KISEP Original Articles 38 2 1999 J Korean Neuropsychiatr Assoc Vol 38, No 2 내측전전두피질에 6-hydroxydopamine 주입후 흰쥐신선조체도파민수용체의변화에관한 자가방사기록법적연구 권준수 * 이동영 * 우종인 * An Autoradiographic Study on the Rat Neostriatal Dopamine Receptor Changes after 6-hydroxydopamine Injection into the Medial Prefrontal Cortex Jun Soo Kwon, M.D.,* Dong Young Lee, M.D.*, Jong Inn Woo, M.D.* 국문초록 중심단어 서 Department of Psychiatry & Institute for Neuroscience, Seoul National University, College of Medicine, Seoul Corresponding author 론 408
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1. 실험동물및재료 연구대상및방법 2. 입체외과수술 (Stereotaxic surgery) Fig. 1. Cursor box placements in brain regions of coronal sections collected 9.2 to 10.2mm anterior to the interaural plane are represented here at anterior. Left hemisphere labels apply to neuroanatomical names according to Paxinos and Watson1986. ac, anterior commissurecc, corpus calosumclau, claustrumcpu, caudate-putamenfrpam, frontal parietal motor parietal motor cortexfrpass, frontal parietal somatosensory cortexlot, lateral olfactory tractna, nucleus accumbensot, olfactory tubercle. 3. 조직준비및보온 (Incubation) 410
Fig. 2. Saturation curve for 3 H-SCH 23390 bindingd1 receptor binding in caudate-putamen sections of rat. Specific and non-specific binding of 3 H- SCH 23390 to striatal sections. Fig. 4. Saturation curve for 3 H-spiperone bindingd2 receptor binding in caudate-putamen sections of rat. Specific and non-specific binding of 3 H- spiperone to striatal sections. Fig. 3. Representive scatchard plot of specific 3 H-SCH 23390 bindingd1 receptor binding in caudateputamen sections of rat. Fig. 5. Representive scatchard plot of specific 3 H-spiperone bindingd2 receptor binding in caudateputamen sections of rat. 411
Fig. 6. Autoradiographic distribution of [ 3 H]SCH 23390 binding in caudate-putamen and nucleus accumbens. Illustrated are a 6-hydroxydopamine injected rat(upper part) and a shamlesioned rat(lower part). Total binding(left side) and nonspecific binding(right part) are shown. cold buffer로 5분간 씻은 후 0 의 증류수에 담근 후 실온에서 건조시켰다. 비특이적 결합을 측정하기 위하 여 특이결합에 사용된 바로 옆 절편을 각각 butaclamol 10μM을 넣고 보온하였다. D2 수용체 결합을 측정하기 위하여 [3H]spiperone 0.3, 0.5, 0.7, 1.0, 1.5, 2.0nM의 6가지 농도를 사용하 였고 역시 5-HT2 수용체 및 α1 아드레날린 수용체를 차단하기 위하여 mianserin 100nM을 넣었다. 같은 방 법으로 ice cold buffer로 5분간 씻은 후 0 의 증류수 에 담근 후 실온에서 건조시켰으며, 비특이적 결합을 측 정하기 위하여 바로 옆 절편을 각각 butaclamol 2μM 을 넣고 보온하였다. 4. 자가방사기록법 및 컴퓨터 영상분석 Fig. 7. Comparison of Bmax and Kd of D1 receptor in caudate-putamen and nucleus accumbens between control and 6-OHDA group. *p<0.05, as compared with control group(by Mann-Whitney U-test) 뇌조직이 있는 유리슬라이드를 빛을 차단하는 X-ray cassette에서 Hyperfilm-3H 위에 순서대로 나란히 배 이 놓은 후 실온의 암실에서 3주([3H]SCH23390), 또 열한다. 광학도를 fmol/mg tissue weight 값으로 전환 는 4주([3H]spiperone)간 보관한 후 Kodak D19로 3 하기 위해 표준 [3H]-microscale(Amersham)도 같 분간 현상한 후 1분 간 증류수에 씻는다. 다시 Kodak 412
