Long-term changes in striatal D1 dopamine receptor distribution after dopaminergic deafferentation

hkwroscience Vol. 32, No. I, pp. 203-212, Printed in Great Britain

0306-4522/89 $3.00 + 0.00 Pergamon Press plc 0 1989 IBRO

1989

LONG-TERM CHANGES IN STRIATAL D, DOPAMINE RECEPTOR DISTRIBUTION AFTER DOPAMINERGIC DEAFFERENTATION M. A. ARIANO Department of Anatomy and Neurobiology, University of Vermont, College of Medicine, Burlington, VT 05405, U.S.A. Abstract-The morphochemical disposition of the adenylate cyclase-linked dopamine receptor (D, type) in the rat striatum has been assessed at various time points after a neurotoxic lesion of the dopaminergic afferent pathway to the caudate nucleus. D, receptor binding sites in the caudate nucleus were determined by in vitro autoradiography of the substituted benzazepine D, antagonists, [3H]SCH 23390 or [‘*‘I]SCH 23982, and contrasted to the pattern of striatal immunohistochemical reactivity of the second messenger

compound, cyclic 3’,5’-adenosine monophosphate. The results demonstrate that the specific association of this dopamine receptor type with cyclic 3’,5’-adenosine monophosphate-stained neurons is abolished at 7 days following chemical interruption of the nigrostriatal pathway, and the receptor disruption is persistent for durations as long as 20 weeks. This investigation suggests that once the postsynaptic receptor pathology is produced by deafferentation, it does not recover the selective morphochemical relationship normally established with the target cell containing the second messenger. This is in contrast to modest biochemical recuperation in D, dopamine receptor binding seen using this experimental paradigm. This change in D, dopamine receptor morphochemistry is discussed in relation to the neurochemical deficits produced by dopaminergic denervation and in Parkinson’s disease.

Two subpopulations of dopamine receptors exist that use different postsynaptic transduction mechanisms in the central nervous system of the rat.20.38 The D, dopamine receptor subtype is positively linked to the activation of the enzyme adenylate cyclase (EC 4.6.1.1), while the D2 receptor type is coupled to a variety of signal mechanisms,15*22 and inhibits cyclic 3’,5’-adenosine monophosphate (cyclic AMP) production in the caudate nucleus.” The regional distribution of the dopaminergic receptors can be morphologically distinguished through application of in vitro autoradiography of specific radiolabeled antagonist ligands. Using this experimental approach, numerous studies have demonstrated that the caudate nucleus has rich concentrations of both dopaminergic receptor subtypes.‘*6*‘3 Additionally, the cellular distribution of the D, dopamine receptor subclass can be related to postsynaptic immunohistochemical localization of the second messenger, cyclic AMP in the rat caudate nucleus.* In this experimental paradigm, a specific association of the D, dopamine receptor binding sites has been shown to exist with cyclic AMPimmunoreactive striatal neuronal cell bodies. A Abbreviations: cpm, counts per minute; cyclic AMP, cyclic 3’,5’-adenosine monophosphate; 6-OHDA, bhydroxydopamine;

SCH

23390,

(R)-( + )-8-chloro-2.3.4.5-

tetrahydro-3-methyl-5-phenyi- l’H~-3:benzazepin-7-b1; SCH 23982, (5R)-8-iodo-2,3,4,5-tetrahydro-3-methyl-5phenyl-lH-3-benzazepin-7-01; SKF 38393, 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-lH-3-benzazepines.

