Brain Research, 528 (1990) 317-322
317
Elsevier BRES 24289
Selective localization of striatal D 1 receptors to striatonigral neurons Madaline
B . H a r r i s o n 1, R o n a l d
G. Wiley 2 and G. Frederick
Wooten 1
1Department of Neurology, University of Virginia Health Sciences Center, Charlottesville, VA 22908 (U.S.A.) and eDepartments of Neurology and Pharmacology, Vanderbilt University, Nashville, TN37212 (U.S.A.)
(Accepted 5 June 1990) Key words: Volkensin; Suicide transport; Striatonigral projection; Quantitative autoradiography; D 1 receptor
A new technique for producing anatomically selective lesions within the brain was used to investigate the cellular localization of the D l and D2 receptor. The cytotoxic lectin, volkensin, is taken up by nerve terminals and retrogradely transported, killing those neurons projecting to the site of injection. Comparison of D 1 and D 2 binding following a unilateral volkensin injection into the substantia nigra has demonstrated that striatal DI binding sites are selectively localized to striatonigral projection neurons. Loss of the nigrostriatal dopaminergic projection is the primary abnormality in Parkinson's disease. Pharmacologic agents affecting dopaminergic transmission are widely used to treat Parkinson's disease and other conditions. Two subtypes of the dopamine receptor are currently recognized, one positively (D1) and the other negatively (D2) linked to adenylate cyclase 26. Use of agonists and antagonists selective for either the D 1 o r D 2 receptor has demonstrated that differing pharmacologic and physiologic effects are mediated by each receptor class 28. These diverse neuropharmacologic effects may result from selective localization of the D~ and D2 receptor to different populations of striatal neurons. The comparative regional distribution of binding sites for the D 1 and D 2 receptors has been described in several species using quantitative film autoradiography 5'7'23. However, their localization to specific populations of neurons within the striatum has not been fully determined. Available evidence suggests that D 2 receptors are present on cholinergic interneurons 11 and dopaminergic nigrostriatal neurons 14. Their proposed localization to corticostriatal neurons has been more controversial 16' 24,27. The D 1 receptor appears to be located on intrinsic striatal neurons 13 including neurons projecting to the substantia nigra pars reticulata 3'21. In one study, a concomitant decrease was seen in G A B A levels and G A D activity suggesting the presence of D 1 binding in areas of GABAergic terminals of the striatonigral projection 21. In addition, axonal transport of the D~ receptor in the striatonigral projection has been demonstrated 1. To test the hypothesis that the D 1 receptor is selec-
tively expressed on the cell bodies of intrinsic striatonigral neurons, we have used a new technique employing volkensin, a suicide transport agent effective in the central nervous system 29. Volkensin is a cytotoxic plant lectin which is taken up by nerve terminals and retrogradely transported to the cell body where it inhibits protein synthesis resulting in cell death 25. Following microinjection of this compound into the substantia nigra, we examined D 1 and D 2 binding in the striatum. Six adult male Sprague-Dawley rats, 250-300 g, received injections of volkensin in the left substantia nigra. Animals were anesthetized with ketamine, 140200 mg/kg, and acepromazine, 3-5 mg/kg, i.p. and positioned in a Kopf small animal stereotaxic apparatus. Injections of volkensin, 27 ng//A in phosphate-buffered saline (PBS), were made using a micropipette with tip diameter of 25-75 /~m, coupled to a 10-~1 Hamilton syringe. Volumes ranged from 0.075 to 0.4 /A. The coordinates were 5.2 mm posterior to bregma, 2.2 mm to the left of midline, and 8.2 mm ventral to the dural surface 2°. After survival times ranging from 17 to 21 days, animals were anesthetized with CO2, and transcardially perfused with cold Krebs-Henseleit buffer. The whole brain was rapidly removed, frozen in n-hexane chilled in dry ice, and stored at -70 °C prior to study. Twenty-/~m sections were cut on an American Optical cryostat at -18 to -20 °C, thaw-mounted on coverslips subbed with poly-D-lysine and stored over dessicant at -70 °C until used in binding experiments. Adjacent pairs of sections through the striatum and substantia nigra were examined for D 1 and D2 binding.
