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THE JOURNAL OF COMPARATIVE NEUROLOGY 498:491–507 (2006)

Cellular Composition and Cytoarchitecture of the Rabbit Subventricular Zone and Its Extensions in the Forebrain GIOVANNA PONTI,1 PATRIZIA AIMAR,1 AND LUCA BONFANTI1,2* Department of Veterinary Morphophysiology, University of Turin, 10095 Grugliasco, Italy 2 Rita Levi Montalcini Center for Brain Repair, 10125 Turin, Italy

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ABSTRACT Persistent neurogenic sites, harboring neurogenic progenitor cells, which give rise to neuronal precursors throughout life, occur in different mammals, including humans. The telencephalic subventricular zone (SVZ) is the most active adult neurogenic site. Despite remarkable knowledge of its anatomical and cellular composition in rodents, detailed arrangement of SVZ in other mammals is poorly understood, yet comparative studies suggest that differences might exist. Here, by analyzing the cellular composition/arrangement in the SVZ of postnatal, young, and adult rabbits, we found a remarkably heterogeneous distribution of its chain and glia compartments. Starting from postnatal stages, this heterogeneity leads to a distinction between a ventricular SVZ and an abventricular SVZ, whereby the former contains small chains and isolated neuroblasts and the latter is characterized by large chains and a loose astrocytic meshwork. In addition to analysis of the SVZ proper, attention has been focused on its extensions, called parenchymal chains. Anterior parenchymal chains are compact chains surrounded by axon bundles and frequently establish direct contact with blood vessels. Posterior parenchymal chains are less compact, being squeezed between gray and white matter. In the shift from neonatal to adult rabbit SVZ, chains occur very early, both in the SVZ and within the brain parenchyma. Comparison of these results with the pattern in rodents reveals different types of chains, displaying a variety of relationships with glia or other substrates in vivo, an issue that might be important in understanding differences in the adaptation of persistent germinative layers to different mammalian brain anatomies. J. Comp. Neurol. 498:491–507, 2006. © 2006 Wiley-Liss, Inc. Indexing terms: neurogenesis; astrocyte; glial tubes; chain migration; neural stem cells

Adult neurogenesis in mammals is a spatially restricted process that has been fully demonstrated to occur in the hippocampus and olfactory bulb of rodents (Gage, 2000; Alvarez-Buylla and Garcia-Verdugo, 2002). During the last few years, several reports, in most cases carried out at the light microscopic level, have confirmed that adult neurogenesis does exist within the same regions of other mammalian species, including rabbits (Luzzati et al., 2003), cattle (Rodriguez-Perez et al., 2003), primates (Gould et al., 1997, 1999a,b, 2001; Kornack and Rakic, 1999, 2001a; Pencea et al., 2001; Bernier et al., 2002), and humans (Eriksson et al., 1998; Bernier et al., 2000; Sanai et al., 2004; Be´dard and Parent, 2004). These studies suggest that important differences might exist concerning both the internal arrangement of mammalian neurogenic regions and, theoretically, the possible destination(s) of the newly generated progeny. © 2006 WILEY-LISS, INC.

The forebrain subventricular zone (SVZ) is a major reservoir of adult neural stem cells (Morshead et al., 1994; Gage, 2000; Alvarez-Buylla and Garcia-Verdugo, 2002) and the source of olfactory bulb neuronal precursors undergoing long-distance, rostral migration through the ros-

Grant sponsor: MURST (Fondo per l’Incentivazione della Ricerca di Base; FIRB); Grant number: RBNE01YRA3; Grant sponsor: Regione Piemonte; Grant number: 1837/27; Grant sponsor: Compagnia di San Paolo; Grant number: 5053SD/PF; Grant sponsor: University of Turin. *Correspondence to: Luca Bonfanti, Department of Veterinary Morphophysiology, Via Leonardo da Vinci 44, 10095-Grugliasco (Turin), Italy. E-mail: [email protected] Received 6 November 2005; Revised 27 February 2006; Accepted 29 March 2006 DOI 10.1002/cne.21043 Published online in Wiley InterScience (www.interscience.wiley.com).

The Journal of Comparative Neurology. DOI 10.1002/cne

492 tral migratory stream (RMS), being unique in allowing an adult-generated neurogenic progenitor cell progeny to reach brain regions located far from the site of origin (Lois and Alvarez-Buylla, 1994; Garcia-Verdugo et al., 1998; Peretto et al., 1999). In rodents, rabbits, and primates, this cell displacement occurs in the form of tangentially oriented “chains” of neuroblasts expressing the polysialylated form of the neural cell adhesion molecule (PSANCAM; Bonfanti and Theodosis, 1994; Rousselot et al., 1995; Bernier et al., 2000, 2002; Kornack and Rakic, 1999, 2001a; Pencea et al., 2001; Luzzati et al., 2003; RodriguezPerez et al., 2003) and doublecortin (DCX; Nacher et al., 2001). In the adult, these chains are separated from the mature brain parenchyma by astrocytic sheaths (Lois et al., 1996; Jankovski and Santelo, 1996; Peretto et al., 1997), referred to as glial tubes in rodents (Peretto et al., 1997), whereas chains and glial tubes are absent at birth and later assemble at specific postnatal stages (Alves et al., 2002; Tramontin et al., 2003; Peretto et al., 1999, 2005). Although a more detailed description is available in rodents, some examples of intriguing structural differences have been described in the SVZ of rabbits (Luzzati et al., 2003), cattle (Rodriguez-Perez et al., 2003), and humans (Sanai et al., 2004; Quinones-Hinojosa et al., 2006), involving both glial and neuronal compartments. In humans, in particular, cells of the RMS do not form chains; the SVZ itself displays a different glial organization not involving well-formed glial tubes (Sanai et al., 2004; Quinones-Hinojosa et al., 2006). On the other hand, in a previous study carried out with rabbits, we reported the existence of large chains of neuroblasts within the SVZ, some of which extend outside the neurogenic area to enter subcortical regions in the frontal and temporal lobes (Luzzati et al., 2003). These latter chains are glia-independent; they are not contained within the typical glial tube arrangement or any glial meshwork, so they are in direct contact with the mature brain parenchyma. The occurrence of “parenchymal chains” (Luzzati et al., 2003) at specific subcortical sites represents further extensions of the SVZ chains, theoretically explaining alternative destinations for SVZ-derived neuronal precursors that do not follow the RMS, although no cortical neurogenesis apart from the subcortical amygdala has been found in the rabbit (Luzzati et al., 2003). On the other hand, cortical neurogenesis has been proposed to occur in primates (Gould et al., 1999a, 2001), there being no detectable extra-SVZ chains other than the temporal stream directed to the amygdala (Bernier et al., 2002). All aspects reported above are different from the pattern described in rodents, in which SVZ neuronal precursors seem to be consistently directed toward the olfactory bulb through the dense astrocytic meshwork of the glial tubes (Lois et al., 1996; Jankovski and Sotelo, 1996; Peretto et al., 1997; AlvarezBuylla and Garcia-Verdugo, 2002). Thus, it appears clear that important differences might occur in SVZ arrangement and extension when different species and different postnatal stages are considered. Further knowledge on the SVZ anatomical and cellular arrangement is required to provide the anatomical basis for future brain repair strategies concerning the possible endogenous mobilization of newly generated cells from the neurogenic progenitor cell compartments (Goldman, 2004; Lie et al., 2004). In this context, the lagomorphs appear to be an interesting model to unravel how the SVZ neuro-

G. PONTI ET AL. genic site has evolved among mammals. In the present study, we have used confocal and electron microscopy to address the following issues: 1) the cellular arrangement of rabbit SVZ, with particular attention on the relationships between the neuronal (neuroblasts’ SVZ chains) and glial (SVZ astrocytes) compartments; 2) the changes occurring in the postnatal rabbit SVZ; and 3) the detailed ultrastructural features of the extensions represented by rabbit parenchymal chains. Finally, the aim of these analyses was to compare the existence of different types of chains and their relationship with glial or other substrates in the SVZ and forebrain of different species, to enhance understanding of their adaptation to different brain anatomies in vivo.

