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Influence of Environment on the Efficacy of Intrastriatal Dopaminergic Grafts
Experimental Neurology 165, 172–183 (2000) doi:10.1006/exnr.2000.7462, available online at http://www.idealibrary.com on
Influence of Environment on the Efficacy of Intrastriatal Dopaminergic Grafts Ma`te´ Da`niel Do¨bro¨ssy, Michel Le Moal, Marie-Francoise Montaron, and Nora Abrous Domaine de Carreire, INSERM U259, Rue Camille Saint Sae¨ns, Bordeaux Cedex 33077, France Received November 17, 1999; accepted April 12, 2000
Functional recovery is influenced by experience. The aim of the present work was to examine the effects of “enriched” environment (EE) versus an “impoverished” environment on the anatomical and functional integration of intrastriatal dopaminergic grafts. These influences were studied using a paradigm where grafting was performed before the dopamine-depleting lesion. Dopaminergic grafts were implanted into the left neostriatum of adult male rats. In the enriched group, grafted rats were housed collectively and were trained on different behavioral tests following grafting. In contrast, impoverished grafted rats were housed individually and not further manipulated. Ten weeks after grafting, the mesotelencephalic dopaminergic pathway was destroyed unilaterally to the grafted side and different behaviors were followed for 7 months. Grafting prior to lesioning had no prophylactic effects on the performance as the graft did not prevent the onset of the lesion-induced impairments. However, under EE conditions, a graft effect was manifested in the reduction of drug-induced rotation and on the indices of bias as tested by a spatial alternation test. No positive graft effects were observed in the skilled paw reaching test. Grafted rats raised under impoverished conditions performed in a fashion indistinguishable from the control lesioned animals on most measures of behavior. A beneficial effect of EE conditions was observed on survival of TH-positive neurons within the grafts. The results suggest that survival of grafted neurons, and the reduction of the magnitude of particular behavioral impairments, can be optimized by increasing the complexity of the subject’s environment. ©
2000 Academic Press
Key Words: dopaminergic grafts; 6-OHDA lesion; rotation; skilled paw reaching; spatial alternation; enriched and impoverished environment.
INTRODUCTION
Neural transplantation addresses the question of functional recovery following brain damage and neurodegenerative disorders, in particular Parkinson’s dis0014-4886/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
ease (PD). Two main experimental avenues are pursued in order to optimize the transplantation-induced benefits. The first approach focuses on aspects related to the microenvironment of the grafts (for review see 9). For this purpose “bridge” grafts (8, 14, 76), coimplants and trophic factors (12, 29), or alternative source of neurotransmitter-producing cells (38, 57) are used to improve the anatomical and functional integration of the grafts. A second approach, at a more integrated level, considers the effects of various environmental factors applied before or after a lesion (for review see 77) on the behavioral recovery induced by the transplants. Experience modulates brain function (23–25). Rearing conditions that increase the level of stimulation, referred to as “enriched” environment, have been shown to improve behavioral performance (22, 31, 41, 42, 53, 59, 61, 74, 78). Complex environments have been implicated in reducing age-related impairments (17, 43, 64) and recovery from brain damage (18, 35, 54). In contrast, animals deprived of environmental stimulation, i.e., raised in an impoverished environment, have exhibited behavioral anomalies (26 –28, 36). Data on reconstructive neurosurgery and neurological rehabilitation in humans are not conclusive (for review see 56). The benefits of behavioral therapy for PD are recognized; however, the effects of such therapy on the efficacy of intrastriatal grafts are unknown (46, 51, 66, 73). In animals, experiments investigating the combined effects of enriched environment and neuronal transplant have focused mainly on housing conditions (39), pretesting experience (9, 10), postgraft training (7, 46 – 47), and priming the graft by pharmacological treatments (70). Dominantly, these studies using striatal, septal, fimbria-fornix, and neocortical or occlusion models have reported beneficial effects of enriched experience and training (20, 39 – 40, 47, 55, 71). Exploration of this question in the context of the 6-hydroxydopamine (6-OHDA) parkinsonian model and dopaminergic grafts is sparse. This question is important regarding the circumstances that maximize the functional benefits of grafts in the parkinsonian model. In
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turn, it could improve the outcome and consistency of the clinical application of neural transplantation. Our study compared the anatomical and functional benefits of dopaminergic grafts implanted into animals subjected to different environmental stimulation involving different housing condition and experience. In the enriched environment (EE) condition rats were collectively housed providing social interaction, regularly handled, and exposed to various behavioral tests before lesioning. In the “impoverished” environment (IE) condition the animals were individually housed, minimally handled, and deprived of prelesion training. The environmental influences were studied using a prophylactic paradigm where grafting was performed in the intact neostriatum before the dopamine-depleting lesion (4, 58, 67). This approach allowed us to test the effect of the environment on the transplant without the lesion-induced alterations. The behaviors studied were drug-induced rotation, skilled forelimb use, and spatial alternation in a two-lever operant chamber. The survival of dopaminergic neurons was examined by tyrosine hydroxylase (TH) immunohistochemistry. METHODS AND MATERIALS
Subjects and Housing Conditions Male Spague–Dawley rats (n ⫽ 60; Iffa-Credo, Lyon, France) weighing 250 g at the start of the experiment were used in the study. Animals in the EE (n ⫽ 30) group were housed collectively with four rats in transparent cages. On the side of the collective cages various drawings of geometrical objects of different hues and sizes were exposed. EE animals were regularly manipulated. The animals in the IE (n ⫽ 30) were housed individually in opaque cages and were not handled other than when the litter was changed. All the animals were kept in the same temperature (24°C) and humidity (50%) controlled room with a 12:12-h light cycle (lights on at 8:00 AM). Transplantation Grafting was performed 3 weeks after the arrival of the animals. The age of the host animals at the time point of transplantation was 3 months. Mesencephalic tissue from embryonic day 14 rat embryos were dissected out under phosphate-buffered saline (PBS, pH 7.2) and collected in cold PBS supplemented with 10 mM glucose. Pooled tissue pieces were submitted to a 5-min enzymatic digestion at 20°C using 1% trypsin (Worthington, UK) in PBS with 0.1% DNase (Sigma, France). After washing with PBS, a synthetic culture medium was added (S-MEM-Hepes, Gibco-BRL, France), supplemented with 0.02% trypsin inhibitor (Sigma, France), and a cellular suspension was obtained by repeated aspiration through a small-bore
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Pasteur pipette. Following a brief centrifugation (600g, 10 min), cells were resuspended in S-MEM-Hepes containing 0.2% bovine serum albumin (Sigma, France). The final suspension was kept on ice in siliconized glass tubes and was used within 6 h. Grafting was carried out under chloral hydrate anesthesia (60 mg/ml of saline, administered at a dose 0.5 ml/100 g). A 2-l aliquot of the cell suspension was injected stereotaxically into the left ventrolateral striatum of the rat using a glass cannula (100 m external diameter) connected to a 10-l Hamilton syringe at the following coordinates: A ⫽ ⫹2.5, L ⫽ ⫹2.5, V ⫽ ⫺6.0. Measurements were taken from the level of the skull, with the interaural line at ⫹5. The graft was delivered over a period of 3.5 min with an additional 4 min prior to the retraction of the cannula. The number of injected viable cells was 150,000 cells/l, as evaluated by dye exclusion. Animals were grafted from the same suspension by two investigators on the same day. The different groups were randomized during the transplantation. Sham-grafted animals received injections of the 2 l culture medium. Lesion Surgery Ten weeks after grafting, animals were anesthetised with chloral hydrate as described above. The left mesotelencephalic dopaminergic pathway was lesioned by stereotaxical injection a total of 12 g of 6-hydroxydopamine (6-OHDA, Sigma) dissolved in isotonic saline containing 0.005% of ascorbic acid (pH 4.0). The neurotoxin was delivered through two 30-gauge stainlesssteel cannula which were at a fixed distance of 1 mm apart with one of the cannula being 1 mm shorter than the other. The double cannula was lowered into the brain with the two cannulae in plane with each other with respect to the AP axis. The coordinates taken from the anterior cannula were A ⫽ 0.0, L ⫽ ⫹1.6, V ⫽ ⫺9.0; the measurements were made from the level of the skull with the interaural line at ⫹5. The rate and volume of delivery of the neurotoxin through each cannula was 0.3 l/min over 5 min, i.e., a total of 3 l of solution. The cannulae were left in place for an additional 3 min to allow for the diffusion of the neurotoxin from the injection site. Sham-lesioned animals received injections of isotonic saline containing ascorbic acid. Rotation Drug-induced turning behavior was studied in rats using transparent hemispheric bowls (diameter 40 cm). Rats were attached with a harness to a flexible wire linked to a photocell system. The number of 360° turns in either direction were recorded automatically during 60-min sessions, and the result expressed as net scores (ipsilateral minus contralateral rotations per minute). D-Amphetamine (Sigma; 5.0 mg/kg) was in-
