Anatomy and connectivity of intrastriatal striatal transplants

Progressin NeurobiologyVol. 38, pp. 611-639, 1992

0301-0082/92/$15.00 © 1992 Pergamon Press pie

Printed in Great Britain. All rights reserved

ANATOMY AND CONNECTIVITY OF INTRASTRIATAL STRIATAL TRANSPLANTS KLAS WICTORIN

Department of Medical Cell Research, University of Lund, Biskopsgatan 5, S-223 62 Lund, Sweden (Received 30 October 1991)

CONTENTS Abbreviations 1. Introduction 2. The striatal excitotoxin lesion model 2.1. The lesioned striatum 2.2. An animal model of Huntington's disease 3. Donor tissue dissection and implantation 3.1. Dissection of the striatal primordium 3.2. Implantation 4. Internal organization of the transplants 4.1. Development of the transplanted tissue 4.2. Striatum-like characteristics of the transplants 4.3. A heterogeneous composition 5. Afferent connections from the host brain 5.1. Afferents from the cortex and thalamus 5.2. Monoaminergic afferents 5.3. Regenerative responses of adult target-deprived CNS axons 5.4. A functional host innervation of the transplanted neurons 5.5. Development of the afferent innervation 6. Efferents to the host brain 6. I. Efferent projections mainly to the host globus pallidus 6.2. Time-course and species-differences 6.3. Specificity and growth along white matter tracts 7. Functional behavioural effects 7.1. Reported behavioural effects 7.2. Functional mechanisms 7.3. Partial reconstruction of striatal circuitry 8. New approaches 9. Clinical perspectives 10. Concluding remarks Acknowledgements References

ABBREVIATIONS ChAT CNS CPu CRL DARPP-32 E FG FITC GAD GP HD IA KA PHA-L SN TH TRITC WGA-HRP

choline acetyl transferase central nervous system caudate-putamen crown-rump length dopamine- and cyclic AMP-regulated phosphoprotein, with a molecular weight of 32 kilodaltons embryonic day Fluoro-Gold fluorescein isothiocyanate glutamic acid decarboxylase globus pallidus Huntington's disease ibotenic acid kalnic acid Phaseolus vulgaris leucoagglutinin substantia nigra tyrosine hydroxylase tetra methylrhodamine isothiocyanate

wheatgerm agglutinin-horseradish peroxidase 611

611 612 612 613 613 613 613 614 614 615 615 616 616 619 62O 620 623 624 625 625 625 631 631 631 631 632 633 634 635 635 635

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K. WICTORIN 1. INTRODUCTION

Transplantation of neuronal or non-neuronal tissue into the mammalian central nervous system (CNS) is today widely used both to address basic neurobiological questions and to develop potential therapies for various neurodegenerative disorders (see reviews by Bjfrklund and Stenevi, 1984; Dunnett, 1990a; Gage and Fisher, 1991; Bj6rklund, 1991, and the symposia volumes of Gash and Sladek, 1988; Dunnett and Richards, 1990, for recent overviews). Among the various animal models used in these experiments, the intrastriatal striatal graft model (with embryonic striatal tissue implanted into an excitotoxically lesioned striatum) is one of the most frequently used. This may in part be due to the well characterized normal anatomy of the striatum (or caudate-putamen), with many chemically identified types of neurons and fibre systems. The intrastriatal striatal graft model has thus provided a good experimental tool to further clarify aspects of anatomy, development and regeneration in this important region. Moreover, since the excitotoxic lesion of the rat striatum is considered as an animal model of the severe neurological disorder Huntington's disease (HD) (see Sanberg and Coyle, 1984; DiFiglia, 1990, for recent reviews), the observed functional behavioural effects of the implants have rendered much interest, and raised hope for a potential clinical application. Overall the documented history of neural transplantation started about one hundred years ago, when the first report on neural transplantation into the brain appeared, describing attempts to move large pieces of neocortical tissue between cats and dogs (Thompson, 1890). Then followed a series of early publications by, e.g. Ranson (1909), Dunn (1917) and LeGros Clark (1940) providing the first real evidence for the general feasability of grafting into the mammalian CNS, and containing findings of, for instance, neurotrophic effects of the grafted neural tissue, and of the importance of donor- and recipient-ages for graft survival (see, e.g. Bj6rklund and Stenevi, 1985, for review). However, it was only about 20 years ago that the modern era of neural transplantation really started, when three different laboratories applied new autoradiographic and transmitter-specific histochemical methods to analyze the grafts. Thus, Das and coworkers (Das and Altman, 1971, 1972) investigated fetal neural grafts implanted into neonatal recipients, Olson and collaborators (Olson and Malmfors, 1970; Olson and Seiger, 1972a) used the anterior eye chamber as a site to examine the development and growth of neural implants, and Bj6rklund and colleagues (Bj6rklund and Stenevi, 1971; Bjfrklund et al., 1971) studied sprouting of central monoaminergic fibres into, e.g. smooth muscle implants. Then, in 1976 came the important obsevations concerning an actual formation of fibre connections between neural grafts and the recipient mammalian CNS. Thus, Lund and Hauschka (1976) demonstrated that host visual afferents of a neonatal recipient innervated a graft of fetal superior colliculus, and Bjfrklund et al. (1976) showed that implanted fetal monoaminergic cells could actually send efferent fibres into the adult deafferented hippocampus. The

next major breakthrough came in 1979, when two different groups could report functional behavioural effects of intracerebral neural grafts, i.e. grafts of dopamine-rich mesencephalic tissue placed into rodents with experimental Parkinsonism (Bj6rklund and Stenevi, 1979; Perlow et al., 1979). The first resport on intrastriatal striatal grafts appeared in 1981, when Schmidt et al. described that grafts of embryonic rat striatum placed into the excitotoxically lesioned adult rat striatum, could show long-term survival and restore striatal levels of the transmitter-related enzymes choline acetyltransferase (CHAT) and glutamic acid decarboxylase (GAD), as compared to lesion-only controls. Then, only a few years later implantation of fetal striatal tissue was shown to result in functional behavioural effects, as both Deckel et al. (1983) and Isacson et al. (1984) reported graft-induced amelioration of lesioninduced locomotor hyperactivity. These early reports have since been followed by a large number of publications describing a remarkable ability of the striatal implants both to integrate anatomically with the recipient brain, as well as to ameliorate behavioural deficits caused by the lesions. The present review deals primarily with work describing the formation of a striatum-like structure by the implanted cells and the anatomical integration between the grafts and the surrounding host brain, with a focus on work from our own laboratory. In addition, the reported functional behavioural effects of the grafts are reviewed, and recent development and new approaches in this transplantation model are discussed.

2. THE STRIATAL EXCITOTOXIN LESION MODEL In most experiments with intrastriatal striatal grafts, the implants have been placed into an excitotoxically damaged adult striatum, and thus for the understanding of the implants and their development it is important to first of all assess the basic features of such a lesion site. Coyle and Schwartz (1976) and McGeer and McGeer (1976) were the first to report that injections of the potent excitatory glutamate analogue kainic acid (KA) into the rat striatum cause neuronal degeneration in the injected area, and behavioural symptoms resembling those observed in patients suffering from the severe neurological disorder Huntington's disease (HD). Since those early observations, KA and other so-called 'excitotoxins' have been widely used to produce selective neuronal lesions in different CNS regions and thus to create animal models of various different neurodegenerative disorders (see Coyle and Schwartz, 1983, for review). Due to the many similarities between the lesion models and the real disorders, excitatory amino acid neurotoxicity has indeed been thought to play a role in several different neurological diseases (see DiFiglia, 1990; Meldrum and Garthwaite, 1991, for recent reviews). Among the glutamate analogues with potent excitotoxic effects, N-methyl-D-aspartic acid (NMDA), ibotenic acid (IA), and quinolinic acid (QA) share a common receptor type (NMDA-receptors), while KA and quisqualic acid (QUIS) bind to

INTRASTRIATALSTRIATALTRANSPLANTS separate kainate- and quisqualate-receptors, respectively (for reviews see Rothman and Olney, 1987; Watkins and Olverman, 1987). 2.1. THE LESIONEDSTRIATUM In experiments with striatal grafts, K A and IA have been the most frequently used excitotoxins, although QA has also been utilized. For striatal KA-infusions, Coyle and Schwartz (1976) described dramatic dose-related effects, with a 95% loss of neurons at the injection site, and sharp increases in glial cell number and size, and although less potent, IA has been shown to have the same effects as K A on the infused striatum (Schwartz et al., 1979; Isacson et al., 1985b). Thus, the IA or K A injections result in long-lasting reductions (70-85%) in striatal levels of the transmitter-related enzymes choline acetyltransferase (CHAT) and glutamic acid decarboxylase (GAD), whereas axons of passage and nerve terminals show no direct changes, at least not acutely after the infusions (Schwartz et al., 1979; Isacson et aL, 1985b). During the first weeks after such excitotoxin injections, thus at the time of neural implantation in most experiments (see below), the total volume of the striatum is unaltered, whereas after about 4 weeks the lesioned region starts to shrink. Then after several months, the whole striatum is severely atrophic, with the lesioned area consisting mainly of condensed bundles of the internal capsule (Schwartz et al., 1979; Isacson et aL, 1985b). Some details on the methods used to lesion the rats are given in Fig. 1. Apart from the more direct focal cell death and related changes, the striatal lesions also cause anterograde trans-synaptic degeneration of large numbers of neurons in the striatal projection areas, such as the substantia nigra (Krammer, 1980; Saji and Reis, 1987), and there are marked increases in size and number of reactive astrocytes also in the globus pallidus and substantia nigra (Isacson et aL, 1987b). Over time, changes appear to occur also in afferent

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systems terminating in an excitotoxically lesioned region (see, e.g. Sofroniew et al., 1986; Peschanski and Besson, 1987), and apparently also in fibres-ofpassage (Coffey et al., 1990). 2.2. AN ANIMALMODEL OF HUNTINGTON'SDISEASE There are many neurochemical and histopathological similarities between the excitotoxin rat model and the changes in brains of patients suffering from Huntington's disease (HD), and since no naturally occurring genetic model is available, the excitotoxin model is today the most widely used animal model of this disorder. Thus, the major pathological changes in the brain of a HD patient are a severe atrophy, and neuronal cell loss and ghosis in the striatum, but there are also severe changes in other parts of the basal ganglia, as well as in other brain regions such as the cerebral cortex (see references in Sanberg and Coyle, 1984). Behaviourally, HD is characterized by constant involuntary and dance-like ("choreic") movements and dementia (Huntington, 1872; see reviews by Bruyn, 1968; Bird, 1980; Martin, 1984). The disorder is genetic, determined by a single autosomal dominant gene on the short arm of chromosome 4 (Gusella et al., 1983), and in persons with the, as yet unknown, genetic abnormality, the symptoms typically begin around the age of 40-50 years. With regard to the behavioural deficits seen in the lesioned rats, such as the increased locomotor activity, regulatory changes and learning impairments, they resemble the symptoms of the HD patients, although involuntary choreiform movements are not seen in the rats (see Sanberg and Coyle, 1984, for review). Recently, Isacson and colleagues have developed a primate excitotoxin lesion model of HD with even closer neuropathological and behavioural similarities to the real human disorder (Isacson et al., 1989; Hantraye et al., 1990; and see below).

