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The Journal of Neuroscience, February 1, 1998, 18(3):1020-1037
Primary Neural Precursors and Intermitotic Nuclear Migration in the Ventricular Zone of Adult Canaries
Arturo Alvarez-Buylla1, Jose Manuel García-Verdugo2, Adria S. Mateo1, and Horacio Merchant-Larios3 1 The Rockefeller University Field Research Center, Tyrrel Road, Millbrook, New York 12545, 2 Universidad de Valencia, Valencia, Burjassot-46100, Spain, and 3 Instituto de Investigaciones Biomédicas, UNAM, Mexico City, 04510 Mexico
ABSTRACT
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Abstract
Introduction
New neurons continue to be born in the ventricular zone (VZ) of the lateral ventricles in the brain of adult birds. On the basis of serial section reconstruction and electron microscopy, we determined that the VZ of the adult canary brain is composed of three main cell types (A, B, and E). Type A cells were never found in contact with the ventricle and had microtubule-rich processes typical of young migrating neurons. Type B cells were organized as a pseudostratified epithelium, all contacted the ventricle, and most had a characteristic single cilium. Type E cells, also in contact with ventricle, were ultrastructurally similar to the mammalian multiciliated ependymal cells. After six injections of [3H]-thymidine (1 every 12 hr), Types A and B cells were found labeled. Type E cells were never [3H]-thymidine labeled. One to two hours after a single injection of [3H]-thymidine, all labeled cells corresponded to Type B cells. At survivals of 5, 24, and 74 hr after [3H]-thymidine injection, the proportion of labeled Type B cells decreased and that of Type A cells increased, indicating that Type B cells were the primary precursors. Most [3H]-labeled nuclei at 1-2 hr after [3H]-thymidine injection were separated from the ventricular cavity, but most of the mitotic cells were adjacent to the ventricle. This observation and measurements of the distance between labeled nuclei and the ventricular surface at 1, 5, 7, and 11 hr after [3H]-thymidine injection indicate that Type B cell nuclei move toward the ventricle to divide. This work reveals the architecture of the VZ in an adult vertebrate brain, identifies the primary precursor of new neurons, and describes nuclear translocation of these precursors during the cell cycle.
Key words: neurogenesis; stem cells; neuronal migration; mitosis; ependyma; songbirds; neuroblasts; cilium
INTRODUCTION
Most neurons in the CNS of vertebrates are born in the ventricular zone (VZ) (Boulder Committee, 1970
), a pseudostratified columnar epithelium adjacent to the brain ventricles. During brain development cells replicate their DNA in the deeper (basal) VZ and move to the apical VZ to undergo mitosis adjacent to the ventricular surface (Sauer, 1935
It is generally believed that the VZ disappears as brain histogenesis comes to an end (The Boulder Committee, 1970
One of the best-studied systems of adult neuronal production is that of the canary brain (Goldman and Nottebohm, 1983
Proliferating cells in the juvenile and adult canary brain are largely restricted to the walls of the lateral ventricles. Within this wall, proliferation occurs at higher rates in regions of the lateral wall of the lateral ventricle that are denominated "hot spots," in which a large number of new neurons are born (Alvarez-Buylla et al., 1990b
Here we have determined the cell types and three-dimensional organization of the VZ in the adult canary brain. We identify the neuronal precursors and show that these cells undergo interkinetic migration within the VZ. This is the first study to identify the primary precursors and to show the interkinetic migration of these cells in the adult vertebrate brain.
MATERIALS AND METHODS
One-year-old female Wasserschlager canaries (Serinus canaria) were used for all experiments. Birds were bred and maintained indoors at the yearly New York State photoperiod. Seeds and water were available ad libitum. Before perfusion with saline or fixatives, birds were deeply anesthetized with 0.5 mg of pentobarbital (Nembutal) injected into the pectoral muscle. All animal procedures were in accordance with institutional guidelines approved by The Rockefeller University.
