- Email: [email protected]
Can fetal neural transplants restore function in monkeys with lesion-induced behavioural deficits?
Neural transplantation has become an established procedure in the study of many regions of the mammalian CNS. Among these regions, the SCN is unique in two respects. (1) The tissue is remarkably resilient: it is not unusual to obtain 80-90% of total restoration of overt rhythmicity in an experiment. (2) Functional restoration is detectable often within a few days of the implantation. These features suggest that just as transplantation is an important new tool for studying circadian rhythms, the circadian system itself may provide a tool for the further development of transplantation procedures in general.
Selected references 1 Moore, R. Y. and Lenn, N. J. (1972) J. Comp. Neurol. 146, 1-14 2 Card, P. J. and Moore, R. Y. (1982) J. Comp. Physiol. 206, 390-396 3 Stephan, F. J. and Zucker, I. (1972) Proc. NatlAcad. Sci. USA 69, 1583-1586 4 Moore, R. Y. and Eichler, V. (1972) Brain Res. 42, 201-206 5 Inouye, S-I. T. and Kawamura, H. (1979) Proc. NatlAcad. Sci. USA 76, 5962-5966 6 Green, D. J. and Gillette, R. (1982) Brain Res. 245, 198--200 7 Groos, G. A. and Hendricks, J. (1982) Neurosci. Left. 34, 283-288 8 Earnest, D. J. and Sladek, C. D. (1986) Brain Res. 382, 129-133 9 Drucker-Colin, R., Aguilar-Roblero, R., Garcia-Hernandez, F., Fernandez-Cancino, F. and Rattoni, F. B. (1984) Brain Res. 311,353-357 10 Sawaki, Y., Nihonmatsu, I. and Kawamura, H. (1984) Neurosci. Res. 1, 67-72 11 Lehman, M. N. et aL (1987)J. Neurosci. 7, 1626-1638 12 DeCoursey, P. J. and Buggy, J. (1988) Comp. Endocrinol. 7, 49--54 13 Boer, G. J. and Griffioen, H. A. (1990) Eur. J. Morphol. 28, 330-345 14 Ralph, M. R., Foster, R. G., Davis, F. C. and Menaker, M. (1990) Science 247, 975-978
15 Rosenwasser, A. N. (1988) Prog. Psychobiol. Physiol. Psychol. 13, 155-226 16 Meijer, J. H. and Reitveld, W. J. (1989) Physiol. Rev. 69, 671-707 17 Rusak, B. (1989)J. BioL Rhythms 4, 121-134 18 Ralph, M. R. and Menaker, M. (1988) Science 241, 1225-1227 19 Sollars, P. J. and Kimble, D. P. (1988) Soc. Neurosci. Abstr. 14, 49 20 Davis, F. C. (1989) Soc. Neurosci. Abstr. 15, 493 21 Zimmerman, N. H. and Menaker, M. (1979) Proc. NatlAcad. Sci. USA 76, 999-1003 22 Van den Pol, A. N. and Tsujimoto, K. L. (1985) Neuroscience 15, 1049-1086 23 Watts, A. G. and Swanson, L. W. (1987) J. Comp. Neuro/. 258, 230-252 24 Van den Pol, A. N. in Suprachiasmatic Nucleus: The Mind's Clock (Klein, D. C., Moore, R. Y. and Reppert, S. M., eds), Oxford University Press (in press) 25 Lehman, M. N., Silver, R. and Bittman, E. L. in Suprachiasmatic Nucleus: The Mind's Clock (Klein, D. C., Moore, R. Y. and Reppert, S. M., eds), Oxford University Press (in press) 26 Weigand, S. J. and Gash, D. M. (1988)J. Comp. NeuroL 267, 562-579 27 Canbelyi, R. S., Lehman, M. N. and Silver, R. Brain Res. (in press) 28 Klein, D. C. eta/. (1983) Brain Res. Bull. 10, 647-652 29 Pickard, G. E. and Turek, F. W. (1983) Neurosci. Left. 43, 67-72 30 Earnest, D. J., Sladek, C. J., Gash, D. M. and Weigand, S. J. (1989) J. Neurosci. 9, 2671-2677 31 Hakim, H. and Silver, R. (1988) Soc. Neurosci. Abstr. 14, 51 32 Roberts, M. H., Bernstein, M. F. and Moore, R. Y. (1987) Dev. Brain Res. 32, 59-66 33 Aebischer, P., Winn, S. R. and Galletti, P. M. (1988) Brain Res. 448, 364-368 34 Silver, R., Lehman, M. N., Gibson, M., Gladstone, W. R. and Bittman, E. L. Brain Res. (in press) 35 Bos, N. P. A. and Mirmiran, M. (1990) Brain Res. 511, 158-162 36 Ralph, M. R. and Torre, E. R. (1990)Soc. Neurosci. Abstr. 16, 769
Can fetal neural transplantsrestore functionin monkeys with lesion-inducedbehaviouraldeficits? R. M. Ridley and H. F. Baker R. M. Ridleyand H, F. Bakerare in the Division of Psychiatry, Clinical Research Centre, Walford Road, Harrow HA1 3UJ, UK.