3. DONOR TISSUE DISSECTION AND IMPLANTATION ~3 EXCITOTOXIC

F16. I. Schematic drawing of the method used to excitotoxically lesion the adult rat striatum. In our own experiments we use ibotenic acid (12-15/~g per rat striatum), which is dissolved in phosphate-buffer and unilaterally injected from a Hamilton syringe with the rat placed in a stereotaxic frame. The coordinates were chosen to result in a neuron-depleting lesion of the head of the striatum, and care was taken to avoid extrastriatal damage. The tail of the striatum as well as thin lateral and medial rims of the head of the striatum are usually spared by the lesion. For details, see Isacson et aL (1985b) and Wictorin et al. (1988).

Embryonic striatal tissue from rat (with embryonic ages ranging from El4 to El8) has been the most frequently used donor material in the studies on intrastriatal striatal grafts. Apart from rat fetal striatum, we have in some experiments used mouse or human tissue to prepare our cell suspensions (see further below). As an alternative to the use of embryonic tissue, genetically modified neuronal or nonneuronal primary cells or immortalized cell lines have recently been introduced as donor material in some of the intracerebral grafting models (Gage et al., 1987; Cattaneo and McKay, 1991; and see further below). 3.1. DISSECTIONOF THE STRIATALPRIMORDIUM Figure 2 illustrates the dissection of the rat ganglionic eminences. In our own experiments, we routinely use rat fetuses with crown-rump lengths (CRL) of l l - 1 4 m m , which correspond to gestational ages E14-15 (with E0 as the day of sperm positivity; E, embryonic day). (See Olson and Sciger (1972b) and

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K. WICTORIN

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. " ~ C P u

ME

i NE' SE"

o,. • o °

I

A

B

FIG. 2. Schematic drawings of the regions of the E14-15 rat forebrain which are dissected to prepare the striatal cell suspension. (A) Medial view of the lateral ventricular wall, with the dotted line indicating the approximate region of the caudate-putamen (CPu) anlage included in our dissections. ME and LE indicate the medial and lateral elevations, respectively, of the ganglionic eminences. (B) Coronal section through the center of the region indicated in A. Here the dotted lines show the estimated minimal (thick arrow) and maximum (arrowhead) extents of our dissections. The lateral ventricular wall consists of a neuroepithelium (NE), a subependymal layer (SE), and the anlages for striatum (S), pallidum (P), amygdala (A), and lateral cortical regions. (Modified from Wictorin et al., 1990a.) Seiger and Olson (1973) for details on the CRL-E correlations.) At this embryonic stage, striatal neurogenesis is very intense, with neurogenesis in the developing rat striatum starting around El3 and continuing until postnatal day P4, with large neurons formed between El3 and El6, the medium-sized neurons born between El4 and El8 (ventrolateral cells) and E18-E22 (dorsomedial cells), and with less than 10% of the cells generated after P0 (Bayer, 1984). The ventricular elevations of the ganglionic eminences (ME and LE in Fig. 2) are the sites of production of neurons and neuroglia of the basal parts of the telencephalon, and the developing striaturn receives contributions from both the elevations (Smart and Sturrock, 1979; Fentress et al., 1981). As discussed further in the next chapter, the striatal grafts have a notably heterogeneous composition, with only about 30-50% of the cross-sectional area showing typical striatum-like characteristics. This could possibly be explained by difficulties in specifically dissecting only striatal tissue, since many other non-striatal cells also originate in the vicinity of, or even inside the ganglionic eminences. Thus, for instance the globus pallidus anlage is partly present within the medial elevation (Smart and Sturrock, 1979), the developing amygdala is situated nearby in the lateral ventricular wall, the lateral parts of the cortex are formed lateral to and underneath the striatal anlage, and furthermore some cells are possibly contributed to the cerebral cortex by the lateral elevation (Smart, 1976; Smart and Sturrock, 1979; Fentress et al., 1981). 3.2. IMPLANTATION

The donor tissue in intracerebral grafting experiments can be implanted into the hosts either as solid tissue pieces or as cell suspensions, and in the intrastriatal striatal graft model both methods have been used. We use the suspension method, with a fine

implantation needle, which has the advantage that it allows a direct and exact stereotaxical placement of the tissue into deep brain sites with minimal damage to the recipient brain due to the implantation procedure (Bj6rklund et al., 1983). Furthermore, the viability of the cells and the exact cell numbers that are implanted can be monitored by way of a viability stain and through cell counting in a haemocytometer prior to implantation (Brundin et al., 1985a). Figure 3 depicts the main steps in the preparation of the tissue before transplantation into the lesioned striatum. 4. INTERNAL ORGANIZATION OF THE TRANSPLANTS Since the main objective of the intrastriatal striatai graft experiments is to try to replace the degenerated host striatum, it is of course important to first of all assess to what extent the well characterized intrinsic striatal structure can actually be re-established by the implanted cells. In the normal intact brain, the striatum (or caudate-putamen) is characterized by at least 15 different putative transmitter types, and many different morphological cell types. The internal structure of the striatum is dominated by the medium sized densely spiny neurons ( > 95% of the total cell number), which use GABA as their transmitter, and in addition contain, e.g. either substance P (striatonigral neurons) or met-enkephalin (striatopailidal neurons). There are furthermore, at least three different types of interneurons; GABAergic, NPY/somatostatincontaining, and cholinergic. Especially in the primate striatum, but to some extent also in the rodent striatum, many of the transmitter-related compounds are heterogenously distributed, and the projection neurons are often organized in clusters, and several of the afferents to the striatum terminate in a patchy manner (Graybiel and Ragsdale, 1978, 1983; Herkenham and Pert, 1981; Gerfen, 1984; and further

INTRASTRIATAL STRIATALTRANSPLANTS

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FIG. 3. Schematic drawings of the steps involved in the preparation of tissue according to the cell suspension technique. In our experiments, as well as in many other studies, the dissected tissue pieces (a), which are taken from the ganglionic eminences of E14-15 striatal embryos (see Fig. 2 for details of the dissection) are first enzymatically (b) (with trypsin) and then mechanically (c) dissociated into a cell suspension (see Bj6rklund et al., 1983; Isacson et al., 1985b, for details). In most of our studies, we then implant approximately 2 striatal anlages per lesioned host striatum, which corresponds to about 6-10 x 105viable cells. The cell suspension (d) is injected from a Hamilton syringe, at the same coordinates as used for the ibotenic acid lesion (e), with the lesion usually preceding the implantation by about 1-2 weeks. references in Graybiel, 1990). Thus, areas of low AChE-activity are called "striosomes" or "patches", whereas the dominating regions of high ACHEactivity are referred to as "matrix". In the rat, above all opiate receptor ligand binding provides a good marker for the patch/matrix arrangement. It is beyond the scope of this review to introduce the complexity of the anatomy of the normal striatum in greater detail, with its many characteristic cell types and transmitters (for reviews, the readers are advised to study, e.g. Graybiel and Ragsdale, 1983; Smith and Bolam, 1990). 4.1.

DEVELOPMENTOFTHETRANSPLANTEDTISSUE

In the vast majority of experiments in this transplantation model, the grafts have been analyzed at several months after implantation. However, in one of our studies we assessed the gradual development of the implants from two days post-grafting and onwards to compare the maturation of the striatal grafts with normal striatal ontogeny, and also to compare the developmental time-course of the grafts with the reported development of graft-induced behavioural effects (Labandeira-Garcia et al., 1991). We found that the striatal grafts develop with a time-course that is fairly close to that of the normal striatum, with the volume of the implants increasing about 5-8 times during the first three weeks, and cell specific markers developing within a few days after implantation. For instance, the first clearly DARPP-32 positive neurons appeared at 4-5 days post grafting (DARPP-32, Dopamine- and Adenosine 3',5'-monophosphate-

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Regulated PhosphoProtein). Altogether, the morphological analysis pointed to an almost completed maturation by 3 weeks, and by 6-8 weeks after implantation, the grafts were not different from previously observed several-month-old implants. Another interesting observation was that the integration between the implanted tissue and various host afferent fibres occurred in two partially overlapping phases, with first, a non-specific intermingling between the growing implant and target-deprived host afferents, and second, from about 4-6 days, a more specific process with, for instance a directed innervation of the striatum-like compartments by adult host dopaminergic afferents (see further below, Section 5.5). Also with regard to the behavioural effects of the implants, most studies have been conducted with several-month-old grafts. However, in the studies of Sanberg et al. (1986) the first functional effects were observed already between three and six weeks after implantation. The early morphological maturation that we observed (Labandeira-Garcia et al., 1991) supports the possibility that the striatum-like characteristics of the grafts and their anatomical integration with the host brain can at least partly underlie also these early behavioural effects (see further below, Section 7.1). Other important questions related to the development of the implanted tissue concern the migration of neurons between graft and host, and the delineation of the graft-host border, which are important both from a general developmental point of view, and for the assessment of the anatomical graft-host integration. In most experiments, the graft-host border has been defined by the sheath of condensed host internal capsule bundles, which characteristically surrounds the grafts in the excitotoxically lesioned striaturn. Several lines of evidence have recently proven that this is indeed the true graft-host border. First, thymidine-prelabelling of either the donor tissue (McAllister, 1987; Wictorin et aL, 1989a; Graybiel et al., 1989) or the recipient (Liu et al., 1990) has shown that no or very little migration occurs from the grafts into the host, although glial cells apparently do migrate from the grafts into the host (Zhou et aL, 1989). Second, the use of mouse-to-rat grafts and a neuronal marker specific for the mouse donor tissue (see Fig. 12) has further proven this point (Wictorin et al., 1991). Thus, synaptic contacts of host afferent fibres onto neuronal elements inside the condensed sheath of internal capsule bundles can be securely identified as contacts onto grafted neurons, rather than onto spared or migrated host neurons.

