Striatal Dopaminergic AfferentsConcentrate in GDNF-Positive PatchesDuring Development and in Developing

Intrastriatal Striatal Grafts

ELENA LOPEZ-MARTIN,1 HECTOR J. CARUNCHO,2

JANNETTE RODRIGUEZ-PALLARES,1 MARIA JOSE GUERRA,1

AND JOSE LUIS LABANDEIRA-GARCIA1*1Department of Morphological Sciences, University of Santiago de Compostela,

Galicia, Spain2Department of Fundamental Biology, University of Santiago de Compostela, Galicia, Spain

ABSTRACTGlial cell line-derived neurotrophic factor (GDNF) has potent trophic action on fetal

dopaminergic neurons. We have used a double immunocytochemical approach with antibodiesthat recognize GDNF and tyroxine hydroxylase (TH) or the phosphoprotein DARPP-32, tostudy the developmental pattern of their interactions in the rat striatum and in intrastriatalstriatal transplants. Postnatally, at one day and also at 1 week, GDNF showed a patchydistribution in the striatum, together with a high level of expression in the lateral striatalborder, similar to that observed for the striatal marker DARPP-32 and also for TH. In theadult striatum, there was diffuse, weak immunopositivity for GDNF, together with wide-spread expression of DARPP-32-positive neurons and TH-immunoreactive (TH-ir) fibers. In1-week-old intrastriatal striatal transplants, there were some GDNF immunopositive patcheswithin the grafts and although there was not an abundance of TH-positive fibers, the ones thatwere seen were located in GDNF-positive areas. This was clearly evident in 2-week-oldtransplants, where TH-ir fibers appeared selectively concentrated in GDNF-positive patches.This pattern was repeated in 3-week-old grafts. In co-transplants of mesencephalic andstriatal fetal tissue (in a proportion of 1:4), TH-ir somata were located mainly at the borders ofareas that were more strongly immunostained for GDNF, and TH-ir fibers were also abundantin these areas and were found in smaller numbers in regions that were weakly positive forGDNF.

These results demonstrate that GDNF-ir is coincident with that for TH and DARPP-32,and suggest that GDNF release by fetal striatal neurons both in normal development and indeveloping striatal grafts may have not only a trophic but also a tropic influence on TH-irfibers and may be one of the factors that regulate dopaminergic innervation of the striatum.J. Comp. Neurol. 406:199–206, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: neurotrophic factors; neuronal transplants; basal ganglia; rat

Glial cell line-derived neurotrophic factor (GDNF) hasrecently been cloned (Lin et al., 1993) and it has alreadybeen extensively shown to exert a trophic effect on fetalmesencephalic dopaminergic neurons both in vivo and invitro (Lin et al, 1993; Beck et al., 1995; Bowenkamp et al.,1995, 1997; Hudson et al., 1995; Kearns and Gash, 1995;Sauer et al., 1995; Tomac et al., 1995a, 1995b; Gash et al.,1996; Winkler et al., 1996; Choi-Lundberg et al., 1997).GDNF is a distant member of the transforming growthfactor-b superfamily (for reviews, see Lindsay and Yanco-poulos, 1996; Unsicker, 1996) which, among other brain

areas, is highly expressed in the developing striatum andthen decreases to reach adult levels (Schaar et al., 1993;Stromberg et al., 1993; Springer et al., 1994; Choi-

Grant sponsor: XUGA; Grant sponsor: The Spanish CICYT.*Correspondence to: Jose Luis Labandeira-Garcıa, Facultad de Medicina,

Dept. Ciencias Morfologicas, 15705—Santiago de Compostela, Galicia,Spain. E-mail: cmlaband@uscmail.usc.es

Received 25 March 1998; Revised 19 October 1998; Accepted 29 October1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 406:199–206 (1999)

r 1999 WILEY-LISS, INC.

Lundberg and Bohn, 1995; Nosrat et al., 1996; Suvanto etal., 1996; Pochon et al., 1997; Trupp et al., 1997). Inaddition, GDNF is also widely distributed in nonneuronaltissues indicating that its functional role is not restrictedto the nervous system (Moore et al., 1996; Nosrat et al.,1996; Pichel et al., 1996; Sanchez et al., 1996; Suvanto etal., 1996). Interestingly, GDNF knockout experimentshave shown that nigral fibers are capable of reaching thestriatum in the absence of GDNF, but the animals die ataround the first day after birth (Moore et al., 1996; Pichelet al., 1996; Sanchez et al., 1996).

Therefore, it was not possible to study the maturationpattern of striatal dopaminergic afferents in these ani-mals, as the tyroxine hydroxylase (TH) immunoreactivityreaches levels similar to those of adults, at 3 or 4 weeksafter birth (Broaddus and Bennett, 1990).

