Quantitative Analysis Reveals Dominance of Gliogenesis Over Neurogenesis in an Adult Brainstem Oscillator € nther K.H. Zupanc Ruxandra F. S^ırbulescu, Iulian Ilies¸, Gu Laboratory of Neurobiology, Department of Biology, Northeastern University, Boston, Massachusetts 02115 Received 14 August 2013; revised 1 February 2014; accepted 12 March 2014

ABSTRACT: Neural stem/progenitor cells in the neurogenic niches of the adult brain are widely assumed to give rise predominantly to neurons, rather than glia. Here, we performed a quantitative analysis of the resident neural progenitors and their progeny in the adult pacemaker nucleus (Pn) of the weakly electric fish Apteronotus leptorhynchus. Approximately 15% of all cells in this brainstem nucleus are radial glia-like neural stem/ progenitor cells. They are distributed uniformly within the tissue and are characterized by the expression of Sox2 and Meis 1/2/3. Approximately 2–3% of them are mitotically active, as indicated by expression of proliferating cell nuclear antigen. Labeling of proliferating cells with a single pulse of BrdU, followed by chases of up to 100 days, revealed that new cells are generated uniformly throughout the nucleus and do not undergo substantial

INTRODUCTION Until several decades ago, the prevalent hypothesis was that brain neurogenesis is restricted to embryonic and early postembryonic stages of development, whereas gliogenesis persists throughout adult life. However, starting in the 1960s, an increasing number of studies demonstrated the generation of new neurons in the adult brain of numerous vertebrates, although large differences have been found between

Correspondence to: G.K.H. Zupanc ([email protected]). Contract grant sponsor: Northeastern University (to G.K.H.Z.). Ó 2014 Wiley Periodicals, Inc. Published online 18 March 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/dneu.22176

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migration. New cells differentiate into S1001 astrocytes and Hu C/D1 small interneurons at a ratio of 4:1, reflecting the proportions of the total glia and neurons in this brain region. The continuous addition of new cells leads to a diffuse growth of the Pn, which doubles in volume and total cell number over the first 2 years following sexual maturation of the fish. However, the number of pacemaker and relay cells, which constitute the oscillatory neural network, remains constant throughout adult life. We hypothesize that the dominance of gliogenesis is an adaptation to the high-frequency firing of the oscillatory neurons in this nucleus. VC 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 934–952, 2014

Keywords: adult neurogenesis; adult gliogenesis; teleost fish; pacemaker nucleus; stem/progenitor cells

different taxa, both in the number of neurogenic regions and in the number of neurons produced (for review, see Lindsey and Tropepe, 2006). In teleost fish, the vertebrate taxon with the highest neurogenic potential, several dozens of distinct proliferation zones exist, and the number of adult-born neurons is at least one, if not two, orders of magnitude higher than in mammals (for recent reviews, see Chapouton et al., 2007; Kaslin et al., 2008; Zupanc and S^ırbulescu, 2011, 2013). This continuous generation of new neurons is closely linked to the continued growth of most teleost fish during adulthood, and to their excellent ability to regenerate after brain injuries (for reviews, see S^ırbulescu and Zupanc, 2013; Zupanc and S^ırbulescu, 2011, 2013). The source of the adult-born neurons and glia are resident neural stem/progenitor cells that exhibit the

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two fundamental properties of stem cells—the capacity to renew themselves through mitotic cell division and the ability to differentiate into various cell types (Gage, 2000; Weissman et al., 2001; Temple, 2001)—both in vitro and in vivo (Reynolds and Weiss, 1992; Palmer et al., 1999; Hinsch and Zupanc, 2006; Bonaguidi et al., 2011). In the mammalian brain, neurogenesis is restricted to two regions: the dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricles, from where new cells migrate into the olfactory bulb (for reviews, see Duan et al., 2008; Zhao et al., 2008; Balu and Lucki, 2009; Basak and Taylor, 2009; Imayoshi et al., 2009; Ma et al., 2009; Whitman and Greer, 2009). Lineage tracing of individual neural stem/progenitor cells in the adult mouse dentate gyrus has shown that astroglia and neurons are generated at similar frequencies (Bonaguidi et al., 2011). Stem/progenitor cells in the adult brain exhibit considerable plasticity. For example, in the hippocampus the lineage fate of their progeny can be modulated by both intrinsic and extrinsic factors, including physical exercise of the animals or their exposure to an enriched environment (Kempermann et al., 1997; van Praag et al., 1999a,b, 2002). Outside of these neurogenic niches, actively proliferating neural stem/progenitor cells are widely distributed in the brain, but their progeny differentiate exclusively into glia, predominantly of oligodendrocyte lineage (for review, see Ninkovic and G€otz, 2013). Traumatic brain injury promotes proliferation of neural stem/progenitor cells both in neurogenic and non-neurogenic regions. However, this effect appears to result only in an increase in the number of glial cells but not that of neurons (Norton et al., 1992; Fawcett and Asher, 1999; Floyd and Lyeth, 2007; Sandhir et al., 2008; Christie and Turnley, 2013; Gao and Chen, 2013). Whereas the increase in the number of glial cells as part of the brain’s response to injury is well documented, less is known about gliogenesis and its functional significance in the healthy central nervous system (CNS). To address this issue, in this study we examined the pacemaker nucleus (Pn) of the weakly electric fish Apteronotus leptorhynchus, a wellcharacterized model system in behavioral neurobiology (for reviews, see Dye and Meyer, 1986; Zupanc and Bullock, 2005). This nucleus located in the medulla oblongata plays a crucial role in the control of the discharges produced by the electric organ of the fish, which in turn are used for orientation in the environment and for communication with conspecifics. Like in all apteronotids, the electric organ of A. leptorhynchus is formed by modified spinal motor axons, whose synchronous depolarizations result in

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electric organ discharges (EODs) at a species-specific frequency of 650–1000 Hz (de Oliveira-Castro, 1955; Bennett, 1971; Waxman et al., 1972). The frequency of these electric organ discharges is controlled, in a one-to-one fashion, by the frequency of the oscillatory network formed by two of the neuronal cell types of the Pn, pacemaker cells and relay cells (for review, see Dye and Meyer, 1986). While the pacemaker cells are restricted to the Pn, the relay cells project down the spinal cord to form electrotonic junctions with the electromotor neurons. Mapping of the proliferation zones in the adult brain of A. leptorhynchus has previously indicated the presence of mitotic cells in the Pn (Zupanc and Horschke, 1995). Moreover, immunohistochemical experiments have revealed an extremely high density of astroglia in this nucleus, compared to other regions in the adult brain (Zupanc et al., in press). In this study, we performed a quantitative analysis of the generation and fate of the adult-born cells in the Pn; characterized the putative neural stem/progenitor cells that give rise to both neurons and glia; and related these findings to the morphometric characterization of the Pn and to its continued growth during adulthood. Taken together, the results of the present investigation define a novel vertebrate model system that offers a unique opportunity to examine the link between adult neurogenesis and adult gliogenesis, and the functional significance that these phenomena play in the neural control of a well-characterized behavior.

METHODS Animals Brown ghost knifefish (A. leptorhynchus; Gymnotiformes, Teleostei) were supplied by tropical fish importers and maintained in the laboratory as described previously (Gama Salgado and Zupanc, 2011). A total of 50 fish (32 males, 18 females), with an average total length of 15.3 cm (6SD: 2.6 cm) and an average weight of 8.4 g (6SD: 3.6 g) were used. The gonadosomatic index averaged 0.0026 (6SD: 0.0009) in males and 0.0381 (6SD: 0.0277) in females. These data indicate that all fish were adults in their second or third year of life. All animal experiments were approved by the Institutional Animal Care and Use Committee of Northeastern University. All efforts were made to reduce the number of animals used and to minimize animal suffering.

BrdU Administration and Tissue Sampling To examine cell proliferation and the fate of newly generated cells, fish were lightly anesthetized in 2% ethyl Developmental Neurobiology

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carbamate (urethane; Fisher Scientific) in aquarium water, and injected intraperitoneally with 20 mL/g body weight of 5-bromo-20 -deoxyuridine (BrdU) solution (3 mg/mL; GE Healthcare). After a postinjection survival time of 2 h, 5 days, 10 days, or 100 days, the fish were deeply anesthetized in a 3% solution of ethyl 3-aminobenzoate methanesulfonate (MS-222; Sigma-Aldrich) in aquarium water, and intracardially perfused with 2% freshly depolymerized paraformaldehyde (Fisher Scientific) in 0.1 M phosphate buffer (PB), pH 7.4, for 30 min. The brain was postfixed in the fixative solution at 4 C for 2 h, and cryoprotected in 1 M sucrose (Fisher Scientific) in 0.1 M PB at 4 C for 36 h. After embedding, the brain was cryosectioned transversally at a thickness of 16 or 30 mm, and the sections were collected on Superfrost Plus Gold slides (Fisher Scientific).

