- Email: [email protected]
Dopamine receptor gene transfer into rat striatum using a recombinant adenoviral vector: rotational behaviour
Neuroscience Letters 291 (2000) 135±138
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Dopamine receptor gene transfer into rat striatum using a recombinant adenoviral vector: rotational behaviour Albert Hung Choy Wong a,c,*, Mark G.L. Knapp b, Hubert H.M. Van Tol a,b,c a Department of Psychiatry, Faculty of Medicine, University of Toronto, Ontario, Canada Department of Pharmacology, Faculty of Medicine, University of Toronto, Ontario, Canada c Institute of Medical Science, Faculty of Medicine, University of Toronto, Ontario, Canada
b
Received 8 March 2000; accepted 15 July 2000
Abstract To study the role of dopamine (DA) receptor expression on dopamine-mediated rotational behaviour, adenovirus expressing the lacZ reporter gene (AdCMVLacZ) or D2R expressing adenoviral vector (AdRSVD2) viruses, mediating expression of b-galactosidase and DAD2 receptors, respectively, were microinjected stereotactically into Sprague± Dawley rat striatum. Apomorphine stimulated rotational behaviour was measured in rats unilaterally injected with either AdCMVLacZ or AdRSVD2. No signi®cant difference in rotational direction was observed until day 14 post-injection, when animals showed a tendency to rotate away from the injected side. Our data indicate that unilateral changes in receptor density mediated by a non-cell type selective adenoviral vector results in minor changes in rotational behavior. This suggests that supersensitivity in dopamine receptor signaling, rather than receptor levels per se, are the major factor in determining rotational response with dopamine agonist stimulation in unilateral striatal dopamine depleted animals. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Recombinant adenovirus; Dopamine receptors; Rotation; Behaviour; Apomorphine; Striatum
The dopamine (DA) system plays an important role in the pathophysiology of Parkinson's and Huntington's disease, and the dopamine receptor is the only neuroreceptor consistently implicated in the therapeutic treatment of psychosis as seen in schizophrenia. Most antipsychotic drugs block the dopamine D2 receptor (D2R) with an af®nity that is correlated with their clinical effect [3,19]. These observations have led to the suggestion that the psychosis seen in schizophrenia is related to an overactive dopamine system, and there are some reports of increased D2R levels in schizophrenics [3,20,27]. The development of an animal model that facilitates the study of the behavioral and biochemical changes resulting from increased dopamine receptor levels is of vital importance to understanding the basis of the antipsychotic action of neuroleptics. Agonists and antagonists that have high af®nity for these receptor subtypes might
* Corresponding author. Room 711, Centre for Addiction and Mental Health, Clarke Division, 250 College Street, Toronto, Ontario, M5T 1R8, Canada Tel.: 11-416-535-8501 ext. 4010; fax: 11-416-979-6936. E-mail address: [email protected] (A. Hung Choy Wong).
mimic changes in the levels of these receptors, but such compounds are not yet generally available. Rotational behaviour in rodents has been studied in the context of lesions in the striatum and the substantia nigra. 6-Hydroxydopamine (6-OHDA) lesions cause degeneration of the nigrostriatal dopamine pathway, and result in a significant laterality of rotation towards the lesioned side spontaneously, and with amphetamine stimulation. Apomorphine stimulation, in contrast, induces contralateral rotational preference [5]. Mild striatal dopamine D2 receptor proliferation occurs in response to the lesion (20±40%), and other authors have questioned whether this upregulation is suf®cient to account for the changes in rotational behaviour [11]. Using a pharmacological dose-response approach, and quantitative modeling of the biochemical and behavioral data, Mandel et al. [11] concluded that simple upregulation of D2 receptors is unlikely to account for supersensitization as measured by rotational behaviour. To date, there has been only one published report of the behavioral changes induced by modifying dopamine receptor expression with viral gene transfer technology [23]. Those authors reported a marked laterality in rotational behaviour 3 and 7 days after injecting the striatum of Spra-
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 40 2- 6
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A.H.C. Wong et al. / Neuroscience Letters 291 (2000) 135±138
