Immunohistochemical localization of the vesicular glutamate transporter VGLUT2 in the developing and adult mouse claustrum

Journal of Chemical Neuroanatomy 31 (2006) 169–177 www.elsevier.com/locate/jchemneu

Immunohistochemical localization of the vesicular glutamate transporter VGLUT2 in the developing and adult mouse claustrum ´ ngeles Real, Jose´ Carlos Da´vila, Salvador Guirado * Ma A Department of Cell Biology, University of Ma´laga, 29071 Ma´laga, Spain Received 28 July 2005; received in revised form 22 December 2005; accepted 22 December 2005 Available online 24 January 2006

Abstract We studied the immunoreactive expression pattern for the vesicular glutamate transporter VGLUT2 in the embryonic, postnatal and adult mouse dorsal claustrum, at the light and electron microscopic levels. VGLUT2 immunoreactivity in the dorsal claustrum starts to be observed at E16.5, with a dramatic increase towards P0. At this age, abundant VGLUT2-immunoreactive axons and puncta are observed in all pallial regions, including the claustral complex. From the first postnatal week, VGLUT2 immunoreactivity declines in several telencephalic areas, including the pallium, but abundant VGLUT2-immunoreactive fine axons and puncta remain in the claustrum. Beginning at E18.5, VGLUT2 immunoreactivity within the claustrum shows a characteristic arrangement: a central part of the region is practically devoid of VGLUT2 immunoreactivity, and it is surrounded by plenty of immunoreactive axon terminals forming a shell around it. This core/shell arrangement of the VGLUT2 immunoreactivity resembles the complementary expression of parvalbumin and calretinin described in the mouse claustrum [Real, M.A., Da´vila, J.C., Guirado, S., 2003. Expression of calcium-binding proteins in the mouse claustrum. J. Chem. Neuroanat. 25, 151–160]. We observed immunoreactive neuronal cell bodies as well in the dorsal claustrum, but only at P0. Electron microscopic analysis reveals that VGLUT2 immunoreactivity in the developing and adult dorsal claustrum consists predominantly of presynaptic boutons making asymmetric synaptic contacts. These VGLUT2-immunoreactive boutons are observed as early as E16.5 and may be related to thalamo-claustral incoming fibers. # 2006 Elsevier B.V. All rights reserved. Keywords: Glutamate; Lateroventral pallium; Development; Neuroanatomy; Neurotransmission

1. Introduction The claustrum is a phylogenetically conserved, poorly understood, pallial territory in mammals. Based on anatomical and topographical criteria, two parts of the claustrum can be distinguished: the dorsal part is usually called claustrum proper and it is located deep to the insular cortex (therefore, it is also named dorsal or insular claustrum); the ventral part is called endopiriform nucleus and is located deep to the piriform cortex (Druga, 1966; Sherk, 1988; Dinopoulos et al., 1992).

Abbreviations: ac, anterior commissure; AI, agranular insular cortex; Cl, claustrum; CPu, caudate-putamen; CxP, cortical plate; ec, external capsule; En, endopiriform nucleus; Ins, insular cortex; lot, lateral olfactory tract; Pir, piriform cortex * Corresponding author at: Departamento de Biologı´a Celular, Gene´tica y Fisiologı´a, Facultad de Ciencias, Campus de Teatinos, Universidad de Ma´laga, 29071 Ma´laga, Spain. Tel.: +34 952131961; fax: +34 952132000. E-mail address: [email protected] (S. Guirado). 0891-0618/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2005.12.002