proportion of the cyclic AMP-stained neurons of the caudate nucleus comprise the striatonigral projection neurons,’ and are classed cytoarchitecturally as medium spiny neurons. The medium spiny neuron constitutes the vast majority .of the projection cells of the caudate nucleus, and it has been reported that the medium spiny striatonigral neurons receive monosynaptic dopamine input from the substantia nigra.2’*33v40 This neuron group exhibits [‘Hldopamine binding sites,4 and expresses dopamine-sensitive adenylate cyclase activity in both the striatum’6+‘8 and the substantia nigra.32,‘6 The selective, clustered association of striatal D, binding sites and cyclic AMP-containing neurons, referred to as a morphochemical relationship, can be altered following dopaminergic deafferentation of the caudate nucleus after 6-hydroxydopamine (6-OHDA) infusion into the substantia nigra.* This treatment produces degeneration of the nigrostriatal pathway and virtual loss of dopamine terminals in the ipsilateral striatum. Using this same experimental paradigm, investigators have reported numerous alterations in behavior’7*‘g.27*28 and biochemistry7*3g in the unilateral, 6-OHDAlesioned rat. The present work has examined the long-term consequence of D, dopamine receptor morphochemical changes associated with this deafferentation paradigm, l-20 weeks after unilateral neurotoxin infusion into the substantia nigra. This animal model mirrors the primary neurochemical deficit reported to underlie Parkinson’s disease, and

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demonstrates many of the observations associated with permanent receptor changes seen post mortem in this neurological disorder.‘2.34.3s EXPERIMENTAL

poration (Bloomfield, NJ). SKF 38393 was obtained from Smith. Kline and French (Philadelphia, PA). 6-OHDA was purchased from Sigma Chemical Co. (St Louis, MO). A11 other chemicals used were of reagent grade and obtained from standard suppliers.

PROCEDURES

Male Sprague-Dawley rats (Charles River, Canada, 200250g) were used as a source of experimental tissue. The animals were maintained in an AAALAC accredited facility, in strict accordance with the USPHS Guide for the Care and Use of Laboratory Animals, and all procedures have been approved by the University Institutional Animal Care and use Committee. Rats were anesthetized using chloral hydrate (3.3 ml/kg, i.p.), placed into a stereotaxic apparatus (D. I. Kopf, Tujunga, CA), and a unilateral infusion of 6-OHDA (16 pg/ml in phosphate-buffered saline-ascorbate, pH 7.4) was given at co-ordinates of AP - 3, L + 2.5, and V - 8.5,” as discussed previously.2 The animals were allowed to survive from 7 days to 20 weeks prior to the morphochemical analysis of striatal D, dopamine receptor binding sites versus cyclic AMP characterized neurons. Depletion of dopamine from the striatum was determined using catecholamine tissue histofluorescence3*‘4 and further substantiated by high-performance liquid chromatography electrochemistry.-“’ Fresh frozen tissue sections were obtained at 8 pm using a cryostat, rostra1 to the decussation of the anterior commissure (AP + 2.2 mm; see Ref. 3 1). These sections were processed for routine cyclic AMP immunohistochemical distribution using the peroxidase-antiperoxidase method” with a well-characterized, polyclonal antisera.4,i Subsequently, the tissue sections were incubated for D, dopamine receptor binding sites using radiolabeled selective antagonists. Autoradiographic localization of D, dopamine receptor binding sites was determined using a modification of an established in vitro assay.4,4’ The tissue sections were exposed typically for 3 weeks (in the tritiated ligand experiments) or 15-18 h (following incubation with the iodinated ligand) at 4”C, developed photographically, and finally, the sections were examindd &ng bright-field microscopy, and nhotoarauhed with Panatomic-X film (ASA 32). D, doiamine receptor binding activiiy was confirmed in caudate homogenates generated from an identically prepared series of animals to the morphochemical studies. Homogenates of caudate nuclei were incubated with varying concentrations of [‘H]SCH 23390 to determine total D, binding. Inclusion of 10pM SKF 38393 (a specific D, receptor agonist), or SCH 23390 were used to establish non-specific D, binding sites as previously reported.* Assays were performed in duplicate and normalized by protein content.23 Total and non-specific receptor binding was plotted as the concentration of radioligand used in the incubation versus counts per minute (cpm) bound in the homogenate, using the program ASYSTANT (Macmillan Software, New York). In each series of biochemical or morphochemical experiments, total binding employed 2-4 nM of the radiolabeled antagonist ligand, while non-specific binding was determined with inclusion of 10 PM of cold competitive ligand in the incubation medium. Specific binding of the radiolabeled ligands was determined by the visual subtraction of non-specific from total binding in the morphochemical paradigm, or as the difference in cpm per milligram of protein in biochemical studies. Materials