4.2. STRIATUM-LIKECHARACTERISTICS OFTHE TRANSPLANTS Following the early observations by Schmidt et al. (1981) of graft-induced recoveries in GAD- and ChAT-levels, most of the normal striatal transmitters and transmitter-related enzymes such as substance P, met-enkephalin, somatostatin, neuropeptide Y, GABA, GAD, ACHE, and CHAT, have been identified in the transplants with either histochemical, immunohistochemical or /n situ hybridization techniques (Isacson et aL, 1985b, 1987a; Walker et al.,

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1987; Roberts and DiFiglia, 1988, 1990; Graybiel 1989; Wictorin et al., 1989c; Zhou and Buchwald, 1989; Clarke and Dunnett, 1990; Giordano et al., 1990; Liu et al., 1990; Sirinathsinghji et al., 1990). The grafts have also been shown to possess high levels of dopamine D 1 and D2 receptors, and muscarinic receptors (Isacson et al., 1987a; Deckel et al., 1988; Liu et al., 1990; Mayer et aL, 1990; Helm et aL, 1991) and to contain typical striatal cell types, such as medium-sized densely spiny neurons (McAllister et al., 1985; Clarke et al., 1988; Helm et al., 1990a). Most of the various normal striatal markers are, however, distributed within the implants in a notably patchy manner, with distinct areas of strong ACHEstaining corresponding to areas of for instance high enkephalin- or substance P-immunoreactivity and dopamine D2-receptor binding, surrounded by areas virtually devoid of these markers (Isacson et al., 1985b, 1987a, and see below). There could be several different explanations for this heterogeneity, such as clustering of neurons, or mixing of clusters of graftderived cells with areas of spared host tissue. However, as already mentioned, the experiments with thymidine-labelling of the donor cells prior to grafting (Graybiel et al., 1989), and cross-species mouseto-rat grafts and the use of a mouse-specific neuronal marker (Wictorin et al., 1991) have evidenced that the graft-derived cells are evenly distributed inside the graft-host border, and that all the tissue inside this border-zone is indeed graft-derived. Another way to explain the patchiness could be to compare it with the patch and matrix arrangement of the normal striatum (Graybiel and Ragsdale, 1983; Graybiel, 1990). In the rat, however, the compartments are not so easily identifiable with, e.g. AChE-histochemistry, and this is therefore not a likely explanation for the very uneven distribution of several markers in the grafts. Isacson et al. (1987a) proposed that the heterogeneity could result from a retention of immature features in the implants, since the correspondence of patches of AChE-staining to areas of high metenkephalin fibre staining, resembled that of the immature rat striatum during the early postnatal period. Although that hypothesis can not be totally ruled out, several more recent reports, however, suggest that the heterogeneity of the implants rather results from a mixing of striatal and non-striatal tissue-types within the implants. et al.,

4.3. A HETEROGENEOUSCOMPOSITION Indeed, as discussed in Section 3.1, the dissection of the striatal primordium most probably involves tissue types other than striatal. The presence of other tissue-types within the implants was initially suggested by Isacson et al. (1987a), Walker et al. (1987) and DiFiglia et al. (1988), who all recognized pallidaMike structures in the grafts, and more recently cortical pyramidal-like cells have been observed in the transplants (D. J. Clarke et al., personal communication). As illustrated in Fig. 4, we have further characterized the implants using antibodies against the neuronal phosphoprotein DARPP-32 (Dopamine- and Adenosine Y,5'-monophosphateRegulated PhosphoProtein) (Wietorin et al., 1989c),

which is normally enriched in dopaminoceptive regions, with neurons possessing dopamine D1 receptors (Ouimet et al., 1984). DARPP-32-immunocytochemistry thus serves as a very suitable way to identify striatal tissue, and indeed the grafts characteristically possess dense DARPP-32-immunoreactive patches, totally corresponding to AChE-positive regions. Other areas of the grafts contained no or very little DARPP-32-positivity, and thus did not resemble striatal tissue. Further evidence for the heterogeneous composition of the grafts came also from the thorough investigation of Graybiel et al. (1989), where the AChE-rich areas or "patch (P)-regions", were found to contain markers of both striosomes and matrix. The AChE-negative areas or "non-patch (NP)-regions", on the other hand, were found to contain calbindin- and somatostatin-immunoreactive neurons with features normally found in the pallidum, basolateral amygdala, and ventrolateral cortex. The presence of such neuronal cell types could easily be explained by difficulties to dissect only striatal tissue from the striatal primordium (see Fig. 2).

5. AFFERENT CONNECTIONS FROM THE HOST BRAIN The striatum-like appearance of large areas of the grafts and the notably heterogeneous composition are very important to keep in mind when assessing the formation of anatomical connections between the implants and the surrounding host brain. McGeer et al. (1984) were the first to actually document the presence of host fibres (dopaminergic) within the striatal implants (in that case from neonatal donors). The first more systematic report on afferents to the gratis was then published by Pritzel et al. (1986) who could, using a combination of dopamine histofluorescence and retrograde tracing, demonstrate a dense and patchy dopaminergic innervation from the substantia nigra, as well as some evidence for afferent inputs also from the cortex and thalamus. Since then, a large number of papers have further characterized the presence and distribution of different host afferent systems within the intrastriatal striatal grafts (see below). There are, in addition, some reports suggesting that no or only minimal host-graft connections are established (e.g. Walker and McAllister, 1987; McAllister et al., 1989). This discrepancy in the anatomical findings could possibly be due to differences in grafting parameters or anatomical techniques, although the large number of recent papers repeatedly documenting host-graft connections leaves no doubt that the grafts indeed integrate and establish connections with the surrounding host brain. When examining the patterns of host-graft connectivity, the complex arrangement of the afferents to the normal striatum should be noted. The striatum receives a large number of inputs from different regions of the brain, with the major projections arising in the cerebral cortex, the intralaminar thalamic nuclei, and the substantia nigra (see, e.g. Graybiel and Ragsdale, 1983, for review). The cortical input to the head of the striatum originates mainly in frontal neocortical

FIG. 4. Photomicrograph from a coronal section through the center of an intrastriatal striatal graft. Th section has been immunohistochemically stained to reveal DARPP-32 immunoreactivity, and the implan shows characteristic striatum-like, DARPP-32-positive, patches. Within these patches, the appearance c the DARPP-32-immunoreactivity, as well as of many other typical striatal markers, is very similar to th~ of the surrounding intact host striatum (H). The non-patch areas, on the other hand, are virtuall DARPP-32-negative and do not show any striatum-like features. The asterisks denote the graft-ho~ border, which is constituted by condensed bundles of the host internal capsule. Scale bar = 250 #n (Modified from Wictorin et al., 1989c.)

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FIG. 6. Photographs illustrating the afferent cortical input to the striatal grafts, as visualized through anterograde PHA-L-tracing from the frontal cortex. (A) Darkfield microphotograph of the distribution of PHA-L-immunoreactive terminals within a striatal transplant (T), after 15-20 injections of PHA-L into the ipsilateral frontal cortex. The dashed line labels the graft-host border. (B) Higher magnification of the square in A. Note the beaded, terminal-like appearance of the cortical afferents, which reach high densities foremost in peripheral graft portions. (C) Electron micrograph illustrating the typical morphology of a PHA-L-positive terminal bouton (asterisk), labelled from the frontal cortex, forming an asymmetric synaptic contact (small arrowhead) onto a spine(s) of a neuron within the transplant, d, dendritic shaft; H, host striatum; L, area of lesion-induced gliosis and packed myelinated fibre-bundles surrounding the graft. Scale bars: A and B = 100/tin; C = 0 . 4 0 p m . (Modified from Wictorin and Bj6rklund, 1989; Wictorin et al., 1989b.)

