Embryonic and postnatal development of GABA, calbindin, calretinin ...

THE JOURNAL OF COMPARATIVE NEUROLOGY 481:42–57 (2005)

Embryonic and Postnatal Development of GABA, Calbindin, Calretinin, and Parvalbumin in the Mouse Claustral Complex ´ CARLOS DA ´ VILA,1 M. A ´ NGELES REAL,1 LUIS OLMOS,1 ISABEL LEGAZ,2 JOSE LORETA MEDINA,2 AND SALVADOR GUIRADO1* 1 Department of Cell Biology, Genetics and Physiology, Faculty of Sciences, University of Ma´laga, 29071 Ma´laga, Spain 2 Department of Human Anatomy, Faculty of Medicine, University of Murcia, 30100 Murcia, Spain

ABSTRACT We analyzed the development of immunoreactive expression patterns for the neurotransmitter ␥-aminobutyric acid (GABA) and the calcium-binding proteins calbindin, calretinin, and parvalbumin in the embryonic and postnatal mouse claustral complex. Each calcium-binding protein shows a different temporal and spatial pattern of development. Calbindin-positive cells start to be seen very early during embryogenesis and increase dramatically until birth, thus becoming the most abundant cell type during embryonic development, especially in the ventral pallial part of the claustrum. The distribution of calbindin neurons throughout the claustrum during embryonic development partly parallels that of GABA neurons, suggesting that at least part of the calbindin neurons of the claustral complex are GABAergic and originate in the subpallium. Parvalbumin cells, on the other hand, start to be seen only postnatally, and their number then increases while the density of calbindin neurons decreases. Based on calretinin expression in axons, the core/shell compartments of the dorsal claustrum start to be clearly seen at embryonic day 18.5 and may be related to the development of the thalamoclaustral input. Comparison with the expression of Cadherin 8, a marker of the developing dorsolateral claustrum, indicates that the core includes a central part of the dorsolateral claustrum, whereas the shell includes a peripheral area of the dorsolateral claustrum, plus the adjacent ventromedial claustrum. The present data on the spatiotemporal developmental patterns of several subtypes of GABAergic neurons in the claustral complex may help for future studies on temporal lobe epilepsies, which have been related to an alteration of the GABAergic activity. J. Comp. Neurol. 481: 42–57, 2005. © 2004 Wiley-Liss, Inc. Indexing terms: calcium-binding proteins; dorsal claustrum; endopiriform nucleus; immunohistochemistry

The claustral complex, or claustrum sensu lato, is a pallial territory anatomically subdivided into a dorsal and a ventral part. The dorsal part is usually referred to as claustrum proper, and it is located deep to the insular cortex (therefore, it is also named dorsal or insular claustrum); the ventral part is named endopiriform nucleus and is located deep to the piriform cortex (Druga, 1966; Sherk, 1988; Dinopoulos et al., 1992). Several studies have revealed that these two anatomically differentiated parts of the claustral complex are involved in a different set of connections. Thus, the dorsal claustrum is extensively and reciprocally connected with the cerebral cortex, including motor and somatosensory areas (Clasca´ et al., 1992; Dinopoulos et al., 1992; Majak et al., 2002), whereas the en© 2004 WILEY-LISS, INC.

dopiriform nucleus is related to the piriform, entorhinal, and perirhinal cortices instead of the isocortex (Druga,

Grant sponsor: Spanish Direccioń General de Investigacioń-Fondo Europeo de Desarrollo Regional (FEDER); Grant number: BFI2003-06453-C02-01/02; Grant sponsor: Fondo de Investigaciones Sanitarias-FEDER; Grant number: 01/0057-02; Grant sponsor: Seńeca; Grant number: PB/50/FS/02; Grant sponsor: Red Centro de Investigacioń de Enfermedades Neurolo´gicas-Nodo 318. *Correspondence to: Salvador Guirado, Department of Cell Biology, Genetics, and Physiology, Faculty of Biology, University of Ma´laga, 29071 Ma´laga, Spain. E-mail: [email protected] Received 7 June 2004; Revised 29 July 2004; Accepted 21 August 2004 DOI 10.1002/cne.20347 Published online in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENT OF MOUSE CLAUSTRAL COMPLEX 1971; Markowitsch et al., 1984; Witter et al., 1988; Dinopoulos et al., 1992). On the other hand, dorsal claustrum and endopiriform nucleus have been proposed to originate from distinct histogenetic compartments of the lateroventral pallium. On the basis of their different mRNA expression of the homeobox gen Emx1, Puelles et al. (2000) suggested that the claustral complex is subdivided into nuclei that derive from either the lateral or the ventral pallial histogenetic divisions. This suggestion has been corroborated recently and expanded based on expression of additional developmental regulatory genes that are expressed distinctly in the lateral or ventral pallial parts of the claustral complex (Medina et al., 2004). Thus, the dorsolateral claustrum, located deep to the insular and dorsal piriform cortices, shows strong mRNA expression of Emx1 and Cadherin 8 (Cad8) and is considered part of the lateral pallium (Puelles et al., 2000; Medina et al., 2004). In contrast, the ventromedial claustrum, located medial to the dorsolateral claustrum and extending ventrally toward the endopiriform nuclei (deep to the ventral piriform cortex), lacks Emx1 and Cad8 expression but shows moderate to strong expression of Semaphorin 5A (Puelles et al., 2000; Medina et al., 2004). Based on this, the ventromedial claustrum and the endopiriform nuclei (except a posterior part) are considered part of the ventral pallium (Puelles et al., 2000; Medina et al., 2004). Calcium-binding proteins are widely distributed in the central nervous system and have proved to be excellent neuroanatomical markers in the adult as well as in the developing brain (for review, see Andressen et al., 1993; Heizmann and Braun, 1995). For this reason, we have analyzed recently the distribution pattern of calciumbinding proteins in the adult mouse claustrum, searching for putative different functional zones within this region (Real et al., 2003a). Specifically, we showed that the parvalbumin and calretinin immunoreactivity displayed a core/shell arrangement within the adult dorsal claustrum (Real et al., 2003a), which appears to correlate to the patch of Cad8 expression observed in the developing dorsolateral claustrum (Obst-Pernberg et al., 2001; Medina et al., 2004). Calcium-binding proteins are also expressed during both early and late development of the nervous system, and it is believed that these proteins may be involved in triggering calcium-dependent processes such as cell movement or process outgrowth (Enderlin et al., 1987; Ellis et al., 1991; Spitzer, 1994; Frassoni et al., 1998), as well as in the maturation of cortical inhibitory circuits (Hendrickson et al., 1991; Solbach and Celio, 1991). To understand how the compartmental organization of the claustrum is achieved, in the present study, we analyzed the expression of the calcium-binding proteins calbindin, calretinin, and parvalbumin during the embryonic and postnatal development of mouse claustral complex. To correlate the core/ shell compartments identified in our previous study with the different histogenetic parts of the claustrum, we compare the expression of calcium-binding proteins with that of Cad8, a marker of the dorsolateral claustrum. Finally, because calcium-binding proteins are expressed in subsets of GABAergic neurons in the claustrum (Druga et al., 1993; Real et al., 2003a; Kowianski et al., 2004), we also analyzed the expression of the inhibitory neurotransmit-

