GABAA receptor subunit expression in intrastriatal striatal grafts

Abstract To compare the expression of GABAA recep-tor subunits in the normal substantia nigra and in fetalmesencephalic neurons ectopically transplanted into thedopamine-depleted striatum, we have employed singleand double immunocytochemical approaches using tyro-sine hydroxylase (TH) and α1, α2, α3, and β2/3 GABAAreceptor subunit specific antibodies. In the substantia ni-gra, α1 and β2/3 GABAA receptor subunits were labeledin processes in the pars compacta (SNc) and, more in-tensely, in both somata and processes in the pars reticul-ata (SNr). There was no clear TH and α1 or β2/3 colo-calization, with the exception of some TH-immunoreac-tive (-ir) neurons that showed a weak immunoreactivityfor β2/3. Sections immunolabeled for α2 showed a faintdiffuse labeling for this subunit both in the SNr and inthe SNc. Scattered somata were immunopositive for α2,and some of them were also TH-ir. The labeling for α3and TH showed that TH-positive neurons expressed in-tense α3 immunoreactivity, although some TH-negativesomata in the SNr expressed weak α3 immunoreactivity.In the transplants, double immunostaining proceduresshowed that the labeling for α1 or β2/3 appeared particu-larly concentrated in patches of intensely immunoreac-tive neuronal processes that surrounded TH-ir cells, butthese processes were not TH-ir. In the case of α2, diffuseimmunostaining was observed all over the graft, withsome scattered positive somata. Only a few of them werealso TH positive. Sections immunoreacted for α3 andTH revealed that TH-ir neurons expressed intense α3immunoreactivity, and that only a few TH-negative neu-rons were weakly positive for α3. These results show

that mesencephalic tissue ectopically grafted into thestriatum develops a pattern of GABAA receptor expres-sion similar to that normally expressed in situ, and par-ticularly that the grafted dopaminergic neurons expresssimilar GABAA receptors, including the α3 subunit. Thismight be due to the similarity of GABAergic afferents tothese neurons in the SNc and the graft, or that at the timeof transplantation this expression had already been deter-mined.

Keywords GABAergic transmission · Parkinson’s disease · Basal ganglia · Neural transplantation · Substantia nigra

Introduction

Intrastriatal mesencephalic grafts have been reported as apossible therapeutic approach for Parkinson’s disease(PD; recently reviewed in Dunnett and Björklund 1999).These grafts have already been performed in some par-kinsonian patients at several hospitals, relieving differentsymptoms of PD (see Olanow et al. 1996). Numerousstudies in animal models of PD [i.e., 6-hydroxydopamine(6-OHDA)-lesioned rats or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice or monkeys]have also shown that intrastriatal ventral mesencephalictransplants compensate, at least in part, the motor dys-function induced by the lesion (Björklund and Stenevi1979; Dunnett et al. 1981a, b; López-Martín et al.1999b; Rozas et al. 1998).

GABAergic connections are an important componentof the basal ganglia circuit. Among the main GABAergicstriatal efferents are those projecting to the substantia ni-gra (mostly to the pars reticulata), and recently the exis-tence of a GABAergic nigrostriatal pathway was alsoshown (Rodríguez and González-Hernández 1999). Inaddition, the functional activity of dopaminergic neuronsin the substantia nigra pars compacta (SNc) is also regu-lated by GABAergic afferents from the pars reticulata(SNr; Tepper et al. 1995). The importance of GABA–

J. Rodríguez-Pallares (✉ ) · A. Muñoz · M.J. Guerra J.L. Labandeira-GarcíaDepartment of Morphological Sciences, Faculty of Medicine, Rúa San Francisco s/n, 15705-University of Santiago de Compostela, Galicia, Spaine-mail: cmjann@uscmail.usc.esTel.: +34-981-563100 Ext. 12223, Fax: +34-981-547078

H.J. CarunchoDepartment of Fundamental Biology, Faculty of Biology, 15706-University of Santiago de Compostela, Galicia, Spain

