Host dopaminergic afferents affect the development of DARPP-32 immunoreactivity in transplanted embryonic striatal neurons

Nemsciance Vol.48,No.4, pp.857-869, 1992

0306-4522/92 $5.00 + 0.00 Pergamon Press Ltd 8 1992 IBRO

Printed in Great Britain

HOST DOPAMINERGIC AFFERENTS AFFECT THE DEVELOPME~ OF DARPP-32 IMM~OREACTIVITY IN TRANSPLANTED EMBRYONIC STRIATAL NEURONS B. DEFONTAINES,M. PFSCHANSKI and B. ONTENIENTE* INSERM CJF 91-02, U.F.R. de M&de&e, Rue du General Sarrail, F-94010 Creteil, France Abetmet-Homotopic transplantation provides an interesting way to observe the relationships between develop~g cells and ingrowing host afferents. We have performed a complete and selective elimination of the mesostriatal dopaminergic system in adult rats to observe the influence of its absence on the development and chemical differentiation of embryonic striatal cells. Cell suspensions from striatal primordia of 14-&day-old embryos were transplanted into host striata that were (if neuron-depleted by kainic acid (control group) or (ii) deprived of dopamine by ti-hydroxydopamine prior to the neuronal depletion by kainic acid (experimental group). The expression of dopamine- and adenosine ~,5’-monophosphate-relate phosphoprotein (DARPP-32) by transplanted cells was observed in correlation with their innervation by host dopaminergic afferents which in turn were identified by tyrosine hydroxylase immunohistochemistry. Observations were made between four days and three months after tr~splan~tion. Four days after transplantation, no i~unor~ctivity for DARPP-32 was observed in transplants of control animals despite the presence of tyrosine hydroxylase-immunopositive fibers growing from the host to discrete cell clusters in the transplant. DARPP-32-labeled cells appeared soon afterwards. Six days after transplantation they displayed varying intensities of immunoreaction, ranging from just detectable to normal levels and were specifically targeted by developing tyrosine hydroxylase-immunopositive fibers. The number of DARPP-3Zlabeled cells increased rapidly and they formed increasingly compact clusters. Fourteen days after transplantation and afterwards, all the DARPP-32-labeled cells displayed an intensity of immunoreaction and a distribution comparable to that observed in long-term transplants. Transplants in the experimental hosts displayed the same organization and developmental features as the control transplants with the exception of DARPP-32 labeling which was not detected before eight days after transplantation. Ten days after transplantation, the distribution and intensity of DARPP-32 labeling was similar to that observed at six days in the control group. The evolution of DARPP-32 labeling after IO days in the experimental group paralleled that observed six days post-transplantation and beyond in the control group. Dopaminergic mesostriatal host afferents are able to provide developing cells in grafted striatal tissues with normal innervation very rapidly. Despite this rapidity, the innervation does not seem to have any trophic influence on the general development of the transplant but does affect the onset time of the expression of neurochemical markers that are directly related to its synaptic function. The expression of DARPP-32 after one week in the absence of dopaminergic afferents suggests that the

influence of extracellular dopamine supplied by other brain structures or extracellular mechanisms unrelated to dopaminergic systems or a genetic program are able to compensate for the lack of an adequate presynaptic element.

On the basis of their diffuse distribution and early onset in emb~ogenesis, it has been suggested that monoaminergic systems are involved in the development of several structures in the central nervous However, experiments performed system. 21,24*2536285639 in vivo have been largely inconclusive due to difficulty in manipulating selective factors in Malawian embryos, and most of the data have been obtained after removal of the monoaminergic systems in the early postnatal period. Alternative experimental paradigms have been developed for structures where histogenesis *To whom correspondence

should be addressed. dopamine- and adenosine 3’,~-monophosphat~~~lat~ pho~hoprotein; -IR, immunoreactive; RA, kainic acid, 60HDA, 6-hydroxydopamine; PB, phosphate buffer; PBST, Triton X-lOOcontaining phosphate-bread saline; TH, tyrosine hydroxylase.

