Thirteen adult specimens of G. omarorum (Richer-de-Forges et al., 2009; 8 males, 2 females and 3 of non-determined sex; weight: 6.26 ± 2.94; total fish length: 12.23 ± 2.04 mean ± SD) were collected from Laguna del Cisne, Uruguay (latitude 35°50 S, longitude 55°08 W). According to the measured length of the specimens, which is about half of the maximal length (Richer-de-Forges et al., 2009), we estimate that most of the fish used in this study have already reached the adult period, corresponding to the first gonadal maturation, at the age of 1 year (Balon, 1975; Barbieri and Cruz, 1983).

Macroscopic gonadal morphology was confirmed in 10 of the specimens studied. Animals were kept in individual tanks on a 12 h:12 h light: dark cycle and daily fed with Tubifex tubifex. Water conductivity was adjusted to 200 μs and temperature maintained at 20–25°C.

All procedures were performed in accordance to the guidelines of CHEA (Comisión Honoraria de Experimentación Animal, ordinance number: 4332-99, Universidad de la República). Experiments were approved by the Animal Ethics Committee of the Instituto de Investigaciones Biológicas Clemente Estable (protocol number 010/09/2011).

For the application of Neurobiotin tracer (Vector Laboratories, Burlingame, CA, USA) in the corpus cerebelli (CCb), animals were anesthetized by immersion in 3-aminobenzoic acid ethyl ester (MS-222, Sigma-Aldrich, St Louis, MO, USA, at a dose of 120 mg/l) maintained by gill perfusion of the same anesthetic solution during surgery.

For transcardial fixation, fish were deeply anesthetized by immersion in MS-222 (500 mg/l, Sigma-Aldrich) followed by gill perfusion of the same anesthetic solution.

Animals were first perfused with 20–30 ml of saline (0.7% NaCl solution) in order to wash blood out from the circulatory system, followed by 10% paraformaldehyde (Sigma-Aldrich) dissolved in phosphate buffer 0.1 M, pH 7.4 (PB; Carlo Erba, Val de Reuil, France). Brains were dissected out and post fixed in the same fixative solution for 24 h at 4°C. After embedding the brains in a gelatin/albumin (Sigma-Aldrich) mixture denatured with glutaraldehyde (Sigma-Aldrich), frontal serial sections (60 μm) were obtained with a vibratome (Leica VT1000S, Wetzlar, Germany) and serially collected in 24 or 48 multiwell plates.

In many cases, sections from most brain regions were processed in parallel, though we focused on certain rostral-caudal levels. At these levels, long distance and/or high pace of migration of newborn cells were previously evidenced (Olivera-Pasilio et al., 2014): the medial (L1) and caudal region (L2) of OB, corresponding to plates 1 of Gymnotus carapo atlas (Corrêa et al., 1998) and plate 38 of A. lepthorhynchyus atlas (Maler et al., 1991); the rostral region of the subpallium (L3) that corresponds to plates 2 and 32 of the mentioned atlases, respectively; the medial region and caudal pole of the midbrain TeO and torus semicircularis (TS; L4, and L5), the rostral pole of the rhombencephalic corpus cerebelli (CCb; L5), and the caudal pole of the rhombencephalic electrosensory lateral line lobe (ELL; L6; Figure 1).

To reveal the structural organization of the brain regions under analysis, mainly those where newborn neurons migrate, some sections were counterstained with DAPI (Sigma) or ToPro3 (Molecular Probes, Grand Island, NY, USA) after immunohistochemistry. Also, serial sections of control animals (N = 2) were counterstained with fluorescent Nissl staining using Neuro Trace® (Molecular Probes) as described below (Supplementary Figure 1) and βIII-tubulin, HuC/HuD or tyrosine hydroxylase (TH; Supplementary Figure 2). For fluorescent Nissl counterstaining, free floating brain sections of a control animal were stained with Neuro Trace® at 1/150 dilution in PB for 20 min at room temperature and rinsed in PB with 0.1% Triton X-100 (10 min) followed by PB (3 × 5 min). For ToPro3 counterstaining, brain sections were immersed in ToPro3 for 10 min at a 1/4,000 dilution, followed by rinsing in PB (3 × 5 min). All sections were mounted in a sealing and anti-fading coverslipping solution containing polyvinyl alcohol (PVA, Sigma) and 1,4 diazabicyclo [2.2.2]octane (DABCO, Sigma) and prepared according Peterson lab protocol (http://neurorenew.com/wp-content/uploads/2014/12/pva2.pdf).