gue±Dawley (SD) rats with an adenoviral (Ad) vector containing the rat dopamine D2 cDNA with the cytomegalovirus (CMV) immediate early promotor. The expression of D2 receptors as measured by autoradiography was increased by .40% 3±5 days after injection and declined to 20% above baseline 7±14 days after injection. They reported the net number of right vs. left rotations and found that injected animals had approximately 100 net rotations away from the AdD2 injected side. The contralateral striatum was injected with control Ad expressing the b-galactosidase gene. These data suggest that, in contrast to Mandel et al. [11] and Dunnett et al. [5], simple D2 receptor upregulation may be suf®cient induce rotational laterality. In our study we use a direct approach to the manipulation of the DA system to address the question of whether simple upregulation of D2 receptors is suf®cient to account for the laterality in rotational behaviour following unilateral 6-OHDA lesions. The construction of the D2R expressing adenoviral vector (AdRSVD2) has been described previously by us [9]. The adenovirus expressing the lacZ reporter gene (AdCMVLacZ) was obtained from Dr Frank Graham (McMaster University, Hamilton, Canada). Microinjection was performed using standard stereotactic neurosurgical procedures, under anesthesia with a single injection of Na pentobarbital (55 mg/kg i.p.), and buprenorphine (0.01±0.05 mg/kg). Each injection consisted of 10 9 plaque-forming units in a single injection of 1 ml over a 5 min interval to minimize tissue damage - using an automated delivery system. The needle was then withdrawn slowly over 30 s, and the skull defect ®lled with bone wax. The scalp was closed with 5.0 proline sutures, and the animals allowed to recover for 2±4 h before being returned to their cages. Throughout the procedure they were kept warm under surgical towels. The study of rotational behaviour involved 12 SD rats (300±325 g) receiving right-sided unilateral injections of either AdCMVLacZ, or AdRSVD2. As we have reported previously [9], injection with AdRSVD2, but not AdCMVLacZ, results in a consistent 20±30% increase in striatal D2R expression, not necessarily con®ned to the neurons that normally express the D2R. There were six animals in each group. After recovering from the surgery, the animals were acclimatized to the Rotorat apparatus (Med Associates Inc., St. Albans, VT) for 1 h daily. On days 3, 7 and 14 after adenoviral injection, the animals were given 0.1 mg/kg (i.p.), doses of apomorphine and placed in the rotorat apparatus. Rotational behaviour was recorded automatically by registering centripetal movement of a ¯exible shaft connected to a jacket ®tted to the thorax. Angular displacements of greater than 458 were recorded directly into computer memory over the 1-h observation period. Unilateral injection of AdRSVD2 or AdCMVLacZ into the striatum resulted in no signi®cant changes in rotational behavior for both groups of animals on either day 3, 7 or 14 post-injection, using analysis of variance (ANOVA) (Fig.
Fig. 1. Rotational behaviour following right unilateral CPu injection of either AdD2 or AdBeta viruses, expressed as a ratio of complete left vs. right or right vs. left rotations.
1). When the data are expressed as absolute numbers of net rotations, (i.e. right minus left complete rotations), only day 14 differences between the AdCMVLacZ and AdRSVD2 groups displayed a trend to a difference in the absolute number of rotations (t-test P , 0:05) as seen in Fig. 2. The direction of rotation ± contralateral to the AdRSVD2 injected side ± was consistent with the expected effect of a unilateral increase in dopamine receptor expression. However, when the data are analyzed as a ratio of left over right rotation (complete 3608 turns), the effects are very small (,10%) and are not signi®cantly different between the two groups. The role of the dopaminergic nigro-striatal system in controlling locomotor activity has been well documented, and is explicitly visible in Parkinson's disease and in the extrapyramidal side-effects induced by neuroleptic medication. Asymmetrical manipulation of the nigro-striatal system in rodent models, with unilateral 6-OHDA substantia nigral lesions, striatal lesions, or pharmacological D2 receptor blockade results in asymmetrical rotational locomotion. After dopamine agonist stimulation, 6-OHDA lesioned animals tend to rotate contralateral to the lesioned side, suggesting that dopamine supersensitivity contributes to this effect. Dopamine supersensitivity may be mediated by the observed receptor up-regulation on the lesioned side and increased ef®cacy of receptor-mediated signaling. It is unknown which of these two effects contribute more significantly to the observed rotational asymmetry after 6-OHDA
Fig. 2. Rotational behaviour following right unilateral CPu injection of either AdD2 or AdBeta viruses, expressed as net number of clockwise minus counterclockwise rotations.
