Neuronal regeneration in the goldfish telencephalon following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) insult

Adult female goldfish (Carassius auratus (Linnaeus, 1758)) were purchased from a commercial supplier (Mt. Parnell Fisheries Inc., Mercersburg, Pennsylvania, USA) and maintained at 18 °C under a natural-simulated photoperiod on standard flaked goldfish food. Fish were kept in 70 L tanks (15–18 fish per tank). All procedures were performed according to the guidelines of the Canadian Council on Animal Care and were approved by the University of Ottawa animal care committee. Goldfish were anesthetized using 3-aminobenzoic acid ethylester (MS-222; 0.05% in water, Sigma-Aldrich, Oakville, Ontario, Canada) for all handling and sampling procedures.

The neurotoxin MPTP (Sigma-Aldrich M0896, Oakville, Ontario, Canada) was dissolved in 0.6% saline to give a dose of 50 μg/g body weight of fish at 2 μL/g. Sexually recrudescent female goldfish (September 2014 experiment; ∼33 g ± 1.6; gonadosomatic index ∼2.8 ± 0.5) either received a single intraperitoneal injection of MPTP or a control 0.6% saline injection at time 0 similar to the treatment schedule used by Pollard’s group (Pollard et al. 1992; Goping et al. 1995). All female goldfish also received a single intraperitoneal injection of BrdU (Life Techonologies B23151, Burlington, Ontario, Canada) dissolved in 0.6% saline at a dose of 150 μg/g body weight at 3 dpi of MPTP prior to sampling at 4 and 7 dpi. Female goldfish were randomly sampled at 4 and 7 dpi when they were perfused intracardially with 0.6% saline (∼10 mL) and then 4% paraformaldehyde (PFA, Sigma-Aldrich, P6148, Oakville, Ontario, Canada) freshly depolymerized in phosphate-buffered saline (PBS; pH = 7.4). Following perfusion, the brains, whole pituitary, and gonads were carefully dissected from the goldfish. The gonadosomatic index was calculated by dividing the gonad weight by body weight of the goldfish × 100.

Female goldfish (December 2014 experiment; 32 g ± 1.4; gonadosomatic index 1 ± 0.1; n = 10) received either a single intraperitoneal injection of MPTP (50 μg/g body weight) or a control 0.6% saline injection at time 0. The mobility measurements were performed once per day at 0, 1, 3, and 7 dpi in the late morning where time 0 was measured immediately after the administration of MPTP. Fish movement and swimming motions were recorded using an Olympus Stylus TG-850 (Olympus, Center Valley, Pennsylvania, USA) camera in individual tanks. The fish were placed in 4 cm of water in a 13 cm × 30 cm transparent aquarium similar to the experiment performed by Weinreb and Youdim (2007). The fish were allowed to acclimatize for 30 min prior to recordings. The total movement of a single fish, which includes the total distance of swimming and time of rest, was measured over a period of 5 min using Logger Pro 3 (Vernier Software & Technology, Beaverton, Oregon, USA). Statistical analysis was performed using SPSS Statistics (version 21, IBM, New York, New York, USA). The data for total distance traveled were normally distributed and compared using one-way analysis of variance (ANOVA) followed by Tukey’s test to compare the effects of MPTP treatment. The resting time data were not normally distributed and were therefore compared using the Kruskal–Wallis test followed by Dunn’s multiple comparison test to determine the effects of MPTP treatment. p-values <0.05 were considered statistically significant.

