Journal of' NeurochemistryRaven Press, Ltd., New York(c) 1995 International Society for Neurochemistry

['H] Paroxetine Binding Is Altered in the Hippocampus butNot the Frontal Cortex or Caudate Nucleus from Subjects

with Schizophrenia

*tBrian Dean, *$Kenneth Opeskin, *Geoffrey Pavey, *tLee Naylor, *Christine Hill,*§Nicholas Keks, and *-{ -David L. Copolov

*The Rebecca L. Cooper Laboratories, The Mental Health Research Institute of Victoria ; tThe NH and MRCSchizophrenia Research Unit, Royal Park Psychiatric Hospital, Parkville ; $The Victorian Institute of Forensic Pathology,

South Melbourne ; and §Department of Psychiatry, The Alfred Hospital, Prahran, Victoria, Australia

Abstract : [ 3H]Paroxetine binding to particulate mem-brane from tissue, obtained at autopsy, from the hip-pocampus, frontal cortex, and caudate nucleus fromsubjects who had or had not had schizophrenia was mea-sured . The density of [ 3H]paroxetine binding to mem-branes from subjects who had or had not had schizophre-nia did not differ. Similarly, the affinity of [H]paroxetinebinding in the frontal cortex and caudate nucleus wasnot different . By contrast, the affinity of [H]paroxetinebinding to hippocampal membrane from subjects whohad schizophrenia was significantly lower than the affinityof binding for the nonschizophrenic subjects (0.40 ± 0 .06vs . 0.26 ± 0 .02 ; p < 0.05) . As [ 3H]paroxetine binds tothe serotonin transporter, these data suggest that theserotonin transporter is altered in the hippocampusin subjects with schizophrenia . Key Words : Schizo-phrenia-Paroxetine-Serotonin transporter-Hippo-campus-Frontal cortex-Caudate nucleus .J. Neurochem. 64, 1197-1202 (1995) .

The drug paroxetine has been shown to bind to theneuronal serotonin transporter (Laruelle et a1 ., 1988 ;Backstrom et al ., 1989 ; Plenge et al ., 1990) . Hence, thedecreased binding of [ 3H]paroxetine to brain tissue,obtained at autopsy from subjects with schizophrenia,suggests that there are changes in the serotonin trans-porter in the schizophrenic brain (Laruelle et al.,1993) . A change in the serotonin transporter wouldbe expected to have effects on serotonergic neuronalfunction . This is significant because changes in seroto-nergic neurotransmission have been postulated to playa role in the pathology of schizophrenia (Kahn andDavidson, 1993) .Only one study has used [ 3H]paroxetine to identify

changes in the serotonin transporter in the schizo-phrenic brain (Laruelle et al ., 1993) . Because thisfinding could be important in understanding biochemi-cal changes underlying schizophrenia, we decided to

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confirm that the serotonin transporter is altered in theschizophrenic brain . Thus, [ 3H]paroxetine binding tobrain tissue, obtained at autopsy, from subjects withschizophrenia was measured .

EXPERIMENTAL PROCEDURES

Tissue donorsWith ethics committee approval, brain tissue was collected

at autopsy from subjects stated to have been or not to havebeen schizophrenic . The subjects who were not schizo-phrenic were selected so that overall, the schizophrenic andnonschizophrenic subjects were of a similar age and of thesame sex ratio . The diagnoses of schizophrenia were con-firmed, by a senior psychiatrist, using DSM-111-R criteria(American Psychiatric Association, 1987), following an ex-tensive case history review .

Tissue collectionTissue was collected, at autopsy, from the hippocampus,

caudate nucleus, and frontal cortex (Brodmann's area 9) ofthe left hemisphere of the brain . Once collected, the tissuewas rapidly frozen to -70°C and stored until required .