A.H.C. Wong et al. / Neuroscience Letters 291 (2000) 135±138
lesions. Our previously published results show reliable overexpression of D2-like receptors in cells infected with recombinant adenoviral strains containing the D2R [9]. In vivo gene transfer into selected rat brain regions using stereotactic microinjection of the various viral strains also results in reliable overexpression. Striatal expression of D2 receptors was increased approximately 20±30% by injection of AdRSVD2 [9], which is comparable to 6-OHDA lesion induced D2 receptor up-regulation. This observation allows us to investigate whether D2 receptor up-regulation in striatum, independent from loss of dopaminergic projections can mediate an asymmetric rotation response. Our experiments suggest that a 20±30% unilateral D2 receptor up-regulation can mediate only a small change in rotational laterality. The method of analysis of the data in¯uences the interpretation of our ®ndings. When our data are presented in terms of net rotations we do con®rm a statistically signi®cant laterality on day 14 with a twotailed t-test. However, this signi®cance is not found when the data are analyzed as a ratio of left vs. right turns, or when an ANOVA (two-factor with replication on 3 days) is used. We feel that examining the ratios is more accurate in re¯ecting the magnitude of the behavioral effects, since the significance of a small difference in laterality could be magni®ed by simply having more animals in each test group. Most groups do not report their results in terms of ratios, so it is unknown whether their results would have been signi®cant in this type of analysis. Although our experiments suggested a trend towards a net contralateral rotation to injection site at day 14 post-injection of AdRSVD2, the effect was extremely small when expressed as a ratio of left over right rotations (,10% of total rotations). The laterality in net rotational behaviour we observed was comparable to that reported by Umegaki et al. [23], but our results differ in several important aspects. Although the laterality expressed as the net number of turns in one direction vs. the other showed a signi®cant tendency to rotate away from the DA-virus injected side, we found this to be signi®cant only at day 14, while Umegaki et al. [23] report these ®ndings on day 3 and day 7. Umegaki et al. [23] did not report the ratio of left vs. right rotations and therefore we cannot compare the magnitude of rotational effects between our experiments. Differences in the promoter may explain the different time-course of behavioral effects on the basis of different peak- expression times. We used the rous sarcoma virus (RSV) promoter, while Umegaki et al. [23] used the CMV promoter. The number of net turns was similar between our two studies. These similar results are in the context of some methodological differences: including the unilateral vs. bilateral injection protocol. Our animals received only unilateral Ad injections, which caused visible damage to the injected striatum. This may have decreased the net effects of increased D2R expression induced by the recombinant adenovirus. Some evidence of the damage effects of Ad injection can be seen in the tendency for control animals injected with AdCMVLacZ to rotate
137
towards the injected side, suggesting that the intact (left) side had a greater number of functioning dopamine receptors. The increase in right rotations in the AdCMVLacZ injected rats from day 3 to 14 seen in the normalized data suggests that there are ongoing decreases in the function of the striatum. In¯ammation follows after intra-cerebral adenovirus injection [2], and this may partly explain the impaired function of the AdCMVLacZ-injected striatum. Another factor to consider in these experiments is the lack of cell-speci®c expression of recombinant D2R genes. We and Umegaki et al. [23] used Ad vectors that may induce expression in cells that do not express D2 receptors endogenously, and the effect of such expression may affect rotational behaviour. Both of these viral vectors will infect the same cell-types, the use of different promoters may alter the pro®le of cell-speci®c expression, which, in principle, might account for the differences in rotational behaviour. The effects of D2R expression in cells that do not normally express this gene have not been well studied experimentally. In cells that co-express D1 and D2 receptors, some authors report synergistic effects based on activation of the arachidonic acid cascade [15], despite the opposing effects of D1 and D2 receptors on stimulating and inhibiting adenylyl cyclase. Forcing D2R to be expressed on non-neuronal cells may have unpredictable effects on the striatal environment that supports the surrounding neurons. The net behavioral effects of these changes remain to be explored. Laterality in net rotation seen with 6-hydroxydopamine (6-OHDA) lesions is usually of the same magnitude as seen in our experiments [5,16,17,24,25]. This is correlated with the relative upregulation in D2 receptor expression seen (20±30%) with 6-OHDA lesions of the substantia nigra [11,26], although these lesions produce a substantial depletion of dopamine (.80%) in the striatum [21] and the caudate (.70%) [11]. However, Moser et al. [13], report the relative ratio of left vs. right rotations, indicating that the rotational effects of 6-OHDA lesioning are far more robust than we have observed. This would suggest that supersensitivity in dopamine receptor signaling, rather than receptor expression levels, is the dominant component in mediating rotational laterality after dopamine agonist stimulation in 6-OHDA lesioned animals. A dissociation between DAD2 upregulation and behavioral supersensitivity has been observed previously [4,6,10,18]. The mechanism controlling such DA supersensitvity is still unresolved, but could involve increased ef®cacy in coupling to downstream effectors such as g-proteins [1,12,14] or adenylyl cyclase [22]. However, contradictory observations have also been reported [7,8]. To our knowledge, possible changes in RGS protein activity may also contribute to the phenomenon of DA receptor supersensitivity. Therefore, we suggest that simple D2 receptor upregulation through adenoviral-mediated gene transfer does not result in an effective upregulation the functional D2 receptor pool.
