Effects of 6-OHDA injections on the zebrafish brain. (A,A′) Tyrosine hydroxylase (TH) immunohistochemistry of sagittal sections of the OB from control, 1-day post-injection (dpi), 3 dpi, 7 dpi, and sham fish. 6-OHDA injection site at the dorsal telencephalic ventricle near the telencephalon (Tel) is shown with a white arrowhead in the top left panel. Dotted lines indicate the OB. (A′) Magnified views of (A). Increased spacing in TH+ staining is indicated with white arrowheads. Green: TH; blue: DAPI. Scale bars: 100 µm in (A); 20 µm in (A′). (B) Quantification of the average number of TH+ cells in sagittal sections of the OB from (A) (n = 6–11). ANOVA: F (4, 36) = 8.34, p < 0.0001. ctrl vs. 1 dpi p = 0.0001, ctrl vs. 3 dpi p = 0.0025, ctrl vs. 7 dpi p = 0.0088. (C) Quantification of the average number of TH+ cells in dopaminergic populations 2, 5/6, 7, and 12/13 from sagittal sections of control and 1 dpi fish (n = 4). For population 2: t = 3.883, df = 7, p = 0.0060. (D) Quantification of swimming responses in control, 1 dpi, 3 dpi, 7 dpi, and sham fish (n = 9–12). (E) (TdT) dUTP Nick-End Labeling (TUNEL) staining in sagittal sections of the OB from control, 1 dpi, 3 dpi, and sham groups. TUNEL+ profiles are indicated with white arrowheads. Red: TUNEL; blue: DAPI. Scale bars: 100 µm in (E); 20 µm in (E′). (F) Quantification of average TUNEL+ profiles in OB sections from (E) (n = 4–5). ANOVA: F (3, 15) = 40.03, p < 0.0001. ctrl vs. 1 dpi p < 0.0001, 1 dpi vs. 3 dpi p < 0.0001, 1 dpi vs. sham p < 0.0001. (G) Tbr2a immunohistochemistry of sagittal sections of the OB from control and 1 dpi groups. Yellow: Tbr2a; blue: DAPI. Scale bar: 100 µm. (H) Quantification of Tbr2a+ cells in OB sections from (G) (n = 4). Box plots indicate mean (+), quartiles (boxes) and range (whiskers). One-way ANOVA or unpaired t-test for (C), * p < 0.005, ** p < 0.001, *** p = 0.0009, **** p < 0.0001.

Dysregulation of synaptic contacts in the OB caused by 6-OHDA. (A) Schematic diagram of a sagittal view of the OB, indicating three glomerular clusters: dorsal (dG), dorsolateral (dlG), and ventromedial (vmG). (B–E) Double immunohistochemistry of synaptic vesicle protein 2 (SV2) and tyrosine hydroxylase (TH) in sagittal sections of the OB from controls, 1 dpi, 3 dpi, and 7 dpi groups. Representative images taken from 5–7 animals analyzed per group. Dotted lines indicate the glomerular clusters selected. Whole bulb (B), and magnified views of: (C) dlG cluster, (D) dG cluster, and (E) vmG cluster. Green: Tbr2a; red: TH; blue: DAPI. Scale bars: 100 µm in (B); 50 µm in (C–E).

6-OHDA injections selectively reduce olfactory-mediated responses. (A) Schematic representation of the behavioral chamber used for studying responses to cadaverine. A rectangular, narrow chamber with a camera located in front was used to study fish’ vertical displacement following cadaverine exposure. (B) Time course of a dye distribution in the behavioral chamber from (A) indicating that odorant solutions remain in half of the chamber (indicated by a dotted line) during 30 s after cadaverine delivery. In (C), the asterisk (*) indicates the position where cadaverine solution was administered. (C) Representative swimming trajectories of zebrafish pre-(30 s) and post-(30 s) cadaverine delivery in controls (upper panels), 1 dpi (middle panels), and 7 dpi (lower panels) fish. (D) Quantification of swimming responses to cadaverine in controls, 1 dpi, 3 dpi, and 7 dpi (n = 5–10 fish). Top panel: time spent darting after cadaverine exposure. ANOVA F (3, 27) = 19.93, p < 0.0001. ctrl vs. control cad p < 0.0001, ctrl cad vs. 1 dpi p = 0.0033, 1 dpi vs. 7 dpi p = 0.0007. Bottom panel: time spent freezing after cadaverine exposure. ANOVA F (3, 26) = 12.28, p < 0.0001. ctrl vs. control + cad p = 0.0206, ctrl cad vs. 1 dpi p = 0.0379, 1 dpi vs. 7 dpi p = 0.0002. (E) Quantification of darting (top panel) and freezing (bottom panel) before odorant exposure (n = 4–9 fish). For freezing: ANOVA F (2, 19) = 4.088, p = 0.0334. 1 dpi vs. 7 dpi p = 0.0261. (F) Schematic representation of the behavioral chamber used for studying responses to alanine. A larger circular chamber with an overhead camera was used to study fish’ swimming distance following alanine exposure. (G) Quantification of swimming responses to alanine compared against pre-odorant exposure in controls, 1 dpi, 3 dpi, 7 dpi, and anosmic fish (n = 6–12 fish). One-sample Wilcoxon test when compared to baseline of 100% (no change). ctrl = p = 0.0005, 1 dpi p = 0.0312, 7 dpi p = 0.0010. * p < 0.005, ** p < 0.001, *** p = 0.0009.

