Mesencephalic Tissue Research Articles

Neural transplantation cannula and microinjector system: experimental and clinical experience. Technical note.

The authors present a simple, reliable, and safe system for performing neural transplantation in the human brain. The device consists of a transplantation cannula and microinjector system that has been specifically designed to reduce implantation-related trauma and to maximize the number of graft deposits per injection. The system was evaluated first in an experimental rat model of Parkinson's disease (PD). Animals in which transplantation with this system had been performed showed excellent graft survival with minimal trauma to the brain. Following this experimental stage, the cannula and microinjector system were used in eight patients with PD enrolled in the Halifax Neural Transplantation Program who received bilateral putaminal transplants of fetal ventral mesencephalic tissue. A total of 16 transplantation operations and 64 trajectories were performed in the eight patients, and there were no intraoperative or perioperative complications. Magnetic resonance imaging studies obtained 24 hours after surgery revealed no evidence of tissue damage or hemorrhage. Transplant survival was confirmed by fluorodopa positron emission tomography scans obtained 6 and 12 months after surgery. As neural transplantation procedures for the treatment of neurological conditions evolve, the ability to deliver viable grafts safely will become critically important. The device presented here has proved to be of value in maximizing the number of graft deposits while minimizing implantation-related trauma to the host brain.

Intrastriatal ventral mesencephalic xenografts of porcine tissue in rats: immune responses and functional effects.

Transplantation of neural tissue from other species has the potential to improve function in patients with neurodegenerative disorders. We investigated the functional effects of embryonic porcine dopaminergic neurons transplanted in a rat model of Parkinson's disease and the immune responses to the grafts in immunosuppressed and nonimmunosuppressed hosts. Twenty-three rats with unilateral 6-hydroxydopamine lesions received dissociated, 27-day-old embryonic porcine ventral mesencephalic tissue in the right striatum. Eighteen rats received cyclosporine (10 mg/kg, IP, daily) during the whole period of 14 weeks, in combination with prednisolone (20 mg/kg, IP, daily) the first 4 days. Five rats served as nonimmunosuppressed controls. All rats were tested for amphetamine-induced rotational behavior at 3-week intervals. Two immunosuppressed rats were excluded due to severe side effects of the treatment. Functional recovery was seen in 9 of 16 immunosuppressed rats at 12 weeks. Six animals remained functionally recovered at 14 weeks and contained an average of 5750+/-1450 (SEM) dopaminergic neurons. Between 9 and 14 weeks, three immunosuppressed rats rejected their grafts, based on rotation scores and immunohistochemical demonstration of cell infiltrates. One additional immunosuppressed rat showed evidence of ongoing rejection at 14 weeks. The striata in animals with ongoing or recent rejection contained large numbers of CD4- and CD8-positive lymphocytes, NK cells, macrophages, and microglia cells, whereas scar tissue was found in rats with grafts rejected at earlier time points (n = 11). Embryonic porcine ventral mesencephalic tissue matures in the adult rat striatum, reinnervates the host brain, and restores behavioral defects. Immunosuppressive treatment was necessary for long-term graft survival and functional recovery, but did not sufficiently protect from rejection mechanisms. Porcine neural tissue is an interesting alternative to embryonic human tissue for intracerebral transplantation in neurodegenerative diseases. However, to achieve stable graft survival in discordant xenogeneic combinations, an appropriate immunosuppressive treatment or donor tissue modifications are needed.

Co-grafts of muscle cells and mesencephalic tissue into hemiparkinsonian rats: behavioral and histochemical effects

Extracts from skeletal muscle cell cultures have been shown to increase levels of the enzyme tyrosine hydroxylase (TH) and promote survival of different types of developing neurons in vitro. To determine the effect of muscle cell co-grafts on the survival of dopamine neurons in a rat model of Parkinson’s disease, we transplanted an embryonic day (ED)-15 rat mesencephalic cell suspension alone or with neonatal muscle cells into 6-hydroxydopamine (6-OHDA) denervated rat striatum. In parallel experiments conducted in vitro, we cultured ED-15 rat mesencephalon or rat striatum in conditioned medium from neonatal rat muscle cultures (MC-CM). Our results showed that: (A) in vitro, MC-CM increased the number of TH-immunoreactive (TH-IR) neurons in embryonic mesencephalic cultures but did not induce expression of TH in embryonic striatal cultures; (B) in vivo, animals with co-grafts of muscle cells and ED-15 mesencephalon had more TH-IR in the grafted striatum compared to animals that received mesencephalic cells grafts alone, although the graft-induced reversal of circling behavior in response to methamphetamine was the same in both transplanted groups; and (C) grafts of muscle cells alone did not induce TH-IR in the denervated striatum and did not reduce methamphetamine-induced circling. These findings suggest that in vivo, neonatal muscle cells secrete factors that promote survival and/or outgrowth of fetal midbrain dopamine cells and improve the levels of TH-IR in grafted striatum.

