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Functional recovery following transplantation of ventral mesencephalic cells in rat subjected to 6-OHDA lesions of the mesolimbic dopaminergic neurons
Brain Research, 304 (1984) 137-141
137
Elsevier BRE 10129
Functional Recovery Following Transplantation of Ventral Mesencephalic Cells in Rat Subjected to 6-OHDA Lesions of the Mesolimbic Dopaminergic Neurons D. NADAUD, J. P. HERMAN, H. SIMON and M. LE MOAL Laboratoire de Neurobiologie des Comportements (1NSERM U. 259), Universit~de Bordeaux II, 146 rue L~o-Saignat, 33600 Bordeaux (France)
(Accepted November 29th, 1983) Key words: grafts - - mesolimbic system - - dopamine - - functional recovery - - open-field - - amphetamine-induced hyperactivity - -
apomorphine
Lesion of mesolimbic dopaminergic neurons induces hypoactivity and hypo-exploration in a new environment, lack of hyperactivity to amphetamine and hyperresponsiveness to apomorphine. Transplantation of embryonic dopaminergic cells from rat embryos into the deafferented nucleus accumbens restores, 8 weeks after grafting, some of the deficits obtained after the lesion. Spontaneous activity and behavioral responses in an open-field are improved and some of the grafted rats show a complete recovery of amphetamine-induced hyperactivity. However, hyperresponsiveness to apomorphine has not been significantly reduced by the treatment. These resuits indicate that transplanted dopaminergic neurons can restore some of the functional activities of the n. accumbens. INTRODUCTION The ability of neuronal grafts to restore functions lost after brain damage has been demonstrated for several neurochemical systems such as the hypothalamic peptidergic 9, septohippocampal cholinergic 13 and dopaminergic ( D A ) nigrostriatal (NS) neurons. Motor deficits such as circling behavior induced in rats after unilateral NS lesion are reduced by implantation of nigral tissue2,16 or adrenal medulla8 or injection of a mesencephalic cell suspension 18 in the vicinity of or into the denervated striatum. However, other deficits such as akinesia and aphagia obtained after bilateral NS lesion are not reduced by the graftsS. Curiously, no investigation has been done on the mesolimbic D A neurons located in the ventral tegmental area (VTA) that project to limbic-related structures (head of the caudate nucleus, nucleus accumbens, septal area, amygdala, hippocampus) and to prefrontal, cingulate and entorhinal cortices6A9, 20,23. These neurons modulate regions the function of which is necessary for adaptive behavior, initiation of action and for regulating cognitive-representational processes~2,15,21. Moreover, mesolimbic transmission has been implicated in the pathogenesis of psychopathological states22. A n impairment of D A transmis0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
sion in the n. accumbens results in deficits such as hypoactivity, hypoexploration, disappearance of amphetamine-induced hyperactivity and supersensitive locomotor responses to apomorphinel0, n. In this paper, we present results indicating that injections of D A cell suspension within the n. accumbens reduce some of the deficits obtained after mesolimbic cell bodies lesions. MATERIALS AND METHODS Male Wistar rats weighing 160--180 g at the beginning of the experiment were used. For the lesions of the mesolimbic D A neurons, animals were injected bilaterally with 6-hydroxydopamine ( 6 - O H D A ) into the V T A (2.5 mm posterior to the bregma, + 0.5 mm laterally from the midline and 8.5 mm under the skull surface) with 6 - O H D A (4.5 pg base in 1 pl, injected over 5 min). Non-lesioned controls (n = 6) received vehicle injection only. Four weeks later, animals displaying a decrease of more than 80% of their amphetamine-induced locomotor activity relative to controls were chosen for transplantation; this degree of behavioral deficit has been shown to be associated with a greater than 90% depletion of the dopamine content of the n. accumbens 11. Grafts consisted of a
138 suspension of embryonic mesencephalic cells prepared from ventral mesencephalic D A areas dissected from rat embryos at 16-17 days of gestation. The brain tissue blocks were put into a tissue culture medium (Hank's MEM) and the cellular suspension was obtained by mechanical dissociation. This suspension (2.5-4 #1) was injected bilaterally into the n. accumbens of the experimental animals (n = 10) at two different vertical coordinates (4 mm anterior to the bregma, 0.8 mm laterally from the midline, 7 and 8 mm vertical from the surface of the skull). Lesioned controls (n = 5) consisted of lesioned animals injected with a cellular suspension prepared from pieces of parietal cortex obtained from rat embryos at 20 days of gestation. Non-lesioned controls did not receive grafts. Spontaneous and drug-induced locomotor activity was measured for 1 h in a circular corridor 12 cm wide and 170 cm long, equipped with 4 photocell beams. Locomotor records were taken 2 weeks before and 4, 6 and 8 weeks after grafting. Drugs used were D-amphetamine (1.5 mg/kg i.p.) and apomorphine (0.1 mg/kg s.c.). The apomorphine test was done 48 h following the amphetamine test. Open-field behavior was observed 7 weeks after the implantation procedure as a measure of emotional response