Electron microscopy (EM)
The brains were fixed by intracardial perfusion with 0.9% saline followed by 50 ml of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.4, followed by overnight (4°C) immersion in the same fixative. Frontal vibratome sections (100 µm thick) at the level of the anterior commissure were collected serially and post-fixed in 2% osmium tetroxide for 1 hr. After dehydration in ascending concentrations of ethanol, sections were transferred to propylene oxide, impregnated overnight with TABB 812 resin (Marivac Limited, Halifax, Canada) or araldite (Durcupan; Fluka, Buchs, Switzerland), and flat-embedded. Small fragments (~0.5 × 1 mm) of the lateral wall of the lateral ventricle (see Fig. 1) were cut and reembedded, and the VZ was cut transversely on a Reichert ultramicrotome. Sections were collected on mesh grids or on single-hole Formvar-coated grids. Ultrathin sections (60 nm) were stained with lead citrate and uranyl acetate, and observed in a Jeol 100CX EM or Philips CM-10. Twenty birds were used for the ultrastructural reconstructions and cell classification studies.[3H]-thymidine autoradiography in 2-µm-thick sections
Experiment A. Two adult canaries received six injections of [3H]-thymidine every 12 hr (100 µl per injection of [3H]-thymidine, 6.7 Ci/mM; New England Nuclear). Birds were killed 1 hr after the last injection, and the brains were fixed and processed for electron microscopy as described above. Semithin sections (2 µm thick) were cut and placed on slides. Slides were then covered with autoradiographic emulsion (NTB2, Kodak) and incubated for 1 month at 4°C. Autoradiograms were developed for 3 min in D19 developer (Kodak) at 17°C. Sections were observed stained either with Giemsa or toluidine blue or unstained under Nomarski illumination (Nikon); [3H]-labeled cells were photographed in these semithin sections at several magnifications. A cell was considered labeled if its nucleus was overlaid by six or more autoradiographic grains and the same cell was labeled in an adjacent section. Semithin sections were then reembedded, detached from the glass slides, and sectioned for EM examination. Labeled cells under the EM were identified on the basis of the light microscope photomicrographs. A total of 66 cells were studied. Experiment B. Sixteen adult canaries received a single 100 µl injection of [3H]-thymidine (6.7 Ci/mM; New England Nuclear) into the pectoral muscle. Groups of birds were killed 1-2, 5, 24, and 74 hr after injection, and their brains were processed for autoradiography and EM as in Experiment A (see Table 2).Position of [3H]-thymidine-labeled cells in the VZ
Twenty female canaries received a single 50 µl intramuscular injection of [3H]-thymidine; 1, 3, 5, 7, and 11 hr after injection, groups of 4 birds were killed and perfused with 0.9% saline followed by 3% paraformaldehyde in PB and embedded in polyethylene glycol (Alvarez-Buylla et al., 1987-particle penetration is greater after polyethylene glycol than in plastic-embedded tissue. Cells were considered labeled if they contained more than six autoradiographic grains overlying their nucleus. This labeling criterion was more than 20 times above background.
Reconstructions
The lateral wall of the lateral ventricle of two canaries was processed for electron microscopy as above. Three different regions of the lateral wall of the lateral ventricle were reconstructed in serial ultrathin sections using the light and electron microscope (Doetsch et al., 1997
RESULTS
Three major cell types coexist in the VZ of the lateral ventricle
The VZ of adult canaries varies in morphology, thickness, and cell arrangement. We concentrated our analysis on the lateral wall of the lateral ventricle at the level of the anterior commissure, a region of high proliferation in the adult canary brain (Fig. 1A) (Alvarez-Buylla et al., 1990b
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Figure 1. A, Hemisection of the adult canary brain at the level of the anterior commissure (AC). The lateral ventricle is indicated by dots, and the approximate locations of regions shown in B-D are indicated by black rectangles. Regions studied in detail are in the dorsal and ventral neurogenic hot spots (Alvarez-Buylla et al., 1990b
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Figure 2. Ultrastructural characteristics of the three main cell types in the adult canary VZ. A, Photomicrograph of Types A and B cells. Type A cells (a) are deeper in the VZ and have very scant cytoplasm. In contrast, Type B cells (b) have a cytoplasm rich in organelles, contact the ventricular lumen (see Results for full description), and frequently show a long basal process (arrows) entering the underlying brain parenchyma. B, Type A (a) cells can be found under Type B (b) or E (not seen here) cells. When cut longitudinally, Type A cells have a spindle shape and tangentially oriented processes (arrows). V, Ventricle. C, A pair of centrioles is observed frequently at the base of Type A (a) cell processes (arrowheads). Some of these centrioles are adjacent to a basal body and an intracellular cilium (large arrow). Processes of Type A cells are linked to neighboring cells by small electron-dense junctional complexes (small arrows). D, Cross section of Type A (a) cell processes (arrows) showing bundles of microtubules. The cell bodies of some of these processes were identified in serial section reconstructions (Table 3, Fig. 3). E, Ultrastructure of apical cytoplasm of Types B (b) and E (e) cells. Both Type B cells contact the ventricle, one of them through a thin process that squeezes between the neighboring B and E cells. Notice the difference in the electron density, number of polyribosomes, and mitochondria between Types B and E cells. Part of the characteristic cilium (arrow) of Type B cells can also be observed in the cell in the middle. V, Ventricle. F, Most apical cytoplasm of Type B cell showing characteristic cilium (arrowhead), basal body (large arrow), and associated centriole (small arrow). In contrast to Type A cells, the centrioles of Type B cells are found close to the ventricular surface; also notice the Golgi apparatus (above the centriole) of Type B cells with flattened sacculae. V, Ventricle. G, Annulate lamellae in the apical cytoplasm of Type B cell. H, Ultrastructure of Type E or ependymal cells (e). Basal processes (asterisks) in Type E cells were electrolucent and rich in intermediate filaments. The luminal surface contains microvilli and clusters of cilia. Three Type A (a) cells are also observed in this micrograph. V, Ventricle. I, Detail of apical cytoplasm in ependymal cells; notice multiple cilia (arrows) and swollen Golgi apparatus sacculae (arrowhead). V, Ventricle.