GDNF has been shown to enhance and promote thesurvival of fetal neural transplants in different areas of thenervous system (Rosenblad et al., 1996; Trok et al., 1996;Granholm et al., 1997; Sautter et al., 1998). In thestriatum, GDNF augments not only the survival andgrowth of nigral dopaminergic grafts, but also their func-tional capability (Rosenblad et al., 1996; Granholm et al.,1997; Sautter et al., 1998). In addition, co-transplantationof striatal and mesencephalic fetal tissue enhances theefficacy of grafts when compared with nigral transplantsalone (De Beaurepaire and Freed, 1987; Yurek et al., 1990;Constantini et al., 1994).

Intrastriatal striatal grafts are interesting experimen-tal models which allow the study of the anatomical integra-tion of fetal neurons in lesioned central nervous system ofadults, and have also been proposed as a possible therapeu-tic approach for Huntington’s disease (DiFiglia et al., 1988;Graybiel et al., 1989; Wictorin et al., 1989a, 1989b; Laban-deira-Garcıa et al., 1991, 1994; Liste et al., 1997). In fact,striatal grafts implanted in the excitotoxically lesionedstriatum, grow, establish connections with the host tissue,and ameliorate the functional deficits induced by thelesion (Deckel et al., 1983; Isacson et al., 1984; Dunnet etal., 1988; Labandeira-Garcıa et al., 1991, 1994, 1995;Labandeira-Garcıa and Guerra, 1994). Detailed studies ofthe afferent innervation of intrastriatal striatal grafts bythe host tissue have shown a well defined time-coursepattern of dopaminergic innervation of the graft that isrestricted to the striatal-like patches of the graft (i.e.,DARPP-32- immunopositive), suggesting the release ofspecific factors responsible for this selectivity (Wictorin etal., 1989a, 1989b; Won et al., 1989; Labandeira-Garcıa etal., 1991).

In this work, we have used a double immunocytochemi-cal approach to demonstrate that GDNF expression fol-lows a developmental pattern similar to DARPP-32 or TH;that striatal dopaminergic (i.e., tyroxine hydroxylase im-munoreactive) terminals specifically innervate intrastria-tal striatal graft areas that produce the highest levels ofGDNF. Furthermore, we have also shown that in co-transplants of mesencephalic and striatal fetal tissue (in aproportion of 1:4) the dopaminergic cells and fibers are alsoassociated with GDNF-positive areas.

MATERIALS AND METHODS

Experimental design

A total of 35 Sprague-Dawley rats were used in thisstudy. All experiments were carried out in accordance with

Principles of laboratory animal care (NIH publication No.86–23, revised 1985), and also in accordance with theprotocols of the animal care committee at the University ofSantiago de Compostela. Groups of five rats were perfusedpostnatally, at day 1 and at day 7 for developmentalstudies, as was a group of five adult rats weighing about250 g. A second group of 15 female adult rats receivedunilateral intrastriatal injections of ibotenic acid (seebelow), and 10 days later, a fetal striatal cell suspensionwas injected in the same area. Rats from this group werekilled at 1, 2, or 3 weeks postgrafting. Finally, in a thirdgroup of five female adult rats, unilateral lesions weremade with intrastriatal injections of ibotenic acid (seebelow). Ten days later, these rats received an injection of amix of mesencephalic and striatal cell suspensions (in aproportion of 1:4) in the same area, and were killed 2weeks postgrafting. All surgery was performed underequithesin (3ml/Kg i.p.).

Excitotoxic lesion and transplantation

A total of 14–16 µg of ibotenic acid (10 µg/µl in 0.1 Mphosphate buffer, pH 7.4) were injected into the rightstriatum, at three injection sites: (I) A 5 10.2, L 5 3, V55.5; (II) A 5 10.2, L 5 3, V 5 4; (III) A 5 11.5, L 5 2.5, V 54.6 (A 5 anterior from Bregma, L 5 lateral from Bregma,V 5 ventral from dura; tooth bar at 22.3; all coordinates inmm). Ten days post-lesion, the rats received intrastriatalinjections of cell suspensions prepared from striatal primor-dia 14–15 days of gestation (E 14–15); or well from bothstriatal primordia (E 14–15) and ventral mesencephalon(E 13–14) for co-transplantation experiments. The lateralganglionic eminences or mesencephalon were dissectedout and incubated in 0.1% trypsin (Sigma, St. Louis, MO),0.05% DNase (Sigma), and DMEM (Gibco, Faisley, Scot-land) for 20 minutes at 37°C. Afterwards, the tissue wasrinsed in DNase/DMEM and mechanically dissociated toproduce a milky cell suspension. This cell suspension wascentrifugated at 600 rpm for 5 minutes, and the superna-tant was carefully removed and re-suspended in 0.05%DNase/DMEM to the final volume required. Approxi-mately one million viable cells (estimated by acridineorange/ethydium bromide) were administered to each ratat three injection sites: (I) A 5 10.2, L 5 3, V 5 4.5; (II) A 510.6, L 5 2.7, V 5 4.5; (III) A 5 11.5, L 5 2.5, V 5 4.7. (SeePakzaban et al., 1993; Olsson et al., 1995; Dunnett andBjorklund,1997, for details).