Abbreviations:DARPP-32,

is largely a prenatal event. Such paradigms include the use of simplified in vitro or invertebrate models and the transplantation of specific embryonic tissues. In vitro investigations on various kinds of vertebrate and invertebrate tissue have provided the strongest evidence that neurotransmitters influence neuronal morphology.24*26Homotypic neurot~nspiantation of embryonic tissue into adult hosts constitutes another approach since it allows the observation of developing embryonic neurons in conditions that are closer to normal than in vitro studies. It also simplifies the manipulation of environments factors, allowing specific deafferentation. Adult monoamine@ central systems are able to provide an extensive innervation to embryonic or newborn transplanted tissue. 5,6,20,30~32,33,3~~40.44,50This sprouting is extremely rapid with mo~hological features similar to those observed during normal

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axogenesis”.” j5 and leads to the formation of synaptic contacts.6 Therefore, transplanted striatal neurons may develop under conditions closely related to normal embryogenesis. We have used this paradigm to investigate the extent to which the striatal dopaminergic system is involved in the maturation of striatal neurons and their acquisition of a specific identity. We have chosen as a phenotypic marker the D,-related phosphoprotein, dopamineand adenosine 3’,5’-monophosphate-regulated phosphoprotein (DARPP-32)18 which is specific to medium spiny neurons in the striatum.‘* The onset of immunohistochemical detection of DARPP-32 in the rat striatum has been dated at embryonic day 14.” We have therefore used striatal primordia from 14-I 5-day-old embryos, dated by crown-rump length. The progress of the immunohistochemical expression of DARPP-32 in embryonic striatal neurons has been followed in the presence or absence of the host dopaminergic nigrostriatal afferents. EXPERIMENTAL

PROCEDURES

Lesion surgery A total of 84 adult female Sprague-Dawley (Charles River, France) rats (250-280g) were divided evenly into experimental and control groups. All surgical steps were carried out under chloral hydrate anesthesia (400 mg/kg). Surgical procedures were performed on three series of 28 animals each. In each series, all the animals received an injection of kainic acid (KA; Sigma, 7.5pg in 0.15~1 of saline) in the right striatum (coordinates A = 0.8 from bregma, L = 1.5 from medial suture and H = 5 from pial surface, tooth bar 3.3 mm below the interaural zero). One month before, half of the animals of each series had received an i.p. injection of desipramine (25 mg/kg, Sigma), followed 45 min later by two injections of 6-hydroxydopamine (6-OHDA; Sigma, 2 x 4 pg in 1 ~1 of saline containing 0.1% ascorbic acid) aimed at the medial forebrain bundle and the ventral tegmental area (coordinates from 4). Animals treated with KA only were considered as the control group and animals with 6-OHDAand KA-lesions were considered as the experimental group. All the animals were supplemented with a subcutaneous injection of 20ml of 5% glucose for four days after the KA lesion. Transplantation Two weeks after the KA injection, experimental and control animals in each series received an intrastriatal injection of the same cell suspension prepared from striatal primordia at 14-15 days’ gestation (fetal crown-rump length of 13-l 5 mm). Striatal primordia were dissected and incubated in a saline solution containing 0.6% D-ghCOSC and 0.1% trypsin for 20min at 37°C. The striatal pieces were further mechanically dissociated and the final cellular concentration adjusted to 100,000 cells/pi. Each host received 2~1 of the cell suspension.

VI d.

Four, six, eight. IO. i4. 21 ~lrld 30 dab,:. JI ~CYI:;IIP plantation in each series, pairs of annnals from both K4lesioned and 6-OHDA~-KA-lesioned groups and processed fbr immunohistochemistr\ hydroxylase (TH) and DARPP-72.