43 ter GABA during claustral development and discussed the possible subpallial origin of these cells. Considering that the claustral complex is involved in propagation of epileptiform activity from the amygdala (Kudo and Wada, 1990; Hoffman and Haberly, 1996; Wada and Kudo, 1997; Mohapel et al., 2001; Zhang et al., 2001), and that the severity of seizures is related to the inhibitory GABAergic activity (Stevens et al., 1988), we hope that our data will help for future studies on the role of each specific GABAergic subpopulation in the development of temporal lobe epilepsies.

MATERIAL AND METHODS Mouse embryos, from 12.5 days post coitum (embryonic day 12.5, E12.5) until E19.5, and neonates, from birth (postnatal day 0, P0) through P23, were used in the present study. 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, 0.2% glutaraldehyde, and 0.2% picric acid in 0.1 M phosphate buffer, pH 7.4, at 4°C. After 1 day of fixation, fetal brains were removed from the cranium and post-fixed for another day in fresh fixative solution. Postnatal animals were anesthetized with diethyl ether and then perfused transcardially with saline solution, followed by 50 ml of a fixative consisting in 4% paraformaldehyde, 0.2% glutaraldehyde, and 0.2% picric acid in 0.1 M phosphate buffer (PB), pH 7.4, for 20 minutes. Brains were removed and post-fixed overnight at 4°C in fresh fixative solution without glutaraldehyde. After extensive washes in PB, the brains were embedded in 4% agarose, and 50-␮m-thick frontal sections were obtained by using a Vibratome. Brain sections were processed for immunohistochemistry following a standard procedure. Briefly, free-floating sections were first incubated in 2% normal goat serum and 0.3% Triton X-100 in 0.1 M phosphate buffered saline (PBS), pH 7.4, at room temperature for 1 hour, to block nonspecific binding of the antibodies and permeate the tissues, and then were transferred to either one of the four rabbit primary antibodies (anti-GABA, 1:2,000, Sigma; anti-calbindin, 1:4,000, Swant; anti-calretinin, 1:2,000, Swant; and anti-parvalbumin, 1:1,000, Swant) for 18 hours at 4°C. After extensive washes in PBS, the sections were incubated in a biotinylated goat anti-rabbit IgG (Sigma) diluted 1:500 for 1 hour, washed again in PBS, and incubated in ExtrAvidin–peroxidase (Sigma) diluted 1:2,000 for 1 hour. The immunolabeling was revealed with 0.05% diaminobenzidine (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, England). 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

´ VILA ET AL. J.C. DA

44 rabbit no immune serum (1:500). No immunostaining could be detected under these conditions. Transverse sections of the mouse telencephalon from each embryonic or postnatal age were stained with toluidine blue (Nissl staining) for cytoarchitectonic details. In addition, to better delineate the different parts of the claustral complex, frontal sections of the developing mouse telencephalon from another group of mice (E12.5 until birth) were hybridized for Cadherin 8 (a specific gene marker of the developing dorsolateral claustrum), following a procedure previously described (Obst-Pernberg et al., 2001; see Medina et al., 2004, for details on the procedure). Finally, digital photographs were taken on a Nikon microscope equipped with a Nikon DXM1200 digital camera. Digital images were adjusted for brightness/contrast by using Adobe Photoshop; no additional filtering or manipulation of the images was performed. Figures were mounted and labeled by using PageMaker 7.0.

RESULTS We describe here the expression (immunoreactivity pattern) of the neurotransmitter GABA and the calciumbinding proteins calbindin, calretinin and parvalbumin in the developing mouse claustral complex, from embryonic day 12.5 until postnatal day 23. The claustral complex is a ventrolateral pallial region that includes the claustrum proper (deep to the insular cortex) and the endopiriform nuclei (deep to the piriform cortex). Based on Nissl staining, the incipient mouse claustrum proper began to be clearly anatomically recognizable between E14.5 and E15.5 (Fig. 1A,B). At E15.5, the dorsolateral subdivision (dorsolateral claustrum) could be distinguished as a small cell aggregate localized deep to the insular cortical plate (Fig. 1A,B), coinciding with a patch of Cad8 expression (Fig. 2C). Ventral to it, the primordia of the endopiriform nucleus could also be identified located deep to the piriform cortical plate, although it was difficult to discern between it and the deep layer of the piriform cortex (Fig. 1B). During later embryonic development (Fig. 1C–F), the claustral complex became more clearly organized and its different cytoarchitectonic subdivisions, including the dorsal and ventral endopiriform nuclei, could be clearly recognized at E18.5 (Fig. 1E,F). Throughout embryonic development, the dorsolateral claustrum was always coincident with a patch of Cad8 expression, which became a useful gene marker of this claustral subdivision in embryos. After birth, the ventralmost part of the dorsolateral claustrum started to show weaker signal of this gene, as described previously (Obst-Pernberg et al., 2001). The claustral complex showed different expression patterns of GABA and calcium-binding proteins during development, with major changes occurring at E13.5–16.5 (mainly an increase in GABA and calbindin expression), E18.5– birth (P0) (incoming of calretinin fibers), and postnatally (beginning of parvalbumin expression). Therefore, we grouped the description of results into three stages: early–intermediate claustral development (E13.5–E16.5), late development (E18.5–P0), and postnatal ages (P4 – P23).