MSO Administration To disrupt astroglial metabolism, fish (n 5 3) were anesthetized and injected intraperitoneally with 1 mM/g L-methionine sulfoximine (MSO; Sigma-Aldrich) dissolved in 0.1 M phosphate-buffered saline. Controls matched for sex and age (n 5 3) were injected with saline. After a survival time of 6 h, the fish were perfused intracardially and the brain was removed for cryosectioning as described earlier.

Immunohistochemistry Tissue sections were processed for immunohistochemistry as previously described (Zupanc et al., 2012). Briefly, the sections were desiccated for 90 min, washed thrice in 0.1 M phosphate-buffered saline, and blocked for 1 h at room temperature in buffer containing 3% sheep serum, 1% bovine serum albumin, 1% teleostean gelatine, and 0.3% Triton X100. Primary antibodies, mouse anti-Hu C/D (clone 16A11; Invitrogen), rabbit anti-caspase-3 (BD Pharmingen), rabbit anti-S100 (Dako), mouse anti-glutamine synthetase (Abcam), chicken anti-glial fibrillary acidic protein (GFAP) (Abcam), rabbit anti-connexin 43 (Cell Signaling), mouse anti-vimentin (clone V9; Sigma-Aldrich), mouse anti-Sox2 (clone 9-9-3; Abcam), rabbit anti-Sox2 (Millipore), mouse anti-Meis 1/2/3 (clone 9.2.7; EMD Millipore), rabbit anti-Pax6 (Covance), mouse anti-proliferating cell nuclear antigen (PCNA; clone PC10; Sigma-Aldrich), rabbit anti-PCNA (Abcam), and rat anti-BrdU (clone BU1/75; AbD Serotec) were applied overnight at 4 C. For antiBrdU and anti-PCNA immunolabeling, sections were incubated in 2 M HCl at 37 C for 30 min and washed twice in 0.1 M borate buffer, pH 8.5, before blocking. For anti-Hu C/D labeling, sections were incubated for 30 min in 50 mM Tris–HCl buffer, pH 8.0, at room temperature before blocking. The secondary antibodies, Alexa 488-, Alexa 546-, or Alexa 635-conjugated goat anti-mouse IgG, Alexa 488-, or Alexa 546-conjugated goat anti-rabbit IgG, Alexa 488conjugated goat anti-chicken IgG (all from Life Technologies), and Cy3-conjugated donkey anti-rat IgG (Jackson Immunoresearch) were applied overnight at 4 C. Where applicable, the sections were counterstained with 40 ,6-diaDevelopmental Neurobiology

midino-2-phenylindole dihydrochloride (DAPI; SigmaAldrich).

Microscopy and Image Analysis Images were acquired using a Zeiss LSM 710 laser scanning microscope (Carl Zeiss) equipped with 203 and 633 objectives, and a Zeiss Axioskop 20 epifluorescence microscope equipped with 203 and 403 objectives and an AxioCam MRc5 digital camera (both from Carl Zeiss). Optical sections were taken at a resolution of 0.08–0.61 mm/pixel and an optical thickness of 1.4–30 mm, using Zen or AxioVision software (both from Carl Zeiss). Cell counts, cell profile measurements, and Pn section area measurements were performed in ImageJ (National Institutes of Health), while GFAP immunolabeling was quantified using MATLAB (The MathWorks, Inc.). All cell counts were adjusted using the methods of Konigsmark (1970) and/or Smolen et al. (1983). For large neurons with low total numbers (pacemaker and relay cells), counts were based on a large number of sections (n 5 10–18 per fish) and were subsequently extrapolated to the entire Pn. For all other cell types, corrected counts (n 5 2–10 sections per fish) were converted to volumetric densities. Three-dimensional (3D) simulations of the observed cell distributions were constructed in MATLAB. To estimate Pn volumes, sections were examined using a Zeiss Axioskop epifluorescence microscope equipped with a camera lucida. Digitized drawings were processed in MATLAB to determine section areas, from which the total Pn volumes were extrapolated (n 5 15 fish, 6–18 sections per fish). All results are reported as average 6 standard error.

RESULTS Morphology of the Pn Morphometric analysis of the Pn in 15 fish with total lengths ranging between 12.0 and 19.2 cm indicated that the volume of the Pn is positively correlated with their size (Pearson’s q 5 0.63, p < 0.05). The volume of the Pn ranged between 0.11 and 0.31 mm3, with an average of 0.21 6 0.01 mm3. In the following, we will refer to a Pn with the latter volume as the “average Pn.” Counterstaining with DAPI indicated a rather uniform distribution of cells within the nucleus. The estimated mean volumetric density of these cells was 960,000 6 90,000/mm3 (n 5 3 fish), and was independent of the size of the fish, indicating that the total number of cells in the Pn can be estimated based on the volume of this brain nucleus [Fig. 1(A)]. Thus, within the entire sample of fish analyzed (n 5 15), the total number of cells in the Pn ranged from approximately 100,000 to 300,000, with an average of 200,000 cells. Regression analysis

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Figure 1 Variation of Pn morphology with total length of fish. A: The estimated total volume of the Pn, as well as the total estimated number of cells in the Pn, increase linearly with the size of the fish (Pearson’s q 5 0.63, p < 0.05). B: By contrast, the total number of pacemaker cells and relay cells do not vary significantly with the total length of the fish (p > 0.1). C: The density of small interneurons shows a nonsignificant increasing trend with the total length of the fish (p > 0.1). Lines indicate the corresponding linear regressions.

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suggests that the total number of Pn cells doubles over the size range examined [Fig. 1(A)]. Previous findings based on analysis of electron micrographs, dye-filling of physiologically characterized cells, and nicotinamide adenine dinucleotide phosphate-diaphorase activity (Elekes and Szabo, 1985; Dye and Heiligenberg, 1987; Turner and Moroz, 1995) identified three classes of neurons in the Pn—relay cells, pacemaker cells, and small interneurons. Our analysis of Hu C/D-immunostained cells in the Pn largely confirmed these observations, but revealed in addition two subclasses of cells among the small interneurons. The four neuronal cell types differed in morphology and size (Figs. 2 and 3). Relay cells were typically spherical or ovoid, with maximum lengths of their major axes of 50–70 mm [Fig. 2(B)]. Pacemaker cells displayed predominantly a spherical morphology, with diameters of the somatic profiles ranging between 25 and 40 mm [Fig. 2(C)]. Small interneurons could be divided into two subclasses, which will be referred to as type-1 and type-2 small interneurons. Type-1 small interneurons had small round or oval somata, with lengths of the major axes of 5–8 mm. Type-2 small interneurons displayed club- or spindle-shaped somata with visible proximal parts of neurites and lengths of the major axes of 8–16 mm [Fig. 2(D)]. Quantitative analysis of Hu C/D1 cells (n 5 3 fish) indicated that, in the average Pn, type-1 and type-2 interneurons are present at a 9:4 ratio. To investigate whether large neurons, such as pacemaker and relay cells, are added during adult growth, we quantified these cell types in a sample of 15 fish with total lengths ranging between 12.0 and 19.2 cm. No significant correlation was found between the number of either pacemaker or relay cells and the length of the fish (p > 0.10). On average, there were 20 6 1 relay cells and 87 6 4 pacemaker cells in the Pn of the fish examined [Fig. 1(B)]. Correspondingly, to assess whether additional small interneurons are generated throughout the lifespan of the fish, the volumetric density of these cells was measured in a sample of 11 fish with total lengths between 12.0 and 20.0 cm. Subsequent correlation analysis did not detect any significant dependence between the density of small interneurons in the Pn and the length of the fish (p > 0.10), indicating that, unlike the larger relay and pacemaker cells, this neuronal population grows at the same rate as the entire nucleus. The overall density of small interneurons reached an average of 26,000 6 1500 cells/mm3 over the examined sample [Fig. 1(C)], corresponding to a total of 3700 type-1 and 1600 type-2 small interneurons in the average Pn. Developmental Neurobiology