Experiments are now being conducted in monkeys to see whether the transplantation of fetal neural tissue, rich in certain neurotransmitter-producing cells, can restore behaviour in animals with movement or learning impairments induced by lesions that have destroyed important neurotransmitter pathways. Transplantation of doDamine neurones in humans may prove to be a useful therapy in Parkinson's disease, in which a severe movement disorder is associated with degeneration of the dopamine system. Transplantation of cholinergic neurones in monkeys can overcome a severe learning impairment induced by lesion of the cholinergic system. Cholinergic transplantation may eventually be of use in a variety of neurodegenerative dementing illnesses. Although most of the work done in developing the techniques of tissue transplantation into the CNS (including the behavioural assessment of such procedures) has been carried out in rodents, there are several reasons for extending these studies to primates prior to clinical application. First, the behav-
366
ioural repertoire of primates might be amenable to more detailed assessment than that of rodents; therefore, more precise measures of restitution of function and, more importantly, more sensitive measures of deleterious effects may be obtained. Second, it might be possible to measure those more complex and cognitive functions that hopefully might be most affected by transplantation. Third, the optimum techniques for transplantation might vary between species. The best estimate, prior to clinical use, of the most appropriate techniques in humans may only be possible in other primates. Work carried out in monkeys has been very limited but the results are important. They are reviewed here in order to demonstrate what has been achieved experimentally as a prelude to possible therapeutic use in humans. Two techniques of fetal neural tissue transplantation into the CNS have been studied in monkeys: transplantation of tissue fragments dissected from fetal brain into previously prepared cavities in recipient brain; and the direct stereotaxic injection of
© 1991,ElsevierSciencePublishersLtd,(UK) O166-2236/91/$02.O0
TINS, Vol. 14, No. 8, 1991
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4-phenyl-1,2, 3, 6-tetrahydropyridine (MPTP) or 6-hydroxydopamine (6-OHDA). Both these models produce the primary pathological condition of Parkinson's disease, i.e. degeneration of the ascending dopaminergic pathways from the substantia nigra to the striatum. The unilateral method produces marked ipsilateral rotation (i.e. towards the lesioned side). This rotation is eminently quantifiable, can be manipulated by dopaminergic drugs, and its diminution can serve as an index of the restitution of impaired function. Moreover, reversal of the direction of rotation (from ipsilateral to contralateral) can indicate over-activity of the transplant 2. In contrast, bilateral lesions produced by peripheral administration of MPTP result in types of behaviour, including bradykinesia, rigidity and poor movement initiation, that are more characteristic of the clinical symptoms of Parkinson's disease, but that are not so easy to quantify. Unlike the unilateral procedure, peripheral MPTP treatment can produce animals that
Fig. 1. Drawing of a marmoset attempting to retrieve pieces of bread placed on a staircase outside a perspex screen. To obtain reward the monkey must reach through the vertical slot at either side of the staircase using only one arm. Monkeys with unilateral lesions of the subtantia nigra tend to neglect the contralateral side, i.e. fail to use the contralateral arm. When they do attempt to reach into the contralateral side of the staircase, they frequently show clumsiness and often attempt to retrieve the most difficult piece at the top of staircase (but nearest to the ipsilateral side) before attempting to retrieve the easiest piece nearest to the slot but in the furthest contralateral position.
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, dissociated fetal brain cell suspensions or solid tissue fragments into deep brain structures. Dopamine-rich tissue for transplantation into the basal ganglia of monkeys with damage to the dopamine pathway is dissected from the fetal ventral mesencephalon using techniques based on those used in rats ~. Acetylcholine-rich tissue from the septal area of the fetal brain is currently used for transplantation into the hippocampus of monkeys that have had the cholinergic projection to that area disrupted by transection of the fornix.
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Assessing behavioural recovery i/| The principal method of assessing effects of intracerebral transplantation in a~nals involves the pro~.o,;,l i duction of a pathological condition similar to that underlying a particular clinical disease. The resulting change in the animal's behaviour can then be measured before and after the transplantation procedure. For rigorous scientific assessment it is preferable that the behavioural change is quantifiable Fig. 2. Drawing of a marmoset performing trials of a on an interval scale, i.e. one in which the numerical visuospatial task in a Wisconsin General Test Apparatus. In values measure real quantities. In practice, however, this task the monkey has to learn to move the stimulus on it is sometimes only possible to produce an arbitrary its left to uncover the foodwell containing reward during trials in which one pair of identical stimuli (corks) are number in order to rate the severity of impairment. Some workers consider it important that the presented. It must also learn to move the stimulus on its behavioural impairment resembles the symptoms of right to obtain reward on trials on which a different pair of the disorder being modelled, but this is not strictly identical stimufi (brackets) are presented. These two types necessary provided the physiological basis of the of trials are presented in a random order until the animal makes a predetermined number of correct responses (e.g. behaviour is understood. For example, in primate 27 correct in 30 consecutive trials). Acquisition of this task models of Parkinson's disease, it is possible to requires that the animal associates different stimuli with produce either a unilateral or a bilateral lesion of the different responses, and is impaired by lesions of the nigrostriatal tract using neurotoxins such as 1-methyl- cholinergic projection to the hippocampus. HNS, Vo1.14, No. & 1991
367
much controversy regarding the mechanism by which grafts influence the host and this has been the subject of an earlier TINS review 5. The behavioural improvement reported by Bankieif) wicz et al. 4 might have been due to regenerative cO 100processes in the host (stimulated by the grafting a3 procedure), which then might have led to reinnervation of the host caudate by dopaminergic fibres not destroyed by the MPTP treatment. However, cO such sprouting is possible only where a proportion of 0 host dopaminergic neurones survives the lesion procedure. It is possible that a better differentiation between the behavioural effects of experimental grafts and control procedures will be demonstrated when lesioning methods are more efficient and when - -100 P the optimum techniques for grafting have been ; 3 4 6 0 determined in primates. time after transplantation (months) Pioneering work on fetal neural tissue transplantation in primates has been carried out by a collaborFig. 3. Following unilateral 6-OHDA lesion of the substantia nigra, marmosets rotate ipsilaterally (towards the lesion ative team based in Yale and Rochester in the US, side) when injected i.m. with amphetamine. In the months and is described most extensively by Taylor et al. 6'7. following transplantation with dopamine-rich tissue into This group examined the behavioural effects of the caudate or caudate and putamen, marmosets show bilateral stereotaxic transplantation of many small progressively less ipsilateral rotation. Marmosets with fragments of fetal dopaminergic tissue into nondopamine-rich tissue transplanted into putamen alone do cavitated sites of the caudate nucleus of five vervet not show any improvement. C), control animals (n = 5); monkeys (Cercopithecus aethiops) with parkinsonism [2, unilateral 6-OHDA lesion of the substantia nigra, no graft (n = 10); 0, nigral lesion plus caudate and putamen grafts (n = 6); I , nigral lesion plus caudate graft only (n = 5); A, nigral lesion plus putamen graft only (n = 5). These results are subject to histological confirmation. 200
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are incapable of self-care, resulting in many practical difficulties. Motor skills can also be measured in unilaterally lesioned animals and in those bilaterally lesioned animals that are not grossly incapacitated during tasks requiring, for example, the retrieval of rewards from awkward or concealed places (Fig. 1). At present there are no primate models of Alzheimer's disease suitable for behavioural assessment. However, one important feature of this disease is the degeneration of the ascending cholinergic projections, which makes a major contribution to cognitive decline3. It is possible to make selective lesions within this system and to assess the effects of cholinergic transplants on the learning impairments that are produced. In this case cognitive ability is usually assessed by determining the number of trials it takes for an animal to learn to discriminate between stimuli in a Wisconsin General Test Apparatus (Fig. 2). Behavioural m e a s u r e s in animal models of Parkinson's disease Bankiewicz et aL 4 have described the effects of
bilateral surgical transplantation of fragments of dopamine-containing fetal ventral mesencephalon into the caudate nucleus of five monkeys (Macaca mulatta) that had been treated unilaterally with MPTP; these effects were compared with animals that were only lesioned, or with MPTP-treated animals with control grafts, or with surgical cavitation only. Although the fetal tissue grafts showed good long-term survival, they did not appear to reinnervate the host tissue. Behavioural recovery was quite good but not better than that produced by nonneural grafts and other control procedures. There is 368
Transplant of dopamine-rich nigral fetal tissue into the putamen of a marmoset with a unilateral 6-OHDA lesion of the substantia nigra. Orange stain shows tyrosine hydroxylase, a marker for dopamine cells. Abbreviations: C, cortex; Ca, caudate; IC, internal capsule; P, putamen. Scale bar is 0.5 mm. Arrows indicate transplanted tissue. Fig. 4.