Exp Brain Res (2000) 135:331–340DOI 10.1007/s002210000537

R E S E A R C H A RT I C L E

Jannette Rodríguez-Pallares · Héctor J. Caruncho Ana Muñoz · María José Guerra José Luis Labandeira-García

GABAA receptor subunit expression in intrastriatal ventral mesencephalic transplants

Received: 20 March 2000 / Accepted: 21 July 2000 / Published online: 12 October 2000© Springer-Verlag 2000

dopamine interactions and their changes in PD have beenstudied with special interest (see Hossain and Weiner1995). In particular, we and others have demonstratedimportant changes in GABAA receptor subunit expres-sion in different basal ganglia nuclei in animal models ofPD (Bernard et al. 1996; Calon et al. 1995; Caruncho et al. 1997; Gagnon et al. 1993; Griffiths et al. 1990; Panet al. 1985; Robertson et al. 1990; Sanna et al. 1998;Stasi et al. 1999). In fact, we suggested that in unilaterallesions of the nigrostriatal pathway by the injection of 6-OHDA, the early changes observed in GABAA recep-tor abundance tend to be compensated over time, as wasalso shown for glutamate receptors (see Caruncho et al.1997; Wüllner et al. 1994). However, this does not hap-pen in mice with hereditary parkinsonism where mesen-cephalic dopaminergic grafts are able to restore, at leastin part, GABAA receptor levels (Stasi et al. 1999).

GABAA receptors are heteropentameric proteins withan intrinsic chloride channel. Up to now, 18 differentsubunits have been cloned (α1–6, β1–3, γ1–3, δ, ε, π,ρ1–3; recently reviewed in Metha and Ticku 1999). Thephysiological and pharmacological properties of GABAAreceptors depend upon their subunit composition (seeDucic et al. 1995; Sieghart 1995 as a review). GABAAreceptor subunits show a different expression in separatebrain areas: in the striatum the most abundant subunitsare α2, α4, β2/3, γ2, and δ, while the most abundant sub-units in the SNr are α1, β2/3, and γ2; and in the SNc areα3 and γ2 (Caruncho et al. 1996b; Fritschy and Möhler1995; Persohn et al. 1992; Wisden et al. 1992).

Fetal ventral mesencephalic cells implanted in the do-pamine-depleted striatum result in a graft that includesboth tyrosine hydroxylase (TH)-positive and -negativeneurons; the former being located mostly in the periph-ery of the graft (see, for example, Chkirate et al. 1993;López-Martín et al. 1999a). While most cellular studieson these grafts have focused on the understanding of thedopaminergic system and its relation to the graft func-tional actions, only a few studies have been dedicated toother systems, particularly in terms of neurotransmitterreceptor expression in the grafted neurons. A recent re-port by Todaka et al. (1998) has demonstrated thatAMPA-glutamate receptor subunits may exert an impor-tant role in the fate of developing TH-positive neuronsboth in the SNc and in intrastriatal mesencephalic trans-plants; the receptor expression being similar in bothcases. However, to our knowledge there are no referenc-es about GABAA receptor subunit expression in ectopicmesencephalic transplants (such as those made in thestriatum) in comparison with their expression in the sub-stantia nigra in situ. In order to further advance our un-derstanding on this subject, we employed a double im-munolabeling of TH and different GABAA receptor sub-units to study their expression in intrastriatal mesence-phalic transplants in comparison with that observed inthe substantia nigra. Our results indicated that the sameGABAA receptor subunits that are well expressed in theSNc or in the SNr are similarly expressed in TH-positiveand -negative areas of the transplant, respectively.

Materials and methods

Experimental design

A total of ten adult Sprague-Dawley rats were used in this study.All experiments were carried out in accordance with Principles ofLaboratory Animal Care (NIH publication number 86–23, revised1985). A group of six rats received a unilateral injection of 6-OHDA in the right medial forebrain bundle (MFB; see below)and 10 days later a fetal mesencephalic cell suspension was inject-ed into the striatum. The rats in this group were killed 6 monthspostgrafting (mature grafts). The remaining rats were used as con-trols and to analyze GABAA receptor subunit expression in the un-lesioned substantia nigra.

