Adult neurogenesis in the short-lived teleost Nothobranchius furzeri: localization of neurogenic niches, molecular characterization and effects of aging

We studied adult neurogenesis in the short-lived annual fish Nothobranchius furzeri and quantified the effects of aging on the mitotic activity of the neuronal progenitors and the expression of glial fibrillary acid protein (GFAP) in the radial glia. The distribution of neurogenic niches is substantially similar to that of zebrafish and adult stem cells generate neurons, which persist in the adult brain. As opposed to zebrafish, however, the N. furzeri genome contains a doublecortin (DCX) gene. Doublecortin is transiently expressed by newly generated neurons in the telencephalon and optic tectum (OT). We also analyzed the expression of the microRNA miR-9 and miR-124 and found that they have complementary expression domains: miR-9 is expressed in the neurogenic niches of the telencephalon and the radial glia of the OT, while miR-124 is expressed in differentiated neurons. The main finding of this paper is the demonstration of an age-dependent decay in adult neurogenesis. Using unbiased stereological estimates of cell numbers, we detected an almost fivefold decrease in the number of mitotically active cells in the OT between young and old age. This reduced mitotic activity is paralleled by a reduction in DCX labeling. Finally, we detected a dramatic up-regulation of GFAP in the radial glia of the aged brain. This up-regulation is not paralleled by a similar up-regulation of S100B and Musashi-1, two other markers of the radial glia. In summary, the brain of N. furzeri replicates two typical hallmarks of mammalian aging: gliosis and reduced adult neurogenesis.


Introduction
The continued production of neurons throughout adulthood, in at least in some circumscribed brain regions, has been observed in all vertebrate species so far studied. In mammals, adult neurogenesis is restricted to two principal stem cell niches in the telencephalon: the subependymal zone (or subventricular zone, SVZ) which generates GABAergic and dopaminergic interneurons of the olfactory bulb and the subgranular zone (SGZ) of the dentate gyrus. These areas contain adult neuronal stem cells (aNSCs) of glial phenotype, which are able to self-renew. aNSCs generate an intermediate precursor, which gives rise to a transient amplifying neuroblast and finally to neurons (Gage et al., 2008). These different stages can be identified by a combination of morphological criteria and the expression of molecular markers. For example, aNSCs in the hippocampus are characterized by the expression of SOX2, glial fibrillary acid protein (GFAP), and activated Notch signaling (Lugert et al., 2010), whereas the amplifying neuroblast expresses a combination of PSA-NCAM, doublecortin (DCX), and the mitotic marker Ki-67 (Gage et al., 2008). Doublecortin expression (but not expression of mitotic markers) persists for some time in young terminally differentiated neurons (Brown et al., 2003). Neurons generated during adulthood are functional, integrate into existing circuits, and are proposed to be involved into some specific aspects of behavioral plasticity (van Praag et al., 2002;Gage et al., 2008).
Adult neurogenesis is a highly plastic process, which can be enhanced by environmental stimulation and physical exercise (Kempermann et al., 1997;van Praag et al., 1999) and it is modulated by epigenetic mechanisms, microRNAs (Cheng et al., 2009;Yoo et al., 2009;Ma et al., 2010), and growth factors.
Neurogenesis in the SGZ decreases exponentially with age in rodents (Kuhn et al., 1996;Ben Abdallah et al., 2010), dogs (Pekcec et al., 2008), and humans (Knoth et al., 2010). This decay is initiated during early postnatal life, and, in adults, neuronal production in the SGZ is < 10% of what observed during puberty (Ben Abdallah et al., 2010;Knoth et al., 2010). Age-dependent decrease in neurogenesis is observed in SVZ as well, but it is less dramatic than in the SGZ (Luo et al., 2006).
Decreased adult neurogenesis is the consequence of quiescence of aNS-Cs, which are still present, but not active, in the old brain and can be reactivated by physiological and pathological stimuli (Lugert et al., 2010).
In contrast to mammals, adult neurogenesis is extensive in teleost fish and is observed basically in all brain regions (Zupanc & Horschke, 1995;Ekstrom et al., 2001). Systematic anatomical studies in zebrafish and medaka (Adolf et al., 2006;Grandel et al., 2006;Kuroyanagi et al., 2010) have identified at least 16 different neurogenic niches. In fish, aNS-Cs are mostly associated with the ventricular system. In the fish telencephalon, aNSCs have the typical morphology of radial glia and share several molecular markers of mammalian aNSCs (Ganz et al., 2010;Marz et al., 2010). A pallial and subpallial neurogenic niche can be identified in fish and these are homolog to the SGZ and SVZ of mammals (Adolf et al., 2006;Mueller & Wullimann, 2009). Analysis of viral tracing has directly demonstrated that adult radial glial is self-renewing and pluripotent, proving its stem cell identity (Rothenaigner et al., 2011). aNSCs in the optic tectum (OT) (Ito et al., 2010) and cerebellum (Kaslin et al., 2009) are not of glial nature and express neuroepithelial markers.
Studies on zebrafish used young adults of age 3-6 months and maximum reported lifespan for this species is 66 months (Gerhard et al., 2002) and there are no quantitative reports on age-dependent modulation of adult neurogenesis. As fish show substantial growth during adult life, it is unclear to what extent adult neurogenesis is responsible for neuronal turnover or rather reflects addition of new cells in the context of continuous brain growth. Gliosis, measured as up-regulation of GFAP in astroglia, is a well-described hallmark of mammalian aging (Riddle, 2007;Park et al., 2009). In fish, however, radial glia is the predominant glia type (Cuoghi & Mola, 2009) and retains its neurogenic function in adulthood. It is therefore of interest to explore whether also radial glia undergoes gliosis as function of age.
To investigate the effects of aging on adult neurogenesis in teleosts, we used a novel animal model: the short-lived fish Nothobranchius furzeri. This species has a captive lifespan of 3-7 months depending on the strain (Terzibasi et al., 2008) and it is an emerging model organism for biological investigations into aging Terzibasi et al., 2009;Graf et al., 2010;Di Cicco et al., 2011;Hartmann et al., 2011). In particular, we demonstrated that N. furzeri undergoes agedependent neurodegeneration and cognitive impairments (Valenzano et al., 2006a,b;Terzibasi et al., 2009), which in mammals often correlate with reduced adult neurogenesis (Gage et al., 2008).
Aims of the present paper were the following: 1 To localize neurogenic niches in the adult brain of N. furzeri.
2 To investigate the expression of conserved molecular markers of neurogenesis in this species. 3 To quantify the effects of age on adult neurogenesis in the OT. 4 To study the effects of aging on the radial glia.