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that had been induced by intramuscular administration of MPTP. These effects were compared with the behaviour of three MPTP-lesioned monkeys with control grafts and three non-lesioned animals with control gaffs. Four moderately to severely affected lesioned monkeys showed substantial restitution of normal behaviour and diminution of behaviour indicative of parkinsonism (e.g. the degree of bradykinesia) when transplanted with fetal dopaminergic tissue. Two of these monkeys, together with another one that had a slightly less severe lesion-induced impairment, were tested in an object retrieval task and showed substantial improvements over time. In contrast, the MPTP-treated animals transplanted with inappropriate control tissue showed no behavioural recovery and were largely recumbent. They were unable to participate in the object retrieval task and were killed earlier than those receiving dopaminergic grafts because they were so severely affected. Thus, these authors have shown that substantial restitution of behavioural competence that is equivalent to a major clinical improvement can be achieved in monkeys with severe parkinsonian symptoms. When these monkeys were killed, the number of surviving graft-derived neurones, which immunostain with antibodies to tyrosine hydroxylase (a marker for dopamine cells), was small, but this in itself demonstrates that pronounced behavioural recovery can be achieved with a very modest increase in the level of dopaminergic activity in the caudate. This effect in severely incapacitated monkeys is important because it is hoped that transplantation will be particularly beneficial to those human patients in whom the disease is so advanced that drug treatment is no longer adequate. Currently, the more usual technique for transplantation of fetal material involves the dissociation of donor tissue into a cell suspension that can then be injected stereotaxicall~¢ with the minimum of trauma into deep target areas'. Freed et al. 8 and Bakay et al. 9 have compared seven monkeys (Macaca mulatta and Macaca radiata) with lesions alone with six animals that had bilateral MPTP-induced lesions and bilateral or unilateral stereotaxically placed fetal cell suspension grafts in either caudate alone or caudate plus putamen. The four monkeys best displaying graft survival showed considerable clinical improvement. The common marmoset (Callithrixjacchus) is a very small primate with a high and reliable reproductive rate. It is therefore possible to use this species for transplantation experiments with larger numbers of subjects using donor tissue of very precise gestational age. Research groups in Britain have used both bilateral MPTP administration and unilateral 6-OHDA injections in this species to produce models of Parkinson's disease (Refs 2, 10 and Annett, L. E. et al., pers. commun.). In these experiments a total of 54 monkeys have been used, 26 of which were given 6-OHDA unilaterally and ten of which were given MPTP bilaterally. Overall, unilaterally lesioned animals showed functional improvement following transplantation of fetal, dopamine-rich cell suspension into the ipsilateral basal ganglia, while the bilaterally lesioned animals improved only after bilateral grafting into the basal ganglia. Marked improvements in activity and decreases in drug-induced rotation were seen in the unilaterally lesioned animals following TINS, Vol. 14, No. 8, 1991
Fig. 5. Transplant of cholinergic-rich septal fetal tissue into the hippocampus of a marmoset with fornix transection, stained for acetylcholinesterase, a marker for cholinergic nerve terminals. The transplant stains darkly, but substantial reinnervation of surrounding host tissue can also be seen. Abbreviations: D, dentate gyrus; E, entorhinal cortex; H, hippocampus. Scale bar is 0.5 mm. Arrows indicate transplanted tissue.
transplantation of dopamine-rich tissue into both the caudate and putamen, or into the caudate only. No improvement in rotation was seen in untransplanted animals or animals with transplants into the putamen (Fig. 3). Preliminary data suggest that monkeys with grafts into the putamen show less contralateral neglect (measured by the latency to start removing a sticky label from the contralateral leg) than monkeys with no grafts or dopamine grafts into the caudate (Annett, L. E., pers. commun.). The importance of the site at which the transplant is placed in determining the form of behavioural recovery strongly suggests that the graft is interacting with adjacent host tissue to alter function. Transplanted dopaminergic cells (revealed by tyrosine hydroxylase immunohistochemistry) were seen in the caudate or the putamen or both, post mortem (Fig. 4). In summary, these experiments suggest that the transplantation of fetal dopamine-fich tissue into monkeys with experimentally induced parkinsonism can be of therapeutic benefit. Researchers are currently seeking to define the optimum methods of transplantation, including the age limit of the donor tissue, that can be used successfully. The use of fetal tissue transplantation in patients with Parkinson's disease is reviewed by Lindvall (p. 376) in a separate article in this issue of TINS.