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Figure 2 Neuronal morphology of the Pn. A: Immunostaining against Hu C/D reveals three neuronal cell types in this brain nucleus, which are distinguished by size, morphology, and abundance. Scale bar 5 100 mm. B: Relay cell. This type of neuron is characterized by its spherical or ovoid morphology and its large size, with the major axis assuming maximal lengths of 70 mm. Scale bar 5 20 mm. C: Pacemaker cell. This predominantly spherical type of neuron is 4–6 times more frequent than relay cells and displays diameters of up to 35 mm. Scale bar 5 20 mm. D: Small interneurons. They are spherical, club-shaped, or spindle-shaped, and distinguished by their abundance and small size, with their major axis typically not exceeding 10 mm. Scale bar 5 20 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Immunostaining against the glial-specific marker protein S100 [Fig. 4(A)] revealed a remarkably dense meshwork of glial fibers in the Pn. A similar pattern was observed after immunolabeling against other glial markers, including GFAP (Fig. 5), glutamine synthetase, and vimentin (not shown). These fibers are closely associated with pacemaker and relay cells [Figs. 4(B) and 5]. Quantitative analysis of S1001 cells indicated that there were roughly 39,000 labeled cell bodies in the average Pn (190,000 6 20,000 cells/mm3). Moreover, the glial meshwork in which the pacemaker and relay cells are embedded expresses high levels of connexin-43, a member of Developmental Neurobiology

the connexin family of transmembrane gap junction proteins (Fig. 5), suggesting that the glial cells form a syncytium.

Cell Proliferation Mitotic cells in the Pn were identified through immunohistochemical detection of PCNA [Figs. 6 and 7(A)] and BrdU. Proliferation zones, that is, areas within the Pn distinguished by a high concentration of PCNA1 cells, were not evident. Instead, the PCNA1 cells appeared to be rather uniformly distributed within the Pn. The average volumetric

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Figure 3 Neuronal classes in the Pn. A: 3D simulation of the number, morphology, and distribution of the three major classes of neurons in the entire Pn, based on average cell counts and cell size measurements (n 5 3 fish). B: Probability distribution functions of neuronal profile diameters. A Gaussian mixture fit of measured average diameters (n 5 372 cells) indicated that the optimal solution consists of four distinct neuronal categories. Note that while the large cells (relay cells and pacemaker cells) form clearly separated size categories, small interneurons appear to comprise two populations with distinct average diameters and variances, but partially overlapping size ranges. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

density of these cells was 11,000 6 1400 cells/mm3, indicating a total number of approximately 2200 PCNA1 cells in the average Pn (n 5 4 fish). Thus, approximately 1% of the total number of cells (as estimated from the counts of DAPI-stained nuclei) was immunopositive for PCNA. Similarly, the distribution of BrdU1 cells within the Pn 2 h after a single BrdU pulse (n 5 3 fish) was uniform. The average volumetric density of BrdU1 cells was 2800 6 600 cells/mm3 or approximately

570 cells in the average Pn. This indicates that approximately 0.3% of the total cells in the Pn are in the S-phase of mitosis within any 2-hour period.

Characterization of Neural Stem/ Progenitor Cells To further explore the sources and dynamics of proliferating cells in the Pn, the expression of the stem

Figure 4 Glial morphology of the Pn. A: Immunostaining against S100 reveals an abundance of glial cells in this nucleus. The meshwork of glial fibers is closely associated with pacemaker cells (P) and relay cells (R). Scale bar 5 200 mm. B: High-magnification image of the boxed area in A. Scale bar 5 10 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Developmental Neurobiology

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Figure 5 Association between glia and neurons in the Pn. Three-dimensional shadow projection of a z-stack comprising 25 confocal images spanning a total thickness of 12 mm. Hu C/D1 neurons (blue) are embedded in a dense meshwork of GFAP1 glial cells (green) which express connexin-43 in a punctate pattern (red). A pacemaker neuron (P) is surrounded by the dense astrocytic syncytium. Scale bar 5 5 mm.

cell markers Sox2, Meis 1/2/3, and Pax6 was studied in conjunction with PCNA. Sox2 immunostaining labeled numerous cells, which were uniformly distributed within the Pn [Figs. 6(A) and 7(B)]. These cells displayed predominantly a club-shaped or sickle-shaped morphology, with a major axis of 7–10 mm. A minority of the cells were spheroidal, with a diameter of approximately 5 mm. The mean volumetric density of the Sox21 cells was estimated at 150,000 6 10,000 cells/mm3, corresponding to approximately 30,000 cells in the average Pn (n 5 2 fish). Double immunostaining revealed coexpression of PCNA and Sox2 [Fig. 6(A–A00 )] in an estimated 900 cells (4500 6 1100 cells/mm3) within the Pn. Of 30,000 Sox21 cells, 3% underwent mitosis, as indicated by colocalization of PCNA. Conversely, of 2200 mitotic cells, 41% expressed Sox2. The morphology and distribution of the Meis 1/2/31 stem cells in the Pn closely resembled those of Sox21 cells. Their mean volumetric density was estimated to be 160,000 6 30,000 cells/mm3, corresponding to approximately 32,000 cells in the average Pn (n 5 2 fish). Double immunostaining revealed coexpression of PCNA and Meis 1/2/3 [Fig. 6(B–B00 )] in an estimated 600 cells (2900 6 900 cells/mm3) within the Pn, indiDevelopmental Neurobiology

cating that approximately 2% of the Meis 1/2/31 progenitors undergo mitosis [Fig. 7(C)]. Conversely, of 2200 mitotic cells 26% expressed Meis 1/2/3. The similarity in morphology and numbers between Sox21 and Meis 1/2/31 progenitors prompted the hypothesis that these markers label the same cell population. Indeed, double immunolabeling showed that, while these two progenitor populations are not identical, they overlap significantly (Fig. 8). Quantitative analysis indicated that, of 1340 Sox21 cells, 1302 (97%) also expressed Meis 1/2/3. Conversely, of 1321 Meis 1/2/31 cells, 1302 (98%) coexpressed Sox2. The morphology and the distribution of the cells that expressed Pax6 differed markedly from those immunopositive for Sox2 and Meis 1/2/3. The vast majority of the Pax61 cells exhibited a spherical shape (diameter approximately 3 mm) or was slightly ovoid (major axis  5 mm). They were not evenly distributed within the Pn but were found more frequently near the ventrolateral surface of the Pn. Their mean volumetric density was estimated to be 3100 6 500 cells/mm3, corresponding to approximately 640 cells in the average Pn (n 5 2 fish). Double immunostaining revealed coexpression of PCNA and Pax6 [Fig. 6(C–C00 )] in an estimated 230 cells (1100 6 400 cells/mm3) within the Pn. Of 640 Pax61 cells, 36% underwent mitosis, as indicated by colocalization of PCNA [Fig. 7(D)]. Conversely, of 2200 mitotic cells, approximately 10% expressed Pax6. To further investigate the degree of identity between the Sox21/Meis 1/2/31 neural stem/progenitor cell population and the Pax61 cells, we combined immunolabeling against Sox2 and Pax6. Of 71 Pax61 cells, only 9 (13%) coexpressed Sox2, indicating that these markers label two largely separate neural stem/progenitor cell populations. To determine the cellular identity of the stem cells, Sox2 immunohistochemistry was combined with immunostaining against the glial marker S100. Only those cells were analyzed in which a clearly visible DAPI-stained nucleus was evident. In 1170 of 1240 Sox21 cells examined by confocal microscopy in two fish, S100 and Sox2 immunoreactivity colocalized (Fig. 9), suggesting a glial identity of the stem/ progenitor cells in the Pn.