Correspondence: M.B. Harrison, Department of Neurology - Box 394, University of Virginia, Health Sciences Center, Charlottesville, VA
22908, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
318 Sections were thawed, air-dried and pre-incubated in PBS for two 5-min intervals at 4 °C. For D 1 receptor binding, sections were incubated in 0.5 nM [3H]SCH 23390 (Dupont-NEN; S.A. 60.4 Ci/mmol) at 37 °C for 90 min. For D 2 receptor binding, sections were incubated in 1 nM [3H]spiperone (Dupont-NEN; S.A. 23.9 Ci/mmol) at room temperature for 90 min. One/aM mianserin was added to both incubation solutions to block binding at the 5-HT2 receptor. Non-specific binding was assessed in each case after the addition of 2/aM (+)-butaclamol. Following incubation, sections were rinsed in PBS at 4 °C
twice for 10 s, followed by a 5-s rinse in distilled H20 at 4 °C. They were dried over desiccant overnight and apposed to [3H]Hyperfilm (Amersham) for 14 days ([3H]SCH 23390) or 21 days ([3H]spiperone). Atrophy of the striatum with ventricular enlargement ipsilateral to the volkensin injections was noted in each rat. The autoradiographs were analyzed on a DUMAS image analysis system to quantitate changes in binding. For purposes of analysis, the striatum was divided into quadrants: dorsomedial (DM), dorsolateral (DL), ventromedial (VM), and ventrolateral (VL). The density of
Fig. 1. Effects of injection of volkensin on the substantia nigra. A: Dt binding: autoradiograph of total binding of [3H]SCH 23390 in the substantia nigra following injection of volkensin into the left side. B: Nissl stain: 20-/zmfrozen section through the substantia nigra fixed with formalin vapor and stained with toluidine blue.
319 binding sites was measured for each quadrant both ipsilateral and contralateral to the substantia nigra injection. Measurements were taken from 5 separate
sections through the b o d y of the striatum from each animal. The effects of the volkensin injection on the substantia
Fig. 2. Autoradiographs of total D 1 and D 2 binding in the striatum following an injection of volkensin into the left substantia nigra. A: D1 ([3H]SCH 23390) receptor distribution in the head of the striatum. B: D2 ([3H]spiperone) receptor distribution in the section adjacent to that shown in (A).
320
250.
*
Contralateral Ipsilateral p(.O01
~-6 m E
~oo50-
Percent of specific binding remaining on lesioned side Comparison of D~ and D 2 receptor binding in the striatum ipsilateral to the nigral lesion. Binding on the lesioned side is expressed as a percentage of that in the corresponding region on the contralateral side.
200-
~ ~ 150I Cm O E m~
TABLE I
1, ill DM
VM
Quadrant
D1 D2
DL
DM
VM
DL
VL
21% 71%
41% 80%
49% 101%
65% 101%
VL
Distribution of D1 Binding by Quadrant N-%~IContreloterol Ipsiloterel 250-
*
P(.O01
200-
~.~ w ~ ~E
150-
~
loo50-
l/ Ii i] H/ DM
VM
DL
VL
Distribution of D2 Binding by Quadrant E ~ Contralateral m Ipsilateral
25°T
*
p(.05
200--r
150-
100-
50. 0
1,i,i, MED
LAT
ALL
D}stribution of 01 Binding in Substantia Nigro
Fig. 3. Distribution of changes in D 1 and D 2 binding. A: distribution of changes in D~ binding with [3H]SCH 23390 by quadrant in the striatum. DM, dorsomedial; DL, dorsolateral; VM, ventromedial; VL, ventrolateral. Values given are of mean _+ S.E. shown by the error bar. B: distribution of changes in D E binding with [3H]spiperone by quadrant in the striatum. C: distribution of changes in D~ binding by region in the substantia nigra. MED, medial pars reticulata; LAT, lateral pars reticulata; ALL, entire pars reticulata.