in a new environment]. The open-field consisted of 1 x 1 m, 50 cm high white arena divided into 25 squares and illuminated with a white 100 W light bulb placed 1 m above the center of the arena. Animals were placed in the central square and total locomotor activity (number of squares entered) and rearings were recorded for 10 min. Results were expressed in locomotor activity and rearing scores by minutes. The animals were sacrificed 10 weeks after grafting. The accumbens nucleus was dissected out and tissue dopamine levels measured by a radioenzymatic assay 7. Data from spontaneous and drug-induced locomotor activity were evaluated by one-way analysis of variance (ANOVA) followed by application of multiple comparison of individual means (NewmanKeuls test). For the open-field scores a Mann and Whitney test was performed. Logarithmic transforms of the data from biochemical analyses were evaluated with an A N O V A followed by a NewmanKeuis test. A criterion of P < 0.05 was used for statistical significance.
RESULTS After treatment with 6-OHDA, spontaneous locomotion in the circular corridor (Fig. 1) decreased to 53.3% and 62.2% of control levels for, respectively, the experimental group and the lesioned controls (P < 0.05 compared to the controls, Newman-Keuls test). Following the grafting procedure spontaneous locomotor activity was restored in experimental animals (time-effect, P < 0.05, A N O V A test) while the lesioned controls showed no consistent change. In the open-field (Fig. 2), the behavior of the lesioned controls was characterized by a marked decrease in the locomotor activity and rearing scores. There was a tendency towards improvement of these scores in some of the grafted animals, but these improvements did not reach the non-lesioned control level. However these scores were higher than those of the lesioned control group both for activity and for rearing measures (P < 0.05, Mann-Whitney test). While non-lesioned animals displayed a locomotor activation upon the injection of amphetamine
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Fig. 1. Effect of grafting on the evolution of spontaneous locomotor activity measured during 1 h in a circular corridor, expressed in % of non-lesioned control level activity. NL, non-lesioned control group; L, lesioned control group; G, grafted group. Time effect: P < 0.05 in grafted group, N.S. in lesioned group (ANOVA test).
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Fig. 2. Open-field behavior: locomotor activity (left) and rearings (right) measured 7 weeks after grafting in non-lesioned control rats (NL), lesioned control rats (L) and grafted rats (G). Open circles in group G represent rats which had recovered in amphetamine-induced activity (see text and Fig. 3C). P < 0.05, grafted rats vs lesioned control rats in locomotor activity and in rearing scores (Mann-Whitney test).
(Fig. 3A, B and C), lesioned controls showed no such a response throughout the period of the experiment. On the other hand, after the grafting of D A cells there was a marked heterogeneity of responses (Fig. 3B). Some animals showed a level of activation similar to non-lesioned controls 8 weeks after the ®
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grafting and others did not show such a recovery and remained at the level of the lesioned controls. Thus, on the basis of the level of amphetamine-induced activity at 8 weeks, we subdivided, a posteriori, the DA-grafted group into two subgroups. Animals showing less than a 50% drug-induced activity with respect to non-lesioned controls were allocated to group G1 (n = 5) and those showing more than 50% level to group G2 (n = 5). Group G1 displayed no responsiveness to amphetamine during the time of the experiment and was not significantly different from the lesioned control group while group G2 showed a gradual improvement of its amphetamine response and in fact reached the level of response of the nonlesioned control group at 8 weeks (Fig. 3C). This evolution was different from that of the lesioned control group (P < 0.05, A N O V A test). This same subdivision within the grafted group did not permit to •differentiate between G1 and G2 for the spontaneous locomotor activity and for the open-field scores. Lesion of the mesolimbic D A neurons induced a post-synaptic dopamine receptor supersensitivity as demonstrated by the increased response to apomorphine (Fig. 4). The hyperresponsiveness to apomorphine remained after grafting• However, a closer examination of the results revealed a difference in the pre-grafting scores of the two subgroups in that before grafting animals of the G2 subgroup displayed a significantly greater response to apomorphine than
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Fig. 3. Locomotor activity measured during 1 h in a circular corridor in response to D-amphetamine (1.5 mg/kg i.p.) in nonlesioned control rats (NL), lesioned control rats (L) and grafted rats (G) before (A) and after (B) grafting• Open circles represent rats which were allocated to the recovered group G2. In C, evolution of amphetamine-induced locomotor activity in the two control groups, NL and L, and in the two grafted groups, G1 (uncompensated) and G2 (compensated). P < 0.05, G2 group vs lesioned control group (ANOVA test).
