Investigating olfactory behaviors in adult zebrafish

Odor-driven behaviors such as feeding, mating and predator avoidance are crucial for animal survival. While the zebrafish olfactory circuitry is well understood, a comprehensive description of odor-driven behaviors is needed to better relate olfactory computations to animal responses. Here, we used a medium-throughput setup to measure the swimming trajectories of 10 zebrafish in response to 17 ecologically relevant odors. By selecting appropriate locomotor metrics, we constructed ethograms systematically describing odor-induced changes in the swimming trajectory. We found that fish reacted to most odorants, using different behavioral programs and that combination of few relevant behavioral metrics enabled to capture most of the variance in these innate odor responses. We observed that monomolecular odors in similar chemical categories were weakly clustered based on the behavioral responses, likely because natural odors elicited stronger reactions than the monomolecular odors. Finally, we uncovered a previously undescribed intra and inter-individual variability of olfactory behaviors and suggest a small set of odors that elicit robust responses. In conclusion, our setup and results will be useful resources for future studies interested in characterizing olfactory responses in aquatic animals.


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appropriate locomotor metrics, we constructed ethograms systematically describing odor-induced changes in 22 the swimming trajectory. We found that fish reacted to most odorants, using different behavioral programs and 23 that combination of few relevant behavioral metrics enabled to capture most of the variance in these innate 24 odor responses. We observed that monomolecular odors in similar chemical categories were weakly clustered

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Here we characterize zebrafish olfactory behavior using a medium-throughput setup allowing for 68 exposure to well-defined odor concentrations. Using this approach, the swimming trajectories of 10 fish were 69 recorded in response to 17 ecologically relevant odors. By selecting 7 appropriate locomotor metrics, we 70 constructed behavioral ethograms systematically describing odor-induced changes in the swimming trajectory.

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We found that fish reacted to most odorants, using different behavioral programs. A combination of few relevant 72 behavioral metrics enabled to capture most of the variance in these innate odor responses. In general, odors 73 belonging to similar categories were weakly clustered based on the behavioral responses. This was likely because 74 natural odor extracts (food, blood, skin extract) have a tendency to elicit stronger reactions than the 75 corresponding individual monomolecular components. Finally, we quantified intra and inter-individual variability 76 of olfactory behaviors and suggest a small set of odors that elicit robust responses. In conclusion, both our setup 77 and our results will be useful resources for future studies interested in characterizing olfactory responses in 78 aquatic animals.

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A vertical olfactory setup with precise control of olfactory cue concentration and fast switching of odors

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To reproducibly measure fish responses to a large variety of odorants, we built a computer-controlled 82 setup automatically recording the position of freely swimming individual fish ( Figure 1A). The arena was 15 cm 83 large, 11.5 cm high, and 3 cm deep (approximately 6 x 5 x 1 fish body lengths) and contained around 400 mL of 84 water, allowing us to investigate zebrafish displacement in both the vertical and horizontal dimensions. The flow 85 rate was adjusted to 90 mL/min, which was fast enough to rapidly clear the arena, but not strong enough to 86 exhaust or stress the fish. In addition, a T-shaped connector deflected the inflow towards the lateral walls ( Figure   87 1A). This was to avoid pushing the fish down and to enable a rapid and homogeneous distribution of the stimuli 88 within the arena. To characterize the onset and dynamic of the olfactory cue delivered to the arena, we replaced 89 it by a dye and measured the change in reflected light overtime ( Figure 1B). The cue reached the arena 8 seconds 90 after the valve opened and rapidly spread through the arena, covering its entire volume within 30 seconds. The 91 cue concentration had returned to pre-stimulus levels within 15 minutes ( Figure 1C). Based on this, we chose an 92 inter-trial duration of 20 minutes to ensure complete clearance of prior cues before the following recording 93 started.

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Characterization of zebrafish behavior in response to diverse olfactory cues 102 Fish rely on different categories of water-soluble olfactory cues to guide fundamental behaviors 103 important for survival. We thus chose ecologically relevant olfactory cues related to one of the four following 104 categories: feeding, social, decay and alarm cues (see Table 1). We used seven different feeding-related

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To characterize zebrafish olfactory behaviors, we then measured the swimming trajectory of 10 adult 116 fish (7 males and 3 females) in response to these 17 odorants and to a water control. Single fish were habituated 117 to the arena and olfactory cues were delivered after a 5 min baseline period. Mapping the fish position after the 118 odor cue delivery yielded occupancy maps that differed markedly across odorants (Figure 2 A,B,C,D). In 119 particular, feeding cues such as food extract, nucleotides and methionine induced exploration of the upper part 120 of the arena (Figure 2A), where the odor cue was first delivered. In contrast, fish swam at the bottom of the tank 121 in response to alarm cues such as blood and skin extract ( Figure 2C). Overall, except increased activity closer to 122 the lateral walls, the average occupancy map in response to feeding ( Figure 2F) and social cues ( Figure 2G) 123 showed no clear differences compared to the water control ( Figure 2E). This was likely due to the important 124 inter-cue variability within these categories. Average occupancy maps in response to alarm cues ( Figure 2H) 125 revealed a consistent increase in bottom diving, that was also observable, although to a lesser extent, in response 126 to decay cues ( Figure 2I).   159 Pfeiffer, 1963). Conspecific blood elicits the whole suite of specific alarm behaviors displayed in response to skin 160 extract and thus seems to be a novel and equally powerful alarm substance in zebrafish.

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To determine whether the behavioral metrics captured independent aspects of the odor response, we 162 calculated their average pairwise correlation during odor response ( Figure 3H). As could be expected, freezing 163 was negatively correlated to most active locomotion indexes (velocity, burst swimming and horizontal swimming,

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Advantages and limitations of the medium-throughput olfactory behavior assay. We describe a medium-243 throughput setup to measure the behavior of individual fish exposed to olfactory cues of known concentrations.

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The automated delivery combined with rapid odor clearance enable to test multiple stimuli a day without  zebrafish. This finding is consistent with the ecology of these odors, given that alarm substances indicate a freshly 318 wounded or killed fish, thus a high probability for an imminent threat, whereas bacteria-mediated production of 319 decay cue takes hours to develop and thus signals a long-gone threat.

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Complex blends versus single compounds. Among feeding cues, the natural and complex food extract elicited 321 the strongest response ( Figure 3A,B), compared to simpler odors containing one or 2 monomolecular odorants, 322 and was in general very different from all these cues (Figure5A,B). Similarly, skin and blood extracts evoked

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We confirmed previously described behavioral responses to classically used odorants and also characterized a 346 new powerful alarm substance (blood). We also provide recommendation for future studies to take into account 347 the inter-and intra-individual reproducibility of odor behaviors. Finally, beyond neuroscience questions, 348 olfaction psychophysics in fish also has important impacts in terms of species conservation. In this context, it is 349 timely and crucial to provide tools and methods to reliably quantify the olfactory behavior in aquatic species to 350 assess and ultimately limit the negative anthropogenic impact on aquatic ecosystems. concentration to which the fish were exposed is documented in Table 1