Immunocytochemistry

Rats were anesthetized with chloral hydrate (400 mg/Kg) and perfused transcardially with a solution of 4%paraformaldehyde in 0.1 M phosphate buffer, pH 7.4.Brains were carefully dissected out, cryoprotected in thesame buffer containing 20% sucrose, and cut into 40-µm-thick sections with a freezing microtome. Sections wereprocessed for GDNF, TH, or DARPP-32 immunostainingas follows. Samples were first pre-incubated with a block-ing solution containing 10% normal serum in PBS (phos-phate-suffered saline) with 1% BSA (bovine serum albu-min) and 0.2 Triton X-100, and sections were then incubatedovernight at room temperature with the correspondingprimary antibody diluted in PBS-BSA (a rabbit polyclonalantibody anti-GDNF [Santa Cruz Biotechnology, SantaCruz, CA], a rabbit polyclonal antibody anti-tyrosine hy-droxylase [Peel-Freez Biologicals, Rogers, Arkansas], anda mouse monoclonal antibody anti-DARPP-32 [a generous

200 E. LOPEZ-MARTIN ET AL.

gift from Drs. P. Greengard and E.L. Gustafson]). Theantibody dilutions used were: 1:150 for GDNF, 1:500 forTH, and 1:20,000 for DARPP-32. The sections were thenincubated for 90 minutes at room temperature with thecorresponding biotynilated secondary antibody diluted1:100, and then for another 90 minutes with avidin-biotin-peroxidase (ABC, Vector, Burlingame, CA, 1:100). Finally,the labeling was revealed with 0.04% hydrogen peroxideand 0.05% 3,3’-diaminobenzidine (DAB).

Other sections were processed for double immunolabel-ing (see Labandeira-Garcıa et al., 1991, for details) of THand GDNF, and some co-transplant sections were immuno-labeled for TH and DARPP-32. Briefly, TH was labeled asabove, but with DAB-nickel sulphate to develop the reac-tion so that the precipitated product was black. Afterwashing in PBS, the sections were incubated in blockingsolutions containing increasing concentrations of avidinand biotin. The samples were then incubated with theGDNF (or DARPP-32) antibody solution, followed by thecorresponding biotynilated secondary antibody. Detectionof the GDNF or DARPP-32 expression was carried outwith DAB alone, giving a brown precipitate. Controlexperiments omitting primary antibodies showed a lack ofimmunoreactivity (ir).

RESULTS

GDNF expression in the normaldeveloping striatum

At postnatal day 1, GDNF-ir was observed in welldefined, irregular patches throughout the striatum, andmore intensely labeled regions were also seen in thelateral striatal border. The GDNF intensely stained areaswere also strongly positive for TH (Fig. 2A,B). At postnatalday 7 there was diffuse staining of the striatum that wasstronger in some irregularly shaped patches and in thelateral border, whereas, for example, the cortical or palli-dal immunolabeling was very faint (Fig. 1). It was difficultto discern individual immunopositive somata or dendritesin the patches, however it was possible to identify someimmunopositive astrocytes in areas outside the patches, aswell as some weakly immunopositive somata close to thelateral border. In this case the labeling was restricted tothe somata but immunopositive dendrites could not beclearly identified. A similar patchy distribution could beseen for DARPP-32 or TH, and in fact with double immuno-labeling it was clear that the GDNF-ir patches, as well asthe lateral border, were coincident with TH-positive areas(Fig. 2C,D). In the adult rat striatum however, there wasweak, diffuse immunostaining for GDNF, similar to thatseen in nonpatchy areas at postnatal day 7.

GDNF expression in developingstriatal grafts

In most 1-week-old transplants, two types of areas,positive or negative for GDNF-ir, could already be seen, aswell as some areas with weak GDNF immunostainingsurrounding the most intensely stained regions. Althoughthe TH immunolabeling of the transplant was not promi-nent at this stage, in some graft areas, generally close tothe graft border, some thin TH immunoreactive fibers,which appeared to be restricted to GDNF-positive areas,could be seen (Fig. 3A). In 2- and 3-week-old transplants,small caliber TH immunoreactive fibers, with some vari-

cosities, were abundant in the graft and located primarilyin GDNF-positive areas (Fig. 3B). In GDNF immunoreac-tive areas most of the labeling was diffuse between thesomata and the neuropil, although immunopositive so-mata , which were more intensely labeled than the sur-rounding areas, were sometimes discerned.

Co-transplants of striataland mesencephalic tissue

Both neuronal somata and TH-ir fibers were seen in2-week-old transplants; mainly in the GDNF-positive ar-eas of the co-grafts (Fig. 4A). TH-positive fibers originatedfrom both the developing graft and the surrounding host,could be observed in GDNF-ir areas, where TH-positivesomata were found—generally in the lateral border ofGDNF patches (Fig. 4B,C). TH-ir fibers were also seen insome areas which stained weakly for GDNF (Fig. 4A,C),while the GDNF negative zones were almost devoid ofdopaminergic innervation (i.e., TH-ir fibers). Double label-ing of co-transplants with TH and DARPP-32, showed thatTH-positive fibers, both from the transplant and the hoststriatum, accumulate in DARPP-32–positive areas. Inaddition, in these co-transplants (using a 1:4 proportion ofmesencephalic and striatal cells in the suspension), mostTH-positive somata tend to be located in or at the border ofDARPP-32–positive patches (Fig. 4D).

