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Quantification of lesion-induced dopaminergic supersensitivity using the rotational model in the house
358
Brain Research, 330 (1985) 358-363 Elsevier
BRE20690
Quantification of lesion-induced dopaminergic supersensitivity using the rotational model in the mouse RONALD J. MANDEL and PATRICK K. RANDALL Departments of Psychology, Physiology and Biophysics, University of Southern California, Andrus Gerontology Center, Los Angeles, CA 90089-0191 (U.S.A.) (Accepted October 30th, 1984) Key words: dopamine - - 6-hydroxydopamine - - nigrostriatal - - supersensitivity - - apomorphine - - striatonigral
A dual lesion technique was used to determine the degree of supersensitivity resulting from nigrostriatal lesions in C57BL/6J mice. Internal capsule lesions encroaching on globus paUidus resulted in reliable ipsilateral rotation both to apomorphine and amphetamine. Dose-response curves to apomorphine were determined before and 21 days after 6-hydroxydopamine lesion of the contralateral nigrostriatal pathway. A 31.5-fold shift to the left was observed following the nigrostriatal lesion, with no change in slope. The extent and placement of the internal capsule lesion, as well as the magnitude of supersensitization correspond closely to those previously reported in the rat.
An important requirement for assessing physiological, rather than binding characteristics of dopamine receptors, is the precise quantification of the functional result of dopamine agonism. The nature of the relationship between physiological response and receptor number is unknown and cannot be obtained without precision of both physiological and biochemical measurement, particularly under conditions where the number of binding sites is altered. Lesion of the dopamine containing nigrostriatal pathway in the rodent is thought to result in supersensitivity in striatum to dopamine agonists 20. Increases in responsiveness of striatal neurons to iontophoresed dopamine 5 and behavioral sensitivity to systemic injection of dopamine agonistst9, 20 are consistent with the denervation supersensitivity hypothesis at a functional level. At a more molecular level, the numbers of binding sites for dopamine agonists and antagonists are increased following either denervation 2 or chronic administration of dopamine antagonists 15. Most investigators report large increases in behavioral sensitivity to agonists following denervation 9,12,14A9,20or chronic blockade 7 whereas increases in density of dopamine binding sites are relatively
modest (20-40%) 10,11.16. Seeman 16 has tried to reconcile this apparent discrepancy on the basis of data from Schwarz et a1.14, who used unilateral kainate lesions of striatum to measure dose-response curves to apomorphine with the rotational model. Seeman 16 suggested that the magnitude of response to dopamine agonists is proportional to the absolute, rather than the fractional occupancy of dopamine receptors. The answers to such questions require a precise evaluation of dose-response curves in both the innervated and denervated preparation. Following unilateral lesion of the nigrostriatal pathway, administration of direct dopamine agonists produces dose-related rotational behavior contralateral to the lesion. This is thought to result from Supersensitization of the denervated striatum 2x. Until recently, lack of a baseline sensitivity measure has precluded estimation of the actual shift in the dose-response curve resulting from the lesion. Ungerstedt and Marshall 22 first described the use of a lesion that blocked the behavioral output of one striatum so that turning could be elicited in an animal without a nigrostriatal lesion. This rotation is thought to be due solely to striatal dopamine agonist stimula-
Correspondence: P. K. Randall, Departments of Psychology and Physiology, University of Southern California, Andrus Gerontology Center, University Park, Los Angeles, CA 90089-0191 (U.S.A.). 0006-8993/85/$03.30© 1985 Elsevier Science Publishers B.V. (Biomedical Division)
359 tion on the unlesioned side, providing a 'pre-supersensitivity' estimate of apomorphine sensitivity. Subsequently, the striatonigral tract has been identified at the most likely functional site of this lesion TM. The current experiment was undertaken first, to localize the output lesion in the C57BL/6J mouse and second, to quantify changes in sensitivity after unilateral striatal denervation, utilizing the basic Marshall and Ungerstedt 9 methodology. A within-animal experimental design was used to estimate the shift of the dose-response curves for the same animals, controlling several nuisance variables I not dealt with in the Marshall and Ungerstedt 9 experiment. Male C57BL/6J mice were obtained at 8-10 weeks of age from Jackson Laboratories, Bar Harbor, ME. Sixteen-to 18-week-old mice, weighing 25 + 0.15 g, were prepared for surgery as reported previously t3 and electrolytic lesions made with a Grass model DCLM5A lesion maker and a 0.02 mm diameter platinum-iridium electrode which was insulated except for 0.1-0.2 mm at the tip with teflon and an additional layer of Stoner-Mudge