nigra were assessed both by changes in D 1 receptor binding and by confirmation of histologic changes with a Nissl stain (Fig. 1). Binding data from 4 animals was analyzed. The substantia nigra was divided into medial and lateral regions, with measurements taken from 3
separate sections for each region and for the entire structure on the lesioned and the intact side. Specific binding was determined by subtracting nonspecific from total binding. Analysis of variance (ANOVA) was used to compare specific binding in the striatum ipsilateral and contralateral to the substantia nigra lesion for the D 1 and D2 receptor subtypes. Differences among the quadrants were further assessed by analysis of the degrees of freedom for quadrants in the ANOVA. The D t binding data in the substantia nigra were analyzed by a paired t-test. In the substantia nigra, injection of volkensin produced a profound, though incomplete loss of D 1 receptor binding, which decreased from 128.6 + 27.8 fmol/mg (mean + S.E.) to 16.7 + 6.1 (P < 0.05). While the change was more pronounced in the medial region, this was not a statistically significant difference (Fig. 3C). In the striatum, the effects of the nigral lesion on receptor binding were most evident in the medial portion of the rostral striatum. For both D 1 and D 2 binding, the dorsomedial quadrant was the most affected; D 1 binding decreased from 176 + 18 (mean + S.E.) to 36.8 + 10.1 (P < 0.001), while D 2 binding went from 138 + 5 to 98.5 + 7.5 (P < 0.001). In the ventromedial quadrant, D 1 decreased from 173.2 _+ 17.3 to 70.7 + 19.5 (P < 0.001) and D 2 decreased from 126.6 + 1.3 to 109.9 + 9.5 (P < 0.001). In the two lateral quadrants, D 2 binding did not change. In the dorsolateral quadrant, D 1 decreased from 182.6 + 19 to 89.6 + 13.4 (P < 0.001), while in the ventrolateral quadrant, the change was from 188.5 + 19.5 to 122 + 22.1 (P < 0.001) (Fig. 3A,B). The difference in the density of binding sites on the intact and lesioned side was greater for D 1 than D 2 across all quadrants (P < 0.001). Specific comparison of the quadrants indicated that overall, the effect of the lesion was greatest in the DM quadrant (P < 0.001); the VM quadrant was more affected than the lateral quadrants (P = 0.01), while the lateral quadrants were not significantly different. When the specific binding remaining on the side ipsilateral to the lesion is expressed as a percentage of that in the
321 corresponding region of the contralateral striatum, the topographic distribution and relative magnitude of the changes is readily apparent (Table I). These data indicate that the D~ receptor in the striatum is selectively expressed by striatonigral neurons, given the almost complete loss of striatal D~ sites after a discrete lesion of the striatonigral projection. The medial location of the changes is consistent with the known topography of striatonigral projections. Striatonigral projection neurons have been shown to maintain their mediolateral topographic relationship in the substantia nigra 15, while the rostral to caudal origins of D 1 bearing cells in the striatum are reflected in a medial to lateral distribution of D l bearing terminals in the substantia nigra 2. The nigral lesion consistently involved the medial substantia nigra with more residual D 1 binding seen laterally, although the difference was not statistically significant (Fig. 3C). Our results confirm the selective localization of the Dj receptor suggested by the decrease in D~ binding in both the striatum and the substantia nigra after excitotoxic lesions of the striatum 3'~2'~3. There are several possible explanations for the smaller change seen in D 2 binding. This finding may reflect the presence of a small population of post-synaptic D 2 receptors on striatonigral neurons. Alternatively, it may result from non-selective cytotoxic effects of volkensin on another population of striatal neurons bearing the D 2 receptor. Finally, it is possible that this small reduction in D 2 sites is a secondary phenomenon, with changes such as dendritic retraction taking place in pre-synaptic neurons following loss of synaptic contact with the affected neurons 9. It is unlikely that the changes seen in either D~ or D 2 binding result from effects of the lesion on the dopaminergic nigrostriatal projection. The prior experimental evidence on D e receptor binding after loss of dopaminergic innervation shows either no change or an increase
1 Aiso, M., Potter, W.Z. and Saavedra, J.M., Axonal transport of dopamine D~ receptors in the rat brain, Brain Research, 426 (1987) 392-396. 2 Altar, C.A. and Hauser, K., Topography of substantia nigra innervation by D~ receptor-containing striatal neurons, Brain Research, 410 (1987) 1-11. 3 Altar, C.A. and Marien, M.R., Picomolar affinity of IzsI-SCH 23982 for D~ receptors in brain demonstrated with digital subtraction autoradiography, J. Neurosci., 7 (1987) 213-222. 4 Ariano, M.A., Striatal D~ dopamine receptor distribution following chemical lesion of the nigrostriatal pathway, Brain Research, 443 (1988) 204-214. 5 Beckstead, R.M., Wooten, G.E and Trugman, J.M., Distribution of D1 and D2 dopamine receptors in the basal ganglia of the cat determined by quantitative autoradiography, J. Comp. Neurol., 268 (1988) 131-145. 6 Bennett, Jr. J.P. and Wooten, G.F., Dopamine denervation does not alter in vivo 3H-spiperone binding in rat striatum: implications for external imaging of dopamine receptors in Parkinson's
in binding 6'1°'19. Studies looking at the response of the D1 receptor to loss of dopaminergic input have produced variable results, with findings of increased binding 8'22, no change 17, or a slight decrease 4'18. Determination of the specific localization of D1 and D 2 receptor subtypes to particular neuronal populations or pathways is critical to a better understanding of the effects of pharmacologically specific agents. This has important implications for understanding the basic pathophysiology of movement disorders, as well as for understanding the mechanism of action of currently available dopaminergic agents used in the treatment of Parkinson's disease. Finally, differential effects at the D 1 and D 2 receptor are thought to be involved in the side effects of dopamine agonist and antagonist agents such as dopainduced dyskinesias and tardive dyskinesia 3°. Increased understanding of the anatomic substrate underlying these effects can potentially contribute to the development of agents with a better therapeutic profile. The demonstration of the selective localization of D~ striatal receptors on striatonigral neurons is an essential and significant step in the process of understanding the basis for these pharmacologic effects. This was made possible by the development of a neurotoxin, volkensin, with which anatomically selective lesions can be made based on the projection of neurons to distant sites. Although still in the early stages of development, it offers a powerful new approach to the understanding of functional anatomic relationships within the central nervous system.