These two parts of the claustrum show distinct sets of connections with the neocortex or the piriform and entorhinal cortices (Druga, 1971; Markowistch et al., 1984; Witter et al., 1988; Clasca´ et al., 1992; Dinopoulos et al., 1992; Majak et al., 2002). Besides, the dorsal claustrum and the endopiriform nucleus have been proposed to originate from distinct histogenetic compartments of the lateroventral pallium. On the basis of their different expression of the homeobox gene Emx1 and other developmental regulatory genes, it has been suggested that the claustral complex is subdivided into regions that derive from either the lateral or the ventral pallial histogenetic divisions (Puelles et al., 2000; Medina et al., 2004). We have recently analyzed a number of neuronal markers that, as a whole, provide a good picture of the inhibitory structures in the developing and adult mouse claustrum, including GABA, three calcium-binding proteins, and the neuronal nitric oxide synthase (Guirado et al., 2003; Real et al., 2003; Da´vila et al., 2005). Nevertheless, to further characterize

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the neurochemical organization of the claustrum it is obviously needed to study the excitatory glutamatergic neurotransmission in the region. Recently, two vesicular glutamate transporters, VGLUT1 and VGLUT2 (formerly known as brain-specific Na+-dependent inorganic phosphate cotransporter and differentiation-associated Na+-dependent inorganic phosphate cotransporter; BNPI and DNPI, respectively), have been identified and together seem to cover all glutamatergic pathways in the CNS (Ni et al., 1995; Bellocchio et al., 1998, 2000; Aihara et al., 2000; Takamori et al., 2000; Fremeau et al., 2001; Sakata-Haga et al., 2001; Kaneko and Fujiyama, 2002; Kaneko et al., 2002). The expression of VGLUT1 and 2 mRNA transcripts appears complementary. VGLUT1 is massively present in excitatory neurons from the cerebral and cerebellar cortices, as well as the hippocampus, whereas most glutamatergic neurons from the diencephalon and rhombencephalon preferentially use VGLUT2 (Fremeau et al., 2001; Herzog et al., 2001; Varoqui et al., 2002). At the subcellular level, VGLUT1 and VGLUT2 are found in synaptic vesicles located in terminals forming asymmetrical contacts (Bellocchio et al., 1998; Fremeau et al., 2001; Fujiyama et al., 2001; Hayashi et al., 2001; Sakata-Haga et al., 2001; Takamori et al., 2001; Varoqui et al., 2002), the hallmark of the glutamatergic terminals (Shepherd and Greer, 1998). The complementary expression of VGLUT1 and VGLUT2 accounts for the release of glutamate by all known excitatory neurons (Fremeau et al., 2002), although another vesicular glutamate transporter, VGLUT3, is expressed by many cells traditionally considered to release a different classical transmitter (see, Fremeau et al., 2004). After some preliminary studies on the expression pattern of VGLUT1 in the murine brain, which resulted in a homogeneous non-area-specific immunostaining of the claustrum (non-published results), we decided to concentrate our efforts in studying the neurochemical distribution of the VGLUT2 protein in the developing and adult mouse dorsal claustrum, at the light and electron microscopic levels.

2.2. Immunohistochemistry Brain sections were processed for immunohistochemistry following a standard procedure. Briefly, free floating sections were first incubated in 0.03% hydrogen peroxide (H2O2) in PBS for 20 min to eliminate the endogenous peroxidase, washed, and incubated in PBS containing 2% normal goat serum and 0.3% Triton X-100 for 1 h to block non-specific binding of the antibodies and permeate the tissues. Then, sections were incubated in the primary antibody for 48–72 h at 4 8C. The primary antiserum was an antiVGLUT2 raised in guinea-pig (Chemicon International; diluted 1:5000; see for example, Kiss et al., 2003; Todd et al., 2003). After extensive washes in PBS, the sections were incubated with a biotinylated goat anti-guinea-pig IgG (Vector Laboratories Inc.; diluted 1:500) for 1 h, washed again in PBS, and incubated in ExtrAvidin-Peroxidase (Sigma; diluted 1:2000) for 1 h. The immunolabeling was revealed with 0.05% diaminobenzidine (DAB, Sigma), 0.05% nickel– ammonium sulphate and 0.03% hydrogen peroxide (H2O2) in PBS. All steps were carried out at room temperature with gentle agitation. After a thorough wash in PBS, the sections were mounted on gelatinized slides, air-dried, dehydrated in ethanol, cleared in xylene, and cover-slipped with DPX (BDH, Poole, UK). From each animal, 4–5 vibratome brain sections were selected for electron microscopy and processed following an immunohistochemical protocol similar to that described above, except that Triton X-100 was not included in the solutions, and the peroxidase reaction was performed without nickel–ammonium sulphate. After the DAB reaction, the sections were osmicated (1% OsO4 in distilled water), dehydrated through a graded series of acetone, contrasted in 1% uranyl acetate, and flat-embedded between two aluminum sheets in Araldite 502 (Sigma). After examination in the light microscope, areas of interest were trimmed and glued on the top of resin polymerized blocks, and then sectioned at 70–90 nm by using an ultramicrotome. As a control of the immunohistochemical method used in the present study, sections were processed as indicated but the corresponding primary antiserum was replaced by guinea-pig normal serum (1:500). No immunostaining could be detected under these conditions.