Proliferating cells were labeled with 2.3 mg/ml 5-Chloro-2′-deoxyuridine thymidine (CldU, Sigma-Aldrich) dissolved in 0.7% sodium chloride, and administered i.p. at 20 μl/g of body weight, according to two procedures: (1) pulse administration: a single injection followed by post-thymidine analog survivals of 1 (N = 3), 7 (N = 2), or 30 days (N = 3); and (2) sequential administration: four daily injections followed by survivals of 90 (N = 2) or 180 days (N = 1).

To reveal the differentiation of cerebellar newborn cells into the neuronal phenotype, Neurobiotin tracer (Vector Laboratories, Inc., Burlingame, CA, USA) was applied in vivo at the molecular layer of the Cb, 86 or 176 days after repetitive CldU administration. Animals were anesthetized and the soft tissue over the rostralmost portion of the sagittal suture was carefully removed with the tip of a scalpel (Supplementary Figure 3A). Then, a small perpendicular and superficial incision was made in order to disrupt this portion of the sagittal suture and expose the dorsal surface of the rostral portion of CCb. After slightly cutting the cerebellar surface, crystals of Neurobiotin were applied with the tip of a needle (Supplementary Figure 3B). Finally, the wound was sealed with Histoacryl® (B. Braun Aesculap AG, Tuttligen, Germany), animals were allowed to recover from anesthesia and returned to their tanks.

To break double-stranded DNA into single strands and expose the proliferation marker CldU to the antibodies, free floating tissue sections were first incubated in 2 N HCl (Baker, Phillipsburg, N.J. USA) in PB containing 0.3% Triton X-100 (Baker; PB-T) for 50 min at room temperature, followed by rinsing in PB (3 × 10 min). Then, sections were incubated for 1–2 days at 4°C in rat anti BrdU-CldU antibody (Accurate Chemical & Scientific Corporation, Westbury, NY, USA) at a dilution of 1:500 in PB-T, along with other primary antibody [rabbit anti doublecortin (Abcam, Cambridge, MA, USA) at 1/2,000 dilution, mouse anti HuC/HuD (Molecular Probes, Eugene, OR, USA) at 1:200 dilution, or rabbit anti TH (ThermoScientific, Waltham, MA USA) at 1/800 dilution]. Sections were rinsed in PB (3 × 10 min) and incubated in a mixture of donkey anti rat Cy5 secondary antibody (Jackson Immuno Research, West Grove, PA, USA) at 1:1,000 dilution and goat anti mouse Alexa 488 (Jackson Immuno Research) at 1:1,000 dilution in PB-T, for 90 min at room temperature. After rinsing in PB (3 × 10 min), sections were mounted in PVA/DABCO cover slipping solution. Negative controls involved the omission of CldU or of primary antibodies incubation; both controls resulted in no detectable staining (data not shown).

In order to evidence the co-localization of CldU and Neurobiotin, sections were first pretreated in HCl and incubated in rat anti BrdU-CldU as described above. After rinsing in PB (3 × 10 min), sections were incubated during 90 min in donkey anti rat Cy5 at 1:1,000 dilution, together with streptavidin Cy3 (VECTOR) at a 1:500 dilution in PB-T. Sections were finally rinsed in PB (3 × 10 min) and mounted in PVA-DABCO coverslipping solution for immunofluorescence.