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INTRASTRIATAL STRIATAL TRANSPLANTS

areas (McGeorge and Faull, 1989), and the cortical projection neurons, which use glutamate as their transmitter, are found mainly in layer V, but also in layer VI and in supragranular layers. These neocortical afferents form asymmetric synaptic contacts foremost onto dendritic spines of the striatal neurons (Frotscher et al., 1981; Somogyi et al., 1981; Dub6 et al., 1988). The thalamic afferents to the striatum originate almost exclusively from the intralaminar nuclei and the parafascicular nucleus, and establish asymmetric synaptic contacts above all onto dendritic shafts of striatal neurons (Dub6 et al., 1988). A clearly different type of afferent input is provided by the dopaminergic neurons of the substantia nigra (pars compacta), which densely innervate the striatum, with a very rich collateralization of the fibres, and form symmetrical synaptic contacts onto both dendritic shafts and spines of the striatal neurons (Freund et al., 1984). As described in detail below, anatomical as well as physiological studies have indicated a remarkable ability of the host afferent fibre systems to innervate the grafts, with the cortical, thalamic and nigral inputs most carefully characterized, although evidence for inputs also from, e.g. the mesencephalic raphe and amygdala has also been presented. In our experiments, as schematically shown in Fig. 5, we have used a large number of different neuroanatomical techniques to visualize the afferent inputs to the implants. 5.1. AFFERENTSFROMTHE CORTEXAND THALAMUS The first evidence suggesting that host neocortical areas innervate the striatal implants came from the observations by Pritzel et al. (1986), who observed

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faintly retrogradely labelled cells in the ipsilateral frontal cortex after WGA-HRP-injections into the grafts. We then substantiated this evidence by injecting Fluro-Gold into the implants, which resulted in labelling of numerous cells in the frontal cortex with a normal distribution in the cortical layers, although even in the best cases the number of labelled cells amounted to only about one third of what was labelled after identical injections into a homotypic area of an intact striatum (Wictorin and Bj6rklund, 1989). We then examined the distribution of the cortical afferents within the grafts by way of anterograde tracing from the frontal cortex with Phaseolus vulgaris leucoagglutinin (PHA-L), and as illustrated in Fig. 6A and B, it was found that the cortical afferents densely innervate foremost the peripheral portions of the implants, and that the afferents have a beaded terminal-like appearance (Wictorin and Bj6rklund, 1989). Using the same anterograde tracing method (Wictorin et al., 1989b; Xu et al., 1989) the labelled cortical terminals were shown to form normal-looking asymmetric synaptic contacts onto neurons within the grafts, as shown in Fig. 6C. However, above all in the study of Xu et al. (1989) the postsynaptic distribution of the synaptic contacts was reported to be the most different in the grafts as compared to the normal host striatum, with only about 50% of the contacts onto spines in the grafts, whereas in the normal striatum the vast majority of the contacts are onto spines (over 90%). In our study 0Victorin et al., 1989b) we also found a somewhat lower proportion of contacts onto spines; 87% of the totally identified contacts onto spines, as compared to 98% in the host striatum. A thalamic input to the striatal implants was first suggested by the appearance of a few retrogradely

WGA-HRP

FIG. 5. Schematic drawing of the techniques used in our experiments to reveal the afferent connections from the host brain to the grafts. In brief, retrograde tracers (Fluoro-Gold, FG, or rhodamine-labelled latex beads, RLB) were injected into the grafts to retrogradely label host cells with terminals present within the grafted tissue. In other animals, the distribution of afferent fibres within the transplants were analyzed either by way of anterograde tracing from the frontal cortex (FCx) (with multiple injections of Phaseohis-vulgaris leucoagglutinin, PHA-L) or thalamus (Th) (with anterograde transport of wheatgermagglutinin horse radish-peroxidase, WGA-HRP) or by way of immunocytochemical detection of tyrosine hydroxylase (TH) (to reveal the dopaminergic afferents from the substantia nigra, SN) or serotonin (5-HT) (for afferents from the mesencephalic raphe, MR).

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labelled thalamic neurons, in one specimen, after the WGA-HRP injections into the implants (Pritzel et al., 1986). Again, as for the cortical afferents, these early findings from WGA-HRP-tracing were substantiated in the experiments with injections of either RLB or FG into the grafts, when numerous retrogradely RLB- or FG-labelled neurons were observed also in the thalamus after such tracer injections (Wictorin et al., 1988; Wictorin and Bj6rklund, 1989). The number of labelled neurons were in the order of 5-30% of what was labelled in control specimens after injections into intact striata, and it was very clear that injections into the peripheral portions of the grafts resulted in several-fold more labelled neurons as compared to injections into central portions of the grafts. The distribution of afferent thalamic fibres within the grafts was analyzed using anterograde transport of WGA-HRP from the ipsilateral thalamus, and this further evidenced that the thalamic afferents to the grafts are concentrated to peripheral graft portions (Wictorin et al., 1988). In addition, Xu et al. (1990) have recently analyzed also the thalamic input at the ultrastructural level, and found that normal-looking synaptic contacts are formed onto neurons in the grafts, although also in this case with a different distribution on synaptic targets, as compared to the normal striatum. 5.2. MONOAMINERGICAFFERENTS As mentioned above, Pritzel et al. (1986) demonstrated, using dopamine histofluorescence, that the host nigral afferents form dense patches within the grafts. Within these patches the fibres reach densities close to those found in normal striatum, whereas areas in between the dense patches contain no or only very few dopaminergic fbres. These observations were then followed by those of Clarke et al. (1988) who analyzed the terminals at the ultrastructural level using tyrosine hydroxylase (TH)-immunohistochemistry, and indeed, normal-looking, symmetric synaptic contacts were identified onto dendritic shafts and spines of Golgi-labelled medium-sized densely spiny neurons within the implants. The patches of nigral afferents were found to be most prominent in peripheral portions of the grafts, although they occurred also in more central portions (Pritzel et al., 1986). The experiments with injections of retrograde tracers (WGA-HRP, RLB, or FG) into the grafts provided evidence that these monoaminergic afferents are indeed derived from the host substantia nigra pars compacta (Pritzel et al., 1986; Wictorin et al., 1988; Wictorin and Bj6rklund, 1989), and the number of retrogradely labelled cells was sometimes around 50-75% of that obtained in the substantia nigra after identical tracer-injections into intact control striata (Wictorin and Bj6rklund, 1989). The major characteristics of the host dopaminergic input are illustrated in Fig. 7. With regard to host afferents from the mesencephalic raphe to the grafts, they have been evidenced using both retrograde tracing from the grafts (Wictorin et al., 1988; Wictorin and Bj6rklund, 1989) and immunohistochemical detection of serotonergic fibres (Wictorin et al., 1988). Their distribution was found to be even throughout the implants, without

any aggregation into dense patches, and with the density of the 5-HT-fibres approaching that normally found in an intact striatum, above all in the peripheral graft portions. 5.3. REGENERATIVERESPONSESOF ADULTTARGETDEPRIVEDCNS AXONS The presence of host afferent fibres and the actual formation of synaptic contacts within the striatal grafts is very interesting, since it demonstrates that also different types of adult CNS axons, which have been target-deprived, can show a regenerative response with several striking features when embryonic cells are implanted into their terminal regions. First of all the reaction of the target-deprived terminals appears to be very specific. For instance, the dopaminergic afferents from the substantia nigra specifically innervate densely just those areas which are striatum-like, i.e. that resemble their normal target area. This feature of the dopaminergic innervation, which was not shared by, e.g. the cortical, thalamic or serotonergic afferents, suggests that there are certain specific mechanisms guiding the dopaminergic axons, such that the implanted striatum-like regions are able to stimulate the growth of the dopaminergic terminals, whereas non-striatum-like regions, which are presumably, e.g. pallidal or cortical can not evoke such responses to the same extent. Indeed control grafts of cortical or cerebellar tissue, placed into the ibotenate lesioned striatum, did not become densely innervated by the host dopaminergic afferents (Wictorin et al., 1990a; Labandeira-Garcia et al., 1991). Similarly, in vitro-experiments have shown that fetal striatal tissue can stimulate dopaminergic fibres to grow (Denis-Donini et al., 1983), and to select and specifically innervate striatal tissue, and to avoid other tissue-types which are not their normal targets (Won et al., 1989). A second type of specificity shown by the ingrowing host afferents is the normal appearance at the ultrastructural level of the synaptic contacts formed onto the grafted neurons. Thus, the dopaminergic afferents form morphologically normal symmetric synaptic contacts onto the normal postsynaptic targets of medium-sized densely spiny neurons within the grafts (Clarke et al., 1988), whereas the cortical afferents form typical asymmetric contacts, which at least in our own study were mostly onto their normal postsynaptic targets (Wictorin et al., 1989b), although in the study of Xu et al. (1989) the postsynaptic distribution was more different. Secondly, it is of interest to assess the importance of a preceding lesion for the regenerative responses to occur. We studied this by comparing the host innervation of striatal grafts put into lesioned striata with other grafts which were implanted into intact brains, and found that the grafts put into the non-lesioned recipients were much smaller, did not increase markedly in size after implantation, intermingled less with the host, and received less host afferent innervation (Labandeira-Garcia et al., 1991). These results are consistent with findings from other analogous grafting models, with either intrahippocampal hippocampal (T6nder et al., 1989) or intrathalamic thalamic (Peschanski and Isacson, 1988) grafts, since also

FIG. 7. Photographs illustrating the afferent input to the striatal grafts from the host substantia nigra. (A) Coronal section through the centre of a transplant stained to reveal tyrosine hydroxylase (TH)immunoreactivity. The TH-positive afferents are concentrated to dense patches with fibre densities similar to that of the normal host striatum (H), surrounding the implants. This section is adjacent to the DARPP-32-immunoreacted section depicted in Fig. 4, and the near total matching of the DARPP-32-positive areas with the regions of dense TH-fibre staining is very clear. (B) Higher magnification of the square in A. (C) Detail from a section which has been reacted to simultaneously reveal both DARPP-32- and TH-immunoreactivities. Note the dense TH-positive innervation (black fibres) of the DARPP-32-positive (grey cell bodies) patch regions (p), and the very few TH-fibres present in the non-patch-regions (np). Scale bars: A = 250/~m; B = 100/zm; C = 25/am. (Modified from Wictorin et al., 1989c.)

