Memantine and reduced time with dyskinesia in Parkinson's Disease.

OBJECTIVES: The partial glutamate antagonist amantadine is currently used in clinical practice, to reduce dyskinesia developing as a side-effect of levodopa treatment in patients suffering from Parkinson's disease (PD). This study was aimed at evaluating the antidyskinetic effect of another glutamate antagonist, memantine. METHODS AND MATERIALS: We performed a randomized, double-blind and placebo-controlled crossover clinical trial of memantine (20 mg), with a 3-week treatment period, and 15 patients completed the study. RESULTS: The primary outcome measure, a change in observed dyskinesia ratings, did not reach significance. Seven of the 15 patients reduced the L-dopa-induced dyskinesias by 32%, whereas for three patients, they increased by 33%, and for five patients, they did not change. Data from the self-administered diaries, as a secondary outcome measure, did show a significant 35% reduction in the percentage of time of the day spent with dyskinesia, from 25% (placebo) to 16% (memantine). Memantine was well tolerated, without any serious adverse events, or worsening in the parkinsonian motor score. CONCLUSION: The results suggest that memantine may be a useful antidyskinetic drug, and a larger clinical study is warranted.

Generation of regionally specified neurons in expanded glial cultures derived from the mouse and human lateral ganglionic eminence.

The specific identity of neuronal precursors within the embryonic brain is, at present, not clear. Here we show that cultures with glial characteristics derived from the embryonic mouse or human lateral ganglionic eminence (LGE) can be expanded over many passages and maintain their glial identity. Interestingly, removal of serum and EGF from the culture medium results in the generation of large numbers of neurons. The neurons derived from these cultures display many characteristic features of striatal neurons, which normally derive from the LGE, even after extensive expansion in vitro. Furthermore, a portion of the neurons generated in these cultures were shown to arise from glial fibrillary acidic protein (GFAP)-expressing cells. These results demonstrate that at least a subpopulation of neurogenic LGE precursors exhibit glial characteristics.

Retinal integration of grafts of brain-derived precursor cell lines implanted subretinally into adult, normal rats.

The ability of in vitro-expanded neural precursor cells or cell lines to differentiate following transplantation has significant implications for current research on central nervous system repair. Recently, interest has been focussed on grafts of such neural precursors implanted also into the eye or retina. Here, we demonstrate with a non-traumatizing subretinal transplantation method, that grafts of the two immortalized brain-derived cell lines C 17-2 (from postnatal mouse cerebellum) and RN33B (from the embryonic rat medullary raphe) survive for at least up to four weeks, after implantation into the adult normal rat retina. For both cell lines, implanted cells gradually integrate into all major retinal cell layers, including the retinal pigment epithelium, and judged by the morphology differentiate into both glial- and neuron-like cells, as shown by thymidine autoradiography, mouse-specific in situ hybridization, and using immunohistochemistry to detect the reporter gene LacZ. Our results suggest that these and other similar neural cell lines could be very useful in the continuous experiments in models of retinal disorders to further assess both the cell replacement and ex vivo gene therapy approaches.

Long-term, EGF-stimulated cultures of attached GFAP-positive cells derived from the embryonic mouse lateral ganglionic eminence: in vitro and transplantation studies.

Long-term attached cultures, prepared from mouse embryonic days 15-17 lateral ganglionic eminence, were grown in a medium including epidermal growth factor and serum, and the survival, differentiation, and migration of cells from either early or late passages were analyzed following transplantation. The cultured cells had the morphology of type I astroglial cells, with the vast majority of the cells immunoreactive for glial fibrillary acidic protein (around 90%), the intermediate filament marker nestin, and also the mouse-specific neural markers M2 and M6. The cultures were kept over 25 passages (7 months). During the first 8 passages, the growth rate gradually declined, but it increased again after passage 9 and thereafter stabilized at values similar to those observed during the initial culture period. After passages 4-6 and 18, cell suspensions were implanted cross-species into the intact or lesioned striatum of adult (passages 4-5 only) or intact striatum of neonatal rats (passages 4-6 or 18). Both early and late passage cells formed M2 (and M6)-positive transplants. In the neonatal recipients, widespread migration was seen from the needle tract throughout most of the striatum, along the internal capsule, and into the globus pallidus. In the adult striatum, the cells remained mostly around the injection tract, or within 0.4-0.6 mm from the graft core. These long-term attached cultures are interesting to compare to nonattached neurosphere cultures, and might also offer a means of propagating relatively pure populations of astroglia-like cells for basic transplantation studies or for use in experimental trials with ex vivo gene transfer.