This work was supported by funds from The National Institutes of Health Grant HD-07325, The Veterans Administration, The Mary Anderson Harrison Endowment of the University of Virginia, The American Parkinson Disease Association, and The United Parkinson Foundation. We appreciate the assistance of Eugene K. Harris, Ph.D. in the statistical analysis and of Rose Powell in manuscript preparation.
disease, Ann. Neurol., 19 (1986) 378-383. 7 Boyson, S.J., McGonigle, P. and Molinoff, P.B., Quantitative autoradiographic localization of the D 1 and D z subtypes of dopamine receptors in rat brain, J. Neurosci., 6 (1986) 31773188. 8 Buonamici, M., Caccia, C., Carpentieri, M., Pegrassi, L., Rossi, A.C. and Di Chiara, G., D-1 receptor supersensitivity in the rat striatum after unilateral 6-hydroxydopamine lesions, Eur. J. Pharmacol., 126 (1986) 347-348. 9 Cowan, W.M., Anterograde and retrograde transneuronal degeneration in the central and peripheral nervous system. In W.J.H. Nauta and S.O.E. Ebbesson (Eds.), Contemporary Research Methods in Neuroanatomy, Springer-Verlag, Berlin, 1970, pp. 215-251. 10 Creese, I. and Snyder, S.H., Nigrostriatal lesions enhance striatal 3H-apomorphine and 3H-spiroperidol binding, Eur. J. Pharmacol., 56 (1979) 277-281. 11 Dawson, V.L., Dawson, T.M., Filloux, F.M. and Wamsley, J.K., Evidence for dopamine D-2 receptors on cholinergic interneu-
322 rons in the rat caudate-putamen, Life Sci., 42 (1988) 1933-1939. 12 Dubois, A., Savasta, M., Curet, O. and Scatton, B., Autoradiographic distribution of the D~ agonist [3H]SKF 38393, in the rat brain and spinal cord. Comparison with the distribution of D 2 dopamine receptors, Neuroscience, 19 (1986) 125-137. 13 Filloux, EM., Wamsley, J.K. and Dawson, T.M., Presynaptic and postsynaptic D 1 dopamine receptors in the nigrostriatal system of the rat brain: a quantitative autoradiographic study using the selective D 1 antagonist [3H]SCH 23390, Brain Research, 408 (1987) 205-209. 14 Filloux, F.M., Wamsley, J.K. and Dawson, T.M., Dopamine D-2 auto- and postsynaptic receptors in the nigrostriatal system of the rat brain: localization by quantitative autoradiography with [3H]sulpiride, Fur. J. PharmacoL, 138 (1987) 61-68. 15 Gerfen, C.R., The neostriatai mosaic, I. Compartmental organization of projections from the striatum to the substantia nigra in the rat, J. Comp. Neurol., 236 (1985) 454--476. 16 Joyce, J.N. and Marshall, J.E, Quantitative autoradiography of dopamine D 2 sites in rat caudate-putamen: localization to intrinsic neurons and not to neocortical afferents, Neuroscience, 20 (1987) 773-795. 