Disruption of Stem/Progenitor Cells To further confirm the glial identity of stem/progenitor cells in the Pn, as well as to investigate the immediate effects of glial disruption on their proliferative activity, fish were injected with the gliotoxin MSO

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Figure 6 Immunohistochemical characterization of stem cells in the Pn. Among the stem cell populations revealed by expression of Sox2 (A), Meis 1/2/3 (B), and Pax6 (C), only a fraction are in the stage of cell proliferation, as indicated by expression of PCNA (A0 –C0 ). Double-labeled cells unambiguously identified in the 1.4-mm-thick optical sections are indicated by arrows (A00 –C00 ). Scale bar 5 10 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

(n 5 3) or saline (n 5 3) and the Pn was examined after a survival time of 6 h. Triple immunolabeling against GFAP, PCNA, and Sox2 revealed a marked reduction in GFAP expression and cellular proliferation in the Pn after MSO administration, but no significant change in the total number of Sox21 stem/progenitor cells (Fig. 10). Total GFAP labeling (defined as the relative area immunostained, multiplied by the average labeling

intensity after background correction) was reduced eightfold in treated fish, as compared to controls (p < 0.05, independent-samples t-test; n 5 3 fish per group). The average volumetric density of PCNA1 cells was of 4200 6 100 cells/mm3 in treated fish, a more than twofold decrease as compared to controls, which showed 9000 6 1100 cells/mm3 (p < 0.05, independent-samples t-test; n 5 3 fish per group). A similar reduction was observed in the number of cells Developmental Neurobiology

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Figure 7 3D simulations illustrating localization and densities of stem/progenitor cells in the Pn. A: Distribution of PCNA1 cells in the Pn. B: Distribution of Sox21 cells (red) and proliferating PCNA1/Sox21 (green) cells in the Pn. C: Distribution of Meis 1/2/31 cells (red) and proliferating PCNA1/Meis 1/2/31 cells (green) in the Pn. D: Distribution of Pax61 cells (red) and proliferating PCNA1/Pax61 cells (green) in the Pn. All simulations are based on cell counts in tissue sections (n 5 2–4 fish). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

coexpressing Sox2 and PCNA, from 6000 6 1000 cells/mm3 in the control group to 2700 6 400 cells/ mm3 in MSO-treated fish (p < 0.05, independent-

samples t-test). However, the total densities of Sox21 cells did not differ significantly between the groups (p > 0.2), reaching 410,000 6 45,000 cells/

Figure 8 Overlap between Sox21 and Meis 1/2/31 stem/progenitor cell populations. The majority of the progenitor cells coexpresses the two markers (arrows). However, the two populations are not completely identical, with some cells expressing exclusively Sox2 (open arrow) or Meis 1/2/3 (arrowheads). Scale bar 5 50 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Developmental Neurobiology

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Figure 9 Cellular identity of stem cells in the Pn. Immunostaining against both Sox2 (A) and S100 (B) demonstrates that all Sox21 cells shown in the confocal image coexpress S100, suggesting a glial identity of the stem cells. C: DAPI counterstain. D: Overlay. Scale bar 5 20 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

mm3 in controls and 350,000 6 20,000 cells/mm3 after MSO administration.

Development of the New Cells To investigate whether the newly generated cells migrate within the Pn, mitotic cells were labeled with a single pulse of BrdU, followed by a chase of 2 h, 5 days, 10 days, or 100 days (n 5 2–3 fish per time point). The distance from each labeled cell to the closest point on the surface of the Pn was measured within transverse sections. The resulting distributions of distances did not differ from each other, or from a uniform distribution (2-sample Kolmogorov–Smirnov tests; p > 0.3, Bonferroni adjustment for multiple comparisons), indicating that no substantial migration occurs in the adult Pn. To examine the long-term survival of the adultborn cells, as well as their fate, mitotic cells were

labeled with a single pulse of BrdU and examined after various chase times. Using triple immunolabeling against BrdU, the neuronal marker Hu C/D, and the glial marker S100, the rate and proportion of differentiation of the new cells could be determined. In both neurons and glia, BrdU immunolabeling was limited to the nuclear region [Fig. 11(A–A000 , B– B000 )]. At all examined time points, BrdU labeled exclusively small interneurons, mostly of type-1, and no BrdU1 pacemaker or relay cells were observed. The average volumetric density of BrdU1 cells in the Pn was 2800 6 600 cells/mm3 at 2h after a single BrdU pulse (n 5 3 fish). This density increased approximately threefold to 8200 6 2000 cells/mm3 at 5 days post-BrdU (n 5 2 fish), and to 9400 6 1900 cells/mm3 at 10 days post-BrdU (n 5 3 fish). After 100 days post-BrdU administration, the BrdU1 cell density decreased to 3600 6 1500 cells/mm3 (n 5 2 fish). The rate of neuronal differentiation, assessed by Developmental Neurobiology

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Figure 10 Effect of glial disruption on stem/progenitor cell proliferation. Administration of MSO, a potent glutamine synthetase inhibitor, has a rapid effect on astroglial metabolism. At 6 h postadministration, GFAP expression is significantly reduced (A–C). Cellular proliferation, as indicated by PCNA expression, is also significantly decreased (A0 , B0 , and D). Overall numbers of Sox21 are not altered significantly (A00 , B00 , and E); however, the number of proliferating Sox21/PCNA1 progenitors decreases significantly after MSO treatment (D). Scale bar 5 100 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

combining BrdU and Hu C/D immunolabeling, showed a similar pattern at early time points, increasing from 330 6 170 cells/mm3 at 2 h to 1120 6 430 cells/mm3 at 5 days, but then decreased to 620 6 260 Developmental Neurobiology

cells/mm3 at 10 days and 470 6 20 cells/mm3 at 100 days. The latter number represents approximately 13% of the surviving new cells. By contrast, the rate of gliogenesis, as indicated by immunolabeling against both

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Figure 11 Differentiation and survival of newly generated cells in the Pn. A–A000 : At 100 days post-BrdU administration, triple immunolabeling against BrdU, the neuronal marker Hu C/D and the glial marker S100, shows that many of the newly generated cells differentiate into glia (arrows). Maximum intensity projection of a z-stack comprising 22 confocal images spanning a total thickness of 9.5 mm. Magenta line in A000 indicates the level of the z-stack orthogonal view shown on the right side. Scale bar 5 25 mm. B–B000 : BrdU1 neurons were also observed at such long survival times (arrow). Maximum intensity projection of a z-stack comprising 16 confocal images spanning a total thickness of 7 mm. Magenta line in B000 indicates the level of the z-stack orthogonal view shown on the right side. Scale bar 5 5 mm. C: Quantitative analysis of cell proliferation and differentiation in the Pn. The overall number of BrdU-labeled cells, as well as the number of BrdU1/Hu C/D1 cells, initially more than doubles, but is reduced by half from 5 to 100 days post-BrdU administration, whereas the absolute number of S1001 cells does not decrease significantly. At 100 days, gliogenesis dominates, with almost four times the number of S1001 glia being present, compared to the number of Hu C/D1 neurons. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

BrdU and S100, exhibited an overall growing trend, increasing from 140 6 80 cells/mm3 at 2 h to 610 6 280 cells/mm3 at 5 days and 460 6 160 cells/ mm3 at 10 days, and further to 1700 6 1100 cells/mm3

at 100 days, the latter figure representing approximately 46% of the surviving new cells [Fig. 11(C)]. To identify apoptotic cells in the Pn, we used immunolabeling against active caspase-3. Remarkably, Developmental Neurobiology

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despite examining a total of 57 Pn sections in nine fish, we could not find any caspase-31 cells, although such cells were found in neighboring brain regions within the same sections. This absence of caspase-31 cells in the Pn indicates that the rate of apoptosis is likely negligible.

DISCUSSION Morphometric Characterization of the Pn Although adult neurogenesis has been studied intensively over the past 2 decades (Bonfanti et al., 2011), little quantitative information is available on the adult neural stem/progenitor cells and the progeny they give rise to. In this study, we performed a comprehensive quantitative analysis of these cells, using the Pn as a well-defined model brain region. For this analysis, we examined fish with total lengths ranging from 12.0 to 20.0 cm, which correspond to individuals at the beginning of their second year of life—when they reach sexual maturity—and to individuals at the end of their third year, respectively (Ilies¸ et al., in press). Since the life span of A. leptorhynchus is 3–5 years (Froese and Pauly, 2013), the sample of fish used in our investigation is representative for a large part of adulthood in this species. A. leptorhynchus is a teleost fish which exhibits continuous growth throughout its life, and proportional growth can be observed in the central nervous system. Both the volume of the Pn and the total number of cells increase with total length/age of the fish [Fig. 1(A)]. In young adults with a total length of 12 cm, the Pn has an estimated volume of 0.14 mm3, corresponding to approximately 140,000 cells. By the time the fish reach the end of their third year, both the volume of the Pn and the total number of cells double. Of the total cell population of the Pn, only a minor fraction—roughly 3%—are immunopositive for Hu C/ D, the vast majority of them exhibiting the morphological characteristics of small interneurons. We propose that these interneurons do not represent a homogenous class, as previously thought (Turner and Moroz, 1995), but instead form two distinct groups differing in morphology and size. However, nothing is known about their possible function. In addition to interneurons, we have estimated that, on average, there are 87 pacemaker cells and 20 relay cells in the Pn. These counts are in excellent agreement with previously determined numbers (Dye and Heiligenberg, 1987). In addition to the Hu C/D-expressing neurons, we have estimated that a further 20% of the total number Developmental Neurobiology