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jected ip, and apomorphine was injected sc (Aguettan, France; 0.05 mg/kg). Paw Reaching Paw reaching was measured using a modified version of the “staircase” test apparatus described by Montoya et al. (50). The paw reaching box consists of two chambers fixed to each other end to end. The first into which the rat is placed is a transparent Perspex chamber (203 mm long by 108 mm high by 103 mm wide) with a hinged lid. The second chamber is smaller (165 mm long by 108 mm high by 60 mm wide) and it contains a central platform running along its length creating a 19-mm-wide trough on either side. The narrowness of the chamber limits the animal’s left paw to reach into the left trough and the right paw only into the right trough. The top surface of the central platform (35 mm wide) overhangs the sides preventing the animal from scraping pellets up on the side of this platform. A removable double staircase is inserted at the end of the chamber into the troughs to either side of the central platform, with the highest stair 13 mm and the lowest 64 mm below the platform. Both sides of the staircase contain eight 3 mm deep wells which were each baited with two 45-mg food pellets, giving the rats an maximum of 16 pellets per side. The animal is able to retrieve the pellets by reaching into the trough. The number of pellets it collects from either side being an index of the rat’s reaching ability, reflecting its success in grasping and retrieving the pellets. Rats were left to habituate to the boxes on the first day of testing and the pellets taken were not counted. On the following days rats were placed into the chamber for 15 min at the end of which the pellets that remained on the staircase on either side were counted. Animals received one trial per day for 16 days. The schedule was the same during the postgrafting and postlesioning testing sessions. Skinner Box Testing: Spatial Alternation Task Training and testing were conducted in operant chambers (Imetronic, Bordeaux, France), with a house light, a central food well, and two retractable levers. The central food well was on the wall opposite to the levers. The Skinner boxes were controlled by a Windows version of Immetronic’s software and linked to the operant chambers by Immetronic’s interface. Rats were habituated to the operant chambers over 7 days and trained to nose poke to collect food from the central food well. Animals learned to press the levers for food pellets over 3 days. The spatial alternation task is a two-choice, continuous nonmatching paradigm. The task involves presenting animals with both sample levers on all trials and rewarding the animal if it selects the lever opposite to the one which was rewarded on the trial before.
On the first trial the reinforced lever was randomly selected, and from then on each following trial a winshift response pattern was rewarded by the delivery of a 45-mg food pellet into the lit central food well located in the opposite wall to the levers. The collection of the pellet triggered off the next trial with the presentation of both levers. Conversely, pressing the same lever to that presented as the sample was counted as an error and resulted in a “time out” during which the house light was extinguished for 5 s. At the end of the time out period the house light came on and the two levers were extended. Following an error trial the rat was required to press the lever opposite to the side prior to the initial error. The session lasted until rats carried out either 51 reinforced trials or until the lapse of 30 min, whichever happened first. Immunohistochemistry At the completion of the behavioral testing rats were given a terminal dose of chloral hydrate (600 mg/ml saline, administered at a dose of 1.5 ml/rat) and perfused transcardially with 100 ml 0.1 M phosphatebuffered saline (pH 7.3) containing 5 ⫻ 10 4 IU/ml heparin, followed by 400 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) at a rate of 60 ml/min (Masterflex pump, U.S.A.). After 24 h of postfixation, 50-m sections were cut using a Vibratome (Vibratome System 1000, U.S.A.). Free-floating sections were processed through a standard immunohistochemical procedure in order to visualize tyrosine hydroxylase (TH) with a rabbit anti-TH primary antibody (dilution of 1/10000, Boy, Paris, France). A 48-h incubation period was followed by incubation with a biotin-labeled goat anti-rabbit secondary antibody (1/200, Dako Laboratories, Glostrup, Denmark). TH-positive immunoreactivity (IR) was visualized by the biotin–streptavidin technique (ABC kit, Dako Laboratories) using 3,3⬘-diaminobenzidine as the chromogen. Using a random start, TH-IR cells were counted in the grafts, one-in-two sections, with the sections being 100 m apart. The total number (N total) of TH cells was calculated according to the equation N total ⫽ (¥Q⫺) ⫻ thickness ⫻ (1/section sampling fraction). Experimental Design and Behavioral Testing The behavioral testing schedule is described schematically in Fig. 1. After the arrival the animals were subjected to either EE (n ⫽ 30) or IE (n ⫽ 30). Three weeks later half of the rats from both housing conditions received either embryonic mesencephalic tissue transplants (GE, n ⫽ 15; GI, n ⫽ 15) or were sham grafted receiving culture medium injections into the central striatum. Ten weeks after the transplantation procedure, the transplanted rats (i.e., GE and GI) received 6-OHDA injections into the medial forebrain bundle on the same side as the grafts. The sham-
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FIG. 1. Experimental groups and general procedures. (A) After the arrival animals were housed, grafted, or received sham grafts; lesioned; or received saline injections, depending on which experimental group they were in. (B) The behavioral procedures the animals were subjected to depended on which environmental condition applied. Animals from the enriched (EE) and the impoverished environment (IE) received mesencephalic grafts at the same time but only the former group was tested prior to the lesion, while all animals received training following the lesion.
grafted animals received either 6-OHDA (LE, n ⫽ 10; LI, n ⫽ 10) or ascorbic acid (SE, n ⫽ 5; SI, n ⫽ 5) injections. Animals were food deprived and kept at 85–90% of their normal body weight during all times apart from 1 week before and 3 days after the grafting and the lesion procedures. At these periods they had ad libitum access to food. The EE animals were tested both before and after the lesion, while the IE animals were tested on their acquisition only after the lesion. Statistical Analysis All data were analysed by ANOVA followed by post hoc comparisons when appropriate using the Newman–Keuls test (Statistica for Windows 5.1). The between factors used were environment conditions effect (two levels, enriched vs impoverished) and group effect (three levels, sham, lesion, and graft). The within fac-
tors were session effect and/or paw side effect, depending on the behavior results analyzed. RESULTS Immunohistochemistry
6-OHDA injection into the medial forebrain bundle resulted in the almost complete loss of TH-positive staining from the substantia nigra and the ventral tegmental area from the same side as where the lesion was performed. Furthermore, total loss of TH-positive staining in the neostriatum and the nucleus accumbens ipsilaterally to the lesion site was observed. The gross morphology of the grafts shown in Fig. 2 was comparable to that described previously in the literature (3, 4, 52). However, the count of TH-positive neurons from the grafted groups with different experiences
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FIG. 2. Influence of environment on dopaminergic graft survival. Animals were raised under (A) enriched or (B) impoverished conditions. TH immunohistochemistry was performed 26 weeks after grafting and 16 weeks after lesioning. G, the graft; S, the striatum; the stars mark the border between the graft and the host. Bar, 250 m.