Results
Distribution of neurogenic niches in the brain of Nothobranchius furzeri

Whole mounts
To label mitotically active cells, we injected the nucleotide analog 5-ethynil-2¢deoxyuridin (EdU). Whole-mount detection of Edu+ cells showed a widespread distribution in the brain of young (5-7 weeks old) subjects 4 h after injection ( Fig. 1A-E). We concentrated on those areas located in telencephalon, OT, and cerebellum, which were subject of in-depth studies in zebrafish and medaka (Adolf et al., 2006;Kaslin et al., 2009;Alunni et al., 2010;Ito et al., 2010).
In the telencephalon, three distinct regions can be recognized: A niche of tightly packed high proliferating cells, which starts quite sharply at the rostro-ventral conjunction of the two telencephalic hemispheres region I). This region has a subpallial origin, is homologous to the SVZ of mammals (Mueller & Wullimann, 2009), and is observed in the telencephalon of other teleost species (Zupanc & Horschke, 1995;Ekstrom et al., 2001;Adolf et al., 2006;Grandel et al., 2006;Alunni et al., 2010;Kuroyanagi et al., 2010). Neurogenic cells are also scattered on the dorsal and lateral surfaces of the telencephalon in various teleost species. Because of developmental eversion of the telencephalic vesicles, this region of pallial origin is facing the third ventricle and it is proposed to be homologous to the subgranular neurogenic niche of mammals (Mueller & Wullimann, 2009). In N. furzeri, the pallial neurogenic region can be subdivided into two different areas region II and III (Fig. 1B,D): Region II is placed dorsally and rostrally and shows a much lower level of proliferation as compared to region I; region III is placed ventro-caudally and shows a level of proliferation intermediate between regions I and II. Region II and region III are separated by a ventro-lateral patch of telencephalic surface devoid of proliferative cells (Fig. 1D, indicated with *). This area is observed also in zebrafish and is suggested to correspond to the pial rather than the ventricular surface (Mueller & Wullimann, 2009). Proliferation in region III is concentrated on its ventro-caudal portion (Fig. 1B,D arrowhead). An area of high proliferative activity is observed in the preoptic segment of the ventricular system (region IV, Fig. 1C,E). Intense neurogenic activity was observed in the dorsomedial, posterior, and ventral margins of the OT (regions V and VI; Fig. 1A,C). A clearly visible neurogenic niche is located along the midline surface of the cerebellum (Fig. 1A, region VII). Further distinct niches visible in whole-mount preparations are located at the caudal margin of the cerebellum (Fig. 1A, region VIII) and lining the margin of the fourth ventricle, (Fig. 1B, region IX).
Sagittal overviews of the N. furzeri brain labeled with EdU are shown in Fig. S1 (Supporting Information).