6-OHDA lesion and fetal mesencephalic transplantation

Rats were anesthetized with equithesin (3 ml/kg i.p.) and unilater-ally lesioned in the right MFB by stereotaxic injection of 12 µg 6-OHDA HBr (to give 8 µg 6-OHDA free base) in 4 µl sterile sa-line containing 0.2% ascorbic acid at the following coordinates:3.7 mm posterior to Bregma, 1.6 mm right of midline, 8.8 mmventral to the skull at the midline, in the flat skull position (Paxinos and Watson 1986). Ten days postlesion the rats receivedintrastriatal injections of cell suspensions prepared from fetal ven-tral mesencephalon at 13–14 days of gestation. The pieces of ven-tral mesencephalon were dissected out and incubated in 0.1% trypsin (Sigma), 0.05% DNase (Sigma), and DMEM (Gibco) for 20 min at 37°C. Afterwards, the tissue was rinsed inDNase/DMEM and mechanically dissociated to produce a milkycell suspension. This cell suspension was centrifuged at 600 rpmfor 5 min and the supernatant was carefully removed and resus-pended in 0.05% DNase/DMEM to the final volume required. Atotal of approximately one million viable cells (estimated by acri-dine orange/ethidium bromide; in about 6 µl) were administered toeach rat at three injection sites: (1) A=1.8, L=2.2, V=4.5, (2)A=0.6, L=2.0, V=4.5, and (3) A=0.6, L=3.2, V=4.5 (see for detailsDunnett and Björklund 1997).

Immunohistochemistry

Rats were anesthetized and perfused transcardially with a solutionof 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4.Brains were carefully dissected out, cryoprotected in the samebuffer containing 20% sucrose, and cut into 40-µm-thick sectionswith a freezing microtome. Sections including the transplant or thesubstantia nigra were processed for TH or GABAA receptor α1,α2, α3, or β2/3 subunits as follows: Sections were firstly preincu-bated with a blocking solution containing 10% normal serum inPBS with 1% BSA and 1% Triton X-100, and then incubatedovernight at 4°C with the corresponding primary antibody dilutedin 1% BSA in PBS. The antibodies used were: a rabbit polyclonalantibody anti-TH (Pel-Freez Biologicals), a rabbit polyclonal anti-body anti-α1 (Fritschy et al. 1992), guinea pig polyclonal antibod-ies anti-α2 or -α3 (Fritschy et al. 1992), and the mouse monoclo-nal antibody bd17 that recognizes a common epitope for the β2and β3 subunits (Ewert et al. 1990, 1992). These antibodies wereused at the following dilutions: 1:500 for TH, 1:10,000 for β2/3,1:20,000 for α1, and 1:40,000 for α2 and α3. The sections werethen washed and incubated for 90 min at room temperature withthe corresponding biotinylated secondary antibody diluted 1:100,and then for another 90 min with avidin-biotin-peroxidase (ABC;Vector; 1:100). Finally, the labeling was revealed with 0.04% hy-drogen peroxide and 0.05% 3,3′-diaminobenzidine (DAB).

Other sections were processed for double immunolabeling ofone of the GABAA receptor subunits cited above and TH. Briefly,one of the GABAA receptor subunits was immunolabeled as de-scribed, but using DAB-nickel sulfate to develop the reaction sothat the precipitated product was black. After washing in PBS, thesections were incubated with the anti-TH antibody, followed by a

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swine anti-rabbit antibody (1:50), and finally a rabbit-PAP anti-body (1:100). Detection of the second labeling was carried outwith DAB alone, giving a brown precipitate. Control experimentsomitting primary antibodies showed a lack of immunoreactivity.