of Pn cells exhibit immunoreactivity for S100. We hypothesize that a significant fraction of these S1001 cells are neural stem/progenitor cells, identified through their immunopositivity against various stem cell markers (see the Neural Stem/Progenitor Cells section). This leaves roughly 75% of the Pn cells unaccounted for in terms of their cellular identity. A similar percentage of unidentified adult-born cells have been found in a quantitative analysis of neurogenesis and gliogenesis in the adult hippocampus of mice (van Praag et al., 2002). As also suggested by the authors of the latter study, a significant number of the unidentified cells might be endothelial cells of capillaries which extend throughout the Pn. Moreover, despite the lack of immunopositivity against Hu C/D or S100, it is possible that some of these cells are neurons or glia. Hu C/D is an RNA-binding protein, which is first detectable in neurons around the time when they exit the cell cycle (Marusich et al., 1994; Barami et al., 1995). Although Hu C/D is also expressed by mature neurons in various systems, it remains unclear whether antibodies against this protein labeled all neurons in the adult Pn. Similarly, antibodies against S100 may not have identified all glia in the Pn. In the subgranular zone of the dentate gyrus, multiple immunostaining against various glial marker proteins indicated a heterogeneity of glial cells (Filippov et al., 2003; Steiner et al., 2004). Despite the lack of complete identification of the various cell types in the Pn, the large ratio of S1001 to Hu C/D1 cells underscores the importance of glia in this brain nucleus.

Neural Stem/Progenitor Cells A remarkable feature of the Pn is the large number of neural stem/progenitor cells uniformly distributed throughout this brain region. We have estimated that about 30,000 Sox21/Meis 1/2/31 cells (15% of the total number of cells) are present in an average Pn of 0.21 mm3. Only a minor fraction (2–3%) of this cell population undergoes mitosis, whereas the vast majority of progenitors is quiescent at any given time. Both Sox2 and Meis 1/2/3 have been shown to play important, and similar, roles in neural stem cell maintenance during development. Sox2 (Sex determining region of Y chromosome-related high mobility group box 2) is expressed at high levels in neural stem and progenitor cells in both embryonic and adult brains (Episkopou, 2005). Constitutive expression of Sox2 inhibits neuronal differentiation, whereas inhibition of this transcription factor results

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in precocious neuronal differentiation (Bylund et al., 2003; Graham et al., 2003). Information available on Meis 1/2/3 (myeloid ecotropic viral integration site 1 homolog 1/2/3), a homeobox protein belonging to the TALE (three amino acid loop extension) family of homeodomaincontaining proteins, is largely restricted to embryonic development. To the best of our knowledge, this study provides the first indication of expression of this protein in neural stem/progenitor cells in the adult brain. In the developing eye of zebrafish, meis1 is expressed throughout the eye primordium (Bessa et al., 2008). Later, as neurogenesis is initiated, its expression is confined to the ciliary margin where the retinal stem cell population resides. Forced maintenance of meis1 expression inhibits normal differentiation of cells, and knocking down meis1 function causes a delay in the G1-to-S-phase transition of the eye cells. These results suggest a role of meis1 in the maintenance of a proliferative, multipotent state of stem cells during embryonic development. A similar regulatory function of the cell cycle may be exerted by the protein that is recognized by the anti–Meis 1/2/3 antibodies in the adult neural stem/progenitor cells of the Pn. The third neural stem/progenitor cell marker examined in this study was Pax6. This highly conserved transcription factor was first identified as a Paired box (Pax) family member (Walther and Gruss, 1991), and has subsequently been shown to be expressed in specific spatiotemporal patterns in stem/progenitor cells of both the embryonic and adult brain. Pax6 plays not only a crucial role in the regulation of proliferation of these cells, but is also involved in downstream processes during development, such as cell migration, adhesion, and differentiation, as well as regionalization of brain structures (for reviews, see Simpson and Price, 2002; Manuel and Price, 2005; Osumi et al., 2008). The notion that Sox2 and Meis 1/2/3 on the one hand, and Pax6 on the other, define different subpopulations of the neural stem/progenitor cells in the Pn is congruent with the observation that only 13% of the Pax61 cells coexpress Sox2. Immunohistochemical staining aimed at a further characterization of the neural stem/progenitor cells in the Pn has shown that the vast majority of them expresses S100. As these cells assume morphological properties of radial glia, we hypothesize that Sox21/ Meis 1/2/31 radial glia-like precursors serve as the adult neural stem/progenitor cells that give rise to the new neurons and glia in the Pn. This hypothesis is in line with evidence from studies of the subventricular zone of the lateral ventricles and the dentate gyrus of the hippocampus in the adult mammalian brain, where radial glia were identified as the self-renewing

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and multipotent endogenous neural stem cells (Doetsch et al., 1999; Laywell et al., 2000; Seri et al., 2001; Bonaguidi et al., 2011). To further examine the role of astroglia as proliferating stem/progenitor cells in the Pn, we used the glutamine synthetase inhibitor and gliotoxin MSO to specifically disrupt astrocyte metabolism (Sarthy and Ripps, 2001; Clasadonte and Haydon, 2012). MSO is a xenobiotic aminoacid derived from methionine which is phosphorylated by glutamine synthetase in the presence of ATP, resulting in an irreversible inhibition of the enzyme (Ronzio and Meister, 1968; Weisbrod and Meister, 1973; Griffith and Meister, 1978). In addition, MSO has also been reported to inhibit c-glutamylcysteine synthetase, leading to a depletion of glutathione in cells (Richman et al., 1973). After 6 h of systemic exposure to MSO, we observed a significant decrease in GFAP expression in the Pn [Fig. 10(A–C)], as well as a significant increase in glutamine synthetase (data not shown), suggesting that astroglial cytoarchitecture had been effectively disrupted. At the same time, MSO treatment caused a significant reduction in cell proliferation, in particular among Sox21 progenitor cells. This result is in agreement with previous findings that suggest that glutamine plays an important role in the regulation of cell division (DeMarco et al., 1999; Kung et al., 2011). Most significantly, this result provides evidence— independent of the coexpression of S100 by Sox2immunolabeled cells—in favor of the hypothesis that at least part of the neural stem/progenitor cells in the Pn have the characteristics of astrocytes.

Development of the Adult-Born Cells Labeling of S-phase cells with a single pulse of BrdU, followed by a survival time of 2 h, shows that new cells are generated uniformly throughout the Pn. Similar distributions were observed for cells labeled with the second proliferation marker used in this study, PCNA, as well as for Sox21 and Meis 1/2/31 neural stem/progenitor cells. These results suggest that there are no defined proliferation zones (i.e., discrete areas distinguished by high density of proliferating cells) in the Pn. This situation differs markedly from many other brain regions in teleost fish, in which neural stem/progenitor cells, concentrated in distinct proliferation zones, give rise to the majority of the newly generated cells (Zupanc et al., 1996; Ekstr€om et al., 2001; Zupanc et al., 2005; Grandel et al., 2006; Fernandez et al., 2011; Teles et al., 2012). Analysis of the distribution of the BrdU-labeled cells at longer post-BrdU administration survival times has failed to provide evidence for migration of Developmental Neurobiology

