Differential distribution of group I metabotropic glutamate receptors in developing human cortex

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www.elsevier.com/locate/brainres

Research Report

Differential distribution of group I metabotropic glutamate receptors in developing human cortex Karin Boer a , Ferechte Encha-Razavi b , Martine Sinico c , Eleonora Aronica a,d,⁎ a

Department of (Neuro)Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Service Histologie-Embryologie-Cytogénétique, Groupe Hospitalier Necker Enfants-Malades, Paris Cedex, France c Service d'anatomie pathologique, CHI de Créteil, Creteil, France d Epilepsy Institute in The Netherlands Foundation (Stichting Epilepsie Instellingen Nederland, SEIN), Heemstede, The Netherlands b

A R T I C LE I N FO

AB S T R A C T

Article history:

Neuronal and glial cells in human cerebral cortex are enriched in group I metabotropic

Accepted 3 February 2010

glutamate receptors (mGluRs). Developmental regulation of mGluRs has been shown in

Available online 10 February 2010

rodent brain and recent studies suggest an involvement of mGluR-mediated glutamate signaling in the proliferation and survival of neural progenitor cells. In the present

Keywords:

study, we have investigated the expression and cell-specific distribution of group I

Metabotropic glutamate receptor

mGluRs (mGluR1α and mGluR5) during prenatal human cortical development. mGluR5

Human cortex

was expressed in developing human cortex from the earliest stages tested (9 gestational

Cortical development

weeks, GW), with strong expression in the ventricular/subventricular zones. mGluR1α

Immunocytochemistry

immunoreactivity (IR) was observed in the cortical plate at 13 GW and persisted

mGluR1

throughout the prenatal development. Both receptors were expressed in pyramidal

mGluR5

neurons in the first postnatal year. Group I mGluRs were also expressed by reelinpositive Cajal–Retzius cells present in the marginal zone/layer I of the developing cortex. mGluR5 IR in these cells was observed in the earliest developmental stages and persisted during the early postnatal period. In contrast, mGluR1α IR was detected in Cajal–Retzius cells during the late phase of prenatal development. These findings show a differential expression pattern of group I mGluR subtypes, suggesting a role for both receptors in the early stages of corticogenesis with, however, a different contribution to human cortical developmental events. © 2010 Elsevier B.V. All rights reserved.

1.

Introduction

Recent experimental evidence suggests critical functions for metabotropic glutamate receptors (mGluRs) during cortical development (Di Giorgi Gerevini et al., 2005; Di Giorgi Gerevini et al., 2004; Schlett, 2006). In particular, a growing body of evidence indicates that mGluR may support basic developmental

processes, such as proliferation, differentiation and survival of neural progenitors (for review see, Catania et al., 2007). The mGluR family includes eight subtypes that have been subdivided into three main groups on the basis of their sequence, second messenger systems and pharmacological profile. Group I includes mGluR1 and mGluR5, which are coupled to phosphoinositide (PI) hydrolysis, whereas the other subtypes

⁎ Corresponding author. Dep. (Neuro) Pathology, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Fax: +31 20 5669522. E-mail address: [email protected] (E. Aronica). 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.02.005