The use of a recombinant lentiviral vector for ex vivo gene transfer into the rat CNS.

A major obstacle in ex vivo gene transfer has been the loss of transgene expression soon after implantation of the grafted transduced cells. Recently, a lentiviral vector system has been developed which has proven to express high levels of transgenes in vivo after direct injection into the tissue. In this study, we have investigated the use of such a vector for ex vivo gene transfer to the brain. A number of neural cell types were found to be permissive to transduction by the lentiviral vector in vitro and a majority of them expressed the transgene after transplantation to the rat brain. Transgene expression was detected up to 8 weeks post-grafting. These findings suggest that recombinant lentiviral vectors may be used for further development of ex vivo gene therapy protocols to the CNS.

Extensive migration and target innervation by striatal precursors after grafting into the neonatal striatum.

Embryonic striatal precursors grafted into the lesioned adult host striatum show limited integration with little migration and restricted efferent projections. In the present study, the influence of an immature striatal environment on the integrative capacity of grafted neuroblasts was examined after transplantation of striatal progenitors into the striatum at different stages of postnatal development. Mouse progenitors, derived from embryonic day 13.5-14 lateral or medial ganglionic eminence or the cerebellar primordium, were transplanted as a single cell suspension into the developing postnatal day 1, 7 and 21 rat striatum. The grafted cells and their axonal projections were visualized using antibodies raised against the mouse-specific neural markers, M6 and M2. Cells from the lateral (but not the medial) ganglionic eminence showed a remarkable capacity to innervate selectively the striatal target structures, globus pallidus, entopeduncular nucleus and substantia nigra, reminiscent of endogenous striatal neurons, which is not observed after grafting into adult hosts. M6 and M2-immunopositive cellular profiles from both the lateral and medial ganglionic eminences were observed to have migrated extensively away from the injection site, in contrast to the cerebellar precursors which remained clustered at the implantation site. Cells from the lateral ganglionic eminence were largely confined within the striatal complex where they developed striatal characteristics, displaying expression of DARPP-32, the 32,000 mol. wt dopamine- and cyclic AMP-regulated phosphoprotein, whereas cells from the medial ganglionic eminence had migrated caudally along the internal capsule and were observed predominantly in the globus pallidus and thalamus, in addition to the striatum. The cells located outside the striatum were all DARPP-32 negative. The improved integration and increased projection capacity of the lateral ganglionic eminence precursors grafted into postnatal day 1 hosts gradually declined as the host advanced into later stages of development (postnatal day 7), and in postnatal day 21 hosts the grafted striatal precursors behaved similarly to grafts implanted into adult recipients. These results demonstrate the specific capacity of embryonic striatal progenitors to integrate into the developing basal ganglia circuitry during early postnatal development, and that the extent of neuronal and glial integration and graft host connectivity declines when the host has developed beyond the first postnatal week.

Enhanced synthesis of platelet-derived growth factor following injury induced by 6-hydroxydopamine in rat brain.

The kinetics of platelet-derived growth factor messenger RNA synthesis in the substantia nigra and in the striatum, before and after unilateral intranigral 6-hydroxydopamine injection, was studied and compared with that after sham operation by a quantitative reverse transcription-polymerase chain reaction. The kinetics of brain-derived neurotrophic factor messenger RNA was studied as a comparison. Furthermore, the expression of platelet-derived growth factor A- and B-chain proteins was analysed by enzyme-linked immunosorbent assay and immunohistochemistry. In the ipsilateral striatum of 6-hydroxydopamine-lesioned rats, the signal density of messenger RNA for both A- and B-chains had already increased at one day and remained at an elevated level during the observation period of four weeks. In the substantia nigra ipsilateral to the lesion, a strongly increased level of B-chain and, to a lesser extent, of A-chain messenger RNA was already detected at 4h, reaching a maximal level at one day. No significant increase was seen either in sham-operated rats or in the contralateral striatum and substantia nigra. Amounts of platelet-derived growth factor proteins were examined separately by enzyme-linked immunosorbent assay in both sides of the substantia nigra, striatum and cortex. Three days after 6-hydroxydopamine lesions the levels of both platelet-derived growth factor A- and B-chains increased in the ipsilateral striatum, substantia nigra, and cortex. An increase in the A-chain was also observed in the contralateral side of the brain. The signal for brain-derived neurotrophic factor messenger RNA increased in the striatum in the lesioned side and, to a lesser extent, in the contralateral side, as well as in the substantia nigra, where a significant difference was observed when compared with the contralateral side. Semiquantitative immunohistochemical analysis on the substantia nigra confirmed the enhanced platelet-derived growth factor expression, revealing that the majority of the platelet-derived growth factor-producing cells were neurons. In summary, we have shown that platelet-derived growth factor messenger RNA as well as its protein are induced after injury to dopaminergic cells. These data indicate an important role of platelet-derived growth factor in the dopaminergic system.