17 Langer, S.Z., Pimoule, C., Reynolds, G.P. and Schoemaker, H., Dopaminergic denervation does not affect [3H]-SCH 23390 binding in the rat striatum: similarities to Parkinson's disease, Br. J. Pharmacol., 87 (1986) 161E 18 Marshall, J.E, Navarrete, R. and Joyce, J.N., Decreased striatal D 1 binding density following mesotelencephalic 6-hydroxydopamine injections: an autoradiographic analysis, Brain Research, 493 (1989) 247-257. 19 Neve, K.A., Altar, C.A., Wong, C.A. and Marshall, J.F., Quantitative analysis of [3H]spiroperidoi binding to rat forebrain sections: plasticity of neostriatal dopamine receptors after nigrostriatal injury, Brain Research, 302 (1984) 9-18. 20 Paxinos, B. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, London, 1986. 21 Porceddu, M.L., Giorgi, O., Ongini, E., Mele, S. and Biggio,
22
23
24 25
26 27
28 29 30
G., 3H-SCH 23390 binding sites in the rat substantia nigra: evidence for a presynaptic localization and innervation by dopamine, Life Sci., 39 (1986) 321-328. Porceddu, M.L., Giorgi, O., De Montis, G., Mele, S., Cocco, L., Ongini, E. and Biggio, G., 6-Hydroxydopamine-induced degeneration of nigral dopamine neurons: differential effect on nigral and striatal D-1 dopamine receptors, Life Sci., 41 (1987) 697-706. Richfield, E.K., Young, A.B. and Penney, J.B., Comparative distribution of dopamine D-1 and D-2 receptors in the basal ganglia of turtles, pigeons, rats, cats, and monkeys, J. Comp. NeuroL, 262 (1987) 446--463. Schwarcz, R., Creese, I., Coyle, J.T. and Snyder, S.H., Dopamine receptors localized on cerebral cortical afferents to rat corpus striatum, Nature (Lond.), 271 (1978) 766-768. Stirpe, E, Barbieri, L., Abbondanza, A., Falasca, A.I., Brown, A.N.E, Sanvig, K., Olsnes, S. and Pihl, A., Properties of volkensin, a toxic lectin from Adenia volkensii, J. Biol. Chem., 260 (1985) 14589-14595. Stool, J.C. and Kebabian, J.W., Two dopamine receptors: biochemistry, physiology and pharmacology, Life Sci., 35 (1984) 2281-2296. Trugman, J.M., Geary, II W.A. and Wooten, G.E, Localization of D-2 dopamine receptors to intrinsic striatal neurones by quantitative autoradiography, Nature (Lond.), 323 (1986) 267269. Waddington, J.L. and O'Boyle, K.M., The D-1 dopamine receptor and the search for its functional role: from neurochemistry to behaviour, Rev. Neurosci., 1 (1987) 157-184. Wiley, R.G. and Stirpe, E, Modeccin and volkensin but not abrin are effective suicide transport agents in rat CNS, Brain Research, 438 (1988) 145-154. Wooten, G.F., Progress in understanding the pathophysiology of treatment-related fluctuations in Parkinson's disease, Ann. NeuroL, 24 (1988) 363-365.
317
Elsevier BRES 24289
Selective localization of striatal D 1 receptors to striatonigral neurons Madaline
B . H a r r i s o n 1, R o n a l d
G. Wiley 2 and G. Frederick
Wooten 1
1Department of Neurology, University of Virginia Health Sciences Center, Charlottesville, VA 22908 (U.S.A.) and eDepartments of Neurology and Pharmacology, Vanderbilt University, Nashville, TN37212 (U.S.A.)
