This study examined the effects of intranasal zinc sulfate exposure on the olfactory system of adult zebrafish. The following results were found: - At 2 days post-exposure, the olfactory organ was inflamed with loss of cilia and olfactory sensory neurons based on decreased anti-calretinin labeling. - By 5 days, the olfactory organ began recovering with thinner epithelium, intermittent sensory neurons, and partially recovered labeling. - By 10 days, the olfactory organ structure and labeling resembled controls, indicating reconstitution. - At 2 days, fish could not detect bile salts (detected by ciliated neurons) but could detect amino acids (detected by microvill

1. Injury-Induced Neuronal Turnover with Zinc Sulfate Affects Ciliated Olfactory Sensory Neurons More Than Microvillous
Olfactory Sensory Neurons in the Adult Zebrafish
James T. Hentig, Jr. and Christine A. Byrd-Jacobs
Department of Biological Sciences, Western Michigan University
Our lab studies plasticity of the olfactory system in the adult zebrafish. The olfactory organ of the
zebrafish is open to the aquatic environment allowing the opportunity for exposure to various
xenobiotics. We have performed a number of studies examining the degenerative effects of Triton
X-100 on the zebrafish olfactory system. We found that olfactory sensory neurons degenerate
within one day of exposure to this detergent, but they regenerate within five days. Resulting
alterations in glomerular structure and behavior have been reported.
Zinc sulfate is a toxicant that causes rapid olfactory degeneration. The effects have been
demonstrated across a wide array of animal models but not in the adult zebrafish. Here we present
an investigation into the effects of this chemical on olfactory organ morphology, sensory neuron
survival, and olfactory acuity in the adult zebrafish.
Introduction
Hypothesis
We hypothesize that zinc sulfate exposure will result in rapid degeneration and regeneration of the
olfactory organ and that olfactory acuity will be affected. We predict that our results will be similar
to the effects of Triton X-100 exposure and that the olfactory epithelium exhibits a universal
response to chemical ablation.
Methods
Zinc Sulfate Dosing:
Adult zebrafish (Danio rerio) were anesthetized in 0.03% MS222 until unresponsive to a tail
pinch. Fish were treated with intranasal administration of 2µl of 1M zinc sulfate in dH2O to the
right olfactory organ, and the left side served as an untreated, internal control. Fish were placed on
ice to allow for a 3-minute exposure to the zinc sulfate before being returned to a recovery tank for
survival times of 2, 5, or 10 days.
Tissue Processing:
Fish were euthanized with an overdose of MS222 before being placed in 4% paraformaldehyde for
24 hours. Whole heads were decalcified with RDO, dehydrated through ascending ethanol washes,
and embedded in paraffin. Semi-serial, 10µm sections were mounted on positively charged slides.
Hematoxylin and Eosin staining:
Sections were stained following typical H & E protocols and coverslipped with DPX.
Anti-Calretinin Labeling:
Sections were rehydrated through descending ethanol and phosphate-buffered saline (PBS)
washes, treated with 3% H2O2, and blocked with 2% BSA and 0.4% Triton X-100 in PBS for 1 h
at room temperature. They were incubated in anti-calretinin (Santa Cruz Biotechnology; 1:1000
made in blocker) in a humid chamber at 4°C for 20 hours. Following rinses, sections were
incubated in biotinylated anti-goat IgG (Vector Laboratories; 1:100 made in blocker) at room
temperature for 1 hour. They were treated with ABC solution (Vector Laboratories) for 1.5 hours
and exposed to DAB Solution (Vector Laboratories) until sufficient staining was observed.
Optical Density Measurements:
The amount of antibody labeling was estimated using SPOT Software 5.0 (Diagnostic
Instruments). Images of anti-calretinin-labeled sections at 20x were converted to 8-bit gray scale.
Using ImageJ software (NIH), the optical density of olfactory sensory neuron staining was
calculated. To do this, the gray area intensity of the sensory area of three lamellae per section from
three alternating semi-serial sections of each fish was measured and averaged. The background
gray area intensity was also measured, and the estimated optical density for that fish was then
calculated using the formula: OD = log(background intensity/average labeling intensity). The
percent difference between sides was calculated and compared using ANOVA with Tukey post hoc
analysis, using a significance level of 0.05.
Scanning Electron Microscopy:
Fish were euthanized with an overdose of MS222 before being placed in 3% glutaraldehyde in
PBS for 48 hours. Whole heads were rinsed with PBS before secondary fixation in 1% osmium
tetroxide in PBS for 1 hour. Heads were rinsed in ascending ethanol solutions before being rinsed
in HDMS for 10 minutes. Following air drying for 24 hours, olfactory organs were removed and
mounted, splattered with gold, and imaged with a Hitachi S-4500 scanning electron microscope.
Behavioral Assay:
Fish were placed individually in a testing tank on the appropriate day following zinc sulfate
exposure and allowed 1.5 hours to acclimate. Fish were exposed to either an amino acids mixture
(alanine, cysteine, histidine, methionine, and valine at 100 µM each) or a bile salts mixture
(taurocholic acid, taurodeoxycholic acid, taurochenodexoycholate, lithocholic acid, glycocholic
acid, and glycochenodeoxycholate at 100 µM each) delivered through a tube on one side of the
testing apparatus, while water was simultaneously delivered through a tube on the opposite side.