Measurement of [ 3H]paroxetine binding toparticulate membrane

Tissue blocks were removed from the -70°C freezer andplaced in 5 volumes (vol/wt) of ice-cold Tris buffer (50mM Tris, 120 mM NaCl, and 5 mM KCl, pH 7.4) andchopped . The chopped tissue was homogenized using three15-s bursts of a Janke and Kunkel Ultra Turrax t25 tissuehomogenizer and centrifuged at 30,000 g for 10 min at 4°C,and the supernatant was discarded . The pellet of particulate

Received May 16, 1994 ; revised manuscript received July 19,1994 ; accepted July 19, 1994.

Address correspondence and reprint requests to Dr . B . Dean atThe Rebecca L. Cooper Laboratories, The Mental Health ResearchInstitute of Victoria, Private Bag 3, Parkville, Victoria, 3052, Aus-tralia.

Abbreviation used: PMI, postmortem interval .

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membrane was suspended in ice-cold Tris buffer, homoge-nized, and centrifuged at 30,000 g for 10 min at 4°C twice,and then the washed membrane was suspended in Tris buffer.The particulate membrane protein concentration was thenmeasured using the Bio-Rad kit method based on the me-thod of Bradford (1976) . Finally, the particulate membranewas diluted to give a final concentration of 2.0 mg of pro-tein/ml .The binding of [ ;H]paroxetine to particulate membrane,

from hippocampus, cortex, or caudate nucleus, at a finalconcentration of 0.2 mg of protein/ml, was measured in thepresence of a range of concentrations of [ 3H]paroxetine(0.2-2 .0 nM) in the absence or presence of 10 -5 M citalo-pram in Tris buffer. After incubating with the particulatemembrane for 2 h at room temperature, the bound [ 3H]-paroxetine was separated from free, after addition of 3 mlof ice-cold Tris buffer, by filtration over GF/C filters on aBrandel cell harvester . Following three washes with 5 ml of0.9% NaCl, the filter paper containing the membrane-bound[ 3H ] paroxetine was transferred into 5 ml of Beckman Readi-protein liquid scintillation cocktail, and the radioactivity oneach filter was counted in a Canberra-Packard model 1500liquid scintillation counter.

Specific binding of [ ;H] paroxetine to particulate membranewas taken as the radioactivity bound to membrane withoutcitalopram minus the radioactivity bound in the presence ofcitalopram. The KD and B�,ax for [ a H]paroxetine binding werecalculated from Scatchard plots of these data using the Ligandcomputer program . Statistical analysis was carried out usingthe ANOVA test in the Sigmastat computer program .

All the schizophrenic individuals from whom tissue wasobtained had been treated with neuroleptic drugs . Thus, todetermine the possible effect of neuroleptic drugs, [ 3H]-paroxetine binding to hippocampul and caudate membraneswas measured as described above in the absence or presenceof 10 -'M haloperidol .

Tissue donorsTissue was collected from 14 (12 male and two

female) subjects with schizophrenia, seven (all male)of whom had committed suicide, and 14 (12 male andtwo female) subjects with no known history of mentalillness, seven (six male and one female) of whom hadcommitted suicide (Table 1) . There was no statisticaldifference between the mean age of the subjects whohad a history of schizophrenia and those who did not(Table 2) . By contrast, the subjects who had beenschizophrenic but had not committed suicide were sig-nificantly older than subjects who had been schizo-phrenic and committed suicide (p < 0.01) and thesubjects who had not been schizophrenic but who did(p < 0.05) or did not (p < 0.01) commit suicide(Table 2) .

There was no difference between the time of deathand tissue collection [postmortem intervals (PMI)] forthe tissue from the schizophrenic and nonschizo-phrenic subjects (Table 2) .

Although all the subjects with schizophrenia fromwhom tissue was obtained at autopsy had been treated

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RESULTS

B. DEAN ET AL.

with typical neuroleptic drugs before death, none ofthem had been treated with antidepressants, and therewas no history of major depression .