Projection neurons in fetal striatal transplants are predominantly derived from the lateral ganglionic eminence.

In the present study, we have characterized aspects of integration, growth and phenotypic differentiation of embryonic grafts derived from the selective dissection of either the lateral or medial portion of the ganglionic eminences of the rodent forebrain. Donor tissues were derived from embryonic day 15 rat, or embryonic day 14 mouse embryos, and injected, as single cell suspensions into the striatum or substantia nigra of adult rats previously subjected to an intrastriatal ibotenic acid lesion. Two to six weeks following grafting, immunocytochemical detection of DARPP-32, the 32,000 mol. wt dopamine- and cyclic AMP-regulated phosphoprotein, was used to identify areas with a striatum-like phenotype within both the intrastriatal and the intranigral grafts. It was thus revealed that all the lateral ganglionic eminence grafts, irrespective of their placement, were dominated by striatum-like tissue (up to 90% of the total graft volume), while the medial ganglionic eminence transplants were only sparsely positive (< 10% of the total graft volume). These striatum-like regions of the grafts were selectively innervated by tyrosine hydroxylase immunopositive fibres from the host substantia nigra. Furthermore, axons derived from the lateral ganglionic eminence mouse grafts placed in the striatum, as detected by the mouse-specific neuronal marker M6, showed a more extensive and directed outgrowth towards the globus pallidus when compared to fibres emanating from the medial ganglionic eminence grafts. Mouse lateral and medial ganglionic eminence grafts placed into the substantia nigra exhibited similar fibre outgrowth patterns; both types of grafts thus innervated the substantia nigra-pars reticulata and extended axons into the cerebral peduncle. These results show that DARPP-32-positive striatal projection neurons are derived, for the most part, from the lateral ganglionic eminence and that the restricted lateral ganglionic eminence dissection provides a more optimal source of striatal tissue for grafting in the rat Huntington model.

Neurotransmitter-related gene expression in intrastriatal striatal transplants--I. Phenotypical characterization of striatal and non-striatal graft regions.

In the present study, we have re-examined the heterogeneous nature of intrastriatal striatal transplants derived from embryonic day 14-15 rat striatal primordia implanted into the previously excitotoxically lesioned striatum of adult rats, using in situ hybridization histochemistry to localize neurotransmitter-related messenger RNAs. These grafts are characterized by discrete patches of DARPP-32 messenger RNA expression, which cover approximately one-third of the cross-sectional graft area. The messenger RNAs encoding for preproenkephalin (the enkephalin precursor), preprotachykinin (precursor to substance P), choline acetyltransferase, as well as the D1 and D2 dopamine receptors, which are abundant in the normal striatum, were all present in the striatal grafts and were expressed almost exclusively in the DARPP-32-positive graft regions. In these graft regions, the expression of the neurotransmitter-related messenger RNAs was generally similar to that seen in the intact striatum, although the level of expression of preproenkephalin and preprotachykinin messenger RNAs varied notably among the patches of expression. Cellular analysis performed on individual patches showed that the expression per cell of preproenkephalin and preprotachykinin messenger RNAs was inversely related, such that patches with higher than normal preproenkephalin messenger RNA levels displayed lower than normal preprotachykinin messenger RNA levels, and vice versa. Moreover, messenger RNA expression for the dopamine D2 receptor was overall lower than that for the dopamine D1 receptor, both with respect to the level per cell and the number of positive cells within the DARPP-32 patches. Glutamate decarboxylase messenger RNA was expressed throughout the grafts, in 98% of all neurons located in the DARPP-32-positive regions and in 75% of all neurons in the non-DARPP-32 regions of the graft. Interestingly, the cellular expression of glutamate decarboxylase messenger RNA was considerably higher in the non-DARPP-32 expressing regions than that in the DARPP-32 messenger RNA-rich areas, where it approximated that of the intact striatum. Furthermore, grafted neurons located outside the DARPP-32-expressing regions displayed similar levels of expression to those found in the overlying cortex and in the closely adjacent globus pallidus. To further characterize the DARPP and non-DARPP graft compartments, messenger RNAs encoding the alpha 1 and beta 2 subunits of the GABAA receptor were studied. These receptor subunits, which exhibit a high expression in the host cortex and pallidum but little in the intact striatum, were found in discrete patches situated outside, but often closely associated with, the DARPP-32-rich areas of the graft.(ABSTRACT TRUNCATED AT 400 WORDS)