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Table 1. Morphological characteristics of different cell types in the adult avian VZ Type A cells (Figs. 1E, 2A-D; also see Fig. 8) had an undifferentiated appearance; usually just a thin rim of cytoplasm was visible around their nuclei. This scant cytoplasm contained a small Golgi apparatus, few rough endoplasmic reticulum but abundant polyribosomes, and few round to oval mitochondria. Pinocytic vesicles (coated pits) were also common in Type A cells (Fig. 8B). The nucleus was round or elongated, with darker chromatin than that of Types B and E cells, and contained one or two large nucleoli. Type A cells were generally found in clusters with other cells of similar morphology between the brain parenchyma and the epithelial layer formed by Type B and E cells (see below). Type A cell processes were most frequently oriented parallel to the ventricular wall (Fig. 2B). These processes were rich in microtubules (Fig. 2D) and established small electron-dense contacts with neighboring cells (Fig. 2C). Frequently, a pair of centrioles was observed in one of these processes close to the nucleus (Fig. 2C; see Fig. 8B), and on occasions the centrioles were associated with the basal body of a cilium (Fig. 2C). In contrast to Types A and B cells, this cilium was not on the ventricular surface but was close to the cell body and probably internalized. Type A cells were never observed in contact with the ventricular lumen (see Table 3).
Type B cells (Figs. 1E, 2A,E-G; also see Figs. 5, 6, and 8) were found adjacent to the ventricular lumen, or their nuclei were deeper in the VZ and arranged as a pseudostratified epithelium. The nuclei were oval or irregular in shape and occasionally had small invaginations. Nuclei of Type B cells had prominent nucleoli and lax chromatin, but in some Type B cells chromatin was found in different states of aggregation (see cell 1 in Fig. 5). The apical cytoplasm contained many polyribosomes, occasional stacks of annulate lamellae (Fig. 2G), and a large Golgi apparatus (Fig. 2F). The dictyosomes of Type B cells, unlike those of Type E cells, had many thin, undulated saccules associated with a rich set of coated vesicles (Figs. 2F, 3). Mitochondria were also smaller and rounder than those observed in Type E cells (Fig. 2E) but more abundant than in Type A cells. Junctional complexes were observed between Type B cells or Type E and B cells close to the luminal surface (Figs. 4B, 5); however, these junctional complexes were smaller than those observed among Type E cells (Fig. 1E). Type B cells had thin and electron-dense basal processes (Fig. 2A), unlike those of Type E cells that were thicker and electrolucent (Fig. 2H). Type B cell basal processes contained microtubules, mitochondria, intermediate filaments, and free ribosomes. In single sections, some Type B cells found deeper in the VZ were apparently not connected to the ventricular lumen. However, three-dimensional reconstructions (see below) revealed that Type B cells had an end foot that reached the ventricle (Figs. 2E, 5B). One striking feature of Type B cells was a single specialized cilium without a rootlet but adjacent to the centriole (Figs. 2F, 3). Transverse and longitudinal serial sections through 10 of these cilia showed that they were 3.0-3.5 µm long and 0.2 µm in diameter. They contained eight or nine pairs of microtubules in the periphery and none in the center (i.e., 8 + 0 or 9 + 0 structure). The distal end of the cilium had an electron-dense enlargement of 0.25 µm (Fig. 3A). The plasma membrane was closer to the microtubules compared with the cilia in ependymal cells. The specialized cilia of Type B cells were straight or slightly curved, suggesting that they are relatively rigid.
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Figure 3. Specialized cilium in Type B cells. A, Longitudinal section of one of these cilia (short arrow). In the top left corner is a cross section of a Type E cell cilium (arrow). Notice the centriole at the base of cilium (arrowhead). B, Cross section (arrow) of a Type B cell cilium showing an 8+0 microtubule structure. The arrowheads point to the Golgi apparatus of two Type B cells.
