Visualizing a set of olfactory sensory neurons responding to a bile salt

Abstract

SUMMARYIn the present study, we exposed the olfactory epithelia of crucian carp, Carassius carassius, and brown trout, Salmo trutta, to dextran coupled with Alexa dyes together with odorants. Dye uptake was severely reduced after pre-exposure to nocodazole, an inhibitor of microtubule polymerization that impairs endocytosis, supporting the hypothesis that odour-activated olfactory receptor molecules undergo endocytosis. Application of the bile acid taurolithocholate, a potent and specific odorant for fish, resulted in the labelling of a sparse (less than 3%) cell population with the typical morphology of ciliated sensory neurons (CSNs) -long dendrites and cell somata deep in the sensory epithelium. The dye was distributed throughout the sensory neuron, also revealing axons and target glomeruli. Stained axons redistribute at the entrance of the olfactory bulb and terminate in two small target areas, a dorsal and a medial one. These results are consistent with the notion that taurolithocholate is detected specifically by a few ciliated sensory neurons. Application of the olfactory epithelium of brown trout to bile acid stained cells with the appearance of CSNs. Application of an alarm agonist, hypxanthine-3-N-oxide, to crucian carp olfactory organ caused staining of another set of sensory neurons. Furthermore, our results show that odour-induced uptake of a dye can serve to identify the subtype of olfactory sensory neurons responding to a particular odorant, and to pinpoint the target regions of these neurons in the olfactory bulb as a first step to elucidating the neuronal network responding to a particular odour.

Mixed input to olfactory glomeruli from two subsets of ciliated sensory neurons does not seem to impede relay neuron specificity in the crucian carp

“…We stained two subsets of ciliated sensory neurons of the olfactory epithelium of crucian carp with the aid of odorant-induced uptake of fluorescent dye (Døving et al, 2011). To this end we perfused the ipsilateral olfactory epithelium, first with 10 nmol l −1 of the bile salt taurolithocholate (TLC) together with 10 µmol l −1 of a dextran conjugated with Alexa 488 in APW for 5 min, rinsed with APW for 5 min, and subsequently exposed the epithelium with 10 nmol l −1 of the alarm substance hypoxanthine-3-Noxide (H3NO) together with 10 µmol l −1 of a dextran conjugated with Alexa 647 in APW for 5 min.…”

“…H3NO is a potent odorant that evokes an alarm reaction in minnows (Brown et al, 2000), black tetra (Pfeiffer et al, 1985) and zebrafish (Mathuru et al, 2012;Parra et al, 2009). Although the odorants are different their putative role as olfactory cues in crucian carp is not yet proved, both TLC and H3NO induce activity-dependent uptake of fluorescent dye into ciliated OSNs (Døving et al, 2011). Previous studies in crucian carp have shown that the ciliated OSNs project to the medial olfactory bulb and synapse with relay neurons with axons in the medial part of the medial olfactory tract (Hamdani and Døving, 2002).…”

“…However, most studies have not combined physiological and anatomical methods to investigate odour mapping in the olfactory bulb while identifying activated primary OSNs. Thus, as previous findings revealed that dextran conjugated with Alexa dye could stain a specific set of primary OSNs (Døving et al, 2011) as a result of activity-dependent uptake of the fluorescent dye, the present study was undertaken with the intention of clarifying whether there are two separate subsets of ciliated OSNs responding to TLC and H3NO, respectively, with axons projecting to different glomeruli in the olfactory bulb. The TLC was applied with a pseudo-green dye and H3NO with a pseudo-magenta dye.…”

Sensitivity and specificity of the olfactory epithelia of two elasmobranch species to bile salts

“…Amino acids, a feeding stimulant in fishes (Zielinski and Hara, 2006), are detected primarily by microvillous ORNs (Lipschitz and Michel, 2002;Sato and Suzuki, 2001), but also possibly by ciliated and crypt ORNs (Hansen et al, 2004;Vielma et al, 2008). In contrast, several studies demonstrated that for teleosts, bile salts are detected only by ciliated ORNs (Døving et al, 2011;Hansen et al, 2003;Sato and Suzuki, 2001). Additionally, cross-adaptation and mixture experiments confirm that teleosts possess ORs for bile salts that are independent from those for amino acids, prostaglandins, gonadal steroids and polyamines (Laberge and Hara, 2004;Michel and Derbidge, 1997;Zhang and Hara, 2009).…”

Multiplex quantification of lamprey specific bile acid derivatives in environmental water using UHPLC–MS/MS

“…These compounds, which possess diverse structures and are relatively stable in aquatic environments, are proposed to function as intraspecific chemical signals [2]. Recent studies have shown that bile acids serve as acute and specific olfactory stimuli for teleost fishes and jawless vertebrates, such as sea lampreys [3][4][5]. Since fish and other aquatic animals produce and release bile alcohols with diverse conjugations and side chains, these particular metabolites were referred to as bile acid derivates.…”