DISCUSSION

This study demonstrated a patchy distribution of GDNFexpression in the developing striatum, while in the adult

Fig. 1. Glial cell line-derived neurotrophic factor (GDNF) immuno-labeling in the striatum of a 1-week-old rat. Note the patchy staining(arrows) and the high level of immunoreactivity in the striatal lateralborder (arrowheads). The non-patchy areas of the striatum show aweak labeling. Scale bar 5 500 µm.

GDNF EXPRESSION IN INTRASTRIATAL STRIATAL GRAFTS 201

Fig. 2. Glial cell line-derived neurotrophic factor (GDNF) expres-sion in developing striatum. A: Double labeling of GDNF and tyrosinehydroxylase (TH) in a coronal section through the striatum of a1-day-old rat. Note patches positive for both GDNF and TH, and theintense labeling at the lateral striatal border. B: High magnification ofthe striatal lateral border area signed by an arrow in A. Note theintense labeling for both TH (black) and GDNF (brown). C: Double

labeling of TH and GDNF in the striatum of a 1-week-old rat. There isa patchy distribution co-localizing GDNF and TH. D: High magnifica-tion of the intensely stained patch signed by an arrow in C. Note thehigh level of labeling for both GDNF (brown) and TH (black), and thatareas outside the patch (upper part of the figure) show a very lowintensity of labeling for both GDNF and TH. Scale bars 5 500 µm forA,C; 50 µm for B; 25 µm for D.

202 E. LOPEZ-MARTIN ET AL.

rat there was a lower level of striatal GDNF and thepatchy distribution was no longer evident. While to ourknowledge patchy distribution of GDNF in the developingstriatum of the rat has not been described, there are anumber of reports showing a high level of expression ofGDNF in the developing striatum, with lower levels ofGDNF in adults, whereas levels of pallidal and corticalGDNF are low during development and higher in adults(Schaar et al., 1993; Stromberg et al., 1993; Springer etal.,1994; Choi-Lundberg and Bohn, 1995; Nosrat et al.,1996; Suvanto et al., 1996; Pochon et al., 1997; Trupp et al.,1997). Interestingly, the patchy development of GDNF inthe rat striatum coincides with that observed for striatalmarkers such as DARPP-32 (Foster et al., 1987; Laban-deira-Garcıa et al., 1994), or other markers such as, forexample, the expression of specific receptor subunits(Fritschy et al., 1994; Liste et al., 1997). Dopaminergicinnervation (i.e., TH-ir fibers) also follows a similar pat-tern (Voorn et al., 1988; Labandeira-Garcıa et al., 1994).This clearly suggests an important role for GDNF in thedopaminergic innervation of the normal developing stria-tum, which could be related to the growth promotingactivity of GDNF both in vivo and in vitro (Lin et al., 1993.,Hudson et al., 1995; Tomac et al., 1995a, 1995b; Yan et al.,1995), and also to the protective role of GDNF againstdegeneration of dopaminergic neurons in animal models ofParkinson’s disease (Beck et al., 1995; Bowenkamp et al.,1995, 1997; Kearns and Gash, 1995; Oppenheim et al.,1995; Sauer et al., 1995; Tomac et al., 1995a, 1995b; Gashet al., 1996; Winkler et al., 1996; Bjorklund et al., 1997;Choi-Lundberg et al., 1997; Lapchak et al., 1997). It is

interesting to note that GDNF does not seem to benecessary for dopaminergic fibers to reach the striatum, asGDNF knockout transgenic mice appear to have normaldopaminergic striatal innervation (Moore et al., 1996;Pichel et al.,1996; Sanchez et al., 1996). However, thesetransgenic mice die at around 1 day old, therefore it is notknown if the patchy development of the dopaminergicinnervation of the striatum and the increase in TH immu-noreactivity to adult levels (at 3 or 4 weeks old), could bedisturbed in these animals (see aforementioned papersand Broaddus and Bennett, 1990).

In developing intrastriatal striatal grafts, we have seenthat GDNF expression in the graft striatal compartment(DARPP-32–positive) precedes the invasion of the hostdopaminergic fibers that will specifically reach the GDNFrich areas. These patches show more intense immunolabel-ing than surrounding areas which are weakly immunoposi-tive, which possibly reflects a GDNF gradient from cellsreleasing GDNF in the GDNF rich patches (i.e. striatum-like)towards nearby zones, gradient that would attract host dopa-minergic fibers to the striatal-like compartment of the graft. Itis also possible that this is a reflection of the patchy distribu-tion of GDNF during striatal development (see RESULTS).