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We would like to acknowledge the support of the Ontario Mental Health Foundation and the Canadian Psychiatric Research Foundation in funding this research. Dr Van Tol is a Career Scientist of the Ontario Ministry of Health. [1] Butkerait, P., Wang, H.Y. and Friedman, E., Increases in guanine nucleotide binding to striatal G proteins is associated with dopamine receptor supersensitivity, J. Pharmacol. Exp. Ther., 271 (1994) 422±428. [2] Cartmell, T., Southgate, T., Rees, G.S., Castro, M.G., Lowenstein, P.R. and Luheshi, G.N., Interleukin-1 mediates a rapid in¯ammatory response after injection of adenoviral vectors into the brain, J. Neurosci., 19 (1999) 1517±1523. [3] Creese, I., Burt, D.R. and Snyder, S.H., Dopamine receptor binding predicts clinical and pharmacological potencies of antischizophrenic drugs, Science, 192 (1976) 481±483. [4] Dewey, K.J. and Fibiger, H.C., The effects of dose and duration of chronic pimozide administration on dopamine receptor supersensitivity, Naunyn Schmiedebergs Arch. Pharmacol., 322 (1983) 261±270. [5] Dunnett, S.B. and Iversen, S.D., Spontaneous and druginduced rotation following localized 6-hydroxydopamine and kainic acid-induced lesions of the neostriatum, Neuropharmacology, 21 (1982) 899±908. [6] Engber, T.M., Marin, C., Susel, Z. and Chase, T.N., Differential effects of chronic dopamine D1 and D2 receptor agonists on rotational behavior and dopamine receptor binding, Eur. J. Pharmacol., 236 (1993) 385±393. [7] Gupta, S.K. and Mishra, R.K., Effects of chronic treatment of haloperidol and clozapine on levels of G-protein subunits in rat striatum, J. Mol. Neurosci., 3 (1992) 197±201. [8] Inoue, A., Ueda, H., Nakata, Y. and Misu, Y., Supersensitivity of quinpirole-evoked GTPase activation without changes in gene expression of D2 and Gi protein in the striatum of hemidopaminergic lesioned rats, Neurosci. Lett., 175 (1994) 107± 110. [9] Knapp, M., Wong, A.H., Schoots, O., Guan, H.C. and Van Tol, H.H., Promoter-independent regulation of cell-speci®c dopamine receptor expression, FEBS Lett., 434 (1998) 108± 114. [10] LaHoste, G.J. and Marshall, J.F., New concepts in dopamine receptor plasticity, Ann. NY Acad. Sci., 702 (1993) 183±196. [11] Mandel, R.J., Hartgraves, S.L., Severson, J.A., Woodward, J.J., Wilcox, R.E. and Randall, P.K., A quantitative estimate of the role of striatal D-2 receptor proliferation in dopaminergic behavioral supersensitivity: the contribution of mesolimbic dopamine to the magnitude of 6-OHDA lesioninduced agonist sensitivity in the rat, Behav. Brain Res., 59 (1993) 53±64. [12] Marcotte, E.R., Sullivan, R.M. and Mishra, R.K., Striatal Gproteins: effects of unilateral 6-hydroxydopamine lesions, Neurosci. Lett., 169 (1994) 195±198. [13] Moser, A., Siebecker, F., Nobbe, F. and Bohme, V., Rotational behaviour and neurochemical changes in unilateral N-methyl-norsalsolinol and 6-hydroxydopamine lesioned rats, Exp. Brain Res., 112 (1996) 89±95.