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are negatively coupled to adenylyl cyclase (Conn and Pin, 1997; Pin and Acher, 2002; Recasens et al., 2007). Several lines of evidence support in particular the role of group I mGluRs in brain development and developmental disorders (for review see (Catania et al., 2007)). PI hydrolysis induced by activation of group I mGluRs is substantial in developing rat brain (Casabona et al., 1997; Catania et al., 2007). mGluR5 mRNA is highly expressed in rat brain during the early postnatal development (Catania et al., 1994), and although progressively decreasing afterwards, it remains high in the adult olfactory bulb, a zone of active neurogenesis in the postnatal rat brain (Minakami et al., 1995; Romano et al., 1996). A more recent study demonstrated expression of the mGluR5 subtype in zones of active neurogenesis in the embryonic and postnatal brain (Di Giorgi Gerevini et al., 2004). In vitro studies support the role of mGluR5 in the maintenance of the undifferentiated state of mouse embryonic stem cells (Cappuccio et al., 2005; Spinsanti et al., 2006) and indicate that group I mGluR subtypes exert different functions in the regulation of neural progenitors cell proliferation and survival (Castiglione et al., 2008; Di Giorgi Gerevini et al., 2005). In particular, the study of Castiglione et al. (Castiglione et al., 2008), suggest that mGluR5 might support specifically the survival of progenitors undergoing differentiation into neurons, whereas mGluR1 might sustain the proliferation of earlier progenitors. Interestingly, group I mGluR expression has also been shown during development in the Cajal–Retzius cells (Lopez-Bendito et al., 2002), a population of transient neurons that critically regulate the laminar organization of the neocortex (Meyer et al., 1999; Mienville, 1999). The expression pattern and cellular localization of mGluRs during corticogenesis in human brain remains uncharacterized. In the present study we examined the expression of group I mGluR subtypes (mGluR1α and mGluR5) in the developing human cerebral cortex. Information about the cellular distribution of mGluRs during human corticogenesis is important to get better insights into the role of the different receptor subtypes in prenatal human development.

2.

Table 1 – Comparison of mGluR1α and mGluR5 expression in developing human cortex. mGluR1α

mGluR5

Age

MZ

CP

VZ

MZ

CP

VZ

9–10 GW 13–17 GW 20–25 GW 20–31 GW 36–40 GW 3 w–7 m 8 years

− − − − + + −

+ + + + + + +

− − − − − − −

+ + + + + + −

+ + + + + + +

+ + + + + − −

GW, gestational weeks; w, weeks; m, months; MZ, marginal zone (Cajal–Retzius cells); CP, cortical plate; VZ, ventricular zone.

tion of these results. One major limitation in studies using post-mortem fetal tissue is the availability of brain tissue. An ideal experimental design with a large number samples from the same prenatal ages is difficult to achieve. The number of cases with permission for brain autopsy at early developmental stages is limited and these cases include often cerebral pathologies. Moreover, frozen representative material is not available in all cases. Thus it was not possible to perform in parallel immunocytochemical and western blot analysis at different developmental ages.

2.2.

mGluR1α shows expression in the cortical plate (CP)

At 9 weeks we did not detect significant levels of immunoreactivity (IR) for mGluR1α (Fig. 2 A and B). At all the prenatal ages examined (GW 9–36) the neuroepithelium of the ventricular zone (VZ) was mGluR1α negative; also the subventricular zone (SVZ), the intermediate zone (IZ) and the subplate (SP) did not display detectable mGluR1α IR (Fig. 2 A, C, E, G, I). In contrast, expression was detected at GW 13 in the CP with a radial staining pattern, which was also observed at 17 GW (Fig. 2 D and F). The mGluR1α IR in the cortex increases at older ages

Results

2.1. Temporal mGluR1α and mGluR5 expression during human cortical development The expression pattern of group I mGluRs was studied immunocytochemically at different prenatal ages, 9, 10, 13, 16, 17, 20, 22, 23, 25, 29, 31, 36 and 40 GW, as well as at postnatal ages of 3 weeks, 2 months, 7 months and 8 years (Table 1). Western blot analysis could be only performed in 3 selected cases (13 GW, 2 months postnatally and adult cortex, 30 yrs) of which frozen cerebral cortex material was available (Fig. 1). mGluR1α and mGluR5 receptor proteins were both detected as a band of approximately 140 kDa, corresponding to receptor monomers (expected range 130–150 kDa). As previously reported (Aronica et al., 2001b; Casabona et al., 1997), the mGluR1α antibody labeled an additional higher molecular weight band which corresponds to receptor dimers (not shown). Expression of both receptors was detected at all ages examined, however their expression appeared to decrease to similar level postnatally (Fig. 1). We acknowledge limitations to the interpreta-

Fig. 1 – Western blot analysis of mGluR5 and mGluR1α in total homogenates of temporal cortex at 13 gestational weeks (GW), 2 months postnatally and adult temporal cortex (30 yrs). The expression of protein β-actin (43 kDA) and synaptophysin (Syn; 38 kDa) are shown in the same protein extracts.