coating. A stainless steel anal probe coated with petroleum jelly completed the circuit. All 6-hydroxydopamine (6-OHDA) lesions were identical to those described in Randall 13. Rotational behavior was assessed using time-lapse video photography as previously described 13. Sixty-five mice were used to ascertain a reliable set of coordinates which were adjusted from those of Slotnick and Leonard 17 and current parameters which would produce discretely localized output lesions as described by Ungerstedt and Marshall 22. The final coordinates (-1.6 mm A - P , +2.0 mm M - L , -4.5 mm D - V ) , and current parameters (2 mA/ 10 s) produced reliable ipsilateral turning to amphetamine challenge with a moderately discrete lesion. Since both the 6-OHDA lesion and the electrolytic lesion of the internal capsule cause transient aphagia and adipsia6, body weight and water intake were monitored for at least 1 week postsurgically. In addition, mice received 1 ml of 0.9% saline subcutaneously daily, and were provided with a highly palatable milk mixture for no less than 1 week. One week following the electrolytic lesion, mice were screened for the effectiveness of the output lesion with a test dose of 2.0 mg/kg D-amphetamine sulfate (Sigma Chemicals, St. Louis, MO). Animals
that did not exhibit robust turning of at least 80% ipsilaterally to their output lesion were sacrificed for histological examination. In order to determine the dose-response curves for apomorphine, 24 animals were administered output lesions and were screened for rotational behavior. Twelve mice that showed appropriate levels of turning were randomly divided into 3 groups for replicates of a 4 x 4 balanced Latin Square with doses of 0.75, 1.5, 3.0 and 6.0 mg/kg apomorphine hydrochloride (Merck, Sharp and Dohme, Rahway, N J). Doses were calculated as the salt of the drug and all injections were administered intraperitoneally in 0.9% saline. Test days were at weekly intervals with the last day of the square replicated on the fifth test day. One week later the same animals underwent 6-OHDA lesions of the nigrostriatal pathway in the opposite hemisphere. Twenty-one days after the second lesion, another dose-response curve was determined using doses of 0.01, 0.02, 0.04 and 0.08 mg/kg apomorphine, and the same balanced Latin Square design. One week later all animals received an additional, higher dose (1.5 mg/kg). Mice were perfused with 10% formalin solution and brains fixed for at least 24 h. Fifty micron frozen sections through the lesion were stained with thionin and examined for lesion verification. For both dose-response curves, net turns toward the side of the output lesion for the 1-h session were used as the dependent variable. Since one cell was missing from two of the balanced Latin Squares, data from the last week and the following replication (fifth) week were arithmetically averaged and a randomized block ANOVA8 was performed on each dose-response curve separately. EDs0 and slope factors were estimated using ALLFIT 3. The location of the most effective lesion for ipsilateral rotation, one week postoperatively to dopamine agonists, was in the ventral internal capsule, lateral to the entopeduncular nucleus, with extensive damage to the caudal globus pallidus. Lesions that extended medially into the entopeduncular nucleus produced contralateral turning in the amphetamine screening test, particularly when the lesion did not extend into the globus pallidus (Fig. 1). The effective lesion reported here is probably the striatal efferent pathway to substantia nigra reticulata TM. In addition,
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I0 rain. period=(p-lwe-drug) Fig. 1. Lesion sites and rotational behavior in amphetamine screening test. Darkened areas represent sites damaged in common between all animals which also corresponds to the minimum effective lesion size, with rotational behavior 1 week after surgery indicated by arrows. More medial sites produced contralateral (right) rotation, especially if the globus pallidus was completely spared. Internal capsule lesions, producing ipsilateral (left) rotation, were most effective if posterior globus pallidus was involved. The hatched region indicates the maximum extent of the functional output lesion. however, damage in the globus pallidus may indicate some damage to other classical striatal efferents. After the output lesion, animals regained body weight within 14 days. Removal of the moist palatable food did not h a m p e r the recovery after this lesion. A f t e r 6 - O H D A lesions, however, body weight d r o p p e d to dangerously low levels when this diet was removed, and promptly recovered upon its return. Net rotation toward the side of the output lesion increased with dose of a p o m o r p h i n e in both pre- and p o s t - 6 - O H D A conditions (F(3,30) = 11.8; F(4,36) = 12.47, respectively; P < 0.01). Both curves had strong log-linear components (F(1,32) = 40.5; F(1,29) = 46.3, respectively; P < 0.01) with no significant residual (both F < 1). The d o s e - r e s p o n s e curves for a p o m o r p h i n e of both intact and d e n e r v a t e d striata are shown on right