revealed a significantly greater number of surviving dopaminergic cells among the EE grafted rats (mean ⫾ SEM, 2515 ⫾ 517) when compared to the IE grafted rats (1283 ⫾ 222) [environmental condition effect, F(1, 13) ⫽ 5.3, P ⬍ 0.05]. Drug-Induced Rotation D-Amphetamine
The results of D-amphetamine-induced rotation are shown in Fig. 3. For the EE group two sessions were conducted between the grafting and the lesioning operation. The unilaterally placed dopaminergic graft on its own did not induce any preferential side bias in the rotation after the D-amphetamine injection. Following the lesion surgery, three drug tests were performed on the EE and the IE animals. For the EE group, the analysis resulted in a significant interaction between the Group and Session factors [Group ⫻ Session, F(8, 60) ⫽ 8.1, P ⬍ 0.001]. Subsequent New-
man–Keuls tests for multiple comparisons indicated that the lesion induced a significant increase in the ipsilateral rotation score for the LE and GE groups, with respect to the sham-lesioned SE group. The increase was apparent during the first two testing sessions (in both testing times SE ⬍ LE, GE, P ⬍ 0.001). At these time points there was no statistical difference between the LE and the GE groups. Nevertheless, on the final session carried out 3 months after the lesion, a graft effect was observed in the GE group as manifested by a decrease in the ipsilateral rotation with respect to the LE (LE ⬍ GE, P ⬍ 0.05) and approached the level of the SE group so as to abolish any statistical difference between them (SE, GE, P ⫽ 0.075, n.s.). For the IE group, the lesion induced an ipsilateral rotation bias resulting in a Group effect, which consequent post hoc analysis showed to be between the GI/LI groups and the sham control [Group, F(2, 16) ⫽ 17.9, P ⬍ 0.001; post hoc, LI ⬍ SI, P ⬍ 0.001; and GI ⬍ SI, P ⬍ 0.001]. Nevertheless, contrary to that reported for the
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FIG. 3. Influence of environment on amphetamine-induced rotation. The two prelesion tests were carried out (A) 2 and (B) 3 weeks following grafting. The three postlesion tests were done (C) 1, (D) 2, and (E) 12 weeks following the lesion. Results are expressed as the number of ipsilateral turns minus contralateral turns per minute (mean ⫾ SEM). **P ⬍ 0.01 compared to the matched lesion group. SE, sham enriched; LE, lesion enriched; GE, graft enriched; SI, sham impoverished; LI, lesion impoverished; GI, graft impoverished.
EE animals, the IE rats with grafts did not manifest a diminution of the ipsilateral rotation on the final Damphetamine rotation session. Overall, the environmental condition on its own did not affect the magnitude of the ipsilateral rotation [Housing, F(1, 31) ⫽ 0.04, n.s.]. However, the reduction in ipsilateral rotation on the final testing session uniquely in the EE grafted animals, accompanied by the absence of any effect in the IE animals, results in a three-way interaction between the environmental condition, treatment, and session that approaches statistical significance [Environment ⫻ Group ⫻ Session, F(4, 62) ⫽ 2.35, P ⫽ 0.06]. Apomorphine The contralateral rotation displayed by the animals treated with apomorphine (0.05 mg/kg) is shown in Fig. 4. For the EE group, apomorphine induced a significant
Group effect [Group, F(2, 15) ⫽ 4, 9, P ⬍ 0.05]. Further post hoc analysis indicated a significant difference between the SE and the LE animals, with the GE animals not differing from either of the two (SC ⬍ LC, P ⬍ 0.05). Among the IE rats, the treatment did not result in any main effects. The rotation was more pronounced in the lesioned EE then among the lesioned IE rats; however, this did not reach significance. Skilled Paw Reaching
Prelesion EE vs Postlesion EE The results are illustrated in Fig. 5. Prior to the lesion (Fig. 5, left panel, prelesion testing) the SE and LE animals showed no significant paw bias, whereas for the GE animals the graft on its own enhanced the performance by favoring the pellet collection by ipsilateral paw with respect to the contralateral paw. This
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FIG. 4. Influence of environment on apomorphine-induced rotation. Results are expressed as the number of ipsilateral turns minus contralateral turns per minute (mean ⫾ SEM). *P ⬍ 0.05 in comparison with SE. For abbreviations see the legend to Fig. 3.
graft effect is more precisely described as beneficial to the ipsilateral paw then as impairing to the contralateral paw, and it does not reach statistical significance. The lesion induced a contralateral deficit as shown by the increase in the number of remaining pellets, effecting the contralateral side in particular [Session, F(1, 30) ⫽ 6.5, and Side, F(1, 30) ⫽ 14.2, both P ⬍ 0.05]. The bias toward the ipsilateral side was present in both the LE and the GE groups equally; i.e., the graft did not prevent the onset of the contralateral impairment in the skilled paw reaching test (post hoc, postlesion, ipsilateral versus contralateral sides within group for LE and GE animals, P ⬍ 0.05).
showed poor overall acquisition of the task which resulted in equally poor performance with both paws. [Group, F(2, 16) ⫽ 2.2, n.s.]. Comparing the performance of the EE and IE animals there was a strong environmental component observed [Environment, F(1, 31) ⫽ 12.5, P ⬍ 0.05]. There was no side bias following the lesion amongst the IE rats [Side, F(1, 16) ⫽ 0.6, n.s.].
Postlesion IE vs Postlesion EE
Prelesion EE vs Postlesion EE
As shown in Fig. 5 (right panel) animals of the different groups raised under impoverished conditions
Time to finish the task (Fig. 6A). Prior to the lesion animals from all three EE groups completed the task at a similar rate. The lesion induced a significant increase in the time to complete the task in the LE and GE groups with respect to the SE [Group ⫻ Session, F(2, 28) ⫽ 6.6, P ⬍ 0.05]. The decreased time for the SE animals reflects the effect of the additional training during the second testing session. Pellets collected and bias index (Figs. 6B and 6C). In neither of these two measurements were there any main effects, although the lesion introduced greater variability with the two groups. Although it did not reach statistical significance, the bias measure showed an ipsilateral tendency for the LE and a contralateral bias for the GE rats. Total lever presses (Fig. 6D). Before the lesion the number of lever presses the animals made were indistinguishable. The surgery introduced a mild increase in the total number of lever presses executed by the LE and GE groups, while the SE animals showed a re-
FIG. 5. Influence of environment on paw reaching. The number of leftover pellets following the testing session out of an initial of 16 per side is represented. The lower the score, the better the performance. The means of the last 5 days are shown (mean ⫾ SEM). *P ⬍ 0.05 in comparison with the ipsilateral paw. For abbreviations see the legend to Fig. 3.
Spatial Alternation in the Skinner Box
Results of the spatial alternation task are summarized in Fig. 6.
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DISCUSSION
FIG. 6. Influence of environment on spatial alternation task. The parameters measured were (A) the time to complete the test, (B) the number of pellets taken during a session, (C) the response bias, and (D) the number lever presses executed per session. The test lasted either 1800 s or until 51 pellets were collected, whichever came first. The results are expressed as the mean of the last five sessions (mean ⫾ SEM). *P ⬍ 0.05 when comparing the groups indicated on the graph. Detailed statistical analysis are included under Results. For abbreviations see the legend to Fig. 3.
duced lever pressing frequency [Group, F(2, 28) ⫽ 3.2, P ⬍ 0.05; post hoc, SE ⬍ LE and GE, P ⬍ 0.05]. Postlesion EE vs Postlesion IE Time to finish the task. The latency to finish the task was not influenced by the environmental conditions, although there was a tendency for the IE rats to be slower. The main factor was the surgical manipulation, with the lesioned and the grafted rats both being equally effected in either group [Group, F(1, 29) ⫽ 16.3, P ⬍ 0.05]. Pellets collected and bias index. The number of pellets collected, reflecting the number of successful responses, had a strong environmental component. All the EE rats performed significantly better then the IE rats on this measurement collecting nearly twice as many pellets [Environment, F(1, 29) ⫽ 5.1, P ⬍ 0.05; Environment ⫻ Group, F(2, 29) ⫽ 1.1, n.s.]. There were no main effects with respect to the bias index. Total presses. The number of lever presses during the session is an index of the animals’ activity and engagement in the task. Animals of the EE condition executed more lever presses then the IE rats [Environment, F(2, 29) ⫽ 5.1, P ⬍ 0.05]. Furthermore, while for the LE and GE rats the lesion augmented the rats of response with respect to the control animals, for the LI and the GI rats the response rates decreased [Environment ⫻ Group, F(2, 29) ⫽ 3.1, P ⬍ 0.05].