Videos of swimming behavior were recorded 30 seconds prior to odorant delivery and 30 seconds
following odorant delivery. Swimming behavior was analyzed by counting the number of turns
before odorant delivery and during the odor trial. Comparisons of pre-odor and odor trial
behaviors were performed using a repeated measures ANOVA, with a significance level of 0.05.
The mean percent difference in anti-calretinin immunoreactivity between treated and internal-
control side olfactory organs was compared using optical density measurements. There was a
significant decrease in anti-calretinin labeling at 2d and 3d after ZnSO4 treatment. By 5d after
chemical ablation and beyond, the amount of anti-calretinin labeling was not different from
controls. * = P<0.05.
Quantification of α-Calretinin Labeling
Summary
Effects on Morphology
•At 2 days following exposure to 1M ZnSO4 the olfactory organ appeared severely
inflamed, the cilia from ciliated sensory neurons were lost, and there was a significant
reduction in olfactory sensory neurons (based on decreased anti-calretinin labeling in
the olfactory organ).
•By 5 days after treatment, the olfactory organ began to recover with a noticeably
thinner olfactory epithelium, intermittent patches of ciliated sensory neurons, and
recovery of anti-calretinin labeling.
•The olfactory organ structure resembled control organs by 10 days following
treatment, and the olfactory epithelium appeared reconstituted by this time point based
on return of anti-calretinin labeling and cilia covering the olfactory epithelium.
Behavioral Effects
•In zebrafish, amino acids are detected by microvillous olfactory sensory neurons
while bile salts are detected by ciliated olfactory sensory neurons.
•At 2 days following exposure to zinc sulfate, fish did not detect bile salts and their
response to amino acids was not significantly different from pre-odor behavior.
•By 10 days following zinc sulfate exposure, the ability to detect amino acids was
regained, but the fish still did not detect bile salts. The ability to perceive bile salts
returned at 14 days following zinc sulfate exposure.
Conclusions
This study demonstrates that chemical ablation of the olfactory epithelium with zinc
sulfate results in severely altered structural morphology and loss of olfactory acuity,
but both recover within 14 days.
Recovery of anatomical structure precedes the return of olfactory-mediated
behavior. Microvillous olfactory sensory neurons regained their ability to detect amino
acids prior to the return of ciliated olfactory sensory neurons and the ability to detect
bile salts.
Thus, zinc sulfate has a differential effect on olfactory sensory neuron subtypes. The
bile salt-sensing ciliated sensory neurons appear to be more susceptible to the chemical
than the amino acid-sensing microvillous sensory neurons. This is similar to our
previous results with detergent exposure, suggesting that there is a universal response
of the olfactory epithelium to chemical lesioning.
This project was supported by NIH-NIDCD #011137 (CBJ) and NSF REU grant to
WMU #DBI 1062883 (JTH).
Acknowledgements
BehavioralAssay
The number of turns fish made pre- and post-delivery of a bile salt mixture was
compared. Control fish responded to the odor with a significant increase in turning
behavior. Fish at 2d and 10d after ZnSO4 lesioning did not appear to detect the bile
salts, but by 14d following chemical ablation fish responded to the odor by increasing
their turning behavior. * = P<0.05.
Behavioral responses were compared before (pre-odor) and after delivery of an amino
acid mixture to the tank (odor trial). Control fish made significantly more turns after
exposure to the odor. At 2d following ZnSO4 treatment, fish did not show a statistically
significant response to amino acids. At 10d and 14d after chemical ablation fish made
more turns following amino acid delivery. * = P<0.05.
Effects of 1M ZnSO4 on Olfactory Organ Morphology
Control 2 Day 5 Day 10 Day
H&E
At 2d after ZnSO4 treatment, the
olfactory organ was inflamed and
the tissue appeared fused (*).
After 5d, the olfactory epithelium
(arrows) was noticeably thinner
than control tissue.
The morphology of the olfactory
organ 10d after ZnSO4 resembled
that of control.
Untreated olfactory organs had a
distinct separation between sensory
and non-sensory epithelia (arrows).
α-Calretinin
Anti-calretinin labeled olfactory
sensory neurons throughout the
olfactory epithelium (arrowheads)
of control fish.
With 2d survival, anti-calretinin
labeling (arrowheads) was
diminished and confined to the
apical surface of the epithelium.
Anti-calretinin labeling showed a
return of olfactory sensory neurons
by 5d, although it appeared to be
less than control levels.
By 10d, anti-calretinin labeling
appeared to resemble control
levels in amount and intensity.
SEM
A) The sensory (S) and non-sensory
(NS) regions of a lamella, with a
defined separation (arrows), are
shown in a control fish. A’) The
surface of control olfactory epithelia
was densely packed with cilia and
microvilli from olfactory sensory
neurons.
B) At 2d following exposure, gross
morphology of the olfactory organ
was intact, although some lamellae
were inflamed. B’) The olfactory
epithelial surface appeared to
contain only microvilli, with
sensory cilia lacking. Non-sensory
cilia remain (*).
C) Lamellae were thin at 5d post-
treatment with ZnSO4. C’) On the
surface of the olfactory epithelium,
intermittent cilia (arrows) were
present across the mat of
microvilli.
D) Given 10d to recover from ZnSO4
exposure, the sensory (S) and non-
sensory (NS) regions, with a clear
separation (arrows) resembled that of
control. D’) The sensory region
appeared to be densely packed with
cilia and microvilli, similar to control
tissue.
**
B’
*
A
NS
S
B C D
S
NS
D’C’A’