Restitution of cognitive function by cholinergic transplants The marmoset is the only primate that has been used for the behavioural assessment of cholinergic grafts. Work done in rodents has already provided evidence that transplants of ACh-rich tissue taken from the fetal septal area can improve performance on a variety of learning and memory tasks when grafted into the hippocampus or cortex of animals whose cholinergic projections to these areas have been damaged by neurotoxic lesionn, surgical transection 369
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Fig. 6. Transplants of fetal cholinergic-rich tissue (but not non-cholinergic-rich tissue) into the hippocampus of fornix-transected monkeys restore their ability to learn visuospatial tasks. This histogram shows learning ability on visuospatial tasks by control monkeys, open bar (n = 12); monkeys with fornix lesions only, crosshatched bar (n = 10); monkeys with fomix lesions and subsequent cholinergic-rich fetal septal transplants into hippocampus, tinted bar (n = 8); and monkeys with fomix lesions and subsequent non-cholinergic fetal hippocampal transplants into hippocampus, crisscrossed bar (n = 4). Pre-lesion data show that the groups did not differ prior to fomix transection. Post-lesion data show that all three groups with fornix transections were significantly impaired on visuospatial tasks. Post-transplant data show that 3-4 months after transplantation, monkeys with cholinergicrich grafts are no longer impaired whereas monkeys with non-cholinergic grafts or no grafts remain severely impaired.
ever, they were not impaired in their ability to carry out tasks in which they always had to choose one of two different objects presented simultaneously (twochoice visual object discrimination tasks). This pattern of impairment in monkeys indicates selective hippocampal dysfunction17. Impairment on visual discrimination tasks occurs after damage to the surrounding cortical tissue. Having ascertained the degree of impairment, we grafted bilaterally ACh-rich cell suspensions prepared from marmoset fetuses into the hippocampus of eight of these lesioned animals, and non-cholinergic hippocampal tissue into a further four lesioned animals. Three months after transplantation, all these animals, together with a total of 12 control animals, began a lengthy programme of testing on a range of tasks. While the fornix-transected, non-grafted animals remained impaired on visuospatial tasks throughout the test period, the performance of the animals with cholinergic grafts was consistently better than that of the fornix-lesioned animals, and was indistinguishable from that of the control animals (Fig. 6). However, those with non-cholinergic grafts showed no improvement on visuospatial tasks. When the brains of the animals with cholinergic grafts were examined histologically at the end of the experiment, grafts staining densely for acetylcholinesterase activity were seen bilaterally within the temporal lobes, with substantial fibre outgrowth into surrounding host tissue 16. These results demonstrate that ACh-rich grafts can restore cognitive abilities. However, although there are a number of human diseases in which cognitive decline is associated with cholinergic dysfunction (e. g. Alzheimer's disease, Parkinson's disease with dementia and Korsakoff's syndrome), there is also severe additional pathology in these conditions. Substantial further study of primates with experimentally induced conditions that more closely resemble these human disease conditions is required before it can be predicted that cholinergic transplantation could have a role in treating the cognitive dysfunction in these disorders.
of the fimbria-fornix12'13, or prolonged alcohol ingestion 14. For the past few years we have been studying the cognitive effects of both neurotoxic and surgical lesions of the cholinergic pathways in marmosets. Lesions within the cholinergic projections produce impairments that are essentially the same as those seen following surgical removal of the target areas. Selected references We have shown that these cognitive deficits induced 1 Bj6rklund, A. eta/. (1983)Acta Physiol. Scand. (Suppl.) 522, 1-7 by cholinergic lesions can be ameliorated by direct 2 Annett, L. E. etaL (1990) Prog. Brain Res. 82, 535-542 administration of cholinergic agonists such as pilocar- 3 Perry, E. K. etal. (1978) Br. Med. J. 2, 1457-1459 4 Bankiewicz, K. S., Plunkett, R. J., Mefford, I., Kopin, I. J. and pine, but we have also argued that normal cognitive Oldfield, E. H. (1990) Prog. Brain Res. 82, 561-572 function does not appear to be specifically dependent 5 Bj6rklund, A. et al. (1987) Trends Neurosci. 