Tracking <em>Drosophila</em> Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

Abstract

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

“…Furthermore, odorants exhibit various levels of volatility, complicating interpretation of behavioral studies, which depends on the formation of stable odor gradients (Monte et al, 1989; Gershow et al, 2012). To activate individual ORNs, we utilized a recently reported optogenetic technique (Hernandez-Nunez et al, 2015; Clark et al, 2018). The optogenetic approach is also advantageous in that one can alter the frequency of light stimulation, enabling us to vary the temporal patterns of ORN activation, which is difficult to achieve using odor stimuli.…”

“…We genetically expressed CsChrimson in individual pairs of ORNs (using individual Or-Gal4s). ORNs expressing CsChrimson were activated by shining red LEDs (630 nm, 1.3 W/m 2 intensity) on larvae navigating in the presence of the anisole gradient (Figure 3A; Clark et al, 2018). We varied our stimulus to alternate between lights OFF and ON for a total of 3 min (Figure 3B).…”

“…Larval movement was tracked in the presence and absence of CsChrimson activation and in the presence and absence of the directional cue. We then measured navigational parameters as described in previous studies (Gershow et al, 2012; Mathew et al, 2013; Newquist et al, 2016; Clark et al, 2018).…”

“…Using the Ubuntu mate operating system, we programmed three temporal variations of red LED light stimulus during the second minute: 0.04 Hz, 1 Hz, and constant (Figure 3B). Detailed descriptions of how to construct the behavior arena and the LED platform, and how to process the data for the optogenetics assay are provided in Clark et al (2018).…”

“…Based on the findings of recent studies (Kreher et al, 2008; Ince et al, 2013), we hypothesized that larval ORNs can be grouped into a small number of functional subsets. Aspects of this study were made possible by the recent development of a technique that enables the researcher to track larval navigation while simultaneously activating individual olfactory neurons using optogenetics (Klapoetke et al, 2014; Hernandez-Nunez et al, 2015; Clark et al, 2018). Optogenetic activation with CsChrimson allows for specific and reliable activation of individual ORNs, and also allows us to vary the temporal patterns of their activation.…”