Labeling intensity was estimated as optical density, in at least three sections per rat and antibody, with the aid of NIH-Image 1.55 image analysis software (Wayne Rasband, NIMH) on a Macintosh personal computer coupled to a video camera(CDD-72; MTI) connected to a Nikon Optiphot 2 microscope with a 4× Nikon Apo-plan objective. For each section, the mea-surement was done by outlining the specific area in the computerscreen. In each case, optical densities were corrected by subtrac-tion of background as observed in the cerebral peduncle (substan-tia nigra) or in the corpus callosum (grafts). Data were expressedas a percentage of optical density of a grafted area with respect tothe SNc or the SNr of control rats. Means were compared usingANOVA followed by the post hoc Tukey test (P<0.05). Data nor-mality and homogeneity of variances were confirmed before eachANOVA. All statistical analyses were performed with the aid ofSigmastat 2.0 from Jandel Scientific.

Results

α1, α2, α3, and β2/3 GABAA receptor subunit expression in the substantia nigra

In the SNr there was an intense labeling of processes im-munoreactive for the α1 and β2/3 subunits, and somescattered immunopositive somata. A faint diffuse immu-nolabeling for the α2 subunit (with some individual α2-positive somata) was distinguished. There was also a dis-crete α3 subunit immunoreactivity, which appeared to beassociated with some large neuronal somata or with dendritic processes from neurons in the SNc (Figs. 1A,B, 2A).

In the SNc the labeling for the α3 subunit was muchstronger that in the SNr, and located in somata and pro-cesses. Membrane regions showing a high degree of α3-immunoreactive (-ir) “hot spots” were clearly seen bothin dendrites and somata. Similarly to that observed inSNc, there was a diffuse and faint immunolabeling of theα2 subunit, with some scattered positive somata. The la-beling for the α1 subunit was less intense than in SNrand was located on neuronal processes that originated inSNr neurons, without apparent labeling of somata. Thelabeling for the β2/3 subunits in the SNc was similar tothat of α1, but in addition there was a faint immunostain-ing of some neuronal somata (Figs. 1A, B, 2A).

If the overall labeling in the SNr and the SNc is con-sidered, α1 and β2/3 subunits were expressed in bothSNr and SNc with some more intense labeling in theSNr. There was a faint diffuse labeling for the α2 subunitin both SNr and SNc. Finally, the immunoreactivity forthe α3 subunit was intense in the SNc and less marked inthe SNr (Figs. 1A, B, 2A).

TH and GABAA receptor subunit labeling in intrastriatalmesencephalic transplants

According to previous studies (López-Martín et al.1999a, b), the grafts consist of TH-positive neurons and

TH-negative (i.e., non-dopaminergic, mostly SNr-like)areas. Neurons immunopositive for TH appeared locatedmostly in small groups in the periphery of the mesence-phalic transplants. TH-ir fibers originating from thegrafted dopaminergic neurons were seen all over thetransplant, and crossed the graft-host border to innervatethe surrounding host striatum (Fig. 2B).

In accordance with that observed in the substantianigra, immunolabeling for the α2 subunit showed afaint diffuse staining all throughout the transplant,without clear distinction of graft compartments (i.e.,the central and border areas; optical densities 105±12.2% and 104±13.7% of those of the SNr and the SNc,respectively). Immunoreactivity for α3 subunit wasseen in processes and neuronal somata, mostly groupedin patches along the transplant border, where most ofthe dopaminergic neurons are located. The immunore-activity for α3 subunit was slightly higher in thesepatches than in the SNc of control rats (136±4.0%),though this increase was not statistically significant.Membrane regions of the neurons immunopositive forα3 subunit showing a high degree of α3-ir “hot spots”were clearly seen both in dendrites and somata. In otherareas of the graft, the immunoreactivity was similar tothat observed in SNr of control rats (107±12.2%). Inaddition, some weak α3-ir somata were observed scat-tered through the graft. In the case of the α1 or the β2/3subunits, most areas of the transplant appeared immu-noreactive (intense immunopositive processes andsome scattered somata), but the labeling was especiallyintense in patches of densely concentrated immunore-active processes, which were located preferentially inthe periphery of the grafts. In these patches the labelingshowed a significant increase with respect to that ob-served in the SNc for α1 and for β2/3 subunits(539±9.0% and 512±14.2%, respectively). In the rest ofthe graft the labeling was 150±15.0% for α1 and178±16.3% for β2/3 of that observed in the SNr of con-trol rats, however, the increases were not statisticallysignificant (Figs. 1C, D, 2C, D).