[ 3H]Paroxetine binding to particulate membraneIn all cases the Scatchard plot of [ 3H]paroxetine

binding to particulate membranes was linear, sug-gesting the presence of a single binding site . Further-more, there was no difference in either the affinity(KD) or density (Bm.,x) of [ 3H]paroxetine binding toparticulate membranes from the frontal cortex or cau-date nucleus from subjects who had schizophrenia andsubjects who had not had schizophrenia (Fig . 1 andTable 2) . Similarly, there was no difference betweenthe density of [ 3H]paroxetine binding to particulatemembrane from the hippocampus of subjects who hador those who did not have schizophrenia . By contrast,there was a significant decrease in the affinity (in-creased KD) of binding of [3H] paroxetine to particulatemembrane from the hippocampus of subjects withschizophrenia compared with that for the nonschizo-phrenic subjects (p < 0.05 ; Fig . 1 and Table 2) .

Further analysis of these data showed that the de-creased affinity of [ 3H]paroxetine binding was mostlyattributable to a change in the subjects who had beenschizophrenic and had committed suicide (p < 0.025 ;Fig . 1 and Table 2) . By contrast, the affinity of [ 3H]-paroxetine binding tended to be lower for the schizo-phrenic subjects who had not committed suicide, butthis difference did not reach significance .

There were no significant correlations between theKD or the Bmax for ['H]paroxetine binding to hippo-campal, caudate nucleus, or frontal cortical tissue andage of the donor or the PMI .

Effect of haloperidol on [ 3H ]paroxetine binding['H]paroxetine binding to particulate membranes

from the hippocampus and caudate from a single indi-vidual was similar in the absence (KD = 0.26 and0.34 nM and B � ,ax = 315 and 420 fmol/mg of protein,respectively) or presence (KD = 0.25 and 0.37 n M andBmax = 310 and 390 fmol/mg of protein, respectively)of 10 -' M haloperidol .

DISCUSSION

This study has shown that the affinity, but not thedensity, of [ 3H]paroxetine binding is altered in thehippocampus but not frontal cortex or caudate nucleusof subjects with schizophrenia . These results differfrom a previous study that showed a decreased densityof [ 3H]paroxetine binding in the frontal but not theoccipital pole from subjects with schizophrenia (Lar-uelle et al ., 1993) . However, both studies suggest thatthere are changes in [ 3H]paroxetine binding in braintissue from subjects with schizophrenia .

In addition to showing that the affinity of [ 3H]-paroxetine binding is altered in the hippocampus fromsubjects who had been schizophrenic, this study has

shown that the change in affinity of binding is mostapparent in subjects who had been schizophrenic andhad committed suicide . This observation is significantas changes in serotonergic function have been sug-gested to play a role in precipitating suicidal behavior(Mann et al ., 1989) . Furthermore, the incidence ofsuicide is thought to increase greatly in subjects withdepressive illness . Significantly, like two prior studies(Lawrence et al ., 1990 ; Hrdina et al ., 1993), this study

[3H]PAROXETINE BINDING IN SCHIZOPHRENIA

TABLE 1. Clinical data on subjects from whom tissue was collected

TABLE 2. The sex and age ofsubjects who had or who had not had schizophrenia and the PMIjbr the collection ofbrain tissue from these individuals as well as the affinity (K D) and density (B_,,) of[3H]paroxetine binding

to particulate membrane from their hippocampus, frontal cortex, and caudate nucleus

did not show differences in the affinity or density of[3H]paroxetine binding in tissue from subjects whohad committed suicide but did not have schizophrenia.Thus, the changes in [ ;H]paroxetine binding demon-strated in the hippocampus from subjects with schizo-phrenia who had committed suicide appear differentfrom the changes associated with suicidal behavior innonschizophrenic individuals . However, because of thelow numbers of individuals in this study who had had

['H]Paroxetine binding

Data are mean -} SEM values . Kp values are in units of nM, and B�,ax values are in units of fmol/mg of protein .

