supported by macro-scopic or microscopic evidence of frontal lobe 2.1. Materialspathology. This has led to the hypothesis that the

3 3 3symptoms of bipolar disorder may result from [ H]8-OH-DPAT, [ H]flumazenil, [ H]Micro-3 changes in the molecular cytoarchitecture of the scales and Hyperfilm- H were obtained from

brain (Kishimoto et al., 1998). Amersham Australia, Sydney, Australia.3 3 3Early studies directed toward identifying molecu- [ H]Ketanserin, [ H]citalopram, [ H]AMPA,3 3 3lar abnormalities in bipolar disorder reported that [ H]kainate, [ H]muscimol and [ H]MK 801 were

plasma g-aminobutyric acid (GABA) was decreased obtained from New England Nuclear via AMRAD(Berrettini et al., 1983), unchanged (Gerner et al., Pharmacia Biotech, Melbourne, Australia. All other1984) or increased (Petty and Schlesser, 1981) in chemicals were obtained from Sigma Aldrich, Castlesubjects with bipolar disorder. Notably, one of these Hill, New South Wales, Australia.studies showed that both plasma and CSF GABAwere only increased in subjects with bipolar disorder

2.2. Tissue collectionbeing treated with lithium (Berrettini et al., 1983)which raised the question as to whether the increased

Brodmann’s Area 9 was collected at autopsy fromplasma GABA levels were drug-induced. However,the left brain hemisphere of subjects who were listedfurther evidence to support a role for GABA abnor-as having bipolar disorder or schizophrenia in reportsmalities in bipolar disorder came from the demon-of death (Table 1). Tissue was also collected fromstration that clonazepam, which binds to the GABAAthe same brain regions from subjects with no historyreceptor, was useful in the treatment of mania (Postof psychiatric illness (controls). In cases where deathet al., 1998). Thus, it is still hypothesised thatwas witnessed, the time between death and autopsyabnormalities in the GABAergic system has a role inwas taken as the postmortem interval (PMI, Tablethe pathology of bipolar disorder (Petty, 1995).1). Where death was not witnessed, tissue was onlyOne study has reported a decrease in 5-hydroxy-taken from individuals who had been seen alive up toindolacetic acid, a metabolite of serotonin (5HT), in5 h before being found dead, in these cases PMI wasthe frontal and other cortical regions obtained post-taken as the interval half way between the donormortem from subjects with bipolar disorder (Youngbeing found dead and last being seen alive. In allet al., 1994). This finding, as is the case withcases, cadavers were refrigerated within 5 h of beingschizophrenia (Dean et al., 1999a), suggests therefound and tissue was rapidly frozen to 2 708Cmay be changes in the serotonergic and GABAergicwithin 30 min of autopsy. The pH of the brain tissuesystems in bipolar disorder. To determine if there arewas measured as described previously (Kingsbury etsimilar changes in serotonergic and GABAergical., 1995).markers in the two illnesses, we have measured

important markers of these systems in BA 9 fromsubjects with bipolar disorder, schizophrenia and 2.3. Diagnostic evaluationsubjects with no history of psychiatric disorders.

Serotonin (Arvanov and Wang, 1998) and GABA Sufficient information needed to be available from(Herrero et al., 1999) have also been shown to clinical case records to enable a psychologist andpotently modulate glutamate function in the frontal psychiatrist to reach a diagnostic consensus using thecortex. Thus, in an attempt to determine if changes in Diagnostic Instrument for Brain Studies, a semi-

B. Dean et al. / Journal of Affective Disorders 66 (2001) 147 –158 149

Table 1Demographic data on subjects with schizophrenia, bipolar disorder and no history of mental illness from which frontal cortex was obtainedfor the study of neurotransmitter receptors

Subject DSM-IV Age Sex DOI PMI pH Cause of death Manner of deathdiagnosis (year) (year) (h)