0
Before Grafting
After
Grafting Fig. 4. Locomotor activity measured during 1 h in a circular corridor in response to apomorphine (0.1 mg/kg s.c.) in non-lesioned control group (NL), lesioned control group (L), grafted group G1 (uncompensated) and grafted group G2 (compensated) before and 8 weeks after grafting. P < 0.05, G2 group vs G1 group before grafting (Newman-Keuls test).
140 TABLE I Dopamine and DO PA C concentrations in nuclei accumbens reinnervated by ventral mesencephalic tegmentum dopamine graft after 6-hydroxydopamine lesions
The concentrations are expressed as ng/mg tissue (mean -+ S.E.M.). DOPAC/DA ratio was calculated from the individual DOPAC and dopamine values for each sample (mean + S.E.M.). For statistical comparison every group was compared with every other: * and ** represent statistical difference (P < 0.05) from non-lesionedcontrols (*) and from lesioned controls
(**).
DA
Non-lesioned controls (n = 6) 7.90_+0.47
DOPAC
DOPAC/ DA ratio
2.15_+0.25 0.27_+0.02
Lesioned controls (n = 5) 0.25+0.07* 0.11+0.03" 0.53_+0.04* Uncompensated grafted rats (group G1) (n = 5)
0.35_+0.05*
0.17_+0.05" 0.44_+0.10
Compensated grafted rats (group G2) (n = 5)
but also of the mesolimbic system as shown by the significant restoration of the amphetamine-induced locomotor response. The tendency towards improvement of spontaneous locomotor activity and of open-field activity and exploration points in the same direction. The slight decrease of locomotor response to apomorphine in the G2 group could also correspond to a partial restoration of post-synaptic DA receptor sensitivity towards normal levels. But this point should be further examined in future experiments. The very low degree of restoration of dopamine level at 10 weeks in the compensated group compared to dopamine level of non-lesioned group is striking. However, it seems, at least in the case of striatal transplant, that a quite slight restoration of dopamine level can be sufficient to restore amphetamine induced behaviors TM. Moreover, there was no significant correlation between the D A level within the nucleus accumbens and the locomotor or the open-field tests in the grafted group (G1 + G2). It is of importance to notice two points at this stage. First, there is no correlation between the recovery scores in the different tests. While all grafted animals showed a tendency to recover spontaneous locomotor activity, only some of them (G2 subgroup) displayed a restored locomotor response to amphetamine. Likewise, the open-field scores were not correlated with the amphetamine scores (see Fig. 2). Some animals having amphetamine-induced locomotor activity like non-lesioned controls showed no improvement at all in their open-field behavior; conversely, some animals having no recuperation in the pharmacological test displayed mild improvement in the open-field test. This is reminiscent of the effects found after striatal grafts, i.e. marked improvement in pharmacological responses (decrease in amphetamine-induced turning) and no improvement in aphagia-adipsia and sensory neglect4, 5. This lack of correlation could indicate that either the grafts in the different animals re-innervated functionally different parts of the n. accumbens, known to be anatomically heterogeneous 3, or that, more complex responses, such as occur in open-field behavior, require more than the simple presence of cells secreting dopamine in a non-regulated way in the n. accumbens. Secondly, it seems that there is a correlation between the hypersensitivity of postsynaptic D A receptors before 4,5,7,16
0.96+0.17".** 0.30_+0.08* 0.32+0.05
animals of the G1 subgroup (P < 0.05, NewmanKeuls test). This difference disappeared at 8 weeks after the grafts and the apomorphine response of the G2 group was at the same level than the G1 group due to a slight decrease of supersensitive response in the G2 group. Biochemical measurements (Table I) revealed a low level of dopamine in the nucleus accumbens of group G1 (uncompensated grafted group) - - a value identical to that of lesioned non-implanted controls - - respectively, 4.4% and 3.2% of the non-lesioned controls. On the other hand the levels of dopamine and D O P A C were increased in the compensated group G2, comparatively to lesioned control values (P < 0.05 for DA, Newman-Keuls test) although they remained far below control levels (12.2%). DISCUSSION The results demonstrate that grafted embryonic D A neurons can compensate some of the deficits resulting from the lesion not only of the nigrostriatal system as previously demonstrated by other groups 2.