Goldfish brains were dissected and collected after intracardial perfusion and postfixed with 4% PFA overnight at 4 °C. The following day, the brains were washed in a series of solutions (1× PBS (2× 30 min); 0.85% NaCl (1× 40 min); Ethanol (EtOH) 70%/NaCl 0.85% 1:1 (1× 30 min); EtOH 70% 2× 20 min; EtOH 85% (1× 40 min); EtOH 95% (1× 40 min); EtOH 100% (2× 30 min)) at 4 °C in preparation for paraffin embedding (toluene (2× 45 min); melted paraffin (3× 60 min)). All the brains were embedded in paraffin prior to sectioning. Six-micrometre sections were obtained for staining using a motorized microtome (HM 350, Microm, Heidelberg, Germany) and the sections were placed on Superfrost plus microscopic glass slides (Fisher Scientific 12-550-15, Toronto, Ontario, Canada). To visualize incorporated BrdU and TH, the paraffin sections were deparaffinized by washes in Xilol (2× 20 min) and serial dilutions of ethanol (2 min per wash) (2× EtOH 100%; EtOH 95%; EtOH 85%; EtOH 70%; EtOH 50%; EtOH 30%). The sections were then washed with 1× PBS (2× 10 min) prior to incubation with 50% formamide/50% 2× saline–sodium citrate (SSC) for 3 h in a 65 °C water bath. Following the incubation, the sections were washed with 2× SSC (20× SSC stock solution: 3 mol/L NaCl, 0.3 mol/L Sodium citrate, pH 7.0) (2× 5 min) prior to DNA denaturation by incubating the sections in 2 N HCl for 30 min at 37 °C. This was followed by washing in sodium borate buffer (0.1 mol/L, pH 8.5) for 2× 5 min at room temperature (RT) and then with 1× PBS with 0.2% TritonX-100 (3× 5 min). Nonspecific protein binding sites were blocked using 1% nonfat milk with 0.2% Triton X-100 in 1× PBS for 45 min at RT. Sections were then incubated with rat anti-BrdU (dilution 1:300; Abcam ab6326, Cambridge, Massachusetts, USA) and rabbit anti-TH antibodies (dilution 1:400; Millipore Ab152, Toronto, Ontario, Canada) in 1% milk with 0.2% Triton X-100 in 1× PBS at 4 °C overnight in a moist chamber. After 1× PBS with 0.2% TritonX-100 rinses of 3× 10 min on a shaker, the sections were incubated in Alexa Fluor 594 goat anti-rat immunoglobulin G (IgG) (dilution 1:300; Invitrogen A11007, Burlington, Ontario, Canada) and Alexa Fluor 488 goat anti-rabbit IgG (dilution 1:300; Life Technologies A11008, Burlington, Ontario, Canada) secondary antibodies in 1× PBS with 0.2% TritonX-100 for 1.5 h at RT. The sections were then washed three times in 1× PBS with 0.2% Triton X-100 for 5 min and mounted with anti-fading medium Vectashield with 4,6-diamino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, California, USA) and kept in the dark until visualization of slides with Nikon A1RsiMP confocal (Nikon, New York, New York, USA) microscope.

Immunocytochemistry for GFAP was used to identify RGCs (Forlano et al. 2001; Xing et al. 2015) in relation to TH-immunoreactivity in the ventral telencephalon. The paraffin sections were deparaffinized as described above and then washed with 1× PBS (2× 10 min). Nonspecific protein binding sites were blocked with 1% milk with 0.2% Triton X-100 in 1× PBS for 45 min at RT. Sections were then covered with mouse anti-pig GFAP antibody (1:500; Chemicon, Temecula, California, USA, Cat. # MAB360), which was previously validated in fish (Forlano et al. 2001; Xing et al. 2017) and rabbit anti-rat TH antibody (1:500; Chemicon, Tamecula, California, USA, Cat. # AB152), which was previously validated in fish (Yamamoto et al. 2011; Xing et al. 2017) overnight at 4 °C. Sections were then washed for 2× 10 min in PBS and incubated with a donkey anti-rabbit Alexa fluor 488 (1:500; Invitrogen Molecular Probes, Invitrogen, Burlington, Ontario Canada) and goat anti-mouse Alexa fluor 596 (1:500; Invitrogen Molecular Probes, Invitrogen, Burlington, Ontario Canada) for 1 h at RT. The sections were then washed three times in 1× PBS with 0.2% Triton X-100 for 5 min and mounted with antifading medium Vectashield with DAPI (Vector Laboratories, Burlingame, California, USA) and kept in the dark until visualization of slides with a confocal microscope.