1 Bipolar 74 F 35 45 6.26 Drug toxicity and ischemic heart disease Uncertain2 58 F 10 41 5.68 Ischemic heart disease Natural3 59 M 24 34 6.46 Rupture aorta Natural4 38 M 3 24 6.42 Carbon monoxide poisoning Suicide5 66 M 2 17 6.41 Aspiration of food Accident6 55 F 14 52 6.46 Unascertained Natural7 60 F 23 50 6.08 Cardiomegaly Natural8 61 M 40 58 6.44 Ischemic heart disease Natural

Mean 59 18.9 40 6.28S.E.M. 3.6 5.0 5.0 0.1

9 Schizophrenic 65 F 18 50 6.35 Ruptured abdominal aneurysm Natural10 69 M 6 48 6.44 Carbon monoxide poisoning Suicide11 57 M 28 24 6.06 Ischemic Heart disease Natural12 48 F 22 53 6.21 Pulmonary thromboembolism Natural13 65 M 35 41 6.57 Ischemic heart disease Natural14 38 M 15 40 5.52 Mediastinitis Natural15 47 F 20 50 6.31 Pneumonia Natural16 79 F 45 26 6.27 Complications of fractured neck of femur Accident

Mean 59 23.6 42 6.22S.E.M 4.8 4.3 3.9 0.11

17 Control 56 F 24 5.88 Coronary artery atheroma Natural18 57 M 27 6.43 Coronary artery atheroma Natural19 68 M 41 6.06 Complications of heart surgery Accident20 47 F 24 5.89 Post-operative pulmonary thromboembolism Accident21 38 M 26 6.42 Traumatic asphyxia Accident22 67 M 32 6.14 Myocardial infarction Natural23 39 F 65 6.38 Mitral valve prolapse Natural24 77 F 64 6.32 Hypertensive heart disease Natural

Mean 56 38 6.19S.E.M. 5.0 6.1 0.08

structured protocol for postmortem assessment (Keks 2.4. Measurement of radioactive drug binding toet al., 1999) allowing the diagnosis of bipolar mood tissue sections by in situ radioligand binding anddisorder or schizophrenia according to DSM-IV autoradiographycriteria to be established (American PsychiatricAssociation, 1994). Subjects with schizoaffective 2.4.1. Preparation of tissuedisorder were excluded. When available, postmortem For this study the binding of the radioligand totoxicology was reviewed to exclude recent substance tissue sections was measured at a single concen-misuse. Duration of illness (DOI) was calculated as tration at approximately three times the binding Kd

the time from first admission to death and a com- for each radioligand. Thus, this represents a singleprehensive medication history was obtained for each point saturation analysis study. In such studies thesubject. Most recently prescribed psychotropic drug specific binding is calculated as radioligand bindingdoses were recorded and then converted to stan- (total binding) minus the binding of that radioliganddardised drug doses (Soulsby, 1999) (Table 2). in the presence of a non-radioactive drug active at


Table 2Pharmacological history of subjects with schizophrenia and bipolar disorder from which frontal cortex was obtained for the study ofneurotransmitter receptors

Subject Drugs recorded as the last dose prescribed close to death Postmortem blood report

Antipsychotic Chlor. eq. Antidep. Amityp. eq. Benzos Diazepam Other

drugs mg/day drugs mg/day eq.