Embryonic days 13.5–16.5 Calbindin-immunoreactive cells were present in pallial regions as early as E12.5, whereas clear GABAergic cells were first observed in the pallium at E14.5 (not shown). At E13.5–E14.5, numerous calbindin-immunoreactive cells were accumulated into the ventrolateral part of the pallium, especially in the region containing the claustral– endopiriform primordium plus the insular and piriform cortical plates (Fig. 2A,B). At E14.5–E15.5, calbindinimmunoreactive cells were clearly more abundant in the endopiriform region than in the dorsolateral claustrum (Fig. 2F; for identification of the dorsolateral claustrum, compare calbindin immunostaining in Fig. 2F with Cad8 expression in Fig 2C). Dorsally, fewer calbindin-positive cells were observed in the developing dorsal pallium, located in the marginal zone and the subplate. At this age, many of the calbindin-immunoreactive cells of the developing claustral– endopiriform region show a morphology resembling migratory neuroblasts or immature neurons. At E14.5–E15.5, the distribution of calbindin and GABAimmunoreactive cells in the ventrolateral pallium was similar, although calbindin-immunoreactive cells were more abundant and more strongly positive than GABAimmunoreactive cells (Fig. 2D,E). At E16.5, the number and immunoreactivity intensity of calbindin cells in the ventrolateral pallium increased notably (Fig. 3A,B). An increase in GABA-immunoreactive cells was also evident, but this finding was less striking than that of calbindin (Fig. 3C). Calbindin-immunoreactive cells were densely concentrated in the piriform region, including the piriform cortex (especially its deeper layer, just deep to the cortical plate) and the endopiriform nucleus (Fig. 3A,B). In addition, calbindin-immunoreactive neurons also were densely concentrated in the marginal zone around the interface between the piriform and insular cortices (Fig. 3A,B; arrow). In the claustrum proper, it was possible to distinguish between the dorsolateral claustrum, containing scattered calbindin-immunoreactive cells, and the ventromedial claustrum, which appeared as a thin band of densely grouped immunoreactive cells continuous ventrally with those of the endopiriform region (Fig. 3A,B). Superficial to the dorsolateral claustrum, the deeper layer of the insular cortex contained numerous calbindin-positive neurons. At this age, most calbindin-immunoreactive cells of the ventrolateral pallium displayed small cell bodies with a few, short, irregular processes. At these embryonic stages (E14.5–E16.5), parvalbumin immunoreactivity was completely absent from pallial regions (not shown), whereas a few calretinin-immunoreactive cells were observed almost restricted to the marginal zone of the cortex (putative Cajal–Retzius cells) and to the piriform cortex (Fig. 3D). At the surface of the piriform cortex, the olfactory tract appeared strongly calretinin-immunoreactive (Fig. 3D), representing an important landmark of this pallial region from early embryonic stages. At E16.5, calretininimmunoreactive axons are very numerous in the internal capsule, and a few, fine and varicose calretinin-positive axons were also observed around the external capsule, but without apparent relation with the claustrum (Fig. 3D, arrows). It is interesting to note the presence of a small patch of lightly stained calretinin-immunoreactive cells immersed in a positive neuropil located in the dorsalmost part of the piriform cortex near the interface between the piriform and insular cortices (asterisk in Fig. 3D). This calretinin-

Fig. 1. A–F: Transverse sections through the telencephalon of mouse embryos at embryonic day (E) 15.5 (A,B), E16.5 (C,D) or E18.5 (E,F), stained with toluidine blue. B, D, and F are enlarged views of the corresponding boxed areas, showing the cytoarchitecture of the embryonic claustral complex. Cl, dorsal claustrum; CPu, caudate-

putamen; DEn, dorsal endopiriform nucleus; En, endopiriform nucleus; Ins, insular cortex; lot, lateral olfactory tract; Pir, piriform cortex; VEn, ventral endopiriform nucleus. Scale bars ⫽ 300 ␮m in A,C,E; 150 ␮m in B,D,F.


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Fig. 2. Transverse sections through the telencephalon of mouse embryos at embryonic day (E) 14.5 (A,B) or E15.5 (C–F). A: Transverse section at a rostral telencephalic level, immunostained for calbindin. B: Detail of the boxed area in A, showing numerous calbindinpositive cells in the lateral and ventral pallia (LP, VP). C: Detail of the lateroventral pallial region, showing a patch of Cad8 expression in the primordium of dorsal claustrum (Cl). D: ␥-Aminobutyric acid (GABA) -immunostained section at a rostral level of the claustral complex.

E: Detail of the boxed area in D. F: Detail of the claustral complex of a calbindin-immunostained section. Calbindin-positive cells appeared more abundant than GABA-positive cells within the claustral complex at this age. CPu, caudate putamen; DP, dorsal pallium; En, endopiriform nucleus; Ins, insular cortex; lot, lateral olfactory tract; Pir, piriform cortex. Scale bar ⫽ 350 ␮m in A; 150 ␮m in B,C; 250 ␮m in D; 75 ␮m in E,F.

immunoreactive cell aggregate was observed from rostral to caudal levels of the telencephalon and from E16.5 until P4 (Figs. 3D, 4C, 7A).

in the ventrolateral pallium than in the dorsal pallium. Also as before, the distribution of GABA and calbindinimmunoreactive neurons in the ventrolateral pallium was generally similar (Fig. 4A,B). GABA- and calbindinpositive cells were abundant in the deep layers of the piriform and insular cortices, as well as in the endopiriform region and the ventromedial claustrum (Fig. 4A,B). In contrast, the dorsolateral claustrum showed fewer, scattered calbindin-positive cells (Fig. 4B). The whole piriform region displayed a high density of calbindin-