and left, respectively, in Fig. 2. EDs0 and logistic slope factors for the two curves were estimated by obtaining the best-fitting p a r a m e t e r s for the generalized logistic function 3. Slope factors did not differ, since fitting with a single, shared slope factor did not yield a significantly greater residual variance than fitting with separate slopes (F(2,4) = 1.39; P > 0.05). In addition, no evidence was found for an alteration in maximum response as d e t e r m i n e d by fitting with separate versus shared maximum response p a r a m eters ( F < 1). The c o m m o n slope and m a x i m u m response p a r a m e t e r s were 1.52 and 180.5, respectively. The EDs0s were 5.06 mg/kg for the intact, and 0.16 mg/kg for the d e n e r v a t e d striatum, representing a 31.5-fold increase in sensitivity. Since the experiment was run following a balanced Latin Square design, an estimate of the residual due to drug dosage can be m a d e by examining total rotations on days following each dose (Fig. 3). A l t h o u g h statistical analysis was impossible due to the loss of cells in the Latin Square, it is unlikely that any residual effect was present. The time course of the response to a p o m o r p h i n e was similar before and after 6 - O H D A treatment (Fig. 4) and was consistent with the short behavioral action of this drug. The output lesion site r e p o r t e d here is consistent with the location of the striatonigral efferent pathway TM. The pattern of recovery from the output lesion
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Fig. 3. Residual effects of the previous day's dose on rotational behavior. A: represents the mean total contralateral rotations across all doses on daysfollowing the value on the abscissa in mice with output lesion only. B: represents similar data following the 6-OHDA lesion. was i n t e r e s t i n g in light of a r e c e n t r e p o r t by D e w a r et al. 4. I m m e d i a t e l y after surgery, virtually 100% of the
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Fig. 4. Time-course of apomorphine induced rotation before and after 6-OHDA lesion. Symbols represent mean net left turns (toward output and contralateral to 6-OHDA lesion). A: open squares = 6.0; open circles = 3.0; open triangles = 1.5; and solid circles = 0.75 mg/kg. B: open squares = 0.08; open circles = 0.04; open triangles = 0.02; and solid circles = 0.01 mg/kg. Solid diamonds represent the pre-drug mean across all animals.
362 continued ipsilateral turning. The lesion that remained effective destroyed the internal capsule and extensively damaged the globus pallidus. Animals with more medial lesions, not encroaching on globus pallidus, did not continue to turn. These data agree well with Dewar et al. 4 who observed similar behavioral patterns after kainic acid lesions in corresponding pathways in the rat. The use of the striatal output lesion is a powerful methodological tool for studying basal gangliar function, providing measurement of the behavioral sensitivity of an intact striatum using the rotational model. The 31.5-fold shift in sensitivity to apomorphine after 6 - O H D A described here, agrees with other reports in rats 9,12. The fact that the two dose-response curves are parallel suggests that there is no contamination of the animals' response by a generalized increase in reactivity due to non-specific surgical damage or repeated drug administration. We did not observe an increase in either the slope of the curve, or the maximum response as predicted by Seeman 16 and reported by Schwarz et a1.14. These data clearly do not support the hypothesis that the magnitude of response to dopamine agonists is proportional to the absolute occupancy of dopamine receptors. The use of a within-animal design allowed compa-
1 Cochran, W. G. and Cox, G. M., Experimental Design, 2nd edn., Wiley, New York, 1957, pp. 132-144. 2 Creese, I., Burt, D. R. and Snyder, S. H., Dopamine receptor binding enhancement accompanies lesion induced behavioral supersensitivity, Science, 197 (1977) 596-598. 3 De Lean, A., Munson, P. J. and Robard, D., Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose-response curves, Amer. J. Physiol., 235 (1978) 97-102. 4 Dewar, D., Jenner, P. and Marsden, C. D., Lesions of the globus pallidus, entopeduncular nucleus and substantia nigra alter dopamine mediated circling behavior, Exp. Brain Res., 52 (1983) 281-292. 5 Feltz, P. and De Champlain, J., Enhanced sensitivity of caudate neurones to microiontophoretic injections of dopamine in 6-hydroxydopamine treated cats, Brain Research, 43 (1972) 601-605. 6 Fibiger, H. C., Zis, A. P. and McGeer, E. G., Feeding and drinking deficits after 6-hydroxydopamine administration in the rat: similarities to the lateral hypothalamic syndrome, Brain Research, 55 (1973) 135-148. 7 Gianutsos, G. and Moore, K. E., Dopaminergic supersensitivity in striatum and olfactory tubercle following chronic administration of haloperidol or clozapine, Life Sci., 20 (1977) 1585-1592. 8 Kirk, R. E., Experimental Design: Procedures for the Behavioral Sciences, Brooks/Cole, Belmont, CA, 1968, pp.