It is known that fetal striatal grafts form extensiveconnections, both efferent and afferent, with the hosttissue (Wictorin et al., 1989a, 1989b; Labandeira-Garcıa etal., 1991). A detailed time-course study of the developmentof dopaminergic afferents to the graft (Labandeira-Garcıaet al., 1991) demonstrated the existence of a striatal-likecompartment in the transplant, as shown by the expres-sion of the phosphoprotein DARPP-32, that was well

Fig. 3. Glial cell line-derived neurotrophic factor (GDNF) expres-sion in developing striatal grafts. A: Double GDNF and tyrosinehydroxylase (TH) immunostaining in a 1-week-old graft. Note thepresence of areas stained for GDNF (brown) while adjacent regions

lack GDNF labeling (asterisk). TH-ir fibers (black) are seen selectivelyconcentrated in GDNF-positive zones (arrows). B: Labeling of GDNF(brown) and TH (black) in a 2-week-old graft. Note that the TH-irfibers are concentrated in the GDNF-labeled patch. Scale bar 5 50 µm.

GDNF EXPRESSION IN INTRASTRIATAL STRIATAL GRAFTS 203

Fig. 4. Glial cell line-derived neurotrophic factor (GDNF) expres-sion in 2-week-old co-transplants of striatal and mesencephalic tissue.A: View of a co-graft immunostained for GDNF (gray) and tyrosinehydroxylase (TH) (black). Note the existence of GDNF-positive patchesand negative (asterisk) areas, and that TH-ir neurons and fibersappear closely associated to the GDNF-positive patches. B: Highmagnification micrograph showing a profuse TH-ir innervation of aGDNF-immunopositive patch, whereas areas weakly stained for GDNF

(asterisk) are almost devoid of TH-ir fibers. C: High magnificationimage of a GDNF-positive patch in a striatal-mesencephalic co-graft(asterisk). Note that TH-ir neuronal somata are located mostly at theborder of a GDNF-positive patch (arrowheads). D: DARPP-32 (gray)and TH (black) immunostaining in a striatal-mesencephalic co-graft.Note that TH-ir fibers are mostly distributed in DARPP-32 positivepatches, while TH-ir neuronal somata tend to be aligned at the borderof a DARPP-32–positive patch. Scale bars 5 50 µm.

204 E. LOPEZ-MARTIN ET AL.

defined before innervation of the graft by host dopaminer-gic fibers: DARPP-32 begins to be expressed around 4–5days postgrafting while the innervation of the graft bydopaminergic fibers begins at the end of the first weekpostgrafting. These authors postulated the existence offactors released by cells from the striatal-like graft compart-ment that would have an important tropic influence inguiding incoming dopaminergic fibers to the striatal-likepatches. In fact, GDNF immunoreactivity is already pre-sent in some graft areas at 1 week postgrafting, suggestingthat GDNF is a factor that could be involved in thisprocess. Interestingly, Wang et al. (1996) have shown thatinjection of GDNF along a tract from the nigral area to thestriatum, after one or two months lesioning of the medialforebrain bundle and transplantation of fetal ventralmesencephalic cells into the lesioned nigral area, inducesfiber outgrowth from the grafts following the GDNF tract.These experiments also indicate an important role forGDNF in guiding dopaminergic fibers towards specifictargets.

Our experiments with co-transplants including fetalstriatal and mesencephalic cells showed that TH-ir neu-rons appeared in GDNF rich areas, agreeing that nigro-striatal graft cells survive and grow better when GDNF isadded to the cell suspensions (Rosenblad et al., 1996;Granholm et al., 1997). This may be of importance in thepossible therapeutic use of nigral transplants in the treat-ment of Parkinson’s disease. Supporting this idea, co-transplants of striatal and nigral tissue showed thatgrafted nigral cells preferently innervated the striatalco-grafts rather than the host striatal tissue (De Beaure-paire and Freed, 1987). Interestingly, striatal-nigral co-transplants show different development to that of nigraltransplants in that, in the latter, the TH-ir neurons aremainly located in the graft-striatum border, whereas in theco-transplants they can be found throughout the trans-plant (Constantini et al., 1994), possibly reflecting therelease of trophic and/or tropic factors, such as, for ex-ample, GDNF, by the fetal striatal cells. These authorsdemonstrated a patchy distribution in the co-grafts of theTH-ir fibers while TH-ir somata were found both insideand outside the patches delineated by the dopaminergicfibers. In our co-grafting experiments TH-positive somataappear mainly in the GDNF immunolabeled patches (pref-erently in their border areas) This may reflect the use inour case of a proportion of 1:4 in nigral-striatal cell,whereas Constantini et al. (1994) used a ratio of 1:1.