Most sections were imaged on a confocal system consisting of an Olympus BX61 microscope equipped with a FV300 confocal module and four lasers lines (405, 488, 543, and 633 nm) and the following objectives: 4x: UPlanFLN 0,13AN, 10x: UplanSAPO 0,40AN, 20x: UPlanFI 0,50AN, 40x: UPlanFI 0.75AN, and 60x: Plan ApoN 1,42AN objectives of the Confocal Microscopy Facility (IIBCE). Others were imaged on a Leica TCS SP5 II microscope equipped with four lasers lines (405, argon multiline 458, 476, 488, and 514 nm, and HeNe 543 and 633 nm), and HC PL FLUOTAR 5x/0.15, HC PL APO 20x/0.70 CS, HCX PL APO 40x/1.25-0.75 Oil, HCX PL APO 63x/1.40-0.60 Oil, HCX PL APO 63x/1.20 W CORR, and PL APO 100x/1.40-0.70 Oil CS objectives of the Confocal Microscopy Unit (Facultad de Medicina, UdelaR).

Acquisition settings were adjusted to ensure the completely dynamic range detection. Images of confocal planes were sequentially scanned with each laser line. Fifteen confocal planes, every 2 μm, were acquired in sections of levels L1–L3. For levels L4–L6, 15, 20, 25 to 30 confocal planes, every 0.5 or 1 μm were acquired. Post-processing of images was limited to small changes in the distribution histogram or background subtraction (Fiji Rolling ball radius: 50 pixels) in most cases.

Co-expression of markers was confirmed by orthogonal projections in the X-Z and Y-Z planes of the z-stacks at X-Y position of presumptive co-localization and/or visualization of the images corresponding to each channel of a single plane separately along with their overlay.

We used the same nomenclature and abbreviations as previously (Maler et al., 1991; Zupanc et al., 1996; Corrêa et al., 1998; Meek and Nieuwenhuys, 1998; Olivera-Pasilio et al., 2014).

To quantify the amount of labeled cells per section in levels L1–L3, we constructed 3-D models of the sections from serial 2D low power confocal microphotographs with BioVis3D (R) as detailed in Iribarne and Castelló (2014). The boundaries of the olfactory bulb (OB), and internal cell layer were drawn and the location of every labeled nucleus was indicated in each plane of the 3D reconstruction with the tool “dot sets.” The spatial distribution of labeled nuclei was evidenced by making transparent the constructed models.

We also used BioVis3D to determine the location of labeled nuclei in the x and y axes. First, we established the crossing of the mid-line and dorsal border of the OB as the origin of the x and y axes (red lines in Supplementary Figure 1). Then, lines extending from each nucleus to the x and y axes (green and blue lines in Supplementary Figure 1) were drawn and their length was calculated by the software. The resulting values were normalized by the width and height of each region of interest at each studied level to calculate the relative distance of every nucleus.

For every level (L1–L3) of each chase duration (1, 7, and 30 days), the mean and standard deviation of the total amount of labeled nuclei per section were calculated. We also quantified the amount of labeled nuclei within and outside the internal cell layer of L1 and L2 (Supplementary Figures 4A,B), and within and outside the ventricular proliferation zone adjacent to the ventral and dorsal subdivisions of the ventral telencephalon of L3 (Supplementary Figure 4C).

Differentiation into the neuronal phenotype by demonstration of the expression of neuronal markers (HuC/HuD or TH) or retrograde Neurobiotin labeling of long term CldU label retaining newborn cells was quantified with the aid of Fiji-ImageJ Point tool. In z stacks of the brain regions of interest, CldU+ nuclei, and double labeled CldU+/HuC/HuD+, CldU+/TH+, or CldU/Neurobiotin cells were marked in selected equally spaced confocal planes to avoid over counting. The total amount of each class was registered and the percentages of CldU+ cells that expressed neuronal markers or were labeled with Neurobiotin were calculated.

We analyzed variations in the amount of CldU labeled cells per section using One-way ANOVA or Mann-Whitney U-test, after determining the variance of the samples with Kolmogorov-Smirnov test. Differences among groups were considered significant when p < 0.05.

The sessile OB of G. omarorum shares the histoarchitecture of teleost's OB (Byrd and Brunjes, 1995; Kermen et al., 2013), consisting of a round core of small and densely packed cells, the internal cell layer (ICL) surrounded by three concentric diffuse layers: the external cell layer (ECL), the glomerular layer (GL), and the outer primary olfactory fiber layer (POFL) adjacent to the OB's surface (L1 and L2 in Supplementary Figure 1).