[14] Olianas, M.C. and Onali, P., Supersensitivity of striatal D2 dopamine receptors mediating inhibition of adenylate cyclase and stimulation of guanosine triphosphatase following chronic administration of haloperidol in mice, Neurosci. Lett., 78 (1987) 349±354. [15] Piomelli, D., Pilon, C., Giros, B., Sokoloff, P., Martres, M.P. and Schwartz, J.C., Dopamine activation of the arachidonic acid cascade as a basis for D1/D2 receptor synergism, Nature, 353 (1991) 164±167. [16] Pycock, C.J., Turning behaviour in animals, Neuroscience, 5 (1980) 461±514. [17] Pycock, C., Tarsy, D. and Marsden, C.D., Inhibition of circling behavior by neuroleptic drugs in mice with unilateral 6-hydroxydopamine lesions of the striatum, Psychopharmacologia, 45 (1975) 211±219. [18] Reddy, P.L., Veeranna, X.X., Thorat, S.N. and Bhargava, H.N., Evidence for the behavioral supersensitivity of dopamine D2 receptors without receptor up-regulation in morphine-abstinent rats, Brain Res., 607 (1993) 293±300. [19] Seeman, P., Lee, T., Chau-Wong, M. and Wong, K., Antipsychotic drug doses and neuroleptic/dopamine receptors, Nature, 261 (1976) 717±719. [20] Seeman, P., Guan, H.-C. and Tol, H.V., Dopamine D4 receptors elevated in schizophrenia, Nature, 365 (1993) 441±445. [21] Sullivan, R.M., Fraser, A. and Szechtman, H., Asymmetrical orientation to edges of an open®eld: modulation by striatal dopamine and relationship to motor asymmetries in the rat, Brain Res., 637 (1994) 114±118. [22] Treisman, G.J., Muirhead, N. and Gnegy, M.E., Increased sensitivity of adenylate cyclase activity in the striatum of the rat to calmodulin and GppNHp after chronic treatment with haloperidol, Neuropharmacology, 25 (1986) 587± 595. [23] Umegaki, H., Chernak, J.M., Ikari, H., Roth, G.S. and Ingram, D.K., Rotational behavior produced by adenovirusmediated gene transfer of dopamine D2 receptor into rat striatum, NeuroReport, 8 (1997) 3553±3558. [24] Ungerstedt, U., Postsynaptic supersensitivity after 6hydroxy-dopamine induced degeneration of the nigrostriatal dopamine system, Acta Physiol. Scand. Suppl., 367 (1971) 69±93. [25] Ungerstedt, U., Striatal dopamine release after amphetamine or nerve degeneration revealed by rotational behaviour, Acta Physiol. Scand. Suppl., 367 (1971) 49±68. [26] Woiciechowsky, C., Guilarte, T.R., May, C.H., Vesper, J., Wagner Jr, H.N. and Vogel, S., Intrastriatal dopamine infusion reverses compensatory increases in D2-dopamine receptors in the 6-OHDA lesioned rat, Neurodegeneration, 4 (1995) 161±169. [27] Wong, D.F., Wagner Jr., H.N., Tune, L.E., Dannals, R.F., Pearlson, G.D., Links, J.M., Tamminga, C.A., Broissolle, E.P., Ravert, H.T., Wilson, A.A., Toung, T.T., Malat, J., Williams, J.A., O'Tuama, L.A., Snyder, S.H., Kuhar, M.J. and Gjedde, A., Positron emission tomography reveals elevated D2 dopamine receptors in drug-naive schizophrenics (published erratum appears in Science 235(4789) (1987) 623), Science, 234 (1986) 1558±1563.