(Accepted 5 June 1990) Key words: Volkensin; Suicide transport; Striatonigral projection; Quantitative autoradiography; D 1 receptor
A new technique for producing anatomically selective lesions within the brain was used to investigate the cellular localization of the D l and D2 receptor. The cytotoxic lectin, volkensin, is taken up by nerve terminals and retrogradely transported, killing those neurons projecting to the site of injection. Comparison of D 1 and D 2 binding following a unilateral volkensin injection into the substantia nigra has demonstrated that striatal DI binding sites are selectively localized to striatonigral projection neurons. Loss of the nigrostriatal dopaminergic projection is the primary abnormality in Parkinson's disease. Pharmacologic agents affecting dopaminergic transmission are widely used to treat Parkinson's disease and other conditions. Two subtypes of the dopamine receptor are currently recognized, one positively (D1) and the other negatively (D2) linked to adenylate cyclase 26. Use of agonists and antagonists selective for either the D 1 o r D 2 receptor has demonstrated that differing pharmacologic and physiologic effects are mediated by each receptor class 28. These diverse neuropharmacologic effects may result from selective localization of the D~ and D2 receptor to different populations of striatal neurons. The comparative regional distribution of binding sites for the D 1 and D 2 receptors has been described in several species using quantitative film autoradiography 5'7'23. However, their localization to specific populations of neurons within the striatum has not been fully determined. Available evidence suggests that D 2 receptors are present on cholinergic interneurons 11 and dopaminergic nigrostriatal neurons 14. Their proposed localization to corticostriatal neurons has been more controversial 16' 24,27. The D 1 receptor appears to be located on intrinsic striatal neurons 13 including neurons projecting to the substantia nigra pars reticulata 3'21. In one study, a concomitant decrease was seen in G A B A levels and G A D activity suggesting the presence of D 1 binding in areas of GABAergic terminals of the striatonigral projection 21. In addition, axonal transport of the D~ receptor in the striatonigral projection has been demonstrated 1. To test the hypothesis that the D 1 receptor is selec-
tively expressed on the cell bodies of intrinsic striatonigral neurons, we have used a new technique employing volkensin, a suicide transport agent effective in the central nervous system 29. Volkensin is a cytotoxic plant lectin which is taken up by nerve terminals and retrogradely transported to the cell body where it inhibits protein synthesis resulting in cell death 25. Following microinjection of this compound into the substantia nigra, we examined D 1 and D 2 binding in the striatum. Six adult male Sprague-Dawley rats, 250-300 g, received injections of volkensin in the left substantia nigra. Animals were anesthetized with ketamine, 140200 mg/kg, and acepromazine, 3-5 mg/kg, i.p. and positioned in a Kopf small animal stereotaxic apparatus. Injections of volkensin, 27 ng//A in phosphate-buffered saline (PBS), were made using a micropipette with tip diameter of 25-75 /~m, coupled to a 10-~1 Hamilton syringe. Volumes ranged from 0.075 to 0.4 /A. The coordinates were 5.2 mm posterior to bregma, 2.2 mm to the left of midline, and 8.2 mm ventral to the dural surface 2°. After survival times ranging from 17 to 21 days, animals were anesthetized with CO2, and transcardially perfused with cold Krebs-Henseleit buffer. The whole brain was rapidly removed, frozen in n-hexane chilled in dry ice, and stored at -70 °C prior to study. Twenty-/~m sections were cut on an American Optical cryostat at -18 to -20 °C, thaw-mounted on coverslips subbed with poly-D-lysine and stored over dessicant at -70 °C until used in binding experiments. Adjacent pairs of sections through the striatum and substantia nigra were examined for D 1 and D2 binding.
Correspondence: M.B. Harrison, Department of Neurology - Box 394, University of Virginia, Health Sciences Center, Charlottesville, VA
22908, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
318 Sections were thawed, air-dried and pre-incubated in PBS for two 5-min intervals at 4 °C. For D 1 receptor binding, sections were incubated in 0.5 nM [3H]SCH 23390 (Dupont-NEN; S.A. 60.4 Ci/mmol) at 37 °C for 90 min. For D 2 receptor binding, sections were incubated in 1 nM [3H]spiperone (Dupont-NEN; S.A. 23.9 Ci/mmol) at room temperature for 90 min. One/aM mianserin was added to both incubation solutions to block binding at the 5-HT2 receptor. Non-specific binding was assessed in each case after the addition of 2/aM (+)-butaclamol. Following incubation, sections were rinsed in PBS at 4 °C
twice for 10 s, followed by a 5-s rinse in distilled H20 at 4 °C. They were dried over desiccant overnight and apposed to [3H]Hyperfilm (Amersham) for 14 days ([3H]SCH 23390) or 21 days ([3H]spiperone). Atrophy of the striatum with ventricular enlargement ipsilateral to the volkensin injections was noted in each rat. The autoradiographs were analyzed on a DUMAS image analysis system to quantitate changes in binding. For purposes of analysis, the striatum was divided into quadrants: dorsomedial (DM), dorsolateral (DL), ventromedial (VM), and ventrolateral (VL). The density of
Fig. 1. Effects of injection of volkensin on the substantia nigra. A: Dt binding: autoradiograph of total binding of [3H]SCH 23390 in the substantia nigra following injection of volkensin into the left side. B: Nissl stain: 20-/zmfrozen section through the substantia nigra fixed with formalin vapor and stained with toluidine blue.
