Ž .Brain Research 808 1998 320–323

Short communication

w3 xAn increase in H CGS21680 binding in the striatum of postmortem brainsof chronic schizophrenics

Akeo Kurumaji a,b,), Michio Toru a

a Department of Neuropsychiatry, Tokyo Medical and Dental UniÕersity School of Medicine, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japanb ( )CREST, Japan Science and Technology JST , Japan

Accepted 11 August 1998

Abstract

w3 xWe measured adenosine 2a receptors in basal ganglia of 13 schizophrenics and 10 controls, using H CGS21680 as a ligand for thew3 xreceptor binding assay. There was a significant increase in the specific H CGS21680 binding in the putamen and caudate, but not in the

globus pallidus of externa, of the schizophrenic patients, compared to those of controls. These results provide evidence suggesting thatadenosine 2a receptors play a role in the pathophysiology of schizophrenia. q 1998 Elsevier Science B.V. All rights reserved.

w3 xKeywords: Schizophrenia; Basal ganglia; Adenosine 2a receptors; H CGS21680; Postmortem brain

Adenosine modulates neuronal functions through recep-tor-mediated mechanism. Four subtypes of adenosine re-ceptors, A1, A2a, A2b and A3, have been identified in the

w xbrain 4 . A2a receptors are coupled stimulatory guanineŽ .nucleotide binding G proteins, which positively couple

w xthe receptor to adenylate cyclase 5,15 . The receptorsexhibited a selective distribution in the basal ganglia of ratw x w x7,18 and human 12,19 brain. It has been recently re-ported that A2a receptor-knockout mice exhibit a reduced

w xexploratory activity and score higher in anxiety tests 9 .Moreover, A2a receptors are largely colocalized with

Ž .dopamine 2 D2 receptors on the striatopallidal GABAer-w xgic neurons in the striatum 3,14,18 . Stimulations of A2a

receptors in the striatum inhibit and their blockades poten-tiate D2 receptors-related GABA release in the globus

w x w xpallidus 2 . A2a receptors agonists induce catalepsy 1 ,while A2a receptors antagonists reverse the behavioral

w xeffect induced by D2 receptors antagonists 8 . Activationsof A2a receptors in rat striatal membranes decrease theaffinity of D2 agonist, and the binding of the D2 antago-nist is not affected by A2a receptors potentiations, suggest-ing an interaction at the receptorrG-protein coupling levelw x2 . Accordingly, these findings indicate there is an ant-agonistic interaction between D2 and A2a receptors in the

w xstriatum 2 .

) Corresponding author. Fax: q81-3-5803-0135; E-mail:0724.pysc@med.tmd.ac.jp

Antipsychotic drugs bind to and block striatal D2-likereceptors in a direct correlation with their clinical effectiveantipsychotic doses, indicating a major role of dopaminer-

w xgic systems in schizophrenia 16 . Many studies had re-ported an increase in D2-like receptor bindings in the

w xstriatum of postmortem brains of schizophrenics 17 .Moreover, D2 receptors have the antagonistic interaction

w xwith A2a receptors in the striatum 2 . Caffeine, a nonse-lective A2a receptor antagonist, exacerbated psychotic

w xsymptoms in schizophrenic patients 11 . Consequently,A2a receptors possibly play a role in the pathophysiologyof schizophrenia.

Ž . . X2-p- 2-Carboxyethyl phenylamino -5 -N-carboxamide-Ž .adenosine CGS21680 has been shown to bind with high

w xaffinity to adenosine A2a receptor in rat 6 and humanw x w3 xbrain 12,20 . We measured H CGS21680 binding in the

basal ganglia of postmortem brains of schizophrenic pa-tients to obtain further evidence supporting that A2a recep-tors are involved in the development of schizophrenia.

The postmortem brains specimens used were from 13Ž .schizophrenic patients eight males and five females and

Ž10 controls subjects free from neurological disease seven.males and three females whose details were described in a

w xprevious report 20 . None of the subjects had been venti-lated before death. All the schizophrenic patients, based onchart review, met the diagnostic criteria for schizophreniaas considered in DSM-IV. Seven of the 13 schizophrenicswere ‘off-drug’ cases, who had not received antipsychoticsfor more than 40 days before death. The age range of the

0006-8993r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 98 00840-3

( )A. Kurumaji, M. TorurBrain Research 808 1998 320–323 321

w3 xFig. 1. Scatchard plots of H CGS21680 binding to membranes ofŽ . Ž . Ž .caudate v , putamen ' and glubus pallidus pars externa B of

w3 xcontrols. Concentrations of 1–80 nM H CGS21680 were incubatedwith approximately 0.4 mg of membrane protein at 238C for 120 min.

ŽValues of B and K in the caudate 120.7 fmolesrmg protein, 28.8max d. Ž .nM , putamen 200.0 fmolesrmg proten, 35.4 nM and globus pallidus

Ž .pars externa 174.9 fmolesrmg protein, 19.4 nM were calculated fromthe first-order equation estimated by means of a computer with nonlinearregression analysis.

Žschizophrenic patients was 41–75 years mean"S.E.M.,. Ž60.1"3.0 years and that controls was 52–74 years 66.7

."2.7 years . The interval from death to freezing of theŽschizophrenics ranged from 3.2 to 24.0 h mean"S.E.M.,

.11.3"2.1 h and that of controls from 1.7 to 13.0 hŽ .5.3"1.2 h ; the former was significantly greater than the

Ž .latter by Mann–Whitney U test p-0.05 . The storagetime at y808C was not different between the schizophren-ics and controls.