Fig. 8. Autoradiographic distribution of [ 3 H]spiperone binding in caudate-putamen and nucleus accumbens. Illustrated are a 6-hydroxydopamine injected rat(upper part) and a sham-lesioned rat(lower part). Total binding(left side) and nonspecific binding(right part) are shown. rapid fixer에 약 4분 간 고정한 후 깨끗이 씻고 건조 D2 수용체의 결합도를 보기 위해 미상핵-피각, 측좌 시킨다. 핵 부위를 분석하였고(그림 1) 정확한 지형학적 위치 감광된 필름의 영상을 분석시스템인 digital autoradio- 는 Paxinos와 Watson의 Atlas(1986)를 참조하였 graphic system인 Amersham RAS-R1000 receptor 다. 총결합에서 바로 옆 뇌절편의 영상인 비특이성결 analysis system을 이용하여 분석을 하였다. Video 합을 뺀 것이 특이성결합인데 fmol/mg tissue weight camera(dage-mti series 68 NEWVICON)에 의해 로 계산하였다. 이와 같이 얻은 결과들을 포화곡선(그 필름을 통과한 빛을 기록한 후 color TV monitor에 영 림 2 for D1 그림 4 for D2)을 산출하였고, Scat- 상화시키게 된다. Video signal은 512X512 pixel에 chard 분석을 시행하여 최대 결합력(maximum number digitize되고 각 pixel의 밝기는 256 gray level로 바뀌 of binding site)인 Bmax과 해리 상수(dissociation 게 된다. constant)인 Kd를 구하였다(그림 3 for D1 그림 5 먼저 거리를 검도(calibration)한 후 microscale이 감 광된 부위의 평균광학도(optical density)를 이용하여 표준곡선을 구한다. 이 표준곡선에 의해 측정하고자 하 는 부위의 감광정도에 따라 광학도를 구한다. 정량분석은 관심영역(area of interest)을 설정하고 관심영역부위에서 마우스(mouse)로 직사각형 부위를 만들고 그 부위의 광학도의 평균을 측정하였다. D1 및 for D2). 5. 통계처리 실험군인 6-OHDA 투여군과 대조군의 Bmax와 Kd 를 비교하기 위하여 SPSS 프로그램(version 7.01)을 이용하여 비모수 통계방법인 Mann-Whitney test를 사용하였다 413
Fig. 9. Comparison of Bmax and Kd of D2 receptor in caudate-putamen and nucleus accumbens between control and 6-OHDA group. 결 1. 내측전전두엽 (MPFC) 에 6-OHDA 투여후흰쥐뇌의 D 1 수용체의변화 2. 내측전전두엽 (MPFC) 에 6-OHDA 투여후흰쥐뇌의 D 2 수용체의변화 과 고찰 414
참고문헌 1) Maas JW, Bowden CL, Miller AL, Javors MA, Funderburg LG, Berman N, Weintraub ST(1997): Schizophrenia, psychosis, and cerebral spinal fluid homovanillic acid concentrations. Schizophr Bull 23 (1):147-154 415
2) Haracz JL(1982):The dopamine hypothesis:an overview of studies with schizophrenic patients. Schizophr Bull 8(3):438-469 3) Hurd YL, Pristupa ZB, Herman MM, Niznik HB, Kleinman JE(1994):The dopamine transporter and dopamine D 2 receptor messenger RNAs are differentially expressed in limbic- and motor-related subpopulations of human mesencephalic neurons. Neuroscience 63 (2):357-362 4) Meltzer HY(1991):The mechanism of action of novel antipsychotic drugs. Schizophr Bull 17(2): 263-287 5) Brunello N, Masotto C, Steardo L, Markstein R, Racagni G(1995):New insights into the biology of schizophrenia through the mechanism of action of clozapine. Neuropsychopharmacology 13(3):177-213 6) Wilson JM, Sanyal S, Van Tol HH(1998):Dopamine D 2 and D 4 receptor ligands:relation to antipsychotic action. Eur J Pharmacol 351(3):273-286 7) Andreasen NC(1979):The clinical assessment of thought, language, and commucation disorders;ii Diagnostic significance. Arch Gen Psychiatry 36: 1315-1321 8) Crow TJ(1980):Positive and negative schizophrenic symptoms and the role of dopamine. Br J Psychiatry 137:383-386 9) Crow TJ(1980):Molecular pathology of schizophrenia;more than one disease process? Br Med J 280:66-68 10) Seeman P, Ulpian C, Bergeron C(1984):Bimodal distribution of dopamine receptor densities in brains of schizophrenics. Science 225:728-731 11) Weinberger DR, Kleinman JE(1986):Observations on the brain in schizophrenia. In:Psychiatry Update. Vol 5, Ed by Hales RE and Frances AJ, American Psychiatric Association Press, pp42-67 12) Wong DF, Wagner HN Jr, Tune LE, Dannals RF, Pearlson GD, Links JM, Tamminga CA, Broussolle EP, Ravert HT, Wilson AA, Thomas Toung JK, Malat J, Williams JA, O Tuama LA, Snyder SH, Kuhar MJ, Gjedde A(1986):Positron emission tomography reveals elevated D 2 dopamine receptors in drugnaive schizophrenics. Science 