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and immunopositive neuronal cell bodies could be detected in cortical layers II–VI (Fig. 2 H, J and K). In agreement with previous observations, mGluR1α IR was mainly seen in neurons with a diffuse somatodentritic distribution in human adult specimens (Aronica et al., 2001c; Ong et al., 1998).

2.3. mGluR5 shows strong expression during early cortical development The expression of mGluR5 was intense and diffuse already at 9 GW (Fig. 3A–C). Immunolabeling was observed in the VZ and the SVZ (Fig. 3 B), as well as within the CP and the SP/IZ (Fig. 3 A and C). Strong expression in the CP was also detected at 13 and 17 GW and was characterized by radially oriented processes (Fig. 3 F, I). Around mid gestation, mGluR5 was observed in the neuropil and in cell somas displaying pyramidal shapes and this pattern of IR (with cytoplasmatic neuronal labeling) persisted at later prenatal and postnatal ages (Fig. 3 K, M and N), in agreement with previous studies in human control specimens (Aronica et al., 2001a; Oka and Takashima, 1999; Tang et al., 2001). The mGluR5 IR in the VZ was prominent at 13 and 17 GW and present in radially oriented fibers, extending through the SVZ; mGluR5 IR was also detected in horizontally oriented fibers within the SVZ and IZ at both ages (Fig. 3 D–E and G–H). Double-labeling experiments with the proliferation marker Ki67 indicated that mGluR5-positive cells in the VZ at early stages of development (9–17 GW) are capable of dividing (Fig. 4 A–C). In addition, we showed co-localization of mGluR5 with neuronal precursor markers (nestin and vimentin) within the VZ (Fig. 4 D–I). At 23 GW, the VZ cells still expressed mGluR5 IR. At older prenatal ages mGluR5 IR was observed in the ependymal zone, as well as in the subependymal zone in clusters of cells variably oriented (SE; Fig. 3 L). Postnatally, mGluR5 immunopositive neuronal cell bodies were present in the cortical layers II–VI (Fig. 3 M–N).

2.4. Group I mGluRs are transiently expressed by Cajal–Retzius cells In the early cortical plate stages (9–17 GW) mGluR5, but not mGluR1α, was expressed in the marginal zone (MZ) by Cajal– Retzius cells, a population of cells that are strongly reelin immunoreactive (Fig. 4 J–O). The expression of mGluR5 was still observed in Cajal–Retzius cells in the perinatal and early postnatal period (<1 year; Fig. 4 P–S); whereas mGluR1α IR in Cajal–

Retzius cells was only detected at 36 GW (data not shown) and early postnatally in residual reelin-positive Cajal–Retzius cells in layer I (Fig. 4 T). However, during postnatal development (>1 year) and adult cortical control specimens, as well as in selected specimens of adult patients with focal cortical dysplasia (FCD) type I (with increased number of reelin-positive cells in layer I; (Garbelli et al., 2001; Thom et al., 2003)) we could not detect any co-localization of reelin and group I mGluRs (data not shown).

3.

Discussion

The development of the human cerebral cortex depends on a precisely orchestrated cascade of events, including proliferation, migration and maturation of neural progenitor cells (Rakic and Lombroso, 1998). Recently, activation of mGluR subtypes by glutamate has been shown to be involved in these developmental processes, supporting proliferation, differentiation, and survival of neural progenitor cells (Catania et al., 2007). Immunocytochemical studies of post-mortem tissue represent one of the few available approaches for studying neurotransmitter receptor expression during human brain development, providing information about their cellular distribution and location that can be used to interpret functional experimental data. This study provides the first description of the expression pattern and cellular localization of group I mGluRs in human neocortex during the pre- and early postnatal development. The period of prenatal development studied (9–40 GW) includes all the critical stages of human cortical development (proliferation, migration and maturation/organization) which are involved in the formation and differentiation of the CP to reach the six-layered structure of mature cerebral cortex (Kostovic and Rakic, 1990; Rakic and Lombroso, 1998). During the period of CP formation (9–17 GW), mGluR1α showed a progressive increase in expression in radially oriented processes of neural cells, positioned in the CP. Expression of mGluR1 mRNA and protein, with localization of the protein in the CP, has been previously shown in developing rat central nervous system (Lopez-Bendito et al., 2002; Shigemoto et al., 1992). The human observations reported here indicate a more prominent expression of mGluR1α during the early prenatal cortical development. At about 13 GW, the consistent expression observed with immunocytochemistry within the CP was confirmed by western blot analysis, revealing