141 grafting and the degree of subsequent restoration of amphetamine response by the graft as judged from the pre-grafting response to a p o m o r p h i n e of the G1 and G2 subgroups (Fig. 4). Although the significance of this correlation is obscure it could relate to the
rons have been shown to stimulate the growth of D A neurons in culture 17. This hypothesis will be tested in subsequent experiments. ACKNOWLEDGEMENTS
mechanism of induction of the differentiation of the graft by the denervated sites. From this point of view, it is interesting to note that the production of a neuronotrophic factor by denervated nervous tissue has
We would like to thank Dr. R. D. Oades for his help with the English version of this manuscript. This work was supported by I N S E R M G r a n t 133030.
been recently demonstrated TM and that striatal neu-
REFERENCES 1 Archer, J., Tests for emotionality in rats and mice: a review, Anita. Behav., 21 (1973) 205-235. 2 Bj6rklund, A. and Stenevi, U., Reconstruction of the nigrostriatal dopamine pathway by intracerebral nigral transplant, Brain Research, 177 (1979) 555-560. 3 Chronister, R. B., Sikes, R. V., Tron, T. V. and De France, J. F., The organization of the nucleus accumbens. In R. B. Chronister and J. F. De France (Eds.), The Neurobiology of the Nucleus Accumbens, Haer Institute for Electrophysioiogical Research, 1981, pp. 97-146. 4 Dunnett, S. B., BjSrklund, A., Stenevi, U. and Iversen, S. D., Behavioural recovery following transplantation of substantia nigra in rats subjected to 6-OHDA lesions of the nigro-striatal pathway. I. Unilateral lesions, Brain Research, 215 (1981) 147-161. 5 Dunnett, S. B., Bj6rklund, A., Stenevi, U. and Iversen, S.D., Behavioural recovery following transplantation of substantia nigra in rats subjected to 6-OHDA lesions of the nigro-striatal pathway. II. Bilateral lesions, Brain Research, 229 (1981) 457--470. 6 Fallon, J. H. and Moore, R. Y., Catecholamine innervation of the basal forebrain and neostriatum, J. comp. Neurol., 180 (1978) 545-580. 7 Fekete, M. I. K., Kanyisca, R. and Herman, J. P., Simultaneous radioenzymatic assay of catecholamines and dihydroxyphenylacetic acid (DOPAC); comparison of the effects of drugs on the tubero-infundibularand striatal dopamine metabolism and on plasma prolactin level, Life Sci., 23 (1978) 1549-1556. 8 Freed, W. J., Morihisa, J. M., Spoor, E., Hoffer, B. J., OIson, L., Seiger, A. and Wyatt, R. J., Transplanted adrenal chromaffin cells in rat brain reduce lesion-induced rotational behaviour, Nature (Lond.), 292 (1981) 351-352. 9 Gash, D. and Sladek, J. R., Jr., Vasopressin neurons grafted into Brattleboro rats: viability and activity, Peptides, 1 (1980) 11-14. 10 Kelly, P. H., Seviour, P. W. and Iversen, S. D., Amphetamine and apomorphine response in the rat following 6OHDA lesions of the nucleus accumbens septi and corpus striatum, Brain Research, 94 (1975) 507-522. 11 Koob, G. F., Stinus, L. and Le Moat, M., Hyperactivity and hypoactivity produced by lesions to the mesolimbic dopamine system, Behav. Brain Res., 3 (1981) 341-359. 12 Le Moal, M., Stinus, L., Simon, H., Tassin, J. P., Thierry, A. M., Blanc, G., Giowinski, J. and Cardo, B., Behavioral effects of a lesion in the ventral mesencephalic tegmentum:
evidence for involvement of A10 dopaminergic neurons. In E. Costa and G. L. Gessa (Eds.), Non-Striatal Dopaminergic Neurons, Advanc. Biochem. Psychopharmacol., Vol. 16, Raven Press, New York, 1977, pp. 237-245. 13 Low, W. C., Lewis, P. R., Bunch, S. T., Thomas, S. R., Iversen, S. D., Bj6rklund, A. and Stenevi, U., Function recovery following neural transplantation of embryonic septal nuclei in adult rats with septo-hippocampal lesion, Nature (Lond.), 300 (1982) 260--262. 14 Nieto-Sampedro, M., Lewis, E. R., Cotman, C. W., Manthorpe, M., Skaper, S. D., Barbin, G., Longo, F. M. and Varon, S., Brain injury causes a time-dependent increase in neurotrophic activity at lesion site, Science, 217 (1982) 860-861. 