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Background: Cell transplantation is expected to be a promising treatment for Parkinson's disease (PD), in which re-innervation of the host striatum by grafted dopamine (DA) neurons is essential. In particular, the dorsolateral part of the striatum is important because it is the target of midbrain A9 DA neurons, which are degenerated in PD pathology. The effect of exercise on the survival and maturation of grafted neurons has been reported in several neurological disease models, but never in PD models., Objective: We investigated how exercise influences cell transplantation for PD, especially from the viewpoint of cell survival and neurite extensions., Methods: Ventral mesencephalic neurons from embryonic (E12.5) rats were transplanted into the striatum of adult 6-OHDA-lesioned rats. The host rats then underwent treadmill training as exercise after the transplantation. Six weeks after the transplantation, they were sacrificed, and the grafts in the striatum were analyzed., Results: The addition of exercise post-transplantation significantly increased the number of surviving DA neurons. Moreover, it promoted neurite extensions from the graft toward the dorsolateral part of the striatum., Conclusions: This study indicates a beneficial effect of exercise after cell transplantation in PD.

Background . Autologous neural cell ecosystem (ANCE) transplantation improves motor recovery in MPTP monkeys. These motor symptoms were assessed using semi-quantitative clinical rating scales, widely used in many studies. However, limitations in terms of sensitivity, combined with relatively subjective assessment of their different items, make inter-study comparisons difficult to achieve. Objective . The aim of this study was to quantify the impact of MPTP intoxication in macaque monkeys on manual dexterity and assess whether ANCE can contribute to functional recovery. Methods . Four animals were trained to perform 2 manual dexterity tasks. After reaching a motor performance plateau, the animals were subjected to an MPTP lesion. After the occurrence of a spontaneous functional recovery plateau, all 4 animals were subjected to ANCE transplantation. Results . Two of 4 animals underwent a full spontaneous recovery before the ANCE transplantation, whereas the 2 other animals (symptomatic) presented moderate to severe Parkinson's disease (PD)-like symptoms affecting manual dexterity. The time to grasp small objects using the precision grip increased in these 2 animals. After ANCE transplantation, the 2 symptomatic animals underwent a significant functional recovery, reflected by a decrease in time to execute the different tasks, as compared with the post-lesion phase. Conclusions . Manual dexterity is affected in symptomatic MPTP monkeys. The 2 manual dexterity tasks reported here as pilot are pertinent to quantify PD symptoms and reliably assess a treatment in MPTP monkeys, such as the present ANCE transplantation, to be confirmed in a larger cohort of animals before future clinical applications.

Biomaterials have been shown to significantly improve the outcome of cellular reparative approaches for Parkinson's disease in experimental studies because of their ability to provide transplanted cells with a supportive microenvironment and shielding from the host immune system. However, given that the margin for improvement in such reparative therapies is considerable, further studies are required to fully investigate and harness the potential of biomaterials in this context. Given that several recent studies have demonstrated improved brain repair in Parkinsonian models when using dopaminergic grafts derived from younger foetal donors, we hypothesized that encapsulating these cells in a supportive biomaterial would further improve their reparative efficacy. Thus, this study aimed to determine the impact of a GDNF-loaded collagen hydrogel on the survival, reinnervation, and functional efficacy of dopaminergic neurons derived from young donors. To do so, hemi-Parkinsonian (6-hydroxydopamine-lesioned) rats received intrastriatal transplants of embryonic day 12 cells extracted from the rat ventral mesencephalon either alone, in a collagen hydrogel, with GDNF, or in a GDNF-loaded collagen hydrogel. Methamphetamine-induced rotational behaviour was assessed at three weekly intervals for a total of 12 weeks, after which rats were sacrificed for postmortem assessment of graft survival. We found that, following intrastriatal transplantation to the lesioned striatum, the GDNF-loaded collagen hydrogel significantly increased the survival (4-fold), reinnervation (5.4-fold), and functional efficacy of the embryonic day 12 dopaminergic neurons. In conclusion, this study further demonstrates the significant potential of biomaterial hydrogel scaffolds for cellular brain repair approaches in neurodegenerative diseases such as Parkinson's disease., (© 2018 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Objective: To describe a technique to approach the anterior inferior basal ganglia., Case Description: A 29-year-old man presented with several episodes of facial droop and a cavernous malformation anterior, inferior, and lateral to the head of the caudate nucleus. Several possible surgical approaches were considered, including a transsylvian approach and a contralateral interhemispheric approach. Ultimately, the patient underwent a contralateral interhemispheric trans-striatocapsular approach to the lesion and was discharged without neurologic deficit within 72 hours., Conclusion: The approach described here is a novel alternative to transsylvian or supracarotid approaches to the anterior inferior basal ganglia and in this patient provided a well-tolerated surgical corridor that allowed complete resection of his cavernoma. We discuss several advantages and disadvantages of the various approaches to the anterior inferior basal ganglia., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum(1). Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra(2). The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions(3,4). These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or βIII tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Huntington's disease (HD) is an inherited neurodegenerative disorder that usually occurs in the third or fourth decades of life. Stem cell therapy is one of the approaches for HD treatment. Since mesenchymal stem cells (MSCs) have the ability to migrate into the lesioned site, we transplanted rat bone marrow-derived MSCs intravenously, following unilateral intrastriatal lesion made by quinolinic acid (QA) in Wistar rats. QA administration caused widespread neuropathological deficits similar to those found in HD, including impairments in motor and cognitive functions. Animals receiving MSCs exhibited significant improvement in motor and cognitive performance compared with sham group animals that did not receive cells. Animals were tested by apomorphine-induced rotations, beam walk, cylinder and hang wire tests at different times after cell transplantation. Results indicate that systemic transplantation of MSCs can significantly reduce the behavioral abnormalities of these animals. This method of systemic injection has a great advantage over invasive surgical techniques for transplantation of cells at the lesioned site.