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Figure 4. Serial section EM analysis of the dorsal (sections A-J, region b of Fig. 1A) and ventral (sections K-T, region c in Fig. 1A) part of the lateral wall of the lateral ventricle showing the composition and architecture of the VZ in adult canaries. Each section is separated 2 µm from the next, and the ventricle is to the left in all drawings. In the dorsal VZ the epithelium is thin and flattened. Clusters of Type A cells are next to Type B cells that form a flattened pseudostratified epithelium. Interspersed among Type B cells are small groups of ependymal cells, each with broad exposure to the ventricle. The basal process of some Type E cells can be observed (sections B and F). In the ventral region (K-T) the VZ is thick and rich in Type B cells, with only small islands of ependymal cells. Type B cells are pseudostratified, and a basal process is observed in some of these cells (sections O, R, and T). Type A cells in this region are more abundant and are loosely organized as clusters at the interface with the underlying parenchyma. Most of the Type A cells have a thin rim of cytoplasm around them; however, some clusters of Type A cells in this region are larger and have more cytoplasm (e.g., section M). These Type A cells were distinguished from Type B cells because they had microtubules, a tangential expansion, or a centriole close to the cell nuclei or in a tangential process.
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Figure 5. Labeled Type B cell (1) 1 hr after [3H]-thymidine injection. Inset shows labeled cell in Giemsa-stained semithin section. A, Notice how the labeled cell (1) extends a process toward the ventricle and a second process in the opposite direction around an adjacent Type B cell. B, This same cell (1) at higher magnification in a different ultrathin section reaches the ventricular lumen (large arrow), squeezing between processes of other Type B cells. This process contains small junctional complexes with neighboring cells (small arrows) close to its apical end. Notice the characteristic cilium of the neighboring Type B cell (arrowhead).
Type E cells had the structure typical of ciliated ependymal cells (Del Brio et al., 1991
In addition to these three cell types, the VZ of adult canaries contained tanycytes, microglial cells, and endothelial cells. Capillaries were particularly abundant in the ventral proliferative hot spot. The cytoplasm of tanycytes was electron-dense with abundant dense-core vesicles and was packed with large elongated mitochondria (not shown). Tanycytes were more abundant in the medial wall of the lateral ventricle, facing the hippocampus. The nucleus of tanycytes was small and irregularly shaped, with large clusters of heterochromatin. These cells had a mitochondria-rich basal process penetrating the adjacent brain parenchyma that occasionally ended on neighboring blood vessels. These cells were clearly different from the Types B and E cells, with characteristics similar to those described previously for mammalian tanycytes (Millhouse, 1972
Serial section analysis of the avian VZ
To understand how the neurogenic VZ of adult canaries was organized, we serially sectioned three different regions of the lateral wall of the lateral ventricle, equivalent to those shown in black rectangles in Figure 1A. One section every 2 µm was photographed under the EM and studied in detail. Individual cells in multiple sections were identified by numbers, and their contours were digitized in different colors (see Materials and Methods): ependymal cells, gray; Type B cells, blue; and Type A cells, red. This analysis is illustrated in Figure 4A-J for the dorsal region (B in Fig. 1A) and in Figure 4K-T for the ventral region (C in Fig. 1A). The difference in organization and composition of distinct VZ regions was evident in these reconstructions. The dorsal region (equivalent to B in Fig. 1A) contained alternating ependymal and Type B cells, with a few flattened Type A cells between the epithelium and underlying parenchyma. Type A cells were found in small clusters usually associated with Type B cells. Although the VZ in this dorsal region was flattened, the pseudostratified organization of clusters of Type B cells could be discerned. In contrast, ependymal cells formed a single layer of heavily interdigitated cells.
In the ventral hot spot (equivalent to C in Fig. 1A) the VZ was largely composed of Type B cells with small islands of ependymal cells (Fig. 4K-T). Type B cells were pseudostratified, and most reached the ventricle. However, it was not possible by studying one section every 2 µm to determine whether all Type B cells reached the ventricle (see below). The contours of Type B cells changed sharply from one section to the next, suggesting a very irregular morphology. Tangentially arranged elongated Type A cells were observed in the interface of the VZ and the underlying brain. Type A cells in this region were loosely organized into clusters associated with ependymal or Type B cells or both. In the region just ventral to this hot spot (equivalent to D in Fig. 1A), the VZ changed again (not shown). Here the VZ was largely composed of ependymal cells with only small islands of Type B cells. Interestingly, Type A cells were also common in this region. They were small and spindle-shaped, and the majority seemed to be cut transversely in our frontal sections.