MATERIALS AND METHODS Tissue preparation Experimentation was conducted in accordance with current EU and Italian laws, with authorization by the Italian Ministry of Health n. 66/99-A. Twelve postnatal (5, 10, 15, 30, 40, and 60 days old; n ⫽ 2 per time point), 10 peripuberal (4 and 6 months old), and six adult (1 and 2 years old) New Zealand white HY/CR female rabbits (Orictolagus cuniculus; Charles River, Milan, Italy) were used. Animals were anesthetized (pentothal sodium; 60 mg/100 g b.w.) and perfused intracardially with a heparinized saline solution (25 IU/ml in 0.9% Ringer solution), followed by 4% paraformaldehyde ⫹ 1% picric acid for light microscopy or 2% glutaraldehyde ⫹ 1% paraformaldehyde and 0.2% picric acid for conventional electron microscopy, in 0.1 M sodium phosphate buffer, pH 7.4. After dissection, brains for light microscopy were postfixed overnight in the same fixative, cryoprotected, frozen at ⫺80°C, and cryostat (25 and 40 ␮m thick) sectioned in series. Brains for electron microscopy (n ⫽ 4, 4 months old) were postfixed for 2 hours, then coronal vibratome sections (200 ␮m) were cut, fixed in osmium-ferrocyanide for 1 hour at 4°C, and then stained en bloc with 1% uranyl acetate, ethanol dehydrated, and embedded in Araldite, as previously described (Peretto et al., 1997). Ultrathin sections were examined with a Philips CM10 transmission electron microscope. The astrocytic glia contact frequencies (percentage of chain surface in direct contact with astrocytic cell bodies and processes) were analyzed at the ultrastructural level within two levels of the SVZ (levels 2 and 3, corresponding to rostral extension and lateral ventricle; see Fig. 1A) and in anterior and posterior parenchymal chains (levels 1 and 5). A semiquantitative analysis was carried out on 10 semithin sections harvested at intervals of 10 ␮m in each level, including at least one of the four types of chain considered (see Fig. 6), in three peripuberal animals (total sections examined ⫽ 120; total chain perimeters examined ⫽ 280). Micrographs were imported on Adobe Photoshop as digital images and were drawn to define the contour of single chains, using different colors for contact with different substrates. Images of the contours were then imported to Image Pro Plus to evaluate their length. The electron microscopic reconstruction of two posterior parenchymal chains was carried out on serial ultrathin sections (80 nm thick) collected onto Formvar-coated grids. Micrographs from the tenth section in the sequence were drawn to define the contour of single cells and added


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in sequence in Adobe Photoshop. Drawings from 35 levels were examined to follow the evolution of the chains across a total length of 27 ␮m.

roxide and FeCN 3% for 1 hour at 4°C, dehydrated, and embedded in araldite as previously described (Luzzati et al., 2003).

Immunohistochemistry

Preparation of figures

Immunohistochemical reactions were carried out by using single peroxidase and double immunofluorescence methods on cryostat sections incubated overnight at 4°C with a single primary antibody or a combination of primary antibodies. The following primary antisera and antibodies were used: 1) anti-PSA-NCAM, diluted 1/3,500 [mouse monoclonal IgM, AbCys AbC0019, clone2-2B, lot 5(99)]; raised against alpha2– 8 linked neuraminic acid (NeuAc alpha2– 8)n, with n ⬎ 10 (polymer usually termed polysialic acid; PSA); 2) anti-BrdU, 1/500 (rat monoclonal, Harlan MAS250 clone BU1/75(ICR1), lot FC03A250-2]; reacts with BrdU in single stranded DNA, BrdU attached to a protein carrier, or free BrdU; this antibody does not cross-react with thymidine; 3) anti-Ki67, 1/300 (mouse monoclonal; Immunotech 0505 clone MIB1); this antibody is raised against a human recombinant peptide corresponding to a 1,002-bp Ki67 cDNA fragment, generated with the synthetic peptide AGGDEKDIKAFMGTPVQKLD; reacts with the Ki67 nuclear antigen (345- and 395-kD double band in Western blot of proliferating cells; manifacturer’s technical information); 4) antiglial fibrillary acidic protein (GFAP), 1/1,000 [rabbit polyclonal, Dako Z0334, lot 096(701)], raised to GFAP isolated from bovine spinal cord; this antibody reacts with GFAP of many species; 5) antivimentin, 1/800 [mouse monoclonal; Dako M0725 clone V9, lot 049(201)]; raised against purified vimentin from porcine eye lens; this antibody reacts with the 57-kDa intermediate filament protein present in cells of mesenchymal origin with a broad interspecies cross-reactivity, displaying no reaction with other closely related intermediate filament proteins; and 6) anti-DCX (C-18), 1/750 (polyclonal goat; Santa Cruz sc-8066, lot J3103); raised against a peptide corresponding to amino acids 385– 402 at the carboxy terminus of doublecortin of human origin. For double staining, a combination of two indirect immunofluorescence procedures with fluorescein isothiocyanate (FITC)- ⫹ Cy3-conjugated antibodies were used. All the antibodies were diluted in a solution of 0.01 M PBS, pH 7.4, containing 0.1% Triton X-100. Fluorescent specimens mounted in Dabco (Sigma, St. Louis, MO) were observed with a laser scanning Olympus Fluorview confocal system, using appropriate filters.

Light photomicrographs were digitized with a digital camera (Coolpix E995; Nikon) fitted to a Zeiss Axioplan microscope. Analysis and digital photography of the double immunofluorescence was carried out with a laser scanning Olympus Fluoview confocal system (Olympus Italia, Milano, Italy). Black-and-white negatives from a Philips CM10 electron microscope were imported into Adobe Photoshop by using an Epson Perfection (3200 Photo) scanner. Digital images were brightness, color, and contrast balanced and assembled into montages in Photoshop 8.0.1.

Immunoelectron microscopy The ultrastructural localization of DCX was obtained by employing a preembedding immunogold reaction (Yang et al., 2004). Rabbits were perfused with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M sodium phosphate buffer, postfixed overnight at 4°C, washed in cold PBS, and vibratome sectioned (70 ␮m thick). After aldehyde blocking in 100 mM NH4Cl for 2 hours, slides were washed in PBS and incubated in 1% NHS⫹ 0.1 Triton X-100 in PBS for 2 hours and then in anti-DCX diluted 1:200 in PBS at 4°C for 48 hours. To detect the reaction, slices were incubated in rabbit anti-goat secondary antibody conjugated with gold particles (5 nm; Ted Pella, Redding, CA) for 2 hours at room temperature, washed in PBS, then washed in distilled water, silver enhanced for 2 minutes (Ted Pella), and washed in tap water. Next, slices were washed in PBS, counterstained in 2% osmium tet-

RESULTS We analyzed the SVZ of late postnatal (40 – 60 days), young peripuberal (4 – 6 months old), and adult (1–2 years old) rabbits. At all the ages studied, the region corresponding to the SVZ was easily recognizable using parameters assessed in a previous report in order to outline its contours (Luzzati et al., 2003), based on the identification of elements typically present in the SVZ of rodents (AlvarezBuylla and Garcia-Verdugo, 2002). These elements involve an area adjacent to the telencephalic ventricular cavities, enriched with neuroblasts and astrocytes, including proliferating and migrating cells, the latter organized in chains (see Fig. 1). The very early postnatal stages (from P5 to P30) leading to this arrangement will be described in a separate section. Because of the persistence of an open olfactory ventricle throughout life (Leonhardt, 1972), the SVZ rostral extension of rabbits maintains direct contact with the ependymal monolayer, covering its dorsal and, to a lesser extent, lateral and medial parts. As a result, the rabbit SVZ rostral extension is a triangular tissue squeezed between the olfactory ventricle (ventrally) and the anterior forceps of the corpus callosum (dorsally; Fig. 1A,B, levels 1, 2). More posteriorly, the topographic location of the SVZ adjacent to the lateral ventricle is roughly similar in rabbits and rodents, being prevalent in the dorsal-lateral part of the ventricular wall (Fig. 1A,B, levels 3, 4).