2.3. Image acquisition Light microscopic images were photographed by using a Leica microscope equipped with a Nikon DXM1200 digital camera. Digital images were loaded into Adobe Photoshop software (Adobe Systems, Mountain View, CA) and converted to grey-scale images. Electron microscopic images were taken by using a Philips EM-100 loaded with Kodak EM film. Electron microscopic black and white negatives were scanned as grey-scale images, and inverted in Adobe Photoshop software. Brightness and contrast were adjusted for the final images. No additional filtering or manipulation for the images was performed. Figures were mounted and labeled by using Adobe PageMaker software.

2. Materials and methods

3. Results 2.1. Animals For the present study we used mouse embryos of E14.5, E16.5, and E18.5; neonates of P0, P7, and P14; and adult mice (3–4 months). Three animals from each stage were studied. Throughout the experimental work, animals were treated according to the European Communities Council Directive (86/609/ EEC) for care and handling of animals in research. Pregnant female mice (OF1 strain) were deeply anesthetized with diethyl ether, and fetuses were removed by caesarean section, cold anesthetized, and fixed by immersion in 4% paraformaldehyde, 0.075 M lysine, and 0.01 M periodate in 0.1 M phosphate buffer, pH 7.4 (PLP; McLean and Nakane, 1974), at 4 8C. After 1 day of fixation, fetal brains were removed from the cranium and post-fixed for another day in fresh fixative solution. Postnatal and adult mice were anesthetized with diethyl ether and then perfused transcardially with saline solution, followed by the PLP fixative. Brains were removed and post-fixed overnight at 4 8C in fresh fixative solution. After extensive washes in 0.1 M phosphate-buffered saline (PBS, pH 7.4), the brains were embedded in 4% agarose, and sectioned at 50 mm-thick by using a vibratome.

We describe in this paper the expression pattern for the VGLUT2 in the developing, from E14.5 until P14, and in the adult mouse dorsal claustrum. A moderate VGLUT2 immunoreactivity is already observed throughout the pallium (including the ventral most region) as early as E14.5, although the incipient dorsal claustrum begins to be clearly recognizable anatomically at E15.5 (Fig. 1(A)–(C)). From this age on, the dorsal claustrum could be easily distinguished as a cell aggregate localized deep to the insular cortical plate in Nisslstained sections (Fig. 1(C), (D) and (G)). At E16.5, a general increase in VGLUT2 immunoreactivity is observed. Numerous VGLUT2-immunoreactive (VGLUT2ir) varicose axons are found around the external capsule, but only a few of immunoreactive structures (mostly consisting of immunopositive puncta) are observed within the claustral