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the new cells over significant distances. Most of the progeny appear to reside in close vicinity to where they were born. This contrasts with the directed migration of young cells found in the cerebellum and the olfactory bulb in the teleostean brain (Zupanc et al., 1996, 2005; Teles et al., 2012). The absence of such migration is likely a direct consequence of the “local” generation of progeny through the widely distributed neural stem/progenitor cells in the Pn, thus making obsolete the need for migration to distant target areas. Immunostaining of BrdU-labeled cells with antiHu C/D or anti-S100 antibodies has enabled us to identify over half of the persisting progeny as small interneurons and glia. Surprisingly, among the differentiated cells, there is a clear dominance of astrocytic S1001 glia. This contradicts the widely held view that neurogenesis dominates in neurogenic regions of the adult brain. Indeed, lineage tracing of individual, quiescent, and nestin-expressing radial glia-like precursors in the adult murine dentate gyrus has revealed similar frequencies of astroglial and neuronal generation (Bonaguidi et al., 2011), indicating that previous studies using different approaches may have underestimated the astroglial fate choice of neuronal precursors by one order of magnitude (Steiner et al., 2004). However, as our analysis also has revealed, the final relative numbers of the neurons and glia are not regulated during early stages of development, when these cells start expressing Hu C/D and S100 but during subsequent stages. While similar generation rates were observed initially for both astrocytes and neurons, the dominance of gliogenesis becomes apparent only if later stages of development are examined. At long term survival intervals, the relative number of persisting new Hu C/D1 small interneurons and S1001 astrocytes match the ratio of the total number of neurons to glia in the Pn. It would be interesting to examine whether the production of neurons and glia at the observed ratio is in direct continuation of embryonic and early postembryonic development, or whether this is a phenomenon characteristic of adult stages of development. Despite our intensive search for newly generated pacemaker and relay cells, we have not found any evidence for de novo generation of these two cells types. No BrdU1 pacemaker or relay cells were observed at any post-BrdU survival time examined. Moreover, as the probability distribution of neuronal profile diameters shows, there are no intermediate-sized neurons between the small interneurons, the pacemaker cells, and the relay cells [Fig. 3(B)]. Such size classes, reflecting transitional stages of development, would be expected if pacemaker cells and/or relay cells are Developmental Neurobiology

generated de novo. Furthermore, we observed no net addition of pacemaker or relay cells with increasing size/age of the fish. We, therefore, conclude that the full complement of pacemaker and relay cells is generated during embryonic, or early postembryonic, development, and thereafter the number of these neurons remains stable throughout adult life. However, small interneurons continue to be added during the growth of the fish, as revealed by the similarity of densities observed across individuals of different sizes and ages. Thus, adult neurogenesis in the Pn is restricted to small interneurons. Interestingly, our previous studies have also failed to provide evidence for the generation of large neurons in the adult teleostean brain. For example, in the cerebellum, the relatively small granule cell neurons are generated continuously and in very large numbers, but we have never found any adult-born Purkinje cells (Zupanc et al., 1996, 2005; Teles et al., 2012). A similar exclusive generation of smaller types of neurons appears to be typical of the adult mammalian and avian brains (Altman and Das, 1965; Altman, 1969; Kaplan and Bell, 1984; Luskin, 1993; Barnea and Nottebohm, 1994; Lois and Alvarez-Buylla, 1994). A remarkable exception of this rule exists in the spinal cord of Apteronotus sp., which grows throughout adulthood by adding new cells, including large spinal motoneurons, to its caudal end (Waxman and Anderson, 1985). However, in contrast to all neurogenic regions in the brain, in the growing caudal tip of the spinal cord of these teleost fish all tissue constituents are formed de novo. Thus, the microenvironment in which neurogenesis takes place likely resembles the one encountered during embryogenesis, rather than during adult stages of development. The continuous generation of new neurons and glia, combined with the absence of a detectable degree of cell death, leads to a continuous growth of the Pn. We have estimated that, after reaching sexual maturity, the total number of Pn cells doubles within 2 years. As the new cells are generated throughout the Pn, and there is no indication that they migrate over significant distances away from the sites of their origin, the entire brain nucleus shows a diffuse growth pattern. A somewhat similar pattern has been found in the granule cell layers of the corpus cerebelli and valvula cerebelli pars lateralis of three species of teleost fish (Zupanc et al., 1996, 2005; Teles et al., 2012). However, in each of these two cerebellar subdivisions, the new cells migrate substantially from the proliferation zones in the molecular layers into the associated granule cell layers, where they spread out evenly. A different growth pattern has been observed in the optic tectum of goldfish, where new

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cells are added predominantly at the caudal pole, in a crescent-shaped zone (Meyer, 1978; Raymond and Easter, 1983).

Proposed Function of the Newly Generated Astrocytes The Pn is a well-studied model system in neuroethology, as it plays a crucial role in the neural control of the EOD, including transient EOD modulations, in weakly electric fish (for review, see Dye and Meyer, 1986). While the normal EOD is used for electrolocation, as well as species and sex recognition, transient EOD modulations serve as communication signals in the context of aggressive encounters and courtship (for reviews, see Assad et al., 1999; von der Emde, 1999; Zupanc, 2002; Zupanc and Bullock, 2005, Silva et al., 2008). Models of the Pn have focused exclusively on the oscillatory properties of the neural network composed of pacemaker and relay cells (Moortgat et al., 2000). Virtually nothing is known about the structure and function of glia in the Pn, although their existence has been known since the early morphological description of this nucleus (Elekes and Szabo, 1985). The results of the present investigation highlight the need to include the influence of glia in any future models of the oscillatory activity of the Pn. Our morphometric analysis indicates that S1001 glial cells outnumber neurons more than fourfold, and the pacemaker and relay cells—the only two types of neurons that have been implicated in the generation of the neural oscillations of the Pn thus far—even more than 200-fold. In addition, we have shown that, among the identified persisting new cells, there are almost four times more S1001 glia than Hu C/D1 neurons. As the glia in the Pn express high levels of connexins, which play a critical role in the formation of a glial syncytium (Giaume and Liu, 2012), we hypothesize that the pacemaker and relay cells are embedded in a dense meshwork of astrocytes interconnected by gap junctions to form an astrocytic syncytium. Research in recent years has shown that glia are involved in a multitude of cellular processes in the brain (for review, see Kettenmann and Ransom, 2013). Intriguingly, during high-frequency firing of neurons, astrocytes play an important role in the clearance of K1 ions (Lux et al., 1986; Somjen, 2004). During low-frequency firing, the action of Na1/K1-ATPase is sufficient to ensure the reuptake of K1 ions released from neurons during the generation of action potentials. During high-frequency firing, however, this mechanism appears to be

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insufficient to restore homeostasis, and then astrocytes surrounding the neurons provide a sink for the excess extracellular K1. Within the astrocytic syncytium, K1 is transported from sites of maximal K1 accumulation to remote areas outside the glia where the extracellular K1 concentration has not yet increased. Failure to clear excess K1 from the extracellular space enhances neuronal excitability and is thought to increase the risk of epileptic seizures, which are characterized by high-frequency oscillations in the range of 100–500 Hz in the mammalian brain (Bragin et al., 1999; Jirsch et al., 2006; Pearce et al., 2013). It is reasonable to assume that the challenges caused by excess extracellular K1 ions are much more severe in the Pn of A. leptorhynchus. Ionic and pharmacological manipulations have indicated that the A-type K1 current is a critical element in the regulation of the oscillations generated by the Pn (Dye, 1991). The oscillatory network of pacemaker and relay cells fires at 650–1000 Hz—frequencies markedly higher than those encountered during epileptic seizures. Even more significantly, the high-frequency firing does not occur transiently but continuously throughout life. We hypothesize that the extensive astrocytic syncytium of the Pn plays a critical role in the removal of excess K1 ions accumulating in the extracellular space during such continuous high-frequency firing. The dominance of gliogenesis, compared to neurogenesis, might be part of the mechanism that ensures homeostatic maintenance of K1 ions, and thus, efficient protection of tissue from damage, during the continuous growth of the adult brain. The authors thank Ian M. Traniello for technical assistance.