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Fig. 1 Microphotographs showing the immunoreactivity forGABAA receptor α2 (A, C) or α3 (B, D) subunit in the substantianigra (A, B) and mesencephalic grafts (C, D). A Both substantianigra pars reticulata (SNr) and substantia nigra pars compacta(SNc) showed a faint diffuse α2 immunoreactivity, with some in-dividual α2-immunoreactive (-ir) somata (arrowheads). B The α3immunoreactivity in the SNc was more intense than in the SNr.Some positive processes and somata can be observed in the SNc(arrowheads) and SNr (arrows). C In intrastriatal mesencephalictransplants, the staining for α2 was also faint and diffuse allthroughout the graft (g; encircled). D The grafts (g; encircled)showed α3-positive processes and somata (arrows), most of themgrouped in patches (asterisks) located in the periphery of thetransplant. Note that some weak α3-ir processes and somata (arrowheads) are also scattered through the graft. Scale bar250 µm in A, B; 160 µm in C; 80 µm in D

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Fig. 1 (Legend see page 333)

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Fig. 2 (Legend see page 336)

Double-immunolabeling of GABAA receptor subunitsand TH in the substantia nigra and in intrastriatal mesencephalic transplants

In the substantia nigra, double immunostaining of α1 orβ2/3 and TH showed that these GABAA receptor sub-units labeled processes both in the SNr and in the SNc.There was no clear colocalization of these markers, withthe exception of some TH-ir neurons that showed a weakimmunoreactivity for β2/3. Sections immunolabeled forα2 and TH showed a faint diffuse labeling for this sub-unit both in the SNr and in the SNc. Scattered somatawere immunopositive for α2, and some of them werealso TH-ir. Double immunostaining procedures clearlyshowed that TH-positive neurons also expressed intenseα3 immunoreactivity. In addition, some TH-negativesomata in the SNr expressed weak α3 immunoreactivity(Fig. 3A–D).

In the transplants, double immunohistochemical stud-ies for GABAA receptor subunits and TH showed thatimmunoreactivity for α1 and β2/3 appeared particularlyconcentrated in patches of immunoreactive neuronal pro-cesses that surrounded TH-ir cells. These intensely la-beled α1- or β2/3-positive processes were not TH-ir.Some scattered somata were immunoreactive for thesesubunits, and some TH-ir neurons showed a faint label-ing for the β2/3 subunits. In the case of α2/TH immuno-stained sections, the above-described diffuse stainingevenly distributed through the grafts with some positivesomata was confirmed, and some of these somata werealso labeled for TH. Sections immunoreacted for α3 andTH confirmed that the TH-positive (i.e., dopaminergic)neurons expressed intense α3 immunoreactivity, and thata few non-dopaminergic neurons were weakly positivefor α3 (Fig. 3E–G).

α1, α2, α3, and β2/3 GABAA receptor subunit expressionin the striatum

No significant changes were observed in α1, α2, α3, andβ2/3 GABAA receptor subunit expression in the striatum

surrounding the grafts with respect to that observed incontrol rats in the present and previous studies (see fordetails Caruncho et al. 1996a, b; Rodríguez-Pallares etal. 2000). The striatum showed an intense labeling forthe α2 and β2/3 subunit (Figs. 1C, 2D, 4A, B) and afaint labeling for the α1 and α3 subunit (Figs. 1D, 2C,4C, D), although some scattered neurons were positivefor these two subunits.

Discussion

This study has focused on the analysis of GABAA recep-tor subunit expression in intrastriatal mesencephalictransplants, and showed that this expression appears tobe similar to that observed in the normal substantia nigradespite the ectopic location of the graft.