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J. Neurochem ., Vol. 64, No. 3, 1995

Group, subject no . SexAge

(years)PMl(h) Cause of death

SchizophrenicI M 47 33 Ischemic heart disease2 F 72 58 Bronchopneumonia3 M 67 39 Aspiration pneumonia4 F 71 28 Chronic obstructive airways disease5 M 71 45 Ischemic heart disease6 M 63 63 Ischemic heart disease7 M 44 32 Ischemic heart disease8 M 27 42 Suicide (burns)9 M 23 25 Suicide (hanging)

10 M 27 53 Suicide (drug overdose)11 M 30 41 Suicide (burns)12 M 22 36 Suicide (carbon monoxide poisoning)13 M 33 32 Suicide (hanging)14 M 47 42 Suicide [multiple injuries (jumping)]

Nonschizophrenic15 M 25 42 Accidental drowning16 M 36 36 Crush accident17 M 23 26 Asphyxiation following asthmatic episode18 M 49 70 Ruptured thoracic aortic aneurysm (natural)19 M 44 36 Acute myocardial infarction20 F 36 22 Pulmonary embolism21 M 44 35 Ischemic heart disease22 M 52 47 Suicide (hanging)23 M 52 48 Suicide (gunshot wound)24 M 31 31 Suicide (hanging)25 M 28 28 Suicide (hanging)26 F 60 22 Suicide (hanging)27 M 18 36 Suicide (hanging)28 M 45 18 Suicide (hanging)

Hippocampus Frontal cortex Caudate nucleus

SubjectsSex(M/F)

Age(years)

PMI(h) KD I Kn B__ Ku

Schizophrenic 12/2 46 ± 5.2 41 ± 3.0 0.46 ± 0.07 185 - 14 0.25 + 0.02 81 ± 6.5 0.I8 ± 0.03 279 ± 20Suicide 7/0 30±3.2 39±3.4 0.56±0.11 192 ± 19 0.22 } 0.03 80 i- 9.9 0.I9 ± 0.05 25I - 18Nonsuicide 5/2 62±4.5 43±5.1 0.35±0.07 177±21 0.28±0.04 82~9.3 0.16-0.04 306-33

Nonschizophrenic 12/2 38±3.8 36±3.9 0.26±0.08 163 ±- 20 0.25 i 0.02 80 ± 7.9 0.20 ± 0.02 257 ± 10Suicide 7/1 41±3.6 35±3.6 0.27±0.03 158 ± 30 0.25 ± 0.02 81 ± 10 0.2I ± 0.03 256 ± 12Nonsuicide 5/1 36±3.7 38±5.9 0.25±0.03 168 - 28 0.25 ± 0.02 79 ± 13 0.I9 ± 0.02 257 + 16

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FIG. 1. Affinity (Kp ; top) and density (B,ax; bottom) of [3Hlparoxetine binding to brain tissue, obtained at autopsy, from subjects whohad or had not had schizophrenia and who did (A) or did not (9) commit suicide.

schizophrenia and either did or did not commit suicide,this observation can only be regarded as preliminary .A much more extensive study of [3H]paroxetine bind-ing to hippocampal tissue from subjects with schizo-phrenia who had or had not committed suicide wouldbe required to confirm this hypothesis .

Several studies have been published reporting thedistribution of serotonin transporters in the human

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B . DEAN ET AL.

brain and have shown a lower number of transportersin the frontal cortex compared with the hippocampusand caudate nucleus (Laruelle et al ., 1988 ; Lawrenceet al ., 1990 ; Plenge et al ., 1990) . The data presentedin this study also show a lower density of serotonintransporter in the frontal cortex compared with thehippocampus and caudate nucleus (Table 2) . Signifi-cantly, both the density and the affinity of [3H]-

paroxetine binding to the serotonin transporter for thenonschizophrenic subjects are in close agreement withthose reported in the published study using tissue fromsubjects with schizophrenia (Laruelle et al ., 1993) .This suggests the differing results obtained in the twostudies are not likely to be due to methodological con-siderations .No correlation was found between the affinity or

density of [ 3H]paroxetine binding and donor age orthe PMl for the tissue . Hence, as in a previous study(Andersson et al ., 1992), these data suggest that theserotonin transporter is a stable molecule that is suit-able for study in postmortem tissue . Therefore, thefact that the schizophrenic individuals who had notcommitted suicide were significantly older than theother subjects should not have influenced the overallfindings because there was no relationship between ageand [ 3H]paroxetine binding .