10, 509-516 on information carried within the cholinergic projec- 6 Taylor, J. R. et al. (1990) Prog. Brain Res. 82, 543-559 tions. Rather, these projections may have a modu- 7 Taylor, J. R. et al. Exp. Brain Res. (in press) latory or enabling effect on cortical or hippocampal 8 Freed, C. R., Richards, J. B., Sabol, K. E. and Reite, M. L. (1988) in Pharmacology and Functional Regulation of Dopfunction, analogous to the enabling function of ascendaminergic Neurones (Beart, P. M., Woodruff, G. N. and ing dopamine projections in the motor systems 15. In Jackson, D. M., eds), pp. 353-360, Macmillan collaboration with Alan Fine at Dalhousie University, 9 Bakay, R. A. E. et al. (1987) Ann. N.Y. Acad. Sci. 495, Canada, we have investigated the behavioural con623-640 sequences of bilateral surgical transection of the 10 Fine, A. etal. (1988) Prog. Brain Res. 78, 479-489 fornix (which carries the cholinergic projection to the 11 Fine, A., Dunnett, S. B., Bj6rklund, A. and Iverson, S. D. (1985) Proc. Natl Acad. Sci. USA 82, 5227-5230 hippocampus) in a total of 22 marmosets, and the 12 Nilsson, O. G., Shapiro, M. L., Gage, F. H., Olton, D. S. and effects of subsequent transplantation of ACh-rich fetal Bj6rklund, A. (1986) Exp. Brain Res. 67, 195-215 tissue into the hippocampus (Fig. 5) in eight of these 13 Low, W. C. etal. (1982) Nature 300, 260-262 14 Arendt, T. et al. (1989) Neuroscience 33,435-462 animals. Monkeys with fornix transections were severely 15 Ridley, R. M., Murray, T. K., Johnson, J. A. and Baker, H. F. Brain Res. 376, 108-116 but selectively impaired in their ability to learn 16 (1986) Ridley, R. M., Thornley, H. D., Baker, H. F. and Fine, A. visuospatial tasks (Fig. 2) (Ref. 16 and Ridley, R. M. (1991) Exp. Brain Res. 83, 533-538 and Baker, H. F., unpublished observations). How- 17 Ridley, R. M. and Baker, H. F. Brain Res. Rev. (in press) 370
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Selected references 1 Moore, R. Y. and Lenn, N. J. (1972) J. Comp. Neurol. 146, 1-14 2 Card, P. J. and Moore, R. Y. (1982) J. Comp. Physiol. 206, 390-396 3 Stephan, F. J. and Zucker, I. (1972) Proc. NatlAcad. Sci. USA 69, 1583-1586 4 Moore, R. Y. and Eichler, V. (1972) Brain Res. 42, 201-206 5 Inouye, S-I. T. and Kawamura, H. (1979) Proc. NatlAcad. Sci. USA 76, 5962-5966 6 Green, D. J. and Gillette, R. (1982) Brain Res. 245, 198--200 7 Groos, G. A. and Hendricks, J. (1982) Neurosci. Left. 34, 283-288 8 Earnest, D. J. and Sladek, C. D. (1986) Brain Res. 382, 129-133 9 Drucker-Colin, R., Aguilar-Roblero, R., Garcia-Hernandez, F., Fernandez-Cancino, F. and Rattoni, F. B. (1984) Brain Res. 311,353-357 10 Sawaki, Y., Nihonmatsu, I. and Kawamura, H. (1984) Neurosci. Res. 1, 67-72 11 Lehman, M. N. et aL (1987)J. Neurosci. 7, 1626-1638 12 DeCoursey, P. J. and Buggy, J. (1988) Comp. Endocrinol. 7, 49--54 13 Boer, G. J. and Griffioen, H. A. (1990) Eur. J. Morphol. 28, 330-345 14 Ralph, M. R., Foster, R. G., Davis, F. C. and Menaker, M. (1990) Science 247, 975-978
15 Rosenwasser, A. N. (1988) Prog. Psychobiol. Physiol. Psychol. 13, 155-226 16 Meijer, J. H. and Reitveld, W. J. (1989) Physiol. Rev. 69, 671-707 17 Rusak, B. (1989)J. BioL Rhythms 4, 121-134 18 Ralph, M. R. and Menaker, M. (1988) Science 241, 1225-1227 19 Sollars, P. J. and Kimble, D. P. (1988) Soc. Neurosci. Abstr. 14, 49 20 Davis, F. C. (1989) Soc. Neurosci. Abstr. 15, 493 21 Zimmerman, N. H. and Menaker, M. (1979) Proc. NatlAcad. Sci. USA 76, 999-1003 22 Van den Pol, A. N. and Tsujimoto, K. L. (1985) Neuroscience 15, 1049-1086 23 Watts, A. G. and Swanson, L. W. (1987) J. Comp. Neuro/. 258, 230-252 24 Van den Pol, A. N. in Suprachiasmatic Nucleus: The Mind's Clock (Klein, D. C., Moore, R. Y. and Reppert, S. M., eds), Oxford University Press (in press) 25 Lehman, M. N., Silver, R. and Bittman, E. L. in Suprachiasmatic Nucleus: The Mind's Clock (Klein, D. C., Moore, R. Y. and Reppert, S. M., eds), Oxford University Press (in press) 26 Weigand, S. J. and Gash, D. M. (1988)J. Comp. NeuroL 267, 562-579 27 Canbelyi, R. S., Lehman, M. N. and Silver, R. Brain Res. (in press) 28 Klein, D. C. eta/. (1983) Brain Res. Bull. 10, 647-652 29 Pickard, G. E. and Turek, F. W. (1983) Neurosci. Left. 43, 67-72 30 Earnest, D. J., Sladek, C. J., Gash, D. M. and Weigand, S. J. (1989) J. Neurosci. 9, 2671-2677 31 Hakim, H. and Silver, R. (1988) Soc. Neurosci. Abstr. 14, 51 32 Roberts, M. H., Bernstein, M. F. and Moore, R. Y. (1987) Dev. Brain Res. 32, 59-66 33 Aebischer, P., Winn, S. R. and Galletti, P. M. (1988) Brain Res. 448, 364-368 34 Silver, R., Lehman, M. N., Gibson, M., Gladstone, W. R. and Bittman, E. L. Brain Res. (in press) 35 Bos, N. P. A. and Mirmiran, M. (1990) Brain Res. 511, 158-162 36 Ralph, M. R. and Torre, E. R. (1990)Soc. Neurosci. Abstr. 16, 769
Can fetal neural transplantsrestore functionin monkeys with lesion-inducedbehaviouraldeficits? R. M. Ridley and H. F. Baker R. M. Ridleyand H, F. Bakerare in the Division of Psychiatry, Clinical Research Centre, Walford Road, Harrow HA1 3UJ, UK.