[3H]-thymidine labeling indicates that Type B cells are the primary precursors
We treated birds with six injections of [3H]-thymidine, one every 12 hr, and killed them 1 hr after the last injection. This procedure resulted in many labeled cells, as identified in autoradiograms at the light microscope level (Fig. 1B,C). Of 66 [3H]-labeled cells then analyzed at the electron microscope, 25 were Type A, 38 were Type B, and 3 were in mitosis. No labeled ependymal cells were found. Although this result indicated that ependymal cells were not actively proliferating, we could not determine whether both Type A and Type B cells were dividing, or whether one cell type gave rise to the other. We therefore treated birds with a single [3H]-thymidine injection and fixed the brains 1, 5, 24, and 74 hr later. This nucleoside remains in the circulation for <2 hr after a single injection (Alvarez-Buylla et al., 1990b
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Table 2. Types of [3H]-labeled cells at different survivals One to two hours after [3H]-thymidine injection, labeled cells had the morphology of Type B cells (Table 2, Fig. 5). At this time labeled nuclei were generally separated from the surface of the ventricle by other cell bodies or processes of neighboring cells; however, 6 of the 27 cells studied at 1-2 hr had a thin extension that reached the ventricular surface (Fig. 5), but none of these cells had broad contacts with the ventricle. It was not possible to determine whether other labeled Type B cells contacted the ventricle. No labeled mitotic cells were encountered in the 1-2 hr survival group, but clusters of heterochromatin (Fig. 5) were noticed in some labeled Type B cells, which suggests that these cells were preparing for mitosis. With increasing survival time, the proportion of labeled Type B cells decreased, whereas that of Type A cells increased (Table 2). By 74 hr, of the 26 cells identified at the EM, only 7 (26.9%) corresponded to Type B cells. Very few of the labeled cells identified at 24 and 74 hr after [3H]-thymidine administration were found to contact the ventricle (Table 2). In contrast, 7 of 29 [3H]-labeled cells identified at the EM at the 5 hr survival contacted the cerebrospinal fluid. These cells were exposed to the ventricle (Fig. 6), and six of them were in mitosis (Table 2, Fig. 7).
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Figure 6. Pair (possibly daughter cells) of [3H]-labeled Type B cells in the dorsal VZ 5 hr after [3H]-thymidine injection. Inset shows the light photomicrograph of a semithin section stained with Giemsa. Notice how cell 2 is exposed to the ventricular lumen (between arrows). The apical borders of this cell are joined by zonulae adherens junctions (arrowheads) to neighboring cells. The dotted line indicates the position of the ventricle. Notice a dark intraventricular cell, possibly a microglia (m).
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Figure 7. Labeled mitotic cell 5 hr after [3H]-thymidine administration. Inset shows semithin section stained with Giemsa. A, Low-magnification electron micrograph of this cell closely associated with two processes (arrows): one rich in intermediate filaments, probably from an ependymal cell projecting into underlying parenchyma, the other electron-dense, probably from a Type B cell. These processes do not seem to originate in the dividing cell but from closely apposed cells. The exposure of this mitotic cell to the ventricular lumen is indicated by arrowheads. B, At higher magnification, abundant pinocytic vesicles (arrows) and microvilli (arrowheads) but no cilia are seen in the exposed surface of the dividing cell. Notice how this dividing cell establishes junctional complexes (open arrows) with neighboring cells.
In contrast to the earlier survival points, many of the labeled cells at 24 and 74 hr corresponded to Type A cells (Table 2, Fig. 8). At 24 hr 37.5% of the labeled cells corresponded to Type A cells, and this proportion increased to 73.1% at the 74 hr survival. [3H]-labeled cells at these longer survivals were frequently found in clusters (Figs. 8, 9), which suggests that some precursors divided several times and gave rise to multiple Type A cells. In the dorsal extent of the VZ where the epithelium is thinner, [3H]-labeled cells were found in groups of no more than two cells. In contrast, we found as many as six [3H]-labeled cells in one cluster in the ventral hot spot. In two clusters of labeled cells in the ventral hot spot, we found three [3H]-labeled pyknotic cells (one at 5.5 hr and two at 24 hr survivals) (Fig. 9). These cells had the ultrastructure of cells undergoing apoptosis, with a light cytoplasm, a highly condensed chromatin, and a wrinkled or broken nuclear envelope. Given the overall low numbers of pyknotic cells observed in the VZ, it was remarkable that these dying cells were labeled by [3H]-thymidine. In two other instances dying cells were found within one or two cell diameters of a mitotic cell. These results suggest that some of the progeny of dividing cells die within the VZ. This is consistent with recent evidence from the developing mammalian brain, indicating that a significant fraction of newly formed cells in the VZ and SVZ die (Thomaidou et al., 1997
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Figure 8. Two labeled Type A cells (1 and 2; see autoradiography in Nomarski photomicrograph in the inset) in the ventral hot spot facing a nonciliated section of the ventricular wall 24 hr after [3H]-thymidine administration. A, Low-magnification electron micrograph showing the multilayered VZ. Labeled cells 1 and 2 are among other Type A cells (a) and are separated from the surface of the ventricular lumen by Type B cells (b). Notice the tangential orientation of Type A cells and how the process in cell 2 (arrows) seems to reach into the underlying parenchyma. B, High-magnification electron micrograph showing ultrastructural characteristics of labeled Type A cell 1: a pair of centrioles (arrowheads), abundant polyribosomes, and coated pits (arrows). Notice also the many cross sections of microtubules.