Neurotransmitter-related gene expression in intrastriatal striatal transplants--II. Characterization of efferent projecting graft neurons.

The phenotypic characteristics of identified graft neurons in intrastriatal striatal transplants which give rise to efferent projections innervating the host brain were examined using a combination of in situ hybridization histochemistry and fluorescent retrograde tracing. Cell suspension grafts of embryonic day 14-15 rat striatal primordia (including both the medial and lateral ganglionic eminences) were implanted into the previously excitotoxically lesioned striatum of adult rats, and after longer than one year the retrograde tracer Fluoro-Gold was injected bilaterally into either the globus pallidus or the substantia nigra. Injections into the globus pallidus resulted in significant retrograde labelling of graft neurons within most of the experimental animals, whereas very few graft cells were labelled after the nigral injections. The vast majority of the neurons retrogradely labelled from the globus pallidus occurred in clusters or patches in the caudal half of the transplants, which corresponded well with DARPP-32 messenger RNA expressing (i.e. striatal) regions of the grafts. Indeed, within these Fluoro-Gold-labelled graft patches, the proportion of retrogradely labelled cells found to contain DARPP-32 messenger RNA was identical to that observed in the intact striatum after similar pallidal injections (93%). In addition, some Fluoro-Gold-labelled cells were found scattered outside the DARPP-32-positive cell clusters; these cells were overall larger and rarely (c. 9%) DARPP-32 messenger RNA-positive. Messenger RNA encoding for glutamate decarboxylase (which was found in 95% of Fluoro-Gold-labelled neurons in the intact striatum) was detected in almost all retrogradely labelled graft neurons located in both the DARPP-32-positive patches of retrograde labelling (93%) and in the DARPP-32-negative regions (82%). In the intact striatum, neurons labelled after pallidal injections of Fluoro-Gold were observed to express preproenkephalin messenger RNA to a greater extent than preprotachykinin messenger RNA (81% vs 21%). Conversely, within the grafts, retrogradely labelled neurons in the patches of Fluoro-Gold-labelled cells were more often found to contain preprotachykinin messenger RNA (50%) than preproenkephalin messenger RNA (21%). The Fluoro-Gold-labelled cells scattered outside the patches of retrograde labelling rarely expressed either preproenkephalin or preprotachykinin messenger RNA. Fluoro-Gold injections into the host substantia nigra resulted in very few retrogradely labelled graft neurons; however, many (85%) of these cells were observed to express glutamate decarboxylase messenger RNA, while only rarely were they observed to contain either DARPP-32, preproenkephalin or preprotachykinin messenger RNAs (c. 10%).(ABSTRACT TRUNCATED AT 250 WORDS)

Retrograde degenerative changes in the substantia nigra pars compacta following an excitotoxic lesion of the striatum.

The retrograde changes induced by an excitotoxic lesion of the striatum (Str) on the neurons in substantia nigra pars compacta (SNc) projecting to the neuron-depleted region were investigated in adult rats. The retrograde tracer Fluoro-Gold (FG) was injected bilaterally into the Str. 2 weeks later, the excitotoxic amino acid ibotenic acid (IA) was injected unilaterally into the same structure. At four different time points after the lesion (1 week and 1, 2 and 3 months, respectively), the size of the FG-labelled cells and number of tyrosine hydroxylase (TH)-positive cells in the SNc were evaluated on the lesioned and control sides. Parallel groups of animals received suspension grafts of fetal striatal tissue into the lesioned striata. At 1 week and 1 month after lesion, there were no changes in cell size, number of TH-positive cells or number of FG-labelled cells expressing TH at the SNc. At 2 and 3 months, however, there was a significant 30% shrinkage of the FG-labelled SNc cells but no evident decrease in TH-positive cell number, or in the expression of the TH protein, on the lesioned side as compared with the non-lesioned control side. Striatal transplants placed into the lesioned Str did not counteract this effect. This finding that an axon-sparing lesion of target cells results in cell shrinkage but no cell loss of the neurons that project to the lesioned area is in line with what has been shown to occur after similar lesions in the cholinergic septohippocampal and basalo-cortical systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular gamma-aminobutyric acid levels in the rat caudate-putamen: monitoring the neuronal and glial contribution by intracerebral microdialysis.