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INTRASTRIATAL STRIATAL TRANSPLANTS

in those graft models, a preceding excitotoxic was found to increase the extent of host afferent innervation. The beneficial effects of the excitotoxic lesions are probably related above all to the creation of space necessary for the growth of the implanted tissue, and to the target deprivation of the host fibre systems, without damage to the axons. In fact it has been observed that the nerve endings in a lesioned-only area show signs of regenerative growth, which have been suggested to be growth-cone-like (Peschanski and Besson, 1987; Nothias et al., 1988). In addition, it has been documented that such lesions contain reactive astrocytes with neurotrophic properties (Lindsay et al., 1979), and increased levels of laminin (Liesi et al., 1984). Thirdly, another interesting feature of the host afferent ingrowth is the marked difference between the various host fibre systems (see Fig. 8 for a schematic overview). The monoaminergic afferents, such as the dopaminergic fibres from the substantia nigra, possess a remarkable capacity to innervate the implants, and to reach terminal densities within the striatum-like areas, which are similar to those observed in the normal striatum. On the other hand, more highly specialized afferents, from the frontal cortex and thalamus, are more restricted to peripheral portions of the grafts, and even in those areas only reach densities which are much lower than those observed in normal striatum. This difference in regenerative capacity between so-called "global systems" (cf. Sotelo and Alvarado-Mallart, 1987a), e.g. the dopaminergic afferents, which are highly collateralized within the striatum, and fibre systems of the so-called "point-to-point" type, e.g. the cortical or thalamic afferents, with more restricted projections, was also pointed out by the results of the above described retrograde tracing experiments. The number of retrogradely labelled cells after tracer-injections into the implants was much higher, and closer to levels obtained after injections into intact control striata, in the substantia nigra as compared to the frontal cortex or thalamus. Similar observations have come also from other experimental models. Thus, monoaminergic or cholinergic afferents have been shown to be especially capable of regeneration also in an adult brain (Bj6rklund and Stenevi, 1984), and for instance, Zimmer et aL (1985) have shown that cholinergic fibres can innervate intrahippocampal hippocampal grafts both in immature and adult recipients without preceding lesions, whereas more specialized afferent systems, such as the perforant path fibres or commissuro-hippocampal fibres, innervate grafts placed into intact animals only if the recipients are immature. Similar differences between global systems and point-to-point-fibres in the ability to innervate embryonic grafts have been documented also in, e.g. the model with fetal thalamic grafts implanted into the kainic acid lesioned adult thalamus (Peschanski et al., 1989). Although less able to innervate the fetal implants, it is clear from the striatal graft model, as well as from the experiments of T6nder et al. (1989) on homotypic hippocampal grafts and Sotelo and Alvarado-Mallart (1987b,c) on cerebellar implants, that also the more highly specialized afferents can indeed innervate such transplants. JPN 38/6---G

,

DA.afferents

n

~ ~'/

$°llT-afferents

FCx

-. _ s Cx-afferents

.~ _ s

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Th-afferents

FIG. 8. Schematic drawings showing the different patterns of innervation of the intrastriatal striatal grafts provided by the various host afferent systems. The dopaminergic (DA) afterents from the substantia nigra (sn) form dense patches of terminals with densities similar to those observed within the normal striatum, and the other monoaminergic projection, the serotonergic (5-HT) input from the mesencephalic raphe (mr), also reaches densities comparable to normal levels within large parts of the implants. On the other hand, the frontal cortical (FCx) and thalamic (Th) inputs to the grafts are concentrated above all to peripheral portions. 5.4. A FUNCTIONALHOST INNERVATIONOF THE TRANSPLANTEDNEURONS Apart from being a sort of in vivo-system for the characterization of regenerative abilities of adult CNS axons, the appearance of host afferents within the implants is of interest since it makes it possible that the implants are in some way regulated or influenced by the host brain. Indeed, direct evidence for a functional input from the frontal cortex has emerged from electrophysiological studies with stimulations of frontal cortical areas and subsequent recordings from cells within the implants. Thus, Rutherford et al. (1987) examined slice preparations and following stimulations of cortical regions found many similarities between the responses in the grafts and in the normal striatum, and also Xu et al. (1991), who performed in vivo-experiments, found electrophysiological evidence for a direct cortical input although the typical responses of neostriatal cells to cortical stimulations were greatly reduced or often absent in the sampled grafted cells. This could, according to Xu et aL (1991) reflect a relative sparsity in the synaptic input to grafted cells and a lack of convergence of inputs from different sources. From the observations in the above described anatomical experiments, with a much denser cortical innervation of more peripheral graft regions, one may speculate that the electrophysiological responses of the grafted neurons to cortical stimulations could vary to a great deal depending on where in the grafts the recording electrodes are placed. Apart from the cortical input, also the anatomically well documented nigral dopaminergic innervation of the implants has been investigated in a more functional way, and in view of the close to normal density of the dopaminergic afferents within the

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K. WICTORIN

striatum-like portions of the grafts (Pritzel et al., 1986; Clarke et al., 1988), and the high levels of dopamine D1 and D2 receptors within these regions (Isacson et al., 1987; Liu et al., 1990; Mayer et al., 1990) there is definitely a strong substrate for a direct functional influence from the host over the transplants through the dopaminergic projection. Indeed, Sirinathsinghji et al. (1988) could demonstrate, using the push-pull perfusion technique, that stimulations of the dopaminergic systems lead to increased release of GABA within the graft and the normal striatal projection areas. To understand the functional aspects of the graft-host integration it is most important to take into account the notable heterogeneity in the composition of the transplants and in the distribution of for instance the host dopaminergic afferents. Thus, more recently, we have studied the expression of the proto-oncogene c-fos in the implanted striatal cells following manipulations of the host dopaminergic afferents, by way of immunocytochemical detection of the protein product Fos, and correlated the changes in Fos-immunoreactivity with the distribution of striatum-like regions within the implants (Mandel et al., 1992). Since it is known that stimulations of dopamine DI receptors increase the expression of c-fos in normal striatal neurons, and thus result in increased production of Fos (Robertson et al., 1989), Fos-immunocytochemistry can be used as a way to monitor a functional influence of the dopaminergic afferent input over the grafted cells. That grafted striatal neurons can show increased Fos-immunoreactivity after manipulations of the dopaminergic system was previously shown by Dragunow et al. (1990), who injected haloperidol into grafted rats, and more recently in the experiments of Liu et al. (1991) with injections of cocaine resulting in a patchy upregulation of Fos-immunoreactivity in the grafts. In our experiments with administration of the dopamine-releasing drug amphetamine to the grafted animals, the number of Fos-immunoreactive neurons increased markedly in the striatumlike portions of the grafts, as in the normal striatum. This was determined in sections double-immunostained for both Fos and DARPP-32, and the specificity of the response and hence confinement to the striatum-like regions was also very clear in specimens with the dopaminergic input removed through a 6-OHDA-lesion of the ascending mesostriatal bundle and with injections of the dopamine agonist apomorphine, In those grafted animals, the dose of apomorphine was chosen so that it would only stimulate supersensitive receptors, which in this situation had been rendered supersensitive through the 6-OHDA-lesion, and the drug injections resulted in sharp increases in Fos-immunoreactivity specifically in the DARPP-32-positive regions. Thus, altogether the striatum-like regions of the grafts responded in a very similar way to what is seen in normal striatum, and the dopaminergic input to the grafted cells appeared to be functional. Altogether the indications of an actual functional influence of the host afferents on the grafts is of interest when discussing how the implants might exert their functional effects (see further below).

5.5. DEVELOPMENTOF THE AFFERENTINNERVATION Other interesting aspects of the afferent innervation of the grafts are the developmental time-course of the innervation and the mechanisms responsible for this graft-host integration. In the already mentioned study on the maturation of the striatal grafts, we found that the grafts develop radidly after implantation, with a time-course similar to that of the normal striatum in situ, and we found that the host afferent innervation develops in two, partially overlapping phases (Labandeira-Garcia et al., 1991). First, there is a period of graft growth and nonspecific intermingling of the implanted cells with adjacent areas of the surrounding host. The increase in volume is approximately 5-fold during the first 2-7 days post-grafting, and the expanding graft tissue becomes gradually mixed with the target-deprived host fibres. Secondly, from about 5-7 days after implantation there is a more specific innervation process with host afferent sprouting and formation of terminal-like patterns, during which different innervation patterns are formed by the various afferent systems (cf. Fig. 8). Due to the interesting specificity shown by the dopaminergic, TH-positive afferents, with a selective innervation of the striatum-like portions of the grafts, as identified, e.g. by DARPP-32immunocytochemistry, we focused much attention on this projection. Figure 9 depicts the development of the innervation from the substantia nigra, and illustrates the two overlapping phases, during which this afferent innervation is established.

stage I (2 days p.g.)

stage II (3.4 days p.g.) .~e~%~'o%°

o o~ne o ° *ma

stage III (5-7 days p.g.) FIG. 9. Schematic representation of the development of the TH-positive afferent innervation and gradual intermingling of the striatal implants with adjacent areas of the host striatum. In the drawings, small ovals represent implanted cells, and those that are black correspond to DARPP-32 positive ceils. The large black circles show the host myelinated internal capsule bundles, and the dashed lines indicate the needle tract. At 2 days post-grafting (stage I) the implanted tissue is mostly confined to the initial needle tract, although the cells have already started to mix with adjacent host fibres. At around 3-4 days post-grafting (stage II), the grafts have grown considerably, and clusters of cells have now clearly intermingled with the host fibres. Then, at 5-7 days post-grafting (stage III) dense patches with DARPP32-positive cells have developed, and thin axons have sprouted from the coarser TH-positive fibres, and have already developed into terminal-like networks within these striatum-like areas. (Modified from Labandeira-Garcia et al., 1991.)