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Figure 9. Cluster of labeled cells in the ventral hot spot 24 hr after [3H]-thymidine administration. The inset shows the autoradiogram of the semithin section photographed with Nomarski optics. Two labeled cells (4 and 5) in this group are undergoing apoptosis.
Mitoses
In all the material analyzed, 18 mitotic cells (labeled and unlabeled) were encountered, 15 of which had chromosomes very close to the ventricle. These cells were frequently exposed to the lumen (Fig. 7), some of them with a large area. Because the ultrastructure of cells was altered during mitosis, we could not identify these cells by type; however, in the ventral VZ, mitotic cells were found in stretches of the VZ containing Type B cells but never in regions where the majority of the neuroepithelium is formed by ependymal cells. Mitotic cells had short, finger-like cytoplasmic extensions to the ventricular lumen, but no cilia (Fig. 7). The orientation of the mitotic aster in all of these cells was parallel or slightly angled with respect to the wall of the ventricle, but never perpendicular to this wall. This suggested that daughter cells in the VZ of adult canaries split tangentially (or close to tangentially) to the ventricular surface. Six [3H]-labeled mitotic cells were encountered 5 hr after [3H]-thymidine (Table 2, Fig. 7). These labeled mitoses were all exposed to the ventricular lumen. Three mitotic cells encountered during serial section reconstruction (Table 3) were also exposed to the ventricle, but no cilium or basal processes were observed on these cells. This suggested that the cilium in Type B cells and probably the basal extension were retracted during mitosis. A similar conclusion has been drawn from serial section analysis in the embryonic mammalian neuroepithelium (Stensaas and Stensaas, 1968
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Table 3. Ultrastructural characteristics of the VZ in serial section reconstructions (intervals of 200 nm). Each cell is represented by a row, and each column shows which of the six ultrastructural features scored were present in each section: contact with ventricle, gray block; no contact with ventricle, white block; cillium, dot; centriole, X; radial process, triangle; tangential process, square. Eighty-four cells were contained within the studied sections. The position of [3H]-labeled VZ nuclei changes with increasing survival
Two of the above observations suggest that Type B cells change position as the cell cycle progresses. (1) The majority of [3H]-labeled nuclei at 1-2, 24, and 74 hr were at a distance from the ventricle. In contrast, a significant number of the [3H]-labeled cells were adjacent to the ventricle 5 hr after [3H]-thymidine; (2) most of the mitotic cells encountered were in contact with the ventricular cavity.
These observations suggest that cells move toward the VZ to undergo mitosis. To provide further support for this hypothesis, we measured the distance from the center of the labeled nuclei to the surface of the ventricle in birds that survived 1, 3, 5, 7, or 11 hr after receiving a single injection of [3H]-thymidine. We did this analysis in the ventral neurogenic hotspot where the thickness of the VZ allowed us to reliably measure changes in the position of labeled cells under the light microscope at different survival times after [3H]-thymidine administration.
Three and five hours after [3H]-thymidine injection, the distribution of [3H]-labeled cells had shifted toward the ventricle as compared with 1 or 7 and 11 hr survivals (Fig. 10). Nuclei moved from an average distance of 6.5 µm at 1 hr to 5.6 and 5.9 µm at 3 and 5 hr, respectively. By 7 and 11 hr, labeled nuclei had moved away from the ventricle to 6.5 and 6.7 µm, respectively. These values are probably an underestimate of the true displacements of nuclei or cells, because they correspond to averages of a nonsynchronous population of dividing cells. To obtain a more accurate representation of this interkinetic migration we determined the average distance from the center of the [3H]-labeled nuclei to the ventricular surface for the 20 cells closest to the ventricle for each bird in the different survival groups. As shown in Figure 11, 3 and 5 hr after [3H]-thymidine injection, cells are significantly closer to the ventricle than at the other survival times studied. Consistent with the ultrastructural findings, these experiments indicate that proliferating Type B cells in the adult avian VZ are changing position as they go through the different phases of the cell cycle.
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Figure 10. Histograms of distance from the center of [3H]-labeled cells to the surface of the ventricle in the ventral hot spot of adult canaries. Each histogram compounds 200 cells counted in four birds (50 cells/bird). A vertical line at 5 µm distance aids in the comparison of distributions. Three and five hours after [3H]-thymidine injection more cells are <5 µm from the ventricle as compared with after 1, 7, and 11 hr, when most of the cells are to the right of the line.