In conclusion, this study demonstrates that in normaldeveloping striatum there is a patchy distribution ofGDNF, and that dopaminergic terminals concentrate inthe striatal areas showing the highest level of GDNF-ir.Furthermore, in intrastriatal striatal grafts, the dopamin-ergic innervation from the host concentrates in the GDNF-rich patches of the graft. These patches coincide with thegraft striatal-like areas, and their time-course of innerva-tion by TH-ir fibers parallels the level of GDNF-ir. Further-more, in striatal and mesencephalic co-grafts, dopaminer-gic neurons and fibers concentrate in the striatal areas ofthe graft instead of the graft-striatum border (i.e., concen-trate in striatal areas showing the highest levels of GDNFexpression). These results suggest that GDNF released bystriatal cells is involved in guiding dopaminergic innerva-tion of the striatum both in normal developing striatumand developing striatal grafts.

LITERATURE CITED

Beck KD, Valverde J, Alexi T, Poulsen K, Moffat B, Vandlen RA, RosenthalA, Hefti F. 1995. Mesencephalic dopaminergic neurons protected byGDNF from axotomy-induced degeneration in the adult brain. Nature373:339–341.

Bjorklund A, Rosenblad C, Winkler C, Kirik D. 1997. Studies on neuropro-tective and regenerative effects of GDNF in a partial lesion model ofParkinson’s disease. Neurobiol Disease 4:186–200.

Bowenkamp KE, Hoffman AF, Gerhardt GA, Henry MA, Biddle PT, HofferBJ, Granholm ACE. 1995. Glial cell line-derived neurotrophic factorsupports survival of injured midbrain dopaminergic neurons. J CompNeurol 355:479–489.

Bowenkamp KE, Lapchak PA, Hoffer BJ, Miller PJ, Bickford PC. 1997.Intracerebroventricular glial cell line-derived neurotrophic factor im-proves motor function and supports nigrostriatal dopamine neurons inbilaterally 6-hydroxydopamine lesioned rats. Exp Neurol 145:104–117.

Broaddus WC, Bennett JP. 1990. Postnatal development of striatal dopa-mine function. I. An examination of D1 and D2 receptors, adenylatecyclase regulation and pre-synaptic dopamine markers. Dev Brain Res52:265–271.

Choi-Lundberg DL, Bohn MC. 1995. Ontogeny and distribution of glial cellline-derived neurotrophic factor (GDNF) mRNA in rat. Dev BrainRes;85:80–88.

Choi-Lundberg DL, Lin Q, Chang YN, Chiang YL, Hay CM, Mohajeri H,Davidson BL, Bohn MC. 1997. Dopaminergic neurons protected fromdegeneration by GDNF gene therapy. Science 275:838–841.

Constantini LC, Vozza BM, Snyder-Keller AM. 1994. Enhanced efficacy ofnigral-striatal cotransplants in bilaterally dopamine-depleted rats: Ananatomical and behavioural analysis. Exp Neurol 127:219–231.

De Beaurepaire R, Freed W. 1987. Embryonic substantia nigra graftsinnervate embryonic striatal co-grafts in preference to mature hoststriatum. Exp Neurol 95:448–454.

Deckel AW, Robinson RG, Coyle JT, Sandberg PR. 1983. Reversal oflongterm locomotor anormalities in the kainic acid model of Hunting-ton’s disease by day 18 fetal striatal implants. Eur J Pharmacol93:287–288.

DiFiglia M, Schiff L, Deckel AW. 1988. Neuronal organization of fetalstriatal grafts in kainate- and sham-lesioned rat caudate nucleus: light-and electron-microscopic observations. J Neurosci 8:1112–1130.

Dunnett SB, Bjorklund A. 1997. Basic neural transplantation techniques. I.Dissociated cell suspension grafts of embryonic ventral mesencephalonin the adult rat brain. Brain Res Protocols 1:91–99.

Dunnett SB, Isacson O, Sirinagthsinghji DHS, Clarke DJ, Bjorklund A.1988. Striatal grafts in rats with unilateral striatal lesions. II. Recoveryform dopamine dependent motor asymmetry deficits in skilled pawreaching. Neuroscience 24:813–820.

Foster GA, Schulzberg M, Hokfelt T, Goldstein M, Hemmings CH, OuimetCC, Walaas SI, Greengard P. 1987. Development of a dopamine- andcyclid adenosine 3’:5’-monophosphate-regulated phosphoprotein(DARPP-32) in the prenatal rat central nervous system, and itsrelationship to the arrival of presumptive dopaminergic innervation. JNeurosci 7:1994–2018.

Fritschy JM, Paysan J, Enna A, Mohler H. 1994. Switch in the expression ofrat GABAA-receptor subtypes during postnatal development and immu-nohistochemical study. J Neurosci 14:5302–5324.

Gash DM, Zhang Z, Ovadia A, Cass WA, Yi A, Simmerman L, Russell D,Martin D, Lapchak PA, Collins F, Hoffer BJ, Gerhardt GA. 1996.Functional recovery in Parkinsonian monkeys treated with GDNF.Nature 380:252–255.

Granholm AC, Mott JL, Bowenkamp K, Eken S, Henry S, Hoffer BJ,Lapchak PA, Palmer MR, Van Horne C, Gerhardt GA. 1997. Glial cellline-derived neurotrophic factor improves survival of ventral mesence-phalic grafts to the 6-hydroxydopamine lesioned striatum. Exp BrainRes 116:29–38.