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immunohistochemi.vtrr Animals were perfused intracardially with 300 ml 01‘ J’!,, paraformaldehyde, 0.5% glutaraldehyde and 0.10/b picric acid in 0.1 M phosphate buffer (PB: pH 7.4). After 24 h 01’ additional fixation, brain slabs containing the striatum and, for 6-OHDA-treated animals, the mesencephalon. were cryoprotected in 30% saccharose for 12 h, frozen. and cut on a cryostat at 20pm. Sections were washed for several hours in PB containing 0.9% sodium chloride and O.?0<, Triton X-100 (PBST) and either processed for perovidase immunohistochemistry using a streptavidin--biotin technique or (one out of four) stained with Cresyl Violet. Free-floating alternate sections were incubated overnight at 4°C with either polyclonal anti-TH (l/2000. J Bw. France) or monoclonal anti-DARPP-32 (l,‘lS,OOO) aniibodies’7.3” in PBST. Subsequent steps included incubations for 1 h in the corresponding biotinylated secondary antibodies (l/250, Vector), 30 min in peroxidase-labeled streptavidin-biotin complex (I/400, Vector) and IS min in 3,3’-diaminobenzidine (Sigma, 0.025% in PB containing 0.001% H,O,). Each step was followed by a 30-min wash in PBST. Sections including the mesencephalic dopammergic nuclei were processed only for TH immunohistochemistry in order to verify the extent of 6-OHDA lesions The sections were mounted on gelatin-coated glass shdes and counterstained with Cresyl Violet to allow hlstological examination. In some cases, photographs were taken either after removal of the Cresyl Violet staining or with the WC of a dark-blue filter.

RFSL LTS

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of the neurotoxins

TH immunohistochemical inspection of the KAlesioned area in control and experimental groups revealed an increase in the size of labeled fibers and varicositics that was evident during the first two weeks after transplantation (four weeks post-KA lesion). In 6-OHDA-lesioned animals. the destruction of the entire dopaminergic mesostriatal projection system was established by the absence of TH-immunoreactive (-IR) cells in both the substantia nigra and ventral tegmental area. After one month, the only remaining TH-IR fibers observed in the striatum ipsilateral to the lesion were scattered straight fibers. Administration of 6-OHDA before the KA lesion had no apparent effect on intrastriatal DARPP-32 labeling in the host since labeled neurons were present in intact striatal tissue (Figs 4A, 5B).

Fig. 1. Cresyl Violet-stained sections showing the development of striatal transplants grown in a KA-lesioned host striatum in the presence (A,C,E) or absence (B,D,F) of host nigrostriatal a!I’erents. (A.B) Four days after transplantation, grafts contain two compartments: clusters of densely packed small cells strongly labeled by Cresyl Violet (star) and more loosely packed larger cells (asterisk). (C,D) Fourteen days after transplantation the two compartments are still visible with an increase in somatic size of the loosely packed cells. (E.F) Thirty days after transplantation. the smaller densely packed cells have disappeared and loosely packed ones have reached their maximal somatic size. Scale bar = 9Opm.

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Observation of the Cresyl Violet -stained sections

Transplants were easily recognized on Cresyl Violet-stained sections in contrast to the neurondepleted area of the host striatum. Transplant survival was similar in KA- and 6-OHDA-KA-lesioned hosts with transplants in both groups displaying similar morphological features and organization (Fig. lA,C,E and B,D,F). Four-day-old transplants (T4) showed a lobular organization characterized by two distinct types of compartments that differed in cell size, density and intensity of Cresyl Violet staining (Fig. IA-B). One compartment contained a high density of small cells strongly labeled with Cresyl Violet. The other compartment contained less densely packed and larger cells with a lighter Cresyl Violet staining. Some cell clusters were found outside the body of the transplant and were completely surrounded by host tissue. Maintaining a lobular organization, the compartments containing large cells progressively increased in size while the compartments with small densely packed cells decreased. The latter completely disappeared between 22 and 30 days post-transplantation (Fig. lE,F). Cells lightly stained by the Cresyl Violet progressively increased in size, in parallel with a decrease in cellular density (Fig. lC-F). Development of the tyrosine hydroxylase -immune reactive innervation in transplants of the control group