Stem cell grafts have been advocated as experimental treatments for neurological diseases by virtue of their ability to offer trophic support for injured neurons and, theoretically, to replace dead neurons. Human embryonic stem cells (HESCs) are a rich source of neural precursors (NPs) for grafting, but have been questioned for their tendency to form tumors. Here we studied the ability of HESC-derived NP grafts optimized for cell number and differentiation stage prior to transplantation, to survive and stably differentiate and integrate in the basal forebrain (neostriatum) of young adult nude rats over long periods of time (6 months). NPs were derived from adherent monolayer cultures of HESCs exposed to noggin. After transplantation, NPs showed a drastic reduction in mitotic activity and an avid differentiation into neurons that projected via major white matter tracts to a variety of forebrain targets. A third of NP-derived neurons expressed the basal forebrain-neostriatal marker dopamine-regulated and cyclic AMP-regulated phosphoprotein. Graft-derived neurons formed mature synapses with host postsynaptic structures, including dendrite shafts and spines. NPs inoculated in white matter tracts showed a tendency toward glial (primarily astrocytic) differentiation, whereas NPs inoculated in the ventricular epithelium persisted as nestin(+) precursors. Our findings demonstrate the long-term ability of noggin-derived human NPs to structurally integrate tumor-free into the mature mammalian forebrain, while maintaining some cell fate plasticity that is strongly influenced by particular central nervous system (CNS) niches.

Transplantation of bone marrow stromal cells (BMSC) has recently been demonstrated to provide neuroprotection in animal models of brain injuries such as ischemia and trauma. The present study was undertaken to explore whether BMSC can promote the survival of dopamine (DA) neurons in neuronal insult models in vitro. We also examined whether BMSC can increase the survival rate of embryonic DA neurons grafted into the striatum of a rat model of Parkinson's disease (PD). Treatment with conditioned media derived from BMSC cultures was found to significantly prevent the death of DA neurons in in vitro cell injury models such as serum deprivation and exposure to the neurotoxin 6-OHDA. In a transplantation study, we also found that the survival of grafted DA cells was significantly enhanced by treating donor cells with the conditioned media at the steps of both cell dissociation and implantation. The results suggest that BMSC may secrete diffusible factors able to protect DA neurons against neuronal injuries. Indeed, BMSC expressed mRNA encoding brain-derived neurotrophic factor, fibroblast growth factor-2 and glial cell line-derived neurotrophic factor, all of which have previously been shown to exhibit potent neurotrophic effects on DA cells. Enzyme-linked immunosorbent assay revealed that the cells release these growth factors into culture media. The present data indicate that BMSC may be a potential donor source of cell-based regenerative therapy for PD where the progressive loss of the midbrain DA neurons takes place.

The present study analyzed whether grafts of the mesencephalic progenitor cell line CSM14.1 into the neonatal rat caudate putamen (CPu) differentiate into neurons and whether this is accompanied by a functional improvement in 6-hydroxydopamine (6-OHDA)-lesioned animals. As in previous studies, a neuronal differentiation of CSM14.1 cells transplanted into the CPu of adult animals could not be observed, so we here used neonatal rats, because graft location and host age seemingly are crucial parameters for neural transplant differentiation and integration. Rats bilaterally lesioned at postnatal day 1 by intraventricular 6-OHDA-injections 2 days later received 100,000 CSM14.1 cells prelabelled with the fluorescent dye PKH26 into the right CPu. Five weeks after grafting, the cylinder test was performed, and the data compared with data from age-matched intact controls and bilaterally lesioned-only animals. Brain slices immunostained for tyrosine hydroxylase (TH) were quantified by optical densitometry. We observed a significant preference of left forelimb use exclusively in transplanted animals. In these rats, TH-containing perikarya were found in the grafted CPu, presumedly leading to the significant increase of TH-immunoreactive fibers in this region. Moreover, confocal laser microscopy revealed a differentiation of transplanted PKH26-labelled CSM14.1 cells into neuronal nuclei antigen or TH-immunoreactive cells. Thus, CSM14.1 cells differentiate into TH-containing neurons, which most probably contribute to the preferred forelimb use, indicating a functional integration of CSM14.1 cells into the host basal ganglia loops during early postnatal development. These findings that are in contrast to observations in adult rats suggest instructive cues for neuronal differentiation and integration given by the neonatal microenvironment.