INTRASTRIATALSTRIATALTRANSPLANTS

6. EFFERENTS TO THE HOST BRAIN

Whereas the striatum receives afferent inputs from widespread cortical and subeortical areas, the efferent projections from the normal striatum are concentrated onto a few output structures, with the major projections terminating in the globus pallidus and entopeduncular nucleus (in primates these two structures are referred to as external and internal segments, respectively, of the globus pallidus), and in the substantia nigra pars reticulata (see, e.g. Graybiel and Ragsdale, 1983, for review). These efferent pathways serve important motor functions, and constitute vital parts of the classical basal ganglia-loops. Gammaamino butyric acid (GABA) is thought to be a transmitter in all these projections, and in addition the striatal projection neurons contain different neuropeptides, most often co-localized with the GABA (see Graybiel, 1990, for a recent review). Thus, met-enkephalin is associated with the striatopallidal pathway and substance P with the striatonigral fibres. In addition, neurotensin and dynorphin have been identified in certain striatal projections neurons. Many of the striatal neurons that project to the substantia nigra also possess collaterals to the globus pallidus, whereas other striatal neurons terminate only in the globus pallidus or only in the substantia nigra (Loopuijt and van der Kooy, 1985). On their way caudally the striatal efferent pathways are myelinated and run inside the internal capsule bundles, and in for instance the globus pallidus, the striatal efferent fibres form symmetric synaptic contacts almost exclusively onto dendritic shafts (Chang et al., 1981). Some more recent findings on the detailed organization of these efferent projections are discussed in Fig. 14. 6.1. EFFERENTPROJECTIONS MAINLY TO THE HOST GLOBUS PALLIDUS

An analysis of possible efferent projections from the grafts into the host brain is important for two major reasons. First, axonal extension within an adult CNS environment has a general neurobiological interest, and the "in vivo graft-model" could help to elucidate potentials and constraints of such growth. Second, for the understanding of the functions of the striatal implants, the occurrence of efferent connections could be of crucial importance for the ways, through which the grafts could influence the host brain. It has indeed been demonstrated in many other models of neural transplantation that graft-derived fibres can extend into the surrounding host brain. This is clear, above all if the recipients are neonatal (see e.g. Lund et al., 1987), or if the grafts are placed directly into their normal target region, which has been denervated prior to grafting (see e.g. Bj6rklund and Stenevi, 1984). The situation for homotypic grafts such as the intrastriatal striatal ones is however different, since in this model the fibres must first grow for some distance through the adult host brain to reach their target regions. The actual development of efferent projections from the striatal grafts into the host brain was first suggested by the biochemical data of Isacson et al. (1985b), where the levels of the

625

GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) in the long-term IA-lesioned rats were found to be reduced in the globus pallidus and substantia nigra to 40% and 64%, respectively, as compared to non-lesioned controls. In implanted animals, the intrastriatal striatal grafts had increased the GAD-levels in the globus pallidus to about 90% of normal levels, however without any effects on nigral GAD-levels. Then, Pritzel et al. (1986) found in one case, with a WGA-HRP injection into an implant, some evidence for fibres extending into the adjacent globus pallidus, and to some extent also into the substantia nigra. These observations were followed by the already mentioned in vivo push-pull perfusion study of Sirinathsinghji et al. (1988), in which cannulae were placed in both the globus pallidus and substantia nigra, to monitor GABA release in intact control animals as well as in lesioned and lesioned-and-grafted rats. The ibotenic acid lesions had reduced GABA release in the globus pallidus and substantia nigra to 5% and 13% respectively of control level, and interestingly, in the grafted rats, GABA release was substantially restored in both these output structures. As schematically described in Fig. 10, we then initialized a more systematic anatomical analysis of the efferent projections from the grafts. Retrograde tracing with Fluoro-Gold (FG) from the globus pallidus labelled patches of graft neurons, which to a large extent matched the patches identified by, for instance, DARPP-32- or AChE-staining (Wictorin et al., 1989a,c). The majority of the retrogradely labelled cells were indeed found to be DARPP-32positive, and it thus appeared that the striatum-like portions of the implants were capable of extending out of the grafts into the adjacent globus pallidus. As shown in Fig. l l, this was further evidenced in experiments with deposits of PHA-L placed in the centre of the transplants (Fig. 11A), which visualized graft-derived fibres that extended caudally across the graft-host border and into the nearby globus pallidus (Fig. liB, C) (Wictorin et al., 1989a). These observations were made in several experimental animals, and in some specimens, labelled fibres could be traced as far caudally as into the entopeduncular nucleus, but no fibres could be found further caudally in, for instance, the substantia nigra. In the retrograde tracing experiments, other animals were injected with RLB or FG into the substantia nigra, and a few labelled cells were indeed identified within the implants. However, the number of cells labelled from the globus pallidus was 30-50 times greater. In our ultrastructural studies (Wictorin et al., 1990), we also examined the region of terminal staining in the rostral host globus pallidus, after the PHA-L-injections into the implants. We found that the graft-derived fibres formed morphologically normal synaptic contacts onto host pallidal neurons, i.e. symmetric synaptic contacts foremost onto dendritic shafts (Fig. liD). 6.2. TIME-CouRSE AND SPECIES-DIFFERENCES

In our cross-species experiments we addressed questions like the time-course of the efferent growth

626

K. WICTORIN PHA-

FG

RLB

t,°

MOUSE FETAL STRIATUM

HUMAN FETAL STRIATUM

MOUSE NEURONSPECIFIC ANTIBODY

B

HUMAN NEUROFILAMENTSPECIFIC ANTIBODY

C

FIG. 10. Schematic drawings of the various methods used in our experiments to reveal the efferent projections from the grafts into the host brain. (A) Our tract tracing experimentsincluded both injections of the anterograde tracer PHA-L into the grafts and injections of the retrograde tracers FG and RLB into the globus pallidus and substantia nigra, respectively. In other animals, cross-species grafting experiments were conducted. Thus, in some specimens(B) embryonic mouse striatum was implanted into rats, and the implants as well as graft-derived fibres were detected using a mouse-specificantiserum. (C) In other rats a similar cross-species approach was used, but in this case embryonic human tissue was used in combination with an antiserum which recognizes human but not rat neurofilaments.

and tissue-type-specificity. We thus implanted mouse fetal striatal tissue into ibotenic acid lesioned rats, and used the mouse neuron specific marker M6, which had previously been successfully used in studies on retinal grafts by R. Lund and his colleagues (e.g. Lund et al., 1985; Hankin and Lund, 1987). As illustrated in Fig. 12, this species-specific marker enabled us to see the graft itself, with no labelling of the surrounding host brain, and thus served as an excellent marker of fibres extending into the host (Wictorin et al., 1991). Already at 3-5 days after implantation, we could see some single efferent fibres, extending for about 0.1-0.2 mm from the caudal tip of the graft. At 8 days post-implantation dense fascicles of fibres were visible in several specimens,

and they were found running inside the white matter tracts of the host internal capsule, as far caudally as into the rostral globus pallidus. At 14 days post implantation, small terminal-like networks were present in the globus pallidus, and some single fibres were found also in the entopeduncular nucleus, but not further caudally. In long-term surviving animals (3-11 weeks post-grafting), the preterminal fibre staining was reduced but small terminal-like networks persisted in the globus pallidus, also at longer survival times. The outgrowth varied between different specimens, with some showing no or very little efferent growth. Additional evidence for the ability of the striatal grafts to extend fibres into the host brains came from

FIG. 11. Photographs demonstrating the efferent projections from the striatal transplants into the host brain, as revealed through anterograde Phaseolus vulgaris leucoagglutinin (PHA-L) tracing. (A) shows a typical injection site inside a transplant (T), with the dashed line indicating the graft-host border. H, host striatum. (B) Arrows point to PHA-L-labelled fibres present within the myelinated bundles of the host internal capsule, from a level just caudal to the transplant in A. (C) PHA-L-labelled terminals, with a notably beaded appearance in the host globus pallidus of the same specimen. (D) Electron micrograph showing a typical PHA-L-labelled terminal bouton in the host globus pallidus, forming a symmetric synapic contact (small arrowhead) onto a dendritic shaft (d) of a host pallidal neuron. Scale bars: A = 200/tm; B = 20 #m; C = 40 #m; D = 0.40 #m. (Modified from Wictofin et al., 1990a.)

627

FIG. 12. Photomicrograph of a sagittal section through a rat brain with an ibotenic acid lesion and an implant of mouse embryonic striatum. The section has been immunocytochemically reacted using a primary antiserum raised against mouse neurons, to reveal the grafts and the graft-derived fibres projecting into the host brain. In this particular case the graft is 8-days-old, and fibres can be followed as they project caudally in dense fascicles inside the host internal capsule fibre bundles, to reach the rostral globus pallidus (gp). co, corpus callosum; H, host; ic, internal capsule; lv, lateral ventricle; T, transplant; Th, thalamus. Scale bar: 500/~m. (Taken from Wictorin et al., 1991.)

628

FIG. 13. Illustrations of the efferent growth from grafts of human ganglionic eminences placed into the ibotenic acid lesioned rat striatum. (A) shows in a schematic sagittal drawing the efferent growth of fibres into the host brain from a 23-week-old graft (hatched area). (B-D) Darkfield micrographs of coronal sections from another grafted rat with human neurofilament-positive fibres; (B) projecting into and through the globus pallidus (gp), and (C) at the level of the subthalamic nucleus (sth), and (D) the substantia nigra (sn). cc, corpus callosum; cp, cerebral peduncle; cpu, host caudate-putamen; ep, entopeduncular nucleus; ic, internal capsule; opt, optic tract; sth, subthalamic nucleus; sn, substantia nigra; th, thalamus; v, lateral ventricle. Scale bars: A = 2 mm; B, C = 200t~m; D = 100 #m. (Modified from Wictorin et al., 1990b.)

