DASPEI staining and imaging

To visualize the impact of the CoCl₂ treatment on the olfactory epithelium, fish were stained with the fluorescent vital dye, DASPEI (2-[4-(dimethylamino)styryl-N-ethylpyridinium iodine; Invitrogen Molecular Probes). DASPEI is a mitochondrial stain taken up by metabolically active cells, can be used as a measure of living or viable cells within a tissue, and its fluorescence intensity is qualitatively correlated with mitochondria activity and number [63]. Other styryl dyes, such as FM1-43, have been show to enter sensory cells via mechanotransduction and ion channels [64], and their fluorescence intensity can be reflective of the amount of dye taken into and retained by the cells. Although we are unsure if the measured fluorescence intensity is representative of the mitochondrial activity or intake through ion channels, the observed fluorescence acts as a measure of overall cell activity and will only stain alive, functioning cells. Fish were immersed in 0.008% DASPEI in RO water for 20 minutes to visualize lateral line neuromasts and cells of the olfactory epithelium. Following staining and tissue collection (see below), all animals were imaged using an eGFP filter set (excitation filter 485/20; 525 LP filter) on a stereo dissection microscope (SteREO Lumar V.12, Zeiss) and images were acquired with Zen Pro software. A. burtoni were stained in groups that always included at least one untreated-control animal to account for any differences in batches of DASPEI solutions and replacement of the fluorescent light source half way through the A. burtoni data collection, but cavefish and goldfish were all stained at one time in the same DASPEI solution to reduce staining variability.

The olfactory epithelia of DASPEI-stained fish were exposed by gently removing the overlaying tissue around the nares. Extended depth of field imaging was used to obtain images of the entire olfactory epithelium. Images were taken at the same magnification and exposure for all animals that were stained together. All animals were left in a dark container prior to imaging, and all images were taken within 5 minutes of exposing fish to light to avoid photobleaching. Fish were imaged in random order and imaging order had no impact on fluorescence intensity (Pearson correlation: A. burtoni: R = 0.078, P = 0.599; goldfish: R = -0.075, P = 0.700; cavefish: R = 0.145, P = 0.469).

To investigate if olfactory abilities recover after CoCl₂ treatment, we examined all fish species immediately following treatment and 18 hours later. Some studies using CoCl₂ to chemically ablate the lateral line system, especially those using cichlids, allow the fish to recover after treatment (from 2 [14,61] to 18 hours [17]). To better mimic these studies, we allowed fish to recover for 18 hours post-treatment, as done in [17]. Further, this allowed us to examine if the impact of CoCl₂ on the olfactory system would be alleviated after recovery or if impairment persisted after treatment, which to our knowledge, has never been tested. Fish assigned to the recovery groups were placed in either normal cichlid-system (A. burtoni; [free Ca²⁺] = 250–500μM) or RO (goldfish and cavefish; [free Ca²⁺] <10μM) water for ~18 hours after which they were quickly netted from their tank and handled as described above for DASPEI staining and imaging.

Following alignment of extended depth of field images on the stereomicroscope, artifacts (small white dots) appeared in all aligned images. Artifacts were consistent across images (each image had 2–3) and were included in fluorescence intensity quantifications. For visualization purposes in Figs ​Figs11–3, artifacts were removed in Photoshop CS6. All images had the same color balance and contrast settings applied.

Representative photomicrographs of: the cichlid A. burtoni immersed in untreated-control (A), low-calcium (B), 0.1mM CoCl₂ (C), and 2mM CoCl₂ (D) solutions; goldfish C. auratus treated with control (E) and 0.1mM CoCl₂ (F) solutions; and cavefish A. mexicanus immersed in control (G) and 0.1mM CoCl₂ (H) solutions. A-D were taken 18-hours post-treatment and E-H were taken immediately after treatment. Small green dots are DASPEI-labeled neuromasts and representative superficial and canal neuromasts are indicated with yellow and white arrows, respectively. Red arrows indicate the olfactory epithelium, which is DASPEI-stained in controls but not in cobalt-treated fish. Scale bars represent 2 mm. Abbreviations: E, eye/eye socket; M, mouth.

Representative photomicrographs of the olfactory epithelium of control (A) and CoCl₂-treated (B) goldfish, C. auratus. C) Quantification of DASPEI fluorescence intensity of the olfactory epithelium of control and CoCl₂-treated C. auratus (N = 7–8 fish per group). Representative photographs of the olfactory epithelium of control (D) and CoCl₂-treated (E) Mexican blind cavefish, A. mexicanus. F) Quantification of DASPEI fluorescence intensity of the olfactory epithelium of control and CoCl₂-treated A. mexicanus (N = 6–7 fish per group). Different letters indicate statistical significance at P<0.05. Scale bars in A, B, D, and E represent 200μm. See Fig 2 for box plot descriptions. Abbreviations: C: caudal, L: lateral, M: medial, R: rostral. Orientation in A also applies to images in B, C, and D.