Anatomical arrangement of the rabbit SVZ Cresyl violet stainings and confocal images of coronal sections cut through the SVZ at different anteriorposterior levels immunostained for antigens allowing the identification of the neuronal (PSA-NCAM, DCX) and glial (GFAP, vimentin) elements revealed the existence of both cellular compartments previously described in rodents, namely, chains of neuroblasts immersed within an astrocytic meshwork (Fig. 1), yet differently organized. The PSA-NCAM⫹ and DCX⫹ chains of neuroblasts appeared quite heterogeneous in size (ranging from 5 to 70 ␮m, in cross-section), mostly appearing larger than in rodents. Moreover, chains were observed to follow a pattern with regard to their topographic distribution within the SVZ area, the larger chains being grouped in the part of the SVZ close to the corpus callosum, whereas only smaller chains (two to four cells) and isolated cells were detectable close to the ventricular wall. Based on differences observed at the confocal level, two distinct portions were identified in the rabbit SVZ, re-


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Figure 1.


RABBIT SUBVENTRICULAR ZONE ferred to as ventricular SVZ (smaller, adjacent to the ventricular surface and containing small aggregates) and abventricular SVZ (larger, relatively distant from the ventricle and containing large chains; Fig. 1). These parts were separated by a layer of tissue, apparently poor in cell bodies (Fig. 1), whose thickness varied according to the levels considered and individual variability. The ultrastructural study confirmed the presence of a band of tissue poor in cell bodies and enriched in astrocytic processes and axons (Fig. 2; see below). In late postnatal animals, this pattern was present both at the rostral extension and at the lateral ventricle levels (Fig. 1D,E), whereas, in peripuberal and adult animals, it was restricted to the rostral extension, the large chains being more clustered, sometimes aggregating into a large mass, at the lateral ventricle levels (Fig. 1F–I). The distinction between ventricular and abventricular SVZ was detectable at all ages studied, the main differences being not qualitative but rather linked to the changing overall anatomy. Indeed, the SVZ appeared slightly reduced in size at increasing ages, and the chains appeared less distinguishable from one another and displaced more laterally compared with the ventricles (Fig. 1D–I). Through the rostral extension, the ventricular SVZ formed an arcade covering the vault of the olfactory ventricle, whereas the abventricular SVZ was a triangle of tissue squeezed between this arcade and the white matter of the corpus callosum anterior forceps (Fig. 1). At the lateral ventricle level, this pattern was maintained, with the ventricular SVZ lining the dorsal-lateral wall of the ventricle and the abventricular SVZ forming a triangle squeezed between corpus callosum and striatum (Fig. 1). Another difference compared with the rodent SVZ concerned the immunocytochemical visualization of the glial meshwork that appeared less dense than the tightly packed glial tubes (Peretto et al., 1997), an aspect to be

Fig. 1. Organization of the rabbit SVZ. A: Schematic representation of the rabbit SVZ whole area (gray) and parenchymal chains (black; modified from Luzzati et al., 2003). In blue, indicated by numbers, are the different levels investigated. LVpr, projection of the lateral ventricle; VLE, ventral-lateral extension. B: Schematic representation of the internal arrangement of SVZ compartments: a ventricular and an abventricular SVZ are distiguishable at the levels both of the rostral extension (representative coronal section at level 1, left; OV, olfactory ventricle) and of the lateral ventricle (LV; representative coronal section at level 3, right); light pink, SVZ area; dark pink, chains of neuronal precursors; white, white matter; gray, gray matter; CC, corpus callosum; AF, its anterior forcep. C: Schematic representation of cell types observed in the ventricular SVZ contacting the OV (left) or the LV (right) on the basis of the ultrastructural analysis (see Fig. 2). Green, astrocytes and their processes; pink, neuroblasts; gray, ependyma; large dark circles, myelinated axons; small, gray circles, unmyelinated axons; asterisk, type C-like cell. Note that astrocytic cell bodies (both contacting the ventricular lumen and forming a row in the ventricular SVZ) are abundant only at the LV level. They are not represented at the OV level because they are rare. D–I: Confocal images after immunostaining with neuronal (DCX, red) and glial (GFAP, green) markers identifying, respectively, the chains and the glial meshwork. J–O: Confocal images after immunostaining with vimentin (red) and GFAP (green), at different ages. Note that vimentin becomes prevalently restricted to a subpopulation of glial cells in the SVZ, particularly in the lateral part. Cell proliferation, detected with Ki67 antigen (P–T, red), is restricted to the SVZ area starting from P15 (P–R). S,T: Double staining for Ki67 (red) and GFAP (green). Scale bars ⫽ 200 ␮m in D–R; 25 ␮m in S; 75 ␮m in T.

495 confirmed by the ultrastructural analysis (see below). At all levels and ages examined, the glial meshwork was present in both portions of the SVZ (ventricular and abventricular), being denser in the ventricular SVZ and, in some cases, on the external perimeter of the SVZ, at the border with the mature brain parenchyma. In addition, by studying the occurrence of the cytoskeletal marker vimentin, which is known to be abundantly present in radial glia and immature astrocytes of adult neurogenic sites (Peretto et al., 1999, 2005; Seri et al., 2004), we observed the persistence of immunoreactive astrocytes in the SVZ, forming a subpopulation decreasing in size at increasing ages (Fig. 1J–O). Very intensely stained astrocytes were mostly localized within the ventricular SVZ, whereas the immunoreaction was fainter in the abventricular SVZ (Fig. 1J–O). In addition, double labelling for vimentin and GFAP (Fig. 1K,M,O) compared with vimentin single staining (Fig. 1J,L,N) showed that astrocytes in the lateral part of the SVZ, an area that contains most of the neuroblasts, is characterized by having less GFAP. This suggests that less mature astrocytes are topographically concentrated around neuronal precursors of the ventricular SVZ and to a lesser extent intermingled with those of the abventricular SVZ. To investigate in greater detail the cellular arrangement of these portions of the rabbit SVZ, we performed an electron microscopic analysis at different anteriorposterior levels, in peripuberal animals. This study was prevalently focused on the SVZ rostral extension (levels 1 and 2), where the compartments formed by glial meshwork and chains are easily identifiable, and on the anterior part of the lateral ventricle (level 3), where most of the stem cell niche was expected to occur.

Cellular arrangement in the rabbit SVZ Cell composition. Electron microscopy confirmed the presence of two main cell compartments at the ultrastructural level: 1) neuroblast-like cells, with a large nucleus and a thin halo of electrondense cytoplasm (type A cells; Doetsch et al., 1997), and 2) protoplasmic astrocytes (type B cells), recognizable by their typically watery-like cytoplasm (Fig. 2). In comparison with rodent neuroblasts, rabbit type A cells were slightly less electron dense in their cytoplasm (Fig. 2E,F), a feature probably resulting from a lower concentration of free ribosomes. Their nuclei also appeared clearer, with very dark, condensed chromatin clusters. To confirm their nature of neuronal precursors, we combined electron microscopy with the immunocytochemical detection of the cytoskeletal marker DCX, which is expressed in newborn neurons (Brown et al., 2003; Yang et al., 2004). All cells showing the cytological features of type A cells in the rabbit SVZ and in its extensions represented by parenchymal chains were intensely marked for DCX (the immunoelectron microscopic localization of DCX in type A cells is shown in Fig. 4, referring to parenchymal chains), whereas the same staining was consistently absent in astrocytes and other cell types or parenchymal structures (see Fig. 4). A common pattern corresponding to the features described above was detectable throughout the SVZ; nevertheless, some differences in the cellular composition were observed in the anterior part (rostral extension, levels 1, 2) vs. the posterior one (lateral ventricle, levels 3, 4). Substantial differences concerned the ventricular SVZ associated to the lateral ventricle (in level 3), which ap-


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Fig. 2. Ultrastructural features of the young/adult rabbit SVZ. A–H: At the level of the rostral extension. I–M: At the level of the lateral ventricle. A: Schematic representation of the topographic location of ultrastructural images (B–D) referring to the abventricular SVZ (B), the ventricular SVZ (D), and the band of tissue interposed between them (C) at the rostral extension level. In the abventricular SVZ (B,B⬘), large chains of neuroblasts (Ch) are in contact with unmyelinated (Ax) and myelinated (arrows) axons and with astrocytic processes (b). Note the tightly packed neuroblasts (a, in B⬘ and E) without the typical intercellular clefts described in rodents (F). Also, the cytoplasm is clearer in rabbit type A cells with respect to those of mice (E,F). Abventricular SVZ chains are frequently associated with blood vessels (bv, in G and H). Neuroblasts can be in direct contact with the blood vessel wall (arrows). Scattered neuroblasts (a) are detectable in the ventricular SVZ (D), squeezed between astrocytic

