Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons.

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Behavior Responses to Chemical and Optogenetic Stimuli in Drosophila Larvae.

An animal's ability to navigate an olfactory environment is critically dependent on the activities of its first-order olfactory receptor neurons (ORNs). While considerable research has focused on ORN responses to odorants, the mechanisms by which olfactory information is encoded in the activities of ORNs and translated into navigational behavior remain poorly understood. We sought to determine the contributions of most Drosophila melanogaster larval ORNs to navigational behavior. Using odorants to activate ORNs and a larval tracking assay to measure the corresponding behavioral response, we observed that larval ORN activators cluster into four groups based on the behavior responses elicited from larvae. This is significant because it provides new insights into the functional relationship between ORN activity and behavioral response. Subsequent optogenetic analyses of a subset of ORNs revealed previously undescribed properties of larval ORNs. Furthermore, our results indicated that different temporal patterns of ORN activation elicit different behavioral outputs: some ORNs respond to stimulus increments while others respond to stimulus decrements. These results suggest that the ability of ORNs to encode temporal patterns of stimulation increases the coding capacity of the olfactory circuit. Moreover, the ability of ORNs to sense stimulus increments and decrements facilitates instantaneous evaluations of concentration changes in the environment. Together, these ORN properties enable larvae to efficiently navigate a complex olfactory environment. Ultimately, knowledge of how ORN activity patterns and their weighted contributions influence odor coding may eventually reveal how peripheral information is organized and transmitted to subsequent layers of a neural circuit.

Differential Contributions of Olfactory Receptor Neurons in a Drosophila Olfactory Circuit.

The ability of an animal to detect, discriminate, and respond to odors depends on the functions of its olfactory receptor neurons (ORNs). The extent to which each ORN, upon activation, contributes to chemotaxis is not well understood. We hypothesized that strong activation of each ORN elicits a different behavioral response in the Drosophila melanogaster larva by differentially affecting the composition of its navigational behavior. To test this hypothesis, we exposed Drosophila larvae to specific odorants to analyze the effect of individual ORN activity on chemotaxis. We used two different behavioral paradigms to analyze the chemotaxis response of larvae to odorants. When tested with five different odorants that elicit strong physiological responses from single ORNs, larval behavioral responses toward each odorant differed in the strength of attraction as well as in the composition of discrete navigational elements, such as runs and turns. Further, behavioral responses to odorants did not correlate with either the strength of odor gradients tested or the sensitivity of each ORN to its cognate odorant. Finally, we provide evidence that wild-type larvae with all ORNs intact exhibit higher behavioral variance than mutant larvae that have only a single pair of functional ORNs. We conclude that individual ORNs contribute differently to the olfactory circuit that instructs chemotactic responses. Our results, along with recent studies from other groups, suggest that ORNs are functionally nonequivalent units. These results have implications for understanding peripheral odor coding.