Telencephalon
Double-labeling with EdU and the proliferation marker proliferating cell nuclear antigen (PCNA) was performed in coronal and horizontal sections of 5-to 7-week-old subjects 4 h after injection Figs S2 and S3). Neurogenic niches identified by the presence of EdU+ cells always correspond to regions labeled with PCNA. In telencephalon, regions I, II, and III can be readily appreciated in horizontal and coronal sections. Region I (Fig. 1I) extends from the niche visible in whole-mount preparations along the third ventricle in caudal direction. Analysis of sections confirms the presence of a region II (Fig. 1G) and a region III. The concentration of dividing cells in the caudal margin of the telencephalon in region III is clearly visible in horizontal sections (Fig. 1F, white arrowhead). In zebrafish, the pallial neurogenic niche contains slow-cycling precursors and the subpallial niche contains faster-dividing precursors (Adolf et al., 2006;Ganz et al., 2010). These can be distinguished based on the percentage of colocalization between PCNA and EdU. High colocalization is diagnostic of fast cycle (Adolf et al., 2006). Conventional microscopy is suggestive of a much higher co-localization in the subpallial area I

Ventricular system
Caudal to region IV in the preoptic region, neurogenic niches were observed widespread along the ventricle, in the hypothalamus and caudally to the medulla oblongata and spinal cord (e.g. Fig. 1M). We do not attempt here to subdivide this into more defined areas.

Optic tectum
Region V corresponds to the medial margin of the OT and is placed at the border with the torus longitudinalis. Proliferating cells are present on both sides of this border in the OT and the torus longitudinalis. Region V shows a typical U-shape in coronal sections (Fig. 1M). Region VI corresponds to the posterior and ventral margins of the OT (Fig. 1H,N). Scattered Edu+, PCNA-positive cells could be detected in the subventricular gray zone of the OT (Figs 1N¢¢ and 5Q).

Cerebellum
Region VII represents the cerebellar neurogenic niche. This niche was characterized in detail in zebrafish (Kaslin et al., 2009). In N. furzeri, region VII has the shape of a dorsal and a ventral wedge of highly active cells connected by a rod of dividing cells in the valvula cerebelli ( Fig. 1N,N¢). The size of the dorsal wedge increases and that of the ventral wedge decreases in caudal direction in corpus cerebelli and in the most posterior portion only the dorsal wedge is visible.
A complete rostro-caudal series of coronal sections to better appreciate the full extent of these neurogenic niches is presented in Fig. S2 (Supporting Information).

Newly generated cells are long-living and differentiate into neurons
Long-term survival To prove the persistence of cells generated during adult life, we analyzed animals 5 weeks after a single injection of EdU. Median lifespan for the MZM-04 ⁄ 10 strain is 27 weeks in our fish colony , so this interval covers a significant proportion of the adult life for this species. In all brain regions, EdU-labeled cells persisted and were found into the brain parenchyma at some distance from the neurogenic niches of origin visualized with PCNA (Fig. 2). This is best appreciated in the OT posterior margin, where the newly generated cells from a dense strip (Fig. 2F,H-M). The distance between this EdU+ strip (red arrows in Fig. 2H,I) and the PCNA+ germinal zone (white arrowhead in Fig. 2H,I) represents the marginal growth of the OT within this period. In the cerebellum, neurogenesis is extensive and cells generated during adulthood migrate in the granule cell layer ( Fig. 2H-I,M-N). At visual inspection, it seems that more cells are generated and integrated into the cerebellum than in any other region of the N. furzeri brain, in line with the extensive cerebellar neurogenesis reported in other fish species (Zupanc & Horschke, 1995;Ekstrom et al., 2001;Adolf et al., 2006;Kaslin et al., 2009;Kuroyanagi et al., 2010).