REFERENCES Altman J. 1969. Autoradiographic and histological studies of postnatal neurogenesis: IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. J Comp Neurol 137:433–458. Altman J, Das GD. 1965. Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J Comp Neurol 124:319–336. Assad C, Rasnow B, Stoddard PK. 1999. Electric organ discharges and electric images during electrolocation. J Exp Biol 202:1185–1193. Balu DT, Lucki I. 2009. Adult hippocampal neurogenesis: Regulation, functional implications, and contribution to disease pathology. Neurosci Biobehav Rev 33:232–252. Developmental Neurobiology

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Barami K, Iversen K, Furneaux H, Goldman SA. 1995. Hu protein as an early marker of neuronal phenotypic differentiation by subependymal zone cells of the adult songbird forebrain. J Neurobiol 28:82–101. Barnea A, Nottebohm F. 1994. Seasonal recruitment of hippocampal neurons in adult free-ranging black-capped chickadees. Proc Natl Acad Sci USA 91:11217211221. Basak O, Taylor V. 2009. Stem cells of the adult mammalian brain and their niche. Cell Mol Life Sci 66:1057– 1072. Bennett MVL. 1971. Electric organs. In: Hoar WS, Randall DJ, editors. Fish Physiology, Vol. 5. Sensory Systems and Electric Organs. New York: Academic Press, pp 347–491. Bessa J, Tavares MJ, Santos J, Kikuta H, Laplante M, Becker TS, Gomez-Skarmeta JL, et al. 2008. meis1 regulates cyclin D1 and c-myc expression, and controls the proliferation of the multipotent cells in the early developing zebrafish eye. Development 135:799–803. Bonaguidi MA, Wheeler MA, Shapiro JS, Stadel RP, Sun GJ, Ming G-l, Song H. 2011. In vivo clonal analysis reveals self-renewing and multipotent adult neural stem cell characteristics. Cell 145:1142–1155. Bonfanti L, Rossi F, Zupanc GKH. 2011. Towards a comparative understanding of adult neurogenesis. Eur J Neurosci 34:845–846. Bragin A, Engel J Jr., Wilson CL, Fried I, Mathern GW. 1999. Hippocampal and entorhinal cortex high-frequency oscillations (100–500 Hz) in human epileptic brain and in kainic acid-treated rats with chronic seizures. Epilepsia 40:127–137. Bylund M, Andersson E, Novitch BG, Muhr J. 2003. Vertebrate neurogenesis is counteracted by Sox1-3 activity. Nat Neurosci 6:1162–1168. Chapouton P, Jagasia R, Bally-Cuif L. 2007. Adult neurogenesis in non-mammalian vertebrates. Bioessays 29: 745–757. Christie KJ, Turnley AM. 2013. Regulation of endogenous neural stem/progenitor cells for neural repair-factors that promote neurogenesis and gliogenesis in the normal and damaged brain. Front Cell Neurosci 6:70. Clasadonte J, Haydon PG. 2012. Astrocytes and epilepsy. In: Noebels JL, Avoli M, Rogawski MA, Olsen RW, Delgado-Escueta AV, editors. Jasper’s Basic Mechanisms of the Epilepsies [Internet]. Bethesda, MD: National Center for Biotechnology Information. de Oliveira-Castro G. 1955. Differentiated nervous fibers that constitute the electric organ of Sternarchus albifrons, Linn. An Acad Bras Cienc 27:557–564. DeMarco V, Dyess K, Strauss D, West CM, Neu J. 1999. Inhibition of glutamine synthetase decreases proliferation of cultured rat intestinal epithelial cells. J Nutr 129:57– 62. Doetsch F, Caille I, Lim DA, Garcıa-Verdugo JM, AlvarezBuylla A. 1999. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97:703– 716. Developmental Neurobiology

Duan X, Kang E, Liu CY, Ming G-l, Song H. 2008. Development of neural stem cell in the adult brain. Curr Opin Neurobiol 18:108–115. Dye J. 1991. Ionic and synaptic mechanisms underlying a brainstem oscillator: An in vitro study of the pacemaker nucleus of Apteronotus. J Comp Physiol A 168:521–532. Dye J, Heiligenberg W. 1987. Intracellular recording in the medullary pacemaker nucleus of the weakly electric fish, Apteronotus, during modulatory behaviors. J Comp Physiol A 161:187–200. Dye JC, Meyer JH. 1986. Central control of the electric organ discharge in weakly electric fish. In: Bullock TH, Heiligenberg W, editors. Electroreception. New York: Wiley, pp 71–102. Ekstr€om P, Johnsson C-M, Ohlin L-M. 2001. Ventricular proliferation zones in the brain of an adult teleost fish and their relation to neuromeres and migration (secondary matrix) zones. J Comp Neurol 436:92–110. Elekes K, Szabo T. 1985. Synaptology of the medullary command (pacemaker) nucleus of the weakly electric fish (Apteronotus leptorhynchus) with particular reference to comparative aspects. Exp Brain Res 60:509–520. Episkopou V. 2005. SOX2 functions in adult neural stem cells. Trends Neurosci 28:219–221. Fawcett JW, Asher RA. 1999. The glial scar and central nervous system repair. Brain Res Bull 49:377–391. Fernandez AS, Rosillo JC, Casanova G, Olivera-Bravo S. 2011. Proliferation zones in the brain of adult fish Austrolebias (Cyprinodontiform[sic]: Rivulidae): A comparative study. Neuroscience 189:12–24. Filippov V, Kronenberg G, Pivneva T, Reuter K, Steiner B, Wang LP, Yamaguchi M, et al. 2003. Subpopulation of nestin-expressing progenitor cells in the adult murine hippocampus shows electrophysiological and morphological characteristics of astrocytes. Mol Cell Neurosci 23:373–382. Floyd CL, Lyeth BG. 2007. Astroglia: Important mediators of traumatic brain injury. Prog Brain Res 161:61–79. Froese R, Pauly D, editors. 2013. FishBase (www.fishbase. org). World Wide Web electronic publication. Gage FH. 2000. Mammalian neural stem cells. Science 287:1433–1438. Gama Salgado JA, Zupanc GKH. 2011. Echo response to chirping in the weakly electric brown ghost knifefish (Apteronotus leptorhynchus): Role of frequency and amplitude modulations. Can J Zool 89:498–508. Gao X, Chen J. 2013. Moderate traumatic brain injury promotes neural precursor proliferation without increasing neurogenesis in the adult hippocampus. Exp Neurol 239: 38–48. Giaume C, Liu X. 2012. From a glial syncytium to a more restricted and specific glial networking. J Physiol Paris 106:34–39. Graham V, Khudyakov J, Ellis P, Pevny L. 2003. SOX2 functions to maintain neural progenitor identity. Neuron 39:749–765. Grandel H, Kaslin J, Ganz J, Wenzel I, Brand M. 2006. Neural stem cells and neurogenesis in the adult zebrafish

Adult Pacemaker Nucleus Development brain: Origin, proliferation dynamics, migration and cell fate. Dev Biol 295:263–277. Griffith OW, Meister A. 1978. Differential inhibition of glutamine and gamma-glutamylcysteine synthetases by alpha-alkyl analogs of methionine sulfoximine that induce convulsions. J Biol Chem 253:2333–2338. Hinsch K, Zupanc GKH. 2006. Isolation, cultivation, and differentiation of neural stem cells from adult fish brain. J Neurosci Methods 158:75–88. Ilies¸ I, Traniello IM, S^ırbulescu RF, Zupanc GKH. Determination of relative age using growth increments of scales as a minimally invasive method in the tropical freshwater Apteronotus leptorhynchus. J Fish Biol, in press, DOI: 10.1111/jfb.12354. Imayoshi I, Sakamoto M, Ohtsuka T, Kageyama R. 2009. Continuous neurogenesis in the adult brain. Dev Growth Differ 51:379–386. Jirsch JD, Urrestarazu E, LeVan P, Olivier A, Dubeau F, Gotman J. 2006. High-frequency oscillations during human focal seizures. Brain 129:1593–1608. Kaplan MS, Bell DH. 1984. Mitotic neuroblasts in the 9day-old and 11-month-old rodent hippocampus. J Neurosci 4:1429–1441. Kaslin J, Ganz J, Brand M. 2008. Proliferation, neurogenesis and regeneration in the non-mammalian vertebrate brain. Phil Trans R Soc B 363:101–122. Kempermann G, Kuhn HG, Gage FH. 1997. More hippocampal neurons in adult mice living in an enriched environment. Nature 386:493–495. Kettenmann H, Ransom BR. 2013. Neuroglia. Oxford/New York: Oxford University Press. Konigsmark BW. 1970. Methods for counting of neurons. In: Nauta WJH, Ebbesson SOE, editors. Contemporary Research Methods in Neuroanatomy. New York: Springer-Verlag, pp 31–40. Kung HN, Marks JR, Chi JT. 2011. Glutamine synthetase is a genetic determinant of cell type-specific glutamine independence in breast epithelia. PLoS Genet 7: e1002229. Laywell ED, Rakic P, Kukekov VG, Holland EC, Steindler DA. 2000. Identification of a multipotent astrocytic stem cell in the immature and adult mouse brain. Proc Natl Acad Sci USA 97:13883–13888. Lindsey BW, Tropepe V. 2006. A comparative framework for understanding the biological principles of adult neurogenesis. Prog Neurobiol 80:281–307. Lois C, Alvarez-Buylla A. 1994. Long-distance neuronal migration in the adult mammalian brain. Science 264: 1145–1148. Luskin MB. 1993. Restricted proliferation and migration of postnatally generated neurons from the forebrain subventricular zone. Neuron 11:173–189. Lux HD, Heinemann U, Dietzel I. 1986. Ionic changes and alterations in the size of the extracellular space during epileptic activity. Adv Neurol 44:619–639. Ma DK, Bonaguidi MA, Ming G-l, Song H. 2009. Adult neural stem cells in the mammalian central nervous system. Cell Res 19:672–682.