SNc dopaminergic neurons showed a high expressionlevel of receptors containing the α3 subunit, which couldindicate the existence of GABAA receptors that show alow affinity and maximal efficacy for GABA (Ducic etal. 1995). On the contrary, most neurons in the SNr ex-pressed mostly receptors containing the α1 and β2/3subunits which, together with the γ2 subunit (Sieghart1995), constitute the GABAA receptor subtype mostwidely distributed in the CNS, and reflect a benzodiaze-pine type I receptor. In addition, in the striatum (i.e., theregion where the graft was implanted) most GABAA re-ceptors contain the α2 or the α4 subunit, indicating thepreponderance of benzodiazepine type II receptors (seeas reviews Mehta and Ticku 1999; Sieghart 1995).

The preponderance of different GABAA receptor sub-unit expression in the above-mentioned brain areas al-lowed us to study if the expression pattern of specificGABAA receptor subunits in fetal ventral mesencephalicneurons implanted ectopically in an adult striatum ischanged with respect to that observed in the normal sub-stantia nigra. Neurotransmitter receptor expression ap-pears to take place before synaptic connections are

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Fig. 2 Substantia nigra (A) and mesencephalic grafts (B, C, D)immunoreacted for GABAA receptor α1 subunit (A, C), β2/3 sub-units (D), or tyrosine hydroxylase (TH; B). The labeling for α1was more intense in the SNr than in the SNc (A); similarly, mostof the grafted tissue (C) was α1-ir, but the labeling was especiallyintense in patches of α1-ir processes located preferentially in theperiphery of the graft (arrows). Some scattered somata (arrow-heads) were seen all throughout the transplant. B In intrastriatalmesencephalic transplants numerous TH-positive neurons and pro-cesses were observed. Neurons were mostly located in smallgroups in the periphery of the graft (arrows), and numerous TH-irfibers crossed the host–graft border to enter into the host striatum.D As observed for α1, the labeling for β2/3 subunits was found allthroughout the grafts (g; encircled), but it was particularly intensein patches (asterisks) of immunoreactive processes concentratedpreferentially in the periphery of the graft (i.e., where the dopami-nergic neurons are usually located; see B). Scale bar 250 µm in A,C; 100 µm in B; 160 µm in D

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Fig. 3 Microphotographs showing double immunolabeling forGABAA receptor subunits (gray-black) and TH (brown) in thesubstantia nigra (A–D) and intrastriatal mesencephalic grafts(E–G). A, B β2/3 subunit-labeled processes both in the SNr andSNc, with no clear colocalization with TH immunoreactivity. Thelabeling in the SNc was less intense than in the SNr, and was ap-parently located in neuronal processes entering from the SNr,where some positive somata (arrowheads) can be distinguished.The area arrowed in A is detailed in B, showing β2/3-positive pro-cesses surrounding TH-positive somata. C Microphotographshowing the faint diffuse labeling for α2 (gray-black) both in theSNr and in the SNc, and some scattered α2-positive neurons in theSNr (arrowheads). D, E High magnification of SNc (D) and graft(E) neurons (arrows) showing double labeling for TH (brown) andα3 subunit (gray-black). F, G In mesencephalic grafts (g; encir-cled) the immunolabeling for β2/3 (F) appeared particularly con-centrated in patches (asterisk) of immunoreactive processes sur-rounding TH-positive neurons (brown; arrows), and the immuno-reactivity for α2 (G) was diffuse and evenly distributed throughthe graft (g; encircled) and less intense than that observed in thesurrounding host striatum (ST). Scale bar 125 µm in A, F; 50 µmin B; 200 µm in C; 25 µm in D, E; 100 µm in G