rison before and after supersensitization in the same animals. The balanced Latin Square design controls the variables of differentially effective output lesions between groups, possible learning effects, habituation to the testing apparatus, and increased sensitivity over time. While the design affords control of many nuisance variables and is extremely efficient statisticallyl,8, it requires a large time investment and is very sensitive to subject mortality. Regardless of the drawbacks associated with a within-animal experimental design, the use of the dual lesion technique is the only precise method available to calculate increases in behavioral sensitivity with the rotational paradigm. A n y hypothesis, above the phenomenological level, regarding physiological function of the striatum after manipulation of numbers of dopamine binding sites, requires the precision available with this or similar experimental methods. This research was supported by N I A Grant AG003272 to P . K . R . R . J . M . is supported by N I A Training Grant AG00037. The authors would like to thank Judith S. Randall for critical reading of the manuscript and excellent preparation of the figures.
131-150. 9 Marshall, J. F. and Ungerstedt, U., Supersensitivity to apomorphine following destruction of the ascending dopamine neurons: quantification using the rotational model, Europ. J. Pharmacol., 41 (1977) 361-367. 10 Mishra, R. K., Wong, R. K., Varmaza, S. L. and Tuff, L., Chemical lesion and drug induced supersensitivity of caudate dopamine receptors, Life Sci., 23 (1978) 443-446. 11 Muller, P. and Seeman, P., Brain neurotransmitter receptors after long term haloperidol: dopamine, acetylch01ine, serotonin, a-noradrenergic, and naloxone receptors, Life Sci., 21 (1977) 1751-1758. 12 Neve, K. A., Kozlowski, M. R. and Marshall, J. F., Plasticity of neostriatal dopamine receptors after nigrostriatal injury: relationship to recovery of sensorimotor functions and behavioral supersensitivity, Brain Research, 244 (1982) 33-44. 13 Randall, P. K., Lesion-induced DA supersensitivity in aging C57BL/6J mice, Brain Research, 308 (1984) 333-336. 14 Schwarz, R., Fuxe, K., Agnati, L. F., Hokfelt, T. and Coyle, J. T., Rotational behaviour in rats with unilateral striatal kainic acid le~ions: a behavioural model for studies on intact dopamine receptors, Brain Research, 170 (1979) 485-495. 15 Schwartz, J.-C., Baudry, M., Martres, M.-P., Constentin, J. and Protais, P., Increased in vivo binding of 3H-pimozide in mouse striatum following repeated administration of ha-
363 loperidol, Life Sci., 23 (1978) 1785-1790. 16 Seeman, P., Brain dopamine receptors, Pharmacol. Rev., 32 (1981) 229-313. 17 Slotnick, B. M. and Leonard, C. M., A Stereotaxic Atlas o] the Albino Mouse Forebrain, U.S.D.H.E.W., Rockville, MD, 1975. 18 Tulloch, I. F., Arbuthnott, G. W. and Wright, A. K., Topographical organization of the striatonigral pathway revealed by anterograde and retrograde neuroanatomical tracing techniques, J. Anat., 127 (1978) 425-441. 19 Ungerstedt, U., Ljundberg, T. and Schultz, W,, Dopamine receptor mechanisms: behavioral and electrophysiological studies. In P. J. Roberts (Ed.), Advances in Biochemical Psychopharmacology, Vol. 19, Raven Press, New York,
1978, pp. 311-321. 20 Ungerstedt, U., Postsynaptic supersensitivity after 6-hydroxydopamine induced degeneration of the nigrostriatal dopamine system, Acta physiol, scand., Suppl. 367 (1971) 69-93. 21 Ungerstedt, U., Striatal dopamine release after amphetamine or nerve degeneration revealed by rotational behavior, Acta physiol, scand., Suppl. 367 (1971) 49-68. 22 Ungerstedt, U. and Marshall, J. F., Nerve degeneration in functional studies: experiments illustrating the problem of lesion specificity and compensatory supersensitivity in the striatum. In G, Johnson, T. Malmfors and C. Sachs (Eds.), Chemical Tools in Catecholamine Research, Vol. 1, NorthH.olland, Amsterdam, 1975, pp. 311-330.