234:1558-1563 13) Chabrol H, Guell A, Bes A, et al(1986):cerebral blood flow in schizophrenic adolescents. Am J Psychiatry 143:130 14) Jernigan TL, Sargent T, Pfefferbaum A, Kusubov N, Stahl SM(1985): 18Fluorodeoxyglucose PET in schizophrenia. Psychiatry Res 16:317-329 15) Kishimoto H, Kuwahara H, Ohno S, Takazu O, Hama Y, Sato C, Ishii T, Nomura Y, Fujita H, Miyauchi T(1987):Three subtypes of chronic schizophrenia identified using 11C-glucose positron emission tomography. Psychiatry Res 21:285-292 16) Weinberger DR, Berman KF, Illowsky BP(1988): Physiological dysfunction of dorsolateral prefrontal cortex in schizophrenia, III:A new cohort and evidence for a monoaminergic mechanism. Arch Gen Psychiatry 45:609-615 17) Benson DF, Kuhl DE, Hawkins RA, Phelps ME, Cummings JL, Tsai SY(1983):The fluorodeoxyglucose 18-F scan in Alzheimer s disease and multiinfarct dementia. Arch Neurol 40:711-714 18) Haberg B, Ingvar DH(1976):Cognitive reduction in presenile dementia related to regional abnormalities of the cerebral blood flow. Br J Psychiatry 128:209-222 19) Brozoski TJ, Brown RM, Rosvold PS(1979): Cognitive deficit caused by regional depletion of dopamine in prefrontal cortex of rhesus monkey. Science 205:929-931 20) Okubo Y, Suhara T, Suzuki K, Kobayashi K, Inoue O, Terasaki O, Someya Y, Sassa T, Sudo Y, Matsushima E, Iyo M, Tateno Y, Toru M(1997): Decreased prefrontal dopamine D 1 receptors in schizophrenia revealed by PET. Nature 385:634-636 21) Weinberger DR(1987):Implications of normal brain development for pathogenesis of schizophrenia. Arch Gen Psychiatry 44:660-669 22) Goldman-Rakic PS(1994):Working memory dysfunction in schizophrenia. J Neuropsychiatry Clin Neurosci 6:348-57 23) Leccese AP, Lyness WH(1987):Lesions of dopamine neurons in the medial prefrontal cortex:effects on self-administration of amphetamine and dopamine synthesis in the brain of the rat. Neuropharmacology 26:1303-1308 24) Martin-Iverson, Szostak C, Fibiger HC(1986): 6-hydroxydopamine lesions of the medial prefrontal cortex fail to influence intravenous self-administration of cocaine. Psychopharmacology 88:310-314 25) Christie MJ, Rowe PJ, Beart PM(1986):Effect of excitotoxin lesions in the medial prefrontal cortex on cortical and subcortical catecholamine turnover in 416
the rat. J Neurochem 47(5):1593-7 26) Pycock CJ, Kerwin RW, Carter CJ(1980):Effect of lesion of cortical dopamine terminals on subcortical dopamine receptors in rats. Nature 286:74-77 27) Rosin DL, Clark WA, Goldstein M, Roth RH, Deutch AY(1992):Effect of 6-hydroxydopamine lesions of the prefrontal cortex on tyrosine hydroxylase activity in mesolimbic and nigrostriatal dopamine systems. Neuroscience 48:831-839 28) Paxinos G, Watson C(1986):The rat brain in stereotaxic coordinates. 