[‘H]SCH 23390 (spec. act. 73 Ci/mmol) was purchased from Amersham, Inc. (Arlington Heights, IL), and [‘251]SCH23982 (spec. act. 220 Ci/mmol) was obtained from New England Nuclear (Boston, MA). NTB-2 emulsion, Panatomic-X black and white film, and D-19 developer were purchased from Eastman Kodak (Rochester, NY). SCH 23390 was a generous gift from Schering-Plough Cor-

RESULTS Unilateral infusion of 6-OHDA into the substantia nigra produced a near complete loss of dopamine in the ipsilateral caudate nucleus, as detected using high-performance liquid chromatography electrochemistrygo (data not shown) or catecholamine tissue histofluorescenceJ4 (Fig. 1). Only animals with 95% or greater depletion of dopamine were employed in these studies. Depletion of dopamine in the caudate nucleus was apparent at the earliest time point investigated 3 days after neurotoxin infusion into the substantia nigra. The biochemical determination of specific D, dopamine receptor binding sites in striatal homogenates demonstrated an initial decrease in the deafferented side 7 days following infusion of the neurotoxin,* with a gradual recovery in binding to control values over the time course of the investigation. At the longest time point examined, 20 weeks post-lesion, specific D, dopamine receptor binding showed a modest elevation above the level on the intact side of the nigrostriatal system (Fig. 2). Cyclic AMP immunoreactivity in the caudate nucleus was unchanged by dopaminergic deafferentation (Figs 3A or 4A vs 3C) as we have reported previously. *s4A population of positively stained cells was readily visible, randomly distributed throughout the neuropil of the caudate nucleus. Somata diameters were approximately 15 pm in the largest dimension. Previous investigations have demonstrated that this cell population projects to the substantia nigra and can be classified as medium spiny by cytoarchitectural characteristics.2,4 The autoradiographic distribution of D, dopamine binding sites was significantly changed following deafferentation of the caudate nucleus. Intact striatal D, dopamine receptor disposition was closely associated with striatal cyclic AMP-reactive neurons (Fig. 3B), while the caudate nucleus ipsilateral to the neurotoxin infusion had lost the clustered D, dopamine receptor localization pattern (Fig. 3D). This disruption of the morphochemistry was visible as early as 3 days following interruption of the nigrostriatal dopaminergic afferents and persisted through the longest time point examined in this investigation of 20 weeks. The pattern of the autoradiogram generated from D, dopamine receptor binding in the deafferented caudate nucleus was similar in appearance to that seen in non-specific D, dopamine receptor binding experiments (Fig. 4), in that the specific morphochemical association with cyclic AMP immunohistochemically characterized neurons could not be distinguished. Specific binding

Long-term changes in dopamine receptor distribution

205

Fig. 1. Dopamine histofluorescence in the caudate nucleus of the rat, determined following catecholamine condensation using glyoxylic acid. Punctate fluorescent terminals are readily apparent as white spots in the caudate neuropil of the control side, while placement of 6-OHDA into the substantia nigra produces loss of dopamine varicosities on the ipsilateral, lesioned side. Data presented are from an animal 18 weeks following neurotoxic lesion placement. Calibration bars = 50 pm. Lesioned striotum

Control strlatum

x IO”

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123 -

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_c-----w-s__.._________

0800

2.40

400

5~)

Nanomolar [‘H]SCH 23390

7~

OBOO

240

400

5.60

720

Nanomolar [~H]SCH 23390

Fig. 2. Assessment of D, dopamine receptor binding in homogenates of the caudate, 20 weeks following a unilateral infusion of 6-OHDA in the substantia nigra. A modest increase in binding activity occurred in the lesioned side versus the control side in a representative experiment. The graphs represent duplicate determinations at the noted ligand concentrations following 1 h incubation at 22°C. The data from the control and lesioned caudate nuclei have been normalized by protein content, and plotted using Asysranr software (Macmillan Software, New York). Specific binding is demonstrated by the broken line in each plot, following subtraction of the non-specific binding from the total binding of the ligand in the homogenates.