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Fig. 1. Photomicrographs of transverse sections through the mouse telencephalon at E14.5 (A and B), E16.5 (E and F), E18.5 (H and I), and P0 (J and K) showing VGLUT2 immunohistochemistry in the claustrum. Panels (C), (D) and (G) are Nissl-stained sections of the rostral telencephalon at E15.5, E16.5, and E18.5, respectively. Panel (F) is a detail of the boxed area in (E). Panels (I) and (K) are higher magnification photomicrographs of the claustrum from the sections showed in (H) and (J), respectively. Asterisk marks the core region of the claustrum in panels (I) and (K). See text for more details. For abbreviations see list. For all photomicrographs in figures, dorsal is to the top and medial is to the left. Scale bars: (A, E, H and J) 400 mm; (B–D and G) 200 mm; (F, I and K) 100 mm.

complex (Fig. 1(E) and (F)). A moderate VGLUT2 immunoreactivity in the piriform region and a high immunoreactivity in the lateral olfactory tract can be observed as well (Fig. 1(E) and (F)).

At E18.5, the claustral complex displays a moderate VGLUT2 immunoreactivity. At this age, a central part of the claustrum begins to be distinguished as a relatively pale zone (asterisk in Fig. 1(I)), contrasting with both the overlaying

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Fig. 1. (Continued ).

(superficial) insular cortex and the deep part of the claustrum, closest to the external capsule, which display abundant immunoreactive axon terminals (Fig. 1(H) and (I)). This immunoreactivity pattern is better observed at intermediate and caudal parts of claustrum. At P0, VGLUT2 immunoreactivity increases dramatically in pallial regions, especially in the deep layers of the medial and dorsal pallium, and a prominent plexus of varicose immunoreactive axons extends superficially throughout layer 1 of the whole pallium. At this age, the arrangement of VGLUT2-ir neuropil within the claustrum shows a characteristic distribution: a dense plexus of positive axon terminals surrounds and forms a shell around a core (the central part) practically devoid of immunoreactivity (Fig. 1(J) and (K)). This plexus of VGLUT2-ir axonal endings extends deeply and dorsally (in relation of the external capsule) as well as superficially over the insular cortex. At P7–P14, a decrease of the VGLUT2 immunoreactivity within the whole pallium is observed, although a prominent plexus of VGLUT2-ir axon terminals remains in the claustrum (Fig. 2(A)–(C)). The VGLUT2 expression within the dorsal claustrum displays the characteristic core/shell arrangement observed in previous ages. This pattern of VGLUT2 immunoreactivity in the claustrum is virtually identical from P14 onward. The adult dorsal claustrum is characterized by the presence of a moderate to dense VGLUT2-ir neuropil, consisting of both very fine varicose axons and puncta (Figs. 2(D)–(F) and 3(D)). VGLUT2 immunoreactivity is denser at intermediate and caudal regions of the dorsal claustrum than rostrally, and displays the characteristic pattern already observed at P0, consisting of a dense plexus of immunoreactive puncta forming a shell around a core practically devoid of positive structures. The core is a VGLUT2-negative neuropil zone, appearing as an oval region in the claustrum, and the shell consists of a thin deep layer of immunoreactive neuropil that separates it from the fibers of the external capsule (arrows in Fig. 2(E) and (F)), and a superficial layer of immunoreactive neuropil continuous with

