Exp Brain Res (2000) 135:331–340 DOI 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
Abstract To compare the expression of GABAA receptor subunits in the normal substantia nigra and in fetal mesencephalic neurons ectopically transplanted into the dopamine-depleted striatum, we have employed single and double immunocytochemical approaches using tyrosine hydroxylase (TH) and α1, α2, α3, and β2/3 GABAA receptor subunit specific antibodies. In the substantia nigra, α1 and β2/3 GABAA receptor subunits were labeled in processes in the pars compacta (SNc) and, more intensely, in both somata and processes in the pars reticulata (SNr). There was no clear TH and α1 or β2/3 colocalization, with the exception of some TH-immunoreactive (-ir) neurons that showed a weak immunoreactivity for β2/3. Sections immunolabeled for α2 showed a faint diffuse labeling for this subunit both in the SNr and in the SNc. Scattered somata were immunopositive for α2, and some of them were also TH-ir. The labeling for α3 and TH showed that TH-positive neurons expressed intense α3 immunoreactivity, although some TH-negative somata in the SNr expressed weak α3 immunoreactivity. In the transplants, double immunostaining procedures showed that the labeling for α1 or β2/3 appeared particularly concentrated in patches of intensely immunoreactive neuronal processes that surrounded TH-ir cells, but these processes were not TH-ir. In the case of α2, diffuse immunostaining was observed all over the graft, with some scattered positive somata. Only a few of them were also TH positive. Sections immunoreacted for α3 and TH revealed that TH-ir neurons expressed intense α3 immunoreactivity, and that only a few TH-negative neurons were weakly positive for α3. These results show J. Rodríguez-Pallares (✉) · A. Muñoz · M.J. Guerra J.L. Labandeira-García Department of Morphological Sciences, Faculty of Medicine, Rúa San Francisco s/n, 15705-University of Santiago de Compostela, Galicia, Spain e-mail:
[email protected] Tel.: +34-981-563100 Ext. 12223, Fax: +34-981-547078 H.J. Caruncho Department of Fundamental Biology, Faculty of Biology, 15706-University of Santiago de Compostela, Galicia, Spain
that mesencephalic tissue ectopically grafted into the striatum develops a pattern of GABAA receptor expression similar to that normally expressed in situ, and particularly that the grafted dopaminergic neurons express similar GABAA receptors, including the α3 subunit. This might be due to the similarity of GABAergic afferents to these neurons in the SNc and the graft, or that at the time of transplantation this expression had already been determined. Keywords GABAergic transmission · Parkinson’s disease · Basal ganglia · Neural transplantation · Substantia nigra
Introduction Intrastriatal mesencephalic grafts have been reported as a possible therapeutic approach for Parkinson’s disease (PD; recently reviewed in Dunnett and Björklund 1999). These grafts have already been performed in some parkinsonian patients at several hospitals, relieving different symptoms of PD (see Olanow et al. 1996). Numerous studies in animal models of PD [i.e., 6-hydroxydopamine (6-OHDA)-lesioned rats or 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP)-treated mice or monkeys] have also shown that intrastriatal ventral mesencephalic transplants compensate, at least in part, the motor dysfunction induced by the lesion (Björklund and Stenevi 1979; Dunnett et al. 1981a, b; López-Martín et al. 1999b; Rozas et al. 1998). GABAergic connections are an important component of the basal ganglia circuit. Among the main GABAergic striatal efferents are those projecting to the substantia nigra (mostly to the pars reticulata), and recently the existence of a GABAergic nigrostriatal pathway was also shown (Rodríguez and González-Hernández 1999). In addition, the functional activity of dopaminergic neurons in the substantia nigra pars compacta (SNc) is also regulated by GABAergic afferents from the pars reticulata (SNr; Tepper et al. 1995). The importance of GABA–
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dopamine interactions and their changes in PD have been studied with special interest (see Hossain and Weiner 1995). In particular, we and others have demonstrated important changes in GABAA receptor subunit expression in different basal ganglia nuclei in animal models of PD (Bernard et al. 1996; Calon et al. 1995; Caruncho et al. 1997; Gagnon et al. 1993; Griffiths et al. 1990; Pan et al. 1985; Robertson et al. 1990; Sanna et al. 1998; Stasi et al. 1999). In fact, we suggested that in unilateral lesions of the nigrostriatal pathway by the injection of 6-OHDA, the early changes observed in GABAA receptor abundance tend to be compensated over time, as was also shown for glutamate receptors (see Caruncho et al. 1997; Wüllner et al. 1994). However, this does not happen in mice with hereditary parkinsonism where mesencephalic dopaminergic grafts are able to restore, at least in part, GABAA receptor levels (Stasi et al. 1999). GABAA receptors are heteropentameric proteins with an intrinsic chloride channel. Up to now, 18 different subunits have been cloned (α1–6, β1–3, γ1–3, δ, ε, π, ρ1–3; recently reviewed in Metha and Ticku 1999). The physiological and pharmacological properties of GABAA receptors depend upon their subunit composition (see Ducic et al. 1995; Sieghart 1995 as a review). GABAA receptor subunits show a different expression in separate brain areas: in the striatum the most abundant subunits are α2, α4, β2/3, γ2, and