2nd ed, San Diego, Academic 29) Iversen SD(1971):The effect of surgical lesions to the frontal cortex and substantia nigra on amphetamine responses in rat. Brain Res 31:295-311 30) Creese I, Burt DR, Snyder SH(1977):Dopamine receptor binding enhancement accompanies lesion-induced behavioral supersensitivity. Science 197(4303): 596-8 31) Bird ED, Spokes EGS, Iversen L(1979):Increased dopamine concentration in limbic areas of brain from patients dying with schizophrenia. Brain 102:347-360 32) Lee T, Seeman P, Tourtellotte WW, Farley IJ, Hornykeiwicz O(1978):Binding of 3 H-neuroleptics and 3 H-apomorphine in schizophrenic brains. Nature 274:897-900 33) Owen F, Cross AJ, Crow TJ, Longden A, Poulter M, Riley GJ(1978):Increased dopamine-receptor sensitivity in schizophrenia. Lancet 2:223-225 34) Mita T, Hanada S, Nishino N, Kuno T, Nakai H, Yamadori T, Mizoi Y, Tanaka C(1986):Decreased serotonin S 2 and increased dopamine D 2 receptors in chronic schizophrenics. Biol Psychiatry 21:1407-1411 35) Mjorndal T, Winblad(1986):Alteration of dopamine receptors in the caudate nucleus and the putamen in schizophrenic brain. Med Biol 64:351-354 36) Joyce JN, Lexow, Bird E, Winokur A(1988): Organization of dopamine D1 and D2 receptors in human striatum:receptor autoradiographic studies in Huntington s disease and schizophrenia. Synapse 2: 546-557 37) Toru M, Watanabe S, Shibuya H, Nishikawa T, Noda K, Mitsushio H, Ichikawa H, Kurumaji A, Takashima M, Mataga N(1988):Neurotransmitters, receptors and neuropeptides in post-mortem brains of chronic schizophrenic patients. Acta Psychiatr Scand 78:121-137 38) Glees P(1944):The anatomic basis of corticostriate connections. J Anat 78:48-51 39) Carmann JB, Cowan WM, Powell TPS, Webster KE(1965):A bilateral corticostriate projection. J Neurol Neurosurg Psychiatry 28:71-77 40) Webster KE(1965):The corticostriatal projection in the cat. J Anat 99:329-337 41) Spencer HJ(1976):Antagonism of cortical excitation of striatal neurons by glutamic acid diethyl ester: Evidence for glutamic acid as an excitatory transmitter in the rat striatum. Brain Res 102:91-101 42) Druce D, Peterson D, De Belleroche J, Bradford HF(1982):Differential amino acid and neurotransmitter release in rat neostriatum following lesioning of the corticostriatal pathway. Brain Res 247:303-307 43) Christie MJ, James LB, Beart PM(1985):An Excitant amino acid projection from the medial prefrontal cortex to the anterior part of nucleus accumbens in the rat. J Neurochem 45:477-482 44) Young AMJ, Bradford HF(1986):Excitatory amino acid neurotransmitters in the corticostriate pathway: Studies using intracerebral microdialysis in vitro. J Neurochem 47:1399-1404 45) Roberts PJ, Anderson SD(1979):Stimulatory effect of L-glutamate and released amino acids on [ 3 H]dopamine release from rat striatum:an in vitro model for glutamate actions. J Neurochem 32:1539-1545 46) Chesselet MF(1984):Presynaptic regulation of neurotransmitter release in the brain. Neuroscience 12: 347-375 47) Cheramy A, Romo R, Godehen G, Baruch P, Glowinski J(1986):In vivo presynaptic control of dopamine release in the cat caudate nucleus. II. Facilitary or inhibitory influence of L-glutamate. Neuroscience 19:1081-1090 48) Carter CJ, L Heureux R, Scatton B(1988):Differential control by N-Methyl-D-Aspartate and kainate of striatal dopamine release in vivo:a trans-striatal dialysis study. J Neurochem 51:462-468 49) Hess EJ, Bracha HS, Kleinman JE, Creese I (1987):Dopamine receptor subtype imbalance in schizophrenia. Life Science 40:1487-1497 50) Memo M, Kleinman J, Hanbauer I(1983):Coupling of dopamine D 1 recognition sites with adenylate cyclase in nuclei accumbens and caudatus of schizophrenics. Science 221:1304-1307 51) Zhang X, Segawa T(1989):Selective blockade of dopamine D 1 receptor by SCH-23390 affects dopamine 417