Intracerebral microdialysis with high pressure liquid chromatography (HPLC) coupled to electrochemical detection was employed to characterize gamma-aminobutyric acid (GABA) release and the effects induced by a preceding neuron-depleting ibotenic acid (IBO) lesion in the rat caudate-putamen (CPu). Extracellular GABA overflow was monitored in the intact and excitotoxically lesioned CPu, either 7-10 days (acute) or more than 3 months post-lesioning (chronic), using loop type dialysis probes perfused at a rate of 2 microliters/min. In the intact CPuu, basal GABA levels were 0.97 pmol/30 microliters of dialysate in the awake animals and 0.76 pmol/30 microliters under halothane anaesthesia. In both the acute and chronic IBO lesioned CPu the extracellular GABA levels were reduced by 80% and 67%, respectively, under halothane anaesthesia. KCl added to the perfusion fluid at a concentration of 100 mM resulted in dramatic increases in GABA overflow from baseline levels in the intact CPu (60- to 70-fold), which were almost totally abolished (> 95%) in the excitotoxically lesioned CPu. Veratridine administered at 75 microM, produced a 45-fold increase in GABA overflow in the intact CPu, but failed to produce any effect in the lesioned CPu. The addition of nipecotic acid (0.5 mM), a GABA uptake blocker, increased basal extracellular GABA levels 6-15-fold in the intact CPu, while GABA overflow in either the acute or chronic lesioned CPu was not significantly altered. Although Ca(2+)-free conditions (with 20 mM Mg2+ added) or tetrodotoxin (TTX, 1 microM) did not alter the basal GABA overflow in the intact CPU under halothane anaesthesia, the omission of Ca2+ resulted in a 47% reduction in basal extracellular GABA levels in awake, freely moving animals. Nipecotic acid-induced GABA overflow was reduced by 22% under Ca(2+)-free conditions, and by 33% in the presence of 1 microM TTX. Moreover, KCl-evoked GABA overflow was reduced by 86% in Ca(2+)-free conditions and by 40% when administered in the presence of 1 microM TTX. These results indicate that the extracellular GABA levels recorded by intracerebral microdialysis in the CPu are derived predominantly from neuronal sources. Under baseline resting conditions only a small fraction (up to 20-30%) of the neuronal release was Ca(2+)-dependent and TTX-sensitive (i.e. possessing the characteristics of impulse-dependent vesicular release).(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of GABA release from intrastriatal striatal transplants: dependence on host-derived afferents.

Extracellular levels of GABA, derived from cell suspension transplants of embryonic day 14-15 rat striatal primordia implanted into the previously excitotoxically lesioned striatum, were measured using intracerebral microdialysis in halothane-anaesthetized rats. GABA overflow was monitored using loop type dialysis probes implanted into grafted, age-matched ibotenic acid-lesioned and intact striata, under baseline conditions and after different pharmacological manipulations. Basal and evoked GABA release, which was reduced by 58 and 96%, respectively, in the excitotoxin-lesioned striatum, was restored by the striatal grafts to levels close to or above those observed in normal striata. The graft-derived release of GABA was most likely of neuronal origin, since the K(+)-evoked (100 mM) GABA overflow was reduced by almost 80% when Ca++ was replaced by 20 mM Mg++ in the perfusion medium, and blockade of GABA uptake by nipecotic acid (0.5 mM), induced a greater than six-fold increase in GABA overflow. However, perfusion of the graft with 1 microM tetrodotoxin in combination with K+ (100 mM) resulted in little if any reduction in the K(+)-evoked overflow. Histological analysis demonstrated a dense tyrosine hydroxylase-positive fibre network in the grafts, which was removed after a 6-hydroxydopamine lesion of the ipsilateral nigrostriatal pathway. The dopamine denervating lesion resulted in an increased K(+)-evoked GABA overflow both in the intact (+76%) and the grafted striata (+181%), suggesting that the tonic dopaminergic inhibitory control of GABA release, seen in the intact striatum, is also present in the grafted striata. The glutamate analogue, kainic acid (1 mM added to the perfusion fluid), evoked a 60-74% increase in GABA overflow both in intact striata (with or without dopaminergic denervation) and in the striatal grafts. This effect seemed to be dependent on an intact corticostriatal projection, since knife-cut transections of the frontal cortex at the level of the forceps minor, abolished the response in both the intact and grafted striata. These results demonstrate that grafts of fetal striatal tissue implanted into the excitotoxically lesioned striatum restore striatal GABA overflow in a neuron-dependent manner, close to or above that seen in the normal intact striatum. Furthermore, the graft-derived GABA release appears to be under normal regulatory control from the host dopaminergic and glutamatergic systems. Since the GABAergic striatal output system is critical for the expression of striatum-related behaviours, it is proposed that the graft-induced behavioural recovery in the striatal lesion model, at least in part, may depend on the restoration of striatal GABAergic neurotransmission.