Influences of environmental manipulations on the efficacy of intrastriatal dopaminernergic grafts were compared between two extreme conditions: (i) EE, i.e., collective housing, intense manipulation and training during the period separating the grafting and the lesion procedures, and (ii) IE, i.e., individually housing, absence of manipulation during the postgraft recovery period. We have shown that: (i) more grafted cells survived in the neostriatum of grafted animals raised under EE when compared to those of IE; (ii) the grafts did not prevent the onset of the lesion-induced impairments in either the EE or the IE held rats; and (iii) the differential environmental experiences resulted in different functional effects for the grafted and the control animals. The results suggest that the behavioral performance reflects the influence of the environment and the training schedule on the animal. Also, it shows that the environment and the training schedule are factors that interact with the transplant. Enriched Environment Increases Survival of Dopaminergic Grafted Neurons It is not the environment per se that influences brain anatomy and chemistry, but the active participation and interaction of the animal with the environment (23, 60, 75). In the context of grafting, animal models of Huntington’s disease indicate that the functional benefits induced by intracerebral grafts can be positively influenced by postgrafting experience (7, 46, 47). In the current experiment the housing conditions and experience differentially affected survival of the grafted TH neurons. Almost twice the number of cells survived in the neostriatum of the EE rats, exposed to more “stimulating” conditions and more intense training schedules, than in the IE rats. The difference is not due to further division after grafting because it has been demonstrated that dopamine cells in nigral grafts do not undergo mitosis after implantation within the striatum (1, 69). Thus, it may be on grounds of enhanced survival of the DA neurons, the majority of which otherwise die the week following grafting in part due to the toxic events within the host environment (15, 68). It has been shown that enriched environment increases the level of trophic factors (72, 78) and can protect neurons from cell death (78). Thus, a more favorable physiological environment for the survival of the transplanted dopaminergic neurons—in terms of availability of trophic factors or/and less toxicity—may be present for the EE animals during the period critical for cell survival. A study comparing the microenvironments of the two experimental groups into which the graft is placed would need to be done to shed light on this question.
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The IE rats were isolated and were manipulated much less during the experiment. Prolonged isolation housing has been characterized by several structural and physiological changes including the alteration of the activity of the hypothalamic–pituitary–adrenocortical axis (HPA) (36, 37, 44, 65). The changes to the activity of the HPA could have occurred during the 3 weeks prior to the grafting. One of the effects of the differential changes to the HPA activity induced by the different housing conditions could have been alterations to the levels of corticosterone secretion or to the mechanisms of stress adaptation. Consequences of impaired or less responsive stress adaptation would be particularly evident following the traumatic event of graft surgery. A hypersecretion of corticosterone has been described following social isolation (5, 19, 21) and an excessive release of corticosterone has been reported to compromise neuronal survival (48) via a blockage of glucose uptake and an induction of hyperpolarization (33, 34). Elevated levels of corticosterone during the early stages following the transplantation could have compromised the survival of the grafts. It has been shown in vitro that mesencephalic glia, which were present in the cell suspension along with the neurons, secrete factors that support the survival of cultured dopaminergic neurons and are regulated by corticosterone in a dose-dependent manner (16). These studies demonstrated that elevated levels of corticosterone reduced dopaminergic cell survival in culture by reducing dopaminergic growth factor secretion by the glia. The presence of glucocorticoid receptors on mesocorticolimbic dopaminergic neurones themselves has been described (30), and corticosterone is known to modulate dopamine release both in vivo (62) and in vitro (63). These data suggest that an excessive release of corticosterone in the IE rats could have had deleterious effects on the survival of the implanted dopaminergic cells. Similar restrictive conditions, including sensory deprivation, have been shown elsewhere to prevent the integration of neocortical grafts with the host brain (6). Functional Effects of Dopaminergic Grafts Are Influenced by Experience Drug-induced rotation. The ability of mesencephalic dopaminergic grafts to reduce and overcompensate for 6-OHDA lesion-induced ipsilateral turning behavior induced by D-amphetamine is well documented (for review see 32). When graft implantation occurs prior to the lesion, reduction and overcompensation in rotation is delayed (4). The current study was terminated 4 months, rather then 7 months, following the lesion. However, extrapolating the data suggests that eventual overcompensation would have happened. The mechanism of action for the delayed graft response is believed to be due to a less vigorous, yet equally exten-
sive, fiber outgrowth from the graft to host. The slower rate of fiber extension could be explained by the age and the conditions under which the grafted neurons emit their projections. The grafts did not offer protection against the development of a drug-induced postural asymmetry. Nevertheless, a graft effect was observed 12 weeks following the lesion uniquely in the EE group. The absence of any effect in the IE group can be explained by the significantly reduced survival rate and functionally active grafted dopaminergic neurons in the neostriatum. Skilled paw reaching test. To date mesencephalic embryonic grafts have not shown the capacity to reverse the 6-OHDA-induced contralateral paw deficit. Again, the graft showed no protective effects with the behavioral profile of the grafted and lesioned EE animals being indistinguishable. Although the double lesion of the neostriatum and the nucleus accumbens yielded a greater deficit implying the involvement of both structures in the function (45), the double grafting of the two aforementioned structures did not result in the restoration of the paw reaching ability (2). It is speculated that for functional recovery to occur in such complex behavioral tasks not only must the tonic dopamine release function be restored, but also dopamine must be available in a spatially and temporally specific manner (49). The IE rats were generally perturbed as revealed by the poor task acquisition of the sham controls. The absence of positive stimuli, the impoverished environment, and the lack of experience resulting from the individual housing could have brought upon the animals a state of low motivation and a lack of explorative behavior necessary to learn the task. Long-term social isolation has been shown to produce learning difficulties in rats. Behavioral rigidity and rule learning deficits have been demonstrated in social isolates and have been linked to the dysfunction of the dopaminergic system (36). Skinner Box Test: Spatial Alternation. The lesion interacted with the animal’s previous experience producing larger deficits amongst the IE animals. However, as opposed to the paw reaching task, the sham animals were equally apt at acquiring the task independent of whether they were kept under the EE or the IE conditions. This would indicate that the IE animals did not lose their ability to learn and that the spatial alternation task represents an environment more conducive to exploration and learning then the paw reaching task. Contrary to the results in the paw reaching and the rotation tests, where the unilateral 6-OHDA lesion induced a bias toward the ipsilateral side, in the Skinner box the LE animals showed a mild bias toward the contralateral lever. The explanation lies in the particularity of the Skinner box and is clarified by a previous
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observation (13): the lesion induces an ipsilateral motor bias which prompts the animal to make a full circular movement, as it completes the rotation it registers a response on the contralateral lever which is the first lever it encounters. The graft alone had no effect on the bias measure as GE rats equally pressed on both levers prior to the lesion. The tendency for the GE rats to press the ipsilateral lever only emerged following the lesion. We speculate that it represents a graftinduced overcompensation, similar to what is seen in D-amphetamine rotation 7 months following the lesion (4). The IE animals all showed a tendency to respond toward the contralateral lever. The results suggests that individual housing and experience deprivation on their own are sufficient to induce a lateralized response deficit.
4.
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7.
8.
CONCLUSION 9.
The aim of the study was to compare the graft viability and the functional effects of the graft in groups housed and trained under enriched or impoverished conditions. Under neither condition did the early grafts provide a protection against the eventual lesion induced impairments. Nevertheless, animals of the EE group receiving more intense training were more prone to positive graft effects then rats of the IE group that received less intense training. Grafts placed into the EE animals showed better survival than grafts implanted into IE rats resulting in a significantly increased dopaminergic neuron count. The results suggest that survival of grafted neurons, and hence the reduction of the magnitude of behavioral impairments, can be optimized by increasing the complexity of the subject’s environment and training protocol. In turn, the consistency of the clinical application of neural transplantation might be boosted by adapting appropriate pre- and posttreatment programs.
10.
11. 12.
13.
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16.
ACKNOWLEDGMENTS This study was supported by a grant from INSERM and the “Institut Electricite´ et Sante´” (Paris, France).