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processes (top) and ependymal cells (e, bottom). Two type C-like cells (asterisks) are indicated in D. OV, olfactory ventricle. A few cell bodies are detectable in the band of tissue between the ventricular and the abventricular SVZ, which is prevalently formed by axons and glial processes (C). I: Schematic representation of the topographic location of ultrastructural images (J–M) referring to the ventricular SVZ at the level of the lateral ventricle. Cellular elements of the stem cell niche described in rodents are present: ependymal cells (e), type A cells (a), astrocytes (b), type C-like cells (asterisks), and mitoses (m). Note that astrocytes do not form a complete ensheathment but rather a row of cells (J). Some of them show a process intermingled between ependymal cells and protruding within the ventricle (M, arrow). Large, oval mitoses (K,L) are probably proliferating type C cells. LV, lateral ventricle. Scale bars ⫽ 3 ␮m in E,F,B⬘; 5 ␮m in B–D,G–M.


RABBIT SUBVENTRICULAR ZONE peared more complex because of the frequent occurrence of astrocytic cell bodies (type B cells) forming an irregular row at the border between the clusters of neuroblasts and the adjacent parenchyma (Fig. 2J,L). Some of these astrocytes displayed a process elongated among ependymal cells and protruding into the ventricle, thus contacting the ventricular lumen (Fig. 2M). In addition, within the row of astrocytes, some type C-like cells (Doetsch et al., 1997) were detectable (Fig. 2). The latter were large, oval, and characterized by a cytoplasm that was darker than astrocytes and lighter than neuroblasts, and their nuclei frequently displayed deep invaginations (Fig. 2J,L). Some large, oval mitoses were also present at the same location (Fig. 2K,L). On the whole, the cellular elements observed within the ventricular SVZ of the lateral ventricle in rabbits were reminiscent of the stem cell niche described in rodents (Doetsch et al., 1997), although their overall organization was different (see below). In the ventricular SVZ of the rostral extension (levels 1, 2), the main cell types were represented by neuroblasts and ependymal cells, whereas astrocytic cell bodies and type C-like cells were rare (Figs. 1C, 2D). Cytoarchitecture. As in the SVZ of rodents, at the electron microscopic level, the rabbit neuroblasts were observed to form typical aggregates corresponding to cross-sections of tangentially oriented chains (Fig. 2). The ultrastructural study confirmed that differently sized chains and astrocytic glia were not uniformly distributed within the SVZ area. In coronal sections of the abventricular SVZ, about 10 –20 medium-large chains were visible, some of which included up to 15–20 nucleated cells when cut transversally (Fig. 2B). Most of these chains consisted of a homogeneous mass of type A cells, although, at the top of some, a few type C-like cells were detectable (Fig. 2B). Their general aspect was frequently compact, with cell membranes of the neuroblasts usually directly juxtaposed (Fig. 2B,B⬘), the occurrence of intercellular clefts being occasional. These chains were frequently observed in strict association with blood vessels, particularly in the rostral extension (Fig. 2). In most cases, neuroblasts were in direct contact with the endothelial cells, without the interposition of astrocytic processes (Fig. 2G,H). Astrocytes of the abventricular SVZ, though more abundant than in the surrounding brain tissue, did not form a continuous sheath around the chains (Fig. 2B). This pattern left wide portions of the neuroblast’s surface in direct contact with parenchymal structures, represented by unmyelinated and, to a lesser extent, myelinated fibers (Fig. 2B⬘). On the other hand, astrocytes and their processes were abundant in the ventricular SVZ, where small clusters of type A cells were extensively ensheathed by the ependymal monolayer on one side (ventrally) and by the glial processes on the other (dorsally; Fig. 2D). This was even more evident at the lateral ventricle level (levels 3, 4), because of the abundance of both astrocytic cell bodies and their processes. As a result, along the narrow area of the ventricular SVZ through the olfactory and lateral ventricles, the contact between type A cells and mature brain parenchyma was less frequent. A broad band of tissue almost devoid of cells and enriched with astrocytic processes and axons (Fig. 2C) filled the area between ventricular and abventricular SVZ, contributing to enhance their distinction. Around the lateral ventricle, the limit between the ventricular SVZ and this band of tissue was sharpened

497 by a row of astrocytes (see above; Fig. 2J–M). At more posterior levels, astrocytes were less abundant, and the limit between the SVZ and the brain parenchyma was frequently filled by bundles of densely packed myelinated axons (not shown).

Anatomical and cellular arrangement in the early postnatal rabbit SVZ The same type of histological and confocal analyses described above for the late postnatal and young/adult rabbit SVZ were carried out on animals at early postnatal ages (from P5 to P30; summarized in Fig. 3). This allowed us to compare the results obtained in a recent study concerning the postnatal assembly of the two SVZ cellular compartments in rodents (Peretto et al., 2005). In early postnatal rabbits (P5), the SVZ appeared quite homogeneous in structure at all the levels examined (Fig. 3A–F). Radial glia, coexisting with a uniform neuroblast cell mass, were prevalent through the SVZ area (Fig. 3B,F), similarly to what had been observed in early postnatal rodents (Peretto et al., 2005). Both in cresyl violetstained (Fig. 3A,C–E) and in immunocytochemically treated (Fig. 3B,F) specimens, no aggregates reminiscent of chains were detectable at this stage, although a slight distinction of the areas corresponding to the ventricular and abventricular SVZ was already visible (Fig. 3F). Starting from P10, and more strikingly at P15, differences were observed at specific levels. Although no chains were present in the anterior part of the olfactory peduncle (anterior to level 1; Fig. 3G,K), a large, homogeneous mass of neuroblasts immunoreactive for DCX was present in the ventral part of the SVZ at level 2 (Fig. 3L–O), whereas, dorsally to the olfactory ventricle, numerous differently sized chains were present (Fig. 3P). Similar chains were also detectable at most posterior levels corresponding to the wall of the lateral ventricle (level 3; Fig. 3J,Q). At about P15–P30, the down-regulation of vimentin was accompanied by the appearance of GFAP in the SVZ glial compartment, although vimentin immunoreactivity continued to be detectable in the SVZ, even in older animals (Fig. 1J–O). With GFAP/DCX double staining, a clear distinction between ventricular and abventricular SVZ was first detectable in P10 –P15 rabbits (Fig. 3J–P). At subsequent stages (P30 –P40), the ventral mass was still present, but coronal sections cut in its anterior and posterior parts revealed that this mass had split into several large chains (Fig. 3S). Even in this case, the glial meshwork was heterogeneous, being more densely packed around the central part of the mass (Fig 3O), and nearly absent around the chains at its anterior and posterior endings (Fig. 3S). In general, notwithstanding wellformed chains in the abventricular SVZ and the ventral cell mass, the glial meshwork was not clearly detectable everywhere (Fig. 3P,S,V). On the whole, from the qualitative point of view, starting from P15, the overall internal arrangement of the SVZ was comparable to that observed in the young/adult animal. Anterior parenchymal chains were not present in P5 rabbits (Fig. 3A–F). They were first observed starting from P10 (Fig. 3H,I), with a highly variable frequency in different animals (about 20% in the period between P10 and P40). By using an antivimentin antibody, radial glial cells were clearly distinguishable in the early postnatal SVZ (P5–P15; Fig. 3B,F,H,J). During this period, no particular


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Fig. 3. Early postnatal development of the rabbit SVZ. Cresyl violet (A,C–E,G,I,Iⴕ,K–N,Q,R,U,Uⴕ), doublecortin (T), vimentin (VIM; green)/doublecortin (DCX; red; B,F,H,J), and GFAP (green)/ DCX (red; O,P,S,V) staining in coronal sections of the SVZ at different anterior-posterior levels. Undefined masses of neuroblasts, not organized to form chains, are intermingled with radial glial fibers at P5 (A–F). The first chains are detectable at P10 (H–J), localized both in the parenchyma (H,I,I⬘) and in the SVZ (J). At this stage, the radial glial structures continue to show no particular relationship with respect to the chains (J). At P15, a large mass of neuroblasts is detect-

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able in the ventral part of the SVZ rostral extension (L–O, arrows). Starting from this stage, dorsally to the olfactory ventricle (OV) and in the dorsolateral corner of the lateral ventricle (LV), a clear distinction between ventricular (Vsvz) and abventricular (Asvz) SVZ is recognizable (P–Q). Qualitatively, this type of organization is present in all subsequent stages (T–V and Fig. 1). No chains were observed in the anterior part of the rostral extension contained within the olfactory peduncle, at any of the postnatal ages examined (A,G,K,R). Scale bars ⫽ 300 ␮m in H,I,Q; 50 ␮m in H(inset); 200 ␮m in A–G,K–P,R– T,U; 100 ␮m in I⬘,U⬘,V; 50 ␮m in J.