Graybiel AM, Liu FC, Dunnett SB. 1989. Intrastriatal grafts derived fromfetal striatal primordia: I. Phenotype and modular organization. JNeurosci 9:3250–3272.

Hudson J, Granholm AC, Gerhardt GA, Henry MA, Hoffma A, Biddle P,Leela NS, Mackerlova L, Lile JD, Collins F, Hoffer BJ. 1995. Glial cellline-derived neurotrophic factor augments dopaminergic circuits invivo. Brain Res Bull 36:425–432.

Isacson O, Brundin P, Gage FH, Bjorklund A. 1984. Functional neuralreplacement by grafted neurons in the ibotenic acid-lesioned striatum.Nature 311:458–460.

GDNF EXPRESSION IN INTRASTRIATAL STRIATAL GRAFTS 205

Kearns CM, Gash DM. 1995. GDNF protects nigral dopamine neuronsagainst 6-hydroxydopamine in vivo. Brain Res 672:104–111.

Labandeira-Garcıa JL, Guerra MJ. 1994. Cortical stimulation induces Fosexpression in intrastriatal striatal grafts. Brain Res 2:87–97.

Labandeira-Garcıa JL, Liste I, Tobio JP, Rozas G, Lopez-Martın E, GuerraMJ. 1995. Intrathalamic striatal grafts survive and affect circlingbehaviour in adult rats with excitotoxically lesioned striatum. Neurosci-ence 68:737–749.

Labandeira-Garcıa JL, Tobio JP, Guerra MJ. 1994. Comparison betweennormal developing striatum and developing striatal grafts using drug-induced Fos expression and neuron-specific enolase immunohistochem-istry. Neuroscience 60:399–415.

Labandeira-Garcıa JL, Wictorin K, Cunningham ET, Bjorklund A. 1991.Development of intrastriatal striatal grafts and their afferent innerva-tion from the host. Neuroscience 42:407–426.

Lapchak PA, Miller PJ, Collins F, Jiao S. 1997. Glial cell line-derivedneurotrophic factor attenuates behavioural deficits and regulates nigro-striatal dopaminergic and peptidergic markers in 6-hydroxydopamine-lesioned adult rats: Comparison of intraventricular and intranigraldelivery. Neuroscience 78:61–72.

Lin LFH, Doherty DH, Lile JD, Bektesh S, Collins F. 1993. GDNF: A glialcell line-derived neurothropic factor for midbrain dopaminergic neu-rons. Science 260:1130–1132.

Lindsay RM, Yancopoulos GD. 1996. GDNF in a bind with known orphan:Accessory implicated in new twist. Neuron 17:571–574.

Liste I, Caruncho HJ, Guerra MJ, Labandeira-Garcıa JL. 1997. GABAAreceptor subunit expression in intrastriatal striatal grafts. Comparisonbetween normal developing striatum and developing striatal grafts.Dev Brain Res;103:185–194.

Moore MW, Klein RD, Farinas I, Sauer H, Armanini M, Phillips H,Reichardt LF, Ryan AM, Carven-Moore K, Rosenthal A. 1996. Renal andneuronal abnormalities in mice lacking GDNF. Nature 382:76–79.

Nosrat CA, Tomac A, Lindqvist E, Lindskog S, Humpel C, Stromberg I,Ebendal T, Hoffer BJ, Olson L. 1996. Cellular expression of GDNFmRNA suggests multiple functions inside and outside the nervoussystem. Cell Tissue Res 286:191–207.

Olsson M, Campbell K, Wictorin K, Bjorklund A. 1995. Projection neuronsin fetal striatal transplants are predominantly derived from the lateralganglionic eminence. Neurocience 69:1169–1182.

Oppenheim RW, Houenou LJ, Johnson JE, Lin LFH, Li L, Lo AC, NewsomeAL, Prevette DM, Wang S. 1995. Developing motor neurons rescuedfrom programmed and axotomy-induced cell death by GDNF. Nature373:344–346.

Pakzaban P, Deacon TW, Burns LH, Isacson O. 1993. Increased proportionof acetylcholinesterase-rich zones and improved morphological integra-tion in host striatum of fetal grafts derived from the lateral but notmeidal ganglionic eminence. Exp Brain Res 97:13–22.

Pichel JG, Shen L, Sheng HZ, Granholm AC, Drago J, Grinberg A, Lee EJ,Huang SP, Saarmas M, Hoffer BJ, Sariola H, Westphal H. 1996. Defectsin enteric innervation and kidney development in mice lacking GDNF.Nature 382:73–76.

Pochon NAM, Menoud A, Tseng JL, Zum AD, Aebischer P. 1997. NeuronalGDNF expression in the adult rat nervous system identified by in situhybridization. Eur J Neurosci 9:463–471.

Rosenblad C, Martinez-Serrano A, Bjorklund A. 1996. Glial cell line-derivedneurotrophic factor increases survival, growth and function of intrastria-tal fetal nigral dopaminergic grafts. Neuroscience 75:979–985.