15 Oades, R. D., Search strategies on a hole board are impaired in rats with ventral tegmental damage: animal model for tests of thought disorder, Biol. Psychiat., 17 (1982) 243--258. 16 Perlow, J. J., Freed, W. J., Hoffer, B. J., Seiger, A., O1son, L. and Wyatt, R. J., Brain grafts reduce motor abnormality produced by destruction of nigro-striatal dopamine system, Science, 204 (1979) 643-647. 17 Prochiantz, A., Diporzio, U., Kato, A., Berger, B. and Glowinski, J., In vitro maturation of mesencephalic dopaminergic neurons from mouse embryo is enhanced in presence of their striatal target cells, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 5387-5391. 18 Schmidt, R. H., Bj6rklund, A. and Stenevi, U., Intracerebral grafting of dissociated CNS tissue suspensions: a new approach for neuronal transplantation to deep brain sites, Brain Research, 218 (1981) 347-356. 19 Simon, H., Le Moal, M., Galey, D. and Cardo, B., Silver impregnation of dopaminergic system after radiofrequency and 6-OHDA lesions of the rat ventral tegmentum, Brain Research, 115 (1976) 215-231. 20 Simon, H., Le Moal, M. and Calas, A., Efferents and afferents of the ventral tegmental A10 region studied after local injection of [3H]leucine and horseradish peroxidase, Brain Research, 178 (1979) 17-40. 21 Simon, H., Scatton, B. and Le Moal, M., Dopaminergic A10 neurons are involved in cognitive function, Nature (Lond.), 286 (1980) 150--151. 22 Stevens, J. R., An anatomy of schizophrenia? Arch. Gen. Psychiat., 29 (1973) 177-189. 23 Thierry, A. M., Blanc, G., Sobel, A., Stinus, L. and Glowinski, J., Dopaminergic terminals in the rat cortex, Science, 182 (1973) 499-501.
137
Elsevier BRE 10129
Functional Recovery Following Transplantation of Ventral Mesencephalic Cells in Rat Subjected to 6-OHDA Lesions of the Mesolimbic Dopaminergic Neurons D. NADAUD, J. P. HERMAN, H. SIMON and M. LE MOAL Laboratoire de Neurobiologie des Comportements (1NSERM U. 259), Universit~de Bordeaux II, 146 rue L~o-Saignat, 33600 Bordeaux (France)
(Accepted November 29th, 1983) Key words: grafts - - mesolimbic system - - dopamine - - functional recovery - - open-field - - amphetamine-induced hyperactivity - -
apomorphine
Lesion of mesolimbic dopaminergic neurons induces hypoactivity and hypo-exploration in a new environment, lack of hyperactivity to amphetamine and hyperresponsiveness to apomorphine. Transplantation of embryonic dopaminergic cells from rat embryos into the deafferented nucleus accumbens restores, 8 weeks after grafting, some of the deficits obtained after the lesion. Spontaneous activity and behavioral responses in an open-field are improved and some of the grafted rats show a complete recovery of amphetamine-induced hyperactivity. However, hyperresponsiveness to apomorphine has not been significantly reduced by the treatment. These resuits indicate that transplanted dopaminergic neurons can restore some of the functional activities of the n. accumbens. INTRODUCTION The ability of neuronal grafts to restore functions lost after brain damage has been demonstrated for several neurochemical systems such as the hypothalamic peptidergic 9, septohippocampal cholinergic 13 and dopaminergic ( D A ) nigrostriatal (NS) neurons. Motor deficits such as circling behavior induced in rats after unilateral NS lesion are reduced by implantation of nigral tissue2,16 or adrenal medulla8 or injection of a mesencephalic cell suspension 18 in the vicinity of or into the denervated striatum. However, other deficits such as akinesia and aphagia obtained after bilateral NS lesion are not reduced by the graftsS. Curiously, no investigation has been done on the mesolimbic D A neurons located in the ventral tegmental area (VTA) that project to limbic-related structures (head of the caudate nucleus, nucleus accumbens, septal area, amygdala, hippocampus) and to prefrontal, cingulate and entorhinal cortices6A9, 20,23. These neurons modulate regions the function of which is necessary for adaptive behavior, initiation of action and for regulating cognitive-representational processes~2,15,21. Moreover, mesolimbic transmission has been implicated in the pathogenesis of psychopathological states22. A n impairment of D A transmis0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