All subjects from whom tissue was collected at au-topsy who were diagnosed as schizophrenic had beentreated with neuroleptic drugs . It therefore remainspossible that the change in [ 3H]paroxetine binding ob-served was drug related rather than a pathophysiologi-cal effect . Unfortunately, there are no studies on theeffects of neuroleptic drugs on the serotonin transporterin animals that could help to support or disprove thishypothesis . However, we have shown that the additionof the neuroleptic drug, haloperidol, at 10-'M, doesnot affect [ 3H]paroxetine binding . This suggests thatif changes in ['H]paroxetine binding were caused byneuroleptic drugs, this is not likely to be due to residualneuroleptic drugs that could be present in the braininterfering with the ['H]paroxetine binding assay .However, further studies of neuroleptic drug treatmenton ['H]paroxetine binding in the rat would be war-ranted .

Significantly, the findings from this study differfrom those reported previously (Laruelle et al ., 1993)in that a change in affinity rather than in density of[ 3H ]paroxetine binding was found . However, the twostudies measured [ 3H]paroxetine binding in differentbrain regions, and thus it could be possible that thereare both regionally and functionally different changesin ['H]paroxetine binding in the schizophrenic brain .These studies, plus those showing changes in serotonin5-HT2 receptors in Brodmann's area 9 (Bennett et al .,1979 ; Mita et al ., 1986 ; Arora and Meltzer, 1991) ofthe frontal cortex but not in other Brodmann's areasassociated with the frontal cortex (Whitaker et al .,1981 ; Reynolds et al ., 1983), suggest that regionalchanges in serotonin neurotransmission occur in theschizophrenic brain . Thus, it will be important to deter-mine if the differing changes in serotonergic receptorsand transporters always occur concurrently in tissuefrom subjects with schizophrenia . If that were the case,this could provide a rationale for why some individualsmay respond to atypical rather than typical neuroleptic

( 3HJPAROXETINE BINDING IN SCHIZOPHRENIA 1201

drugs, as the atypical neuroleptic drugs are more activeon serotonergic neurons (Matsubara et al ., 1993) .

In summary, [3H ] paroxetine has been shown to bindwith high affinity and specificity to the serotonin trans-porter (Laruelle et al ., 1988 ; Backstrom et al ., 1989 ;Plenge et al ., 1990) . The changes in [ 3H]paroxetinebinding to brain tissue from subjects with schizophre-nia, reported in this and a previous study (Laruelle etal ., 1993), suggest a change in the serotonin trans-porter in the brains of subjects with schizophrenia .Such changes in the serotonin transporter suggest thatchanges in serotonergic neurotransmission occur in theschizophrenic brain . Hence, these findings seem to sup-port the hypothesis that, at least in some individuals,changes in serotonergic neurotransmission are in-volved in the pathogenesis of schizophrenia (Kahn andDavidson, 1993) .

Acknowledgment : We would like to thank Professor Co-lin Masters for his support and advice . In addition, we wouldlike to thank Professor Stephen Cordner, Ms . PatriciaO'Brien, and the staff at the Victorian Institute of ForensicPathology for their support and cooperation . Finally, thisstudy would not have been possible without financial supportfrom the Rebecca L. Cooper Medical Research Foundation,the State Government of Victoria, and the Theodore VadaStanley Foundation .

REFERENCES

American Psychiatric Association ( 1987) Diagnostic and StatisticalManual ofMental Disorders, 3rd edit ., revised . American Psy-chiatric Press, Washington, D.C .

Andersson A ., Sundman L, and Marcusson J . (1992) Age stabilityof human brain 5-HT terminals studied with I'HIparoxetinebinding . Gerontology 38, 127-132 .