One day after a pulse of CldU, labeled cells were rare in the OB. Few and scattered proliferating cells were located at any of the layers of the rostral and medial part of the OB (red dots in L1 and L2, Figure 1: double arrows in AL1 and AL2, Figure 2; red dots in AL1 and AL2, Figure 3A). Conversely, densely packed CldU labeled cells were found at the lining of the most rostral portion of the telencephalic ventricle, nearby the caudal OB and the rostral region of the ventral (Vv) and dorsal (Vd) subdivisions of the subpallium. There, proliferating cells formed an extended ventricular proliferation zone (zone 1b, Olivera-Pasilio et al., 2014; red dots lining the ventricle at the zones indicated by the green rectangles in L2 and L3, Figures 1, 3A; arrows in AL2 and AL3, Figure 2). This rostral-caudal gradient in the amount of proliferating cells was confirmed quantitatively as the number of proliferating cells in the rostral and caudal part of the OB was significantly lower than in the rostral portion of the subpallium 1 day after CldU administration (Figure 3B).

Seven days after CldU administration, proliferating cells were also located predominantly at ventricular proliferation zones in the caudal portion of the OB and rostral subpallium (BL1–L3, Figure 2). Only few CldU labeled nuclei were placed throughout the OB layers or the subpallium. The number of labeled cells increased in all levels at this chase duration, but only significantly at L3. The rostral-caudal gradient was preserved but steeper (Figure 3B).

Thirty days after CldU administration, some proliferating cells remained within the boundaries of the proliferation zone 1b adjacent to the caudal OB (CL2, Figure 2) and rostral subpallium (CL3, Figure 2). Unlike shorter chases, some CldU labeled nuclei were located at considerable distances from the rostral region of the proliferation zone 1b, reaching the ICL of caudal and, in a less extent, rostral OB (CL2 and CL1, Figure 2). The amount of CldU labeled cells in L1 and L2 increased up to 2.4 and 3.7 times the values found 1 day after CldU administration, respectively. However, only L1 showed significant differences in the amount of CldU labeled cells between both chases. Conversely, the amount of CldU labeled cells decreased significantly in L3 from 7 to 30 days (Figure 3B). Few CldU labeled nuclei were found lateral to the caudal zone of 1b (adjacent to the subpallium), reaching Vd, Vv, MOTF, and medial zone of FB (CL3, Figure 2).

CldU label retaining cells spilled out the boundaries of the ICL of the caudal OB at a 90 day chase (Figure 2, EL2) whereas most labeled cells remain within the ICL boundaries of the ICL of the rostral OB (Figure 2, EL1). At 180 day chase the spilled out of labeled cells from the ICL of the rostral OB to the surrounding layer was evident (Figure 2, FL1).

The amount of CldU label retaining cells in the lateral zone of the subpallium increased considerably 90 and 180 day after CldU administration. At 90 days newborn cells predominate in the ventral zone, whereas at 180 days were distributed along all the dorso-ventral extension of the subpallium.

All the divisions of the Cb of G. omarorum (CCb, valvula cerebelli -VCb, and caudal lobe, including the eminentia granularis anterior -EGa- and posterior -EGp-), are organized in three layers, molecular, ganglionic, and granular, as exemplified in Supplementary Figure 5A. The ganglionic layer consists of Purkinje and eurydendroid cells. Eurydendroid cells, which are the origin of Cb efferents, are not aggregated in nuclei unlike other vertebrates (Finger, 1978).

Remarkable migrations processes both for the abundance of migrating cells as well as for their final destination, were detected in all cerebellar divisions as previously described (Olivera-Pasilio et al., 2014). Proliferation zones were circumscribed to a single cerebellar layer or even a region within a single cerebellar layer (red dots in L4 and L5, Figure 1).

The proliferation zone of the CCb, evidenced by the distribution of proliferating cells at 1 day chase, occupied the medial zone of its molecular layer (CCb-mol; red dots in L4, Figure 1). Thirty days after CldU administration, derived cells were mainly found at the granular layer of CCb (CCb-gra; blue dots in L4, Figure 1). However, scarce and intensely labeled CldU cells remained within the proliferation zone's boundaries.

