Optogenetic manipulation of neural circuits and behavior in Drosophila larvae

Abstract

Optogenetics is a powerful tool that enables the spatiotemporal control of neuronal activity and circuits in behaving animals. Here, we describe our protocol for optical activation of neurons in Drosophila larvae. As an example, we discuss the use of optogenetics to activate larval nociceptors and nociception behaviors in the third-larval instar. We have previously shown that, using spatially defined GAL4 drivers and potent UAS (upstream activation sequence)-channelrhodopsin-2∷YFP transgenic strains developed in our laboratory, it is possible to manipulate neuronal populations in response to illumination by blue light and to test whether the activation of defined neural circuits is sufficient to shape behaviors of interest. Although we have only used the protocol described here in larval stages, the procedure can be adapted to study neurons in adult flies--with the caveat that blue light may not sufficiently penetrate the adult cuticle to stimulate neurons deep in the brain. This procedure takes 1 week to culture optogenetic flies and ~1 h per group for the behavioral assays.

Figure 1. Workflow for the optogenetic activation of larval nociception

The chart summarizes procedures and necessary materials for this protocol.

Figure 2. Efficacy of behavioral response compared amongUAS-channelrhodopsin-2 lines

(a) Comparison of Channelrhodopsin-2∷YFP fluorescence in various UAS-channelrhodopsin-2∷YFP insertion lines generated by Hwang et al. (2007). YFP signal intensities are shown as normalized to line C. (b) Frequency of larval nociception behavior seen when ppk1.9-GAL4 was crossed to the various UAS-channelrhodopsin-2∷YFP lines. Note the frequency of triggered nociception behavior correlates with the expression level of Channelrhodopsin∷YFP Reprinted with modifications from Hwang et al. (2007) with permission.

Figure 3. Setup of videorecording and illuminating devices

The photograph shows the stereofluorescence microscope with a digital video recorder where optogenetic behavioral experiments are performed in our laboratory.

Figure 4. Optogenetically triggered nociceptive responses in all-trans-retinal fed ppk1.9-GAL4/UAS-channelrhodopsin-2∷YFP and control animals

Blue light induced nocifensive escape locomotion was seen in 83±2.8% of all-trans-retinal fed larvae expressing Channelrhodopsin-2∷YFP in nociceptive sensory neurons (ppk × UAS-ChR2∷YFP, ATR+; n = 181, p < e2-16 compared to the controls with Fisher's exact test with Boferroni correction). In contrast, neither the all-trans-retinal fed controls (ppk × UAS-ChR2∷YFP, ATR-; n = 112) nor the driverless control with all-trans-retinal supplementation (w¹¹¹⁸ × UAS-ChR2∷YFP, ATR+; n = 102) showed nocifensive responses to light stimulation (0.9±0.9% and 2±1.4%, respectively). Error bars represent the standard error. The graph was made based on the data originally published in Hwang et al. (2007) with permission.