Fig. 2 – mGluR1α immunoreactivity (IR) at different ages (9, 13, 17, 23, and 36 gestational weeks (GW)) and at 2 and 7 months postnatally. Panels A–B: 9 GW. A: detectable mGluR1α IR is not observed in the VZ, SVZ, SP/IZ and CP. B: high magnification photograph shows the absence of mGluR1α IR in the VZ. Panels C–D: 13 GW. C: detectable mGluR1α expression is not observed in the SVZ and the VZ (insert in C). D: mGluR1α IR is detected in the CP with positive radial processes (insert in D). Panels E–F: 17 GW. E: detectable mGluR1α expression is not observed in the SVZ and the VZ. F: the mGluR1α IR in the CP increases, with clear positive processes (insert in F). Panels G–H: 23 GW. G: detectable mGluR1α expression is not observed in the SVZ and the VZ. H: mGluR1α cytoplasmic IR is observed in neuronal cells within the CP. Panel I: 36 GW: detectable mGluR1α expression is not observed in the subependymal zone (SE). Panels J–M: at 2- (J) and 7- (K) months, as well as in adult cortex (L–M; 30 years), mGluR1α IR is observed in pyramidal cells of the postnatal cerebral cortex. Panel P: mGluR1α IR in human adult cerebellum, showing strong IR in Purkinje cells. Pre-absorption of primary antibody with the synthetic peptide (N, Q) or omission of the primary antibody (O) resulted in an absence of IR. VZ: ventricular zone; SVZ: subventricular zone; SP/IZ: subplate/intermediate zone; CP: cortical plate. Scale bar in A: A, C–F, I: 100 µm; B, K: 30 µm; G: 200 µm; H: 80 µm; J: 60 µm; L, N–Q: 80 µm; M: 40 µm.

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relative high expression levels. Throughout the prenatal human cortical development, mGluR1α could not be detected in neuroepithelial cells of the VZ/SVZ. This is in agreement with previous immunocytochemical studies in rat brain, showing no detectable mGluR1α IR in zones of active neurogenesis (Di Giorgi Gerevini et al., 2004; Lopez-Bendito et al., 2002). Expression of a functional mGluR1 was, however, observed in neurospheres obtained from SVZ of adult mouse (Castiglione et al., 2008). To our knowledge, expression and function of mGluR subtypes have not been studied in human neurospheres. mGluR5 showed a prominent expression in the developing human cortex from the early stages of cortical plate formation (9 GW). mGluR5 was expressed in radially arranged cellular processes during the period of CP formation and later in neuropil and neocortical neuronal somata. Thus, in the later prenatal development both group I subtypes are expressed in pyramidal cells. The high levels of mGluR5 receptor expression observed at about 13 GW by western blot analysis in total cortical homogenates may reflect the expression of mGluR5 in the deep compartments of the cortical wall (VZ/SVZ and IZ) observed at early stages of corticogenesis. In both the VZ and the SVZ actively proliferating progenitors cells expressed mGluR5 (co-localization with Ki67) and co-localization was also observed with neural progenitors markers, such as vimentin and nestin within zones of active neurogenesis. These observations are in line with the high expression in developing rodent central nervous system (Catania et al., 1994; LopezBendito et al., 2002; Shigemoto et al., 1992) as well as in zones of active neurogenesis in the postnatal rat brain (Di Giorgi Gerevini et al., 2004; Minakami et al., 1995; Romano et al., 1996). An attractive suggestion, provided by in vitro studies using neurospheres, is that activation of mGluR5 may positively regulate proliferation and survival of neural progenitor cells (Cappuccio et al., 2005; Castiglione et al., 2008; Di Giorgi Gerevini et al., 2005; Spinsanti et al., 2006). In particular activation of mGluR5 has been shown to specifically support the survival of progenitors undergoing differentiation into neurons (Castiglione et al., 2008). This regulation may involve the generation of oscillatory increases in intracellular calcium waves induced by activation of mGluR5, but not mGluR1 (Kawabata et al., 1998). In addition, regulation of cell proliferation may involve other pathways, such as the mitogenactivated protein kinase (MAPK) signaling cascade (Mao et al., 2005) and the maintenance of the undifferentiated state of stem cells has been suggested to involve an interaction with