Chapter 13 Clinical observations after neural transplantation in Parkinson's disease

This chapter discusses critical analysis of the clinical findings in Parkinson's disease (PD) patients grafted with embryonic mesencephalic tissue. The emphasis is on the magnitude, pattern and time course of clinical recovery and the relation between the graft-induced symptomatic relief and the restoration of striatal dopaminergic neurotransmission. In addition, the chapter discusses some methodological issues and different strategies to improve the functional recovery following neural transplantation. The clinical observations after neural transplantation clearly documents that cell replacement has the potential to become a novel and effective therapeutic strategy for large numbers of patients with PD. However, neural transplantation currently remains an experimental approach and can only be applied to small groups of patients. The urgent problem is to develop the new sources of dopaminergic cells that are available in large amounts and suitable for transplantation in humans. It is conceivable that these cells, to give rise to maximal symptomatic relief, must exhibit several of the properties characteristic of human embryonic mesencephalic dopamine neurons, such as formation of specialized synaptic contacts with host neurons, regulated dopamine release, and functional integration into the patient's brain.

Monitoring neuroprotection and restorative therapies in Parkinson's disease with PET.

In this article the role of functional imaging (PET and SPECT) as a surrogate marker for following the progression of Parkinson's disease (PD) is discussed. The potential value of PET and SPECT for assessing the efficacy of putative neuroprotective agents in PD is considered and a review of 18F-dopa PET findings in transplantation trials involving implantation of human and procine fetal mesencephalic tissue is presented. It is concluded that functional imaging provides a valuable adjunct to clinical assessment when judging the efficacy of neuroprotective and restorative approaches to PD.

Trasplantes neurales en la enfermedad de Parkinson: resultados clínicos tras 10 años de experiencia

At the end of the 1970s people considered the possibility that transplants might be useful to replace degenerate specific cell populations, such as the mesencephalic dopaminergic neurones in Parkinson's disease (PD). Since then this has become an experimental alternative treatment for patients with degenerative diseases. The history of transplants of catecholamine producing tissues within the brain of patients with PD started in 1985, when Backlund et al published the results of the first implants of autologous adrenal medulla in two patients with Parkinsonism. Since then, many patients throughout the world have benefited from the results obtained using this method. Two main types of tissue have been used in this method: autologous adrenal medulla and human foetal ventral mesencephalic tissue. In this paper we first review the clinical effects of the diverse types of transplant done to date. Then in the second part we give a summary of the clinical results obtained by our group with the different types of transplant carried out. We explain their evolution, original hypothesis and justify the reasons which led us to use three different types of donor material: autologous adrenal medulla, fetal tissue and adrenal medulla co-incubated with peripheral nerve. Then, after showing that the clinical improvement is different depending on the type of tissue transplanted, we comment on the probable reason for the improvement seen in patients with implants. The transplantation of nervous tissue seems to us to be no longer an experimental alternative for the treatment of PD but has become an effective, lasting treatment for patients with Parkinson's disease.

Dopamine cells in nigral grafts differentiate prior to implantation.