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our studies on grafts of human fetal forebrain cells placed into the ibotenic acid-lesioned rat striatum (Wictorin et al., 1990b). In these experiments, the ganglionic eminences were dissected from the brains of 8-10-week-old (post-menstrurn) human embryos from routine elective abortions, and following implantation, the grafts and graft-derived fibres could be identified immunocytochemically by using antibodies recognizing human but not rat neurofilaments. Interestingly, fibres were found to extend for very long distances into the host brain. As shown in Fig. 13, also in these im~plants did the fibres extend along the host internal capsule, into and through the globus pallidus, and in this case large numbers of fibres continued further caudally. Thus, human neurofilament-positive fibres were found in the substantia nigra (about 5-6 mm from the grafts), and some fibres continued along the cerebral peduncle, and could be traced as far caudally as in the spinal cord (i.e. about 20 mm from the grafts). 6.3. SPECIFICITYANDGROWTHALONGWroTE MATTER TRACTS With regard to the specificity of the efferent growth, it was mentioned previously that the axonal growth was specific in the sense that it followed the normal trajectories of striatal output neurons. In addition, we addressed another aspect of specificity by grafting tissue taken from different brain regions into the ibotenic acid lesioned striatum. Thus, in the experiments with mouse-to-rat grafts, we placed either neocortical or cerebellar tissue into the lesioned area, and found that both these tissue types developed well and showed many characteristics of their respective regions. The neocortical tissue projected densely along the host internal capsule bundles into the host brain, for about 3 mm, whereas the cerebellar implants did not extend any significant projections into the host (Wictorin et aL, 1991). The same observations were made with the human-to-rat grafts when control tissue from cerebellum was used, and only very sparse projections could be detected into the host (Wictorin et al., 1990b), whereas on the other hand, control grafts of human neocortical or spinal cord tissue readily extended fibres into the host rat brains (Wictorin et al., unpublished observations). A very interesting aspect of the growth of the graft-derived fibres into the host brain was the choice of growth trajectory. In all experiments it was obvious that the fibres were specifically directed caudally, along the host internal capsule bundles. In crosssections through these bundles, just caudal to the implants, the graft-derived fibres were extending as tight fascicles inside the bundles (see e.g. Fig. 11B), and at the ultrastructural level, we could see that the fibres were indeed myelinated (Wictorin et al., 1990a). This apparent growth along adult myelinated tracts, and actual myelination of the extending fibres is very interesting to consider in the context of the well documented inhibitory influence of adult white matter and myelin on the extension of neurites (see Schwab, 1990, for review). The special properties of the immature grafted neurons could possibly be explained by a relative insensitivity to the growth inhibiting influence of an adult CNS environment, at

631

least during a limited period of development. The long extension of fibres from the human tissue implants could then be explained by their more protracted development and longer growth phase. Alternatively, one could speculate that the human cells could grow for such long distances since the length of the projections that they would form/n situ in a human brain is about 10 times longer than the corresponding projections in the rodent brains.

7. FUNCTIONAL BEHAVIOURAL EFFECTS 7.1. REPORTED BEHAVIOURALEFFECTS As already mentioned Deckel et al. (1983) and Isacson et al. (1984) were the first to report positive functional effects of the intrastriatai striatal grafts. These early findings have since been followed by many other reports, suggesting that the grafts can have functional effects on several different kinds of behaviour (see also Norman et al., 1989b; Dunnett, 1990, for recent reviews). Thus, the striatal grafts have, first of all, been found to ameliorate simple motor disturbances, such as spontaneous locomotor hyperactivity (Deckel et al., 1983, 1986a, b, 1988; Isacson et al., 1984, 1986; Sanberg et al., 1986, 1989b; Giordano et al., 1988), amphetamine-induced locomotor-hyperactivity (Sanberg et aL, 1986), changes in haloperidol-induced catalepsy (Isacson et al., 1985a; Giordano et al., 1988), and amphetamine- or apomorphine-induced rotational behaviour (after unilateral lesions) (Dunnett et al., 1988; Norman et aL, 1989a). In more advanced behavioural tests, the grafts have been shown to ameliorate more complex motor disturbances, i.e. deficits in skilled paw reaching (Dunnett et al., 1988; Montoya et al., 1989; Valouskova et al., 1990). Furthermore, the implants have been reported to influence cognitive disturbances, i.e. deficits in spatial alternation learning (Deckel et al., 1986a; Isacson et al., 1986). 7.2. FUNCTIONALMECHANISMS These observed behavioural effects of the implants have raised interesting questions regarding the mechanisms by which the grafts might exert their functional effects. Overall, grafts placed into the CNS have been shown to be able to influence the host in several different ways (see Bj6rklund et al., 1987, for review). With regard to the intrastriatal striatal grafts one could imagine atrophic influence on the host brain, with prevention of further degeneration following the IA-lesion and also some stimulation of regeneration of severed host neurons. As a second mechanism, the grafts could influence the host brain by simple diffusion of transmitters or other substances. On a more complex level there could be an actual interaction with the recipient brain through established anatomical connections. From other graft models it is well known that embryonic tissue implants can exert trophic effects on, for instance, target-deprived adult neurons. Thus, for instance in models with neural grafts into the spinal cord (Bregrnan and Reier, 1986) or the neocortex (Sofroniew et al., 1986), grafts have been

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shown to counteract lesion-induced cellular loss or shrinkage. One could think of a similar effect exerted by the striatal implants, which could possibly reduce secondary degenerative processes in the structures that project to the lesioned area, at least when implantation occurs early after the excitotoxic lesion. Moreover, the implants could possibly influence the striatal output areas in atrophic fashion, since it has been shown that infusions of the GABAergic agonist muscimol can to a large extent prevent neuronal celt loss, that otherwise occurs in the substantia nigra pars reticulata after striatal excitotoxic lesions (Saji and Reis, 1987). Trophic effects of embryonic implants in this model have also been suggested by, e.g. Giordano et al. (1990), who observed some behavioural effects also when tectal or cortical tissue was implanted into the kainate lesioned striatum. In the context of atrophic influence of the grafts on the host brain, there are also several reports suggesting that intrastriatai transplants of either fetal striatai tissue or of other cell types or trophic factor producing cell lines, prior to lesioning, can prevent, or at least ameliorate the neuronal degeneration which results from striatal infusions of excitotoxic amino acids (Pearlman et al., 1990; Schumacher et al., 1991). When it comes to actually replacing the neurons and related transmitters, which have been lost due to the lesion, one can on the simplest level think of transplants acting as local drug delivery systems or biological pumps. Thus, in the rat model of Parkinson's disease, it has been shown that apart from embryonic grafts which form synaptic contacts onto host neurons and synaptically release dopamine, also grafting of, for instance, genetically modified fibroblasts that simply secrete L-DOPA into the striatum can exert functional effects (Wolff et al., 1989; Horellou et al., 1990). For the striatal grafts, one could similarly think of the embryonic grafts influencing the host brain through a diffuse release of G A B A and other transmitters into the adjacent host brain, and indeed local infusions of GABA-receptor activating drugs into the brain, have been shown to be able to influence locomotor activity (Mogenson and Nielsen, 1983). However, from a theoretical point of view, it is difficult to imagine that deficits in behaviours, which are normally dependent on very advanced and specific anatomical connections could be significantly improved by much simpler mechanisms than through a re-establishment of some of the lost anatomical connections. Indeed, in the light of the reported intricate anatomical development of the grafts, and the establishment of at least partial anatomical connections with the surrounding host brain, there is reason to believe, that the grafts exert their behavioural effects, at least in part, by way of the established afferent and efferent axonal projections. First of all, findings that the behavioural effects are dependent on the homotypic placement of the grafts, i.e. in the lesioned striatum (Isacson et al., 1986; Sanberg et al., 1989b), and on a continuous presence of the graft tissue (Sanberg et al., 1989b), suggest that some direct interaction with the striatal circuitry is crucial. The effects of the excitotoxic lesion of the striatum, i.e. the removal of the inhibitory input to the globus pallidus and the substantia nigra, can be

measured as increases in glucose utilization in these two striatal output structures (Isacson et al., 1984). Behaviourally, this can be observed as an increase in the locomotor activity of the lesioned animals, and the reported graft-mediated functional effects on this behavioural deficit are likely to depend on some kind of reinstatement of the inhibitory control over the striatal output structures. Thus, as mentioned above Isacson et al. (1985b) could measure graftinduced recoveries in G A D activity in the globus pallidus of lesioned-and-grafted animals. Moreover, Sirinathsinghji et aL (1988) reported graft-induced recoveries in GABA-release in both the globus pallidus and the substantia nigra, using the push-pull perfusion technique. Of course, these findings could at least partly be explained by a diffuse release of GABA and other transmitters from the implants into the adjacent host brain, since as already mentioned, local infusions of GABA-receptor activating drugs into the brain, have been shown to be able to influence locomotor activity (Mogenson and Nielsen, 1983). However, the actual existence of efferent projections to at least the rostral globus pallidus, with synaptic contacts onto the host pallidal neurons, suggest that the re-innervation could play a vital role in the behavioural effects of the implants. Evidence that also the afferent anatomical connections to the implants are of importance for the functional effects, comes from the observations that also a complex behaviour such as the skilled paw reaching is influenced by the implants (Dunnett et al., 1988; Montoya et al., 1989; Valouskova et al., 1990). The fine movements of the paw reaching task are known to be dependent on an intact nigrostriatal dopaminergic innervation (Whishaw et al., 1986). As already mentioned, indeed the actual functional influence of the dopaminergic afferents on neurons in the grafts has been evidenced by the way in which the levels of the protein Fos can be altered in the striatal grafts, just as in the intact host striatum, after manipulations with different dopaminergic drugs (Liu et al., 1991; Mandel et al., 1992). Moreover, that the afferent fibres form actual synaptic contacts onto the grafted neurons has been shown for the dopaminergic afferents (Clarke et al., 1988) and the afferents from the frontal cortex (Wictorin et al., 1989b; Xu et al., 1989) and thalamus (Xu et al., 1990). As described above, the functionality of the afferent inputs from the cortex and thalamus has been evidenced in electrophysiological studies, where stimulations of host neurons have led to electrophysiological responses in neurons inside the grafts (Rutherford et al., 1987; Xu et al., 1991). 7.3. PARTIAL RECONSTRUCTION OF STRIATAL CIRCUITRY

Thus, our hypothesis is that the grafts can indeed exert at least some of their functional effects by way of established anatomical connections, and this is further discussed in Fig. 14. It can of course be pointed out that the anatomical integration is certainly not complete, with for instance only a partial re-establishment of the efferent projections from the lesioned part of the striatum. However, there are examples that certain projections of the CNS can

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(b)