Embryonic day 18.5–P0 At these stages, the staining pattern for both GABA and calbindin in the mouse claustrum was similar to the precedent stages, but the number of immunoreactive cells and the positive neuropil increased. As before, calbindinand GABA-immunoreactive neurons were more abundant

Fig. 3. A–D: Transverse sections through the telencephalon of mouse embryos at embryonic day (E) 16.5, immunostained for calbindin (rostral, A; intermediate, B), ␥-aminobutyric acid (GABA, C), or calretinin (D). Calbindin-immunoreactive cells were concentrated in the ventromedial claustrum (Clvm) and endopiriform region, including the endopiriform nucleus (En). GABA-positive cells were also numerous within the claustral complex. Some calretinin-

immunoreactive axons are observed around the external capsule (arrows), but without apparent relation with the claustrum. Note the presence of a patch of lightly stained calretinin-immunoreactive cells in the dorsalmost part of the piriform cortex (asterisk). Cld, dorsolateral claustrum; Ins, insular cortex; En, endopiriform nucleus; CPu, caudate putamen; Pir, piriform cortex; lot, lateral olfactory tract; CL, dorsal clostrum. Scale bars ⫽ 150 ␮m in A–D.

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´ VILA ET AL. J.C. DA immunoreactive cells and processes, making it difficult to differentiate the endopiriform nucleus from the deep layer of the piriform cortex (Fig. 4B). On the contrary, the dorsolateral claustrum appeared as a relatively pale zone, contrasting with the overlaying (superficial) insular cortex and the deep ventromedial claustrum (Fig. 4B). The relatively pale zone of the dorsolateral claustrum overlaps an expression domain of Cadherin 8 (Cad8; Fig. 5A,C,E), confirming that it represents the specific part of the claustrum suggested to derive from the lateral pallium (Medina et al., 2004). At these stages, most calbindin-immunoreactive cells in the ventrolateral pallium displayed morphological features typical of immature neurons. Heavily stained (Golgilike) as well as lightly stained calbindin neurons were observed. At E18.5–P0, major changes in the developing claustrum concerned to calretinin immunoreactivity, especially in relation to the development of a positive neuropil in the claustrum (Figs. 4C, 5D). Some calretinin-immunoreactive cells were present in the piriform cortex (more abundant in the ventral part of its deeper layer), and a few positive cells were also observed in the ventral endopiriform nucleus (Fig. 4C). As before, the dorsalmost part of the piriform cortex showed a small patch of calretinin neurons immersed in a positive neuropil (Figs. 4C, 5D). Very few, small, lightly stained, calretinin-positive cells were also present in the claustrum (in relation to a positive neuropil that is described below) and insular cortex. Numerous and strongly immunoreactive axons were observed in both the internal and external capsules (Figs. 4C, 5D), and these axons appeared to arise in immunoreactive cells of the dorsal thalamus. The axons of the external capsule appeared to be the source of a calretininpositive neuropil that started to develop in the dorsal claustrum at E18.5, reaching a great development at birth (P0; Fig. 5D). Numerous immunoreactive fibers invaded the ventromedial and dorsolateral claustrum beginning at E18.5. Thick smooth fibers as well as fine varicose axons were observed. From E18.5 to P0, the number of calretinin fibers increased enormously in both the ventromedial and dorsolateral claustrum and they spread to the insular cortex, whereas the piriform cortex and the endopiriform nucleus were almost devoid of fibers (except for the positive fibers related to the olfactory tract that ended in the superficial layer of the piriform cortex, and the positive patch of the dorsalmost part of the piriform cortex noted above). At P0, the arrangement of calretinin fibers/neuropil within the claustrum showed a characteristic distribution: a dense plexus of varicose, immunoreactive axons surrounded (and form a shell around) a core practically devoid of positive fibers (Fig. 5D). This plexus of calretinin-positive varicose

Fig. 4. A–C: Transverse sections through the telencephalon of mouse embryos at embryonic day (E) 18.5 (A,B) and E19.5 (C), immunostained for ␥-aminobutyric acid (GABA, A), calbindin (B), or calretinin (C). GABA- and calbindin-positive cells were especially abundant in the piriform region. Numerous calretinin-positive varicose processes can be observed in the dorsal claustrum. Also, note the high immunoreactivity in the lateral olfactory tract (lot) and the patch of lightly stained calretinin-immunoreactive cells in the dorsalmost part of the piriform cortex (asterisk in C). Cld, dorsolateral claustrum; Clvm, ventromedial claustrum; Ins, insular cortex; En, endopiriform nucleus; CPu, caudate putamen; Pir, piriform cortex; lot, lateral olfactory tract; CL, dorsal clostrum; DEn, dorsal endopiriform nucleus; VEn, ventral endopiriform nucleus. Scale bars ⫽ 125 ␮m in A–C.

DEVELOPMENT OF MOUSE CLAUSTRAL COMPLEX

Fig. 5. Transverse sections through the telencephalon of mouse at postnatal day (P) 0. A: Low-magnification view of a section hybridized for Cad8. B–D: A region similar to that included in the boxed area in A is shown immunostained for calbindin (B), double-stained for Cad8 and calbindin (C), and immunostained for calretinin (D). Note the partially complementary staining for Cad8 and both calbindin and calretinin in the dorsal claustrum. E: Transverse section of the telen-

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cephalon at the level of the anterior commissure (ac) hybridized for Cad8. F: Detail of the endopiriform region in a transverse section immunostained for parvalbumin. Cld, dorsolateral claustrum; Clvm, ventromedial claustrum; Ins, insular cortex; En, endopiriform nucleus; CPu, caudate putamen; Pir, piriform cortex; lot, lateral olfactory tract; CL, dorsal clostrum; VEn, ventral endopiriform nucleus. Scale bars ⫽ 200 ␮m in A,E; 100 ␮m in B–D,F.