Axon outgrowth from grafts of human embryonic spinal cord in the lesioned adult rat spinal cord.

Adult rats with acute partial lesions of their upper thoracic spinal cords were implanted bilaterally with cell suspensions of 6-7 week-old embryonic human spinal cord tissue one segment above or below the lesions. After 14-19 weeks, the animals were perfusion-fixed and the tissue analysed with a light microscope after immunocytochemical labelling with an antiserum recognizing human, but not rat, intermediary neurofilaments. Using this method, extensive efferent projections were demonstrated extending longitudinally from the grafts into the host spinal cord, both in the caudal and rostral directions. Within the white matter tracts, dense bundles of fibres extended for about 3-4 mm, and single fibres were identified up to 10 mm away from the implants. Axonal growth of this length within host white matter has not previously been observed from intraspinal grafts of rat CNS tissue.

Differential regulation of neuropeptide mRNA expression in intrastriatal striatal transplants by host dopaminergic afferents.

The effects of dopamine-specific manipulations on neuropeptide gene expression in intrastriatal grafts of fetal striatal tissue were studied by quantitative in situ hybridization histochemistry, using 35S-labeled oligonucleotide probes. Messenger RNA transcripts for the striatal neuropeptides preproenkephalin (PPE) and preprotachykinin (PPT) were detected in neurons forming discrete patches in the striatal grafts. The relative abundance of PPE and PPT mRNA-expressing neurons within the graft patches (51-54%) was similar to that found in normal caudate-putamen. In specimens with intact dopamine afferents the expression of PPE mRNA in grafted neurons was similar to that found in normal caudate putamen, whereas the hybridization signal for PPT mRNA was 27% higher in the graft neurons than in the normal caudate-putamen. Removal of host dopaminergic afferents by 6-hydroxydopamine lesions of the ipsilateral mesostriatal dopamine pathway increased the hybridization signal for PPE mRNA both in the grafts (+84%) and in the spared ipsilateral host caudate-putamen (+125%), whereas the PPT signal was reduced by 53% in the grafts and by 51% in the remaining host caudate-putamen. Similarly, chronic treatment of grafted animals with the dopamine receptor antagonist haloperidol (2 mg/kg per day for 10 days) produced a 146% increase in the PPE signal in the grafts and a 175% increase in the intact contralateral caudate-putamen, whereas the signal for PPT mRNA was again decreased by 52% and 51% in the grafts and host caudate-putamen, respectively. These results show that the host nigrostriatal dopamine pathway differentially regulates enkephalin and substance P gene expression within striatal grafts and thereby exerts a tonic functional influence over grafted striatal neurons.

Long distance directed axonal growth from human dopaminergic mesencephalic neuroblasts implanted along the nigrostriatal pathway in 6-hydroxydopamine lesioned adult rats.