mg/day

Bipolar disorder

1 Fluphenazine 250 Dothiepin 150 Oxazepam 5 Amitriptyline 4.9 mg/ l

Nortriptyline 3.1 mg/ l

Dothiepin 4.0 mg/ l

2 Moclobemide 8 mg/ l

Salicylate 20 mg/ l

Paracetamol 6 mg/ l

3 Lithium 1 g/day Not available

4 Chlorpromazine 300 Paroxetine 100 Lithium 1.5 g/day Ethanol 0.12 g/ l

Codeine 0.2 mg/ l

Paracetamol 46 mg/ l

Doxylamine 0.2 mg/ l

5 Fluphenazine 250 Temazepam 5 Nothing detected

6 Chlorpromazine 300 Temazepam 10 Carbamazepine 200 mg/day Carbamazepine 4 mg/ l

Lithium 1.5 g/day Chlorpromazine 0.3 mg/ l

Lithium 0.8 mg/ l

7 None for 2 months Nitrazepam 10 Lithium 1 g/day Gliclazide | 2 mg/ l

Lithium 1.2 mmol / l

7-Aminoclonazepam 0.05 mg/ l

Thioridazine | 0.3 mg/ l

8 Flupenthixol 2000 Temazepam 160 Benztropine 2 mg/day Frusemide | 0.2 mg/ l

Midazolam Sodium valproate 2.5 mg/day Lignocaine | 0.6 mg/ l

Valproate 44 mg/ l

Diazepam , 0.1 mg/ l

Nordiazepam , 0.1 mg/ l

Schizophrenic

9 Fluphenazine 550 Temazepam 10 Benztropine 2 mg/day Not available

Haloperidol

10 Haloperidol 650 Ethanol 1.4 g / l

Oxazepam 0.1 mg/ l

7-aminonitrazepam 0.3 mg/ l

11 Fluphenazine 150 Not available

12 Fluphenazine 700 Lithium 0.75 g/day Chlorpromazine 0.75 mg/ l

Chlorpromazine Lithium 1.9 mmol / l

Paracetamol 5.0 mg/ l

13 Fluphenazine 150 Nothing detected

14 Haloperidol 160 Procyclidine 15 mg/day Morphine 0.2 mg/ l


Nordiazepam , 0.1 mg/ l

Paracetamol 3 mg/ l

Frusemide 5 mg/ l

15 Flupenthixol 600 Clonazepam 20 Thioridazine 2.8 mg/ l

Chlorpromazine Mesoridazine 0.8 mg/ l

Sulforidazine 0.5 mg/ l


16 Fluphenazine 208 Artane 2 mg/day Nothing detected


the same binding site (non-specific binding). Im- the absence or presence of spiperone (10 mM) afterportantly, because these measures would be obtained incubating for 30 min at room temperature in 170under conditions where available, binding sites are mM Tris–HCl (pH 7.7) (Dean and Hayes, 1996).

3 3saturated by radioligand, specific binding gives a The density of [ H]8-OH-DPAT and [ H]citalopramgood estimate of the density of binding sites (Rod- binding was measured as described previously (Dean

3bard, 1981). et al., 1999b). Hence, prior to measuring [ H]8-OH-For this study 5 3 20 mm frozen tissue sections DPAT binding, all sections were washed in assay

(2208C) were cut from BA 9 from each subject for buffer for 30 min at room temperature. Specific3each of the eight radioligand binding assays and binding of [ H]8-OH-DPAT (1 nM) was then taken

mounted on gelatin-coated slides. For each of the as the difference in the binding in the absence andradioligand binding assays, the sections from all the presence of 5HT (1 mM) after incubating for 60subjects were processed in one batch with all sam- min at room temperature in 170 mM Tris–HCl (pH

3ples being coded so the assayist was blind to donor 7.7) containing 4 mM CaCl . For [ H]citalopram,2

classification. When layering of radioligand binding the tissue sections were incubated for 15 min at roomto BA 9 was clearly apparent, autoradiographs and temperature in assay buffer. The difference between

3Nissl-stained sections were compared to determine the binding of [ H]citalopram (4 nM) alone and inwhich cortical laminae were contained within a layer the presence of 10 mM paroxetine after incubating inof radioligand binding. Layers of radioligand binding 50 mM Tris–HCl (pH 7.4) containing 120 mM NaClwere denoted alphabetically, commencing at the and 5 mM KCl for 60 min at room temperature wasouter layer and moving systematically through the taken as the measure of specific binding.deeper layers.