sion in the n. accumbens results in deficits such as hypoactivity, hypoexploration, disappearance of amphetamine-induced hyperactivity and supersensitive locomotor responses to apomorphinel0, n. In this paper, we present results indicating that injections of D A cell suspension within the n. accumbens reduce some of the deficits obtained after mesolimbic cell bodies lesions. MATERIALS AND METHODS Male Wistar rats weighing 160--180 g at the beginning of the experiment were used. For the lesions of the mesolimbic D A neurons, animals were injected bilaterally with 6-hydroxydopamine ( 6 - O H D A ) into the V T A (2.5 mm posterior to the bregma, + 0.5 mm laterally from the midline and 8.5 mm under the skull surface) with 6 - O H D A (4.5 pg base in 1 pl, injected over 5 min). Non-lesioned controls (n = 6) received vehicle injection only. Four weeks later, animals displaying a decrease of more than 80% of their amphetamine-induced locomotor activity relative to controls were chosen for transplantation; this degree of behavioral deficit has been shown to be associated with a greater than 90% depletion of the dopamine content of the n. accumbens 11. Grafts consisted of a
138 suspension of embryonic mesencephalic cells prepared from ventral mesencephalic D A areas dissected from rat embryos at 16-17 days of gestation. The brain tissue blocks were put into a tissue culture medium (Hank's MEM) and the cellular suspension was obtained by mechanical dissociation. This suspension (2.5-4 #1) was injected bilaterally into the n. accumbens of the experimental animals (n = 10) at two different vertical coordinates (4 mm anterior to the bregma, 0.8 mm laterally from the midline, 7 and 8 mm vertical from the surface of the skull). Lesioned controls (n = 5) consisted of lesioned animals injected with a cellular suspension prepared from pieces of parietal cortex obtained from rat embryos at 20 days of gestation. Non-lesioned controls did not receive grafts. Spontaneous and drug-induced locomotor activity was measured for 1 h in a circular corridor 12 cm wide and 170 cm long, equipped with 4 photocell beams. Locomotor records were taken 2 weeks before and 4, 6 and 8 weeks after grafting. Drugs used were D-amphetamine (1.5 mg/kg i.p.) and apomorphine (0.1 mg/kg s.c.). The apomorphine test was done 48 h following the amphetamine test. Open-field behavior was observed 7 weeks after the implantation procedure as a measure of emotional response in a new environment]. The open-field consisted of 1 x 1 m, 50 cm high white arena divided into 25 squares and illuminated with a white 100 W light bulb placed 1 m above the center of the arena. Animals were placed in the central square and total locomotor activity (number of squares entered) and rearings were recorded for 10 min. Results were expressed in locomotor activity and rearing scores by minutes. The animals were sacrificed 10 weeks after grafting. The accumbens nucleus was dissected out and tissue dopamine levels measured by a radioenzymatic assay 7. Data from spontaneous and drug-induced locomotor activity were evaluated by one-way analysis of variance (ANOVA) followed by application of multiple comparison of individual means (NewmanKeuls test). For the open-field scores a Mann and Whitney test was performed. Logarithmic transforms of the data from biochemical analyses were evaluated with an A N O V A followed by a NewmanKeuis test. A criterion of P < 0.05 was used for statistical significance.