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receptor

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Fig. 4. A. Cyclic AMP immunohistoehemically reactive elements in 8-brn-thick fresh frozen section made from the control striatum. B. Autoradiogram of non-specific tritiated SCH 23390 binding on the emulsioncoated coverslip, following inclusion of 10 PM SKF 38393 in the incubation. The panel is complementary to the tissue section in A. Arrows and asterisks are for orientation only. Calibration bar = SOpm.

for the D, dopamine receptor, determined by the visual subtraction of the pattern and distribution of exposed autoradiographic grains in Fig. 4B from either Fig. 3B or D, would demonstrate a marked difference in binding site association with the second messenger-containing neurons for the intact versus the denervated striatum. The D, dopamine binding site distribution, assessed using the iodinated radiolabeled antagonist ligand SCH 23982, demonstrated the same disruption in receptor localization after dopaminergic deafferentation of the caudate nucleus (Fig. 5). The use of the iodinated probe produced an autoradiogram with more dispersion in the pattern of exposed grains in the nuclear track emulsion coating the coverslip because of the higher emission energy.’ However, the loss of specific clustering of the D,

dopamine binding sites with the cyclic AMP neurons is still evident (Fig. SD) on the side ipsilateral to the neurotoxin infusion. Use of an iodinated probe controls for the potential problem of quench of the autoradiographic signal by the myelinated fiber bundles’,4’ that perforate the substance of the rat caudate nucleus. Non-specific localization of D, dopamine receptor binding using the iodinated ligand with the inclusion of 10 pm SCH 23390 produces an exposed autoradiogram that is analogous to that displaced in the [3H]SCH 23390 and SKF 38393 incubation (Fig. 6), and also similar in exposed silver grain pattern distribution to that seen in the deafferented caudate nucleus. Cyclic AMP immunohistochemical staining patterns were unchanged in the denervated caudate nucleus, when compared to the control side.

Fig. 3. Morphochemical determination of striatal D, dopamine receptor binding 16 weeks after a unilateral infusion of 6-OHDA into the substantia nigra. A. Cyclic AMP immunohistochemically reactive elements in an 8-pm fresh frozen tissue section from the control side. B. Autoradiographic exposure of total binding of [‘H]SCH 23390, at 4 nM in the NTB-2 emulsion-coated coverslip atop the section in panel A. Exposure time was for 4 weeks at 4°C. C. Cyclic AMP immunohistochemically reactive elements in the caudate nucleus ipsilateral to the dopamine lesion. D. Autoradiogram showing total [3H]SCH 23390 binding on the emulsion-coated coverslip complementary to the section shown in C. Parameters of exposure are identical to those in panel B. Arrows and asterisks are for orientation in the paired photomicrographs. Calibration bar = 50 pm.

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Fig. 6. A. Cyclic AMP immunohistochemically reactive elements in S-pm-thick fresh frozen section made from the control striatum. B. Autoradiogram of non-specific iodinated SCH 23982 binding on the emulsion-coated coverslip, following inclusion of 10nM SCH 23390 in the incubation. The panel is complementary to the tissue section in A. Arrows and asterisks are for orientation only. Calibration bar = 50 pm.

DISCUSSION

The present findings confirm and extend our previous work demonstrating a selective, clustered cellular association pattern of D, dopamine receptor binding sites with striatal cyclic AMP reactive neurons.* The current investigation has determined that this morphochemical organization of the D, dopamine receptor with the cyclic AMP, second messenger-containing striatal neurons does not reappear in the rat caudate nucleus following long-term dopamine deafferentation in this structure. These

results suggest that once the ordered distribution of striatal D, dopamine receptor binding sites with cyclic AMP-immunoreactive target neurons is altered after loss of the nigrostriatal presynaptic terminals, the postsynaptic morphochemical association pattern is not re-established. This finding is in contrast to the modest biochemical recovery in receptor binding that can be measured using the same experimental paradigm. A number of observations support a varied and highly complicated interaction between the two different receptor subtypes following perturbation of the