Brain Research, 330 (1985) 358-363 Elsevier
BRE20690
Quantification of lesion-induced dopaminergic supersensitivity using the rotational model in the mouse RONALD J. MANDEL and PATRICK K. RANDALL Departments of Psychology, Physiology and Biophysics, University of Southern California, Andrus Gerontology Center, Los Angeles, CA 90089-0191 (U.S.A.) (Accepted October 30th, 1984) Key words: dopamine - - 6-hydroxydopamine - - nigrostriatal - - supersensitivity - - apomorphine - - striatonigral
A dual lesion technique was used to determine the degree of supersensitivity resulting from nigrostriatal lesions in C57BL/6J mice. Internal capsule lesions encroaching on globus paUidus resulted in reliable ipsilateral rotation both to apomorphine and amphetamine. Dose-response curves to apomorphine were determined before and 21 days after 6-hydroxydopamine lesion of the contralateral nigrostriatal pathway. A 31.5-fold shift to the left was observed following the nigrostriatal lesion, with no change in slope. The extent and placement of the internal capsule lesion, as well as the magnitude of supersensitization correspond closely to those previously reported in the rat.
An important requirement for assessing physiological, rather than binding characteristics of dopamine receptors, is the precise quantification of the functional result of dopamine agonism. The nature of the relationship between physiological response and receptor number is unknown and cannot be obtained without precision of both physiological and biochemical measurement, particularly under conditions where the number of binding sites is altered. Lesion of the dopamine containing nigrostriatal pathway in the rodent is thought to result in supersensitivity in striatum to dopamine agonists 20. Increases in responsiveness of striatal neurons to iontophoresed dopamine 5 and behavioral sensitivity to systemic injection of dopamine agonistst9, 20 are consistent with the denervation supersensitivity hypothesis at a functional level. At a more molecular level, the numbers of binding sites for dopamine agonists and antagonists are increased following either denervation 2 or chronic administration of dopamine antagonists 15. Most investigators report large increases in behavioral sensitivity to agonists following denervation 9,12,14A9,20or chronic blockade 7 whereas increases in density of dopamine binding sites are relatively
modest (20-40%) 10,11.16. Seeman 16 has tried to reconcile this apparent discrepancy on the basis of data from Schwarz et a1.14, who used unilateral kainate lesions of striatum to measure dose-response curves to apomorphine with the rotational model. Seeman 16 suggested that the magnitude of response to dopamine agonists is proportional to the absolute, rather than the fractional occupancy of dopamine receptors. The answers to such questions require a precise evaluation of dose-response curves in both the innervated and denervated preparation. Following unilateral lesion of the nigrostriatal pathway, administration of direct dopamine agonists produces dose-related rotational behavior contralateral to the lesion. This is thought to result from Supersensitization of the denervated striatum 2x. Until recently, lack of a baseline sensitivity measure has precluded estimation of the actual shift in the dose-response curve resulting from the lesion. Ungerstedt and Marshall 22 first described the use of a lesion that blocked the behavioral output of one striatum so that turning could be elicited in an animal without a nigrostriatal lesion. This rotation is thought to be due solely to striatal dopamine agonist stimula-
Correspondence: P. K. Randall, Departments of Psychology and Physiology, University of Southern California, Andrus Gerontology Center, University Park, Los Angeles, CA 90089-0191 (U.S.A.). 0006-8993/85/$03.30© 1985 Elsevier Science Publishers B.V. (Biomedical Division)
359 tion on the unlesioned side, providing a 'pre-supersensitivity' estimate of apomorphine sensitivity. Subsequently, the striatonigral tract has been identified at the most likely functional site of this lesion TM. The current experiment was undertaken first, to localize the output lesion in the C57BL/6J mouse and second, to quantify changes in sensitivity after unilateral striatal denervation, utilizing the basic Marshall and Ungerstedt 9 methodology. A within-animal experimental design was used to estimate the shift of the dose-response curves for the same animals, controlling several nuisance variables I not dealt with in the Marshall and Ungerstedt 9 experiment. Male C57BL/6J mice were obtained at 8-10 weeks of age from Jackson Laboratories, Bar Harbor, ME. Sixteen-to 18-week-old mice, weighing 25 + 0.15 g, were prepared for surgery as reported previously t3 and electrolytic lesions made with a Grass model DCLM5A lesion maker and a 0.02 mm diameter platinum-iridium electrode which was insulated except for 0.1-0.2 mm at