Neuronal differentiation
To study the fate of the newly generated cells, we sacrificed animals 1 week after a single injection of EdU and performed a double-labeling with the neuronal marker HuC ⁄ D (Marusich et al., 1994). Sections were then analyzed by confocal microscopy. As expected, extensive co-localization was observed in telencephalon, OT, and cerebellum (Fig. S4).

Molecular characterization of the neuronal precursors
MicroRNA expression MicroRNAs are emerging as important regulators of neurogenesis and in particular miR-9 and miR-124 (Leucht et al., 2008;Cheng et al., 2009;Yoo et al., 2009). miR-124 is expressed by differentiated neurons while miR-9 was reported, in zebrafish, to be up-regulated in the regions where adult neurogenesis occurs (Kapsimali et al., 2007).
MicroRNAs are extremely conserved in their sequence. A preliminary study of small RNA sequencing (M. Baumgart, M. Grothe, M. Platzer, A. Cellerino, paper in preparation) revealed that the sequences of mature miR-124 and miR-9 in N. furzeri are identical to the sequences of Danio rerio. We therefore used locked nucleic acid (LNA) in situ probes designed for D. rerio. miR-124 expression was detected in nearly all neurons throughout the adult brain, but not in the neurogenic niches. This was particularly evident in the subpallial area I of the telencephalon (Fig. 3A,B). The expression pattern of miR-9 in the telencephalon was complementary to that of miR-124 and was concentrated in the neurogenic niches (Fig. 3C,D). Confocal microscopy (Fig. 3E,F) revealed partial colocalization of miR-9 and EdU 4 h postinjection, indicating that miR-9 is expressed in mitotically active neuronal progenitors.
In the OT, expression of miR-124 ( Fig. 3G) was lower in the neurogenic niche (arrow) and in the neighboring area that contains newly differentiated cells. Expression of miR-124 was specifically reduced in a strip of cells in the most medial portion of the OT (arrowheads). This area corresponds to the location of radial glia (Fig. 5, see below), where expression of miR-9 is particularly high (Fig. 3H). miR-9, however, was not up-regulated in the germinal layer of the OT (arrow), indicating that miR-9 is not associated with neuroepithelial progenitors. Single-channel visualizations of in situ hybridization for miR-124 and miR-9 in the telencephalic structure (Fig. 3B,D,F) are shown in Fig. S6 (Supporting Information, A ⁄ B, D ⁄ E, G ⁄ H, respectively).

Doublecortin
In mammals, neuroblasts and newly generated neurons express DCX (Brown et al., 2003). Because of excellent labeling of neuronal processes, this marker is widely used to quantify neurogenesis (Pekcec et al., 2008;Ben Abdallah et al., 2010;Knoth et al., 2010). A DCX ortholog is present neither in the assembled zebrafish genome nor in the collections of zebra-fish expressed sequence tags. We recovered a transcript, which we call NfuDCX, in a database of N. furzeri  . NfuDCX shows 86.5% aminoacidic sequence similarity to mouse DCX (Data S2). To confirm that NfuDCX is the ortholog of DCX, we performed a phylogenetic analysis aligning all DCX and DCX-like sequences present in the ENSEMBL database. NfuDCX clusters with all other DCX sequences from other vertebrate species and not with DCX-like kinase 1, or DCX-like kinase 2 (Fig. S5). This analysis also revealed that DCX is present in the genomes of the Actynoptegyian fishes Gastrosteleus aculeatus (stickleback), Tetraodon nigroviridis, and Takifugu rubipes (pufferfish) (Table S1, Fig. S5).
Immunolabeling with DCX in the telencephalon (Fig. 3I-M, green) revealed a strip of strongly labeled cells 3-4 cell diameters in distance from the telencephalic surface that is adjacent, but does not overlap, with the ribbon of PCNA+ progenitors (Fig. 3I-M,red). Double-labeling with PCNA and DCX in the OT (Fig. 3N-O, PCNA red and DCX green) and the retina (Fig. 3P-Q, PCNA green and DCX red) revealed that a column of processes labeled for DCX can be found juxtaposed to the germinal margins of active neurogenesis. One week after EdU injections, the column of DCX-labeled processes in the OT (Fig. 3R, red) was in register with the front of EdU+ cells close to the germinal margin (Fig. 3R, green). Two weeks postinjection, the column of DCX-labeled process is found more medial with respect to the strip of EdU+ cells. In the telencephalon, the timing of DCX expression was slower. Only few cells were double-labeled with DCX (red) and EdU (green) 1 week after injection (data not shown), but double-labeling of EdU and DCX is observed 2 weeks after injection ( Fig. 3S-T).
It should be remarked that DCX does not represent a marker of newly generated neurons throughout the brain. Doublecortin expression was not observed in association with the subpallial neurogenic niche (area I) (Fig. 3L), and strong DCX labeling was observed in the olfactory nerve, in fibers entering the olfactory bulb (data not shown) and in fibers entering the medial portion of the OT (Fig. 4H,N).