In the VCb a conspicuous proliferation zone occupied all the extension of the molecular layer (VCb-mol). Proliferating cells were more densely packed near the dorsal-medial region of VCb-mol and migrating cells were sparsely and homogeneously found in all the extension of the granular layer (VCb-gra).

The proliferation zone of the caudal lobe of the Cb occupied the granular layer of the medial granular eminence (EGm-gra) while derived cells were located at the granular layer of the posterior granular eminence (EGp-gra) as shown in Olivera-Pasilio et al. (2014).

The TeO is a multimodal integration center which is interconnected with the torus longitudinalis forming a Cb-like structure (Bell, 2002). In G. omarorum, it is a hollow hemispheric shaped structure. It has a cortical histoarchitecture and consists of seven cellular and fibrillary alternating layers (Supplementary Figure 5B; Meek and Nieuwenhuys, 1998). Proliferating cells were located at the dorsal-medial and ventral-lateral border of the caudal pole of TeO, forming a horseshoe shaped ribbon-like proliferation zone. One day after CldU administration, proliferating cells were distributed in all TeO layers (red dots in L4 and L5, Figure 1), though appeared to be more densely packed in deeper layers as the stratum periventriculare (SPV). At longer chases, CldU label cells occupied more superficial layers of TeO, also extending beyond the proliferation zones' boundaries in the rostral direction (blue dots in L4 and L5, Figure 1).

One hundred eighty days after CldU administration, derived cells were located ~100 μm away from the dorsal region of the proliferation zone at rostral levels of the caudal TeO. These long labeled retaining cells were grouped mainly at the SPV and SAC layers, and less frequently reached the SGC (L5, Figure 1).

The TS of G. omarorum is a spherical protrusion of the mesencephalic tegmentum beneath the TeO. It is a layered structure which receives octavolateral inputs from rhombencephalic primary brain centers (Meek and Nieuwenhuys, 1998). One day after CldU administration proliferating cells were located at the medial and lateral borders of the caudal pole of TS (red dots in L5, Figure 1), forming a ring-shaped proliferation zone. At longer chases derived cells appeared diffusely distributed at the caudal pole of TS (blue dots in L5, Figure 1).

The ELL is the primary relay nucleus of electrosensory pathways. It is a layered cerebellum-like structure (Supplementary Figure 5C; Meek and Nieuwenhuys, 1998) in which principal cells project to contralateral rhombencephalic and mesencephalic structures (praeminentialis nucleus and TS) which project back to the ELL, either directly or indirectly through Cb. At 1 day chase duration, proliferating cells were mainly located at the lateral-caudal border of cellular layers of ELL, and showed a medial-lateral and rostral-caudal gradient (red dots in L6, Figure 1). At longer chases, ELL CldU label retaining cells were extensively and diffusely distributed (blue dots in L6, Figure 1).

One day after thymidine analog administration, DCX labeled a net of processes beneath the ventricular proliferation zone adjacent to the medial and dorsal-medial zone of the telencephalic proliferation zone 1b (inset, Supplementary Figure 6A). We only found CldU-DCX double labeled cells adjacent to the ventral region of this proliferation zone (Supplementary Figure 6B) as evidenced by the overlay of CldU and DCX signals corresponding to a single confocal plane (Supplementary Figure 6C) or by orthogonal projections of a z stack (Supplementary Figure 6D). At this chase duration, we did not find CldU-DCX or CldU-HuC/HuD double labeled cells in any other brain region.

Seven days after CldU administration, some cells at the ventral part of the subventricular zone of the rostral subpallium were CldU-HuC/HuD double labeled (Figure 4), but not at the rostral or caudal zones of the OB. Unexpectedly, we did not find any CldU-DCX double labeled cell at this chase duration.

After a 30 day chase, CldU label retaining cells expressed HuC/HuD at the ICL of the caudal OB (Figure 5A) and the ventral region of the subventricular zone of the rostral subpallium (Figure 5B). CldU-HuC/HuD double labeled cells' nuclei were round and the chromatin either densely (Figure 5A) or loosely (Figure 5B) packed. Some newborn CldU labeled cells with fusiform shape and elongated nuclei with densely packed chromatin also express DCX (Figures 5C,D).