Fig. 5. Morphochemical determination of striatal D, dopamine receptor binding 20 weeks after a unilateral infusion of 6-OHDA into the substantia nigra. A. Cyclic AMP immunohistochemically reactive elements in an S-pm fresh frozen tissue section from the control side. A shadowing of the autoradiogram appears in the photomicrograph due to the slight separation of the focal planes of the tissue section and the coverslip. B. Autoradiographic exposure of total binding of [‘251]SCH23982 in the emulsion-coated coverslip atop the section in panel A. Exposure time was for 17 h at 4°C. C. Cyclic AMP immunohistochemically reactive elements in the caudate nucleus ipsilateral to the dopamine lesion. D. Autoradiogram showing total [‘*‘I]SCH 23982 binding on the emulsion-coated coverslip complementary to the section shown in C. Parameters of exposure are identical to those in panel B. Arrows and asterisks are for orientation in the paired photomicrographs. Calibration bar = 50 pm.

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dopaminergic afferent system. A simple additive or inhibitory relationship linking the two dopamine receptor functions together is unlikely to exist within the intact striatum. Communication between the dopamine receptor subtypes could be severely (un)masked after the abrupt, massive neurochemical insult produced by loss of the neurotransmitter in the caudate nucleus due to nigral 6-OHDA infusion. Moreover, methodological differences in experimental design of various studies have further reduced the clarity of the findings using the unilateral 6-OHDA animal model. However, the ability to reproduce the principal neurochemical deficit of Parkinson’s disease has provided valuable insights into the potential receptor pathologies associated with the human neurological disorder, and into general aspects of dopamine receptor function. Another avenue of research has elucidated differential patterns of glucose utilization following activation of the two receptor subtypes in the unilaterally lesioned, 6-OHDA animal. Striatal D, dopamine receptor stimulation augments the energy responsiveness to projection sites ipsilateral to the substantia nigra lesion, while D, dopamine receptor activation does not demonstrate this change in glucose metabolism in the basal ganglia.” The increased glycolysis in the substantia nigra and entopeduncular nucleus associated with D, dopamine receptor stimulation replicates the experimental elevations seen after the application of L-DOPA or apomorphine. It is well known that changes in the rate of glycolysis measured by alteration in central nervous system 2-deoxyglucose uptake, can be partly explained by changes in the function of the sodium pump associated with nerve terminals.24 This finding has further relevance within the nigrostriatal system, where it has been substantiated that the postsynaptic response produced by D, dopamine receptor occupancy reduces a tetrodotoxin-sensitive, voltage-dependent inward current.” The electrophysiological alterations measured following dopamine application in naive versus catecholamine depleted animals has clearly shown that voltage-dependent processes of striatal neurons are differentially influenced by dopamine receptor subtype specific agents.*x9 The absence of normal dopamine tonus within the dopamine depleted caudate nucleus, together with an increased sensitivity of the dopamine receptors, alters the physiology of independent D, and D2 receptor physiology. These data argue for a complex functional linkage of the two dopamine receptor populations which can be more readily studied following unilateral dopamine depletion. A potential explanation for these findings based on the morphochemical alterations reported here, is that the redistribution of D, dopamine receptors within the denervated striatum makes these binding sites more available to agonists, and consequently changes their physiological profile. However, the lack of discrete anatomical correspondence on the postsynaptic target