Environmental conditions and behavioural experience can affect neuronal function and morphology. It is less well known whether such factors also influence the growth, integration and functional recovery provided by neural grafts placed within the damaged brain. Here we report on the effects of differential housing conditions on striatal graft morphology and functional recovery after striatal lesions. Rats were pretrained on a skilled bilateral forelimb task, the staircase test, and lesioned unilaterally in the lateral dorsal striatum with quinolinic acid. One group of lesioned animals was given suspension grafts of E15 whole ganglionic eminence implanted into the lesioned striatum. Following transplantation, the animals were housed either in standard cages (four per cage) or in enriched environment housing conditions (10 per cage) with tunnels, ladders and increased living space available for exploration, social interaction and play. The differentially housed animals were retested on the skilled staircase test at two separate time points. Repeated testing, environmental enrichment and transplantation positively influenced behavioural recovery. Partial recovery was observed bilaterally amongst the grafted animals in both housing conditions. Nevertheless, the grafted animals housed in the enriched environment performed significantly better in the final test compared with all of the other experimental groups. The grafts survived equally well under both housing conditions but the grafts of animals housed in the enriched environment contained larger projection neurons and were somewhat better reinnervated by dopaminergic afferents. An increased level of striatal brain-derived neurotrophic factor was observed in the control animals housed under the enriched compared with the standard conditions. The results indicate that an enriched environment can affect both graft function and graft morphology through as yet unknown mechanisms.

We assessed the developmental potential of human telencephalic progenitor cells, with and without previous amplification in vitro, following grafting into the nonlesioned adult mouse CNS. Cell suspensions, shown to contain neuroepithelium-like and neuroblast-like cells, were injected into the subventricular zone (SVZ) and the striatum. These two regions were selected for comparative studies because one, the SVZ, is mitotically active, whereas the other, the striatum, is mitotically inactive. In situ hybridization with a human-specific Alu probe showed that the cells survived for up to 30 weeks in both targets and migrated away from the injection site. Fresh cells continued to proliferate and gave rise to very extended grafts before differentiating into neurons and glia. We further show that, when grown in vitro prior to grafting, human cells acquired new properties: Their proliferation was very limited, and they differentiated more rapidly. This study therefore provides new information about the use of these cells, which are a potential tool for both cellular and gene therapy., (Copyright 2002 Wiley-Liss, Inc.)

Parkinson's disease is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra projecting to the striatum. One therapeutic approach to this disease has been the intrastriatal transplantation of dopamine-secreting cells. We have investigated the suitability of glomus cells of the carotid body for dopamine-cell replacement in animal models of Parkinson's disease. Carotid body glomus cells are physiologic arterial oxygen sensors that release large amounts of dopamine in response to hypoxia. We have used hemi-Parkinsonian rats, induced by injection of 6-hydroxydopamine into the substantia nigra, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treated monkeys with chronic Parkinsonism. In both cases we made transplants of carotid body cell aggregates into the putamen. Functional recovery of the grafted animals was observed after the surgery and was stable for several months. Although the study was more detailed in the rat, in the two animal models the amelioration of the motor deficits was paralleled by striatal dopaminergic reinnervation and survival of grafted glomus cells. Our results suggest that intrastriatal autotransplants of carotid body tissue could be a feasible technique to treat some cases of Parkinson's disease in humans.

Excitotoxicity has been involved in the pathogenesis of several neurodegenerative disorders. Using intrastriatal quinolinic acid (QUIN) injection as an animal model of Huntington's disease, we attempt to identify the neurotransmitter phenotype of striatal projection neurons protected by neurturin (NRTN). Control or NRTN-secreting cell lines were grafted in the striatum before QUIN injection and striatal projection neurons were examined by retrograde Fluorogold labeling and in situ hybridization. Intrastriatal grafting of NRTN-secreting cell line selectively prevented the loss of striatopallidal neurons and also the decrease in the mRNA levels for their markers (glutamic acid decarboxylase 67 and preproenkephalin) induced by QUIN, without affecting striatonigral neurons. Thus, our findings show that NRTN is a selective neuroprotective factor for striatopallidal neurons, suggesting that it might be a candidate for the treatment of movement disorders in which this neuronal population is affected.