17.
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Influence of Environment on the Efficacy of Intrastriatal Dopaminergic Grafts Ma`te´ Da`niel Do¨bro¨ssy, Michel Le Moal, Marie-Francoise Montaron, and Nora Abrous Domaine de Carreire, INSERM U259, Rue Camille Saint Sae¨ns, Bordeaux Cedex 33077, France Received November 17, 1999; accepted April 12, 2000
Functional recovery is influenced by experience. The aim of the present work was to examine the effects of “enriched” environment (EE) versus an “impoverished” environment on the anatomical and functional integration of intrastriatal dopaminergic grafts. These influences were studied using a paradigm where grafting was performed before the dopamine-depleting lesion. Dopaminergic grafts were implanted into the left neostriatum of adult male rats. In the enriched group, grafted rats were housed collectively and were trained on different behavioral tests following grafting. In contrast, impoverished grafted rats were housed individually and not further manipulated. Ten weeks after grafting, the mesotelencephalic dopaminergic pathway was destroyed unilaterally to the grafted side and different behaviors were followed for 7 months. Grafting prior to lesioning had no prophylactic effects on the performance as the graft did not prevent the onset of the lesion-induced impairments. However, under EE conditions, a graft effect was manifested in the reduction of drug-induced rotation and on the indices of bias as tested by a spatial alternation test. No positive graft effects were observed in the skilled paw reaching test. Grafted rats raised under impoverished conditions performed in a fashion indistinguishable from the control lesioned animals on most measures of behavior. A beneficial effect of EE conditions was observed on survival of TH-positive neurons within the grafts. The results suggest that survival of grafted neurons, and the reduction of the magnitude of particular behavioral impairments, can be optimized by increasing the complexity of the subject’s environment. ©
2000 Academic Press
Key Words: dopaminergic grafts; 6-OHDA lesion; rotation; skilled paw reaching; spatial alternation; enriched and impoverished environment.
INTRODUCTION
Neural transplantation addresses the question of functional recovery following brain damage and neurodegenerative disorders, in particular Parkinson’s dis0014-4886/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
ease (PD). Two main experimental avenues are pursued in order to optimize the transplantation-induced benefits. The first approach focuses on aspects related to the microenvironment of the grafts (for review see 9). For this purpose “bridge” grafts (8, 14, 76), coimplants and trophic factors (12, 29), or alternative source of neurotransmitter-producing cells (38, 57) are used to improve the anatomical and functional integration of the grafts. A second approach, at a more integrated level, considers the effects of various environmental factors applied before or after a lesion (for review see 77) on the behavioral recovery induced by the transplants. Experience modulates brain function (23–25). Rearing conditions that increase the level of stimulation, referred to as “enriched” environment, have been shown to improve behavioral performance (22, 31, 41, 42, 53, 59, 61, 74, 78). Complex environments have been implicated in reducing age-related impairments (17, 43, 64) and recovery from brain damage (18, 35, 54). In contrast, animals deprived of environmental stimulation, i.e., raised in an impoverished environment, have exhibited behavioral anomalies (26 –28, 36). Data on reconstructive neurosurgery and neurological rehabilitation in humans are not conclusive (for review see 56). The benefits of behavioral therapy for PD are recognized; however, the effects of such therapy on the efficacy of intrastriatal grafts are unknown (46, 51, 66, 73). In animals, experiments investigating the combined effects of enriched environment and neuronal transplant have focused mainly on housing conditions (39), pretesting experience (9, 10), postgraft training (7, 46 – 47), and priming the graft by pharmacological treatments (70). Dominantly, these studies using striatal, septal, fimbria-fornix, and neocortical or occlusion models have reported beneficial effects of enriched experience and training (20, 39 – 40, 47, 55, 71). Exploration of this question in the context of the 6-hydroxydopamine (6-OHDA) parkinsonian model and dopaminergic grafts is sparse. This question is important regarding the circumstances that maximize the functional benefits of grafts in the parkinsonian model. In
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turn, it could improve the outcome and consistency of the clinical application of neural transplantation. Our study compared the anatomical and functional benefits of dopaminergic grafts implanted into animals subjected to different environmental stimulation involving different housing condition and experience. In the enriched environment (EE) condition rats were collectively housed providing social interaction, regularly handled, and exposed to various behavioral tests before lesioning. In the “impoverished” environment (IE) condition the animals were individually housed, minimally handled, and deprived of prelesion training. The environmental influences were studied using a prophylactic paradigm where grafting was performed in the intact neostriatum before the dopamine-depleting lesion (4, 58, 67). This approach allowed us to test the effect of the environment on the transplant without the lesion-induced alterations. The behaviors studied were drug-induced rotation, skilled forelimb use, and spatial alternation in a two-lever operant chamber. The survival of dopaminergic neurons was examined by tyrosine hydroxylase (TH) immunohistochemistry. METHODS AND MATERIALS
Subjects and Housing Conditions Male Spague–Dawley rats (n ⫽ 60; Iffa-Credo, Lyon, France) weighing 250 g at the start of the experiment were used in the study. Animals in the EE (n ⫽ 30) group were housed collectively with four rats in transparent cages. On the side of the collective cages various drawings of geometrical objects of different hues and sizes were exposed. EE animals were regularly manipulated. The animals in the IE (n ⫽ 30) were housed individually in opaque cages and were not handled other than when the litter was changed. All the animals were kept in the same temperature (24°C) and humidity (50%) controlled room with a 12:12-h light cycle (lights on at 8:00 AM). Transplantation Grafting was performed 3 weeks after the arrival of the animals. The age of the host animals at the time point of transplantation was 3 months. Mesencephalic tissue from embryonic day 14 rat embryos were dissected out under phosphate-buffered saline (PBS, pH 7.2) and collected in cold PBS supplemented with 10 mM glucose. Pooled tissue pieces were submitted to a 5-min enzymatic digestion at 20°C using 1% trypsin (Worthington, UK) in PBS with 0.1% DNase (Sigma, France). After washing with PBS, a synthetic culture medium was added (S-MEM-Hepes, Gibco-BRL, France), supplemented with 0.02% trypsin inhibitor (Sigma, France), and a cellular suspension was obtained by repeated aspiration through a small-bore
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Pasteur pipette. Following a brief centrifugation (600g, 10 min), cells were resuspended in S-MEM-Hepes containing 0.2% bovine serum albumin (Sigma, France). The final suspension was kept on ice in siliconized glass tubes and was used within 6 h. Grafting was carried out under chloral hydrate anesthesia (60 mg/ml of saline, administered at a dose 0.5 ml/100 g). A 2-l aliquot of the cell suspension was injected stereotaxically into the left ventrolateral striatum of the rat using a glass cannula (100 m external diameter) connected to a 10-l Hamilton syringe at the following coordinates: A ⫽ ⫹2.5, L ⫽ ⫹2.5, V ⫽ ⫺6.0. Measurements were taken from the level of the skull, with the interaural line at ⫹5. The graft was delivered over a period of 3.5 min with an additional 4 min prior to the retraction of the cannula. The number of injected viable cells was 150,000 cells/l, as evaluated by dye exclusion. Animals were grafted from the same suspension by two investigators on the same day. The different groups were randomized during the transplantation. Sham-grafted animals received injections of the 2 l culture medium. Lesion Surgery Ten weeks after grafting, animals were anesthetised with chloral hydrate as described above. The left mesotelencephalic dopaminergic pathway was lesioned by stereotaxical injection a total of 12 g of 6-hydroxydopamine (6-OHDA, Sigma) dissolved in isotonic saline containing 0.005% of ascorbic acid (pH 4.0). The neurotoxin was delivered through two 30-gauge stainlesssteel cannula which were at a fixed distance of 1 mm apart with one of the cannula being 1 mm shorter than the other. The double cannula was lowered into the brain with the two cannulae in plane with each other with respect to the AP axis. The coordinates taken from the anterior cannula were A ⫽ 0.0, L ⫽ ⫹1.6, V ⫽ ⫺9.0; the measurements were made from the level of the skull with the interaural line at ⫹5. The rate and volume of delivery of the neurotoxin through each cannula was 0.3 l/min over 5 min, i.e., a total of 3 l of solution. The cannulae were left in place for an additional 3 min to allow for the diffusion of the neurotoxin from the injection site. Sham-lesioned animals received injections of isotonic saline containing ascorbic acid. Rotation Drug-induced turning behavior was studied in rats using transparent hemispheric bowls (diameter 40 cm). Rats were attached with a harness to a flexible wire linked to a photocell system. The number of 360° turns in either direction were recorded automatically during 60-min sessions, and the result expressed as net scores (ipsilateral minus contralateral rotations per minute). D-Amphetamine (Sigma; 5.0 mg/kg) was in-