Arora R. C . and Meltzer H . Y . (1991 ) Serotonin, (5-HT Z ) receptorbinding in the frontal cortex ofschizophrenic patients . .l. NeuralTransm. 85, 19-29 .

Backstrom l ., Bergstrom M ., and Marcusson J . (1989) High affinity['H] paroxetine binding to serotonin uptake sites in human braintissue . Brain Res. 486, 261-268 .

Bennett J . P ., Enna S . J ., Bylund D . B ., Gillin J . C ., Wyatt R . J .,and Snyder S . H . (1979) Neurotransmitter receptors in frontalcortex of schizophrenics . Arch. Gen . Psychiatry 36, 927-934 .

Bradford M . M . (1976) A rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye binding . Anal. Biochem . 72, 248-254 .

Hrdina P . D ., Demeter E ., Vu T. B ., Sotonyi P ., and PalkovitsM . (1993) 5-HT uptake sites and 5-HTZ receptors in brain ofantidepressant-free suicide victims/depressives : increase in 5-HT, sites in cortex and amygdala . Brain Res . 614, 37-44 .

Kahn R . S . and Davidson M . ( 1993) Serotonin, dopamine and theirinteractions in schizophrenia . Psychopharmacologv Suppl.(Berlin) 112, SI-S4 .

Laruelle M ., Vanisberg M . A ., and Maloteaux J . M . (1988) Regionaland subcellular localization in human brain of ['H]paroxetinebinding, a marker of serotonin uptake sites . Biol. Psychiatry24, 299-309 .

Laruelle M ., Abi-Dargham A ., Casanova M . F ., Toti R ., WeinbergerD. R ., and Kleinman J. E . (1993) Selective abnormalities ofprefrontal serotonergic receptors in schizophrenia : a post-mor-tem study . Arch. Gen . Psychiatrv 50, 810-818 .

.1 . Neurochem., Vol. 64, No . .3, 1995

1202

Lawrence K. M., De Paermentier F ., Cheetham S . C ., CromptonM . R., Katona C . L., and Horton R . W . (1990) Brain 5-HTuptake sites, labelled with ['H]paroxetine, in antidepressant-free depressed suicides . Brain Res. 526, 17-22.

Mann J . J ., Arango, V ., Marzuk P . M ., Theccanat S., and Reis D . J .(1989) Evidence for the 5-HT hypothesis of suicide. Br. J.Psychiatry 155 (Suppl . 8), 7-14 .

Matsubara S ., Matsubara R ., Kusumi I ., Koyama T ., and Yamashita1 . (1993) Dopamine D, , D2 and serotoninz receptor occupationby typical and atypical antipsychotic drugs in vivo. J . Pharma-col. Exp . Ther. 265, 498-508 .

Mita T ., Hanada S ., Nishino N ., Kuno T., Nakai H ., Yamadori Y .,and Tanaka C . (1986) Decreased serotonin S2 and increased

J. Neurochem., Vol. 64, No. 3, 1995

B. DEAN ET AL.

dopamine D2 receptors in chronic schizophrenics . Biol . Psychi-atry 21, 1407-1414.

Plenge P ., Mellerup E . T., and Laursen H . (1990) Regional distribu-tion of the serotonin transport complex in human brain, identi-fied with 'H-paroxetine, 'H-citalopram and 'H-imipramine.Prog. Neuropsychopharmacol. Biol . Psychiatry 14, 61-72 .

Reynolds G. P., Rossor M . N ., and Iversen L . L . (1983) Preliminarystudies of human cortical 5-HT2 receptors and their involve-ment in schizophrenia and neuroleptic drug action . J. NeuralTransm. Suppl . 18, 273-277 .

Whitaker P . M ., Crow T . J., and Ferrier I . N . (1981) Tritiated LSDbinding in frontal cortex in schizophrenia . Am . J. Psychiatry38, 278-280 .