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Dopamine receptor gene transfer into rat striatum using a recombinant adenoviral vector: rotational behaviour Albert Hung Choy Wong a,c,*, Mark G.L. Knapp b, Hubert H.M. Van Tol a,b,c a Department of Psychiatry, Faculty of Medicine, University of Toronto, Ontario, Canada Department of Pharmacology, Faculty of Medicine, University of Toronto, Ontario, Canada c Institute of Medical Science, Faculty of Medicine, University of Toronto, Ontario, Canada
b
Received 8 March 2000; accepted 15 July 2000
Abstract To study the role of dopamine (DA) receptor expression on dopamine-mediated rotational behaviour, adenovirus expressing the lacZ reporter gene (AdCMVLacZ) or D2R expressing adenoviral vector (AdRSVD2) viruses, mediating expression of b-galactosidase and DAD2 receptors, respectively, were microinjected stereotactically into Sprague± Dawley rat striatum. Apomorphine stimulated rotational behaviour was measured in rats unilaterally injected with either AdCMVLacZ or AdRSVD2. No signi®cant difference in rotational direction was observed until day 14 post-injection, when animals showed a tendency to rotate away from the injected side. Our data indicate that unilateral changes in receptor density mediated by a non-cell type selective adenoviral vector results in minor changes in rotational behavior. This suggests that supersensitivity in dopamine receptor signaling, rather than receptor levels per se, are the major factor in determining rotational response with dopamine agonist stimulation in unilateral striatal dopamine depleted animals. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Recombinant adenovirus; Dopamine receptors; Rotation; Behaviour; Apomorphine; Striatum
The dopamine (DA) system plays an important role in the pathophysiology of Parkinson's and Huntington's disease, and the dopamine receptor is the only neuroreceptor consistently implicated in the therapeutic treatment of psychosis as seen in schizophrenia. Most antipsychotic drugs block the dopamine D2 receptor (D2R) with an af®nity that is correlated with their clinical effect [3,19]. These observations have led to the suggestion that the psychosis seen in schizophrenia is related to an overactive dopamine system, and there are some reports of increased D2R levels in schizophrenics [3,20,27]. The development of an animal model that facilitates the study of the behavioral and biochemical changes resulting from increased dopamine receptor levels is of vital importance to understanding the basis of the antipsychotic action of neuroleptics. Agonists and antagonists that have high af®nity for these receptor subtypes might
* Corresponding author. Room 711, Centre for Addiction and Mental Health, Clarke Division, 250 College Street, Toronto, Ontario, M5T 1R8, Canada Tel.: 11-416-535-8501 ext. 4010; fax: 11-416-979-6936. E-mail address: [email protected] (A. Hung Choy Wong).
mimic changes in the levels of these receptors, but such compounds are not yet generally available. Rotational behaviour in rodents has been studied in the context of lesions in the striatum and the substantia nigra. 6-Hydroxydopamine (6-OHDA) lesions cause degeneration of the nigrostriatal dopamine pathway, and result in a significant laterality of rotation towards the lesioned side spontaneously, and with amphetamine stimulation. Apomorphine stimulation, in contrast, induces contralateral rotational preference [5]. Mild striatal dopamine D2 receptor proliferation occurs in response to the lesion (20±40%), and other authors have questioned whether this upregulation is suf®cient to account for the changes in rotational behaviour [11]. Using a pharmacological dose-response approach, and quantitative modeling of the biochemical and behavioral data, Mandel et al. [11] concluded that simple upregulation of D2 receptors is unlikely to account for supersensitization as measured by rotational behaviour. To date, there has been only one published report of the behavioral changes induced by modifying dopamine receptor expression with viral gene transfer technology [23]. Those authors reported a marked laterality in rotational behaviour 3 and 7 days after injecting the striatum of Spra-
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 40 2- 6
136
A.H.C. Wong et al. / Neuroscience Letters 291 (2000) 135±138