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jected ip, and apomorphine was injected sc (Aguettan, France; 0.05 mg/kg). Paw Reaching Paw reaching was measured using a modified version of the “staircase” test apparatus described by Montoya et al. (50). The paw reaching box consists of two chambers fixed to each other end to end. The first into which the rat is placed is a transparent Perspex chamber (203 mm long by 108 mm high by 103 mm wide) with a hinged lid. The second chamber is smaller (165 mm long by 108 mm high by 60 mm wide) and it contains a central platform running along its length creating a 19-mm-wide trough on either side. The narrowness of the chamber limits the animal’s left paw to reach into the left trough and the right paw only into the right trough. The top surface of the central platform (35 mm wide) overhangs the sides preventing the animal from scraping pellets up on the side of this platform. A removable double staircase is inserted at the end of the chamber into the troughs to either side of the central platform, with the highest stair 13 mm and the lowest 64 mm below the platform. Both sides of the staircase contain eight 3 mm deep wells which were each baited with two 45-mg food pellets, giving the rats an maximum of 16 pellets per side. The animal is able to retrieve the pellets by reaching into the trough. The number of pellets it collects from either side being an index of the rat’s reaching ability, reflecting its success in grasping and retrieving the pellets. Rats were left to habituate to the boxes on the first day of testing and the pellets taken were not counted. On the following days rats were placed into the chamber for 15 min at the end of which the pellets that remained on the staircase on either side were counted. Animals received one trial per day for 16 days. The schedule was the same during the postgrafting and postlesioning testing sessions. Skinner Box Testing: Spatial Alternation Task Training and testing were conducted in operant chambers (Imetronic, Bordeaux, France), with a house light, a central food well, and two retractable levers. The central food well was on the wall opposite to the levers. The Skinner boxes were controlled by a Windows version of Immetronic’s software and linked to the operant chambers by Immetronic’s interface. Rats were habituated to the operant chambers over 7 days and trained to nose poke to collect food from the central food well. Animals learned to press the levers for food pellets over 3 days. The spatial alternation task is a two-choice, continuous nonmatching paradigm. The task involves presenting animals with both sample levers on all trials and rewarding the animal if it selects the lever opposite to the one which was rewarded on the trial before.
On the first trial the reinforced lever was randomly selected, and from then on each following trial a winshift response pattern was rewarded by the delivery of a 45-mg food pellet into the lit central food well located in the opposite wall to the levers. The collection of the pellet triggered off the next trial with the presentation of both levers. Conversely, pressing the same lever to that presented as the sample was counted as an error and resulted in a “time out” during which the house light was extinguished for 5 s. At the end of the time out period the house light came on and the two levers were extended. Following an error trial the rat was required to press the lever opposite to the side prior to the initial error. The session lasted until rats carried out either 51 reinforced trials or until the lapse of 30 min, whichever happened first. Immunohistochemistry At the completion of the behavioral testing rats were given a terminal dose of chloral hydrate (600 mg/ml saline, administered at a dose of 1.5 ml/rat) and perfused transcardially with 100 ml 0.1 M phosphatebuffered saline (pH 7.3) containing 5 ⫻ 10 4 IU/ml heparin, followed by 400 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) at a rate of 60 ml/min (Masterflex pump, U.S.A.). After 24 h of postfixation, 50-m sections were cut using a Vibratome (Vibratome System 1000, U.S.A.). Free-floating sections were processed through a standard immunohistochemical procedure in order to visualize tyrosine hydroxylase (TH) with a rabbit anti-TH primary antibody (dilution of 1/10000, Boy, Paris, France). A 48-h incubation period was followed by incubation with a biotin-labeled goat anti-rabbit secondary antibody (1/200, Dako Laboratories, Glostrup, Denmark). TH-positive immunoreactivity (IR) was visualized by the biotin–streptavidin technique (ABC kit, Dako Laboratories) using 3,3⬘-diaminobenzidine as the chromogen. Using a random start, TH-IR cells were counted in the grafts, one-in-two sections, with the sections being 100 m apart. The total number (N total) of TH cells was calculated according to the equation N total ⫽ (¥Q⫺) ⫻ thickness ⫻ (1/section sampling fraction). Experimental Design and Behavioral Testing The behavioral testing schedule is described schematically in Fig. 1. After the arrival the animals were subjected to either EE (n ⫽ 30) or IE (n ⫽ 30). Three weeks later half of the rats from both housing conditions received either embryonic mesencephalic tissue transplants (GE, n ⫽ 15; GI, n ⫽ 15) or were sham grafted receiving culture medium injections into the central striatum. Ten weeks after the transplantation procedure, the transplanted rats (i.e., GE and GI) received 6-OHDA injections into the medial forebrain bundle on the same side as the grafts. The sham-
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FIG. 1. Experimental groups and general procedures. (A) After the arrival animals were housed, grafted, or received sham grafts; lesioned; or received saline injections, depending on which experimental group they were in. (B) The behavioral procedures the animals were subjected to depended on which environmental condition applied. Animals from the enriched (EE) and the impoverished environment (IE) received mesencephalic grafts at the same time but only the former group was tested prior to the lesion, while all animals received training following the lesion.
grafted animals received either 6-OHDA (LE, n ⫽ 10; LI, n ⫽ 10) or ascorbic acid (SE, n ⫽ 5; SI, n ⫽ 5) injections. Animals were food deprived and kept at 85–90% of their normal body weight during all times apart from 1 week before and 3 days after the grafting and the lesion procedures. At these periods they had ad libitum access to food. The EE animals were tested both before and after the lesion, while the IE animals were tested on their acquisition only after the lesion. Statistical Analysis All data were analysed by ANOVA followed by post hoc comparisons when appropriate using the Newman–Keuls test (Statistica for Windows 5.1). The between factors used were environment conditions effect (two levels, enriched vs impoverished) and group effect (three levels, sham, lesion, and graft). The within fac-
tors were session effect and/or paw side effect, depending on the behavior results analyzed. RESULTS Immunohistochemistry
6-OHDA injection into the medial forebrain bundle resulted in the almost complete loss of TH-positive staining from the substantia nigra and the ventral tegmental area from the same side as where the lesion was performed. Furthermore, total loss of TH-positive staining in the neostriatum and the nucleus accumbens ipsilaterally to the lesion site was observed. The gross morphology of the grafts shown in Fig. 2 was comparable to that described previously in the literature (3, 4, 52). However, the count of TH-positive neurons from the grafted groups with different experiences
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FIG. 2. Influence of environment on dopaminergic graft survival. Animals were raised under (A) enriched or (B) impoverished conditions. TH immunohistochemistry was performed 26 weeks after grafting and 16 weeks after lesioning. G, the graft; S, the striatum; the stars mark the border between the graft and the host. Bar, 250 m.