the tip with teflon and an additional layer of Stoner-Mudge coating. A stainless steel anal probe coated with petroleum jelly completed the circuit. All 6-hydroxydopamine (6-OHDA) lesions were identical to those described in Randall 13. Rotational behavior was assessed using time-lapse video photography as previously described 13. Sixty-five mice were used to ascertain a reliable set of coordinates which were adjusted from those of Slotnick and Leonard 17 and current parameters which would produce discretely localized output lesions as described by Ungerstedt and Marshall 22. The final coordinates (-1.6 mm A - P , +2.0 mm M - L , -4.5 mm D - V ) , and current parameters (2 mA/ 10 s) produced reliable ipsilateral turning to amphetamine challenge with a moderately discrete lesion. Since both the 6-OHDA lesion and the electrolytic lesion of the internal capsule cause transient aphagia and adipsia6, body weight and water intake were monitored for at least 1 week postsurgically. In addition, mice received 1 ml of 0.9% saline subcutaneously daily, and were provided with a highly palatable milk mixture for no less than 1 week. One week following the electrolytic lesion, mice were screened for the effectiveness of the output lesion with a test dose of 2.0 mg/kg D-amphetamine sulfate (Sigma Chemicals, St. Louis, MO). Animals
that did not exhibit robust turning of at least 80% ipsilaterally to their output lesion were sacrificed for histological examination. In order to determine the dose-response curves for apomorphine, 24 animals were administered output lesions and were screened for rotational behavior. Twelve mice that showed appropriate levels of turning were randomly divided into 3 groups for replicates of a 4 x 4 balanced Latin Square with doses of 0.75, 1.5, 3.0 and 6.0 mg/kg apomorphine hydrochloride (Merck, Sharp and Dohme, Rahway, N J). Doses were calculated as the salt of the drug and all injections were administered intraperitoneally in 0.9% saline. Test days were at weekly intervals with the last day of the square replicated on the fifth test day. One week later the same animals underwent 6-OHDA lesions of the nigrostriatal pathway in the opposite hemisphere. Twenty-one days after the second lesion, another dose-response curve was determined using doses of 0.01, 0.02, 0.04 and 0.08 mg/kg apomorphine, and the same balanced Latin Square design. One week later all animals received an additional, higher dose (1.5 mg/kg). Mice were perfused with 10% formalin solution and brains fixed for at least 24 h. Fifty micron frozen sections through the lesion were stained with thionin and examined for lesion verification. For both dose-response curves, net turns toward the side of the output lesion for the 1-h session were used as the dependent variable. Since one cell was missing from two of the balanced Latin Squares, data from the last week and the following replication (fifth) week were arithmetically averaged and a randomized block ANOVA8 was performed on each dose-response curve separately. EDs0 and slope factors were estimated using ALLFIT 3. The location of the most effective lesion for ipsilateral rotation, one week postoperatively to dopamine agonists, was in the ventral internal capsule, lateral to the entopeduncular nucleus, with extensive damage to the caudal globus pallidus. Lesions that extended medially into the entopeduncular nucleus produced contralateral turning in the amphetamine screening test, particularly when the lesion did not extend into the globus pallidus (Fig. 1). The effective lesion reported here is probably the striatal efferent pathway to substantia nigra reticulata TM. In addition,
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I0 rain. period=(p-lwe-drug) Fig. 1. Lesion sites and rotational behavior in amphetamine screening test. Darkened areas represent sites damaged in common between all animals which also corresponds to the minimum effective lesion size, with rotational behavior 1 week after surgery indicated by arrows. More medial sites produced contralateral (right) rotation, especially if the globus pallidus was completely spared. Internal capsule lesions, producing ipsilateral (left) rotation, were most effective if posterior globus pallidus was involved. The hatched region indicates the maximum extent of the functional output lesion. however, damage in the globus pallidus may indicate some damage to other classical striatal efferents. After the output lesion, animals regained body weight within 14 days. Removal of the moist palatable food did not h a m p e r the recovery after this lesion. A f t e r 6 - O H D A lesions, however, body weight d r o p p e d to dangerously low levels when this diet was removed, and promptly recovered upon its return. Net rotation toward the side of the output lesion increased with dose of a p o m o r p h i n e in both pre- and p o s t - 6 - O H D A conditions (F(3,30) = 11.8; F(4,36) = 12.47, respectively; P < 0.01). Both curves had strong log-linear components (F(1,32) = 40.5; F(1,29) = 46.3, respectively; P < 0.01) with no significant residual (both F < 1). The d o s e - r e s p o n s e curves for a p o m o r p h i n e of both intact and d e n e r v a t e d striata are shown on right