cells would severely attenuate the meaningful translation of the neurotransmitter signal within the nigrostriatal system. Biochemical characterization of striatal D, dopamine receptor function after loss of the dopaminergic input has also yielded conflicting information. Early work described a profound supersensitivity in dopamine sensitive adenylate cyclase activity,26 while current investigations have extended these findings to show that D, dopamine receptor affinity does not shift, but receptor number gradually recovers and increases above the control values.’ This latter effect is dependent on the extent of the interruption in the dopaminergic nigrostriatal pathway.29,30The results described in the current work suggest that the discrete morphochemical pattern of D, distribution in the intact caudate nucleus provides a monitored cellular message such that receptor numbers are maintained at a constant value. When the dopamine pre/postsynaptic complex is abolished after infusion of 6-OHDA into the substantia nigra, the physical change in receptor distribution could disrupt the routine feedback mechanism to the cellular synthetic machinery and more D, dopamine receptor molecules are produced. Behavioral investigations are also far from lucid in determining dopamine receptor sensitivities after dopaminergic deafferentation of the striatum. Selective inhibition at one dopamine receptor binding site appears to significantly influence functioning of the other receptor population.‘9~27~39An intriguing priming phenomenon has also been described in which single D, dopamine receptor agonist exposure alters the rotational behavior in the unilaterally dopamine denervated rat.28 The timing of the priming effect is exquisitely sensitive to the lesion interval and drug dosage employed. These findings strongly suggest that investigations that determine the extent of striatal dopamine deafferentation by apomorphine injection and consequent rotational competency will profoundly and significantly change the properties of the D, type of dopamine receptor. The data presented here have employed drug-naive rats, and therefore should not be compared to studies in which dopamine receptors have been challenged with agonists to determine the extent of the neurotransmitter depletion. Additionally, it has been reported that a significant morphological retraction occurs in the dendritic tree of the medium spiny neuron in the putamen in advanced Parkinson’s disease.25 The truncation in the receptor target area for the nigrostriatal afferents may provide an explanation for the reduced efficacy of I.-DOPA therapy seen as the course of the disease proceeds. The data obtained in the present study provides that the deterioration of L-DOPA therapy in Parkinsonism may be further exacerbated by the alteration in the postsynaptic morphochemical receptor pattern necessary for appropriate translation of the neurotransmitter signal.

Long-term changes in dopamine receptor distribution CONCLUSION

The commonality in all of these observations is that D, dopamine receptor characteristics cannot be isolated and studied separately from D, dopamine receptor activity. The application of 6-OHDA to deafferent the nigrostriatal system seems to alter the interactions between the two dopamine receptor populations within the basal ganglia. The results of this investigation show that this experimental paradigm produces a tremendous and long-lasting change in D, dopamine receptor morphochemistry. Also,

D, dopamine

receptors outnumber

the D, type of

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receptor by ratios of at least 5: 1 in the various regions of the basal ganglia. While the morphochemical relationships of the D, dopamine receptor can be more easily detected, future studies need to examine the cellular D, dopamine receptor distribution in this animal model. Acknowledgernenrs-I would like to thank Dr Michael Zigmond for determining residual striatal dopamine content

using electrochemical-HPLC detection after the 6-OHDA infusion. The helpful assistance of MS Jennifer McNichol in the preparation of the illustrations is also noted. This work was supported by USPHS grants NS 23079 and RCDA NS 00864--

REFERENCES 1. Altar C. A. and Marien M. R. (1987) Picamolar affinity of iz51-SCH 23982 for D-l receptors in brain demonstrated

with digital subtraction autoradiography. J. Neurosci. 7, 213-222. 2. Ariano M. A. (1988) Striatal D, dopamine receptor distribution following chemical lesion of the nigrostriatal pathway. Brain Res. 443, 204-214.