The BrdU- and TH-positive cells were visualized using a fluorescent microscope (Zeiss Axiophot Microscope, Carl Zeiss Canada Ltd., Toronto, Ontario, Canada) at 20× magnification with the aid of an Olympus DP70 (Olympus, Center Valley, Pennsylvania, USA) camera. Positive cells were counted in the 4 and 7 dpi goldfish brains to quantify cellular proliferation using ImageJ1.43I software (Rasband 1997–2014). The total number of TH- and BrdU-positive cells in goldfish brain sections were counted using unbiased, single-blind, counting of cells in comparable sections using the disector technique (Guillery 2002). For the TH and BrdU counting, five sections of the ventral telencephalic area at 4 and 7 dpi were analyzed per fish (n = 5 per group) where cells were counted the entire length of the ventricular surface along the ventral-dorsal axis of the left brain hemisphere using a specific counting frame of a width of ∼ 680 ± 80 μm stretching laterally from the ventricular surface to the apical surface. Labelled cells were localized according to the goldfish brain atlas of Peter and Gill (1975) and Hornby et al. (1987). Cell counting was confined within neuroanatomical borders of the target area. The total number of positive cells between the treatment group and control group were first tested for normality and then statistically compared using a Student’s t test (two-tailed) in SPSS Statistics (version 21). p-values ≤0.05 were considered statistically significant. All data are presented as mean + SEM.

Female goldfish were treated with MPTP and their swimming movements were measured following MPTP injection. As shown in Fig. 1, control goldfish traveled an average of ∼1250 cm/5 min and rested for ∼14 s during the test period. However, there was an observable decrease in total distance traveled and an increase in resting time following the injection of MPTP. At 3 dpi of MPTP goldfish decreased distance traveled by ∼85% (p < 0.0001) (Fig. 1). There was a significant increase in resting time (p = 0.0011) at 3 dpi (Fig. 1b). Thereafter, the total distance traveled by MPTP-treated goldfish started to recover towards control levels at 7 dpi but was still significantly different compared with control (p = 0.0178). Reversible changes were evident for the resting time, which approached control levels of 7 dpi (p = 0.2981).

Confocal imaging of control and MPTP-treated female goldfish brain sections at 4 and 7 dpi revealed TH- and BrdU-positive cell labelling in the Vd of the telencephalon. Figure 2b shows BrdU-positively labelled cells (red) located laterally and dorsally from the ventricular surface to the Vd area at 4 dpi of MPTP. Confocal imaging also revealed that BrdU-positive cells are colocalized with DAPI (blue nuclear stain) thereby indicating that BrdU was incorporated into newly generated cells in the female goldfish brain (Fig. 3a). Several double-labelled BrdU- and TH-positive cells were also found in the 4 dpi of MPTP fish group (Figs. 3b and 3c).

The total number of TH-positive neurons in the ventral telencephalon, which includes the Vd, was counted. At 4 dpi, TH-positive cells significantly decreased by 27% compared with control fish (p = 0.02) (Fig. 4a). No significant differences in TH-immunolabelling were observed at 7 dpi (p = 0.69) compared with controls (Fig. 4c). At 4 dpi, BrdU-positive cells significantly increased ∼1.9-fold compared with control fish (p = 0.02) (Fig. 4b). In contrast, no significant differences in BrdU immunolabelling were observed at 7 dpi (p = 0.69) (Fig. 4d).

Immunoreactivity of TH and GFP in control goldfish ventral telencephalon, a region negatively impacted by MPTP toxicity (Goping et al. 1995) and one of which contains large amounts of RGCs (Pellegrini et al. 2016), reveals a close anatomical relationship between catecholamine neurons (TH) and GFAP fibers (∼5 μm) (Fig. 5). Dense GFAP immunoreactivity (red) was observed along the surface of the telencephalon ventricle with fibers extending ventrally towards the Vd where TH-immunoreactive catecolaminergic cells (green) are located.