the leukaemia inhibitory factor (LIF), regulating the expression of c-Myc (Spinsanti et al., 2006). Interestingly, group I mGluRs were also expressed by Cajal– Retzius cells. They represent a population of cells, present in the marginal zone (future layer I) during the early stages of human neocorticogenes, which critically regulate the organization of the layering of the CP (Frotscher, 1998; Meyer et al., 1999). As the cortical development proceeds, the marginal zone (MZ) becomes layer I, the number of Cajal–Retzius cells progressively decreases during early postnatal stages and Cajal– Retzius cells are rarely detected in adult cortex (Fonseca and Soriano, 1995; Martin et al., 1999). Reelin expression in large cells has been shown to detect a phenotype restricted to the period of cortical migration (Meyer et al., 1999). Our observational study shows expression of mGluR5 by large reelinpositive Cajal–Retzius cells in the MZ during the early stages of human corticogenesis. In contrast, mGluR1α IR was detected only transiently in residual reelin-positive cells at late prenatal and early postnatal ages. A similar sequence of expression has been reported in rat brain with expression of mGluR5, but not mGluR1α, in Cajal–Retzius cells present in the MZ during the early stages of corticogenesis (Lopez-Bendito et al., 2002). In addition, Cajal–Retzius cells have been shown to express functional mGluRs in early postnatal mouse cortex (MartinezGalan et al., 2001). In our study both receptor subtypes were expressed in residual large reelin-positive Cajal–Retzius cells in layer I during the first year of life. Since an increased number of reelin-positive Cajal–Retzius cells in layer I has been previously reported in patient with focal cortical dysplasia and microdysgenesis (Garbelli et al., 2001; Thom et al., 2003), we analyzed few selected adult cases of FCD type I with residual Cajal–Retzius cells, however, we could not show detectable mGluR1α or mGluR5 IR in these cases. Whether activation of group I mGluRs in Cajal–Retzius cells (in particular mGluR5) during the critical period of cortical organization regulates the production and/or secretion of reelin trough intracellular calcium signaling is an attractive hypothesis which requires further investigation. In summary, we have shown that the mGluR1α and mGluR5 expression and cellular distribution is differentially regulated during human cortical development. This differential expression is consistent with the recent experimental studies suggesting a prominent role for mGluR5 in the regulation of proliferation, differentiation and survival of neural progenitor cells. In addition we confirmed the expression of group I mGluRs by Cajal–Retzius cells in human brain,

Fig. 3 – mGluR5 immunoreactivity (IR) at different ages (9, 13, 17, 23, and 36 gestational weeks (GW)) and at 2 and 7 months postnatally. Panels A–C: 9 GW. A: mGluR5 IR is observed in the VZ, SVZ and the CP. High magnification photographs show mGluR5 positive cells in the VZ (B, insert in B) and in the CP (C). Panels D–F: 13 GW. The number of mGluR5 immunoreactive cells in the SVZ (D) and VZ (D, E), as well as the positive radial processes in the CP (F), increases. Insert in F shows mGluR5 IR in large cells with Cajal–Retzius-like morphology (arrows) located in the marginal zone (MZ). Panel G–I: 17 GW with strong mGluR5 IR in the SVZ (G) and VZ (G, H) and the CP (I). Panels J–K: 23 GW with strong mGluR5 in the VZ (J) and the CP (K). Panel L: 36 GW: the subependymal zone (SE) contains mGluR5 positive cells (high magnification in insert). Panels M–P: at 2- (M) and 7- (N) months, as well as in adult cortex (O–P; 30 yrs), mGluR5 IR is observed in pyramidal cells of the postnatal cerebral cortex. Panel S: mGluR5 IR in human adult cerebellum. Pre-absorption of primary antibody with the synthetic peptide (Q, T) or omission of the primary antibody (R) resulted in an absence of IR. VZ: ventricular zone; SVZ: subventricular zone; SP/IZ: subplate/intermediate zone; CP: cortical plate. Scale bar in A: A, D, G: 100 µm; B, C, E, F, H, I, N: 30 µm; J–M: 60 µm; Q–T: 80 µm; P: 40 µm.