the deep layers of the neighboring insular cortex. The VGLUT2-negative core (asterisk in Fig. 2(E) and (F)) is more sharply delimited at most caudal regions of the claustrum (Fig. 2(F)). A distinctive feature of the VGLUT2 expression during the development of telencephalon is the presence of immunoreactive cell bodies. VGLUT2-ir cell bodies are observed in the dorsal claustrum, as well as in other pallial regions, only at P0 (Fig. 3(A)–(C)). These positive neurons were consistently observed in the three animals studied at P0 but not in other embryonic ages or in the adult mice. The number of VGLUT2ir cells is considerably lower in lateroventral pallium, including the dorsal claustrum, than in other pallial regions. In the dorsal claustrum, a few positive cells are present in both the core and shell regions (Fig. 3(A) and (B)). 3.1. Electron microscopy The ultrastructural analysis demonstrates that at least part of the immunoreactive varicosities or puncta characteristic of the VGLUT2 immunoreactivity, observed at light microscopic level (Fig. 3(D)) in the developing and adult mouse claustrum, are actually presynaptic boutons. VGLUT2-ir profiles are frequently observed in close apposition to dendritic profiles making asymmetric synaptic contacts. Synaptic contacts are observed as early as E16.5 (Fig. 3(E)), but a prominent postsynaptic density, the most distinctive feature of a mature asymmetrical contact, is only clearly apparent at E18.5–P0 (Fig. 3(F) and (G)). Synaptic vesicles are scarce within VGLUT2-ir boutons at E16.5, but at later embryonic ages and in the adult VGLUT2-ir presynaptic boutons are characterized by the presence of numerous round and clear synaptic vesicles (Fig. 3(G) and (H)). In all cases observed during prenatal development, the postsynaptic target is always a dendritic profile and never a cell body. In the adult, VGLUT2-ir boutons frequently contact small dendritic profiles or spines (Fig. 3(H)). The synaptic contacts are always of the asymmetric type.

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Fig. 2. Photomicrographs of transverse sections through the mouse telencephalon at postnatal ages (P7: A and B; P14: C) in comparison with the adult (D–F), showing VGLUT2 immunohistochemistry in the claustrum. Panel (B) is a detail of the boxed area in (A). Panels (E) and (F) are photomicrographs at intermediate and caudal regions of the claustrum, respectively. Asterisk marks the core region of the claustum, and arrows label the deep shell of the claustrum. For abbreviations see list. Scale bars: (A and D) 400 mm; (B and E) 100 mm; (C and F) 50 mm.

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Fig. 3. Details of VGLUT2 immunoreactivity in cells or varicosities either at light (A–D) or electron microscopic level (E–I). Panel (A) is a photomicrograph of a transverse section through the telencephalon at the level of the anterior commissure at P0. Panels (B) and (C) are details of the claustrum (boxed area in A) and insular cortex, respectively, showing VGLUT2-ir cells (asterisks). Panel (D) is a high power magnification photomicrograph showing VGLUT2 immunoreactivity in the adult claustrum. Arrows label an immunoreactive varicose axon. Panels (E)–(H) show VGLUT2 immunoreactive boutons making asymmetric synaptic contacts on putative dendritic profiles (postsynaptic densities are labeled with asterisks), at embryonic (E), postnatal (F and G), and adult (H) ages. A portion of an immunoreactive cell body (CB), adjacent to a non-immunoreactive cell, is showed in (I). See text for more details. For abbreviations see list. Scale bars: (A) 150 mm; (B and C) 50 mm; (D) 20 mm; (E, F and H) 0.2 mm; (G) 0.5 mm; (I) 1 mm.