For all radioligand binding assays the amount of 32.4.4. The measurement of [ H]AMPA,radioactivity in the diluted radioligand was measured 3 3[ H]kainate and [ H]MK 801 bindingto confirm the concentration of radioligand on each 3 3 3[ H]AMPA, [ H]kainate and [ H]MK 801 bindingsection was correct.was measured in tissue sections that had beenincubated for 30 min at 48C in assay buffer. The32.4.2. Measurement of [ H]muscimol and 3specific binding of [ H]AMPA was taken as the3[ H]flumazenil binding 3difference in [ H]AMPA (100 nM) binding to BA 93Prior to the measurement of [ H]muscimol andin the absence and presence of quisqualic acid (1003[ H]flumazenil binding tissue was washed 3 3 5 minmM) after incubating in 50 mM Tris–HCl (pH 7.4)in assay buffer. The specific binding ofcontaining 2.5 mM CaCl and 0.1 M potassium3 2[ H]muscimol (100 nM) was taken as the differencethiocyanate for 45 min at 48C (Jansen et al., 1990).in binding of the radioligand in the absence and 3Specific binding of [ H]kainate to BA 9 was taken aspresence of 10 mM SR-95531 after incubating in 50the difference in the binding of radioligand (40 nM)mM Tris citrate (pH 7.1) for 60 min at 48C (Dean etin the absence or presence of L-glutamic acid HCl (1al., 1999a). Preliminary experiments withmM) in 50 mM Tris–acetate (pH 7.4) for 60 min at3[ H]flumazenil showed it bound to human cortex48C (Greenamyre and Young, 1989). Specific bind-with a K 5 3 nM (data not shown). Therefore in this 3d ing of [ H]MK 801 (20 nM) was taken as the3study, the specific binding of [ H]flumazenil (12difference in radioligand binding in the absence andnM) was taken as the difference in binding in thepresence of MK-801 (100 mM) after incubating inabsence and presence of clonazepam (1 mM) after50 mM Tris–acetate (pH 7.4) containing 100 mMincubation in 170 mM Tris–HCl buffer (pH 7.4) forglutamate, 50 mM glycine and 50 mM spermidine for60 min at 48C (Hand et al., 1997).60 min at room temperature (Johnson et al., 1993).

32.4.3. Measurement of [ H]8-OH-DPAT,3 3[ H]ketanserin and [ H]citalopram binding 2.4.5. Autoradiography

3[ H]ketanserin binding was measured as the dif- In preparation for autoradiography, all sections3ference in the binding of [ H]ketanserin (10 nM) in were washed twice in ice-cold assay buffer, dipped


into ice-cold distilled water and then thoroughly 3. Resultsdried.

3 3For [ H]ketanserin, [ H]8-OH-DPAT, 3.1. Tissue collection3 3 3[ H]citalopram, [ H]muscimol and [ H]flumazenil

binding, the sections were fixed overnight in parafor- There were no significant differences in the meanmaldehyde fumes in a desiccator. The sections and a age (Table 1, df 5 2,21; F 5 0.11; P 5 0.9), DOI

3set of [ H]micro-scales were then apposed against a (P 5 0.48) or sex ratio (4 M/4 F) for the bipolar,BAS-TR2025 imaging plate until an image of appro- schizophrenic or control subjects. In addition, therepriate intensity was obtained for scanning in the BAS were no significant differences in the mean PMI5000 phosphoimager. Exposure time related to both (df 5 2,21; F 5 0.13; P 5 0.9) or pH (df 5 2,21; F 5

the density of binding sites and the specific activity 0.20; P 5 0.82) for the tissue from the three cohortsof the radioligand utilised. The intensity of the of subjects.images was then measured by comparison to the Five subjects with bipolar disorder and eight

3intensity of the [ H]microscales using AIS image subjects with schizophrenia were recorded as beinganalysis software to obtain results as dpm/mg esti- prescribed antipsychotic drugs close to death (Tablemated tissue equivalents (ETE). 2). Two bipolar subjects were recorded as receiving

Due to the other radioligands used in this study antidepressant drugs, five were prescribed benzo-having the propensity to contaminate phosphoimag- diazepines and five were prescribed mood stabilisers.ing plates, tissue sections that had been exposed to Two schizophrenic subjects were recorded as receiv-

3 3 3[ H]AMPA, [ H]kainate or [ H]MK 801 were ap- ing benzodiazepines and one was prescribed a mood3 3posed to Hyperfilm- H , along with [ H]micro- stabiliser. Post-mortem serum drug screens were

scales, until images of appropriate optical density available on seven of eight subjects with bipolarwere obtained. Exposure time related to both the disorder and six of eight schizophrenic subjects.density of binding sites and the specific activity of These drug screens detected antipsychotic drugs inthe radioligand utilised. Resulting images were then serum from two schizophrenic and two bipolaranalysed with an MCID M1 image analysis system disorder subjects. Antidepressant drugs were de-where the optical density of the images was com- tected in serum from a subject with bipolar disorder,