Neurogenesis
To quantify the effects of age on neurogenesis, 25-week-old animals were analyzed 4 h after EdU injection and compared to 11-week-and 5week-old animals. Neurogenic niches are still present in old fish, but the mitotic activity in telencephalon and OT is visibly reduced (Fig. 4A,B). We quantified the number of Edu+ cells in the germinal layer of the OT using unbiased stereological estimates (see Experimental procedures). A significant age-dependent reduction in proliferating cells was detected (Fig. 4C, ANOVA, P < 0.0001). The absolute number of EdU+ cells monotonically decreased between 5, 11, and 25 weeks. Reduced neurogenesis resulted also in visibly reduced DCX staining in the telencephalon (Fig. 4I-L) and on the posterior margin of the OT (Fig. 4M) of 25-week-old fish when compared to 5-week-old fish (Fig. 4E-G). No differences were seen in the central region of the OT (Fig. 4H-N), where DCX expression is not associated with neurogenesis.
Comparison of neurogenesis decay with somatic growth reveals that there is no direct relationship between these two processes (Fig. 4D). Indeed, neurogenesis is already reduced at age 11 weeks, when fish are still in the growing phase, and it is present -even if reduced -at 25 weeks of age, when the fish have practically ceased to grow.
Gliosis, Musashi-1, S100B, and GFAP Age-dependent up-regulation of GFAP is a well-characterized hallmark of aging in mammals (Riddle, 2007;Park et al., 2009). We therefore studied age-dependent regulation of glial markers in N. furzeri.
The expression of glial markers in telencephalic aNSCs was studied in great detail in zebrafish (Ganz et al., 2010;Marz et al., 2010). In zebrafish, pallial aNSCs, but not the aNSCs in the subpallial region, expresses the typical glial markers S100B and GFAP. Cells on the germinal zone of the OT do not express glial markers and have a neuroepithelial phenotype (Ito et al., 2010).
Glial fibrillary acid protein is not detected in the telencephalon of young N. furzeri. Only faint labeling of GFAP+ radial fibers is observed in the OT of young N. furzeri (Fig. 5A). S100B is expressed in the neurogenic telencephalic areas II and III in cells with the typical morphology of radial glia, but not in the subpallial area I (Fig. 5I-L, Fig. S8). In the OT, S100B marks the radial glia, which constitutes the most medial margin of the OT and is excluded from the germinal layer (Fig. 5M, Fig. S8). Double-labeling with EdU 4 h postinjection and confocal analysis revealed a partial colocalization, demonstrating that at least some S100B+ cells in the pallium are mitotically active (Fig. 5L white arrows, Fig. S8).
Mushasi-1 (Msi-1) is an RNA-binding protein necessary for neurogenesis and enriched in aNSC (Sakakibara et al., 1996). The expression domain of S100B largely overlaps with that of Msi-1. Msi-1, however, also labels the germinal layer of the subpallial neurogenic niche and OT (Fig. 5N-Q), which are not labeled for S100B. At a cellular level, confocal analysis revealed complete co-localization between Msi-1 and Edu 4 h postinjection, revealing that Msi-1 labels both the radial glia and the amplifying neuroblasts ( Fig. 5N-Q, Fig. S8). This analysis also revealed that scattered EdU+ cells in the OT are Msi-1+ (Fig. 5Q).
A dramatic up-regulation of GFAP immunoreactivity is observed in the OT of 25-week-old fish (Fig. 5D) when compared to 5-week-old fish (Fig. 5A). A dense network of fibers running tangentially to the medial margin of the OT is observed, and also, radial processes become strongly positive. These changes are not accompanied by upregulation of S100B (Fig. 5B,E) or Msi-1 (Fig. S5), although GFAP immunoreactivtiy co-localizes with S100B (Fig. 5C,F) and Msi-1 (Fig. S7).
Quantification of GFAP ⁄ S100B ratio revealed a $ 20-fold increase between 5-weeks and 25-weeks of age (Fig. 5G). Up-regulation of GFAP mRNA in the entire brain could also be detected by real-time PCR (Fig. 5H). The panel on the right represents the growth curve of N. furzeri males. The analyzed age-steps for the quantification are marked by red arrows. Forty-five animals were measured to produce this curve. Error bars represent standard errors of means. Scale bar for magnification panels (A¢, A¢¢, B¢¢) is the same, as indicated in panel B¢¢. (E-N) Comparison of doublecortin (DCX) expression (green) in telencephalic and tectal areas of young (5-week-old) vs. old (25-week-old) N. furzeri brain; confocal images (projection of seven optical sections for 6 lm thickness) A. (D) Representation of the telencephalic subpallial (E) and pallial (F) neurogenic niches, and the germinal layer (G) and central region (H) of the OT of a 5-week-old brain: DCX+ processes are present in all telencephalic and tectal margins, as well as in the central tectum. Representation of the subpallial (I) and pallial (L) neurogenic niches of the telencephalon, and the germinal layer (M) and central region (N) of the OT of a 25-week-old brain: DCX+ processes are clearly reduced in the neurogenic areas (I-M) when compared to the corresponding regions (E-G) in the 5-week-old brain. On the other hand, DCX staining in the central tectum shows similar pattern and intensity at both ages and can be regarded as a control for labeling quality.