50 axons extended deeply and dorsally (in relation to the external capsule) as well as superficially over the insular cortex. This pattern of calretinin fibers was especially evident at P0 (Fig. 5D), although in precedent stages was also distinguished (Fig. 4C). Compared with calbindin and Cad8, it appeared that the claustral core poor in calretinin neuropil included only the central part of the dorsolateral claustrum (pale for calbindin and rich in Cad8 expression; Fig. 5A–D). Thus, the rich calretinin neuropil in the claustrum included a “shell” region of the dorsolateral claustrum as well as the ventromedial claustrum. The density of calretinin fibers decreased at intermediate and caudal levels of the claustrum. In addition to the calretinin-positive varicose fibers in the region of the dorsal claustrum/insular cortex, the rest of the pallium showed scarce calretinin innervation, except for another prominent plexus of calretinin axons in the most medial cortex and some staining in layer 1 in relation to Cajal– Retzius cells (not shown). At E18.5–P0, parvalbumin immunoreactivity was virtually absent from the pallium, except for the piriform region, which showed light immunoreactivity. A small patch of positive parvalbumin neurons and neuropil was observed in the dorsalmost part of piriform cortex, in addition to numerous extremely lightly stained parvalbumin neurons through the ventrodorsal extension of the cell layer of the piriform cortex (Fig. 5F). The dorsal claustrum and dorsal endopiriform nucleus were devoid of parvalbumin immunoreactivity, whereas the ventral endopiriform nucleus displayed several barely distinguishable lightly stained parvalbumin-immunoreactive cell bodies (Fig. 5F).

Postnatal days P4 –P23 Some important changes were observed in relation to the abundance and distribution of GABA, calbindin, and parvalbumin in the pallium at these postnatal ages. At P4 –P7, the number of GABA-immunoreactive neurons increased dramatically in the deeper layers of the dorsal pallium and became similar to the levels observed in the ventrolateral pallium. In the ventrolateral pallium, the claustral complex, and the piriform and insular cortices continued to have a similar distribution of GABAergic neurons, although these were less densely grouped (Fig. 6A). As before, GABAergic neurons were more abundant in the piriform cortex (especially the deeper layer, but some cells were also present in the cortical plate and the marginal zone), endopiriform nuclei and ventromedial claustrum than in the insular cortex and dorsolateral claustrum (Figs. 6A, 8A). The distribution of GABAimmunoreactive neurons in the claustrum at P4 –P7 was similar to that seen at P23 and in the adult. The number of calbindin-positive cells in the dorsal pallium was considerably lower than the number of GABAergic neurons. However, calbindin immunoreactivity continued to be remarkably strong in the ventrolateral pallium. This was mainly due to the high number of calbindin-immunoreactive neurons and the strongly positive neuropil observed in the deepest layer of the piriform cortex and endopiriform region (Fig. 6B). Nevertheless, the density of calbindin-immunoreactive neurons observed in the ventrolateral pallium at P4 –P7 appeared to be lower than during embryonic development. At this age, calbindin immunoreactivity in the claustral complex and piriform cortex was generally similar to that of GABA, except for some differences observed in the marginal zone

(Fig. 6A,B). Apparently, the number of GABAergic cells in the endopiriform nuclei, ventromedial claustrum, and deep layer of the piriform cortex was higher than the number of calbindin-positive cells, although the latter cells were more darkly stained (both cell bodies and processes). As before, a few GABAergic cells were present in the marginal zone of the piriform cortex, but no calbindinpositive cells were observed in this zone. At P4 –P7, the distribution and morphological features of calbindin neurons in the claustrum were similar to those of the adult (Figs. 6B, 8B; Real et al., 2003a). Two patterns of cell staining for calbindin were observed throughout the claustrum: darkly stained multipolar neurons, and lightly stained cell bodies with barely visible processes. The pattern of calretinin innervation in both the claustrum (ventromedial plus dorsolateral parts) and the insular cortex at P4 –P7 was similar to that of P0 (Fig. 7A,B), although the plexus of calretinin-positive axons appeared less distinctive at P7 (Fig. 8C). Also, the number of calretinin-immunoreactive neurons increased throughout the dorsolateral claustrum (mainly in the shell region), the ventral endopiriform nucleus, and the deep layer of the piriform cortex at these ages (Figs. 7A, 8C). Most calretinin cells displayed small and rounded cell bodies with few dendrites, like calretinin neurons in this region of the adult mouse (Real et al., 2003a). In addition to the patch of calretinin immunoreactivity observed in the dorsal part of the piriform cortex, a conspicuous group of calretinin-positive cells was observed superficially in the insular cortex, just in the same region innervated by calretinin axons (Figs. 7A, 8C). A major change occurred at these postnatal ages regarding to parvalbumin expression in the claustrum. In contrast to the precedent developmental stages of the claustrum, in which parvalbumin immunoreactivity was virtually absent (although some light staining was present in the piriform cortex), from P4 onward, several parvalbumin-immunoreactive neurons were observed throughout the claustrum, from rostral to caudal levels. At these ages, claustral parvalbumin-immunoreactive neurons showed small and rounded cell bodies with few irregular processes, appeared embedded in a moderately stained neuropil (Fig. 7C,D). The ventral endopiriform nucleus and the whole piriform cortex displayed a very dense neuropil staining (Fig. 7C). At P14, a general increase in the number of parvalbumin-immunoreactive neurons in the whole cortex was apparent, although the region of the dorsal claustrum and the ventral insular cortex appeared relatively pale with only few positive cells. At P23, the last studied stage, the parvalbumin expression pattern in the claustrum was very similar to the adult (Real et al., 2003a), with several multipolar parvalbumin-immunoreactive neurons embedded in a patch of stained neuropil surrounded by a clear zone (Fig. 8D, inset).