At T4, TH-IR fibers had already entered the transplants. They were found exclusively among compartments of cells lightly stained by Cresyl Violet, avoiding compartments of densely packed and strongly Cresyl Violet-stained cells. TH-IR fibers were mainly coarse with enlarged varicosities (Fig. 2A,B). Thinner fibers with smaller varicosities

were also observed, mainly in cell clusters which had extended outside the graft and were completely surrounded by host tissue (Fig. 2C,D). Other cell clusters, just adjacent to the ones receiving numerous fibers, contained only rare TH-IR fibers at their periphery (Fig. 2C,E). The same features were observed at T6, with a great increase in number of TH-IR fibers (Fig. 3A). Both thin and coarse fibers were seen in the center of the graft. This increase enhanced the detection of two groups of cell clusters: one invaded by a rich network of developing TH-IR fibers distinct from the other that contained few or no fibers. Both types of fibers (thick and thin) were still observed at T8 (Fig. 3B), while at TlO thick fibers were only occasionally observed (Fig. 3C) and had disappeared at T14 (Fig. 3D). Few changes were observed after T14 in the organization of TH-IR fibers provided by the host, except for a progressive increase in fiber density (Fig. 3E). Scattered TH-IR cell bodies were present within the transplants (see Fig. 2). At T4, these neurons were very intensely labeled and had already developed a neuritic arborization. They were no longer observed after TlO. DARPP-32 immunoreactivity in transplants control group

of the

At T4, no DARPP-32-IR was observed within transplants of the control group (Fig. 4A). At T6, DARPP-3ZIR neurons appeared within the transplant among groups of differentiated neurons. They displayed various intensities of labeling, from just-detectable to levels comparable to that observed in normal host tissue (Fig. 4B). Strongly immunoreactive neurons were packed in small clusters of

Fig. 2. TH-immunoreactive fibers growing into striatal transplants four days after transplantation. (A) Fibers sprouting (arrows) toward the graft (G) are observed from the host parenchyma (H). (B) Most of the TH-IR fibers are thick and exhibit large varicosities (arrow). Thinner fibers with smaller vaticosities (arrowhead) are also observed. (C--E) A high density of thin tibers is observed in some of the transplanted cell clusters (asterisks) which have extruded from the main bulk of the graft and are completely surrounded by host tissue (H). Adjacent clusters of transplanted cells (open circle) have no or few (arrow in E) TH-IR fibers. Scale bars = l05pm in A; IOpm in B; 30pm in C: 20pm in D; 25pm in E. Fig. 3. Progressive growth of hosts’ TH-IR afferents into striatal transplants. (A) Six days after transplantation both coarse (arrows) and thinner (arrowhead) fibers are present in some of the transplanted cell clusters, avoiding other adjacent clusters (open circles). (B) Eight days after transplantation coarse fibers (arrow) are less frequent while thin ones (arrowheads) increase in number. (C-E) Ten, I4 and 30 days after transplantation. Thin TH-IR fibers progressively establish a dense network in discrete cell clusters. Open circles indicate areas of the transplant with no or few TH-IR fibers. Scale bars = 39pm for A-D: I IOpm for E. Fig. 4. Evolution of the DARPP-32 immunolabeling in striatal transplants grown in the presence of the host’s nigrostriatal innervation. (A) Four days after transplantation no labeling is observed in the graft (G). Example taken in a transplant located at the border of the KA lesion, close to normal striatum (H) in order to compare with the normal level of immunoreaction. (B) Six days after transplantation strongly immunoreactive cells (arrows) are observed packed in clusters while other clusters contain less intensely labeled cells (arrowheads) or no labeled cells at all (open circle). (C) Eight days after transplantation the variations in intensity of immunolabeling are less frequent and the dendritic arborization of DARPP-IR cells begins to obscure the observation of individual cells (arrow). (D,E) From IO days after transplantation DARPP-IR cells tend to form rounded clusters distinct from unlabeled surroundings (open circle). This clear-cut separation is kept at longer survival times (E. 30 days after transplantation) Scale bars = .50/rm in A and B: 30pm in C: 40,tm in I>: 95 !~rn in F