RESULTS After treatment with 6-OHDA, spontaneous locomotion in the circular corridor (Fig. 1) decreased to 53.3% and 62.2% of control levels for, respectively, the experimental group and the lesioned controls (P < 0.05 compared to the controls, Newman-Keuls test). Following the grafting procedure spontaneous locomotor activity was restored in experimental animals (time-effect, P < 0.05, A N O V A test) while the lesioned controls showed no consistent change. In the open-field (Fig. 2), the behavior of the lesioned controls was characterized by a marked decrease in the locomotor activity and rearing scores. There was a tendency towards improvement of these scores in some of the grafted animals, but these improvements did not reach the non-lesioned control level. However these scores were higher than those of the lesioned control group both for activity and for rearing measures (P < 0.05, Mann-Whitney test). While non-lesioned animals displayed a locomotor activation upon the injection of amphetamine
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Fig. 1. Effect of grafting on the evolution of spontaneous locomotor activity measured during 1 h in a circular corridor, expressed in % of non-lesioned control level activity. NL, non-lesioned control group; L, lesioned control group; G, grafted group. Time effect: P < 0.05 in grafted group, N.S. in lesioned group (ANOVA test).
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Fig. 2. Open-field behavior: locomotor activity (left) and rearings (right) measured 7 weeks after grafting in non-lesioned control rats (NL), lesioned control rats (L) and grafted rats (G). Open circles in group G represent rats which had recovered in amphetamine-induced activity (see text and Fig. 3C). P < 0.05, grafted rats vs lesioned control rats in locomotor activity and in rearing scores (Mann-Whitney test).
(Fig. 3A, B and C), lesioned controls showed no such a response throughout the period of the experiment. On the other hand, after the grafting of D A cells there was a marked heterogeneity of responses (Fig. 3B). Some animals showed a level of activation similar to non-lesioned controls 8 weeks after the ®
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grafting and others did not show such a recovery and remained at the level of the lesioned controls. Thus, on the basis of the level of amphetamine-induced activity at 8 weeks, we subdivided, a posteriori, the DA-grafted group into two subgroups. Animals showing less than a 50% drug-induced activity with respect to non-lesioned controls were allocated to group G1 (n = 5) and those showing more than 50% level to group G2 (n = 5). Group G1 displayed no responsiveness to amphetamine during the time of the experiment and was not significantly different from the lesioned control group while group G2 showed a gradual improvement of its amphetamine response and in fact reached the level of response of the nonlesioned control group at 8 weeks (Fig. 3C). This evolution was different from that of the lesioned control group (P < 0.05, A N O V A test). This same subdivision within the grafted group did not permit to •differentiate between G1 and G2 for the spontaneous locomotor activity and for the open-field scores. Lesion of the mesolimbic D A neurons induced a post-synaptic dopamine receptor supersensitivity as demonstrated by the increased response to apomorphine (Fig. 4). The hyperresponsiveness to apomorphine remained after grafting• However, a closer examination of the results revealed a difference in the pre-grafting scores of the two subgroups in that before grafting animals of the G2 subgroup displayed a significantly greater response to apomorphine than
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q
weeks
Fig. 3. Locomotor activity measured during 1 h in a circular corridor in response to D-amphetamine (1.5 mg/kg i.p.) in nonlesioned control rats (NL), lesioned control rats (L) and grafted rats (G) before (A) and after (B) grafting• Open circles represent rats which were allocated to the recovered group G2. In C, evolution of amphetamine-induced locomotor activity in the two control groups, NL and L, and in the two grafted groups, G1 (uncompensated) and G2 (compensated). P < 0.05, G2 group vs lesioned control group (ANOVA test).
0
Before Grafting
After
Grafting Fig. 4. Locomotor activity measured during 1 h in a circular corridor in response to apomorphine (0.1 mg/kg s.c.) in non-lesioned control group (NL), lesioned control group (L), grafted group G1 (uncompensated) and grafted group G2 (compensated) before and 8 weeks after grafting. P < 0.05, G2 group vs G1 group before grafting (Newman-Keuls test).