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supporting the potential role of these receptor subtypes in the regulation of Cajal–Retzius cell functions during human corticogenesis.

4.

Experimental procedures

4.1.

Human material

The subjects included in this study were obtained from the databases of the Department of Neuropathology of the Academic Medical Center (University of Amsterdam; UVA) in Amsterdam, the Netherlands and the Service HistologieEmbryologie-Cytogénétique Hôpital Necker-Enfants malades, Paris, France. Informed consent was obtained for the use of brain tissue and for access to medical records for research purposes. Tissue was obtained and used in a manner compliant with the Declaration of Helsinki. The expression of mGluR1α and mGluR5 was evaluated at the following ages: 9, 10, 13, 16, 17, 20, 22, 23, 25, 29, 31, 36 and 40 gestational weeks (GW) obtained from spontaneous or medically induced abortions with appropriate maternal written consent for brain autopsy. Additionally, we obtained normal-appearing control cortex/white matter at autopsy from 4 controls (3 weeks, 7 months, 8 years and 30 years), without a history of neurological disease. Surgical material obtained from 3 patients (age: 16, 20 and 28 yrs) with focal cortical dysplasia (FCD) type I (Palmini et al., 2004) was also included. All autopsies were performed within 12 h after death.

4.2.

Tissue preparation

Tissue was fixed in 10% buffered formalin and embedded in paraffin. Paraffin-embedded tissue was sectioned at 6 µm, mounted on pre-coated glass slides (StarFrost, Waldemar Knittel Glasbearbeitungs GmbH, Braunschweig, Germany) and used for immunocytochemical staining as described below.

4.3.

Antibody characterization

For the detection of group I mGluRs, we used antibodies specific for the mGluR subtypes 1α (polyclonal rabbit, Chemicon, Temecula, CA; 1:100; raised in rabbit against a 20-amino acid peptide, PNVTYASVILRDYKQSSSTL, corresponding to the C-terminus of mGluR1α) and 5 (polyclonal rabbit, Upstate Biotechnology, Lake Placid, NY; 1:100; raised against a 21-residue peptide (KSSPKYDTLIIRDYTNSSSSL) corresponding to the C-terminal of mGluR5). Characterization of these Abs in human brain tissue has been documented previously (Aronica et al., 2001b; Aronica et al., 2001c; Aronica et al., 2003; Boer et al., 2008; Geurts et al., 2003; Geurts et al., 2005). Pre-absorption of an optimal concentration (1:100) of primary antibody with an excess of synthetic peptide or omission of the primary antibody resulted in an absence of immunoreactivity (Figs. 1 and 2).

4.4.

Immunocytochemical analysis

For single-labeling, paraffin-embedded sections were deparaffinized, re-hydrated, and incubated for 20 min in 0.3% H2O2

diluted in methanol to quench the endogenous peroxidase activity. Antigen retrieval was performed by incubation for 10 min at 121 °C in citrate buffer (0.01 M, pH 6.0). Sections were washed with phosphate-buffered saline (PBS), and incubated for 30 min in 10% normal goat serum (Harlan Sera-Lab, Loughborough, Leicestershire, UK). After incubation with the primary antibodies overnight at 4 °C, the sections were washed in PBS and we used the ready-for-use Powervision peroxidase system (Immunologic, Duiven, The Netherlands) and 3,3′diaminobenzidine (DAB; Sigma) as chromogen to visualize the antibodies. Sections were counterstained with hematoxylin, dehydrated and coverslipped. Sections incubated without the primary antibody were essentially blank. For double-labeling studies we combined mGluR5 (or mGluR1α) with Ki67 (mouse clone MIB-1; DAKO; 1:200), nestin (monoclonal mouse, MAB1259; R&D Systems, Abingdon UK), vimentin (mouse clone V9; DAKO; 1:1000) or reelin (monoclonal mouse, Calbiochem, San Diego, CA; 1:500). After incubation overnight at 4 °C with the primary antibodies, sections were incubated for 2 h at room temperature with Alexa Fluor® 568conjugated anti-rabbit IgG and Alexa Fluor® 488 anti-mouse IgG (1:100, Molecular Probes, The Netherlands). Sections were mounted with Vectashield containing DAPI (targeting DNA in the cell nucleus; blue emission) and analyzed by means of a laser scanning confocal microscope (Leica TCS Sp2, Wetzlar, Germany).