The frozen brains were sectioned as previously reportedw x20 . Twenty-four hours before dissection, brains stored in

airtight packages at y808C were transferred to a cold boxmaintained at y158C to allow sectioning. The frozenbrains were sectioned coronally in 10 mm thick slices,mounted on a freezing microtome, and the frozen blockswere cut into 1.5-mm thick slices. Each specific brain areawas dissected out from these slices in a cold box aty158C. Dissected tissues were homogenized with a glass-Teflon homogenizer in three volumes of chilled 0.32 Msucrose and stored at y808C until being assayed.

w3 xMembrane preparation and the H CGS21680 bindingassay were performed according to the method of Jarvis et

w xal. 6 , with minor modification. The brain homogenateswere thawed at room temperature and rehomogenized us-

Ž .ing an Ultra-Turrax T25 homogenizer 13,500 rpm, 15 sŽ .in 50 volumes of ice-cold 50 mM Tris–HCl pH 7.4

containing 10 mM MgCl . The membrane homogenate2

was then centrifuged at 48,000=g for 10 min at 48C. Theresulting pellet was resuspended in buffer containing 2

ŽIUrml of adenosine deaminase Type II, Sigma, St. Louis,.MO to 20 mgrml of original tissue weight and incubated

at 378C for 30 min to remove endogenous adenosine. ThisŽmembrane homogenate was recentrifuged 48,000=g, 10

.min, 48C and final pellet was frozen at y808C until thetime of assay.

w3 x ŽThe tissue membranes and 5 nM H CGS21680 30.Cirmmol, New England Nuclear, Boston, MA were incu-

Žbated in incubation buffer 50 mM Tris–HCl and 10 mM.MgCl , pH 7.4 at 238C for 120 min. The incubations2

were terminated by filtration through Whatman GFrBfilters followed by three washes with 4 ml of chilledTris–HCl buffer. Nonspecific binding was defined in the

Žpresence of 20 mM 2-chloroadenosine Sigma, St. Louis,.MO . For Scatchard analysis, seven regularly increasing

w3 xconcentrations of H CGS21680 from 1 to 80 nM wereused. Protein concentrations were determined according to

w xthe method of Lowry et al. 10 .

w3 x Ž . Ž . Ž .Fig. 2. Specific binding of H CGS21680 in the basal ganglia of control C and schizophrenics S . ^ ‘Off-drug’ cases, who had received noŽ .antipsychotics for more than 40 days before death; ' ‘On-drug’ cases, who had been treated with antipsychotics until immediately before death.

Horizontal bars indicate mean values. Each comparison was carried out by means of multiple regression analysis adjusted for age at death and intervalfrom death to freezing. a p-0.001 vs. controls, bp-0.05 Off-drug cases vs. controls, c p-0.05 On-drug cases vs. controls.

( )A. Kurumaji, M. TorurBrain Research 808 1998 320–323322

Data from the schizophrenics and controls were com-pared statistically by means of multiple regression analy-sis, regressor variables being diagnosis, age at death, andinterval from death to freezing.

w3 xThe results of H CGS21680 saturation binding stud-ies, ranging from 1 to 80 nM, on the caudate, putamen and

Ž .globus pallidus pars externa of controls are shown in Fig.1. Scatchard plots calculated from the first-order equationestimated by means of computer with nonlinear regressionanalysis were turned out to be a single-site model ratherthan multisite models in the brain areas.

w3 xThe specific binding of 5 nM H CGS21680 wasmeasured in the areas of the schizophrenics and the con-trols. A statistically significant increase was observed inthe caudate and putamen of schizophrenic patients, com-

Ž .pared to the controls Fig. 2 . In addition, the increase wasalso prominent in the off-drug cases of schizophrenia whohad not received antipsychotic drugs more than 40 days

Ž .before death Fig. 2 , although it has been reported thatchronic treatment with typical neuroleptics, but not a typi-cal ones, produces an increase in the receptors in the rat

w xstriatum 13 . Thus, it is less likely that medications ofantipsychotics induced the increased density of the recep-tors in the present study.

There were no correlation between the binding valuesand age at death or between the bindings and the intervalfrom death to freezing in any brain areas. It is also unlikelythat the factors were related to the changes in the recep-tors.

A2a receptors in the striatum are largely expressed withD2 receptors in the GABAergic striatopallidal neuronsw x14,18 . The neurons may be a main locus for the ant-

w xagonistic interaction between the two receptors 2 . Anincrease in D2-like receptors in the striatum ofschizophrenic patients had been reported by many studies

w xusing postmortem brains 17,20 , suggesting hyper-dopaminergic transmission through D2 receptors inschizophrenia. The increased A2a receptors in the striatumof schizophrenics might be a compensatory change to thehyperfunction of D2 receptors, taking account of the coun-teracting interaction between each other. It awaits, how-ever, further studies to clarify the mechanism whereby A2areceptors are increased in the striatum of schizophrenics.Finally, the results of the present study imply a possibilityof therapeutic approaches of A2a receptors-related com-pounds in schizophrenia.

Acknowledgements

We thank Drs. T. Kanaya, N. Naito, H. Yamazaki, K.Nomura, T. Fujimori, O. Matsuda, S. Ando, S. Takahashi,H. Shimada, H. Morioka, T. Nakamura, and J. Semba fortheir help in collecting specimens, and the doctors in thedivision of Mental Disorder Research, National Institute ofNeuroscience, National Center of Neurology and Psychia-

try, for subdividing the brains. We are also grateful toProfs. T. Maeda and K. Kishi for their advice on subdivid-ing the brain. This study was supported by a grant from theNational Center of Neurology and Psychiatry of the Min-istry of Health and Welfare and a Grant-in-Aid for Science

Ž .Research C from the Ministry of Education, Science,Sports and Culture, Japan.

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