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Dopaminergic afferents and striatal transplants five to 10 cells, while larger groups contained cells of different intensities of immunoreaction intermingled with unlabeled ones. Cell clusters that did not contain any DARPP-32-IR neurons were also present (Fig. 4B). At T8, variations in intensity of immunolabeling had almost disappeared (Fig. 4C). The increase in the number and the dendritic development of strongly labeled cells made the observation of individual cells difficult in some cases. However, more loosely packed DARPP-3ZIR cells were still frequent. At TlO, individual DARPP-32-IR cells were rare. Development of the dendritic arborization resulted in the formation of compact and darkly immunolabeled zones contrasting with the completely unlabeled ones (Fig. 4D). Subsequent stages showed no dramatic evolution of the DARPP-M-IR except for an increase in size of the labeled zones (Fig. 4E). At each time-point, similar results were observed in all four animals in each series of the control group. DARPP-32-immunoreactivity experimental group

in transplants of the

No DARPP-32-IR was observed at T4 and T6 in transplants of the 6-OHDA-KA-lesioned hosts (Fig. SB). Staining of adjacent sections for TH confirmed the virtual absence of TH-IR fibers in the transplants (Fig. SA). The first DARPP-32-IR cells were observed at T8, with a low intensity of immunolabeling (Fig. SC). At TlO (Fig. 5D), the pattern of DARPP-3ZIR was similar to that observed at T6 in the KA-lesioned control group, with cells of varying intensities of immunolabeling. Clusters of unlabeled cells were apparent. Differences in intensities of immunolabeling disappeared at T14 (Fig. 5E) although DARPP-32-IR cells seemed to be less packed than in transplants of the control group. At T30, DARPP-32-IR labeling was similar in the transplants of the control and experimental groups (Fig. 5F). At T8 and TlO, the intensity of DARPP-32 immunolabeling in the transplants was more variable in the experimental group than in the control group. In two cases, the DARPP-32Gmmunolabeling at T8 was too faint to be considered specific. DARPP-32IR cells were never observed before T8. Differences

between individual after T14.

animals were no longer detected

DJSCUSSION

Homotopic intrastriatal transplants receive a dense dopaminergic innervation from the host substantia nigra and ventral tegmental area.6,20*40*50 Extra-striatal tissue is unavoidably included in these transplants.% The dopaminergic innervation provided by the host shows a strict specificity for striatal parts of the transplants, and collaterals to the adjacent nonstriatal tissue are sparse. A previous report23 has shown that the development of this innervation begins during the first days following transplantation. In this context, homotypic intrastriatal transplantation becomes a good means of observing the relationships between developing transplanted neurons and ingrowing host afferents. We made a complete and selective lesion of the mesostriatal dopaminergic system in adult rats and observed the repercussions of its absence on the development and neurochemical differentiation of embryonic transplanted cells. Our observations confirm that adult dopaminergic fibers are able to enter and grow within transplanted embryonic striatal tissue very rapidly. They furthermore show that sprouting dopaminergic fibers are able to select their specific striatal targets immediately. The absence of dopaminergic afferents does not alter the general development and organization of the graft but does delay the normal onset of a neurochemical synthesis that is directly related to the dopaminergic receptive function of striatal cells. Both of these results support the argument that there is an early dialog between the transplant and the host that is sustained by reciprocal trophic and tropic interactions.2 Influence of striatal transplanted cells on the growth of host dopaminergic aflerents