3pared to that of the [ H]micro-scales to obtain results benzodiazepines were detected in three bipolar disor-as dpm/mg estimated tissue equivalents (ETE). der and three schizophrenic subjects. Finally, lithium

After analysing either the phospho- or film image, was detected in serum from three bipolar subjectsthe results obtained as dpm/mg ETE were converted and one schizophrenic subject whilst one bipolarto fmol radioligand bound per mg ETE (fmol /per disorder subject had lithium and carbamazepine inmg ETE). her serum and another had sodium valproate.

2.4.6. Statistical analysis 3.2. Radioligand binding studiesAll statistical analyses were carried out using

GRAPHPAD PRISM which showed that each data set in In comparing variation in binding data from eachthe study was normally distributed. Hence, signifi- radioligand, inter-section variation of total and non-cant differences between cohorts and between corti- specific binding for a single radioligand within tissuecal layering of radioligand binding were identified from a single donor did not vary more than 10%.using an ANOVA followed by a Bonferroni test to

3identify specific significant differences between the 3.3. Measurement of [ H]muscimol and3groups. When comparisons between data from only [ H]flumazenil binding

two cohorts were made, as was the case for DOI, and3 3drug dosages, a student t-test was used to test for In all subjects, [ H]muscimol and [ H]flumazenil

significance. Correlations between radioligand bind- binding was present in all cortical laminae. Markeding, demographic data and confounding factors were differences between binding in cortical laminae wereexpressed as a Pearson product–moment assuming a not detected. There was a significant difference

3straight-line best fit. between the mean density of [ H]flumazenil binding


between the diagnostic groups (Table 3, df 5 2,21; df 5 2,21; F 5 24.8; P , 0.001; schizophrenic df 5

F 5 4.16; P 5 0.03) which was due to a significant 2,21; F 5 226; P , 0.001; controls df 5 2,21; F 5

increase in binding to BA 9 from the bipolar subjects 93; P , 0.001) with Layer A having the highest level(P , 0.05). There were no significant differences in of binding. As described previously (Dean et al.,

3 3[ H]muscimol binding between the diagnostic groups 1999b) Layer A of [ H]8-OH-DPAT binding con-(df 5 2,21; F 5 0.36; P 5 0.7). tained cortical laminae I and II, Layer B cortical

lamina III and Layer C cortical laminae IV to VI.3 33.4. Measurement of the [ H]8-OH-DPAT, The binding of [ H]ketanserin was present in a

3 3[ H]ketanserin and [ H]citalopram binding single layer that included cortical laminae II, III and3IV whereas the binding of [ H]citalopram appeared

3In all subjects, the binding of [ H]8-OH-DPAT as a single layer that was ubiquitous throughout thewas distributed in three layers in BA 9 (Table 3, BP cortical laminae.

Table 3The density of radioligand binding to Brodmann’s Area 9 from subjects with bipolar disorder, schizophrenia and control subjects

3 3 3 3 3 3 3 3Subject [ H]8-OH-DPAT [ H]Ketanserin [ H]Citalopram [ H]AMPA [ H]Kainate [ H]MK801 [ H]Muscimol [ H]Flumazenil