Adult C57BL/6 mice were injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Intrastriatal transplantation of bone marrow stromal cells (MSCs) was performed 1 week after MPTP administration. MSCs were harvested from donor adult mice, and then cultured and prelabeled with bromodeoxyuridine (BrdU). MPTP-Parkinson's disease (PD) mice treated with intrastriatal injection of phosphate-buffered saline (PBS), and normal non-MPTP mice were used as controls. MPTP-PD mice with MSC intrastriatal transplantation exhibit significant improvement on the rotarod test (P<0.05) at day 35 compared with PBS controls. Immunohistochemistry shows that BrdU reactive cells survive in the transplanted areas in the MPTP-PD striatum at least 4 weeks after administration. Scattered BrdU reactive cells express tyrosine hydroxylase (TH) immunoreactivity. Our findings suggest that MSCs injected intrastriatally survive, express dopaminergic protein TH immunoreactivity, and promote functional recovery.

Bilateral astroglial transplantation into the neostriatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys resulted in significant performance improvement in a spatial delayed response task, but failed to modify perseveration in an object retrieval detour task, or to improve motor clinical rating. Results suggest that brain circuits subserving various motor and cognitive performances can be functionally dissociated, and that remaining resources for the reorganization of neural circuits involved in spatial working memory performance in parkinsonian monkeys, appear to be responsive to striatal transplantation of subcultured, fetal striatal astroglial cells.

Embryonic neurons transplanted to the adult CNS extend axons only for a developmentally defined period. There are certain intercellular factors that control the axonal extension, one of which may be the expression of the bcl-2 protein. In this study, rats with complete striatal dopamine fiber denervation received embryonic day 14 mouse ventral mesencephalon cells overexpressing human bcl-2 or control wild-type ventral mesencephalon cells. All rats were treated with cyclosporine to prevent rejection and the surviving grafts were analyzed for cell survival and outgrowth of dopaminergic fibers. The results demonstrate that bcl-2 overexpression does not enhance neuronal graft survival. However, the bcl-2 overexpressing neurons had a higher number of dopaminergic fibers that grew longer distances. These results show that overexpression of bcl-2 can result in longer distance axonal growth of transplanted fetal dopaminergic neurons and that genetic modification of embryonic donor cells may enhance their ability to reinnervate a neuronal target territory.

Intracerebral neural xenografts elicit a host immune response that results in their rapid rejection. This forms a key barrier to the therapeutic use of xenogeneic tissue transplantation for conditions such as Parkinson's disease. The current study sought to provide insight into the cellular components of donor cell suspensions that are important in stimulating the host rejection response and thereby to suggest rational manipulations of xenogeneic donor tissue that might ultimately enhance its clinical utility. The neural stem cell mitogens, epidermal growth factor and fibroblast growth factor-2, have been used to isolate and expand populations of primordial neural precursor cells from the embryonic pig brain. The immune response elicited by these cells on transplantation into the non-immunosuppressed rat has been fully characterised. In the first experiments, expanded neural precursors were grafted into the hemi-parkinsonian, non-immunosuppressed Sprague-Dawley rat and graft status and host response examined 10, 21, 35 and 60 days post-transplantation. While equivalent primary tissue grafts were completely eliminated at 35 days, grafts of expanded neural precursors with healthy neurofilament-positive projections were present at all time-points, and two large grafts remained even at 60 days. Some grafts appeared to elicit minimal host immune responses at the time-points they were examined, although most did appear to be undergoing a rejection process since a co-ordinated response involving host cytotoxic T-lymphocytes, microglia/macrophages, immunoglobulin M and complement could be demonstrated to varying degrees. Subsequent experiments went on to demonstrate further that expanded precursor populations and primary tissue suspensions differed in their immunogenic profile. Firstly, when primary tissue was injected intraperitoneally into immunocompetent rats a vigorous primary humoral response was generated. No such response was detected following injection of expanded neural precursors. Secondly, flow cytometric analysis revealed small but significant levels of class II porcine major histocompatibility complex expression in primary cell suspensions but no such expression in expanded precursor populations.The results of this study therefore demonstrate that the immunogenicity of porcine neural cell suspensions used for intracerebral grafting is reduced when neural stem cell mitogens are used to expand precursor cells. The implications of these findings in the development of novel xenogeneic cellular therapies for neurodegenerative conditions such as Parkinson's disease are discussed.