The sprouting ability of adult central monoaminergic systems has been demonstrated by their remarkable response to axotomy.‘v” Transplantation studies have shown that a similar sprouting ability is expressed without major axonal injury in the presence of embryonic tissues.3,6*20,32-“,35~~~~,49~~ As suggested by morphological observations, similar mechanisms may underly both types of response and involve a

Fig. 5. Evolution of the DARPP-32 immunolabeling in intrastriatal transplants grown in the absence of the host’s nigrostriatal innervation. (A) Virtually no TH-immunoreactive fibers are observed within the transplant. (B) Six days after transplantation no DARPP-32 immunoreactivity is observed within the transplant. Example taken in a transplant located at the border of the KA lesion in order to show the normal level of DARPP-32 immunoreaction (H). Star indicates red blood cells. (C) Eight days after transplantation, low levels of DARPP-32 labeling are seen in some of the transplanted cells (arrow). (D) Ten days after transplantation, clusters of labeled cells with varying intensities of immunoreaction are seen, close to clusters with no labeled cells at all. (E) Fourteen days after transplantation all the labeled cells have reached their normal level of immunoreactivity. (F) Thirty days after the transplantation the distribution of the DARPP-32 immunoreactivity is similar to that observed in transplants grown in the presence of host TH-IR afferents. Scale bars = 80 pm in A; 73 pm in B-E; 65 pm in F.

return to an immature developmental stage with re-expression of the corresponding genes.B”.f’The role of the transplant in such a mechanism would thus parallel that exerted by developing cells on their afferents during embryogenesis. Previous results have shown the rapidly ingrowing monoamincrgic afferents to be thick in diameter with enlarged varicosities.33.34However, the KA lesion also appears to affect the ingrowing fibers in the striatum because fibers with this same mo~hology have been seen in the KA-lesioned area of striatum in this work and previousiy.7.30 This is not the case for the monoaminergic input to the KA-lesioned thalamus.3i.3J Since a similar sprouting is observed in striatal transplants without previous neuronal depletion.2” it appears that in the striatum both the neurochemical and morphological modifications resulting from the iesion’9 as well as the presence of transplanted fetal neurons can stimulate the sprouting process in dopaminergic# afferents. Co-culture of embryonic mesencephalic and striatal neurons has provided evidence for targetdependent stimulation of dopamine uptake or of neurite elongation.‘“*‘6,4’,42 This effect has been attributed to a ceILcell interaction rather than to a diffusible trophic factor because it was also observed when cells were replaced by particulate membrane fractions from young animals4’ This conclusion is chalIeng~ by other studies showing that soluble striatal extracts from embryonic day 14 (Ei4) striata can dramatically enhance neuritic growth and differentiation of mesencephalic neurons.47 Because there was a difference between the ages of the donors in these two experiments, the growth of dopaminergic af!ferents toward embryonic striatai tissue could actually be the result of two consecutive events. One, during the first stages of re-axogenesis, would be led by diffusible factors and the other, at a slightly later stage, would depend upon cell-cefl membrane interactions. In vitro neurite elongation stops when dopaminergic growth cones encounter a target’ and subsequent differentiation into a nerve terminaf or a synaptic varicosity takes place only if the target is adequate.9,‘9 Information relevant to the issue of target-dependent stimulation can be found in this work because there was a selective interaction of TH-positive fibers from the host with two different types of transplanted neurons. Cresyl Violet staining revealed the existence of two cellular compartments in transplants during the first three weeks of development. Cell clusters with a high density of small cells strongly Labeled with Cresyl Violet progressively disappeared while loosely packed lightly stained cells progressively increased in size. Clusters of small densely packed cells have been described as immature proliferative areas progressively giving rise to loosely packed groups of differentiating neurons and glial cells.23The evolution of these two clusters was comparable in the control and experimental groups. Growing ~~-positive