A B C A B C A B C A B

Bipolar disorder

1 26.6 8.0 9.7 14.9 2.4 327 210 219 31 26 41 175 131 477 464

2 25.1 4.8 7.0 40.5 7.4 205 156 185 27 22 38 133 95 501 414

3 52.6 13.2 16.6 66.4 7.3 237 194 197 33 26 48 187 150 522 508

4 16.7 4.9 7.4 35.8 4.3 200 140 151 21 18 36 168 129 453 410

5 48.3 10.6 18.0 47.7 9.3 208 138 150 24 19 33 175 134 475 530

6 46.3 11.3 17.8 36.8 8.2 274 147 164 21 17 31 190 140 359 425

7 41.7 9.7 16.7 51.3 9.4 190 139 162 23 20 40 169 137 451 409

8 48.8 16.4 20.9 52.0 11.4 208 164 178 32 25 47 211 163 468 446

Mean 39.2 9.9 14.3 43.2 7.5 231 161 176 26.5 21.6 39.3 176 134 463 451

S.E.M 4.8 1.4 1.9 5.4 1.0 17 9.6 8.5 1.8 1.3 2.2 7.9 6.9 17 17

Schizophrenia

9 47.1 9.4 17.2 44.3 10.0 245 171 177 28 24 44 126 108 480 413

10 47.7 11.0 14.2 60.9 10.1 301 164 175 34 30 43 191 141 588 428

11 56.4 10.5 15.7 25.6 7.1 328 224 206 38 35 53 206 154 415 375

12 44.6 8.4 14.4 71.4 11.2 278 199 186 26 21 41 178 143 515 341

13 41.6 5.3 7.3 41.6 4.4 227 152 166 30 27 46 180 125 490 408

14 39.7 8.1 13.8 76.0 6.3 240 151 174 26 21 36 152 116 403 252

15 49.2 8.9 14.8 22.8 8.8 307 210 220 26 18 35 203 145 489 409

16 54.1 10.5 17.5 43.8 8.5 238 170 194 16 12 30 202 135 516 457

Mean 47.6 9.0 14.4 48.3 8.3 271 180 187 27.9 23.5 40.9 180 133 487 385

S.E.M 2.0 0.6 1.1 7.0 0.8 14 9.7 6.5 2.3 2.5 2.6 9.9 5.6 21 44

Controls

17 48.7 8.8 13.2 71.0 9.4 171 133 140 23 18 32 185 141 499 389

18 50.5 11.5 16.5 72.8 9.4 284 192 194 37 27 48 196 144 526 384

19 51.0 10.7 18.0 53.5 13.9 189 109 122 26 19 36 165 116 388 355

20 32.1 4.5 10.3 19.8 4.6 228 165 177 19 15 33 207 148 367 335

21 48.4 8.1 4.2 68.7 9.4 329 203 210 26 24 51 245 191 447 358

22 32.0 8.5 12.1 49.7 8.3 143 115 140 14 12 27 150 117 526 340

23 54.2 9.0 12.2 68.7 12.9 333 199 205 25 23 41 217 154 595 472

24 38.7 4.9 9.6 80.3 14.8 218 150 161 29 24 43 176 122 537 431

Mean 44.5 8.3 12.0 60.6 10.3 237 158 169 24.6 20.2 38.8 193 142 486 383

S.E.M 3.1 0.9 1.5 6.8 1.2 25 13 12 2.4 1.8 3.0 11 8.8 28 17


3There were no differences in the [ H]8-OH-DPAT with age in layers A and B (Fig. 1). In the schizo-binding between the cohorts (Layer A: df 5 2,21; phrenic subjects, but not in the bipolar disorder or

3F 5 1.84; P 5 0.18; Layer B: df 5 2,21; F 5 0.62; control subjects, [ H]flumazenil binding increasedP 5 0.55; Layer C: df 5 2,21; F 5 0.74; P 5 0.49). with age (Fig. 2). There were no correlations be-

3[ H]Ketanserin (df 5 2,21; F 5 1.94; P 5 0.17) and tween between any of the other radioligand binding3 data and age and there were no correlations between[ H]citalopram (df 5 2,21; F 5 2.12; P 5 0.15) bind-

any of the other confounding factors (DOI, PMI, pH,ing did not differ between the groups.final prescribed drug doses or levels of drugs inblood) and any of the measures of radioligand3 33.5. The measurement of [ H]AMPA, [ H]kainatebinding.3and [ H]MK 801 binding

3The binding of [ H]AMPA was distributed in threedistinct layers in BA 9 from all subjects (Table 3, BP 4. Discussiondf 5 2,21; F 5 9.31; P 5 0.001; schizophrenic df 5