Experiments are now being conducted in monkeys to see whether the transplantation of fetal neural tissue, rich in certain neurotransmitter-producing cells, can restore behaviour in animals with movement or learning impairments induced by lesions that have destroyed important neurotransmitter pathways. Transplantation of doDamine neurones in humans may prove to be a useful therapy in Parkinson's disease, in which a severe movement disorder is associated with degeneration of the dopamine system. Transplantation of cholinergic neurones in monkeys can overcome a severe learning impairment induced by lesion of the cholinergic system. Cholinergic transplantation may eventually be of use in a variety of neurodegenerative dementing illnesses. Although most of the work done in developing the techniques of tissue transplantation into the CNS (including the behavioural assessment of such procedures) has been carried out in rodents, there are several reasons for extending these studies to primates prior to clinical application. First, the behav-
366
ioural repertoire of primates might be amenable to more detailed assessment than that of rodents; therefore, more precise measures of restitution of function and, more importantly, more sensitive measures of deleterious effects may be obtained. Second, it might be possible to measure those more complex and cognitive functions that hopefully might be most affected by transplantation. Third, the optimum techniques for transplantation might vary between species. The best estimate, prior to clinical use, of the most appropriate techniques in humans may only be possible in other primates. Work carried out in monkeys has been very limited but the results are important. They are reviewed here in order to demonstrate what has been achieved experimentally as a prelude to possible therapeutic use in humans. Two techniques of fetal neural tissue transplantation into the CNS have been studied in monkeys: transplantation of tissue fragments dissected from fetal brain into previously prepared cavities in recipient brain; and the direct stereotaxic injection of
© 1991,ElsevierSciencePublishersLtd,(UK) O166-2236/91/$02.O0
TINS, Vol. 14, No. 8, 1991
®
®
i il~ill';J:, "/" "\.,/i,, ~
..... '"11_1 ",, i
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4-phenyl-1,2, 3, 6-tetrahydropyridine (MPTP) or 6-hydroxydopamine (6-OHDA). Both these models produce the primary pathological condition of Parkinson's disease, i.e. degeneration of the ascending dopaminergic pathways from the substantia nigra to the striatum. The unilateral method produces marked ipsilateral rotation (i.e. towards the lesioned side). This rotation is eminently quantifiable, can be manipulated by dopaminergic drugs, and its diminution can serve as an index of the restitution of impaired function. Moreover, reversal of the direction of rotation (from ipsilateral to contralateral) can indicate over-activity of the transplant 2. In contrast, bilateral lesions produced by peripheral administration of MPTP result in types of behaviour, including bradykinesia, rigidity and poor movement initiation, that are more characteristic of the clinical symptoms of Parkinson's disease, but that are not so easy to quantify. Unlike the unilateral procedure, peripheral MPTP treatment can produce animals that
Fig. 1. Drawing of a marmoset attempting to retrieve pieces of bread placed on a staircase outside a perspex screen. To obtain reward the monkey must reach through the vertical slot at either side of the staircase using only one arm. Monkeys with unilateral lesions of the subtantia nigra tend to neglect the contralateral side, i.e. fail to use the contralateral arm. When they do attempt to reach into the contralateral side of the staircase, they frequently show clumsiness and often attempt to retrieve the most difficult piece at the top of staircase (but nearest to the ipsilateral side) before attempting to retrieve the easiest piece nearest to the slot but in the furthest contralateral position.
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, dissociated fetal brain cell suspensions or solid tissue fragments into deep brain structures. Dopamine-rich tissue for transplantation into the basal ganglia of monkeys with damage to the dopamine pathway is dissected from the fetal ventral mesencephalon using techniques based on those used in rats ~. Acetylcholine-rich tissue from the septal area of the fetal brain is currently used for transplantation into the hippocampus of monkeys that have had the cholinergic projection to that area disrupted by transection of the fornix.
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Assessing behavioural recovery i/| The principal method of assessing effects of intracerebral transplantation in a~nals involves the pro~.o,;,l i duction of a pathological condition similar to that underlying a particular clinical disease. The resulting change in the animal's behaviour can then be measured before and after the transplantation procedure. For rigorous scientific assessment it is preferable that the behavioural change is quantifiable Fig. 2. Drawing of a marmoset performing trials of a on an interval scale, i.e. one in which the numerical visuospatial task in a Wisconsin General Test Apparatus. In values measure real quantities. In practice, however, this task the monkey has to learn to move the stimulus on it is sometimes only possible to produce an arbitrary its left to uncover the foodwell containing reward during trials in which one pair of identical stimuli (corks) are number in order to rate the severity of impairment. Some workers consider it important that the presented. It must also learn to move the stimulus on its behavioural impairment resembles the symptoms of right to obtain reward on trials on which a different pair of the disorder being modelled, but this is not strictly identical stimufi (brackets) are presented. These two types necessary provided the physiological basis of the of trials are presented in a random order until the animal makes a predetermined number of correct responses (e.g. behaviour is understood. For example, in primate 27 correct in 30 consecutive trials). Acquisition of this task models of Parkinson's disease, it is possible to requires that the animal associates different stimuli with produce either a unilateral or a bilateral lesion of the different responses, and is impaired by lesions of the nigrostriatal tract using neurotoxins such as 1-methyl- cholinergic projection to the hippocampus. HNS, Vo1.14, No. & 1991
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much controversy regarding the mechanism by which grafts influence the host and this has been the subject of an earlier TINS review 5. The behavioural improvement reported by Bankieif) wicz et al. 4 might have been due to regenerative cO 100processes in the host (stimulated by the grafting a3 procedure), which then might have led to reinnervation of the host caudate by dopaminergic fibres not destroyed by the MPTP treatment. However, cO such sprouting is possible only where a proportion of 0 host dopaminergic neurones survives the lesion procedure. It is possible that a better differentiation between the behavioural effects of experimental grafts and control procedures will be demonstrated when lesioning methods are more efficient and when - -100 P the optimum techniques for grafting have been ; 3 4 6 0 determined in primates. time after transplantation (months) Pioneering work on fetal neural tissue transplantation in primates has been carried out by a collaborFig. 3. Following unilateral 6-OHDA lesion of the substantia nigra, marmosets rotate ipsilaterally (towards the lesion ative team based in Yale and Rochester in the US, side) when injected i.m. with amphetamine. In the months and is described most extensively by Taylor et al. 6'7. following transplantation with dopamine-rich tissue into This group examined the behavioural effects of the caudate or caudate and putamen, marmosets show bilateral stereotaxic transplantation of many small progressively less ipsilateral rotation. Marmosets with fragments of fetal dopaminergic tissue into nondopamine-rich tissue transplanted into putamen alone do cavitated sites of the caudate nucleus of five vervet not show any improvement. C), control animals (n = 5); monkeys (Cercopithecus aethiops) with parkinsonism [2, unilateral 6-OHDA lesion of the substantia nigra, no graft (n = 10); 0, nigral lesion plus caudate and putamen grafts (n = 6); I , nigral lesion plus caudate graft only (n = 5); A, nigral lesion plus putamen graft only (n = 5). These results are subject to histological confirmation. 200
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are incapable of self-care, resulting in many practical difficulties. Motor skills can also be measured in unilaterally lesioned animals and in those bilaterally lesioned animals that are not grossly incapacitated during tasks requiring, for example, the retrieval of rewards from awkward or concealed places (Fig. 1). At present there are no primate models of Alzheimer's disease suitable for behavioural assessment. However, one important feature of this disease is the degeneration of the ascending cholinergic projections, which makes a major contribution to cognitive decline3. It is possible to make selective lesions within this system and to assess the effects of cholinergic transplants on the learning impairments that are produced. In this case cognitive ability is usually assessed by determining the number of trials it takes for an animal to learn to discriminate between stimuli in a Wisconsin General Test Apparatus (Fig. 2). Behavioural m e a s u r e s in animal models of Parkinson's disease Bankiewicz et aL 4 have described the effects of
bilateral surgical transplantation of fragments of dopamine-containing fetal ventral mesencephalon into the caudate nucleus of five monkeys (Macaca mulatta) that had been treated unilaterally with MPTP; these effects were compared with animals that were only lesioned, or with MPTP-treated animals with control grafts, or with surgical cavitation only. Although the fetal tissue grafts showed good long-term survival, they did not appear to reinnervate the host tissue. Behavioural recovery was quite good but not better than that produced by nonneural grafts and other control procedures. There is 368
Transplant of dopamine-rich nigral fetal tissue into the putamen of a marmoset with a unilateral 6-OHDA lesion of the substantia nigra. Orange stain shows tyrosine hydroxylase, a marker for dopamine cells. Abbreviations: C, cortex; Ca, caudate; IC, internal capsule; P, putamen. Scale bar is 0.5 mm. Arrows indicate transplanted tissue. Fig. 4.