revealed a significantly greater number of surviving dopaminergic cells among the EE grafted rats (mean ⫾ SEM, 2515 ⫾ 517) when compared to the IE grafted rats (1283 ⫾ 222) [environmental condition effect, F(1, 13) ⫽ 5.3, P ⬍ 0.05]. Drug-Induced Rotation D-Amphetamine
The results of D-amphetamine-induced rotation are shown in Fig. 3. For the EE group two sessions were conducted between the grafting and the lesioning operation. The unilaterally placed dopaminergic graft on its own did not induce any preferential side bias in the rotation after the D-amphetamine injection. Following the lesion surgery, three drug tests were performed on the EE and the IE animals. For the EE group, the analysis resulted in a significant interaction between the Group and Session factors [Group ⫻ Session, F(8, 60) ⫽ 8.1, P ⬍ 0.001]. Subsequent New-
man–Keuls tests for multiple comparisons indicated that the lesion induced a significant increase in the ipsilateral rotation score for the LE and GE groups, with respect to the sham-lesioned SE group. The increase was apparent during the first two testing sessions (in both testing times SE ⬍ LE, GE, P ⬍ 0.001). At these time points there was no statistical difference between the LE and the GE groups. Nevertheless, on the final session carried out 3 months after the lesion, a graft effect was observed in the GE group as manifested by a decrease in the ipsilateral rotation with respect to the LE (LE ⬍ GE, P ⬍ 0.05) and approached the level of the SE group so as to abolish any statistical difference between them (SE, GE, P ⫽ 0.075, n.s.). For the IE group, the lesion induced an ipsilateral rotation bias resulting in a Group effect, which consequent post hoc analysis showed to be between the GI/LI groups and the sham control [Group, F(2, 16) ⫽ 17.9, P ⬍ 0.001; post hoc, LI ⬍ SI, P ⬍ 0.001; and GI ⬍ SI, P ⬍ 0.001]. Nevertheless, contrary to that reported for the
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FIG. 3. Influence of environment on amphetamine-induced rotation. The two prelesion tests were carried out (A) 2 and (B) 3 weeks following grafting. The three postlesion tests were done (C) 1, (D) 2, and (E) 12 weeks following the lesion. Results are expressed as the number of ipsilateral turns minus contralateral turns per minute (mean ⫾ SEM). **P ⬍ 0.01 compared to the matched lesion group. SE, sham enriched; LE, lesion enriched; GE, graft enriched; SI, sham impoverished; LI, lesion impoverished; GI, graft impoverished.
EE animals, the IE rats with grafts did not manifest a diminution of the ipsilateral rotation on the final Damphetamine rotation session. Overall, the environmental condition on its own did not affect the magnitude of the ipsilateral rotation [Housing, F(1, 31) ⫽ 0.04, n.s.]. However, the reduction in ipsilateral rotation on the final testing session uniquely in the EE grafted animals, accompanied by the absence of any effect in the IE animals, results in a three-way interaction between the environmental condition, treatment, and session that approaches statistical significance [Environment ⫻ Group ⫻ Session, F(4, 62) ⫽ 2.35, P ⫽ 0.06]. Apomorphine The contralateral rotation displayed by the animals treated with apomorphine (0.05 mg/kg) is shown in Fig. 4. For the EE group, apomorphine induced a significant
Group effect [Group, F(2, 15) ⫽ 4, 9, P ⬍ 0.05]. Further post hoc analysis indicated a significant difference between the SE and the LE animals, with the GE animals not differing from either of the two (SC ⬍ LC, P ⬍ 0.05). Among the IE rats, the treatment did not result in any main effects. The rotation was more pronounced in the lesioned EE then among the lesioned IE rats; however, this did not reach significance. Skilled Paw Reaching
Prelesion EE vs Postlesion EE The results are illustrated in Fig. 5. Prior to the lesion (Fig. 5, left panel, prelesion testing) the SE and LE animals showed no significant paw bias, whereas for the GE animals the graft on its own enhanced the performance by favoring the pellet collection by ipsilateral paw with respect to the contralateral paw. This
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FIG. 4. Influence of environment on apomorphine-induced rotation. Results are expressed as the number of ipsilateral turns minus contralateral turns per minute (mean ⫾ SEM). *P ⬍ 0.05 in comparison with SE. For abbreviations see the legend to Fig. 3.
graft effect is more precisely described as beneficial to the ipsilateral paw then as impairing to the contralateral paw, and it does not reach statistical significance. The lesion induced a contralateral deficit as shown by the increase in the number of remaining pellets, effecting the contralateral side in particular [Session, F(1, 30) ⫽ 6.5, and Side, F(1, 30) ⫽ 14.2, both P ⬍ 0.05]. The bias toward the ipsilateral side was present in both the LE and the GE groups equally; i.e., the graft did not prevent the onset of the contralateral impairment in the skilled paw reaching test (post hoc, postlesion, ipsilateral versus contralateral sides within group for LE and GE animals, P ⬍ 0.05).
showed poor overall acquisition of the task which resulted in equally poor performance with both paws. [Group, F(2, 16) ⫽ 2.2, n.s.]. Comparing the performance of the EE and IE animals there was a strong environmental component observed [Environment, F(1, 31) ⫽ 12.5, P ⬍ 0.05]. There was no side bias following the lesion amongst the IE rats [Side, F(1, 16) ⫽ 0.6, n.s.].
Postlesion IE vs Postlesion EE
Prelesion EE vs Postlesion EE
As shown in Fig. 5 (right panel) animals of the different groups raised under impoverished conditions
Time to finish the task (Fig. 6A). Prior to the lesion animals from all three EE groups completed the task at a similar rate. The lesion induced a significant increase in the time to complete the task in the LE and GE groups with respect to the SE [Group ⫻ Session, F(2, 28) ⫽ 6.6, P ⬍ 0.05]. The decreased time for the SE animals reflects the effect of the additional training during the second testing session. Pellets collected and bias index (Figs. 6B and 6C). In neither of these two measurements were there any main effects, although the lesion introduced greater variability with the two groups. Although it did not reach statistical significance, the bias measure showed an ipsilateral tendency for the LE and a contralateral bias for the GE rats. Total lever presses (Fig. 6D). Before the lesion the number of lever presses the animals made were indistinguishable. The surgery introduced a mild increase in the total number of lever presses executed by the LE and GE groups, while the SE animals showed a re-
FIG. 5. Influence of environment on paw reaching. The number of leftover pellets following the testing session out of an initial of 16 per side is represented. The lower the score, the better the performance. The means of the last 5 days are shown (mean ⫾ SEM). *P ⬍ 0.05 in comparison with the ipsilateral paw. For abbreviations see the legend to Fig. 3.
Spatial Alternation in the Skinner Box
Results of the spatial alternation task are summarized in Fig. 6.
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DISCUSSION
FIG. 6. Influence of environment on spatial alternation task. The parameters measured were (A) the time to complete the test, (B) the number of pellets taken during a session, (C) the response bias, and (D) the number lever presses executed per session. The test lasted either 1800 s or until 51 pellets were collected, whichever came first. The results are expressed as the mean of the last five sessions (mean ⫾ SEM). *P ⬍ 0.05 when comparing the groups indicated on the graph. Detailed statistical analysis are included under Results. For abbreviations see the legend to Fig. 3.
duced lever pressing frequency [Group, F(2, 28) ⫽ 3.2, P ⬍ 0.05; post hoc, SE ⬍ LE and GE, P ⬍ 0.05]. Postlesion EE vs Postlesion IE Time to finish the task. The latency to finish the task was not influenced by the environmental conditions, although there was a tendency for the IE rats to be slower. The main factor was the surgical manipulation, with the lesioned and the grafted rats both being equally effected in either group [Group, F(1, 29) ⫽ 16.3, P ⬍ 0.05]. Pellets collected and bias index. The number of pellets collected, reflecting the number of successful responses, had a strong environmental component. All the EE rats performed significantly better then the IE rats on this measurement collecting nearly twice as many pellets [Environment, F(1, 29) ⫽ 5.1, P ⬍ 0.05; Environment ⫻ Group, F(2, 29) ⫽ 1.1, n.s.]. There were no main effects with respect to the bias index. Total presses. The number of lever presses during the session is an index of the animals’ activity and engagement in the task. Animals of the EE condition executed more lever presses then the IE rats [Environment, F(2, 29) ⫽ 5.1, P ⬍ 0.05]. Furthermore, while for the LE and GE rats the lesion augmented the rats of response with respect to the control animals, for the LI and the GI rats the response rates decreased [Environment ⫻ Group, F(2, 29) ⫽ 3.1, P ⬍ 0.05].