gue±Dawley (SD) rats with an adenoviral (Ad) vector containing the rat dopamine D2 cDNA with the cytomegalovirus (CMV) immediate early promotor. The expression of D2 receptors as measured by autoradiography was increased by .40% 3±5 days after injection and declined to 20% above baseline 7±14 days after injection. They reported the net number of right vs. left rotations and found that injected animals had approximately 100 net rotations away from the AdD2 injected side. The contralateral striatum was injected with control Ad expressing the b-galactosidase gene. These data suggest that, in contrast to Mandel et al. [11] and Dunnett et al. [5], simple D2 receptor upregulation may be suf®cient induce rotational laterality. In our study we use a direct approach to the manipulation of the DA system to address the question of whether simple upregulation of D2 receptors is suf®cient to account for the laterality in rotational behaviour following unilateral 6-OHDA lesions. The construction of the D2R expressing adenoviral vector (AdRSVD2) has been described previously by us [9]. The adenovirus expressing the lacZ reporter gene (AdCMVLacZ) was obtained from Dr Frank Graham (McMaster University, Hamilton, Canada). Microinjection was performed using standard stereotactic neurosurgical procedures, under anesthesia with a single injection of Na pentobarbital (55 mg/kg i.p.), and buprenorphine (0.01±0.05 mg/kg). Each injection consisted of 10 9 plaque-forming units in a single injection of 1 ml over a 5 min interval to minimize tissue damage - using an automated delivery system. The needle was then withdrawn slowly over 30 s, and the skull defect ®lled with bone wax. The scalp was closed with 5.0 proline sutures, and the animals allowed to recover for 2±4 h before being returned to their cages. Throughout the procedure they were kept warm under surgical towels. The study of rotational behaviour involved 12 SD rats (300±325 g) receiving right-sided unilateral injections of either AdCMVLacZ, or AdRSVD2. As we have reported previously [9], injection with AdRSVD2, but not AdCMVLacZ, results in a consistent 20±30% increase in striatal D2R expression, not necessarily con®ned to the neurons that normally express the D2R. There were six animals in each group. After recovering from the surgery, the animals were acclimatized to the Rotorat apparatus (Med Associates Inc., St. Albans, VT) for 1 h daily. On days 3, 7 and 14 after adenoviral injection, the animals were given 0.1 mg/kg (i.p.), doses of apomorphine and placed in the rotorat apparatus. Rotational behaviour was recorded automatically by registering centripetal movement of a ¯exible shaft connected to a jacket ®tted to the thorax. Angular displacements of greater than 458 were recorded directly into computer memory over the 1-h observation period. Unilateral injection of AdRSVD2 or AdCMVLacZ into the striatum resulted in no signi®cant changes in rotational behavior for both groups of animals on either day 3, 7 or 14 post-injection, using analysis of variance (ANOVA) (Fig.
Fig. 1. Rotational behaviour following right unilateral CPu injection of either AdD2 or AdBeta viruses, expressed as a ratio of complete left vs. right or right vs. left rotations.
1). When the data are expressed as absolute numbers of net rotations, (i.e. right minus left complete rotations), only day 14 differences between the AdCMVLacZ and AdRSVD2 groups displayed a trend to a difference in the absolute number of rotations (t-test P , 0:05) as seen in Fig. 2. The direction of rotation ± contralateral to the AdRSVD2 injected side ± was consistent with the expected effect of a unilateral increase in dopamine receptor expression. However, when the data are analyzed as a ratio of left over right rotation (complete 3608 turns), the effects are very small (,10%) and are not signi®cantly different between the two groups. The role of the dopaminergic nigro-striatal system in controlling locomotor activity has been well documented, and is explicitly visible in Parkinson's disease and in the extrapyramidal side-effects induced by neuroleptic medication. Asymmetrical manipulation of the nigro-striatal system in rodent models, with unilateral 6-OHDA substantia nigral lesions, striatal lesions, or pharmacological D2 receptor blockade results in asymmetrical rotational locomotion. After dopamine agonist stimulation, 6-OHDA lesioned animals tend to rotate contralateral to the lesioned side, suggesting that dopamine supersensitivity contributes to this effect. Dopamine supersensitivity may be mediated by the observed receptor up-regulation on the lesioned side and increased ef®cacy of receptor-mediated signaling. It is unknown which of these two effects contribute more significantly to the observed rotational asymmetry after 6-OHDA
Fig. 2. Rotational behaviour following right unilateral CPu injection of either AdD2 or AdBeta viruses, expressed as net number of clockwise minus counterclockwise rotations.