Type B cells maintain an end foot on the ventricle
The above results indicate that Type B cells divide and that their position shifts during the cell cycle from the basal to the apical VZ. This movement could be attributable to the migration of the cell and the formation of a process that reaches the ventricle, or to the migration of the nucleus within a preformed process. The irregular shape of Type B cells made the identification of the apical and basal processes in single sections, or reconstructions of sections separated by 2 µm, difficult or impossible. To determine whether Type B cells maintain an end foot on the ventricle and to determine the three-dimensional morphology of all cell types in more detail, we reconstructed part of the ventral hot spot (the region rich in Type B cells), analyzing 1 out of every 2 thin sections (a separation of ~200 nm between sections). Eighty-four cells were completely contained within the 42 ultrathin sections that were analyzed (Table 3). Of these, 33 corresponded to Type A cells, 44 to Type B cells, 3 to mitoses, and 4 to Type E cells. All Type B cells were directly in contact with or had an end foot on the ventricle. Notice (Table 3) that the number of sections in which Type B cells contacted the ventricle was variable; cells close to the lumen had a large area exposed (e.g., cells 27 and 28), whereas those far from the ventricle (e.g., cell 44) had a thin end foot on the ventricle. Except for four cells, all Type B cells had the characteristic cilium with an associated centriole (Fig. 2F). Also the majority of Type B cells had a basal process present in one or several sections. There was no apparent correlation between the presence of the cilium in a section and the presence of a basal process. The cilium was also not necessarily centered in the exposed surface of a Type B cell, but could appear to one side of the cell (e.g., cell 25). This variability in structure most likely reflects the dynamic nature of Type B cells. The structure of Type B cells indicates that interkinetic movements are probably caused by the migration of the nuclei within the cell. The three mitotic cells encountered in this reconstruction were exposed to the ventricle.
The contact with the ventricular lumen was evident in sections transecting an ependymal cell (Table 3). This indicates that ependymal cells, unlike Type B cells, are widely exposed to the ventricle and have a more homogeneous morphology. In every section in which Type E cells contacted the ventricle, multiple cilia were observed. The morphology of ependymal cells was regular, cuboidal, and not pseudostratified (Figs. 2E, 3).
The serial section analysis confirmed that Type A cells had a very different structure and orientation than Types B and E cells. Of the 33 Type A cells identified, none had contact with the ventricle (Table 3). Instead, the majority of these cells had tangential extensions that correspond to the microtubule-rich processes described previously (Fig. 2B,D). Unlike that in Type B cells, the centriole in Type A cells was far from the ventricular surface and generally was associated with one of the tangential processes. These tangential processes were woven between basal processes from Type B or E cells. It was therefore difficult to reconstruct all of these processes, and this may account for the Type A cells in which a centriole could not be found.
DISCUSSION
Type B cells are the primary precursors
Proliferation in the adult avian VZ is related to neurogenesis. In adult canaries, cell division is largely restricted to the walls of the lateral ventricles that face regions of the telencephalon that receive new neurons. Within these walls, neurogenesis is most prominent in proliferative hot spots where the majority of migrating neurons arise. Our EM and [3H]-thymidine analysis showed that the primary proliferating cells within the hot spots corresponded to Type B cells. Among Type B cells, mitotic figures, annulate lamellae, and different stages of chromatin aggregation were observed. These are all characteristics of actively dividing cells. In addition, Type B cells had ultrastructural characteristics of undifferentiated cells similar to those in the embryonic neuroepithelium (Hinds and Ruffett, 1971
Among the characteristics of Type B cells, of most interest was the atypical cilium associated with a centriole in their apical cytoplasm. The three-dimensional reconstruction (Table 3) indicated that most Type B cells had this cilium. A similar single cilium has been observed in the developing neuroepithelium (Sotelo and Trujillo-Cenóz, 1958
Radial cells divide (Misson et al., 1988
Type A cells
With increasing survival after [3H]-thymidine administration, the number of labeled Type B cells decreased, but that of Type A cells increased. Unlike Type B cells, these smaller cells did not contact the VZ, and their processes were oriented parallel to the surface of the ventricle. Type A cells were clustered between the VZ and the underlying parenchyma. Barami et al. (1995)
We do not know why Type A cells accumulate at the interface of the VZ and the underlying parenchyma. It has been observed before that very few young migrating neurons emerge from the walls of the lateral ventricle sooner than 3 d after [3H]-thymidine treatment (Alvarez-Buylla and Nottebohm, 1988
Interkinetic migration in the adult avian brain
Interkinetic movements are well known in the early neuroepithelium (Sauer, 1935
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Figure 11. Average distances between the center of the nuclei and the ventricular surface of the 20 [3H]-labeled cells closest to the ventricle in the ventral hot spot of adult canaries. Each bar is the average of four birds. At 3 and 5 hr survivals, a significant (p = 0.02; Mann-Whitney U) reduction in this distance was observed compared with 1 or 11 hr survivals. Averages for 1, 7, and 11 hr were not significantly different. One-factor ANOVA also showed significant differences (p < 0.05) between 1 hr and 3 or 5 hr survivals. Likewise, 3 and 5 hr survivals were significantly different from 11 hr survivals. The dashed line indicates the average VZ cell radius perpendicular to the ventricle in the ventral hot spot.