and left, respectively, in Fig. 2. EDs0 and logistic slope factors for the two curves were estimated by obtaining the best-fitting p a r a m e t e r s for the generalized logistic function 3. Slope factors did not differ, since fitting with a single, shared slope factor did not yield a significantly greater residual variance than fitting with separate slopes (F(2,4) = 1.39; P > 0.05). In addition, no evidence was found for an alteration in maximum response as d e t e r m i n e d by fitting with separate versus shared maximum response p a r a m eters ( F < 1). The c o m m o n slope and m a x i m u m response p a r a m e t e r s were 1.52 and 180.5, respectively. The EDs0s were 5.06 mg/kg for the intact, and 0.16 mg/kg for the d e n e r v a t e d striatum, representing a 31.5-fold increase in sensitivity. Since the experiment was run following a balanced Latin Square design, an estimate of the residual due to drug dosage can be m a d e by examining total rotations on days following each dose (Fig. 3). A l t h o u g h statistical analysis was impossible due to the loss of cells in the Latin Square, it is unlikely that any residual effect was present. The time course of the response to a p o m o r p h i n e was similar before and after 6 - O H D A treatment (Fig. 4) and was consistent with the short behavioral action of this drug. The output lesion site r e p o r t e d here is consistent with the location of the striatonigral efferent pathway TM. The pattern of recovery from the output lesion
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Fig. 4. Time-course of apomorphine induced rotation before and after 6-OHDA lesion. Symbols represent mean net left turns (toward output and contralateral to 6-OHDA lesion). A: open squares = 6.0; open circles = 3.0; open triangles = 1.5; and solid circles = 0.75 mg/kg. B: open squares = 0.08; open circles = 0.04; open triangles = 0.02; and solid circles = 0.01 mg/kg. Solid diamonds represent the pre-drug mean across all animals.
362 continued ipsilateral turning. The lesion that remained effective destroyed the internal capsule and extensively damaged the globus pallidus. Animals with more medial lesions, not encroaching on globus pallidus, did not continue to turn. These data agree well with Dewar et al. 4 who observed similar behavioral patterns after kainic acid lesions in corresponding pathways in the rat. The use of the striatal output lesion is a powerful methodological tool for studying basal gangliar function, providing measurement of the behavioral sensitivity of an intact striatum using the rotational model. The 31.5-fold shift in sensitivity to apomorphine after 6 - O H D A described here, agrees with other reports in rats 9,12. The fact that the two dose-response curves are parallel suggests that there is no contamination of the animals' response by a generalized increase in reactivity due to non-specific surgical damage or repeated drug administration. We did not observe an increase in either the slope of the curve, or the maximum response as predicted by Seeman 16 and reported by Schwarz et a1.14. These data clearly do not support the hypothesis that the magnitude of response to dopamine agonists is proportional to the absolute occupancy of dopamine receptors. The use of a within-animal design allowed compa-
1 Cochran, W. G. and Cox, G. M., Experimental Design, 2nd edn., Wiley, New York, 1957, pp. 132-144. 2 Creese, I., Burt, D. R. and Snyder, S. H., Dopamine receptor binding enhancement accompanies lesion induced behavioral supersensitivity, Science, 197 (1977) 596-598. 3 De Lean, A., Munson, P. J. and Robard, D., Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose-response curves, Amer. J. Physiol., 235 (1978) 97-102. 4 Dewar, D., Jenner, P. and Marsden, C. D., Lesions of the globus pallidus, entopeduncular nucleus and substantia nigra alter dopamine mediated circling behavior, Exp. Brain Res., 52 (1983) 281-292. 5 Feltz, P. and De Champlain, J., Enhanced sensitivity of caudate neurones to microiontophoretic injections of dopamine in 6-hydroxydopamine treated cats, Brain Research, 43 (1972) 601-605. 6 Fibiger, H. C., Zis, A. P. and McGeer, E. G., Feeding and drinking deficits after 6-hydroxydopamine administration in the rat: similarities to the lateral hypothalamic syndrome, Brain Research, 55 (1973) 135-148. 7 Gianutsos, G. and Moore, K. E., Dopaminergic supersensitivity in striatum and olfactory tubercle following chronic administration of haloperidol or clozapine, Life Sci., 20 (1977) 1585-1592. 8 Kirk, R. E., Experimental Design: Procedures for the Behavioral Sciences, Brooks/Cole, Belmont, CA, 1968, pp.