319 binding sites was measured for each quadrant both ipsilateral and contralateral to the substantia nigra injection. Measurements were taken from 5 separate
sections through the b o d y of the striatum from each animal. The effects of the volkensin injection on the substantia
Fig. 2. Autoradiographs of total D 1 and D 2 binding in the striatum following an injection of volkensin into the left substantia nigra. A: D1 ([3H]SCH 23390) receptor distribution in the head of the striatum. B: D2 ([3H]spiperone) receptor distribution in the section adjacent to that shown in (A).
320
250.
*
Contralateral Ipsilateral p(.O01
~-6 m E
~oo50-
Percent of specific binding remaining on lesioned side Comparison of D~ and D 2 receptor binding in the striatum ipsilateral to the nigral lesion. Binding on the lesioned side is expressed as a percentage of that in the corresponding region on the contralateral side.
200-
~ ~ 150I Cm O E m~
TABLE I
1, ill DM
VM
Quadrant
D1 D2
DL
DM
VM
DL
VL
21% 71%
41% 80%
49% 101%
65% 101%
VL
Distribution of D1 Binding by Quadrant N-%~IContreloterol Ipsiloterel 250-
*
P(.O01
200-
~.~ w ~ ~E
150-
~
loo50-
l/ Ii i] H/ DM
VM
DL
VL
Distribution of D2 Binding by Quadrant E ~ Contralateral m Ipsilateral
25°T
*
p(.05
200--r
150-
100-
50. 0
1,i,i, MED
LAT
ALL
D}stribution of 01 Binding in Substantia Nigro
Fig. 3. Distribution of changes in D 1 and D 2 binding. A: distribution of changes in D~ binding with [3H]SCH 23390 by quadrant in the striatum. DM, dorsomedial; DL, dorsolateral; VM, ventromedial; VL, ventrolateral. Values given are of mean _+ S.E. shown by the error bar. B: distribution of changes in D E binding with [3H]spiperone by quadrant in the striatum. C: distribution of changes in D~ binding by region in the substantia nigra. MED, medial pars reticulata; LAT, lateral pars reticulata; ALL, entire pars reticulata.
nigra were assessed both by changes in D 1 receptor binding and by confirmation of histologic changes with a Nissl stain (Fig. 1). Binding data from 4 animals was analyzed. The substantia nigra was divided into medial and lateral regions, with measurements taken from 3
separate sections for each region and for the entire structure on the lesioned and the intact side. Specific binding was determined by subtracting nonspecific from total binding. Analysis of variance (ANOVA) was used to compare specific binding in the striatum ipsilateral and contralateral to the substantia nigra lesion for the D 1 and D2 receptor subtypes. Differences among the quadrants were further assessed by analysis of the degrees of freedom for quadrants in the ANOVA. The D t binding data in the substantia nigra were analyzed by a paired t-test. In the substantia nigra, injection of volkensin produced a profound, though incomplete loss of D 1 receptor binding, which decreased from 128.6 + 27.8 fmol/mg (mean + S.E.) to 16.7 + 6.1 (P < 0.05). While the change was more pronounced in the medial region, this was not a statistically significant difference (Fig. 3C). In the striatum, the effects of the nigral lesion on receptor binding were most evident in the medial portion of the rostral striatum. For both D 1 and D 2 binding, the dorsomedial quadrant was the most affected; D 1 binding decreased from 176 + 18 (mean + S.E.) to 36.8 + 10.1 (P < 0.001), while D 2 binding went from 138 + 5 to 98.5 + 7.5 (P < 0.001). In the ventromedial quadrant, D 1 decreased from 173.2 _+ 17.3 to 70.7 + 19.5 (P < 0.001) and D 2 decreased from 126.6 + 1.3 to 109.9 + 9.5 (P < 0.001). In the two lateral quadrants, D 2 binding did not change. In the dorsolateral quadrant, D 1 decreased from 182.6 + 19 to 89.6 + 13.4 (P < 0.001), while in the ventrolateral quadrant, the change was from 188.5 + 19.5 to 122 + 22.1 (P < 0.001) (Fig. 3A,B). The difference in the density of binding sites on the intact and lesioned side was greater for D 1 than D 2 across all quadrants (P < 0.001). Specific comparison of the quadrants indicated that overall, the effect of the lesion was greatest in the DM quadrant (P < 0.001); the VM quadrant was more affected than the lateral quadrants (P = 0.01), while the lateral quadrants were not significantly different. When the specific binding remaining on the side ipsilateral to the lesion is expressed as a percentage of that in the