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STRIATAL IBO LESION CORTEX

CORTEX

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6LOBUS PALLIOUS

6LOBUS PALLIDUS

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~

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STRIATAL TRANSPLANT CORTEX

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~E) MOTOR OUTPUT

FIG. 14. Schematic and simplified drawings of some of the major features of the normal basal ganglia circuitry (A) and proposed changes in the excitotoxically lesioned rats (B) and in lesion-and-grafted specimens (C). Recent findings about the intricate connections and complexity of transmitters and receptors in the basal ganglia have provided some new ideas about the organization and interconnections of the involved structures (see, e.g. Alexander and Crutcher, 1990; Gerfen et al., 1990). Thus, the efferent projections from the striatum can be divided into two major pathways, with partly different transmittercontents, receptors and termination areas. First, there is an indirect pathway, which mainly uses GABA and enkephalin as transmitters, and provides an inhibitory projection to the globus pallidus, which in turn provides an inhibitory input to the substantia nigra pars reticulata. These neurons receive an excitatory input (glutamate) from the frontal cortex, and an inhibitory influence from the nigral dopaminergic system, mediated via dopamine D2 receptors. Second, there is a direct pathway from the striatum to the substantia nigra, with GABA and substance P as major transmitters, and which is under the control of the excitatory cortical input and an excitatory input from the nigrostriatal dopaminergic terminals, mediated via dopamine D1 receptors. The postsynaptic cells of the substantia nigra pars reticulata in turn project (inhibitory pathway) on to the thalamus, which projects (excitatory pathway) to the neocortex, which then provides the motor output. (B) In this drawing the consequences of a striatal ibotenic acid (ibo) lesion (shaded area) are shown. The degenerated neurons of the indirect and direct pathways are denoted by dashed lines. The net result of the lesion can be thought of as an increased inhibition of the neurons of the substantia nigra pars reticulata, which eventually leads to an increased motor output (here symbolized by thicker arrows). (C) The striatal transplant (grey) receives major inputs from the frontal cortex and the substantia nigra, and projects to the adjacent globus paUidus. One can therefore suggest that the grafts can reinstate some of the inhibitory input onto the globus pallidus, and mediate its behavioural effects via the indirect pathway, and that the activity of the grafted neurons are under some kind of control from the cortical and dopaminergic inputs. Indeed, as discussed in the text, there are several recent reports suggesting a functional integration of the transplants. maintain behavioural functions although they are much reduced. One example of this is the nigrostriatal projection, in which more than 90% of the dopaminergic neurons can degenerate before any severe impairments emerge (Bernheimer et al., 1973; Stricker and Zigmond, 1976). This does of course not rule out the possibility that the grafts can also influence the host brain in other ways, such as through trophic mechanisms or through simple diffusion of transmitters into adjacent host areas. 8. N E W A P P R O A C H E S Apart from a further characterization of the anatomy and functional effects of the intrastriatal striatal

neuronal transplants into rodents, two major new approaches which seem very promising and relevant for this grafting model, have recently been introduced. First, a primate model with excitotoxic lesions of the striatum has been developed by Isacson, Hantraye and colleagues (Isacson et al., 1989; Hantraye et al., 1990), with infusions of ibotenic acid or quinolinic acid into the monkey striatum resulting in morphological and behavioural deficits closely resembling those observed in HD-patients. In addition, Isacson and co-workers have implanted embryonic rat neurons into the lesioned primate striatum, and observed positive behavioural effects of

K. WICTOR~N

634

these cross-species implants (Isacson et al., 1989). Recently, Helm et al. (1990b) also reported preliminary evidence for survival and anatomical differentiation of grafts of embryonic monkey striatum placed into adult ibotenic acid lesioned monkeys. Another major new approach is the use of genetically manipulated or immortalized cell lines for transplantation. There are many problems with the use of fetal ganglionic eminence-tissue for transplantation, such as the already mentioned difficulties to dissect pure striatal tissue, without contamination of, e.g. cortical, pallidal or amygdaloid tissue. The presence of these other tissue types in the grafts leads to a smaller proportion of, for instance, GABAergic projection neurons in the implants, and to elaborate intrinsic connections inside the grafts, which are likely to reduce the amount of fibres that project out into the host. Therefore, it would be of great value if purer striatal tissue could be obtained. Maybe this problem could be solved through cell sorting or through other methods, which would increase the proportion of striatal cells. When thinking of a potential use of fetal neuronal tissue transplantation in patients suffering from Huntington's disease, there are also the difficulties in obtaining embryonic human tissue, and the ethical problems with using tissue from aborted fetuses. One approach to overcome these problems, which is currently being investigated in many laboratories, is the use of neuronal or nonneuronal cells which have been genetically manipulated to produce, e.g. a trophic factor or transmitter-related substance (see Gage et al., 1991, for recent review). The use of fibroblasts, which can be grown in tissue cultures to large numbers, and which produce N G F or L-DOPA has already been tried in the rat model of Parkinson's disease, with very promising results (Wolffet al., 1989; Horellou et al., 1990). With regard to the intrastriatal striatal graft model, there have been some initial reports on the use of genetically manipulated cells, with insertions of the GAD-gene into fibroblasts (Cheng and Zhou, 1989; Chen et al., 1990). As mentioned earlier, it has been reported that local infusions of GABA or GABAergic drugs can actually influence the behaviour of the experimental animals. One can thus speculate that, e.g. GABA-producing fibroblasts could influence at least some aspects of the behavioural deficits. However as discussed above, for a more complete behavioural restoration it is likely that at least a partial anatomical reconstruction is necessary. In the intrastriatal striatal graft model, perhaps the most viable new approach could be the use of immortalized neuronal cell lines, which can be expanded in vitro, and then implanted into the brain (Cattaneo and McKay, 1991). Recent reports from such experiments suggest that for instance, immortalized hippocampal cell lines can first be expanded in vitro, and then transplanted into the brain, with signs of differentiation into hippocampal cell types (Renfranz et al., 1991).

9. CLINICAL PERSPECTIVES The many similarities between the excitotoxin rat model and the real Huntington's disease (HD),

together with the positive morphological and behavioural reports from animal experiments with transplantation, of course highlights the question of whether transplantation could actually be of benefit in humans suffering from HD. That neural transplantation in general is a potential therapy for neurodegenerative disorders has been encouraged by the gradual development of transplantation trials in patients with Parkinson's disease (PD) (see Lindvall, 1989, 1991, for reviews). After many years of careful animal experiments in the PD rodent model (see Brundin and Bj6rklund, 1987, for review), it was shown that human fetal ventral mesencephalic tissue can survive and give rise to positive functional effects when grafted into rats with an animal model of PD (Strfmberg et al., 1986; Brundin et al., 1986). Recently, Lindvall et al. (1990) presented the first strong evidence that human fetal ventral mesencephalic tissue can indeed also survive implantation into the human brain, and improve motor functions in patients with severe PD. This, of course raises hope that also human fetal striatal tissue could be successfully implanted into the human brain. Our initial experiments with grafting of human fetal striatal tissue into rats with excitotoxic striatal lesions have shown that also this tissue type can indeed survive in the rodent brain and extend fibres into the host (Wictorin et al., 1990b). There are however several reasons why transplantation of human fetal striatal tissue into patients suffering from HD should be approached with great caution. First of all, there are the already mentioned ethical problems associated with the use of human embryonic tissue (see Hoffer and Olson, 1991, for discussion). Although it seems clear that today human embryonic tissue is the only realistic tissue source available to use, there is the already mentioned ongoing and promising development of alternative tissue sources, such as genetically manipulated cells or immortalized cell lines, and it is important that such development continues in parallel with the research on the use of material from aborted fetuses. There are also other more practical problems with the use of embryonic human tissue, such as those of obtaining enough tissue for transplantation, and of performing a reproducible tissue dissection, from the often severely disrupted forebrains of the aborted fetuses. A completely different problem concerns to what extent the rat excitotoxin model is a good model of HD when developing and evaluating the potential use of intracerebral grafting in this disorder. In the excitotoxin model, the degeneration of the striatum occurs very rapidly, whereas in HD it occurs gradually over time, and it is moreover possible that the as yet unknown causative mechanism of HD will also kill any implanted neurons. Although many unsolved problems remain, there are nevertheless several strong reasons in favour of a clinical application of neural transplantation in HD. First of all, there is today no other effective treatment for this severe disorder, and secondly the application of transplantation in Parkinson patients have not pointed to any negative effects of intracerebral grafting of embryonic tissue into the human brain (Lindvail et al., 1990). Animal experiments should continue to create a more solid experimental basis, and factors

INTRASTRIATALSTRIATALTRANSPLANTS like the timing of the grafting in the disease process should be evaluated. Probably the best would be to graft relatively early in the HD patients, before too much degeneration has occurred within the striatum, as well as in other brain areas, such as the neocortex. The excitotoxic primate model of HD by Isacson and colleagues (Isacson et al., 1989), should be of great interest in optimizing grafting parameters, since it appears to provide a closer correlate to the real disorder.

10. CONCLUDING REMARKS Experiments with neural transplantation into the adult mammalian central nervous system is of major importance due to the poor intrinsic regenerative capacity of the brain and spinal cord, and neural grafting can be viewed both as a potential therapy in various neurodegenerative disorders as an experimental tool to investigate regenerative mechanisms. Taken together, the experiments summarized in this review, on the anatomical organization of the intrastriatal striatal grafts, have demonstrated a striking ability of embryonic implants to at least partly reconstruct an excitotoxically disrupted circuitry. The implanted embryonic striatal cells develop rapidly after implantation and mature into a striatum-like structure. Most of the various nuclei that normally provide a dense input to the striatum also innervate the striatal grafts, with partly specific distributions, and with interesting differences in regenerative capacifies between different types of afferent systems. The grafted neurons, in turn, project into the host brain, and show a remarkable ability to grow for at least some distance through adult myelinated fibre tracts, to reach adjacent normal target areas. Continuing research in this and other models of neural transplantation could lead to the development of new methods to improve regeneration in the adult CNS. Acknowledgements--The author would like to thank the

Medical Faculty in Lund, the Crafoord Foundation, "Kungliga Fysiografiska S~illskapeti Lund", and the European Science Foundation for financial support of the experiments carried out by the author and his collaborators.

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