Dissociated ventral mesencephalon of 6 to 8-week-old human embryos were implanted by stereotaxic injection at different sites along the nigrostriatal pathway in adult rats, previously subjected to a 6-hydroxydopamine lesion of the intrinsic mesotelencephalic dopamine pathways. The recipients were immunosuppressed by daily injections of cyclosporin A to prevent rejection. At 13-20 weeks after transplantation, the implanted human neurons and their associated fiber outgrowths were visualized with a species-specific antibody recognizing human, but not rat, intermediary neurofilaments (HNF). From implants placed in the host rostral mesencephalic region, HNF-positive axonal projections were seen to extend in large numbers rostrally along the medial forebrain bundle and the internal capsule, and ramify within the caudate putamen, the ventral striatum and the amygdaloid nuclei (a distance of about 5-6 mm), and more sparsely in the frontal cortex and the olfactory bulb (a distance of about 10 mm). From implants placed in the internal capsule, abundant HNF-positive axons extended in the rostral, but not caudal, direction along the myelinated fiber bundles into the caudate putamen and the ventral striatum. Tyrosine hydroxylase (TH) immunohistochemistry revealed that the vast majority of the rostrally projecting HNF-positive axons were also TH-positive, and that the graft-derived axons gave rise to dense TH-positive terminal networks, above all in large areas of the previously denervated caudate putamen. From control implants of cortical neuroblasts, axonal projections were seen along the medial forebrain bundle and the internal capsule, but the axons were TH-negative and showed only sparse projections to the striatal areas. Instead, dense projections were seen, e.g., in the frontal cortex. The results demonstrate a remarkable ability of human mesencephalic neuroblasts to extend axons along the trajectories of the nigrostriatal and mesolimbocortical pathways to reach and innervate the principal striatal and limbic target areas in the forebrain. This shows that the basic requirements for the formation of long axonal pathways may be present in the adult mammalian central nervous system (CNS) at least for certain types of projection neurons. Furthermore, it implies that the developing human neuroblasts can escape the inhibitory features known to be present along myelinated growth trajectories in the adult mammalian brain. In addition, the present approach may offer new possibilities for functional neural grafting in the rat Parkinson model, since transplanted nigral neurons placed in their natural position within the rostral mesencephalon could provide an anatomically and functionally more integrated system than the standard model with ectopically placed intrastriatal nigral grafts.

Fos expression in intrastriatal striatal grafts: regulation by host dopaminergic afferents.

Previous studies have shown that transplants of fetal striatum, implanted into the ibotenic acid-lesioned striatum of adult rats, become innervated from the host nigrostriatal dopamine (DA) pathway. In the present study we have used DA-receptor-mediated expression of the Fos protein (i.e. the product of the immediate-early c-fos gene) as a cellular marker for functional dopaminergic host-graft interactions in the striatal grafts. Amphetamine (5 mg/kg; 2 h) induced Fos-like immunoreactivity in clusters of cells located mainly within the DARPP-32-positive areas within the transplants, i.e. within the striatum-like graft compartment which is preferentially innervated by the host DA afferents. As in the normal striatum, this effect was largely, although not completely, abolished by a 6-hydroxydopamine lesion of the ipsilateral nigrostriatal DA pathway. Apomorphine (0.25 mg/kg; 2 h) had no detectable effect in grafts with an intact host DA system. Two to 3 weeks after a 6-OHDA lesion of the host DA pathway (i.e. a time sufficient for DA receptor supersensitivity to develop), apomorphine-induced extensive Fos-activation selectively within the DARPP-32-positive areas of the graft. The magnitude of the response was similar to that seen in the DA-denervated host striatum. Dual Fos/DARPP-32 immunostaining revealed that the activated graft neurons were, at least in part, DARPP-32-positive. In intrastriatal grafts of fetal neocortical tissue, which were studied for comparison, the amphetamine- and apomorphine-induced effects on Fos expression were much smaller and similar to that seen in the DARPP-32-negative, non-striatal compartment within the striatal grafts.(ABSTRACT TRUNCATED AT 250 WORDS)

Dopaminergic transplants normalize amphetamine- and apomorphine-induced Fos expression in the 6-hydroxydopamine-lesioned striatum.