A.H.C. Wong et al. / Neuroscience Letters 291 (2000) 135±138
lesions. Our previously published results show reliable overexpression of D2-like receptors in cells infected with recombinant adenoviral strains containing the D2R [9]. In vivo gene transfer into selected rat brain regions using stereotactic microinjection of the various viral strains also results in reliable overexpression. Striatal expression of D2 receptors was increased approximately 20±30% by injection of AdRSVD2 [9], which is comparable to 6-OHDA lesion induced D2 receptor up-regulation. This observation allows us to investigate whether D2 receptor up-regulation in striatum, independent from loss of dopaminergic projections can mediate an asymmetric rotation response. Our experiments suggest that a 20±30% unilateral D2 receptor up-regulation can mediate only a small change in rotational laterality. The method of analysis of the data in¯uences the interpretation of our ®ndings. When our data are presented in terms of net rotations we do con®rm a statistically signi®cant laterality on day 14 with a twotailed t-test. However, this signi®cance is not found when the data are analyzed as a ratio of left vs. right turns, or when an ANOVA (two-factor with replication on 3 days) is used. We feel that examining the ratios is more accurate in re¯ecting the magnitude of the behavioral effects, since the significance of a small difference in laterality could be magni®ed by simply having more animals in each test group. Most groups do not report their results in terms of ratios, so it is unknown whether their results would have been signi®cant in this type of analysis. Although our experiments suggested a trend towards a net contralateral rotation to injection site at day 14 post-injection of AdRSVD2, the effect was extremely small when expressed as a ratio of left over right rotations (,10% of total rotations). The laterality in net rotational behaviour we observed was comparable to that reported by Umegaki et al. [23], but our results differ in several important aspects. Although the laterality expressed as the net number of turns in one direction vs. the other showed a signi®cant tendency to rotate away from the DA-virus injected side, we found this to be signi®cant only at day 14, while Umegaki et al. [23] report these ®ndings on day 3 and day 7. Umegaki et al. [23] did not report the ratio of left vs. right rotations and therefore we cannot compare the magnitude of rotational effects between our experiments. Differences in the promoter may explain the different time-course of behavioral effects on the basis of different peak- expression times. We used the rous sarcoma virus (RSV) promoter, while Umegaki et al. [23] used the CMV promoter. The number of net turns was similar between our two studies. These similar results are in the context of some methodological differences: including the unilateral vs. bilateral injection protocol. Our animals received only unilateral Ad injections, which caused visible damage to the injected striatum. This may have decreased the net effects of increased D2R expression induced by the recombinant adenovirus. Some evidence of the damage effects of Ad injection can be seen in the tendency for control animals injected with AdCMVLacZ to rotate
137
towards the injected side, suggesting that the intact (left) side had a greater number of functioning dopamine receptors. The increase in right rotations in the AdCMVLacZ injected rats from day 3 to 14 seen in the normalized data suggests that there are ongoing decreases in the function of the striatum. In¯ammation follows after intra-cerebral adenovirus injection [2], and this may partly explain the impaired function of the AdCMVLacZ-injected striatum. Another factor to consider in these experiments is the lack of cell-speci®c expression of recombinant D2R genes. We and Umegaki et al. [23] used Ad vectors that may induce expression in cells that do not express D2 receptors endogenously, and the effect of such expression may affect rotational behaviour. Both of these viral vectors will infect the same cell-types, the use of different promoters may alter the pro®le of cell-speci®c expression, which, in principle, might account for the differences in rotational behaviour. The effects of D2R expression in cells that do not normally express this gene have not been well studied experimentally. In cells that co-express D1 and D2 receptors, some authors report synergistic effects based on activation of the arachidonic acid cascade [15], despite the opposing effects of D1 and D2 receptors on stimulating and inhibiting adenylyl cyclase. Forcing D2R to be expressed on non-neuronal cells may have unpredictable effects on the striatal environment that supports the surrounding neurons. The net behavioral effects of these changes remain to be explored. Laterality in net rotation seen with 6-hydroxydopamine (6-OHDA) lesions is usually of the same magnitude as seen in our experiments [5,16,17,24,25]. This is correlated with the relative upregulation in D2 receptor expression seen (20±30%) with 6-OHDA lesions of the substantia nigra [11,26], although these lesions produce a substantial depletion of dopamine (.80%) in the striatum [21] and the caudate (.70%) [11]. However, Moser et al. [13], report the relative ratio of left vs. right rotations, indicating that the rotational effects of 6-OHDA lesioning are far more robust than we have observed. This would suggest that supersensitivity in dopamine receptor signaling, rather than receptor expression levels, is the dominant component in mediating rotational laterality after dopamine agonist stimulation in 6-OHDA lesioned animals. A dissociation between DAD2 upregulation and behavioral supersensitivity has been observed previously [4,6,10,18]. The mechanism controlling such DA supersensitvity is still unresolved, but could involve increased ef®cacy in coupling to downstream effectors such as g-proteins [1,12,14] or adenylyl cyclase [22]. However, contradictory observations have also been reported [7,8]. To our knowledge, possible changes in RGS protein activity may also contribute to the phenomenon of DA receptor supersensitivity. Therefore, we suggest that simple D2 receptor upregulation through adenoviral-mediated gene transfer does not result in an effective upregulation the functional D2 receptor pool.
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