The three-dimensional reconstructions indicate that all Type B cells maintain contact with the ventricle, which suggests that this interkinetic migration is attributable to the displacement of the nucleus and cytoplasm within the processes of Type B cells and not to the formation of a process and migration of entire cells. Interkinetic migration in the adult avian brain is therefore similar to that described during development. However, unlike the embryonic VZ, the adult avian VZ is thin and compact. For this reason, the nuclear displacements in the adult VZ are small compared with those in the embryo. The function of intermitotic nuclear migration is not known. The present results indicate that intermitotic migration continues even in the adult VZ, where the epithelium has become compacted and intermixed with ependymal cells. This process probably plays an important role during proliferation or early neuronal differentiation. The cerebrospinal fluid may contain factors that are required for mitosis or early stages of differentiation. Interestingly, most of the mitotic cells in this study were exposed to the cerebrospinal fluid and had endocytic vesicles (Fig. 6) on their surface, which suggests internalization of substances present in the ventricular lumen.
The position of the cell with respect to the ventricular cavity may also allow segregation of factors within the cytoplasm that are important for cell differentiation (Chenn and McConnell, 1995
Neurogenesis versus ependymal function
Ependymal cells in canaries showed morphology similar to the simple ependymal cells described previously in mammals (Tennyson and Pappas, 1962
Ependymal cells shared the walls of the lateral ventricle with Type B cells. As discussed above, Type B cells are organized and behave similarly to those of the embryonic neuroepithelium. Clearly the adult germinal epithelium retained the functional and anatomical characteristics of a VZ. The fact that ependymal cells were also present in the same epithelium with Type B cells suggests that ependymal functions and neurogenesis can coexist. Our results indicate that these two functions are segregated among two different kinds of cells within the same epithelial layer.
Our study concentrated on the VZ of the lateral wall of the lateral ventricle at the level of the anterior commissure. Our ultrastructural analysis included three distinct regions of this wall. Differences in cell composition and cell types may occur at other rostrocaudal levels of the telencephalon not included in this study. Preliminary observations indicate that the VZ in other regions of the telencephalon also contained the three main cell types described here (not shown). The criteria for cell identification established by the present study should aid future work in determining regional variation of the VZ in the developing and adult avian brain. In particular, the characteristic multiple cilia in ependymal cells versus a single short cilium characteristic of Type B cells may allow the identification (e.g., by scanning EM) of patches of neuronal stem cells in the walls of the brain ventricles.
Conclusion
The present study has determined the architecture of the adult canary VZ (summarized in Fig. 12) and has identified the primary precursors as Type B cells (blue). These cells are organized as a pseudostratified epithelium and maintain an end foot on the ventricular surface. Similar to the neuroepithelial cells in the embryo, Type B cells moved toward the ventricle to undergo mitosis. Type B cells gave rise to Type A cells (red) that move away from the ventricular surface and become oriented parallel to this wall. These cells seemed to correspond to young migrating neurons. Ependymal cells (gray) shared the ventricular surface with Type B cells but did not divide. Our results revealed the dynamic nature of the VZ and have provided the morphological basis for identification of neuronal precursors in the VZ of an adult vertebrate brain.
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Figure 12. Schematic summary of results. Three main cell types comprise the VZ of adult canaries: Type A (red), Type B (blue), and ependymal cells (gray). Ependymal cells are multiciliated and do not divide. Type B cells are pseudostratified, and all contact the ventricle that has a single short cilium on this lumen. These cells are the primary precursors. Their nuclei migrate from basal to apical VZ to round up and divide adjacent to the ventricular cavity (bottom). Type A cells do not contact the ventricle, are oriented largely parallel to the ventricular surface, and have characteristics of young migrating neurons. Results suggest that Type A cells are derived from Type B cells and may move tangentially to the VZ before radial migration.
FOOTNOTES Received Sept. 5, 1997; revised Nov. 12, 1997; accepted Nov. 17, 1997.
This work was supported by National Institutes of Health Grants HD32116 and NS 24478. We thank Jose G. Baltazar and Marie Therese Merchant for technical assistance, and Fernando Nottebohm for providing the birds for this study. We are also grateful to Fiona Doetsch, Steven Goldman, and S. Rasika for their comments on this manuscript.
A.A.-B. and J.M. G.-V. contributed equally to this work.
Correspondence should be addressed to Dr. Alvarez-Buylla, The Rockefeller University Field Research Center, Tyrrel Road, Millbrook, New York 12545.
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