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that had been induced by intramuscular administration of MPTP. These effects were compared with the behaviour of three MPTP-lesioned monkeys with control grafts and three non-lesioned animals with control gaffs. Four moderately to severely affected lesioned monkeys showed substantial restitution of normal behaviour and diminution of behaviour indicative of parkinsonism (e.g. the degree of bradykinesia) when transplanted with fetal dopaminergic tissue. Two of these monkeys, together with another one that had a slightly less severe lesion-induced impairment, were tested in an object retrieval task and showed substantial improvements over time. In contrast, the MPTP-treated animals transplanted with inappropriate control tissue showed no behavioural recovery and were largely recumbent. They were unable to participate in the object retrieval task and were killed earlier than those receiving dopaminergic grafts because they were so severely affected. Thus, these authors have shown that substantial restitution of behavioural competence that is equivalent to a major clinical improvement can be achieved in monkeys with severe parkinsonian symptoms. When these monkeys were killed, the number of surviving graft-derived neurones, which immunostain with antibodies to tyrosine hydroxylase (a marker for dopamine cells), was small, but this in itself demonstrates that pronounced behavioural recovery can be achieved with a very modest increase in the level of dopaminergic activity in the caudate. This effect in severely incapacitated monkeys is important because it is hoped that transplantation will be particularly beneficial to those human patients in whom the disease is so advanced that drug treatment is no longer adequate. Currently, the more usual technique for transplantation of fetal material involves the dissociation of donor tissue into a cell suspension that can then be injected stereotaxicall~¢ with the minimum of trauma into deep target areas'. Freed et al. 8 and Bakay et al. 9 have compared seven monkeys (Macaca mulatta and Macaca radiata) with lesions alone with six animals that had bilateral MPTP-induced lesions and bilateral or unilateral stereotaxically placed fetal cell suspension grafts in either caudate alone or caudate plus putamen. The four monkeys best displaying graft survival showed considerable clinical improvement. The common marmoset (Callithrixjacchus) is a very small primate with a high and reliable reproductive rate. It is therefore possible to use this species for transplantation experiments with larger numbers of subjects using donor tissue of very precise gestational age. Research groups in Britain have used both bilateral MPTP administration and unilateral 6-OHDA injections in this species to produce models of Parkinson's disease (Refs 2, 10 and Annett, L. E. et al., pers. commun.). In these experiments a total of 54 monkeys have been used, 26 of which were given 6-OHDA unilaterally and ten of which were given MPTP bilaterally. Overall, unilaterally lesioned animals showed functional improvement following transplantation of fetal, dopamine-rich cell suspension into the ipsilateral basal ganglia, while the bilaterally lesioned animals improved only after bilateral grafting into the basal ganglia. Marked improvements in activity and decreases in drug-induced rotation were seen in the unilaterally lesioned animals following TINS, Vol. 14, No. 8, 1991
Fig. 5. Transplant of cholinergic-rich septal fetal tissue into the hippocampus of a marmoset with fornix transection, stained for acetylcholinesterase, a marker for cholinergic nerve terminals. The transplant stains darkly, but substantial reinnervation of surrounding host tissue can also be seen. Abbreviations: D, dentate gyrus; E, entorhinal cortex; H, hippocampus. Scale bar is 0.5 mm. Arrows indicate transplanted tissue.
transplantation of dopamine-rich tissue into both the caudate and putamen, or into the caudate only. No improvement in rotation was seen in untransplanted animals or animals with transplants into the putamen (Fig. 3). Preliminary data suggest that monkeys with grafts into the putamen show less contralateral neglect (measured by the latency to start removing a sticky label from the contralateral leg) than monkeys with no grafts or dopamine grafts into the caudate (Annett, L. E., pers. commun.). The importance of the site at which the transplant is placed in determining the form of behavioural recovery strongly suggests that the graft is interacting with adjacent host tissue to alter function. Transplanted dopaminergic cells (revealed by tyrosine hydroxylase immunohistochemistry) were seen in the caudate or the putamen or both, post mortem (Fig. 4). In summary, these experiments suggest that the transplantation of fetal dopamine-fich tissue into monkeys with experimentally induced parkinsonism can be of therapeutic benefit. Researchers are currently seeking to define the optimum methods of transplantation, including the age limit of the donor tissue, that can be used successfully. The use of fetal tissue transplantation in patients with Parkinson's disease is reviewed by Lindvall (p. 376) in a separate article in this issue of TINS.
