Probing Synaptic Transmission and Behavior in Drosophila with Optogenetics: A Laboratory Exercise

Abstract

Optogenetics is possibly the most revolutionary advance in neuroscience research techniques within the last decade. Here, we describe lab modules, presented at a workshop for undergraduate neuroscience educators, using optogenetic control of neurons in the fruit fly Drosophila melanogaster. Drosophila is a genetically accessible model system that combines behavioral and neurophysiological complexity, ease of use, and high research relevance. One lab module utilized two transgenic Drosophila strains, each activating specific circuits underlying startle behavior and backwards locomotion, respectively. The red-shifted channelrhodopsin, CsChrimson, was expressed in neurons sharing a common transcriptional profile, with the expression pattern further refined by the use of a Split GAL4 intersectional activation system. Another set of strains was used to investigate synaptic transmission at the larval neuromuscular junction. These expressed Channelrhodopsin 2 (ChR2) in glutamatergic neurons, including the motor neurons. The first strain expressed ChR2 in a wild type background, while the second contained the SNAP-25^ts mutant allele, which confers heightened evoked potential amplitude and greatly increased spontaneous vesicle release frequency at the larval neuromuscular junction. These modules introduced educators and students to the use of optogenetic stimulation to control behavior and evoked release at a model synapse, and establish a basis for students to explore neurophysiology using this technique, through recapitulating classic experiments and conducting independent research.

Keywords: Drosophila; SNAP-25; behavior; electrophysiology; neuromuscular junction; optogenetics.

Figure 1

LED and power supply assembly. (A) A voltage controlled current source is mounted in an enclosure that holds input, output, power, and switch components. (B) Blue (460 nm) or Red (625 nm) LED is mounted on a heatsink. (C) Port for voltage control. (D) Power output ports for LED. (E) Switching power supply and input. LED light source apparatus modified and upgraded from Pulver et al., 2011.

Figure 2

Drosophila adult and larval CNS. (A) Larval OK371-GAL4 driving expression of mCherry-tagged channelrhodopsin2 in motor neurons of the ventral ganglion (VG) and GFP expression in muscle. Note expression of channelrhodopsin2 in motor neuron axons. Location of muscles 6 and 7 are indicated. The inset has increased brightness of mCherry to show the innervation of the neurons at the muscles. (B) Confocal image of Moonwalker-GAL4 driving expression of GFP in adult brain (green) and the presynaptic marker BRP (magenta).

Figure 3

Sample student recordings from the Drosophila third install larva neuromuscular junction. Evoked potentials are elicited by brief pulses of blue light from a high power LED in transgenic strains expressing Channelrhodopsin 2 in glutamatergic neurons. (A) Wild type evoked potential and miniature endplate potentials. (B) SNAP-25^ts, showing characteristically elevated frequency of miniature endplate potentials indicated by arrowheads.