321 corresponding region of the contralateral striatum, the topographic distribution and relative magnitude of the changes is readily apparent (Table I). These data indicate that the D~ receptor in the striatum is selectively expressed by striatonigral neurons, given the almost complete loss of striatal D~ sites after a discrete lesion of the striatonigral projection. The medial location of the changes is consistent with the known topography of striatonigral projections. Striatonigral projection neurons have been shown to maintain their mediolateral topographic relationship in the substantia nigra 15, while the rostral to caudal origins of D 1 bearing cells in the striatum are reflected in a medial to lateral distribution of D l bearing terminals in the substantia nigra 2. The nigral lesion consistently involved the medial substantia nigra with more residual D 1 binding seen laterally, although the difference was not statistically significant (Fig. 3C). Our results confirm the selective localization of the Dj receptor suggested by the decrease in D~ binding in both the striatum and the substantia nigra after excitotoxic lesions of the striatum 3'~2'~3. There are several possible explanations for the smaller change seen in D 2 binding. This finding may reflect the presence of a small population of post-synaptic D 2 receptors on striatonigral neurons. Alternatively, it may result from non-selective cytotoxic effects of volkensin on another population of striatal neurons bearing the D 2 receptor. Finally, it is possible that this small reduction in D 2 sites is a secondary phenomenon, with changes such as dendritic retraction taking place in pre-synaptic neurons following loss of synaptic contact with the affected neurons 9. It is unlikely that the changes seen in either D~ or D 2 binding result from effects of the lesion on the dopaminergic nigrostriatal projection. The prior experimental evidence on D e receptor binding after loss of dopaminergic innervation shows either no change or an increase
1 Aiso, M., Potter, W.Z. and Saavedra, J.M., Axonal transport of dopamine D~ receptors in the rat brain, Brain Research, 426 (1987) 392-396. 2 Altar, C.A. and Hauser, K., Topography of substantia nigra innervation by D~ receptor-containing striatal neurons, Brain Research, 410 (1987) 1-11. 3 Altar, C.A. and Marien, M.R., Picomolar affinity of IzsI-SCH 23982 for D~ receptors in brain demonstrated with digital subtraction autoradiography, J. Neurosci., 7 (1987) 213-222. 4 Ariano, M.A., Striatal D~ dopamine receptor distribution following chemical lesion of the nigrostriatal pathway, Brain Research, 443 (1988) 204-214. 5 Beckstead, R.M., Wooten, G.E and Trugman, J.M., Distribution of D1 and D2 dopamine receptors in the basal ganglia of the cat determined by quantitative autoradiography, J. Comp. Neurol., 268 (1988) 131-145. 6 Bennett, Jr. J.P. and Wooten, G.F., Dopamine denervation does not alter in vivo 3H-spiperone binding in rat striatum: implications for external imaging of dopamine receptors in Parkinson's
in binding 6'1°'19. Studies looking at the response of the D1 receptor to loss of dopaminergic input have produced variable results, with findings of increased binding 8'22, no change 17, or a slight decrease 4'18. Determination of the specific localization of D1 and D 2 receptor subtypes to particular neuronal populations or pathways is critical to a better understanding of the effects of pharmacologically specific agents. This has important implications for understanding the basic pathophysiology of movement disorders, as well as for understanding the mechanism of action of currently available dopaminergic agents used in the treatment of Parkinson's disease. Finally, differential effects at the D 1 and D 2 receptor are thought to be involved in the side effects of dopamine agonist and antagonist agents such as dopainduced dyskinesias and tardive dyskinesia 3°. Increased understanding of the anatomic substrate underlying these effects can potentially contribute to the development of agents with a better therapeutic profile. The demonstration of the selective localization of D~ striatal receptors on striatonigral neurons is an essential and significant step in the process of understanding the basis for these pharmacologic effects. This was made possible by the development of a neurotoxin, volkensin, with which anatomically selective lesions can be made based on the projection of neurons to distant sites. Although still in the early stages of development, it offers a powerful new approach to the understanding of functional anatomic relationships within the central nervous system.
This work was supported by funds from The National Institutes of Health Grant HD-07325, The Veterans Administration, The Mary Anderson Harrison Endowment of the University of Virginia, The American Parkinson Disease Association, and The United Parkinson Foundation. We appreciate the assistance of Eugene K. Harris, Ph.D. in the statistical analysis and of Rose Powell in manuscript preparation.
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