Transplantation of embryonic dopaminergic neurons is an experimental therapy for Parkinson's disease, but limited tissue availability and suboptimal survival of grafted dopaminergic neurons impede more widespread clinical application. Glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-4/5 (NT-4/5) exert neurotrophic effects on dopaminergic neurons via different receptor systems. In this study, we investigated possible additive or synergistic effects of combined GDNF and NT-4/5 treatment on rat embryonic (embryonic day 14) nigral explant cultures grown for 8 days. Contrary to cultures treated with GDNF alone, cultures exposed to NT-4/5 and GDNF+NT-4/5 were significantly larger than controls (1.6- and 2.0-fold, respectively) and contained significantly more protein (1.6-fold). Treatment with GDNF, NT-4/5 and GDNF+NT-4/5 significantly increased dopamine levels in the culture medium by 1.5-, 2.5- and 4.7-fold, respectively, compared to control levels, and the numbers of surviving tyrosine hydroxylase-immunoreactive neurons increased by 1.7-, 2.1-, and 3.4-fold, respectively. Tyrosine hydroxylase enzyme activity was moderately increased in all treatment groups compared to controls. Counts of nigral neurons containing the calcium-binding protein, calbindin-D28k, revealed a marked increase in these cells by combined GDNF and NT-4/5 treatment. Western blots for neuron-specific enolase suggested an enhanced neuronal content in cultures after combination treatment, whereas the expression of glial markers was unaffected. The release of lactate dehydrogenase into the culture medium was significantly reduced for GDNF+NT-4/5-treated cultures only. These results indicate that combined treatment with GDNF and NT4/5 may be beneficial for embryonic nigral donor tissue either prior to, or in conjunction with, intrastriatal transplantation in Parkinson's disease.

After striatal injury, sprouting dopaminergic fibres grow towards and intimately surround wound macrophages which, together with microglia, express the dopaminergic neurotrophic factors glial cell line-derived neurotrophic factor (GDNF) and brain derived neurotrophic factor (BDNF). To evaluate the importance of these endogenously secreted neurotrophic factors in generating striatal peri-wound dopaminergic sprouting, the peri-wound expression of BDNF or GDNF was inhibited by intrastriatal infusion of antisense oligonucleotides for 2 weeks in mice. Knock-down of both BDNF and GDNF mRNA and protein levels in the wounded striatum were confirmed by in situ hybridization and enzyme-linked immunosorbent assay, respectively. Dopamine transporter immunohisto-chemistry revealed that inhibition of either BDNF or GDNF expression resulted in a marked decrease in the intensity of peri-wound sprouting. Quantification of this effect using [H3]-mazindol autoradiography confirmed that peri-wound sprouting was significantly reduced in mice receiving BDNF or GDNF antisense infusions whilst control infusions of buffered saline or sense oligonucleotides resulted in the pronounced peri-wound sprouting response normally associated with striatal injury. BDNF and GDNF thus appear to be important neurotrophic factors inducing dopaminergic sprouting after striatal injury.

The caudate nucleus and putamen form part of a complex but topographically connected circuitry that links the cortex, the basal ganglia and the thalamus. Within this complex system lie a series of functionally and anatomically segregated loops that allow the concurrent processing of a wide range of cognitive and motor information (Alexander et al., 1986; Alexander and Crutcher, 1990). As a constituent of these loops it has been shown that the striatum is involved in movement initiation, response selection and attentional processes (Robbins and Brown, 1990; Alexander, 1994; Lawrence et al., 1998). Although it is the medium spiny GABAergic projection neurones that are primarily lost in HD, it is not sufficient merely to replace the GABA. Instead it is crucial for striatal tissue transplants to integrate with the host tissue in such a way that the cortico-striatal-thalamic circuitry is restored and is functional. Rodent studies have progressed a long way in establishing the principle that striatal grafts can, at least partially, restore function and integrate appropriately with the host (Dunnett and Svendsen, 1993; Björklund et al., 1994; Sanberg et al., 1998) but the limited behavioural repertoire and the undifferentiated striatum meant that it was inevitable that studies should progress into primate models. Anatomical tracing studies have demonstrated that motor, premotor and somatosensory cortical areas send corticostriatal projections primarily to the putamen region in primates, whereas the head and body of the caudate nucleus mostly receive efferent input from associative cortical areas (Kemp and Powell, 1970; Kunzle, 1975, 1977, 1978; Selemon and Goldman-Rakic, 1985). Based on such anatomical, and functional, studies Alexander and colleagues have proposed the existence of at least five cortico-striatal-thalamic loops including a motor, a dorsolateral-prefrontal and an orbito-frontal loop (Alexander et al., 1986). The concentration of motor inputs to the putamen region suggests a particular involvement of this structure in the motor loop. Indeed, unilateral lesions of the putamen disrupt motor performance in the marmoset and generate apomorphine-induced dyskinesias in larger primates (Burns et al., 1995; Kendall et al., 2000). The implantation of striatal grafts into marmosets that had previously received unilateral putamen lesions ameliorated some of the motor impairments, which suggested at least partial restoration of the motor loop. In support of this we found direct evidence of host-graft cortico-striatal connectivity using an anterograde tracer injected in the primary motor cortical region (Kendall et al., 1998a). In larger primates, with lesions of the caudate and putamen, striatal [figure: see text] allografts and xenografts have been shown to reduce apomorphine-induced dyskinesias (Isacson et al., 1989; Hantraye et al., 1992; Palfi et al., 1998). The mechanism by which dyskinesias are elicited is not fully understood but alterations in firing patterns within both segments of the globus pallidus have been identified during dyskinetic movements (Matsumura et al., 1995). It seems likely that it would actually require re-establishment of afferent connections between the implanted putamen and the globus pallidus as well as of functioning dopamine receptors within the graft for the reduction in the dyskinetic profile to be observed. Certainly there is evidence, from rodent studies and the marmoset study described here, that close proximity of the graft to the globus pallidus yields better functional recovery (Isacson et al., 1986). In addition, anatomical tracing studies in rats have demonstrated connections between the implanted tissue and the host globus pallidus (Wictorin et al., 1989b, 1990) However, the relationship between graft placement and functional recovery remains to be fully substantiated.