RABBIT SUBVENTRICULAR ZONE spatial relationship was detectable between radial glia and the chains/masses of neuroblasts. The latter were oriented orthogonally to the radial glia fibers, which simply appeared bent at the passage of the chains (Fig. 3J). At stages between P10 and P30, radial glia fibers were observed to change their orientation to a more tangential direction at the border between the SVZ and the surrounding brain parenchyma (as previously described for rodents; Peretto et al., 2005). The change in radial glia process orientation occurs parallel to their retraction, leading to the transformation of radial glial cells into astrocytes. Starting from the second month of life, the vimentin-immunoreactive astrocytes were a subpopulation of all (GFAP⫹) SVZ glial cells (Fig. 1J–O). Local cell proliferation, detected with Ki67 antigen (Fig. 1P–T), was widely dectectable in the brain of early neonatal animals (Fig. 1P), and, starting from P15 (Fig. 1Q), it became restricted to the SVZ area. From this stage onward, local cell proliferation was prevalently concentrated along the external edges of the SVZ (Fig. 1P,Q), particularly in the ventricular SVZ (Fig. 1R,S). Ki67-positive nuclei were nearly absent within large chains of the abventricular SVZ, although they were sometimes clustered at their periphery (Fig. 1T).

Peripuberal rabbit parenchymal chains In a previous study, the combination of several approaches, including the immunocytochemical detection of different markers, semithin sections of resin-embedded material, and electron microscopy, had allowed the identification of parenchymal chains located outside the SVZ (Luzzati et al., 2003). In the present study, the occurrence of these chains in animals of different ages was assessed, and further analysis by means of an ultrastructural approach was carried out. As described in the previous paragraph, parenchymal chains are detectable after the second week of life, in about 20% of animals, then reaching a higher frequency in peripuberal animals (they were detected in 50% of animals studied), whereas no anterior chains were detectable in fully adult rabbits. Thus, the detailed ultrastructural study was performed on young, peripuberal rabbits (Figs. 4, 5). As previously described (Luzzati et al., 2003), two groups of parenchymal chains are present in the rabbit brain: 1) anterior chains, within the anterior forceps of the corpus callosum, and 2) posterior chains, between the striatum and the external capsule. The ultrastructural analysis revealed that both groups of chains are composed of cells sharing the morphology and cytology of those found in the SVZ (see Figs. 2, 4, 5). In addition, to confirm the neuronal nature of the precursor cells forming these chains, we combined electron microscopy and immunocytochemistry for the microtubule-binding protein DCX (Nacher et al., 2001; Brown et al., 2003; Yang et al., 2004; Rao and Shetty, 2004) and found that it is specifically associated to neuroblasts in the chains (Fig. 4A⬘⬘⬘). Similarly to chains of the abventricular SVZ, and unlike those observed in the ventricular SVZ of the lateral ventricle, all parenchymal chains consisted of a morphologically homogeneous cell population (Figs. 4, 5). The absence of C-like cells and mitoses in parenchymal chains supports the hypothesis that they are bulks of cells containing migratory elements that originate in the SVZ. This was previously shown for posterior chains by using bromodeoxyuridine (BrdU) treatment, followed by several days’ survival

499 (Luzzati et al., 2003), and confirmed here for anterior chains with the same temporal pattern: scattered BrdUimmunoreactive cells were detectable 5 and 10 days after the last of five injections (Fig. 4B,C), whereas no locally generated cells (Ki67 antigen or 2 hours’ survival after BrdU treatment) could be detected. In addition, a semiquantitative estimate of the number of neuroblast cell processes observed in single sections of parenchymal chains compared with the number of nucleated cell bodies indicated a 4/1 ratio, suggesting that leading and traling processes in these chains are very elongated. Besides common features, important differences were observed concerning the overall organization of the parenchymal chains, as well as their contact with the surrounding tissue. Highly variable was the size of the chains (summarized in Fig. 6), in terms of the number of nucleated cells in a cross-section. Some maintained this large size typical of SVZ chains or were even larger (Fig. 4E), whereas others were very small. Large parenchymal chains, including up to 150 nucleated cells in a transverse section, were observed in the anterior forceps of the corpus callosum (Fig. 4E⬘). The anterior chains appeared compact and were frequently associated with blood vessels (Fig. 4). On the other hand, the posterior parenchymal chains appeared less compact, frequently displaying a “laminar” (a sheet of cells, either U-shaped or forming a ring) or “clustered” (small clusters linked to each other by cellular bridges) appearance (Fig. 5). The serial reconstruction of a tract of two posterior chains [one of these was partially shown in the study by Luzzati et al. (2003)] confirmed that the size of parenchymal chains can vary a great deal in different tracts (Fig. 5). Some of these posterior parenchymal chains were large, including up to 30 – 40 nucleated cells in a transverse section (Fig. 5). The number of cells in each chain and at different levels of the same chain was more constant in the anterior than in the posterior group. This was visible in serial reconstructions of tracts of the posterior parenchymal chains, showing that the number of cells can drop to two or three elements, to increase once again in subsequent tracts (Fig. 5). Moreover, on following distinct posterior parenchymal chains, some were observed to join others to form larger clusters (Fig. 5), showing that the apparent regular arrangement noted along the striatum/external capsule interface (see Luzzati et al., 2003) would seem to correspond to a network of intermingled, nearly parallel chains. This network was not observed for the large anterior chains, which run either nearly parallel to one another or in divergent directions. Serial reconstruction with light microscopy showed that anterior chains are usually contained within the white matter. Nevertheless, in young animals (e.g., 30 days old; Fig. 4H) some of them reach the cortical gray matter beyond the forceps of the corpus callosum, before dissolving as single cells. Similarly to SVZ chains, most of the neuroblasts forming the parenchymal chains were directly juxtaposed to one another, without the interposition of any intercellular clefts (Fig. 4G⬘–G⬘⬘⬘). The semiquantitative analysis of the contact between the external surface of the parenchymal chains and other elements in the surrounding tissue confirmed that they are glia independent, this value remaining below 20% for posterior chains and slightly above this percentage for anterior chains (Fig. 6). This analysis showed that astrocytic contacts were lower in parenchymal chains than in those of the abventricular SVZ.


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Figure 4.


RABBIT SUBVENTRICULAR ZONE A difference was also observed with regard to the type of substrate surrounding the two groups of chains. Unlike anterior chains, which are always in contact with unmyelinated and myelinated axons, the posterior chains were in contact with both gray and white matter, being localized at the border between the striatum/amygdala and the external capsule (Figs. 5, 6). However, in both cases, a common pattern consisted of their arrangement along white matter axon bundles. In the case of dispersed (laminar and clustered) chains, many processes too large to be considered axons and containing the same type of cytoplasm of the chain cells were observed on both sides of the row formed by nearby neuroblasts, in close contact with them (Fig. 5D,E). These elements, as confirmed by the serial reconstructions, are the leading and trailing processes belonging to neuroblasts whose cell bodies are not included in the section.