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Fig. 4 Immunolabeling for GABAA receptor α2 (A), β2/3 (B), α1(C), and α3 (D) subunit in the striatum of control rats. Note an in-tense labeling for α2 and β2/3 subunit (A, B), and a faint labeling

for α1 and α3 subunit (C, D). The globus pallidus (GP) showedmore intense immunoreactivity for α1 subunit than the striatum(ST; C). Scale bar 500 µm

formed (see as reviews Goridis and Brunet 1999; Hynesand Rosenthal 1999), therefore the expression of specificGABAA receptor subtypes could be governed by intrin-sic neuronal factors that might be independent of the en-vironment where the neuron is located. However, thisexpression could also be influenced by external factorssuch as the release of trophic factors, or modified by theimpinging of specific synaptic contacts (Bhisitkul et al.1990). In fact, GABAA neurotransmitter receptor expres-sion can be altered by protracted treatment with alloste-ric modulators (see, for example, Impagnatiello et al.1996; Pesold et al. 1997).

In a previous work (Liste et al. 1997) we have demon-strated that GABAA receptor subunit expression in stri-atal and pallidal regions (i.e., DARPP-32 positive ornegative, respectively) of intrastriatal striatal transplantsis similar to that of striatal or pallidal neurons of the de-veloping and adult striatum, suggesting that contacts be-tween neurons in the two graft regions could contributeto the maturation of the graft and also to the expressionof those receptors. However, in the present report we arereferring to an ectopic transplant, which perhaps couldimply differences in GABAergic innervation of graftedneurons affecting its physiological behavior.

The existence of host afferents into intrastriatal graftsof fetal mesencephalic tissue has already been document-ed: while raphe and cortical projections are well repre-sented throughout the graft, striatal fibers are few and re-stricted to the graft peripheral areas where they synapsemostly with TH-ir neurons (Chkirate et al. 1993; Doucetet al. 1989). Interestingly, local graft circuits are well de-veloped and they could be responsible in part of the graftfunctional activity. In this sense, graft TH-ir neurons re-ceive a high number of GAD-ir contacts from GABA-ergic neurons inside the graft (probably SNr neurons; seeBolam et al. 1987). Our results also indicate a high de-gree of interaction between the different graft compart-ments (i.e., border region TH-ir neurons and central re-gion TH-negative neurons) as is shown by the presenceof patches of α1- and β2/3-immunopositive processes inclose association with TH-ir somata, similar to what isseen in the substantia nigra of control rats where α1- andβ2/3-positive processes from SNr neurons are seen enter-ing the SNc and located closely to TH-ir neurons. How-ever, optical density data revealed that the density of α1-and β2/3-immunoreactive processes in the areas of thegraft containing dopaminergic neurons is much higherthan that observed in the normal substantia nigra. Thissuggests that the dopaminergic neurons may have a tropicinfluence on the α1- and β2/3-ir processes (presumablyfrom grafted SNr neurons). Similarly, it was observed(Labandeira-García et al. 1991; Wictorin et al. 1989) thatin striatal grafts host dopaminergic terminals concentrateselectively in the patches of grafted striatal tissue, andthat GDNF release by fetal striatal neurons appears tohave a tropic influence on the dopaminergic terminals(López-Martín et al. 1999a).

The existence of membrane areas in both somata andprocesses that showed a high level of α3-ir “hot spots”

in TH-ir neurons may indicate that these receptors are lo-cated mostly at postsynaptic sites and not at extrasynap-tic locations as with other GABAA receptor subtypes(see Rodríguez-Pallares et al. 2000). These receptors willprobably mediate the direct interaction between the twosubstantia nigra components; and their presence in graftTH-ir neurons would be important for their regulation bygraft GABAergic neurons.

In conclusion, the present results show that mesence-phalic tissue ectopically grafted into the striatum devel-ops a pattern of GABAA receptor expression similar tothat normally expressed in situ, and that graft local cir-cuits may be important for the regulation of the function-al activity of the graft.

Acknowledgements The authors wish to thank Drs. H. Möhler(University of Zürich) and A. De Blas (University of Storrs, Con-necticut) for the generous gift of the GABAA receptor subunit an-tibodies. This work was supported by grants from XUGA and theSpanish DGESIC (PGC).

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