The transplantation of foetal mesencephalic cells (FMC) into the brain striatal system is an emerging treatment for Parkinson's disease, despite of the relatively poor survival of implanted cells. The ability of neurotrophic factors to regulate neurone survival and differentiation suggests they could be used to enhance the success of cerebral grafts. We analyzed the effect of pre-treatment of FMC suspensions with bone morphogenetic protein-2 (BMP-2) (50 ng/ml) prior to grafting into the striatum of 6-hydroxydopamine lesioned rats. The viability of a FMC suspension was enhanced in vitro by BMP-2. Four weeks after transplantation, the number of dopaminergic neurones was higher and their morphology more developed in grafts pre-treated with BMP-2, compared with non-pre-treated grafts and rats showed a significant reduction in the turning behaviour test. Thus, the pre-treatment of FMCs with BMP-2 should be considered, together with other neurotrophic factors, as a procedure for transplantational treatment of Parkinson's disease.

The present study examined the effects of lesions to the dorsal striatum (DS) in Sprague-Dawley rats, when tested on the acquisition and successive shifts in the position of a goal arm in an eight-arm radial maze. In the procedure we used, rats had to retrieve the location of one baited arm among the eight arms of the maze after it had just been presented as a sample during a forced trial. After attainment of a fixed learning criterion, rats were submitted to five successive shifts in the goal location. Results showed that DS rats were able to learn the position of the goal arm during the acquisition phase as efficiently as sham-operated rats. In contrast, when the position of the goal arm was shifted, although DS rats were able to learn its new position, they made significantly more errors and required more sessions to reach criterion than sham-operated rats. These results suggested that both groups did not solve the task using the same behavioral strategy. The analysis of responses made suggested that sham-operated rats solved the task using the pairing rule between the forced and the free run (matching-to-sample rule), while DS rats solved the task using only visuo-spatial processing. These data therefore suggest that the dorsal striatum plays an important role in rule-learning ability., (Copyright 1999 Academic Press.)

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.

Cell transplantation therapy for Parkinson's patients, although seen to bring benefit to some patients during first clinical trials, remains impracticable on a large scale, in part because of the poor survival of the dopaminergic neurones transplanted. The loss of dopaminergic neurones occurs rapidly over the first 1-2 days after transplantation, in response to factors intrinsic to the host brain. Here we investigated whether contamination of the grafted cell suspension with blood during the transplantation procedure may be one factor responsible for the poor survival of DA neurons within the graft, possibly through factors such as free iron or complement. 6-Hydroxydopamine lesioned rats were grafted with 2 microl suspension of dissociated E14 ventral mesencephalon to which 1 microl blood or 1 microl grafting medium was added. After 6 weeks, there was no significant difference in the number of surviving DA neurones in the two groups. We conclude that contamination of grafts with blood is not a major factor responsible for the extensive death of dopaminergic neurones within them., (Copyright 1999 Academic Press.)

Intrastriatal transplantation of fetal ventral mesencephalon (VM) is currently explored as a potential clinical therapy in Parkinson's disease (PD). Although providing substantial benefit for the patient, behavioral recovery so far obtained with intrastriatal VM grafts is not complete. Using the 6-hydroxydopamine lesion model of PD, we show here that near-complete restoration of the striatal dopamine (DA) innervation can be achieved by multiple intrastriatal microtransplants of fetal DA cells; nevertheless, complete recovery in complex sensorimotor behaviors was not obtained in these animals. In line with the current model of basal ganglia function, this suggests that the lesion-induced overactivity of the basal ganglia output structures, i.e., the substantia nigra (SN) and the entopeduncular nucleus, may not be completely reversed by intrastriatal VM grafts. In the present study, we have transplanted fetal VM tissue or fetal striatal tissue, as a source of DA and GABA neurons, respectively, into the SN of DA-depleted rats. Intranigral VM grafts induced behavioral recovery in some sensorimotor behaviors (forelimb akinesia and balance tests), but the effect did not exceed the recovery observed after intrastriatal VM grafts. Intranigral grafts of striatal tissue induced a pattern of functional recovery which was distinctly different from that observed after intranigral VM grafts, and recovery in coordinated forelimb use in the paw-reaching test was even more pronounced than after intrastriatal transplantation of VM cells. Combined transplantation of DA neurons into the striatum and GABA-rich striatal neurons into the SN induced additive effects of behavioral recovery observed in the forelimb akinesia test. We propose that intranigral striatal transplants, by a GABA-mediated inhibitory action, can reduce the overactivity of the host SN projection neurons and can induce significant recovery in complex motor behavior in the rat PD model and that such grafts may be used to increase the overall functional efficacy of intrastriatal VM grafts., (Copyright 1999 Academic Press.)