2,21; F 5 24; P , 0.001; controls df 5 2,21; F 5 This study has shown an increase in5.75; P 5 0.01) with Layer A showing the highest 3 3[ H]flumazenil but not [ H]muscimol binding in BA3level of binding. Layer A of [ H]AMPA binding 9 from subjects with bipolar disorder compared tocontained cortical laminae I to III, Layer B laminae that in BA 9 from the schizophrenic and the controlIV and V whilst Layer C contained lamina VI. The subjects. Under the conditions used in this study3binding of [ H]kainate was also in three layers 3[ H]flumazenil (Mohler and Richards, 1981) and(Table 3, BP df 5 2,21; F 5 26; P , 0.001; schizo- 3[ H]muscimol (Dean et al., 1999a) would bind tophrenic df 5 2,21; F 5 13; P , 0.001; controls df 5

two different sites on the GABA receptor (Zhao etA2,21; F 5 16; P , 0.01) with the highest density ofal., 1998). It has been shown that changing the3binding being in Layer C. Layer A of [ H]kainateexpression of the g subunit of the GABA receptor2 Abinding contained cortical laminae I and II, Layer Bchanges the density of benzodiazepine binding with-

laminae III and IV whilst Layer C contained laminae 3out changing the density of [ H]muscimol binding3V and VI. Finally, the binding of [ H]MK 801(Zhao et al., 1998). Therefore our data could indicate

appeared in two layers in all subjects (Table 3, BPa change in the relative levels of GABA receptorAdf 5 2,21; F 5 15; P 5 0.002; schizophrenic df 5sub-units in BA 9 from subjects with bipolar disor-

2,21; F 5 16; P 5 0.001; controls df 5 2,21; F 5 13;der. However, we will need to carry out a more

P 5 0.002); Layer A containing cortical laminae I tofocussed study on tissue from a larger number of

III whilst Layer B contained laminae IV to VI.subjects with bipolar disorder to fully address this

There were no significant differences in the bind-hypothesis.3ing of [ H]AMPA (Layer A: df 5 2,21; F 5 1.24;

We have previously reported an increase inP 5 0.31; Layer B: df 5 2,21; F 5 1.18; P 5 0.33; 3[ H]muscimol binding in BA 9 from subjects with3Layer C: df 5 2,21; F 5 1.06; P 5 0.36), [ H]kainate

schizophrenia (Dean et al., 1999a). The absence of(Layer A: df 5 2,21; F 5 2.36; P 5 0.12; Layer B:

such a change in this study could be due to the lowerdf 5 2,21; F 5 0.74; P 5 0.49; Layer C df 5 2,21;

number of individuals studied or the subjects in this3F 5 0.18; P 5 0.84) or [ H]MK 801 (layer A: df 5study being in different medication states.

2,21; F 5 0.82; P 5 0.45 and layer B: df 5 2,21;An important question relates to whether the

F 5 0.37; P 5 0.69) between the groups.change in benzodiazepine binding sites in the tissuefrom subjects with bipolar disorder could be due to

3.6. Radioligand binding: correlations with drug treatment prior to death. It would appear thatdemographic data and confounding factors this effect is unlikely to be due to antipsychotic drug

treatment as subjects with bipolar disorder andIn the tissue from the control subjects, but not that schizophrenia both received such drugs. In addition,

from the subjects with schizophrenia or bipolar it has been shown that treating rats with antipsy-3disorder, the level of [ H]MK801 binding decreased chotic drugs decreases the levels of cortical GABAA


3Fig. 1. The relationship between age and the density of [ H]MK Fig. 2. The relationship between age and the density of3801 binding in BA 9 from subjects with bipolar disorder, subjects [ H]Flumazenil binding in BA 9 from subjects with bipolar

with schizophrenia and control subjects. disorder, subjects with schizophrenia and control subjects.