Discussion
In the present paper, we analyzed the niches of adult neurogenesis in N. furzeri. We found that the neurogenic niches have a similar distribution to that described in detail in zebrafish. In particular, in the telencephalon of both species, there is a clear distinction between a less active pallial and a more active subpallial niche (Adolf et al., 2006;Ganz et al., 2010;Marz et al., 2010). The pallial zone contains slow-cycling mitotically active radial glia positive for S100B, while the subpallial zone contains fast-cycling aNSCs lacking S100B labeling.
Organization of stem cell niches in the OT of N. furzeri is also conserved and almost identical to what described in medaka (Alunni et al., 2010;Ito et al., 2010): a clearly defined, continuous, and very active germinal layer is observed in the lateral, posterior, and inferior border of the OT. Progenitors in the OT are not labeled with S100B antibodies. In addition, a sparse neurogenic activity can be observed throughout the medial margin of the OT. This position corresponds to the location of the radial glia in the OT and double-labeling of EdU, and Msi-1 indicates that also the radial glia in the OT is mitotically active, although at a much lower level as compared to the telencephalon. This is a very similar situation to what described in the retina, where there is a neuroepithelial ciliary marginal zone which can produce all cells with exception of rods and neurogenic Mü ller glia which produces only rods. It remains to be investigated whether radial glia in the OT produces a specific neuronal type or not.
We further characterized the neurogenic niches in telencephalon and OT to define expression of conserved molecular markers. The microRNAs, miR-9 and miR-124, are known to control neurogenesis and neuronal differentiation (Leucht et al., 2008;Cheng et al., 2009;Yoo et al., 2009;Bonev et al., 2011). Here, we report that miR-9 is highly expressed in the radial glia in telencephalon and OT but not in the neuroepithelial precursors of the OT. miR-124, on the other hand, is specifically excluded in neuronal precursors and newly generated neurons. This result is in line with the expression domains reported in adult zebrafish brain (Kapsimali et al., 2007) and suggests that miR-9 controls the activity of the neurogenic radial glia, while miR-124 is necessary for differentiation of postmitotic neurons. The RNA-binding protein Musashi-1 (Msi-1) was found to be expressed in all neurogenic niches in telencephalon and OT and its expression was not restricted to radial glia. All cells labeled with EdU after short intervals were found also to be positive for Msi-1; therefore, this essential neurogenic gene is expressed both in the aNSCs and in the amplifying progenitors in N. furzeri.
An interesting finding was the identification of DCX in N. furzeri. This gene is necessary for adult neurogenesis in mammals (Jin et al., 2010) and is the marker of choice to label newly generated neurons (Brown et al., 2003), but it is absent from the zebrafish genome. In N. furzeri, DCX labels newly generated neurons in pallium and OT and this strongly suggests a conservation of DCX function between mammals and Actinopterygian fishes. Doublecortin expression in N. furzeri is dramatically down-regulated with age, as in all mammalian species studied thus far (Gage et al., 2008;Pekcec et al., 2008;Ben Abdallah et al., 2010;Knoth et al., 2010).
The most important finding of the present paper is the demonstration of a strong age-dependent regulation of neurogenesis in N. furzeri. This age-dependent regulation does not directly correlate with somatic growth and was observed already during early adult life, as observed in mammals (Pekcec et al., 2008;Ben Abdallah et al., 2010;Knoth et al., 2010).
Gliosis is a widespread phenomenon in the aging mammalian brain, which is easily detected as up-regulation of GFAP (Riddle, 2007;Park et al., 2009). Aging-dependent gliosis was not investigated in any fish species so far. We could detect massive gliosis in the radial glia of the OT as a consequence of aging in N. furzeri. Gliosis is the typical response of the brain to injury and is observed in many neurodegenerative diseases. Age-dependent gliosis is therefore a further indication of age-dependent neurodegeneration in N. furzeri. This increase in GFAP is not paralleled by a similar dramatic increase in S100B or Msi-1, indicating that the number of radial glia does not change dramatically but these cells up-regulate GFAP expression.
In summary, we have shown conserved aspects of adult neurogenesis in N. furzeri both in the location of neurogenic niches and in the expression of molecular markers. More importantly, we report that N. furzeri shows age-dependent decrease in neurogenesis and age-dependent gliosis. Intervention studies in aging mammals are associated with high costs and long experimental times. Our data qualify N. furzeri as an alternative model organism to study mechanisms underlying age-dependent regulation of neurogenesis and gliosis.