3. Ariano M. A., Butcher L. L. and Appleman M. M. (1980) Cyclic nucleotides in rat caudate-putamen complex: hi&chemical characterization and effects of deafferentation and kainic acid infusion. Neuroscience 5, 126%1276. 4. Ariano M. A. and Kenny S. L. (1985) Neurotransmitter receptor autoradiography in immunohistochemically identified neurons. J. Neurosci. Meth. 15, 4961. 5. Ariano M. A. and Utkes S. K. (1983) Cyclic nucleotide distribution within rat striatonigral neurons. Neuroscience 9, 23-29. 6. Boyson S. J., McGonigle P. and Molinoff P. (1986) Quantitative autoradiographic localization of the D-l and D-2 subtypes of dopamine receptors in rat brain. J. Neurosci. 6, 3177-3188. 7. Buonamici M., Caccia C., Carpentieri M., Pegrassi L., Ross A. C. and DiChiara G. (1986) D-l receptor supersensitivity in the rat striatum after unilateral 6hydroxydopamine lesions. Eur. J. Pharmuc. 126, 347-348. 8. Calabresi P., Benedetti M., Mercuri N. B. and Bemardi G. (1988) Depletion of catecholamines reveals inhibitory effects of bromocryptine and lysuride on neostriatal neurons recorded intracellularly in vitro. Neuropharmacology 27,579-588. 9. Calabresi P., Benedetti M., Mercuri N. B. and Bemardi G. (1988) Endogenous dopamine and dopaminergic agonists modulate synaptic excitation in neostriatum: intracellular studies from naive and catecholamine-depleted rats. Neuroscience 27, 145-157. 10. Calabresi P., Mercuri N., Stanizone P., Stefani A. and Bemardi G. (1987) Intracellular studies on the dopamine-induced firing inhibition of neostriatal neurons in vitro: evidence for D, receptor involvement. Neuroscience 20, 757-771. 11. Cooper D. M. F., Bier-Laning C. M., Halford M. K., Ahlijanian M. K. and Zahnister N. R. (1986) Dopamine, acting through D-2 receptors, inhibits rat striatal adenylate cyclase by a GTP-dependent process. Molec. Pharmac. 29, 113-119. 12. Crossman A. R. (1987) Primate models of dyskinesia: the experimental approach to the study of basal ganglia-related involuntary movement disorders. Neuroscience 21, l-40. 13. Dawson T. M., Barone P., Sidhu A., Wamsley J. K. and Chase T. N. (1986) Quantitative autoradiographic localization of D-l donamine recenters in the rat brain: use of the iodinated liaand ‘251-SCH 23982. Neurosci. L&r. 68. 261-266. using the SPG method for tissue monoamines. 14. De la Torie J. C. (1980) An improved approach to histofluoresce& J. Neurosci. Meth. 3, l-5. 15. Enjalbert A., Sladeczed F., Guillon G., Bertrand P., Shu C., Epelbaum J., Garcia-Sainz A., Jard S., Lombard C., Kordon C. and Bockaert J. (1986) Angiotensin II and dopamine modulate both CAMP and inositol phosphate productions in anterior pituitary cells. J. Biol. Chem. 261, 4071-4075. 16. Filloux F. M., Wamsley J. K. and Dawson T. M. (1987) Presynaptic and postsynaptic D, dopamine receptors in the nigrostriatal system of the rat brain: a quantitative autoradiographic study using the selective D, antagonist ‘H-SCH 23390. Brain Res. 408, 205-209. 17. Gandolfi O., Roncada P., Dall’Olio R. and Montanaro N. (1988) Behavioral and biochemical expression of D,-receptor supersensitivity following SCH 23390 repeated administrations. Brain Res. 455, 390-393. 18. Gehlert D. R., Dawson T. M., Filloux F. M., Sasnna E., Hanbauer I. and Wamsley J. K. (1987) Evidence that ‘H-forskolin binding in the substantia nigra is intrinsic to a striatal-nigral projection: an autoradiographic study of rat brain. Neurosci. L&f. 73, 114-l 18. 19. Karlsson G., Jaton A.-L. and Vigouret J.-M. (1988) Dopamine D,- and D,-receptor interaction in turning behaviour induced by dopamine agonists in 6-hydroxydopamine-lesioned rats. Neurosci. L.ert. 88, 69-74. 20. Kebabian J. W. and Calne D. B. (1979) Multiple receptors for dopamine. Nature 227, 93-96. 21. Kocsis J. D. and Kitai S. T. (1977) Dual excitatory inputs to caudate spiny neurons from substantia nigra stimulation. Brain Res. 138, 271-283. 22. Lacey M. G., Mercuri N. N. and North R. A. (1987) Dopamine acts on Dz receptors to increase potassium conductance in neurons of the rat substantia nigra zona compacta. J. Physiol. 392, 397-416. 23. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. 24. Mata M., Fink D. J., Gainer H., Smith C. B., Davidsen L., Savaki H., Schwartz W. J. and Sokoloff L. (1980) Activity-dependent energy metabolism in rat posterior pituitary primarily reflects sodium pump activity. J. Neurochem. 34, 213-215.