fibers were observed exclusively among d#eer~cntlatcd neurons, avoiding clusters of immature. still prolifcr ating cells. In addition. proliferation of TH-positivt, afferents lasted for three weeks. which corrcspondcd to the disappearance of undifferentiated cells in !he transplants. Both of these observations buppori ihe idea that molecules with tropic activity could he released by transplanted ceils in the first stages of post-mitotic differentiation. The production cli‘~eh factors would srop when neurons reach ;i more advanced developmental stage or receive their inncrvation and shift to a class of trophic molecules that allows the maintenance of synaptic contacts. The early selection of specific targets among the available transplanted cells further emphasizes the specificity of the factor released by striatal cells for the dopaminergic afferents. Among factors liberated by striatal tissue is SDNF ~str~atal-derived neurntrophic factor’) and possibly bFGF.” but no L%dence for their liberation during embryogenesis or by transplanted cells is yet available.

It has been suggested that catecholamines play several roles in brain development (for review see Ref. 24), including control of cell proliferation,” migration*’ and neuritic growth.‘2.2RIn control animals, transplanted striatai cells were immediately in close contact or proximity to host dopaminergic systems. After six days. tong before the proliferation of transplanted cells was over, the most central part of the transplant had already received some dopaminergic fibers. Respite the highly probabie availability of dopamine in their envjronme~t, transplants of the control group displayed similar characteristics to transplants of the 6%OHDA-KA group in terms of overall size and organization. In addition, the proportion of DARPP-32-labeled cefls in long-term transplants of both groups was similar, indicating an equal survival of striatal tissue. A more refined quantitative analysis might have revealed some smalf effects but reliable quantification is not possible with immunohistochemical techniques. in any case these findings support the conclusion that host mesostriatal afferents do not have a specific influence on the general development of transplanted striatal cells. The role oJ‘dopaminergic aferents in the expression of

L?ARPP-32 Despite the presence of dopaminergic fibers, no immunolabeling for DARPP-32 could be detected in the first days following transplantation in the KAlesioned hosts. This cannot be related to a normal developmental state since some embryonic striatal cells begin to synthesize immunohistochemicaliy detectable levels of DARPP-32 from day 14,‘” i.e. before their dissection for transplantation. The lack of DARPP-32 may be related either to a Mure in sensitivity of the ~mmunoh~stochemicai t~chn~que

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Dopaminergic afferents and striatal transplants used, or to the transplantation procedure, including dissociation of the extra-cellular matrix, impairments in the membrane integrity by trypsin and axotomyinduced death of the more differentiated cells which have not yet received mesostriatal input. According to Foster et al.,” the onset of immunoreactivity to DARPP-32 in the striatum at El4 precedes the arrival of dopaminergic striatal afferents at their target site by two days. They concluded that the expression of DARPP-32 is unrelated to the presence of presynaptic elements. In fact, other investigators report dopaminergic striatal fibers from El4 to El4 l/2.3’*45946 If this is the case, the onset of DARPP-32 would correlate exactly with the appearance of dopaminergic elements resulting in the possibility of an interaction between the pre- and postsynaptic elements. This work cannot address this question because of the lack of DARPP-32 labeling in transplants of the KA-lesioned hosts in the first four days following transplantation. Despite the apparent lack of a specific effect on the development of the transplants, the absence of dopaminergic input did result in a delay in the onset of DARPP-32. In the striatum, DARPP-32 is characteristic of medium spiny neurons38 where it acts as a third messenger (cyclic AMP-dependent) for dopaminergic D, receptorsU However, chronic blockade of dopaminergic receptorsI or chemically induced or pathologic’4,43 dopaminergic striatal denervation in

adults does not modify DARPP-32 immunolabeling or the phosphorylation state. This suggests a surprising independence of the phosphoprotein from synaptically released dopamine. Similar data have been obtained in vitro, with a normal content of DARPP-32 observed in striatal cells after four weeks of monoculture in a dopamine-free medium.“s5’ In addition, DARPP-3Zpositive patches were present in long-term transplants grown in 6-OHDAdeafferented hosts.23 Our results are consistent with these observations, since normal levels of DARPP-32 immunoreactivity were observed both in the adult host striatum surrounding the KA-lesioned area and, after 10 days, in transplants of the 6-OHDA-lesioned animals. However, the delay observed in the expression of the phosphoprotein in the absence of dopaminergic afferents supports a developmental regulation of the protein by these afferents. The subsequent appearance of DARPP-32 after one week would suggest that either extracellular dopamine supplied by other brain structures, extracellular mechanisms unrelated to dopaminergic systems, or genetic programming, are able to compensate for the lack of adequate presynaptic element.