agonist binding to 3 H-spiperone labeled D 2 receptors in rat striatum. Jpn J Pharmacol 50:333-345 52) Stewart J, Vezina P(1989):Microinjection of SCH- 23390 into the ventral tegmental area and substantia nigra reticulata attenuata the development of sensitization to locomotor activating effects of systemic amphetamine. Brain Res 495:401-406 53) Kuhar M, Unnerstall J(1985):Quantitative receptor mapping by autoradiography:some current technical problems. Trends Neurosci Feb, pp49-53 54) Savasta M, Dubois A, Scatton B(1986):Autoradiographic localization of D 1 dopamine receptors in the rat brain with [ 3 H]SCH23390. Brain Res 375: 291-301 55) Richfield EK, Young AB, Penney JB(1986): Properties of D 2 dopamine receptor autoradiography: High percentage of high-affinity agonist sites and increased nucleotide sensitivity in tissue sections. Brain Res 383(1-2):121-8 56) Richfield EK, Penney JB, Young AB(1989): Anatomical and affinity state comparisons between dopamine D 1 and D 2 receptors in the rat central nervous system. Neuroscience 30(3):767-77 57) Cortes R, Gueye B, Pazos A, Probst B, Palacios JM(1989):Dopamine receptors in human brain: Autoradigraphic distribution of D 1 sites. Neuroscience 28:263-273 58) Altar CA, Kim H, Marshall JF(1985):Computer imaging and analysis of dopamine(d 2) and serotonin (S 2) binding sites in basal ganglia or labeled by [ 3 H] spiperidol. Pharmacol Exp Ther 233:527-538 ABSTRACT J Korean Neuropsychiatr Assoc Vol 38, No 2, March 1999 An Autoradiographic Study on the Rat Neostriatal Dopamine Receptor Changes after 6-hydroxydopamine Injection into the Medial Prefrontal Cortex Jun Soo Kwon, M.D., Dong Young Lee, M.D., Jong Inn Woo, M.D. Department of Psychiatry & Institute for Neuroscience, Seoul National University, College of Medicine, Seoul The frontal cortex of rat is innervated by dopaminergic pathwaymesocortical pathway arising from ventral tegmental area. Several studies have suggested that mesocortical dopaminergic neurons may modulate the function of dopaminergic neurons at subcortical sites. The effect of lesions of the dopaminergic nerve terminals in the medial prefrontal cortex of the rat on dopamine D 1 and D 2 receptors within the striatum and olfactory tubercle has been investigated. Bilateral 6-hydroxydopamine lesions were stereotaxically placed in the medial prefrontal cortex. Animal were pretreated with desipramine to block the uptake of neurotoxin into noradrenergic terminals and to make it more selective for dopamine terminal. After 2weeks later, we examined the changes of D 1 and D 2 receptors in caudate-putamen and nucleus accumbens by quantitative autoradiography using the specific D 1 antagonist [ 3 H]SCH23390 and D 2 antagonist [ 3 H]spiperone. The results shows that D 1 receptor at striatum was up regulated 2weeks after destruction of dopamine terminals within medial prefrontal vortex of the rat. This findings suggest that frontal cortical dopamine system may regulate the dopamine system in corpus striatum. KEY WORDSPrefrontal cortex Striatum D 1 and D 2 Receptors 6-hydroxydopamine. 418