DISCUSSION Although basic neurogenic processes contributing to CNS assembly during development follow a common pattern in different mammalian species (Garel and Rubenstein, 2004; Rakic et al., 2004), mature brain anatomy and functions differ greatly in mammals. Persistent neurogenic areas do retain common embryonic features, but they also remarkably change their shape and cellular arrangement throughout postnatal development (Alves et al., 2002; Tramontin et al., 2003; Peretto et al., 2005) and show features peculiar to the species during adulthood (Bernier et al., 2002; Luzzati et al., 2003; Sanai et al., 2004; Quinones-Hinojosa et al., 2006). In this context, despite very detailed knowledge concerning the morpho-

Fig. 4. Analysis of rabbit anterior parenchymal chains (PC). Most of them are in association with blood vessels (bv; A,B,E,F,H). Numbers indicate the level of the section (see Fig. 1A). A: A parenchymal chain (rectangle) within the white matter (WM) of the anterior forcep of the corpus callosum, in a vibratome slice processed for preembedding localization of doublecortin (DCX; higher magnification in Aⴕ). (Aⴕⴕ), Semithin section of the same tissue embedded in araldite. Aⴕⴕⴕ: Immunoelectron microscopic visualization of DCX in the chain neuroblasts (a); b, astrocyte; P, parenchyma. B,C: BrdU-immunoreactive cells are detectable in anterior parenchymal chains 5 and 10 day after the last of five injections. D: Schematic drawing indicating the position of the parenchymal chains showed in E and G, respectively. White, white matter; dark gray, chains. E: Very large chain (about 150 nucleated cells, indicated by yellow dots in the semithin section in Eⴕ) associated with three blood vessels. F: Thin chain surrounding most of a large blood vessel; an ultrastructural detail (rectangle, Fⴕ) shows the chain (Ch) squeezed between endothelial cells (arrows) on one side and astrocytic processes (b) or parenchyma (P) on the other. G: Semithin section of a parenchymal chain not associated with blood vessels. Electron microscopy (Gⴕ) shows that it is surrounded mainly by axons of the white matter (detail in Gⴕⴕ), apart from a contacting astrocyte (b). Note that all the cells forming the chain share the same morphological features of neuroblasts, without any clefts among their membranes (detail in Gⴕⴕⴕ). H: Reconstruction of a long parenchymal chain (PC; red, in drawings) in cresyl-violet-stained coronal sections of a postnatal (P30) animal (drawings: top, sagittal view; bottom, representative coronal sections from H-I to H-IV). This chain reaches the frontal cortex (Cx) beyond the white matter of the corpus callosum (H-IV). Note that the chain is associated with a blood vessel in the SVZ (H-I), following it outside the SVZ. Middle, a section immunostained for DCX (brown). ov, Olfactory ventricle. Scale bars ⫽ 10 ␮m in B,C,E⬘,G,G⬘; 2 ␮m in A⬘⬘⬘,F⬘,G⬘⬘,G⬘⬘⬘; 50 ␮m in A,H-I,-II,-IV; 20 ␮m in A⬘,A⬘⬘,E,F,H (insets at higher magnification).

501 logical and functional aspects of rodent SVZ (for review see Peretto et al., 1999; Gage, 2000; Alvarez-Buylla and Garcia-Verdugo, 2002), few data are available on the extension and internal arrangement of homologue neurogenic regions in other mammals. In addition, recent findings suggest that adult neurogenic processes, namely, the product of neurogenic sites, can vary in their rate and topographical distribution, if different species are considered (Eriksson et al., 1998; Gould et al., 1999a, 2001; Bernier et al., 2002; Luzzati et al., 2003; Sanai et al., 2004). In studies proposing that newborn elements can be found in nonneurogenic brain areas, the real origin of these cells and their possible relationship with the SVZ remain obscure (Gould et al., 1999a, 2001; Kornack and Rakic, 2001b; Luzzati et al., 2003; Lie et al., 2002; Zhao et al., 2003; Frielingsdorf et al., 2004). In primates and rabbits, reasonable evidence that some cells generated within the SVZ can be added within subcortical areas is at present limited to the amygdala (Bernier et al., 2002; Luzzati et al., 2003). This cell displacement is supposed to occur through a “temporal stream” in primates (Bernier et al., 2002) or a “ventral lateral extension” in rabbits (Luzzati et al., 2003), in both cases implying an anatomical link with the SVZ. Thus, differences in the structure and topographic extension(s) of the SVZ could reflect differences in the efficiency, rate, and destination of newly generated cell precursors in mammals. In this context, detailed knowledge of the existing differences could help in understanding the potentialities hidden in the brain of different mammals, helping us to outline strategies for mobilizing and directing endogenous neuronal precursors in the perspective of brain repair. The SVZ, as a major source of multipotent neural neurogenic progenitor cells and a unique site for long-distance cell migration in the adult mammalian brain, is an outstanding candidate for such a perspective (Peretto et al., 1999; Gage, 2000; Alvarez-Buylla and Garcia-Verdugo, 2002). Here, relying on light and electron microscopy, we have addressed the issue of rabbit SVZ internal arrangement in order to perform a comparison with other mammalian species. We extended our study to the changes occurring in the early postnatal period, which are known to lead to assembly of this adult neurogenic site in rodents (see Alves et al., 2002; Tramontin et al., 2003; Peretto et al., 2005). Finally, we have addressed in more detail the analysis of the so-called parenchymal chains, as structures located outside the SVZ area but functionally representing an extension of the SVZ itself in the rabbit brain parenchyma, as previously described (Luzzati et al., 2003).

The rabbit SVZ: comparison with other mammals The SVZ region has been well studied in rodents with regard to its topographical extension, internal arrangement, and cellular/molecular composition, leading to the characterization of its stem cell niche and its unique longdistance, tangential cell migration toward the olfactory bulb (for review see Peretto et al., 1999; Gage, 2000; Alvarez-Buylla and Garcia-Verdugo, 2002). This migration is made possible by the occurrence of neuroblast chains sliding within an almost complete sheath of astrocytic processes (glial tubes), thus outlining two main cellular compartments of the SVZ (Bonfanti and Theodosis, 1994; Rousselot et al., 1995; Doetsch and Alvarez-Buylla,


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Fig. 5. Ultrastructural features of rabbit posterior parenchymal chains. Serial reconstruction of two parenchymal chains (C-post1 and C-post2), localized in the temporal lobe (level 5, colored blue in the schematic representation in A), identified in a toluidine blue-stained semithin section (Aⴕ) between the striatum (St) and the white matter of the external capsule (EC). The chains were ultrastructurally reconstructed through a 27-␮m-long tract. Part of the reconstruction of C-post1 was shown by Luzzati et al. (2003; levels also refer to this reference). Astrocytes (green), oligodendrocytes (yellow), and neurons (blue) directly contacting the chain are indicated; the remaining perimeter of the chain is in contact with mature neuropil and white matter. After 20 ␮m (from level 4 to level 28), the two chains contact each other and fuse into a unique, larger chain. The neuroblasts

originally belonging to each chain are indicated by the inversion of dark and light gray filling their cytoplasm and nuclei. After a further 4 ␮m (from level 28 to level 35), the chain starts to separate again. At level 4, C-post1 shows a clear laminar arrangement (B), here forming a ring, whereas C-post2 is a typical clustered chain (C). Note that the organization of these cell aggregates ramarkably changes as the chains progress within tissue. D,E: Details of the chain/parenchyma interface. Leading and trailing processes (asterisks) are squeezed between neuroblast cell bodies, on the external or internal side of laminae and clusters. a, Type A cells (neuroblasts); b, type B cells (astrocytes); P, brain parenchyma. Scale bars ⫽ 50 ␮m in A; 10 ␮m in B,C; 2 ␮m in D,E.