Acknowledgements-We

thank Prof. S. Rapisardi for his helpful advice and criticism and Dr J.-A. Girault for kindly providing the anti-DARPP-32

antibody.

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Neurosci. 10, 509-516.

3. Bregman B. S. and Reier P. J. (1986) Neural tissue transplants rescue axotomized rubrospinal cells from retograde death. J. camp. Neural. 244, 86-95. 4. Brundin P., Strecker R. E., Widner H., Clarke D. J., Nilsson 0. G., Astedt B., Lindvall 0. and Bjiirklund A. (1988) Human fetal dopamine neurons grafted in a rat model of Parkinson’s disease: immunological aspects, spontaneous and drug-induced behaviour, and dopamine-release. Expl Brain Res. 70, 192-208. 5. Castro A. J., Tiinder N., Sunde N. A. and Zimmer J. (1988) Fetal necortical transplants grafted to the cerebral cortex of newborn rats receive afferents from the basal forebrain, locus coeruleus and midline raphe. Expl Brain Res. 69, 613-622.

6. Clarke D. J., Dunnett S. B., Isacson O., Sirinathsinghji D. J. S. and Bjorklund A. (1988) Striatal grafts in rats with unilateral neostriatal lesions-I. Ultrastructural evidence of afferent synaptic inputs from the host nigrostriatal pathway. Neuroscience 24, 791-801. 7. Coyle J. T. and Schwartz R. (1976) Lesion of striatal neurones with kainic acid provides a model for Huntington’s chorea. Nature 263, 244-246. 8. Dal Toso, Giogi O., Soranzo C., Ferrari G., Favaron M., Benvegnd D., Presti D., Vicini S., Toffano G., Azzone G. F. and Leon A. (1988) Development and survival of neurons in dissociated fetal mesencephalic serum-free cell cultures. I. Effects of cell density and of adult mammalian striatal-derived neuronotrophic factor (SDNF). J. Neurosci. 8, 733-745. 9. Denis-Donini S., Glowinski J. and Prochiantz A. (1983) Specific influence of striatal target neurons on the in oitro outgrowth of mesencephalic dopaminergic neurites: a morphological quantitative study. J. Neurosci. 3, 2292-2299. 10. Di Porzio U., Daguet M. C., Glowinski J. and Prochiantz A. (1980) Effect of striatal cells on in vitro maturation of mesencephalic dopaminergic neurons grown in serum-free conditions. Nature 288, 370-373. 11. Ehrlich M. E., Rosen N. L., Kurihara T., Shalaby I. A. and Greengard P. (1990) DARPP-32 development in the caudate nucleus is independent of afferent input from the substantia nigra. Deul Brain Res. 54, 257-263. 12. Felten D. L., Halhnan H. and Jonsson G. (1982) Evidence for a neurotrophic role of noradrenaline neurons in the postnatal development of rat cerebral cortex. J. Neurocytol. 11, 119-135. 13. Foster A. G., Shultzberg M., Hijkfelt T., Goldstein M., Hemmings H. C. Jr, Ouimet, C. C., Walaas S. I. and Greengard P. (1987) Development of a dopamine- and cyclic adenosine 3’,5’-monophosphate-regulated phosphoprotein (DARPP-32) in the prenatal rat central nervous system, and its relationship to the arrival of presumptive dopaminergic innervation. J. Neurosci. 7, 1994-2018.

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