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25 McNeil1 T. H., Brown S. A., Rafols J. A. and Shoulson I. A. (1988) Atrophy of medium spiny 1 striatal dendrites rn advanced Parkinson’s disease. Brain Res. 455, 148..152. 26. Mishra R. K., Marshall A. M. and Varmuza S. L. (1980) Supersensitivity in rat caudate nucleus: effects of 6-hydroxydopamine on the time course of dopamine receptor and cyclic AMP changes. Bruin Res. 200, 47-57. 21. Morelli M. and DiChiara G. (1987) Agonist-induced homologous and heterologous sensitization to D-l and D-2 dependent contraversive turning. Eur. J. Pharmac. 141, 101-107. 28. Morelli M., Fenu S., Garau L. and DiChiara G. (1989) Time and dose dependence of the “priming” of the expression of dopamine receptor supersensitivity. Eur. J. Pharmac. 162, 329~.336. 29. Nisenbaum E. S., Stricker E. M., Zigmond M. J. and Bcrger T. W. (1986) Long-term effects of dopaminedepleting brain lesions on spontaneous activity of type II striatal neurons: relation to behavioral recovery. Brain Res. 398, 221-230. 30. Onn S. P., Berger T. W., Stricker E. M. and Zigmond M. J. (1986) Effects of intraventricular 6-hydroxydopamine on the dopaminergic innervation of striatum: histochemical and neurochemical analysis. Bruin Res. 376, 8-19. 31. Pellegrino L. S., Pellegrino A. S. and Cushman A. J. (1979) A Stereotaxic Atlas ofthe Rat Brain. Plenum, New York. 32. Porceddu M. L., Giorgi O., Ongini E., Mele S. and Biggio G. (1986) ‘H-SCH 23390 binding sites in the rat substantia nigra: evidence for a presynaptic localization and innervation by dopamine. Life Sci. 39, 321-328. 33. Preston R. J., Bishop G. A. and Kitai S. T. (1980) Medium spiny neuron projection from the rat striatum: an intracellular horseradish peroxidase study. Bruin Res. 183, 253-263. 34. Reisine T. D., Fields J. Z., Yamamura H. I., Bird E. D., Spokes E., Schreiner P. S. and Enna S. J. (1977) Neurotransmitter receptor alterations in Parkinson’s disease. L$k Sci. 21, 335344. 35. Seeman P., Bzowej N. H., Gaun H. C., Bergeron C., Reynolds G. P., Bird E. D., Riederer P., Jellinger K. and Tourtelotte W. W. (1987) Human brain D, and D, dopamine receptors in Schizophrenia, Alzheimer’s, Parkinson’s, and Huntington’s diseases. Neuropsychopharmacology 1, 5-15. 36. Spano P. F., DiChiara G., Tonon G. C. and Trabucchi M. (1976) A dopamine stimulated adenlate cyclase in rat substantia nigra. J. Neurochem. 27, 15651568. 37. Sternberger L. A. (1979) Immunocytochemistry. John Wiley, New York. 38. Stoof J. C. and Kebabian J. W. (1984) Two dopamine receptors: biochemistry, physiology and pharmacology. L@ Sci. 35, 2281-2296. 39. Trugman J. M. and Woolen G. F. (1987) Selective D, and D, agonists differentially alter basal ganglia glucose utilization in rats with unilateral 6-hydroxydopamine substantia nigra lesions. J. Neurosci. 7, 2927-2935. 40. Wilson C. J., Chang H. T. and Kitai S. T. (1982) Origins of postsynaptic potentials evoked in identified rat neostriatal neurons by stimulation in substantia nigra, Expl Brain Resy 45, -157-167. 41. Young W. S. and Kuhar M. J. (1979) A new method for receptor autoradiography: ‘H-opioid receptors in rat brain. Brain Res, 179, 255-270. (Accepted

13 April 1989)