DISCUSSION In the present study, we describe the development of immunoreactive expression patterns for the neurotransmitter GABA and the calcium-binding proteins calbindin, calretinin, and parvalbumin in the embryonic and postnatal mouse claustral complex. In the adult rodent claustrum, each calcium-binding protein appears mainly localized to a differ-

DEVELOPMENT OF MOUSE CLAUSTRAL COMPLEX

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Fig. 6. A,B: Transverse sections through the telencephalon of mouse at postnatal ay (P) 4, immunostained for ␥-aminobutyric acid (GABA, A) or calbindin (B) showing the claustral complex. Note the higher number of GABAergic cells compared with calbindin-positive cells in both the endopiriform nucleus (En) and ventromedial claus-

trum (Clvm), whereas the number of calbindin-positive cells in the core of the dorsolateral claustrum (Cld) appears higher than that of GABAergic cells. Ins, insular cortex; En, endopiriform nucleus; CPu, caudate putamen; Pir, piriform cortex; lot, lateral olfactory tract; ec, external capsule. Scale bars ⫽ 100 ␮m in A,B.

ent cell population, and part of these cells are likely GABAergic interneurons (Druga et al., 1993; Real et al., 2003a; Kowianski et al., 2004). Our results indicate that each calcium-binding protein shows a different temporal and spatial pattern of development. Calbindin (CB) was the earliest and most abundant calcium-binding protein expressed by claustral neurons, and the distribution of calbindin neurons throughout the claustrum during embryonic development partly parallels that of GABA neurons. The density of calbindin-immunoreactive neurons in the claustral complex increases during embryonic development but decreases postnatally. Calretinin, on the other hand, displayed a very different distribution and temporal

pattern of development within the claustral complex: calretinin started to be observed within the complex during intermediate–late embryonic development and was present only in a few cells, but was mainly expressed by axons, which were clearly visible in the dorsal claustrum from embryonic day 18.5 onward. Finally, parvalbumin immunoreactivity started to be observed postnatally in neurons of the mouse claustral complex, similarly to the findings in the rodent cerebral cortex (Alcańtara et al., 1996) and amygdala (Berdel and Morys, 2000). We will separately discuss our results for each calcium-binding protein in the different parts of the claustral complex (with the help of Cadherin 8, a specific gene marker of the

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Fig. 7. A–D: Transverse sections through the telencephalon at the level of the anterior commissure of mouse at postnatal day (P) 4, immunostained for calretinin (A,B) or parvalbumin (C,D). The pattern of calretinin innervation in the claustrum (ventromedial plus dorsolateral parts) at P4 is virtually identical to P0. A: The patch of lightly stained calretinin-immunoreactive cells in the dorsalmost part of the piriform cortex is observed (asterisk). B: A central region


virtually devoid of calretinin immunoreactivity is clearly observed. C: Parvalbumin immunoreactivity is more abundant in the ventral endopiriform nucleus (VEn). D: Several parvalbumin-immunoreactive cells, with small cell bodies and irregular processes, embedded in a moderately stained neuropil are observed in the claustrum. Ins, insular cortex; Pir, piriform cortex; CL, dorsal clostrum; Cld, dorsolateral claustrum. Scale bars ⫽ 150 ␮m in A,C; 25 ␮m in B,D.

Fig. 8. A–D: Transverse sections through the telencephalon of mouse at postnatal day (P) 7 (A–C) or P23 (D), immunostained for ␥-aminobutyric acid (GABA, A), calbindin (B), calretinin (C), and parvalbumin (D). At P23, the claustrum displays a pattern of parvalbumin expression similar to the adult, with several multipolar neurons embedded in a patch of stained neuropil surrounded by a clear

zone (D; inset). Ins, insular cortex; En, endopiriform nucleus; CPu, caudate putamen; Pir, piriform cortex; lot, lateral olfactory tract; CL, dorsal clostrum; VEn, ventral endopiriform nucleus; DEn, dorsal endopiriform nucleus, ac, anterior commissure. Scale bars ⫽ 100 ␮m in A–D; 50 ␮m in D inset.


54 developing dorsolateral claustrum) and compare the development of the neurons expressing these proteins with some data on their partial embryonic origin in the subpallium (Sussel et al., 1999; Legaz et al., 2004).

GABA and calbindin Calbindin-immunoreactive (CB⫹) neurons were first observed very early in the development in the mouse claustrum (E13.5–E14.5; Legaz et al., 2004; present results). The abundance of CB⫹ neurons in the claustral complex (as well as in the superficial piriform cortex) dramatically increased throughout embryonic development. Comparison with Cadherin 8 mRNA, a marker of the developing dorsolateral claustrum (Obst-Pernberg et al., 2001; Medina et al., 2004), indicates that, during embryonic development, CB⫹ neurons are more abundant in the ventromedial claustrum and endopiriform nuclei (dorsal and ventral endopiriform nucleus; ventral pallial derivatives) than in the dorsolateral claustrum (lateral pallial derivative; Puelles et al., 2000; Medina et al., 2004). The density of CB⫹ neurons in the ventral pallial parts of the claustrum decreased postnatally, and the striking differences in the number of CB⫹ cells between the dorsal and ventral parts of the claustrum tended to disappear (Real et al., 2003a). Nevertheless, in adult rodents, the dorsolateral claustrum continued to show lighter CB immunoreactivity than the ventral, endopiriform parts of the claustrum (Celio, 1990; Druga et al., 1993). Our results showed a parallelism between GABA and calbindin in the distribution of immunoreactive cells in the embryonic complex, which is consistent with the possibility that the CB⫹ cells represent a subpopulation of GABAergic cells. This issue was addressed recently by Legaz et al. (2004), who showed experimentally that at least part of the CB⫹ neurons observed in the developing claustral complex originate in the subpallium (ganglionic eminences). This observations resembles the findings of a subpallial origin of most of the calbindin and other GABAergic interneurons of the cerebral cortex (Anderson et al., 1997, 2001; Lavdas et al., 1999; Del Rio et al., 2000; Pleasure et al., 2000; Marıń and Rubenstein, 2001; Nery et al., 2002). This finding is also consistent with the observation of a dramatic decrease in calbindin immunoreactivity observed in the ventrolateral pallium and neocortex of Nkx2.1 knockout mice, which lack a medial ganglionic eminence (Sussel et al., 1999). Our results also showed that the number of CB⫹ neurons appears higher than that of GABAergic neurons in the claustral complex during embryonic development (but not postnatally), suggesting that not all CB⫹ neurons observed in the claustral complex during embryonic development are GABAergic (see also Legaz et al., 2004). Although these data must be taken with caution (because differences may be due to different penetration of antibodies in the sections), they are consistent with data on transient expression of calbindin in the cerebral cortex during development, and with the finding that, in the adult cerebral cortex and basolateral pallial amygdala, calbindin is found in GABAergic interneurons as well as in pyramidal neurons (Celio, 1990; Alcańtara and Ferrer, 1995; Berger and Alvarez, 1996; McDonald, 1997; Hof et al., 1999; Yoon et al., 2000). The possible existence of GABAergic and nonGABAergic CB⫹ neurons in the developing claustral complex may be related to the observation of two calbindin-