The yield of surviving dopamine cells in nigral grafts is typically low. It is unclear whether the dopamine neurons that do survive are postmitotic at the time of implantation, or are precursor cells that differentiate into dopamine neurons following transplantation in the host brain. We have therefore compared the survival of dopamine neurons in grafts that have been labelled with BrdU at different times prior to or following implantation in order to identify those cells that undergo final cell division at each stage of the procedure. Seven groups of rats were prepared with unilateral nigrostriatal lesions. Three groups received nigral grafts derived from E14 embryos labelled with BrdU in utero on either E12, E13 or E14 days of embryonic age (the E14 injection made 2 h prior to preparation of the graft cell suspension). Three further groups received nigral grafts from untreated E14 embryos, and then dividing cells within the grafts were labelled by injection of BrdU into the host lateral ventricle, 2 h, 1 day or 2 days after implantation (equivalent to E14, E15 and E16 days of embryonic age). The control group received standard (unlabelled) E14 grafts. Five weeks after the transplantation surgery, the host brains were processed using double immunohistochemical techniques to detect tyrosine hydroxylase (TH)-positive neurons which had incorporated BrdU. In the grafts labelled with BrdU prior to implantation, there was an increasing proportion of double-labelled cells (out of the total TH-positive cells surviving in the grafts) with birth dates on E12, E13 and E14 (1%, 12% and 10% per day, respectively). By contrast, grafts labelled following implantation, although containing many dividing neurons, had very few of these BrdU-labelled cells expressing a dopaminergic phenotype; < 1% surviving TH-positive cells were double-labelled from the 2 h post-transplant injection, and < 0.1% from each subsequent injection. This suggests not only that the great majority of TH-positive neurons in nigral grafts were already differentiated at the time of implantation, but also that transplantation of E14 ventral mesencephalic tissue either kills dopaminergic precursors or (more likely in our opinion) prevents their differentiation into a dopaminergic phenotype. Precursor cells that would differentiate into dopaminergic neurons beyond E14 if left in situ in the intact ventral mesencephalon do not readily differentiate into mature dopamine neurons following transplantation. If we are to enhance yields of functional dopamine-rich transplants, then we must identify strategies both to protect predifferentiated dopamine neurons in the grafts and to promote differentiation of a dopaminergic phenotype in precursor cells that continue to divide within the grafts following transplantation into an adult host environment.

Patterns of Cell Death and Dopaminergic Neuron Survival in Intrastriatal Nigral Grafts

Previous studies indicate that 80–95% of grafted dopamine neurons die following implantation of embryonic ventral mesencephalic tissue into the striatum. It is believed that the majority die within the first 1–3 weeks after surgery. The aim of this study was to study when and where the implanted neurons die, using the novel fluorescent stain Fluoro-Jade. Fluoro-Jade has recently been shown to stain cell bodies, dendrites, axons, and terminals of degenerating neurons. We transplanted dissociated ventral mesencephalic tissue from embryonic day 14 rat embryos into intact adult rat striatum. After perfusion and sectioning of the implanted rat brains, the number and distribution of Fluoro-Jade and tyrosine hydroxylase-positive neurons were evaluated at 6, 10, 14, and 42 days posttransplantation. Intensely Fluoro-Jade stained neurons were numerous in the grafts at 6 and 10 days after graft surgery; appeared in reduced numbers at 14 days; and had disappeared by the 42-day time point. The number of surviving tyrosine hydroxylase-positive, dopaminergic neurons in the grafts did not change between 6 and 42 days and the low survival rate confirmed that over 90% of these neurons had died during the first week. Assessment of the distribution of neurons positive for Fluoro-Jade or tyrosine hydroxylase revealed higher numbers of neurons stained for these markers located at the periphery than the center of the grafts, and this pattern did not change over time. This study indicates that transplanted neurons continue to die up to 14 days after grafting. Since the majority of transplanted tyrosine hydroxylase-positive neurons most probably die before 6 days after transplantation, neuroprotective strategies should primarily focus on the transplantation procedure and the first week after implantation.

Fetal Porcine Dopaminergic Cell Survival in Vitro and its Relationship to Embryonic Age

One of the critical factors in the survival of embryonic neural grafts is the age at which the population of donor neurons is harvested. This is especially the case for the developing dopaminergic neurons in the embryonic ventral mesencephalon, which are used for neural grafts in Parkinson's disease (PD). The donor age for optimal harvesting of these cells has been well characterized in the mouse, rat, and marmoset, and to a lesser extent in humans. However, the best donor age for porcine ventral mesencephalic tissue has not been ascertained, even though the use of this tissue for xenografts has been explored both experimentally and clinically. In this study the effect of donor age on dopaminergic cell survival was assessed in vitro, from a range of fetal pigs aged from E24 to E35. The number of tyrosine hydroxylase (TH)-positive cells per ventral mesencephalon was then calculated after 1 and 7 days in culture. E26-E27 embryos gave the highest yield of such cells at both survival time points, suggesting that this will be the optimal age for harvesting tissues whether for experimental or clinical nigral xenograft programs.