4.5.

Western blot analysis

For immunoblot analysis we used frozen brain specimens of 13 GW, 2 months postnatally and histologically normal adult cortex (30 yrs). We carefully resected two adjacent specimens of the temporal cortex (including the cortical plate and the VZ in the 13 week brain and equal grey/white matter tissue components for the 2 months and adult brain); one specimen was immediately snap frozen and the other was routinely processed for histological and immunocytochemical examination, as described above. The frozen specimens were homogenized in lysis buffer containing 10 mM Tris (pH 8.0), 150 mM NaCl, 10% glycerol, 1% NP-40, 0.4 mg/ml Na-orthevanadate, 5 mM EDTA (pH 8.0), 5 mM NaF and protease inhibitors (cocktail tablets, Roche Diagnostics, Mannheim, Germany). Protein content was determined using the bicinchoninic acid method (Smith et al., 1985). For electrophoresis, equal amount of proteins (50 μg/lane) were separated by sodium dodecylsulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis (7.5% acrylamide). Separated proteins were transferred to nitrocellulose paper by electroblotting for 1 h and 30 min (BioRad, Transblot SD, Hercules, CA). After blocking for 1 h in TBST (20 mM Tris, 150 mM NaCl, 1% Tween, pH 7.5)/5% non fat dry milk, blots were incubated overnight at 4 °C with rabbit anti-mGluR1α or -mGluR5 (both 1:750), or mouse anti-β-actin (clone AC-15, Sigma; 1:50,000) and rabbit anti-synaptophysin (DAKO; polyclonal rabbit, 1:2000). After several washes in TBST, the membranes were incubated in TBST/5% non fat dry milk, containing horseradish peroxidase (HRP)-labeled goat anti-rabbit or anti-mouse (Dako; 1:2500) for 1 h at room temperature. Immunoreactivity was visualized using Lumi-light PLUS western blotting substrate (Roche Diagnostics, Mannheim, Germany) and digitized using a Luminescent Image Analyzer (LAS-3000, Fuji Film, Japan).

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Fig. 4 – Confocal double-immunofluorescence analysis of mGluR5 in fetal brain. Confocal images. Panels A–C and G–H: 13 GW; D–F and J–L: 17 GW; M–O: 36 GW and P–Q: 7 months. Panels A–C: proliferating cells (with Ki67 positive nuclei; green, A) expressing mGluR5 (red, B) in the ventricular zone; C: merged image (high magnification in insert). Panels D–F: nestin positive cells (green, D) expressing mGluR5 (red, E) in the VZ. Panel F: merged image (high magnification in insert). Panels G–I: vimentin positive cells (green, G) expressing mGluR5 (red, H) in the VZ. Panel I: merged image (nuclei were stained with DAPI; blue). Panels J–T: Cajal–Retzius cells (reelin-positive; green) expressing mGluR5 (red) in the molecular zone (MZ) at 13 GW (J–L), 17 GW (M–O), 36 GW (P–R) and 7 months (S). Panels L, O, R, S: merged images (insert in O: high magnification). Insert in L shows no detectable expression of mGluR1α (red) in Cajal–Retzius cells (reelin-positive; green) in the MZ at 13 GW. Panel T: merged image showing co-localization of reelin (green) and mGluR1α (red) in residual Cajal–Retzius cells at 7 months. Scale bar in A: A–F, J–L: 50 µm; G–I: 20 µm; M–Q: 15 µm.

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Acknowledgments This work was supported by the National Epilepsy Fund (NEF 05-11) and Stichting Michelle (M07.016).

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