Influences of environmental manipulations on the efficacy of intrastriatal dopaminernergic grafts were compared between two extreme conditions: (i) EE, i.e., collective housing, intense manipulation and training during the period separating the grafting and the lesion procedures, and (ii) IE, i.e., individually housing, absence of manipulation during the postgraft recovery period. We have shown that: (i) more grafted cells survived in the neostriatum of grafted animals raised under EE when compared to those of IE; (ii) the grafts did not prevent the onset of the lesion-induced impairments in either the EE or the IE held rats; and (iii) the differential environmental experiences resulted in different functional effects for the grafted and the control animals. The results suggest that the behavioral performance reflects the influence of the environment and the training schedule on the animal. Also, it shows that the environment and the training schedule are factors that interact with the transplant. Enriched Environment Increases Survival of Dopaminergic Grafted Neurons It is not the environment per se that influences brain anatomy and chemistry, but the active participation and interaction of the animal with the environment (23, 60, 75). In the context of grafting, animal models of Huntington’s disease indicate that the functional benefits induced by intracerebral grafts can be positively influenced by postgrafting experience (7, 46, 47). In the current experiment the housing conditions and experience differentially affected survival of the grafted TH neurons. Almost twice the number of cells survived in the neostriatum of the EE rats, exposed to more “stimulating” conditions and more intense training schedules, than in the IE rats. The difference is not due to further division after grafting because it has been demonstrated that dopamine cells in nigral grafts do not undergo mitosis after implantation within the striatum (1, 69). Thus, it may be on grounds of enhanced survival of the DA neurons, the majority of which otherwise die the week following grafting in part due to the toxic events within the host environment (15, 68). It has been shown that enriched environment increases the level of trophic factors (72, 78) and can protect neurons from cell death (78). Thus, a more favorable physiological environment for the survival of the transplanted dopaminergic neurons—in terms of availability of trophic factors or/and less toxicity—may be present for the EE animals during the period critical for cell survival. A study comparing the microenvironments of the two experimental groups into which the graft is placed would need to be done to shed light on this question.
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The IE rats were isolated and were manipulated much less during the experiment. Prolonged isolation housing has been characterized by several structural and physiological changes including the alteration of the activity of the hypothalamic–pituitary–adrenocortical axis (HPA) (36, 37, 44, 65). The changes to the activity of the HPA could have occurred during the 3 weeks prior to the grafting. One of the effects of the differential changes to the HPA activity induced by the different housing conditions could have been alterations to the levels of corticosterone secretion or to the mechanisms of stress adaptation. Consequences of impaired or less responsive stress adaptation would be particularly evident following the traumatic event of graft surgery. A hypersecretion of corticosterone has been described following social isolation (5, 19, 21) and an excessive release of corticosterone has been reported to compromise neuronal survival (48) via a blockage of glucose uptake and an induction of hyperpolarization (33, 34). Elevated levels of corticosterone during the early stages following the transplantation could have compromised the survival of the grafts. It has been shown in vitro that mesencephalic glia, which were present in the cell suspension along with the neurons, secrete factors that support the survival of cultured dopaminergic neurons and are regulated by corticosterone in a dose-dependent manner (16). These studies demonstrated that elevated levels of corticosterone reduced dopaminergic cell survival in culture by reducing dopaminergic growth factor secretion by the glia. The presence of glucocorticoid receptors on mesocorticolimbic dopaminergic neurones themselves has been described (30), and corticosterone is known to modulate dopamine release both in vivo (62) and in vitro (63). These data suggest that an excessive release of corticosterone in the IE rats could have had deleterious effects on the survival of the implanted dopaminergic cells. Similar restrictive conditions, including sensory deprivation, have been shown elsewhere to prevent the integration of neocortical grafts with the host brain (6). Functional Effects of Dopaminergic Grafts Are Influenced by Experience Drug-induced rotation. The ability of mesencephalic dopaminergic grafts to reduce and overcompensate for 6-OHDA lesion-induced ipsilateral turning behavior induced by D-amphetamine is well documented (for review see 32). When graft implantation occurs prior to the lesion, reduction and overcompensation in rotation is delayed (4). The current study was terminated 4 months, rather then 7 months, following the lesion. However, extrapolating the data suggests that eventual overcompensation would have happened. The mechanism of action for the delayed graft response is believed to be due to a less vigorous, yet equally exten-
sive, fiber outgrowth from the graft to host. The slower rate of fiber extension could be explained by the age and the conditions under which the grafted neurons emit their projections. The grafts did not offer protection against the development of a drug-induced postural asymmetry. Nevertheless, a graft effect was observed 12 weeks following the lesion uniquely in the EE group. The absence of any effect in the IE group can be explained by the significantly reduced survival rate and functionally active grafted dopaminergic neurons in the neostriatum. Skilled paw reaching test. To date mesencephalic embryonic grafts have not shown the capacity to reverse the 6-OHDA-induced contralateral paw deficit. Again, the graft showed no protective effects with the behavioral profile of the grafted and lesioned EE animals being indistinguishable. Although the double lesion of the neostriatum and the nucleus accumbens yielded a greater deficit implying the involvement of both structures in the function (45), the double grafting of the two aforementioned structures did not result in the restoration of the paw reaching ability (2). It is speculated that for functional recovery to occur in such complex behavioral tasks not only must the tonic dopamine release function be restored, but also dopamine must be available in a spatially and temporally specific manner (49). The IE rats were generally perturbed as revealed by the poor task acquisition of the sham controls. The absence of positive stimuli, the impoverished environment, and the lack of experience resulting from the individual housing could have brought upon the animals a state of low motivation and a lack of explorative behavior necessary to learn the task. Long-term social isolation has been shown to produce learning difficulties in rats. Behavioral rigidity and rule learning deficits have been demonstrated in social isolates and have been linked to the dysfunction of the dopaminergic system (36). Skinner Box Test: Spatial Alternation. The lesion interacted with the animal’s previous experience producing larger deficits amongst the IE animals. However, as opposed to the paw reaching task, the sham animals were equally apt at acquiring the task independent of whether they were kept under the EE or the IE conditions. This would indicate that the IE animals did not lose their ability to learn and that the spatial alternation task represents an environment more conducive to exploration and learning then the paw reaching task. Contrary to the results in the paw reaching and the rotation tests, where the unilateral 6-OHDA lesion induced a bias toward the ipsilateral side, in the Skinner box the LE animals showed a mild bias toward the contralateral lever. The explanation lies in the particularity of the Skinner box and is clarified by a previous
INTERACTION OF DA GRAFTS WITH THE ENVIRONMENT AND TRAINING
observation (13): the lesion induces an ipsilateral motor bias which prompts the animal to make a full circular movement, as it completes the rotation it registers a response on the contralateral lever which is the first lever it encounters. The graft alone had no effect on the bias measure as GE rats equally pressed on both levers prior to the lesion. The tendency for the GE rats to press the ipsilateral lever only emerged following the lesion. We speculate that it represents a graftinduced overcompensation, similar to what is seen in D-amphetamine rotation 7 months following the lesion (4). The IE animals all showed a tendency to respond toward the contralateral lever. The results suggests that individual housing and experience deprivation on their own are sufficient to induce a lateralized response deficit.
4.
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6.
7.
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CONCLUSION 9.
The aim of the study was to compare the graft viability and the functional effects of the graft in groups housed and trained under enriched or impoverished conditions. Under neither condition did the early grafts provide a protection against the eventual lesion induced impairments. Nevertheless, animals of the EE group receiving more intense training were more prone to positive graft effects then rats of the IE group that received less intense training. Grafts placed into the EE animals showed better survival than grafts implanted into IE rats resulting in a significantly increased dopaminergic neuron count. The results suggest that survival of grafted neurons, and hence the reduction of the magnitude of behavioral impairments, can be optimized by increasing the complexity of the subject’s environment and training protocol. In turn, the consistency of the clinical application of neural transplantation might be boosted by adapting appropriate pre- and posttreatment programs.
10.
11. 12.
13.
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16.
ACKNOWLEDGMENTS This study was supported by a grant from INSERM and the “Institut Electricite´ et Sante´” (Paris, France).
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