1996; Doetsch et al., 1997; Lois et al., 1996; Jankovski and Sotelo, 1996; Peretto et al., 1997). Here, we have shown that the rabbit SVZ, compared with that of rodents, contains similar cell types but is characterized by their different topographical distribution and overall architecture. The main differences consist of 1) a heterogeneous distri-

bution of the chain and glia compartments, 2) the simultaneous occurrence of isolated neuroblasts and very large chains, and 3) the existence of a loose astrocytic meshwork. On the basis of these differences, two distinct parts can be detected all along the extension of the rabbit neuro-


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Fig. 6. Schematic representation of chain/substrate relationships in rodents and rabbit. Chains of neuroblasts (in pink) belonging to different species (rat and rabbit) and different anatomical locations (SVZ and brain parenchyma), when put in relationship with their substrates [including different types of astrocytic glia organization (in green), white matter (in white), blood vessels (black circles) and gray matter (in gray)], reveal different types of morphological organization. Single chain types (in the second row of drawings) are in scale apart from the anterior parenchymal chain associated with three blood

vessels (this is the chain composed of 150 cells showed in Fig. 4E). The red lines mark the direct contact between chains and brain parenchyma, without the interposition of astrocytic glia. In cells and chains of the ventricular SVZ, dark green lines mark the contact between neuroblasts and the ependyma (in gray). Bottom: Histograms showing the percentages of direct contact between different types of chains and astrocytic glia. Each histogram column corresponds to a drawing above.

genic site: a “ventricular SVZ,” adjacent to the ventricular wall and containing small aggregates of neuroblasts immersed within a relatively dense glial/ependymal sheath, and an “abventricular SVZ,” detached from the ventricles and containing large chains of neuroblasts immersed in a looser network of astrocytic processes. Thus, the SVZ appears to be structurally more heterogeneous in lagomorphs than in rodents, suggesting that both chain and glia compartments can differ greatly, with regard to their architecture and mutual relationship, in relatively close

mammalian species. The distinction between rabbit ventricular and abventricular SVZ is reinforced by the occurrence of a thick band of tissue, poor in cells and enriched in nerve fibers and glial processes, which could be reminiscent of the “hypocellular gap” of the human SVZ (Quinones-Hinojosa et al., 2006). In addition to this aspect, another common feature with humans is the absence of well-organized glial tubes, replaced by an incomplete “astrocyte ribbon” (Sanai et al., 2004; Quinones-Hinojosa et al., 2006). Something similar has also been described


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for the bovine SVZ (Rodriguez-Perez et al., 2003). On the other hand, a striking difference between human and rabbit SVZ consists of the absence of chains on both sides of the astrocyte ribbon in the former, in contrast with the large chains characterizing the abventricular SVZ of the latter. In all nonrodent mammals studied so far, the glial meshwork appears to be less tightly packed and less compartmentalized than that in rodents. The glial meshwork could represent a physical separation between the neurogenic area and the mature brain parenchyma, more or less complete according to the species. In this context, it is important to underscore that astrocytic glia in the rabbit SVZ forms a relatively “open” structure, displaying a gradient of neuroblast/glial contact (summarized in Fig. 6). From a comparative point of view, such a pattern may be considered intermediate between two extremes, namely, the rodents’ glial tubes and the humans’ astrocyte ribbon. Noteworthy is that most rabbit SVZ chains occur in the abventricular SVZ, which is characterized by a low density of astrocytic processes, a feature that could allow some of them to leave the SVZ as parenchymal chains (see below). A certain degree of heterogeneity has also been described for rodents, in which chains of neuroblasts and isolated neuroblasts can leave the RMS before reaching the main olfactory bulb (Bonfanti et al., 1997; Yang et al., 2004), and species differences in the arrangement of SVZ compartments have been described to occur in rats and mice (Peretto et al., 2005). In a previous paper (Luzzati et al., 2003), we hypothesized a possible relation between SVZ differences and brain size, because large brains are accompanied by a more complex extension of the ventricular cavities (McFarland et al., 1969). Although the rabbit SVZ appears more complex and provided with parenchymal chains, the analyses carried out on humans found no evidence of similar structures leaving the SVZ, chains being rare even within the SVZ itself (Quinones-Hinojosa et al., 2006). However, the hypothesis remains that, if a higher complexity of cerebral ventricles is related to an increased complexity of the brain in different species, the shape and architectural complexity of periventricular germinative layers could also change accordingly.

Differences concerning SVZ postnatal development in rabbits and rodents Studies carried out on rodents showed that embryonic/ neonatal germinal layers are remarkably different from their counterpart forming the adult neurogenic sites (Peretto et al., 1999, 2005; Alves et al., 2002; Tramontin et al., 2003). We have recently described in detail the progressive morphological and molecular changes occurring in the SVZ of rats and mice, placing this shift during the first month of life (Peretto et al., 2005). Thus, from a comparative perspective, we extended the analysis of the rabbit SVZ internal arrangement to the early postnatal period. Both peculiar features linked to the rabbit neurogenic site, namely, the distinction between ventricular and abventricular SVZ and the existence of parenchymal chains, do appear very early during the second postnatal week. Even chain formation within the SVZ is clearly detectable at P10, whereas rodent chains assemble between the third and fourth weeks (Peretto et al., 2005). Thus we can conclude that the overall pattern of SVZ assembly and chain formation is remarkably different in rodents and lago-

morphs, being much earlier in the latter (see Peretto et al., 2005). This difference is strengthened by the fact that postnatal development is temporally different in rodents and rabbits, being earlier in the former (puberty occurs in the first and second postnatal months in rodents and at about the fourth month in rabbits). On the other hand, notwithstanding the very early assembly of rabbit SVZ chain compartment, its glial counterpart will never attain the degree of organization typical of rodent glial tubes (Peretto et al., 1997, 2005). Putting together these facts, one could speculate that the rabbit SVZ assembles very early but retains a certain degree of “morphological immaturity” for quite a long period. This vision is supported by several details, reminiscent of features described in the early postnatal rodent SVZ, such as 1) the tendency of adult rabbit newly generated cells to form large masses in which single neuroblasts are tightly packed with their siblings, 2) the absence of the typical clefts described among neuroblasts of rodent chains (Lois et al., 1996; Jankovski and Sotelo, 1996; Peretto et al., 1997), and 3) the ultrastructural evidence of direct contact with the wall of blood vessels, without the interposition of astrocytic processes (Peretto et al., 2005). Taken together, these elements could explain the occurrence of a high degree of structural plasticity in the brain, including the existence of parenchymal chains in young animals. For example, the incomplete assembly of rabbit SVZ glial structures during postnatal development leaves the entire neurogenic area in a relatively “open” state for a long period exceding the age of puberty. Similarly to what was observed in the RMS of rodents, the early-migrating masses of neuroblasts are orthogonal to radial glia, thus appearing to be independent of their presence. Nevertheless, even in early postnatal rabbits, this aspect involves well-formed tangential chains, which are not immersed in the glial meshwork but coexist with radial glial cells and processes. This confirms our hypothesis that chain formation and glial tubes/meshwork assembly are not directly related (even in rodents, where they occur nearly at the same stage; see Peretto et al., 2005) and indicates that the two main SVZ compartments can assemble at various postnatal stages in different species. On the other hand, an aspect that seems to be well preserved in different species consists of an evident asymmetry, with a prevalence of well-formed chains in the lateral part of the SVZ. In rodents, this asymmetry is restricted to postnatal development (the distribution of chains in the SVZ then becoming rather homogeneous), whereas in rabbits the same feature continues to be present in young/adult animals.