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Confirming data of light microscopy, a few immunoreactive cell bodies with neuronal phenotype are also observed at P0 (Fig. 3(I)). 4. Discussion Our results indicate that the onset of VGLUT2 immunoreactivity in the telencephalon occurs at intermediate embryonic ages, with a dramatic increase towards P0. VGLUT2 immunoreactivity declines in several telencephalic areas from the first postnatal week, although abundant VGLUT2-ir axons remain in the claustrum. Also in rats, the onset of VGLUT2 expression occurs in the prenatal brain and shows a dramatic increase in its expression levels toward P7, and then declines toward adulthood (Boulland et al., 2004). 4.1. VGLUT2 immunoreactivity delineates a core and a shell within the dorsal claustrum Distinct regions in the developing and postnatal mouse claustrum have been previously described on the basis of cadherin expression patterns (Korematsu and Redies, 1997; Obst-Pernberg et al., 2001). Our results show that VGLUT2 immunoreactivity within the claustrum shows a characteristic core/shell arrangement from E18.5 on. A plexus of VGLUT2ir fine varicose axons and puncta forms a thin band deep in the claustrum, and another plexus runs dorsolaterally in the dorsal claustrum. These terminal fields avoid a central zone that is virtually VGLUT2-immunonegative. This arrangement of VGLUT2 immunoreactivity within the dorsal claustrum is similar to that of calretinin-ir fibers (see below; Real et al., 2003; Da´vila et al., 2005). Moreover, we reported that parvalbumin immunoreactivity appears restricted to the core of the dorsal claustrum in adult mice (Real et al., 2003). Besides, the pattern of nNOS neuropil staining in the developing and adult dorsal claustrum also shows these distinct subdivisions (core/shell) of the dorsal claustrum (Guirado et al., 2003), and the core of the mouse claustrum shows strong Cad8 mRNA expression (Medina et al., 2004; Da´vila et al., 2005). Thus, claustral core is characterized by strong parvalbumin and Cad8 expression, whereas the claustral shell shows strong calretinin and VGLUT2 expression. The distinct subdivisions within the dorsal claustrum may well be related to a differential origin from distinct histogenetic compartments of the lateroventral pallium. On the basis of their different mRNA expression of the homeobox gene Emx1, Puelles et al. (2000) suggested that the claustral complex (dorsal claustrum plus endopiriform nuclei) is subdivided into nuclei that derive either from the lateral or the ventral pallial histogenetic divisions. In fact, the dorsal claustrum shows weak expression of Ngn2/Sema5A but express selectively and strongly Cad8 plus Emx1, and appears to mostly belong to the lateral pallium, but a small (thin) part of it appears to belong to the ventral pallium (Medina et al., 2004). It is tempting to relate the deep VGLUT2-ir plexus with the part of the claustrum derived from the ventral pallium.

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4.2. VGLUT2 innervation of the dorsal claustrum Our results indicate that VGLUT2 immunoreactivity in the developing and adult dorsal claustrum is localized predominantly to very fine axons and varicosities, which is consistent with the presence of the transporter in the membrane of synaptic vesicles (Fujiyama et al., 2001). In addition, our electron microscopy data reveal that VGLUT2 immunoreactivity is present in presynaptic boutons making asymmetric synapses which most likely use glutamate as a neurotransmitter (Ni et al., 1994; Bellocchio et al., 1998, 2000; Aihara et al., 2000; Takamori et al., 2000, 2002; Bai et al., 2001; Fremeau et al., 2001; Fujiyama et al., 2001; Gras et al., 2002; Schafer et al., 2002). VGLUT2-ir boutons are present from E16.5 but it is not likely that contacts made by these boutons are functional synapses at this stage since they contain very few synaptic vesicles and the postsynaptic density is not apparent until E18.5. During development, calretinin-ir axons are observed in the shell of the mouse dorsal claustrum at E18.5, and become especially prominent during early postnatal development (Da´vila et al., 2005). Calretinin immunoreactivity resembles the VGLUT2-ir plexuses of axon terminals observed in the same developmental ages. The calretinin positive axons within the dorsal claustrum are supposed to be extrinsic, most likely arising from thalamic neurons (Gonza´lez et al., 2002; Real et al., 2003; Da´vila et al., 2005) since thalamic nuclei projecting to the claustrum display calretinin immunoreactivity (Gonza´lez et al., 2002). On the other hand, virtually all thalamic nuclei, with the exception of the reticular nucleus, are rich in neurons expressing VGLUT2 mRNA (Hur and Zaborszky, 2005), therefore, the most probable source of the VGLUT2 innervation of the dorsal claustrum is the glutamatergic thalamic neurons, as is the case for the neocortex (Ni et al., 1994, 1995; Fremeau et al., 2001; Fujiyama et al., 2001; Hur and Zaborszky, 2005). It is expected that VGLUT2-ir thalamo-claustral fibers also contain calretinin, although demonstration of this issue will require combined tract-tracing methods and double-labeling immunohistochemistry. VGLUT2-ir axon terminals in the developing dorsal claustrum could also originate from other sources than thalamic neurons, for example the neocortex. In this sense, cortical neurons displaying VGLUT2 immunoreactivity at P0 could be related to the postnatal increase in the number of VGLUT2-ir axonal endings in all pallial regions including the dorsal claustrum (see below). Nevertheless, at E16.5–E18.5 the VGLUT2-ir axons observed in the dorsal claustrum should mostly be of thalamic origin, since thalamic nuclei projecting to the claustrum express this transporter (Boulland et al., 2004; Hur and Zaborszky, 2005), whereas the arrival of VGLUT2 cortical axons to the claustrum should occur from P0 on. 4.3. Neuronal VGLUT2-ir cell bodies In the present study, immunoreactive neuronal cell bodies were found in the claustrum (and other telencephalic regions) at P0. The immunostaining in these cell bodies may represent