951

Manuel M, Price DJ. 2005. Role of Pax6 in forebrain regionalization. Brain Res Bull 66:387–393. Marusich MF, Furneaux HM, Henion PD, Weston JA. 1994. Hu neuronal proteins are expressed in proliferating neurogenic cells. J Neurobiol 25:143– 155. Meyer RL. 1978. Evidence from thymidine labeling for continuing growth of retina and tectum in juvenile goldfish. Exp Neurol 59:99–111. Moortgat KT, Bullock TH, Sejnowski TJ. 2000. Gap junction effects on precision and frequency of a model pacemaker network. J Neurophysiol 83:984–997. Ninkovic J, G€otz M. 2013. Fate specification in the adult brain—Lessons for eliciting neurogenesis from glial cells. Bioessays 35:242–252. Norton WT, Aquino DA, Hozumi I, Chiu FC, Brosnan CF. 1992. Quantitative aspects of reactive gliosis: A review. Neurochem Res 17:877–885. Osumi N, Shinohara H, Numayama-Tsuruta K, Maekawa M. 2008. Concise review: Pax6 transcription factor contributes to both embryonic and adult neurogenesis as a multifunctional regulator. Stem Cells 26:1663–1672. Palmer TD, Markakis EA, Willhoite AR, Safar F, Gage FH. 1999. Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS. J Neurosci 19:8487–8497. Pearce A, Wulsin D, Blanco JA, Krieger A, Litt B, Stacey WC. 2013. Temporal changes of neocortical highfrequency oscillations in epilepsy. J Neurophysiol 110: 1167–1179. Raymond PA, Easter SS Jr. 1983. Postembryonic growth of the optic tectum in goldfish. I. Location of germinal cells and numbers of neurons produced. J Neurosci 3:1077– 1091. Reynolds BA, Weiss S. 1992. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255:1707–1710. Richman PG, Orlowski M, Meister A. 1973. Inhibition of gamma-glutamylcysteine synthetase by L-methionine-Ssulfoximine. J Biol Chem 248:6684–6690. Ronzio RA, Meister A. 1968. Phosphorylation of methionine sulfoximine by glutamine synthetase. Proc Natl Acad Sci USA 59:1642170. Sandhir R, Onyszchuk G, Berman NE. 2008. Exacerbated glial response in the aged mouse hippocampus following controlled cortical impact injury. Exp Neurol 213:372– 380. Sarthy V, Ripps H. 2001. The Retinal M€uller Cell: Structure and Function. New York, NY: Kluwer Academic/ Plenum Publishers. Seri B, Garcıa-Verdugo JM, McEwen BS, Alvarez-Buylla A. 2001. Astrocytes give rise to new neurons in the adult mammalian hippocampus. J Neurosci 21:7153–7160. Silva A, Quintana L, Perrone R, Sierra F. 2008. Sexual and seasonal plasticity in the emission of social electric signals: Behavioral approach and neural bases. J Physiol Paris 102:272–278. Developmental Neurobiology

952

S^ırbulescu et al.

Simpson TI, Price DJ. 2002. Pax6; a pleiotropic player in development. Bioessays 24:1041–1051. S^ırbulescu RF, Zupanc GKH. 2013. Neuronal regeneration. In: Evans DH, Claiborne JB, Currie S, editors. The Physiology of Fishes, 4th ed. Boca Raton: CRC Press, pp 405– 441. Smolen AJ, Wright LL, Cunningham TJ. 1983. Neuron numbers in the superior cervical sympathetic ganglion of the rat: A critical comparison of methods for cell counting. J Neurocytol 12:739–750. Somjen GG. 2004. Ions in the Brain. Oxford/New York: Oxford University Press. Steiner B, Kronenberg G, Jessberger S, Brandt MD, Reuter K, Kempermann G. 2004. Differential regulation of gliogenesis in the context of adult hippocampal neurogenesis in mice. Glia 46:41–52. Teles MC, S^ırbulescu RF, Wellbrock UM, Oliveira RF, Zupanc GKH. 2012. Adult neurogenesis in the brain of the Mozambique tilapia, Oreochromis mosambicus. J Comp Physiol A 198:427–449. Temple S. 2001. The development of neural stem cells. Nature 414:112–117. Turner RW, Moroz LL. 1995. Localization of nicotinamide adenine dinucleotide phosphate-diaphorase activity in electrosensory and electromotor systems of a gymnotiform teleost, Apteronotus leptorhynchus. J Comp Neurol 356:261–274. van Praag H, Christie BR, Sejnowski TJ, Gage FH. 1999a. Running enhances neurogenesis, learning, and long-term potentiation in mice. Proc Natl Acad Sci USA 96: 13427213431. van Praag H, Kempermann G, Gage FH. 1999b. Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat Neurosci 2:266–270. van Praag H, Schinder AF, Christie BR, Toni N, Palmer TD, Gage FH. 2002. Functional neurogenesis in the adult hippocampus. Nature 415:1030–1034. von der Emde G. 1999. Active electrolocation of objects in weakly electric fish. J Exp Biol 202:1205–1215. Walther C, Gruss P. 1991. Pax-6, a murine paired box gene, is expressed in the developing CNS. Development 113:1435–1449. Waxman SG, Anderson MJ. 1985. Generation of electromotor neurons in Sternarchus albifrons: Differences between normally growing and regenerating spinal cord. Dev Biol 112:338–344. Waxman SG, Pappas GD, Bennett MVL. 1972. Morphological correlates of functional differentiation of nodes of

Developmental Neurobiology

Ranvier along single fibers in the neurogenic electric organ of the knife fish Sternarchus. J Cell Biol 53:210– 224. Weisbrod RE, Meister A. 1973. Studies on glutamine synthetase from Escherichia coli: Formation of pyrrolidone carboxylate and inhibition by methionine sulfoximine. J Biol Chem 248:3997–4002. Weissman IL, Anderson DJ, Gage F. 2001. Stem and progenitor cells: Origins, phenotypes, lineage commitments, and transdifferentiations. Annu Rev Cell Dev Biol 17: 387–403. Whitman MC, Greer CA. 2009. Adult neurogenesis and the olfactory system. Prog Neurobiol 89:162–175. Zhao C, Deng W, Gage FH. 2008. Mechanisms and functional implications of adult neurogenesis. Cell 132:645– 660. Zupanc GKH. 2002. From oscillators to modulators: Behavioral and neural control of modulations of the electric organ discharge in the gymnotiform fish, Apteronotus leptorhynchus. J Physiol Paris 96:459–472. Zupanc GKH, Bullock TH. 2005. From electrogenesis to electroreception: An overview. In: Bullock TH, Hopkins CD, Popper AN, Fay RR, editors. Electroreception. New York: Springer, pp 5–46. Zupanc GKH, Hinsch K, Gage FH. 2005. Proliferation, migration, neuronal differentiation, and long-term survival of new cells in the adult zebrafish brain. J Comp Neurol 488:290–319. Zupanc GKH, Horschke I. 1995. Proliferation zones in the brain of adult gymnotiform fish: A quantitative mapping study. J Comp Neurol 353:213–233. Zupanc GKH, Horschke I, Ott R, Rascher GB. 1996. Postembryonic development of the cerebellum in gymnotiform fish. J Comp Neurol 370:443–464. Zupanc GKH, Ilies¸ I, S^ırbulescu RF, Zupanc MM. Largescale identification of proteins involved in the development of a sexually dimorphic behavior. J Neurophysiol, in press, DOI: 10.1152/jn.00750.2013. Zupanc GKH, S^ırbulescu RF. 2011. Adult neurogenesis and neuronal regeneration in the central nervous system of teleost fish. Eur J Neurosci 34:917–929. Zupanc GKH, S^ırbulescu RF. 2013. Teleost fish as a model system to study successful regeneration of the central nervous system. Curr Top Microbiol Immunol 367:193–233. Zupanc GKH, S^ırbulescu RF, Ilies¸ I. 2012. Radial glia in the cerebellum of adult teleost fish: Implications for the guidance of migrating new neurons. Neuroscience 210: 416–430.