immunoreactive cell types, darkly and lightly stained neurons (also described in the adult mouse claustrum; Real et al., 2003a), whose distinct staining intensity for calbindin may be related to the GABAergic phenotype. In this context, it is interesting to note that, in the cerebral cortex and pallial amygdala, the lightly CB⫹ cells are pyramidal neurons, whereas the strongly CB⫹ cells are interneurons (Celio, 1990; McDonald, 1997). In addition, as shown in a recent study using a transgenic mouse harboring the zebrafish dlx4/6-LacZ enhancer/reporter (Stu¨hmer et al., 2002), these two types of CB⫹ neurons in the cortex have a different embryological origin: the vast majority of the strongly CB⫹ cells coexpressed ␤-galactosidase (and were, therefore, considered GABAergic and subpallial in origin), whereas the weakly CB⫹ cells did not express ␤-galactosidase (and were non-GABAergic and possibly originate within the pallium; Stu¨hmer et al., 2002). As noted above, during development of the claustral complex, CB⫹ and GABAergic cells become more abundant in the ventral pallial part of the complex: the ventromedial claustrum plus the endopiriform nuclei. Because the CB⫹, GABAergic neurons originate in the subpallium (Sussel et al., 1999; Legaz et al., 2004), it appears that, during claustral development many of these migratory CB⫹ cells tend to accumulate within (or are attracted to) the ventral pallium, whereas these cells show lower affinity for the lateral pallium. In relation to this, it is interesting to note that the ventral pallium is characterized by expression of Semaphorin 5A (Sema5A; Medina et al., 2004), a member of the semaphorin family of transmembrane proteins involved in axonal pathfinding and cell migration (De Castro, 2003). This finding could indicate that the ventral pallial territory expressing Sema5A may constitute a preferential route for the migration of CB⫹, GABAergic neurons to the pallium. In this context, it is also interesting to note that the ventrolateral pallial regions in the developing mouse telencephalon display a moderate expression of neuropilin2, a semaphorin receptor, which is colocalized to calbindin migrating cells (Marıń et al., 2001). At least some of the CB⫹ cells showing a migratory neuroblast aspect observed in the ventrolateral pallium during early development (E13.5–E14.5) may represent neurons en route to the cerebral cortex. Nevertheless, most of the CB⫹ or GABAergic cells observed in the ventrolateral pallium (and, in particular, in the claustral complex) at E16.5 and later, show an aspect of maturing neurons and appear to be differentiating at their final destination. The expression of calbindin in these immature neurons suggests that this protein plays an important role in their development. This suggestion is not surprising, taking into account that calbindin appears involved in triggering calcium-dependent processes such as cell movement or the process of outgrowth during development of the nervous system (Komuro and Rakic, 1996).

Calretinin Our results confirm previous data on the existence of calretinin-immunoreactive (CR⫹) cells in the subpial layer of the cerebral cortex, including the piriform cortex, since early embryonic stages in rodents and other mammals (Yan et al., 1995; Berger and Alvarez, 1996; Meyer et al., 1998; Jimeńez et al., 2003). Later in development (E16.5–E18.5), a few lightly CR⫹ cells start to be seen in the claustral complex, but only in its ventralmost aspect

DEVELOPMENT OF MOUSE CLAUSTRAL COMPLEX (the prospective ventral endopiriform nucleus), adjacent to a group of positive cells in the ventralmost part of the piriform cortex. The number of CR⫹ cells and their intensity increase moderately during postnatal development until they reach adult levels (Real et al., 2003a). In the rat somatosensory cortex, calretinin is expressed very early during cortical development (E11), with a transient increase during early postnatal development (Vogt Weisenhorn et al., 1994, 1996; Fonseca et al., 1995). Most calretinin-immunoreactive neurons within the developing cortical plate and its derivatives (layers VIa to II) are nonpyramidal and display GABA immunoreactivity (Fonseca et al., 1995). We suggest that, as in the developing rat cortex, at least part of the CR⫹ neurons in the claustral complex may represent GABAergic interneurons, which originate in the subpallium, similar to the CB⫹ GABAergic neurons discussed above (Legaz et al., 2004). Although calretinin-immunoreactive neurons are inconspicuous in the claustral complex of the developing and adult mouse, a prominent plexus of calretinin-positive axons has been described in the adult dorsal claustrum (Real et al., 2003a). The calretinin-positive axons within the dorsal claustrum are supposed to be extrinsic, mainly arising from thalamic neurons since this calcium-binding protein is expressed in thalamic nuclei projecting to the claustrum (see below; Gonza´lez et al., 2002). During development, calretinin-positive axons were observed in the dorsal claustrum at E18.5. The calretinin-positive plexus in the mouse dorsal claustrum became especially prominent around birth and during early postnatal development (P0 –P4), but appeared less distinctive during later postnatal ages and in the adult (Real et al., 2003a). This finding is similar to the findings in the rat neocortex and thalamus, in which several fiber systems display distinctive calretinin immunostaining during the prenatal and early postnatal development, but the immunoreactivity becomes less conspicuous at later postnatal stages (Fonseca et al., 1995; Frassoni et al., 1998). The increase in the density of calretinin axons innervating the mouse dorsal claustrum during late embryonic stages and early postnatal ages correlates with an increase in the intensity of calretinin expression in certain dorsal thalamic nuclei (unpublished observations), which is consistent with the apparent thalamic origin of the calretinin-immunoreactive innervation of the claustrum. The calretinin-positive axons within the dorsal claustrum displayed a characteristic pattern, which is maintained from E18.5 until the adult, consisting in a dense plexus of varicose axons forming a shell around a core practically devoid of positive fibers. This organization was observed previously in the adult mouse dorsal claustrum on the basis of distinct chemoarchitecture and cortical connections, suggesting that the dorsal claustrum consists of a central region or “core” and a belt region or “shell” (Real et al., 2003a,b). Of interest, only the “shell” of the dorsal claustrum in the adult mouse (but not the “core region”) is reciprocally connected with certain midline and intralaminar dorsal thalamic nuclei that contain CR⫹ neurons (Matas et al., 2003). Comparison of CR immunoreactivity and Cadherin 8 mRNA (Cad8, a marker of the dorsolateral claustrum) at P0 (Fig. 4), indicates that the claustral core (poor in calretinin) includes only the central part of the dorsolateral claustrum (rich in Cad8 expression and pale for calbindin), whereas the calretinin-rich