δ, while the most abundant subunits in the SNr are α1, β2/3, and γ2; and in the SNc are α3 and γ2 (Caruncho et al. 1996b; Fritschy and Möhler 1995; Persohn et al. 1992; Wisden et al. 1992). Fetal ventral mesencephalic cells implanted in the dopamine-depleted striatum result in a graft that includes both tyrosine hydroxylase (TH)-positive and -negative neurons; the former being located mostly in the periphery of the graft (see, for example, Chkirate et al. 1993; López-Martín et al. 1999a). While most cellular studies on these grafts have focused on the understanding of the dopaminergic system and its relation to the graft functional actions, only a few studies have been dedicated to other systems, particularly in terms of neurotransmitter receptor expression in the grafted neurons. A recent report by Todaka et al. (1998) has demonstrated that AMPA-glutamate receptor subunits may exert an important role in the fate of developing TH-positive neurons both in the SNc and in intrastriatal mesencephalic transplants; the receptor expression being similar in both cases. However, to our knowledge there are no references about GABAA receptor subunit expression in ectopic mesencephalic transplants (such as those made in the striatum) in comparison with their expression in the substantia nigra in situ. In order to further advance our understanding on this subject, we employed a double immunolabeling of TH and different GABAA receptor subunits to study their expression in intrastriatal mesencephalic transplants in comparison with that observed in the substantia nigra. Our results indicated that the same GABAA receptor subunits that are well expressed in the SNc or in the SNr are similarly expressed in TH-positive and -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 of Laboratory Animal Care (NIH publication number 86–23, revised 1985). 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 injected into the striatum. The rats in this group were killed 6 months postgrafting (mature grafts). The remaining rats were used as controls and to analyze GABAA receptor subunit expression in the unlesioned substantia nigra. 6-OHDA lesion and fetal mesencephalic transplantation Rats were anesthetized with equithesin (3 ml/kg i.p.) and unilaterally 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 saline containing 0.2% ascorbic acid at the following coordinates: 3.7 mm posterior to Bregma, 1.6 mm right of midline, 8.8 mm ventral to the skull at the midline, in the flat skull position (Paxinos and Watson 1986). Ten days postlesion the rats received intrastriatal injections of cell suspensions prepared from fetal ventral mesencephalon at 13–14 days of gestation. The pieces of ventral 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 in DNase/DMEM and mechanically dissociated to produce a milky cell suspension. This cell suspension was centrifuged at 600 rpm for 5 min and the supernatant was carefully removed and resuspended in 0.05% DNase/DMEM to the final volume required. A total of approximately one million viable cells (estimated by acridine orange/ethidium bromide; in about 6 µl) were administered to each 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 details Dunnett and Björklund 1997). Immunohistochemistry Rats were anesthetized 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 the same buffer containing 20% sucrose, and cut into 40-µm-thick sections with a freezing microtome. Sections including the transplant or the substantia nigra were processed for TH or GABAA receptor α1, α2, α3, or β2/3 subunits as follows: Sections were firstly preincubated with a blocking solution containing 10% normal serum in PBS with 1% BSA and 1% Triton X-100, and then incubated overnight at 4°C with the corresponding primary antibody diluted in 1% BSA in PBS. The antibodies used were: a rabbit polyclonal antibody anti-TH (Pel-Freez Biologicals), a rabbit polyclonal antibody anti-α1 (Fritschy et al. 1992), guinea pig polyclonal antibodies anti-α2 or -α3 (Fritschy et al. 1992), and the mouse monoclonal antibody bd17 that recognizes a common epitope for the β2 and β3 subunits (Ewert et al. 1990, 1992). These antibodies were used 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 were then washed and incubated for 90 min at room temperature with the 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% hydrogen peroxide and 0.05% 3,3′-diaminobenzidine (DAB). Other sections were processed for double immunolabeling of one of the GABAA receptor subunits cited above and TH. Briefly, one of the GABAA receptor subunits was immunolabeled as described, but using DAB-nickel sulfate to develop the reaction so that the precipitated product was black. After washing in PBS, the sections were incubated with the anti-TH antibody, followed by a
333 swine anti-rabbit antibody (1:50), and finally a rabbit-PAP antibody (1:100). Detection of the second labeling was carried out with DAB alone, giving a brown precipitate. Control experiments omitting 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 NIHImage 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 measurement was done by outlining the specific area in the computer screen. In each case, optical densities were corrected by subtraction of background as observed in the cerebral peduncle (substantia nigra) or in the corpus callosum (grafts). Data were expressed as a percentage of optical density of a grafted area with respect to the SNc or the SNr of control rats. Means were compared using ANOVA followed by the post hoc Tukey test (P