A segment of tibial nerve was autografted to the right corpus striatum of deeply anesthetized adult rats; the distal graft was left beneath the scalp. Horseradish peroxidase (HRP) conjugates were injected into the distal graft after 2-30 weeks, and the animals were killed 2-3 days later. Small numbers of neostriatal perikarya were HRP labeled at all survival times; most were large (ca. 20 microm in diameter), and many contained acetycholine esterase (AChE). Many more neurons were labelled in the substantia nigra pars compacta (SNpc) 4 weeks or more after grafting. When the graft encroached on the globus pallidus, numerous pallidal neurons, most of them AChE positive, were also labeled. Nigrostriatal neurons, a population of pallidal cholinergic neurons, and a subclass (or classes) of neostriatal neurons, including cholinergic interneurons, thus can be classified as central nervous system (CNS) neurons with a relatively strong regenerative response. In a second experimental series, animals were killed 1-4 weeks after grafting, and sections were probed for the expression of mRNAs encoding growth-associated protein 43 (GAP-43) and the cell adhesion molecules N-CAM and L1. Subpopulations of mostly large neurons scattered throughout the neostriatum gave moderate signals for GAP-43 and N-CAM mRNAs and a stronger signal for L1 mRNAs. Most SNpc neurons were strongly labeled with all three probes. Neostriatal grafts had no apparent effect on the expression of any of the mRNAs in the SNpc or on L1 and N-CAM mRNAs in the striatum. However, GAP-43 mRNA levels were increased in a few, mainly large neostriatal neurons around the graft tip, resembling the HRP-labeled cells. In contrast, previous work has shown upregulation (from an undetectable level) of GAP-43 and L1 mRNAs in neurons regenerating axons into grafts placed in the thalamus and cerebellum. Thus, GAP-43 and L1 mRNA expression, but not necessarily marked upregulation, may correlate with, and be intrinsic determinants of, the ability of CNS neurons to regenerate their axons.

The relevance of the cholinergic system in mnemonic processes has been repeatedly demonstrated. In addition to the cholinergic systems that project to the telencephalon, there are subcortical nuclei with intrinsic cholinergic cells which appear to be involved in memory consolidation; among these is the striatum. Intrastriatal administration of anticholinergic drugs, as well as excitotoxic and electrolytic lesions have been shown to disrupt the acquisition and retention of instrumentally conditioned behaviors. In the present study male Wistar rats were used to confirm the reported detrimental effects of striatal lesions produced by the cholinotoxin AF64A on long-term retention (LTR) of inhibitory avoidance and spontaneous locomotor activity, to determine its effects on short-term retention (STR) and to investigate whether intrastriatal homotopic transplants can reverse the AF64A-induced behavioral deficits. AF64A-striatal lesions did not interfere with STR but disrupted LTR of the inhibitory avoidance task, and striatal transplants prevented this deficit. Spontaneous locomotor activity increased after the lesion but promptly returned to baseline levels. These results support previous findings showing striatal involvement in long-term but not short-term retention and indicate that homotopic transplants induce behavioral recovery of a learning task in striatal lesioned rats.

Recently, we have shown that rapid eye movement sleep deprivation (REM-SD) in animals with lesions of the nigro-striatal pathway facilitates turning behavior and such increase still occurred even in the presence of dopaminergic grafts. The objective of this work was to determine which DA receptors are preferentially involved. The results showed that the D2 receptor antagonist sulpiride decreases significantly turning behavior of lesioned animals, with no effect whatsoever of the D1 antagonist SCH 23390. When lesioned animals were REM sleep deprived, the D1 but not the D2 receptor antagonist prevented the increase of turning induced by REM-SD. This work suggests that the increase of post-synaptic supersensitivity induced by REM-SD in nigro-striatal lesioned animals is mediated by D1 receptors.

