Plasticity of glomeruli and olfactory-mediated behavior in zebrafish following detergent lesioning of the olfactory epithelium
Introduction
The olfactory system is a useful model for studies on neuroplasticity because of its ability to recover from lesion, in part due to the inherent neuronal turnover seen in the olfactory organ. Various methods of chemical lesioning have been used to examine the mechanisms by which the olfactory system responds to damage. Exposure of the olfactory epithelium to a variety of chemicals can eliminate the sensory input to the olfactory bulb by destroying the olfactory sensory neurons (OSNs). The olfactory epithelium can replenish itself, reinnervate the olfactory bulb, and restore function (Schwob et al., 1995, Schwob et al., 1999, Herzog and Otto, 1999, Paskin and Byrd-Jacobs, 2012). While a number of toxic chemicals have been used, Triton X-100 application is a common technique in studies examining the degeneration and regeneration of the olfactory system. Application of the detergent to the nasal cavity destroys OSNs, which temporarily reduces afferent input to the olfactory bulb (Nadi et al., 1981, Baker et al., 1983, Cummings et al., 2000).
A number of studies have examined the effects of chemicals on the fish olfactory system, due to concerns about pollution and toxins in the aquatic environment (Tierney et al., 2010). Application of Triton X-100 to the olfactory organ of catfish damages the olfactory epithelium to various extents, depending on the concentration (Cancalon, 1982, Cancalon, 1983). Low doses of the detergent affect only the superficial portions of the cells of the olfactory epithelium, while high doses destroy both sensory and non-sensory regions of the olfactory organ. In zebrafish, intranasal infusion of Triton X-100 causes immediate disruption of the olfactory epithelium (Iqbal and Byrd-Jacobs, 2010). One day post-lesion the olfactory epithelium is significantly thinner and has an apparent loss of most OSNs. The thickness of the epithelium progresses with return of epithelial depth and density of OSNs by 5 days post-lesion, and rosette morphology returns to near control levels within 7 days. This time course is more rapid than in mammals (Verhaagen et al., 1990, Cummings et al., 2000) and larger fish (Cancalon, 1983). Chronic treatment with Triton X-100 severely disrupts rosette morphology and removes most of the OSNs, although some subsets of OSNs appear more affected than others (Paskin et al., 2011, Paskin and Byrd-Jacobs, 2012).
Zebrafish possess three physiologically distinct OSNs, which are dispersed throughout the olfactory epithelium (Hansen and Zieske, 1998). In general, ciliated OSNs detect bile salts and pheromones (Koide et al., 2009), microvillous OSNs detect amino acids and nucleotides (Lipschitz and Michel, 2002), and crypt OSNs appear to detect pheromones, although these cells are much less understood (Germana et al., 2004, Hamdani et al., 2008). Interestingly, chronic Triton X-100 exposure appears to affect ciliated OSNs primarily, while some microvillous and crypt neurons survive the treatment (Paskin et al., 2011, Paskin and Byrd-Jacobs, 2012).
The axons of the OSNs project to the olfactory bulb in the brain, where they relay sensory information to projection neurons and interneurons in discrete glomeruli. Adult zebrafish have approximately 140 glomeruli per olfactory bulb, a subset of which are highly stereotyped and distinguishable (Baier and Korsching, 1994, Braubach et al., 2012). Glomeruli in the olfactory bulb contain the axonal projections of a single OSN subtype and group in functional zones (Li et al., 2005, Sato et al., 2007, Yaksi et al., 2007). Ciliated OSNs project to the dorsal and medial regions of the olfactory bulb, microvillous OSNs project to the lateral and ventro-lateral regions of the bulb (Friedrich and Korsching, 1997, Sato et al., 2005), and crypt neurons project to a single glomerulus in the dorsomedial group (Ahuja et al., 2013). Consequently, the medial bulb regions process social and reproductive odors (Li et al., 2005, Yaksi et al., 2007) whereas the lateral region of the olfactory bulb tend to process feeding behavior (Li et al., 2005, Yaksi et al., 2007, Koide et al., 2009). Thus, the three zebrafish OSN subtypes are distinct in anatomy, physiology, and behavior.
The olfactory bulb is also affected by detergent application to the peripheral olfactory organ, since its afferent input is reduced. Following chronic application of Triton X-100 over 3 weeks, there is a reduction in olfactory bulb volume (Paskin et al., 2011). The glomeruli in the olfactory bulb that receive innervation from ciliated OSNs are lost, while those containing the axons of the other OSN subtypes show less damage (Paskin and Byrd-Jacobs, 2012). These fish lose the ability to detect bile salts but retain the ability to perceive amino acids. It is unclear if these results are due to accumulated damage from repeated exposure to the detergent.
In the current study, we examined whether a single intranasal infusion with Triton X-100 would yield results similar to the chronic exposure to the detergent. We hypothesized that the axonal projections of ciliated OSNs, and the glomeruli they innervate, would be most affected by detergent exposure, with degradation and recovery within a week. We also wanted to see if regeneration of the OSNs would result in regeneration of glomeruli in the same place and with the same morphology. From this, we also hypothesized that if chemical lesioning causes glomerular disruption, olfactory-mediated behavior would also be affected, with responses to odors mediated by ciliated OSNs most affected. Our work provides a model for rapid degeneration and regeneration of olfactory innervation patterns and of odorant-mediated behavior in a vertebrate.
Section snippets
Experimental procedures
Adult zebrafish of both sexes were generously donated by R. Warga and D. Kane. Fish were maintained in 10–15 gallon aquaria and fed flake food twice daily. All experimental procedures were approved by the WMU Institutional Animal Care and Use Committee.
Time course of the effects of detergent ablation of the olfactory epithelium on glomeruli in the olfactory bulb
Anti-KLH allowed visualization of most, if not all, OSN axons from the olfactory rosette as they spread over the surface of the bulb and terminated in the glomerular layer. Only the axonal component of the glomerulus was considered in this study. In untreated control fish, each glomerulus consisted of a distinct axonal bundle terminating in a spheroidal structure made of axonal fibers (Fig. 1A, B). The size, shape, and location of specific glomeruli appeared to be the same in the left and right
Discussion
Results of this study show that chemical lesioning of the olfactory organ in adult zebrafish with Triton X-100 results in significant, but temporary, damage to the olfactory bulb followed by rapid regeneration of structure and function. Confocal analysis of labeling with an antibody to KLH allowed us to perform a detailed investigation into structural alterations in OSN projections following detergent application to the olfactory organ. Anti-KLH binds an unknown epitope associated with OSN
Acknowledgments
This work was supported by the National Institutes of Health-NIDCD grant #011137 (CBJ), Western Michigan University Lee Honors College research award (EJW), and the National Science Foundation REU DBI-1062883 grant to WMU (SKK). We are grateful to Taylor Paskin for technical advice.
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