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

Behavioral Assay

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-C

alre

tinin

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.

SE

M

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’