55 neuropil in the dorsal claustrum includes a “shell” region of the dorsolateral claustrum as well as the ventromedial claustrum (as defined in Puelles et al., 2000; Medina et al., 2004). It is at present unclear how the internal compartmentalization of the dorsolateral claustrum is established during development, although the arrival of the thalamic or cortical axons may play a role.

Parvalbumin In the developing claustral complex, the onset of parvalbumin expression occurs postnatally, much later than that of GABA and the two other calcium-binding proteins, calbindin or calretinin. This late, postnatal expression of parvalbumin in the claustrum is consistent with other studies concerning different pallial structures. In the rat somatosensory cortex, parvalbumin immunoreactivity appeared at the end of the first postnatal week (Sańchez et al., 1992). Also, in the developing mouse neocortex, parvalbumin appeared at the second week after birth (Del Rio et al., 1994), and in the rat basolateral amygdaloid complex, well immunostained parvalbumin-positive neurons were not observed before P17 (Berdel and Morys, 2000). In the developing mouse claustrum, we observed parvalbumin-immunoreactive neurons (PV⫹) from P4 onward, although the adult pattern was not displayed until around P23. At this stage and in the adult, the mouse dorsal claustrum contains a higher number of PV⫹ neurons than the endopiriform nuclei, as also described in rat (Celio, 1990). The pattern of parvalbumin expression in the mouse dorsal claustrum also delineates a core and a shell region intensely or lightly PV⫹ (Real et al., 2003a), coincident with those observed with calretinin immunoreactivity or Cad8 expression, although the parvalbumin immunoreactivity expression pattern is displayed later. Due to the characteristic late expression of parvalbumin during cortical development, this calcium-binding protein has been implicated in the maturation of cortical inhibitory circuits and/or the onset of experience-dependent activity (Stichel et al., 1987; Seto-Ohshima et al., 1990; Hendrickson et al., 1991; Solbach and Celio, 1991; Sańchez et al., 1992; Alcańtara et al., 1993; Hogan and Berman, 1994). It is interesting to note that the time of onset and increase of PV⫹ neurons in the claustral complex coincides with the postnatal reduction in the number of CB⫹ neurons. This finding has also been observed in the rat cerebral cortex, and it appears that this is related to a phenotypic shift of some nonpyramidal neurons (or interneurons) from CB⫹ to PV⫹ (Alcańtara et al., 1996). This phenotypic shift passes through a stage during the second postnatal week in which 80 –100% of the CB⫹ neurons also express PV, but the two proteins become segregated to mostly nonoverlapping neuronal populations in the adult (Alcańtara et al., 1996). Double-labeling studies will be needed to determine whether a similar phenotypic change occurs in the neurons of the claustral complex during postnatal development. In contrast to the CB⫹ neurons and the CR⫹ neurons, in the cerebral cortex and pallial amygdala of adult rats, all PV⫹ neurons are GABAergic (Kemppainen and Pitka¨nen, 2000) and possibly all have a subpallial origin. This finding is likely to be true also in the claustral complex, although further studies will be required to investigate the exact origin of these neurons within the subpallium.


56

Conclusions Briefly, the calcium-binding proteins CB, CR, and PV are observed in neurons during development of the claustral complex following distinct spatiotemporal patterns. CB⫹ cells start to be seen very early during embryogenesis and increase dramatically until birth, thus becoming the most abundant cell type during embryonic development, especially in the ventral pallial part of the claustrum. PV⫹ cells start to be seen only postnatally, and their number then increases while the density of CB⫹ neurons decreases. CR is observed only in few neurons of the claustral complex but it is very abundant in axons that apparently originate in the thalamus. Based on CR expression, the core/shell compartments of the dorsal claustrum starts to be clearly seen at E18.5 and may be related to the development of the thalamoclaustral input. Comparison with Cad8 expression indicates that the core includes a central part of the dorsolateral claustrum, whereas the shell includes a peripheral area of the dorsolateral claustrum, plus the adjacent ventromedial claustrum. At least part of the CB⫹ and CR⫹ neurons, and possibly all the PV⫹ neurons of the claustral complex are GABAergic and appear to originate in the subpallium. Future studies will need to address whether part of the CB⫹ neurons of the claustral complex originate within the pallium, and whether the postnatal decrease of the CB⫹ neuronal density in the claustral complex is due to transient expression and/or phenotypic shift. Considering that the claustral complex is involved in propagation of temporal lobe epilepsies and that seizures are related to an alteration of the GABAergic inhibitory activity, our results on the development of several subtypes of GABAergic neurons in the claustral complex may be useful for future studies on this disease.

ACKNOWLEDGMENT We thank Dr. Christoph Redies for kindly providing us with the Cadherin 8 cDNA.

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