GDNF partially protects grafted fetal dopaminergic neurons against 6-hydroxydopamine neurotoxicity

Rats were given unilateral 6-hydroxydopamine (6-OHDA) lesions and subsequently received transplants of fetal ventral mesencephalic tissue into the denervated striatum. Four weeks later transplanted animals were tested for graft-mediated reduction of amphetamine-induced rotational behavior. Subsequently, transplanted animals received an intrastriatal injection of either GDNF (10 μg) or citrate buffer into a site lateral to the transplant, and then 6 h later received an injection of either 4.0 μg of 6-OHDA, 8.0 μg of 6-OHDA, or vehicle using the same stereotaxic coordinates that were used for the GDNF/citrate buffer injection. Animals were re-tested for amphetamine-induced rotational behavior 2 weeks later. Histological analysis revealed a significant reduction in the number of cell bodies immunostained for tyrosine hydroxylase (TH+) within the transplant for those animals pretreated with an intrastriatal injection of citrate buffer and subsequently given either dose of 6-OHDA. Transplanted animals pretreated with GDNF and subsequently administered 8.0 μg of 6-OHDA showed a significant reduction of TH+ neurons within the transplant compared to controls, however TH+ cell counts for this group remained significantly higher than the TH+ cell counts for the group of animals receiving the same dose of 6-OHDA but pretreated with citrate buffer. GDNF pretreatment completely protected TH+ cell bodies against 4.0 μg of 6-OHDA. Rotational scores indicated that GDNF provided only partial protection against 6-OHDA neurotoxicity in terms of transplant function. For both groups of transplanted animals receiving GDNF pretreatment and 6-OHDA injections, amphetamine-induced rotational scores dropped below the scores for animals pretreated with citrate buffer but remained significantly higher than the scores for transplanted animals that were not injected with 6-OHDA. Both histological and behavioral measures indicate GDNF partially protects integrated transplants against neurotoxic insult.

Enhancement of graft survival and sensorimotor behavioral recovery in rats undergoing transplantation with dopaminergic cells exposed to glial cell line-derived neurotrophic factor

The goal of this study was to investigate the ability of fetal dopaminergic neurons to improve complex sensorimotor behavior. The authors obtained ventral mesencephalic tissue from 14-day-old rat fetuses. The cells were exposed to glial cell line-derived neurotrophic factor (GDNF) prior to transplantation into rats with unilateral 6-hydroxydopamine lesions of the dopaminergic nigrostriatal pathway. Animals that received 400,000 cells exposed to GDNF demonstrated significant improvement in contralateral forelimb function and showed improvement in rotational behavior faster than animals that received cells not exposed to GDNF. Increasing the number of implanted cells to 800,000 exposed to GDNF did not result in any additional improvement in functional recovery. As neural grafting procedures in the nervous system evolve and genetically engineered cells or stem cells replace fetal tissue, crucial questions about cell number and trophic regulation will need to be addressed. This study demonstrates that grafting of 400,000 cells exposed to GDNF before transplantation has a beneficial effect in the restoration of complex sensorimotor behavior.

A neural transplantation cannula and microinjector system: experimental and clinical experience

The authors present a simple, reliable, and safe system for performing neural transplantation in the human brain. The device consists of a transplantation cannula and microinjector system that has been specifically designed to reduce implantation-related trauma and to maximize the number of graft deposits for each injection. The system was evaluated first in an experimental rat model of Parkinson's disease (PD). Animal transplantation with this system showed excellent graft survival with minimal trauma to the brain. Following this experimental stage, the cannula and microinjector system was used in eight patients with PD enrolled in the Halifax Neural Transplantation Program who received bilateral putaminal transplants of fetal ventral mesencephalic tissue. A total of 16 transplantation operations and 64 trajectories were performed in the eight patients, and there were no intra- or perioperative complications. Magnetic resonance imaging studies obtained 24 hours after surgery revealed no evidence of tissue damage or hemorrhage. Transplant survival was confirmed on fluorodopa positron emission tomography scans 6 and 12 months after surgery. As neural transplantation procedures for the treatment of neurological conditions evolve, the ability to deliver viable grafts safely will become of critical importance. The device presented here has been proven to be of value in maximizing the number of graft deposits while minimizing implantation-related trauma to the host brain.