6-OHDA injections lead to astroglial activation in the OB. (A–C) Glial fibrillary acidic protein (GFAP) immunohistochemistry in sagittal sections of the OB from controls, 1 dpi, 3 dpi, 7 dpi, and 6-OHDA with the anti-inflammatory drug pranlukast (PRAN). Whole bulb (A) and magnified views of: (B) the olfactory nerve and anterior OB, and (C) the dorsolateral (dlG) glomerular cluster (indicated by dotted lines). GFAP+ staining along the olfactory nerve layer (ONL) is indicated with white arrowheads. Green: GFAP; blue: DAPI. Scale bars: 100 µm in (A); 50 µm in (B,C). (D) Quantification of GFAP O.D. in OB sections from (A) (n = 5–9). ANOVA: F (4, 27) = 8.478, p = 0.0001. ctrl vs. 1 dpi p = 0.0306, ctrl vs. 3 dpi p < 0.0001, ctrl vs. 7 dpi p = 0.0275. 1 dpi vs. 3 dpi p < 0.0001. 3 dpi vs. PRAN p = 0.0017. Box plots indicate mean (+), quartiles (boxes) and range (whiskers). One-way ANOVA, * p < 0.005, ** p < 0.001, **** p < 0.0001.

Microglial activation and leukocyte migration in the OB following 6-OHDA injection. (A–E) Lymphocyte cytosolic protein 1 (Lcp1) immunohistochemistry in sagittal sections of the OB from controls, 1 dpi, 3 dpi, 7 dpi, and 6-OHDA with the anti-inflammatory drug pranlukast (PRAN). Whole bulb (A) and magnified views of: (B) middle region of the OB, (C) dlG cluster region, (D) ventral region, and (E) the interphase between the telencephalon and the OB. Yellow: Lcp1; blue: DAPI. Scale bars: 100 µm in (A); 20 µm in (B–E). (F) Quantification of Lcp1+ cells in OB sections from (A) (n = 4–5). ANOVA: F (4, 17) = 10.66, p = 0.0002. ctrl vs. 1 dpi p = 0.0074, ctrl vs. 3 dpi p = 0.0151, ctrl vs. 7 dpi p = 0.0028, 1 dpi vs. 7 dpi p = 0.0028, 1 dpi vs. PRAN p = 0.0019, 3 dpi vs. 7 dpi p = 0.0061, 3 dpi vs. PRAN p = 0.0046. Box plots indicate mean (+), quartiles (boxes) and range (whiskers). One-way ANOVA, * p < 0.005, ** p < 0.001.

6-OHDA injections in the OB cause retrograde degeneration in the OE. (A,A′) HuC/D immunohistochemistry of OE sections from controls, 1 dpi, 3 dpi, and 7 dpi groups. (A’) are magnified views of the OSN-containing sensory lamellae (HuC/D+) from (A). Scale bars: 100 µm in (A); 20 µm in (A′). (B) Quantification of percent change (vs. control) in optical density (O.D.) of HuC/D in the OE from (A) (n = 11–12). ANOVA F (3, 41) = 16.78, p < 0.0001. ctrl vs. 1 dpi p < 0.0001, ctrl vs. 3 dpi p < 0.0001, 1 dpi vs. 7 dpi p = 0.0005, 3 dpi vs. 7 dpi p = 0.0286. (C,C′) Double immunohistochemistry of BrdU and HuC/D in sections of the OE of controls, 1 dpi, 3 dpi, and 7 dpi fish. (C′) Magnified views of (C), showing the sensory and non-sensory regions of the OE lamellae (indicated by dashed lines and HuC/D staining). BrdU+ cells found in the sensory area (HuC/D+) are indicated with white arrowheads. Red: BrdU; green: HuC/D; blue: DAPI. Scale bars: 100 µm in (C); 20 µm in (C′). (D) Quantification of BrdU+ cells in whole lamellae from (A) (n = 4–8). ANOVA F (3, 21) = 21.26, p < 0.0001. ctrl vs. 1 dpi p < 0.0001, ctrl vs. 3 dpi p < 0.0001, ctrl vs. 7 dpi p = 0.0002. (E) Quantification of BrdU+ cells in the non-sensory lamellae from (A) (n = 4–8). ANOVA F (3, 21) = 25.83, p < 0.0001. ctrl vs. 1 dpi p < 0.0001, ctrl vs. 3 dpi p < 0.0001, ctrl vs. 7 dpi p = 0.0073, 3 dpi vs. 7 dpi p = 0.0075. (F) Quantification of BrdU+ cells in the sensory lamellae (HuC/D+) from (A) (n = 4–8). ANOVA F (3, 21) = 14.86, p < 0.0001. ctrl vs. 1 dpi p = 0.0046, ctrl vs. 3 dpi p = 0.0004, ctrl vs. 7 dpi p < 0.0001, 3 dpi vs. 7 dpi p = 0.0075, 1 dpi vs. 7 dpi p = 0.0205. Box plots indicate mean (+), quartiles (boxes) and range (whiskers). One-way ANOVA, * p < 0.005, ** p < 0.001, *** p = 0.0009, **** p < 0.0001.

Leukocytic migration within the OE caused by 6-OHDA injections in the OB. (A–D) Lymphocyte cytosolic protein 1 (Lcp1) immunohistochemistry in sections of the OE from controls, 1 dpi, 3 dpi, and 7 dpi. Whole OE (A) and magnified views of the following: (B) distal epithelial fold region, where the OSN-containing sensory region is indicated by HuC/D staining and a dotted line; (C) medial sensory region (HuC/D+) adjacent to the central raphe; and (D) sensory epithelium (HuC/D+). Migration of Lcp1+ cells is indicated by white arrowheads. White: Lcp1; blue: DAPI. Scale bars: 100 µm in (A); 20 µm in (B–D). (E) Quantification of Lcp1+ cells in OE sections from (A) (n = 4). ANOVA: F (3, 12) = 1.42, p = 0.2840. Box plots indicate mean (+), quartiles (boxes), and range (whiskers).