Rabbit parenchymal chains: comparison with SVZ chains The existence of parenchymal chains in the rabbit brain is itself another remarkable feature; it is well known that in rodents most chains are specifically contained within the glial tubes, thus driving the majority of neuronal precursors to converge toward the core of the olfactory bulb (Lois et al., 1996; Jankovski and Sotelo, 1996; Peretto et al., 1997). At this level, neuroblasts can spread out of the mainstream to reach the main (Lois and Alvarez-Buylla, 1994) and accessory (Bonfanti et al., 1997) olfactory bulb layers. For the rabbit, the occurrence of two groups of parenchymal chains was described: anterior and posterior (Luzzati et al., 2003). In that study, most work was done


RABBIT SUBVENTRICULAR ZONE on the posterior parenchymal chains, located in the temporal lobe of the telencephalon and detectable throughout adulthood. Here, the attention has been focused mostly on the anterior parenchymal chains, directed through the corpus callosum toward the frontal cortex. Unlike posterior parenchymal chains, those of the anterior group are present in peripuberal animals, then disappear in the adult. Similarly to abventricular SVZ chains, parenchymal chains were also large, abundantly passing the average number of cells found in rodents (see schematic representation in Fig. 6). This feature was observed both for anterior and for posterior chains. Nevertheless, whereas the former were generally compact (similarly to SVZ chains), the latter were prevalently laminar or clustered. The ultrastructural serial reconstruction showed that chains of both the laminar (see chain C1 in Fig. 5, level 4) and the clustered (chain C2 in Fig. 5, level 4) types can change their shape along tracts of some micrometers, also merging together (C1, C2 in Fig. 5, from level 29 onward), thus forming anastomosing networks instead of parallel chains. As previously described (Luzzati et al., 2003), these chains appear to be quite variable in size along their length, because of the different numbers of cells that form them at different levels. The compact or noncompact arrangement of parenchymal or other types of chains could be related to the different substrates they encounter outside the SVZ (see also below). All parenchymal chains were mostly glia independent, as previously described (see Luzzati et al., 2003) and confirmed here (Fig. 6) by the semiquantitative analysis showing that they are only occasionally contacted by astrocytic processes, which are part of the usual glial cell populations of gray and white matter. The nonglial substrate in which parenchymal chains are immersed is different in the two groups: exclusively white matter for anterior chains and a mixture of gray and white matter for posterior chains. In this context, it is worth noting that a parallel arrangement with axon bundles is a common pattern of parenchymal chains leaving the SVZ, both anteriorly (in the corpus callosum) and posteriorly (contacting the external capsule on one side). In addition, the anterior parenchymal chains were frequently in contact with blood vessels, the same chains already contacting the vessels in the SVZ rostral extension (as demonstrated in a 1-monthold animal; see Fig. 4H) and using such a substrate as a pathway to leave the neurogenic region. The issue of cell migration in the rabbit SVZ and its extensions was addressed in our previous study (Luzzati et al., 2003). A rostral migration within the rabbit rostral migratory stream comparable to that described in rodents has been previously demonstrated by means of BrdU injection, followed by its immunocytochemical detection at different survival times. When the same BrdU injection protocols were applied to the study of posterior parenchymal chains, a few labelled cells could be observed to appear in the interval between 5 and 10 days following the treatment, suggesting the existence of low levels of cell displacement (Luzzati et al., 2003). In the present study, the same pattern was found to be present in anterior parenchymal chains, indicating that only a small proportion of cells forming these large chains actually migrates. The occurrence of migrating cells is also indirectly confirmed by the high expression of molecules strictly linked to neuronal precursor displacement and plasticity (PSA-

505 NCAM and DCX; Hu et al., 1996; Brown et al., 2003), which can be found in all types of rabbit chains (Luzzati et al., 2003; this study). Finally, the occurrence of large parenchymal chains containing a number of processes several times greater than that of cell bodies indicates that leading and trailing processes of neuroblasts forming these chains are very long. This feature is different from the appearance of chains in the SVZ of rodents, where neuroblasts have shorter leading and trailing processes and chain migration is known to be very fast (Lois and Alvarez-Buylla, 1994; Lois et al., 1996). Thus, the general arrangement of rabbit parenchymal chains does not agree with high-speed cell migration. Taken together, these data suggest that a migratory process does occur in the rabbit brain, although it is far less evident in parenchymal chains than in the SVZ.

Contact between chains and substrates All the chains described here were prevalently in contact with three types of substrates: astrocytic glia, white matter, and blood vessels, with different proportions according to the type of chain and its location. Thus, it might be interesting to examine the link between the different types of chain observed in vivo and the area in which they are located, as well as the substrates they encounter, to compare the results of this analysis with our knowledge of chains in rodents (summarized in Fig. 6). We have to consider several categories of cell aggregates (chains): 1) small SVZ chains (in rat SVZ and, to a lesser extent, in rabbit ventricular SVZ), which are surrounded by a tightly packed astrocytic meshwork separating them from the mature parenchyma; 2) large SVZ chains (in rabbit abventricular SVZ), coming in contact with a medium-dense astrocytic meshwork mixed with axonal fibers; 3) large parenchymal chains (anterior chains), completely immersed within large white matter bundles and frequently in contact with blood vessels; and 4) parenchymal chains, highly heterogeneous in size and shape (posterior chains) located at the interface between gray and white matter, occasionally contacting neurons and oligodendrocytes. These observations lead to the conclusion that specific types of chains can be found in brain regions characterized by certain substrates, suggesting that different types of chain migration could take place in vivo according to the substrates encountered. Such a hypothesis leads us to consider 1) the possible role of glia in cell migration inside and from the adult neurogenic sites and 2) the possible role of alternative substrates. Several lines of evidence, including the pattern of chain formation during the early postnatal period (Peretto et al., 2005; this study), in vitro studies (Wichterle et al., 1997), and the occurrence of rabbit parenchymal chains (Luzzati et al., 2003), along with results of the present study, strongly suggest that chain migration in the adult mamalian brain can be relatively glia independent both inside and outside the SVZ. As discussed above, this view agrees with the current hypothesis that astrocytes in the adult neurogenic sites would play a prevalent role directly as stem cells (Doetsch et al., 1999) and/or indirectly by exerting their influence within the stem cell niche (Doetsch, 2003), rather than a substrate or guidance for cell migration. From a hypothetical therapeutic point of view, the relative glial independence of neuronal precursor chains could be of paramount importance in allowing the possibility of cell displacement through mature brain areas


506 that are devoid of specialized glia structures. On the other hand, several lines of evidence suggest that the existence of an astrocytic meshwork could positively regulate chain migration by creating a favorable enviroment [e.g., by taking up released ␥-aminobutyric acid (GABA), thus reducing its inhibitory effect on cell migration; Bolteus and Bordey, 2004; Ma et al., 2005]. In this context, it is useful to know the second aspect mentioned above, namely, the alternative substrates on which chain migration could occur. The in vivo observations reported here show that chains of neuroblasts can be found in various contexts but suggest that cell migration outside the SVZ and its glial meshwork, though possible, could be made more difficult and inefficient by the occurrence of nonglial substrates. In other words, if a glial substrate is not essential for the occurrence of chain migration, when occurring in the form of sheaths (glial tubes), it could provide a favorable environment that does not exist in the mature parenchyma. Alternatively, it has been suggested that blood vessels and large axonal tracts might provide a pathway for migration of transplanted cells (Wichterle et al., 1999). Our results suggest that rabbit chains are frequently in contact with these substrates. In particular, blood vessels can play a role in directing the rabbit parenchymal chains of newly generated cells through the anterior forceps of the corpus callosum, allowing them to reach brain areas located outside the SVZ. These chains, which are immersed in white matter either in association with blood vessels or not, frequently display a compact aspect. On the other hand, posterior parenchymal chains, which lie at the interface between the white matter of the external capsule and striatal or subcortical gray matter, frequently display a clustered or laminar arrangement. Most of the surface of abventricular SVZ chains, anterior parenchymal chains, and to a lesser extent the posterior ones is in direct contact with axonal fibers, with an orientation parallel to the chains themselves. Although we do not have direct functional data on their migration, it is possible to hypothesize that unmyelinated and myelinated axons could be the main substrate used by chains outside the SVZ, in the absence of well-organized glial structures or blood vessels. Indeed, it is well known that neuronal precursors migrating in the developing nervous system preferably follow “gliophilic” and “neurophilic” substrates (Rakic, 1990). Furthermore, it is worth noting that GABA levels in white matter are thought to be about 50% higher than those in gray matter (Jensen et al., 2005). In conclusion, from a speculative point of view, we think that neuronal precursors generated in the adult brain possess an intrinsic capability to migrate, but can extrinsecate this attitude in different ways according to the substrates they encounter in vivo. Physiologically, the balance between these aspects can be variable in different species according to different brain anatomy and different internal arrangement of the neurogenic zones. Rodentia and Lagomorpha being two relatively closely related orders (Douzery and Houchon, 2004), the peculiar neurogenic system and structural plasticity described in the rabbit brain may be considered a good model for gaining further understanding of the anatomical and molecular regulation of emigration, even under an evolutionary profile.

G. PONTI ET AL.

ACKNOWLEDGMENTS We thank Paolo Peretto and Federico Luzzati for kindly providing help in animal care and experimentation as well as for the use of the confocal microscope.

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