Diminished Viability, Growth, and Behavioral Efficacy of Fetal Dopamine Neuron Grafts in Aging Rats with Long-Term Dopamine Depletion: An Argument for Neurotrophic Supplementation

We examined the behavioral and morphological correlates of the response to a single intrastriatal dispersed cell graft of fetal rat ventral mesencephalic tissue in male Fischer-344 rats of varying age (4, 17, and 24-26 months old) and history of mesostriatal dopamine (DA) depletion (1 or 14 months). Our goal was to determine the impact of advancing age and duration of DA depletion in the host on DA graft viability and function. The findings can be summarized as follows. (1) Fetal DA neuron grafts that were effective in completely ameliorating amphetamine-induced rotational behavior in young rats with short-term lesions were virtually without effect in aged rats with long-term lesions. Middle-aged rats with long-term lesions responded to these grafts with partial behavioral recovery. (2) Age of the host at the time of transplantation, and not duration of DA depletion, was the primary determinant of response to DA grafts. (3) Diminished efficacy of grafts in lesioned aging rats was related to decreased survival and neurite extension of transplanted DA neurons. (4) Co-grafts of DA neurons with Schwann cells as a source of neurotrophic support improved the behavioral outcome of grafts in aged lesioned rats. These findings support the view that the DA-depleted striatum of aged rats is an impoverished environment for survival, growth, and function of DA grafts. Consistent with this view, local supplementation of the neurotrophic environment of grafted DA neurons with products of co-grafted Schwann cells, a demonstrated source of neurotrophic activity for embryonic DA neurons, improved graft outcome.

Discordant xenografts: different outcome after mouse and rat neural tissue transplantation to guinea-pigs

Embryonic neural tissue obtained from other species has been considered as a donor tissue source in repair strategies for human neurodegenerative disorders. The neuro- and immunobiology of distantly related species combinations, discordant xenografts, need to be characterised. For this purpose, a small animal model would be an important research tool. Adult guinea-pigs, and adult rats as controls, received intrastriatal grafts of either mouse or rat embryonic ventral mesencephalic tissue. The survival rates and types of host immune response were assessed at 2 weeks after grafting using stereological techniques and semi-quantitative evaluations. In the mouse-to-guinea-pig group, all transplants were rejected and no tyrosine hydroxylase-immuno reactive (TH-IR) cells remained. In the rat-to-guinea-pig group, there was good survival of TH-IR cells (5050 SEM ± 1550), similar to that in the rat-to-rat group (4900 SEM ± 1540). In the mouse-to-rat group, half of the animals had no surviving TH-IR cells (520 SEM ± 230 for the whole group). These species combinations offer inexpensive, efficient, and suitable conditions to study important survival factors for discordant xenogeneic neural tissue transplants. The factors responsible for the divergent graft outcomes between the two combinations might provide clues on how to manipulate xenogeneic tissue to increase survival rates in the future.

Sequential bilateral transplantation in Parkinson's disease: effects of the second graft.

Five parkinsonian patients who had received implants of human embryonic mesencephalic tissue unilaterally in the striatum 10-56 months earlier were grafted with tissue from four to eight donors into the putamen (four patients) or the putamen plus the caudate nucleus (one patient) on the other side, and were followed for 18-24 months. After 12-18 months, PET showed a mean 85% increase in 6-L-[18F]fluorodopa uptake in the putamen with the second graft, whereas there was no significant further change in the previously transplanted putamen. Two patients exhibited marked additional improvements after their second graft: 'on-off' fluctuations virtually disappeared, movement speed increased, and L-dopa could be withdrawn in one patient and reduced by 70% in the other. The improvement in one patient was moderate. Two patients with atypical features, who responded poorly to the first graft, worsened following the second transplantation. These findings indicate that sequential transplantation in patients does not compromise the survival and function of either the first or the second graft. Moreover, putamen grafts that restore fluorodopa uptake to normal levels can give improvements of major therapeutic value.

Neurturin enhances the survival of intrastriatal fetal dopaminergic transplants

We investigated here the effect of the novel glial cell line-derived neurotrophic factor (GDNF)-family member neurturin (NTN) on transplanted fetal dopamine (DA) neurons. Three groups of rats with complete unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal DA system received intrastriatal grafts of embryonic ventral mesencephalic tissue. Following transplantation animals received repeated injections of vehicle or NTN (0.3 microg or 3.0 microg) over three weeks posttransplantation. NTN-treated animals had significantly (1.8-fold) more tyrosine hydroxylase-immunoreactive (TH-IR) neurons. Graft volume, TH-IR cell volume and overall dopaminergic host reinnervation remained unchanged. Amphetamine-induced rotation was rapidly compensated in all grafted rats. We conclude that administration of NTN may be a powerful way to increase survival of transplanted fetal DA neurons.

