(A) Illustration of the wax plug insertion model. The adult zebrafish olfactory organ (OO) is where olfactory sensory neurons (small black dot) reside and project their axons (black line) through the olfactory nerve to glomeruli (gray oval) in the olfactory bulb (OB) of the brain, which sits rostral to the telencephalon (tel). The wax plug (wax) occludes the olfactory organ causing degeneration of olfactory sensory neuron axons (dashed line) and glomeruli (dotted gray oval). (B) Dorsal view of the adult zebrafish head. The wax plug was inserted into the right nasal cavity (arrow) under the nasal flap. The left naris (arrowhead) was unoccluded and served as an internal control.

The effects of wax plug insertions on rosette morphology were examined at several time points. (A) Morphology of the control rosette showed several lamellae bearing sensory epithelium (arrow) and a central raphe consisting primarily of connective tissue (asterisk). (A’) Higher magnification morphology of control sensory epithelium from boxed region showed typical pseudostratified columnar epithelium with dense cell nuclei and a rough apical surface indicative of cilia. (A”) Lamina propria from the boxed region of the central raphe shown at higher magnification shows the typical connective tissue morphology. (B) With 1 day of wax plug insertion, the lamina propria in the central raphe and lamellae appeared to increase in thickness, but the sensory epithelium at low magnification looked unchanged. (B’) The sensory epithelium at higher magnification appeared to lack cilia on the surface and had less cell density. (B”) The lamina propria at higher magnification appeared paler and included more round cells than control connective tissue. (C) After 3 days of wax plug insertions the lamina propria appeared to continue expanding, but lamellae remained covered in sensory epithelium. (C’) Higher magnification of the sensory epithelium revealed a smooth apical layer and diffuse cell nuclei. (C”) The lamina propria in the central raphe had more extracellular matrix between cells than control connective tissue. (D) One week of wax plug insertions caused the rosette to lose its typical rosette morphology and showed no evidence of lamellae. (D’) The cell nuclei of the remaining epithelium seemed disorganized and the apical surface continued to show a smooth morphology. (D”) The expansive connective tissue beneath the epithelium lacked the pale round cells noted at the earlier time points. (E) After allowing the rosette to recover for 1 week following wax plug insertions, lamellae had returned although the lamina propria still seemed enlarged. (E’) The sensory epithelium seemed to regain its nuclear density and pseudostratified appearance, but the apical surface remained smooth. (E”) The connective tissue of the lamina propria remained expansive. (F) Three weeks of recovery after wax plug insertions allowed the rosette to return to typical morphology (F’) The sensory epithelium appeared recovered in nuclear density and ciliated surface. (F”) The lamina propria remained paler than controls, but the cell shapes were similar to the control connective tissue. Scale bar = 100 μm for A–F and 10 μm for A’-F”.

Anti-KLH immunoreactivity in the olfactory bulb revealed alterations in innervation. (A) Axonal labeling in control olfactory bulbs showed typical patterns with equal innervation on both sides. (B) The affected olfactory bulb (asterisk) had less anti-KLH labeling after 1 week of wax plug insertions, and the label was restricted to the superficial region of the rostral portion of the bulb. (C) Three weeks of recovery allowed the affected bulb (asterisk) to regain similar anti-KLH labeling as the internal control bulb and untreated control bulbs. Scale bar = 100 μm for all. (D) Anti-KLH immunoreactivity was significantly reduced in the affected bulb compared to the internal control bulb after 1 week of damage, but staining returned to control levels after 1 week of recovery. ANOVA was used for comparison; *=P < 0.05.

Confocal imaging of olfactory sensory neuron projections in the olfactory bulbs revealed wax plug-induced alterations. (A) Whole-mounts of control olfactory bulbs labeled with anti-KLH showed axons and glomeruli with equivalent labeling in both bulbs. (B) Higher magnification of the vmGx glomerulus showed tightly packed axons (arrow) projecting into a highly organized glomerulus (asterisk). (C) Twenty-four hours of wax plug insertions produced dramatic alterations in the innervation to the ipsilateral bulb (star), with many fewer axonal projections in all areas except the rostralmost point of the bulb. (D) The vmGx glomerulus was highly disorganized, with the axons entering the glomerulus (arrow) remaining bundled but the rest of the projections (arrowhead) appearing truncated. (E) After 1 week of wax plug insertions, anti-KLH labeling was noticeably reduced in the affected bulb (star) compared to the internal control bulb. (F) Defasciculation of the vmGx axonal bundle (arrow) was seen after 1 week of wax plug insertions, and very few axonal projections were apparent in the glomerular region (arrowhead). (G) One week of recovery allowed most of the axons to return to the affected bulb (star), although not to the levels of the internal control bulb. (H) Reinnervation of the vmGx glomerulus (asterisk) by the axon bundle (arrow) appeared to be similar to control morphology after 1 week of recovery from wax plug insertions. (I) After 3 weeks of recovery, anti-KLH labeling fully returned to the affected bulb (star). (J) Three weeks of recovery allowed the vmGx glomerulus (asterisk) to appear completely recovered with discreet borders. The axon bundle for this specimen is not visible in this plane. Scale bar = 100 μm for A, C, E, G, and I. Scale bar = 20 μm for B, D, F, H, and J.

Comparison of axonal projections in the olfactory bulbs of animals treated with three deafferentation methods. (A) Control olfactory bulbs labeled with anti-KLH showed axonal labeling throughout the bulbs. (B) Higher magnification of the vmGx glomerulus showed tightly packed axons (arrow) projecting into a highly organized glomerulus (asterisk). (C) After 1 week of wax plug insertions the affected bulb (star) showed less anti-KLH staining compared to the internal control bulb. (D) Anti-KLH labeling of afferent axons (arrow) was obviously reduced in the vmGx glomerulus (arrowhead) after 1 week of wax plug insertions. (E) One week after cautery ablation of the rosette, the affected bulb (star) had less anti-KLH labeling than the internal control bulb. (F) The vmGx glomerulus appeared noticeably disorganized with a sparse axonal stalk (arrow) and diffuse projections within the glomerular region (asterisk) 1 week following cautery ablation. (G) After 1 week of continuous Triton X-100 intranasal infusions to ablate the sensory epithelium, the ipsilateral olfactory bulb (star) had no obvious reduction in anti-KLH labeling over the majority of the bulb. (H) Higher magnification of the vmGx glomerulus showed a densely packed and clearly labeled axonal stalk (arrow) going into this glomerulus (asterisk). Scale bar = 100 μm for A, C, E, and G. Scale bar = 20 μm for B, D, F, and H.