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VGLUT2 proteins incorporated either into the membrane of the endoplasmic reticulum, Golgi cisternae, or vesicles. In mature cells, the fast transport of these vesicles down the axon is the main reason why VGLUT2 (and many other) proteins are not easily detected in cell bodies, whereas they accumulate within axon terminals as synaptic vesicles that usually are recycled. However, during development it is likely that neurons engaged in active processes of axogenesis or synaptogenesis show a great increase in the synthesis of specific proteins. In this case, the rate of translation into protein could be higher than the rate of axonal transport and, therefore, the VGLUT2 protein will accumulate in the cell body, being detectable by immunohistochemistry. In this regards, there should be an transient increase in the VGLUT2 protein synthesis between E18.5 and P0, since at P0 we observe VGLUT2-ir cell bodies in claustrum, but not at all later during the postnatal development or in the adult. We suggest that claustral neurons displaying VGLUT2-ir cell bodies are forming connections at this age, for example with the cortex. Note that the claustrum is one of the largest VGLUT2 sources innervating the neocortex: for example, cells in the rat claustrum projecting to the medial prefrontal or primary somatosensory cortices express VGLUT2 mRNA (Hur and Zaborszky, 2005). In conclusion, we observe VGLUT2-ir fine axons and puncta in the developing and adult dorsal claustrum displaying a core/ shell arrangement similar to the complementary expression of calretinin and parvalbumin patches of neuropil, which also appears to correlate with the patch of Cad8 expression observed in the developing claustrum. Based on VGLUT2 expression, the core/shell compartments of the dorsal claustrum starts to be clearly seen at E18.5, and may be related to thalamo-claustral incoming fibers. In addition, we suggest the possibility that, from P0 onwards, some VGLUT2-ir axon terminals in the claustrum could arise from cortical neurons expressing VGLUT2. Acknowledgements We would like to thank Luis Olmos for his excellent technical assistance. This work was supported by Spanish CGI grant BFI2003-06453-C02-01. References Aihara, Y., Mashima, H., Onda, H., Hisano, S., Kasuya, H., Hori, T., Yamada, S., Tomura, H., Yamada, Y., Inoue, I., Kojima, I., Takeda, J., 2000. Molecular cloning of a novel brain-type Na+-dependent inorganic phosphate cotransporter. J. Neurochem. 74, 2622–2625. Bai, L., Xu, H., Collins, J.F., Ghishan, F.K., 2001. Molecular and functional analysis of a novel neuronal vesicular glutamate transporter. J. Biol. Chem. 276, 36764–36769. Bellocchio, E.E., Hu, H., Pohorille, A., Chan, J., Pickel, V.M., Edwards, R.H., 1998. The localization of the brain-specific inorganic phosphate transporters suggests a specific presynaptic role in glutamatergic neurotransmission. J. Neurosci. 18, 8648–8659. Bellocchio, E.E., Reimer, R.J., Fremeau Jr., R.T., Edwards, R.H., 2000. Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter. Science 289, 957–960.

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