Double labeled CldU-HuC/HuD cells were found at the ICL (Figure 6A) and the dorso-medial zone (Figure 6B) of the rostral OB 90 days after CldU administration. All CldU-HuC/HuD double-labeled cells had ovoid to round nuclei surrounded by a thin cytoplasm without evident cellular processes. At 90 days CldU-HuC/HuD double-labeled were also found at L3 (Figure 6C).

At 90 days, double labeled CldU-DCX cells were only found at the dorsal-medial zone of the rostral level of OB (data not shown).

At this chase duration CldU labeled cells also express TH at the ventricular or sub ventricular zone of the rostral subpallium (Figure 7). Some of these cells showed a thin cytoplasmic halo without cellular processes (Figures 7A,A′) whereas others had more expanded cytoplasm from which thin and short cellular processes with few branching points emerge (Figure 7B, arrowheads in Figure 7B′).

One hundred eighty days after CldU administration, double labeled CldU-TH cells were found in the rostral subpallium, far from the proliferation zone (Figure 8), and at both, caudal (Figure 9) and rostral (Figure 10) levels of the OB. Some newborn cells had an ovoid nucleus surrounded by a thin cytoplasmic halo without cellular processes, as those found lateral to the subpallial proliferation zone (Figures 8B,C). Many double labeled cells showed a larger cytoplasmic halo from which two or three relatively thick cellular processes emerged from opposite regions of the cell body. These cells resembling bipolar or multipolar neurons were located at the ICL of caudal (Figures 9B,B′,C,C′, Supplementary Video 1) and rostral (Figures 10C,C′,C″) OB. In other cells at the ICL of the rostral OB, a relatively large nucleus occupied an asymmetric location respect to a very thin cytoplasmic halo, resembling migrating neuroblasts (Figures 10A,B). All double labeled CldU-TH cells were intermingled with cells with single labeled CldU+ nuclei or TH+ cytoplasm.

According to the quantitative analysis, almost 0.5% of long term CldU retaining new born cells at the lateral regions of the subpallium (L3) expressed the mature neuronal marker TH+ after a 180 day chase. The fraction of CldU labeled cells that also expressed TH reached almost 1% and more than 1.5% of the total amount of CldU labeled cells at the caudal (L2) rostral (L1; Table 1).

After a short thymidine analog survival (7 days), we did not find HuC/HuD expression in CldU labeled cells. However, longer survivals (30, 90, and 180 days) allowed us to evidence the differentiation of derived cells into neurons in the CCb and other Cb-like structures.

Repetitive CldU administration allowed the long term retention of CldU in numerous cells at the CCb-gra, both 90 (Figure 15A) and 180 days (Figure 15B) after thymidine analog administration. These derived cells probably correspond to granule cells, both because of their location, as well as the shape and size of their nuclei. Cortical application of Neurobiotin labeled many cells with small round, ovoid, or polygonal soma, at the CCb-gra at both chase durations, even far from the site of tracer application. Note that the site of injection is not visible in the topographic images of Figures 15A,B, and thus is located more than 200 μm from the soma of the Neurobiotin labeled cells. This indicates that the tracer was incorporated into the axons or end terminals of the labeled cells at the CCb-mol and retrogradely transported up to the cell's soma and perisomatic dendrites at the CCb-gra. This, as well as the known histoarchitectural organization of the Cb, further supports the identification of these cells as granule cells. Many long term CldU retaining cells were labeled with Neurobiotin (Figures 15A′,B′,B″, Supplementary Video 2), indicating that the newborn cells acquired the mature granular morphology in 90 days. Also indicates that these newborn granule cells very probably were incorporated into the local cerebellar circuit and might be already functional. Conversely, Purkinje cells were also labeled with Neurobiotin but not CldU.

The fraction of CldU CCb-gra labeled cells that were retrogradely labeled represented almost 1% and more than 9% after chase durations of 90 and 180 days, respectively (Table 1).