Restitution of cognitive function by cholinergic transplants The marmoset is the only primate that has been used for the behavioural assessment of cholinergic grafts. Work done in rodents has already provided evidence that transplants of ACh-rich tissue taken from the fetal septal area can improve performance on a variety of learning and memory tasks when grafted into the hippocampus or cortex of animals whose cholinergic projections to these areas have been damaged by neurotoxic lesionn, surgical transection 369
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Fig. 6. Transplants of fetal cholinergic-rich tissue (but not non-cholinergic-rich tissue) into the hippocampus of fornix-transected monkeys restore their ability to learn visuospatial tasks. This histogram shows learning ability on visuospatial tasks by control monkeys, open bar (n = 12); monkeys with fornix lesions only, crosshatched bar (n = 10); monkeys with fomix lesions and subsequent cholinergic-rich fetal septal transplants into hippocampus, tinted bar (n = 8); and monkeys with fomix lesions and subsequent non-cholinergic fetal hippocampal transplants into hippocampus, crisscrossed bar (n = 4). Pre-lesion data show that the groups did not differ prior to fomix transection. Post-lesion data show that all three groups with fornix transections were significantly impaired on visuospatial tasks. Post-transplant data show that 3-4 months after transplantation, monkeys with cholinergicrich grafts are no longer impaired whereas monkeys with non-cholinergic grafts or no grafts remain severely impaired.
ever, they were not impaired in their ability to carry out tasks in which they always had to choose one of two different objects presented simultaneously (twochoice visual object discrimination tasks). This pattern of impairment in monkeys indicates selective hippocampal dysfunction17. Impairment on visual discrimination tasks occurs after damage to the surrounding cortical tissue. Having ascertained the degree of impairment, we grafted bilaterally ACh-rich cell suspensions prepared from marmoset fetuses into the hippocampus of eight of these lesioned animals, and non-cholinergic hippocampal tissue into a further four lesioned animals. Three months after transplantation, all these animals, together with a total of 12 control animals, began a lengthy programme of testing on a range of tasks. While the fornix-transected, non-grafted animals remained impaired on visuospatial tasks throughout the test period, the performance of the animals with cholinergic grafts was consistently better than that of the fornix-lesioned animals, and was indistinguishable from that of the control animals (Fig. 6). However, those with non-cholinergic grafts showed no improvement on visuospatial tasks. When the brains of the animals with cholinergic grafts were examined histologically at the end of the experiment, grafts staining densely for acetylcholinesterase activity were seen bilaterally within the temporal lobes, with substantial fibre outgrowth into surrounding host tissue 16. These results demonstrate that ACh-rich grafts can restore cognitive abilities. However, although there are a number of human diseases in which cognitive decline is associated with cholinergic dysfunction (e. g. Alzheimer's disease, Parkinson's disease with dementia and Korsakoff's syndrome), there is also severe additional pathology in these conditions. Substantial further study of primates with experimentally induced conditions that more closely resemble these human disease conditions is required before it can be predicted that cholinergic transplantation could have a role in treating the cognitive dysfunction in these disorders.
of the fimbria-fornix12'13, or prolonged alcohol ingestion 14. For the past few years we have been studying the cognitive effects of both neurotoxic and surgical lesions of the cholinergic pathways in marmosets. Lesions within the cholinergic projections produce impairments that are essentially the same as those seen following surgical removal of the target areas. Selected references We have shown that these cognitive deficits induced 1 Bj6rklund, A. eta/. (1983)Acta Physiol. Scand. (Suppl.) 522, 1-7 by cholinergic lesions can be ameliorated by direct 2 Annett, L. E. etaL (1990) Prog. Brain Res. 82, 535-542 administration of cholinergic agonists such as pilocar- 3 Perry, E. K. etal. (1978) Br. Med. J. 2, 1457-1459 4 Bankiewicz, K. S., Plunkett, R. J., Mefford, I., Kopin, I. J. and pine, but we have also argued that normal cognitive Oldfield, E. H. (1990) Prog. Brain Res. 82, 561-572 function does not appear to be specifically dependent 5 Bj6rklund, A. et al. (1987) Trends Neurosci. 10, 509-516 on information carried within the cholinergic projec- 6 Taylor, J. R. et al. (1990) Prog. Brain Res. 82, 543-559 tions. Rather, these projections may have a modu- 7 Taylor, J. R. et al. Exp. Brain Res. (in press) latory or enabling effect on cortical or hippocampal 8 Freed, C. R., Richards, J. B., Sabol, K. E. and Reite, M. L. (1988) in Pharmacology and Functional Regulation of Dopfunction, analogous to the enabling function of ascendaminergic Neurones (Beart, P. M., Woodruff, G. N. and ing dopamine projections in the motor systems 15. In Jackson, D. M., eds), pp. 353-360, Macmillan collaboration with Alan Fine at Dalhousie University, 9 Bakay, R. A. E. et al. (1987) Ann. N.Y. Acad. Sci. 495, Canada, we have investigated the behavioural con623-640 sequences of bilateral surgical transection of the 10 Fine, A. etal. (1988) Prog. Brain Res. 78, 479-489 fornix (which carries the cholinergic projection to the 11 Fine, A., Dunnett, S. B., Bj6rklund, A. and Iverson, S. D. (1985) Proc. Natl Acad. Sci. USA 82, 5227-5230 hippocampus) in a total of 22 marmosets, and the 12 Nilsson, O. G., Shapiro, M. L., Gage, F. H., Olton, D. S. and effects of subsequent transplantation of ACh-rich fetal Bj6rklund, A. (1986) Exp. Brain Res. 67, 195-215 tissue into the hippocampus (Fig. 5) in eight of these 13 Low, W. C. etal. (1982) Nature 300, 260-262 14 Arendt, T. et al. (1989) Neuroscience 33,435-462 animals. Monkeys with fornix transections were severely 15 Ridley, R. M., Murray, T. K., Johnson, J. A. and Baker, H. F. Brain Res. 376, 108-116 but selectively impaired in their ability to learn 16 (1986) Ridley, R. M., Thornley, H. D., Baker, H. F. and Fine, A. visuospatial tasks (Fig. 2) (Ref. 16 and Ridley, R. M. (1991) Exp. Brain Res. 83, 533-538 and Baker, H. F., unpublished observations). How- 17 Ridley, R. M. and Baker, H. F. Brain Res. Rev. (in press) 370
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