Sanchez MP, Silos-Santiago I, Frisen J, He B, Lira SA, Barbacid M. 1996.Renal agenesis and the absence of enteric neurons in mice lackingGDNF. Nature 382:70–73.

Sauer H, Rosenblad C, Bjorklund A. 1995. Glial cell line-derived neuro-thropic factor but not transforming growth factor b3 prevents delayed

degeneration of nigral dopaminergic neurons following striatal 6-hy-droxydopamine lesion. Proc Natl Acad Sci 92:8935–8939.

Sautter J, Tseng JL, Braguglia D, Aebischer P, Spenger C, Seiler RW,Widmer HR, Zurn AD. 1998. Implants of polymer-encapsulated geneti-cally modified cells releasing glial cell line-derived neurotrophic factorimprove survival, growth, and function of fetal dopaminergic cells. ExpNeurol 149:230–236.

Schaar DG, Sieber BA, Dreyfus CF, Black IB. 1993. Regional and cell-specific expression of GDNF in rat brain. Exp Neurol 124:368–371.

Springer JE, Mu X, Bergmann LW, Trojanowski JQ. 1994. Expression ofGDNF mRNA in rat and human nervous tissue. Exp Neurol 124:167–170.

Stromberg I, Bjorklund L, Johansson M, Tomac A, Collins F, Olson L, HofferB, Humpel C. 1993. Glial cell line-dervied neurotrophic factor isexpressed in the developing but not adult striatum and stimulatesdeveloping dopamine neurons in vivo. Exp Neurol 124:401–412.

Suvanto P, Hiltunen JO, Arumae U, Moshnyakov M, Sariola H, Sainio K,Saarma M. 1996. Localization of glial cell line-derived neurotrophicfactor (GDNF) mRNA in embryonic rat by in situ hybridization. Eur JNeurosci 8:816–822.

Tomac A, Lindqvist E, Lin LFH, Ogren SO, Young D, Hoffer BH, Olson L.1995a. Protection and repair of the nigrostriatal dopaminergic systemby GDNF in vivo. Nature 373:335–339.

Tomac A, Widenfalk J, Lin LFH, Kohno T, Ebendal T, Hoffer BJ, Olson L.1995b. Retrograde axonal transport of glial cell line-derived neuro-trophic factor in the adult nigrostriatal system suggests a trophic role inthe adult. Proc Natl Acad Sci USA 92:8274–8278.

Trok K, Hoffer B, Olson L. 1996. Glial cell line-derived neurotrophic factorenhances survival and growth of prenatal and postnatal spinal cordtransplants. Neuroscience 71:231–241.

Trupp M, Belluardo N, Fukanoshi H, Ibanez CF. 1997. Complementary andoverlapping expression of glial cell line-derived neurotrophic factor(GDNF), c-ret proto-oncogene, and GDNF receptor-a indicates multiplemechanisms of thropic actions in the adult rat CNS. J Neurosci17:3554–3567.

Unsicker K. 1996. GDNF: a cytokine at the interface of TGF-bs andneurothropins. Cell Tissue Res 286:175–178.

Voorn P, Kalsbeek A, Jorritsma-Byham B, Groenewegen HJ. 1988. The pre-and postnatal development of the dopaminergic cell groups in theventral mesencephalon and dopamine innervation of the striatum ofthe rat. Neuroscience 25:857–887.

Wang Y, Tien LT, Lapchak PA, Hoffer BJ. 1996. GDNF triggers fiberoutgrowth of fetal ventral mesencephalic grafts form nigra to striatumin 6-OHDA-lesioned rats. Cell Tissue Res 286:225–234.

Wictorin K, Ouimet CC, Bjorklund A. 1989a. Intrinsic organization andconnectivity of intrastriatal striatal transplants as revealed byDARPP-32 immunohistochemistry: specificity of connections with thelesioned host brain. Eur J Neurosci 1:690–701.

Wictorin K, Simmerly RB, Isacson O, Swanson LW, Bjorklund A. 1989b.Connectivity of striatal grafts implanted into ibotenic acid lesionedstriatum. III. Efferent projecting graft neurons and their relation tohost afferents within the grafts. Neuroscience 30:313–330.

Winkler C, Sauer H, Lee CS, Bjorklund A. 1996. Short-term GDNFtreatment provides long-term rescue of lesioned nigral dopaminergicneurons in a rat model of Parkinson’s disease. J Neurosci 16:7206–7215.

Won L, Heller A, Hoffmann PC. 1989. Selective association of dopamineaxons with their striatal target cells in vitro. Dev Brain Res;74:93–100.

Yan Q, Matheson C, Lopez OT. 1995. In vivo neurothropic effects of GDNFon neonatal and adult facial motor neurons. Nature;373:341–344.

Yurek D, Collier T, Sladek J. 1990. Embryonic mesencephalic and striatalco-grafts: Development of grafted dopamine neurons and functionalrecovery. Exp Neurol 109:191–199.

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