140 TABLE I Dopamine and DO PA C concentrations in nuclei accumbens reinnervated by ventral mesencephalic tegmentum dopamine graft after 6-hydroxydopamine lesions
The concentrations are expressed as ng/mg tissue (mean -+ S.E.M.). DOPAC/DA ratio was calculated from the individual DOPAC and dopamine values for each sample (mean + S.E.M.). For statistical comparison every group was compared with every other: * and ** represent statistical difference (P < 0.05) from non-lesionedcontrols (*) and from lesioned controls
(**).
DA
Non-lesioned controls (n = 6) 7.90_+0.47
DOPAC
DOPAC/ DA ratio
2.15_+0.25 0.27_+0.02
Lesioned controls (n = 5) 0.25+0.07* 0.11+0.03" 0.53_+0.04* Uncompensated grafted rats (group G1) (n = 5)
0.35_+0.05*
0.17_+0.05" 0.44_+0.10
Compensated grafted rats (group G2) (n = 5)
but also of the mesolimbic system as shown by the significant restoration of the amphetamine-induced locomotor response. The tendency towards improvement of spontaneous locomotor activity and of open-field activity and exploration points in the same direction. The slight decrease of locomotor response to apomorphine in the G2 group could also correspond to a partial restoration of post-synaptic DA receptor sensitivity towards normal levels. But this point should be further examined in future experiments. The very low degree of restoration of dopamine level at 10 weeks in the compensated group compared to dopamine level of non-lesioned group is striking. However, it seems, at least in the case of striatal transplant, that a quite slight restoration of dopamine level can be sufficient to restore amphetamine induced behaviors TM. Moreover, there was no significant correlation between the D A level within the nucleus accumbens and the locomotor or the open-field tests in the grafted group (G1 + G2). It is of importance to notice two points at this stage. First, there is no correlation between the recovery scores in the different tests. While all grafted animals showed a tendency to recover spontaneous locomotor activity, only some of them (G2 subgroup) displayed a restored locomotor response to amphetamine. Likewise, the open-field scores were not correlated with the amphetamine scores (see Fig. 2). Some animals having amphetamine-induced locomotor activity like non-lesioned controls showed no improvement at all in their open-field behavior; conversely, some animals having no recuperation in the pharmacological test displayed mild improvement in the open-field test. This is reminiscent of the effects found after striatal grafts, i.e. marked improvement in pharmacological responses (decrease in amphetamine-induced turning) and no improvement in aphagia-adipsia and sensory neglect4, 5. This lack of correlation could indicate that either the grafts in the different animals re-innervated functionally different parts of the n. accumbens, known to be anatomically heterogeneous 3, or that, more complex responses, such as occur in open-field behavior, require more than the simple presence of cells secreting dopamine in a non-regulated way in the n. accumbens. Secondly, it seems that there is a correlation between the hypersensitivity of postsynaptic D A receptors before 4,5,7,16
0.96+0.17".** 0.30_+0.08* 0.32+0.05
animals of the G1 subgroup (P < 0.05, NewmanKeuls test). This difference disappeared at 8 weeks after the grafts and the apomorphine response of the G2 group was at the same level than the G1 group due to a slight decrease of supersensitive response in the G2 group. Biochemical measurements (Table I) revealed a low level of dopamine in the nucleus accumbens of group G1 (uncompensated grafted group) - - a value identical to that of lesioned non-implanted controls - - respectively, 4.4% and 3.2% of the non-lesioned controls. On the other hand the levels of dopamine and D O P A C were increased in the compensated group G2, comparatively to lesioned control values (P < 0.05 for DA, Newman-Keuls test) although they remained far below control levels (12.2%). DISCUSSION The results demonstrate that grafted embryonic D A neurons can compensate some of the deficits resulting from the lesion not only of the nigrostriatal system as previously demonstrated by other groups 2.
141 grafting and the degree of subsequent restoration of amphetamine response by the graft as judged from the pre-grafting response to a p o m o r p h i n e of the G1 and G2 subgroups (Fig. 4). Although the significance of this correlation is obscure it could relate to the
rons have been shown to stimulate the growth of D A neurons in culture 17. This hypothesis will be tested in subsequent experiments. ACKNOWLEDGEMENTS
mechanism of induction of the differentiation of the graft by the denervated sites. From this point of view, it is interesting to note that the production of a neuronotrophic factor by denervated nervous tissue has
We would like to thank Dr. R. D. Oades for his help with the English version of this manuscript. This work was supported by I N S E R M G r a n t 133030.
been recently demonstrated TM and that striatal neu-
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