rison before and after supersensitization in the same animals. The balanced Latin Square design controls the variables of differentially effective output lesions between groups, possible learning effects, habituation to the testing apparatus, and increased sensitivity over time. While the design affords control of many nuisance variables and is extremely efficient statisticallyl,8, it requires a large time investment and is very sensitive to subject mortality. Regardless of the drawbacks associated with a within-animal experimental design, the use of the dual lesion technique is the only precise method available to calculate increases in behavioral sensitivity with the rotational paradigm. A n y hypothesis, above the phenomenological level, regarding physiological function of the striatum after manipulation of numbers of dopamine binding sites, requires the precision available with this or similar experimental methods. This research was supported by N I A Grant AG003272 to P . K . R . R . J . M . is supported by N I A Training Grant AG00037. The authors would like to thank Judith S. Randall for critical reading of the manuscript and excellent preparation of the figures.
131-150. 9 Marshall, J. F. and Ungerstedt, U., Supersensitivity to apomorphine following destruction of the ascending dopamine neurons: quantification using the rotational model, Europ. J. Pharmacol., 41 (1977) 361-367. 10 Mishra, R. K., Wong, R. K., Varmaza, S. L. and Tuff, L., Chemical lesion and drug induced supersensitivity of caudate dopamine receptors, Life Sci., 23 (1978) 443-446. 11 Muller, P. and Seeman, P., Brain neurotransmitter receptors after long term haloperidol: dopamine, acetylch01ine, serotonin, a-noradrenergic, and naloxone receptors, Life Sci., 21 (1977) 1751-1758. 12 Neve, K. A., Kozlowski, M. R. and Marshall, J. F., Plasticity of neostriatal dopamine receptors after nigrostriatal injury: relationship to recovery of sensorimotor functions and behavioral supersensitivity, Brain Research, 244 (1982) 33-44. 13 Randall, P. K., Lesion-induced DA supersensitivity in aging C57BL/6J mice, Brain Research, 308 (1984) 333-336. 14 Schwarz, R., Fuxe, K., Agnati, L. F., Hokfelt, T. and Coyle, J. T., Rotational behaviour in rats with unilateral striatal kainic acid le~ions: a behavioural model for studies on intact dopamine receptors, Brain Research, 170 (1979) 485-495. 15 Schwartz, J.-C., Baudry, M., Martres, M.-P., Constentin, J. and Protais, P., Increased in vivo binding of 3H-pimozide in mouse striatum following repeated administration of ha-
363 loperidol, Life Sci., 23 (1978) 1785-1790. 16 Seeman, P., Brain dopamine receptors, Pharmacol. Rev., 32 (1981) 229-313. 17 Slotnick, B. M. and Leonard, C. M., A Stereotaxic Atlas o] the Albino Mouse Forebrain, U.S.D.H.E.W., Rockville, MD, 1975. 18 Tulloch, I. F., Arbuthnott, G. W. and Wright, A. K., Topographical organization of the striatonigral pathway revealed by anterograde and retrograde neuroanatomical tracing techniques, J. Anat., 127 (1978) 425-441. 19 Ungerstedt, U., Ljundberg, T. and Schultz, W,, Dopamine receptor mechanisms: behavioral and electrophysiological studies. In P. J. Roberts (Ed.), Advances in Biochemical Psychopharmacology, Vol. 19, Raven Press, New York,
1978, pp. 311-321. 20 Ungerstedt, U., Postsynaptic supersensitivity after 6-hydroxydopamine induced degeneration of the nigrostriatal dopamine system, Acta physiol, scand., Suppl. 367 (1971) 69-93. 21 Ungerstedt, U., Striatal dopamine release after amphetamine or nerve degeneration revealed by rotational behavior, Acta physiol, scand., Suppl. 367 (1971) 49-68. 22 Ungerstedt, U. and Marshall, J. F., Nerve degeneration in functional studies: experiments illustrating the problem of lesion specificity and compensatory supersensitivity in the striatum. In G, Johnson, T. Malmfors and C. Sachs (Eds.), Chemical Tools in Catecholamine Research, Vol. 1, NorthH.olland, Amsterdam, 1975, pp. 311-330.