The main strategy in experimental and clinical neural transplantation in Parkinson's disease has been to place fetal nigral grafts not in their ontogenic site (substantia nigra) but in their target area (striatum). The reason for this ectopic placement is the apparent inability of nigral grafts placed in the ventral mesencephalon (VM) of the adult host to grow axons for long distances that are capable of reaching the ipsilateral striatum and thus restoring the nigrostriatal pathway. The present study demonstrates for the first time that simultaneous dopaminergic transplants (double grafts) placed in the substantia nigra and ipsilateral striatum of rats bearing unilateral 6-hydroxydopamine lesions reconstruct the dopaminergic nigrostriatal pathway in the adult rat brain. Numerous tyrosine hydroxylase-immunoreactive axons were observed arising from the intranigral graft and growing rostrally along the internal capsule and medial forebrain bundle to reinnervate the ipsilateral striatum, which also had received a dopaminergic graft. These double grafts achieved not only greater striatal reinnervation than the standard intrastriatal graft but also a faster and more complete rotational recovery to amphetamine challenge 6 weeks after transplantation. These results suggest strongly that embryonic nigral transplants implanted in the striatum are capable of promoting growth and providing guidance to axons arising from a dopaminergic graft placed homotopically in the VM, resulting in restoration of the dopaminergic nigrostriatal projection. Reconstruction of the nigrostriatal pathway by double grafts may not only achieve substantial striatal reinnervation but may also contribute to the reestablishment of dopaminergic regulation of the nigrostriatal circuitry.

The physical repair and restoration of a completely damaged pathway in the brain has not been achieved previously. In a previous study, using excitatory amino acid bridging and fetal neural transplantation, we demonstrated that a bridged mesencephalic transplant in the substantia nigra generated an artificial nerve pathway that reinnervated the striatum of 6-hydroxydopamine (6-OHDA)-lesioned rats. In the current study, we report that a bridged mesencephalic transplant can anatomically, neurochemically, and functionally reinstate the 6-OHDA-eradicated nigro-striatal pathway. An excitatory amino acid, kainic acid, laid down in a track during the transplant generated a trophic environment that effectively guided the robust growth of transplanted neuronal fibers in a bundle to innervate the distal striatum. Growth occurred at the remarkable speed of approximately 200 microm/d. Two separate and distinct types of dopamine (DA) innervation from the transplant have been achieved for the first time: (1) DA innervation of the striatum, and (2) DA innervation of the pars reticularis of the substantia nigra. In addition, neuronal tracing revealed that reciprocal connections were achieved. The grafted DA neurons in the SNr innervated the host's striatum, whereas the host's striatal neurons, in turn, innervated the graft within 3-8 weeks. Electrochemical volt- ammetry recording revealed the restoration of DA release and clearance in a broad striatal area associated with the DA reinnervation. Furthermore, the amphetamine-induced rotation was attenuated, which indicates that the artificial pathways were motor functional. This study provides additional evidences that our bridged transplantation technique is a potential means for the repair of a completely damaged neuronal pathway.

The substantia nigra of gestation day 14 was transplanted into the striatum of 3-4-month-old rats to investigate the transplants ultrastructurally at the end of 2 years, as a follow-up to our previous studies. Transplants were of small size in all 10 specimens taken for this study. The changes observed in the transplant and in the interface region with the host striatum were: thickening of the blood vessel walls, perivascular cuffing with lymphocytes and macrophages loaded with tissue debris, degenerating neurons and hypertrophied astroglia containing dense granules indicating ageing or reaction to degeneration and glial processes. The number of surviving neurons in the transplants was small. These were smaller in size and had very few intracytoplasmic membraneous organelles. A higher content of intracytoplasmic ageing lipofuscin pigment was present than in host neurons and age-matched nigral neurons. Synapses were few, and their number varied among transplants. Generally, the synapses were at the interface with the host tissue. The changes observed in all the 2-year-old transplants suggest premature ageing or a slow rejection process. Slow rejection is a possibility, because these rats are only stock-bred, not inbred, and hence they are not completely immunologically compatible.

L-3,4-Dihydroxyphenylalanine (L-DOPA) inhibits the activity of tryptophan hydroxylase (TRH) and thus serotonin synthesis. This inhibitory effect of L-DOPA may be related to some side effects in the patients under L-DOPA therapy. The effects of transplantation of fetal ventral mesencephalon (VM) on extracellular 5-hydroxytryptophan (5-HTP) accumulation was examined by microdialysis as an index of in vivo activity of TRH in the striatum of 6-hydroxydopamine (6-OHDA)-lesioned rats. In the rat striatum perfused with m-hydroxybenzylhydrazine (NSD-1015; an inhibitor of aromatic L-amino acid decarboxylase), L-DOPA and 5-HTP in dialysate were measured simultaneously. In response to NSD-1015, 5-HTP levels were substantially elevated in the lesion plus VM-grafted striata as compared with those in the lesion plus sham-grafted striata. The results indicate that implantation of dopamine-rich VM grafts into the 6-OHDA-lesioned striatum of rats induces hyperactivity of TRH.