Autotransplantation for Parkinson's disease goes a step further.

Autotransplantation for Parkinson's disease goes a step further.

Striatal dopaminergic afferents concentrate in GDNF-positive patches during development and in developing intrastriatal striatal grafts.

Glial cell line-derived neurotrophic factor (GDNF) has potent trophic action on fetal dopaminergic neurons. We have used a double immunocytochemical approach with antibodies that recognize GDNF and tyroxine hydroxylase (TH) or the phosphoprotein DARPP-32, to study the developmental pattern of their interactions in the rat striatum and in intrastriatal striatal transplants. Postnatally, at one day and also at 1 week, GDNF showed a patchy distribution in the striatum, together with a high level of expression in the lateral striatal border, similar to that observed for the striatal marker DARPP-32 and also for TH. In the adult striatum, there was diffuse, weak immunopositivity for GDNF, together with widespread expression of DARPP-32-positive neurons and TH-immunoreactive (TH-ir) fibers. In 1-week-old intrastriatal striatal transplants, there were some GDNF immunopositive patches within the grafts and although there was not an abundance of TH-positive fibers, the ones that were seen were located in GDNF-positive areas. This was clearly evident in 2-week-old transplants, where TH-ir fibers appeared selectively concentrated in GDNF-positive patches. This pattern was repeated in 3-week-old grafts. In co-transplants of mesencephalic and striatal fetal tissue (in a proportion of 1:4), TH-ir somata were located mainly at the borders of areas that were more strongly immunostained for GDNF, and TH-ir fibers were also abundant in these areas and were found in smaller numbers in regions that were weakly positive for GDNF. These results demonstrate that GDNF-ir is coincident with that for TH and DARPP-32, and suggest that GDNF release by fetal striatal neurons both in normal development and in developing striatal grafts may have not only a trophic but also a tropic influence on TH-ir fibers and may be one of the factors that regulate dopaminergic innervation of the striatum.

Successful survival of grafted transgenic neural plate cells in adult central nervous system environment.

1. Accumulating evidence indicates that damaged brain functions can be ameliorated in a variety of animal models by the grafting of fetal neuronal cell or tissue into damaged brain. Clinical trials are under way to determine whether human fetal mesencephalic tissue can ameliorate motor functions in patients with Parkinson's disease. 2. Autopsy findings of parkinsonian patient implanted with human fetal mesencephalic tissue clearly revealed that the fetal neuronal graft can survive for an extended period of time in the human brain and densely reinnervate the surrounding host striatal tissue. 3. It is, however, still important to obtain more practical, effective, and ethically justifiable donor material for the future clinical application of the procedures. Desirable properties for the donor cells include long-term survival in the brain, neuronal cell type for the reconstruction of damaged neural circuits, and susceptibility to genetic manipulation for the practical use. 4. With the development of molecular biology techniques, genetic modification and transplantation of the donor neuronal cells might be a feasible way to cure many kinds of central nervous system diseases toward a "graft-gene therapy."

Functional imaging of Parkinson's disease: is it possible to detect brain areas for specific symptoms?

H2(15)O PET activation studies enable the brain systems involved in controlling different aspects of motor function to be defined. In Parkinson's disease (PD) freely chosen limb movements are performed slowly. This bradykinesia is associated with selective underactivity of the supplementary motor area and dorsal prefrontal cortex, frontal association areas that receive subcortical input principally from the basal ganglia. At the same time there is compensatory overactivity of the lateral premotor and parietal cortex, areas that have a primary role in facilitating motor responses to visual and auditory cues. This finding explains why PD patients find it easier to perform cued as opposed to freely chosen actions. Levels of activation of the supplementary motor area and dorsal prefrontal cortex in PD can be restored with dopaminergic medication, implants of fetal mesencephalic tissue, internal pallidotomy or high frequency electrical subthalamic stimulation. Activation studies suggest that Parkinsonian rest tremor arises from a combination of inappropriate overactivity of cerebellar connections and loss of dopaminergic function. When tremor is relieved by ventral thalamotomy or thalamic stimulation this cerebellar overactivity is corrected but at the expense of reducing levels of primary motor cortex activation. It has been hypothesised that dyskinesias in PD arise due to altered dopamine receptor binding following chronic exposure to levodopa stimulation. Functional imaging findings, however, are against this hypothesis and rather suggest that downstream increases in basal ganglia opioid neurotransmission are more likely to be relevant.