receptors (Farnbach-Pralong et al., 1998). In com- and NMDA receptors (Deakin et al., 1989; Simpsonparison to the schizophrenic subjects, more subjects et al., 1991) in BA 11 but not BA 10 from subjectswith bipolar disorder had been exposed to benzo- with schizophrenia. Thus our study could be seen todiazepines and mood stabilisers. However, such add weight to the hypothesis that there are regionallytreatments have been shown to cause a decrease in specific changes in glutamate receptors in the cortexbenzodiazepine binding in rat cortex (Hetmar et al., from subjects with schizophrenia.1983; Miller et al., 1988). Hence, available data As in a previous study (Piggott et al., 1992), ourwould suggest that the increase in benzodiazepine study has shown an age-related decrease in NMDAbinding sites in the tissue from subjects with bipolar receptors in the cortex of the controls. This age-disorder is not related to drug treatment prior to related decrease in NMDA receptors was not appar-death and could therefore be associated with the ent in the tissue from subjects with bipolar disorderneuropathology of the illness. or schizophrenia. In addition, an age-related increase

Under the conditions used in this study in the benzodiazepine binding sites was detected in3 3 3[ H]ketanserin, [ H]8-OH-DPAT or [ H]citalopram BA 9 from subjects with schizophrenia. Significant-

would bind to the 5HT receptor (Dean et al., ly, a previous study using tissue from subjects with2A

1996a), 5HT receptor (Dean et al., 1999b) and the no history of psychiatric illness showed an increase1A

serotonin transporter (Dean et al., 1999b) respective- in benzodiazepine binding with age early in lifely. Thus the data in this study, like another using (Reichelt et al., 1991) with such an increase notfrontal cortex obtained postmortem (Leake et al., being detected in adult cortex (Freund and Ballinger,1991), do not support the hypothesis that frontal 1988; Zezula et al., 1988). Thus, our data couldcortical serotonergic dysfunction occurs in bipolar indicate that there is a continuing increase in benzo-disorder. In addition, the absence of change in the diazepine binding sites in the frontal cortex of5HT receptor and serotonin transporter in BA9 subjects with schizophrenia that is absent in subjects1A

from schizophrenic subjects is in agreement with with bipolar disorder or subjects with no history ofother studies of that brain region (Joyce et al., 1993; psychiatric illness.Dean et al., 1995, 1999b). By contrast, our finding of In conclusion, we have shown an unexpected

3no change in the 5HT receptor differs from studies discrepancy between the binding of [ H]flumazenil2A3of BA 9 in schizophrenia by ourselves (Dean and and [ H]muscimol to the GABA receptor in theA

Hayes, 1996; Dean et al., 1999a) and others (Arora frontal cortex from subjects with bipolar disorder.and Meltzer, 1991; Bennett et al., 1979; Mita et al., Even though we cannot identify the cause of this1986). However, this finding is in agreement with phenomenon our data do add weight to the hypoth-other studies that failed to show a change in cortical esis that abnormalities in GABAergic function is5HT receptors in schizophrenia (Dean et al., 1996; important in the pathology of bipolar disorder (Petty,2A

Joyce et al., 1993; Whitaker et al., 1981). It remains 1995). In addition, our data support the argumentpossible that our inability to show a significant that there are abnormalities in age-related changes indecrease in 5HT receptors in BA 9 from subjects the NMDA receptor in both bipolar disorder and2A

with schizophrenia is due to the low number of schizophrenia whereas age-related changes in benzo-individuals studied. diazepine binding are only present in subjects with

Our study also shows that the binding of bipolar disorder. Further studies in a greater number3 3[ H]AMPA to the AMPA receptor, [ H]kainate to the of subjects and in other brain regions are warranted

3kainate receptor and [ H]MK 801 to the NMDA to confirm the findings of our study in BA 9 fromreceptor does not differ between the three cohorts. subjects with bipolar disorder and schizophrenia.We are not aware of any other studies on glutamatereceptors in the frontal cortex of subjects withbipolar disorder. However, our data showing no Acknowledgementschange in AMPA receptors are consistent with thosein a previous study on schizophrenia (Kurumaji et This work was supported in part by The Carrollal., 1992). Other studies have reported regionally Family Trust, the National Alliance for Research intospecific changes in the kainate (Deakin et al., 1989) Schizophrenia and Depression and the National


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A change in the density of [3H]flumazenil, but not [3H]muscimol binding, in Brodmann’s Area 9 from subjects with bipolar disorder