Dopamine receptor-mediated Fos protein expression in the striatum has been used to monitor dopamine receptor activation at the cellular level after dopaminergic denervation and reinnervation by fetal nigral transplants. The pattern of striatal Fos expression after systemic administration of either the dopamine receptor agonist, apomorphine, or the dopamine-releasing agent, amphetamine, was studied in rats which had received cell suspension grafts of fetal ventral mesencephalic neurons into the striatum after a complete 6-hydroxydopamine lesion of mesostriatal dopaminergic projection. Grafted animals, and normal and lesioned controls were killed 2 h after administration of either D-amphetamine (5 mg/kg, i.p.) or apomorphine (0.25 mg/kg, s.c.). Fos protein was detected immunohistochemically, and the density of Fos-immunoreactive cell nuclei was measured in 12 selected areas of caudate-putamen, nucleus accumbens and globus pallidus by computerized image analysis. Consistent with previous studies, amphetamine induced high Fos expression in the medial and dorsal parts of the intact caudate-putamen and significantly lower expression in the denervated caudate-putamen. A significant difference between lesioned and intact striata was present also in globus pallidus, but not in nucleus accumbens. In grafted rats, amphetamine-induced Fos activation was restored to normal or supranormal levels in the anterior and central caudate-putamen (i.e. close to the graft deposits), whereas in the tail of caudate-putamen Fos expression was significantly lower than normal. The side-to-side difference in globus pallidus seen in lesioned rats was no longer present in the grafted animals. Apomorphine led to high Fos activation throughout the dopamine-depleted caudate-putamen, whereas only very few immunopositive cells were observed in the intact caudate-putamen. Also in globus pallidus and nucleus accumbens, a significantly higher number of Fos-immunoreactive cells was detected on the denervated side. In the grafted rats, apomorphine-induced Fos activation was similar to normal in all striatal areas sampled, as well as in the globus pallidus. The graft-induced effect extended over a considerably larger area than that covered by the graft-derived tyrosine hydroxylase-immunoreactive innervation. These findings indicate that fetal ventral mesencephalic transplants normalize dopamine receptor-mediated function in the 6-hydroxydopamine-lesioned caudate-putamen and nucleus accumbens, as well as in a primary target of the striatal output neurons, the globus pallidus. The results support the idea that dopamine released from the grafted neurons, both under baseline conditions and after amphetamine administration, exerts functional effects over a larger volume of the striatum than that reached by the graft-derived fibers.

Efferent Projections to the Host Brain from Intrastriatal Striatal Mouse-to-rat Grafts: Time Course and Tissue-type Specificity as Revealed by a Mouse Specific Neuronal Marker.

The developmental time-course and growth characteristics of efferent graft-to-host projections were studied from mouse fetal striatal grafts (E13 - 14) implanted as a cell suspension into the ibotenate-lesioned striatum of immunosuppressed adult rats. A cell surface monoclonal antibody specific for mouse neurons (M6) was used to identify the donor cells and their projections into the host brain. At 3 - 5 days after implantation, sparse fascicles of M6-positive graft-derived fibres extended for approximately 0.3 - 0.4 mm across the graft - host border into the surrounding host striatum. From the beginning they were selectively orientated in one direction, i.e. caudally along the myelinated fibre bundles of the internal capsule. At 8 days, the graft-derived fibres were more numerous and more densely labelled. They ran in dense fascicles inside the myelinated bundles of the host internal capsule and reached the rostral host globus pallidus, a distance of approximately 1.2 mm from the caudal tip of the graft. Two weeks after grafting, the M6-positive fibre fascicles were clearly seen to branch within the globus pallidus to form terminal-like networks. From this time onwards, the immunoreactivity of the outgrowing fibre fascicles gradually diminished, although small but dense terminal-like networks could be found in the host globus pallidus in most, but not all, of the rats at longer survival times (3 - 15 weeks). This is consistent with previous work showing that outgrowing axons lose their M6 immunoreactivity as they mature and become myelinated. Control grafts of fetal neocortical and fetal cerebellar tissue were used to assess the tissue-type specificity of the efferent fibre growth. The neocortical implants projected densely up to about 3 mm into the host brain, along the internal capsule and the corpus callosum and into the overlying cortex. By contrast, although the cerebellar grafts survived well, they showed very little efferent fibre growth. Double immunostaining for DARPP-32 and M6 revealed that all M6-positive fibre fascicles extending from the striatal (but not neocortical) grafts also showed DARPP-32 positivity, and thus that it was the DARPP-32-positive regions of the striatal grafts that projected to the host brain. It is concluded that graft-to-host projections, running along and inside host myelinated bundles, are formed from intrastriatal striatal grafts within 1 - 2 weeks of implantation. Grafts of neocortical tissue grew well along the same trajectory, whereas neurons of a type not normally projecting along the internal capsule, i.e. cerebellum, failed to extend axons over any significant distance along this trajectory.