Experimental procedures
Detailed experimental procedures are provided as Data S1.

Animal experimentation
All experiments were performed on group-house N. furzeri of the MZM-04 ⁄ 10 strain. Details can be found in Terzibasi et al. (2009). The protocols of fish maintenance were approved by the local authority in the State of Thuringia (Veterinä r-und Lebensmittelü berwachungsamt).

Immunohistochemistry and in situ hybridization
Immunohistochemistry was performed using commercially available antibodies (Table S2). In situ hybridization was performed using LNA probes labeled with DIG at 5¢ and 3¢ at an hybridization temperature of 42°C. EdU (50 lL of a 10 lM solution) was injected intraperitoneally and detected according to manufacturer's instruction (Invitrogen, Grand Island, NY, USA).

Cloning of GFAP and quantitative PCR
Glial fibrillary acid protein (Data S3) was cloned using Rapid Amplification of cDNA Ends (RACE; Clonthech, Mountain View, CA, USA), and expression levels were quantified using the Qiagen protocols on a Rotorgene (Qiagen, Hilden, Germany).

Unbiased estimates of cell numbers
To quantify EdU+ cells, brains were serially sectioned and every third section was analyzed. To count cells, two confocal planes at a distance of 4 lm were superimposed and cells present in both planes were excluded. Unbiased cell estimates were so derived: Let the ordered series of sections for a specimen be SðnÞ; n 2 ½1; Q, and let the sampling density be 1 ⁄ X, where Q = M · X, then a random number R; R 2 ½0; X À 1 is extracted and every section Sðn Â X þ RÞ; n 2 ½1; M is analyzed. Then, the unbiased estimate of cell numbersÑ can be calculated by the formulã

Supporting Information
Additional supporting information may be found in the online version of this article:          Data S1 Experimental procedures.
Data S2 Alignment of NfuDCX with mouse DCX.
Data S3 Alignment of NfuGFAP with mouse GFAP.
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