Three-wavelength light control of freely moving Drosophila Melanogaster for less perturbation and efficient social-behavioral studies

Abstract

Abstract:We developed a real-time automated laser-tracking system combined with continuous wave 1064-nm infrared or 473-nm blue lasers to provide punishment for studying memory in Drosophila Melanogaster. Combining optogenetic tools with laser properties, such as 473-nm and 593-nm lasers that activate light sensitive proteins in artificial transgenic flies, we can manipulate the specific neuron of an assigned fly among multiple flies to investigate neuron circuit relationships in social interactions. In restraining condition assay or optogenetic experiments, a ventral irradiated system would be more efficient due to higher ventral cuticle transmissions and neuron ganglia locations. Therefore, ventral irradiated systems cause less perturbation during behavior studies.

SkinnerTrax: high-throughput behavior-dependent optogenetic stimulation ofDrosophila

“…Existing systems that allow such behavior-dependent stimulation have not been designed for high throughput. Two recent advanced systems, FlyMAD 7 and ALTOMS 8,9 , target a laser to flies in real-time using mirror galvanometers, which in principle allows sharing the laser between several flies, as suggested in the discussion section of Bath et al 7 But the scalability of sharing the laser is limited (Supplementary Note 1), and laser-targeting systems require expensive components and complex setups. Thus, for a typical lab laser-targeting systems likely make achieving high throughput prohibitively difficult.…”

Proprioceptive feedback determines visuomotor gain inDrosophila

“…For comparison, Sinha et al used a train of approximately hundred UV laser pulses (193 nm) with an energy of 170µ J each (∼17 mJ total energy) in order to perforate the head cuticle in Drosophila for functional brain imaging [35]. Considering the ∼4.3 times smaller photon energy and ∼10 times larger cuticle transmission at 830 nm compared to 193nm wavelength [36], total thermal energy applied to the wing cuticle is only ∼2.3% of the critical value suggested by [35]. The risk of heat-induced cuticle weakening was thus negligibly small (see also section below).…”

Non-invasive manipulation of Drosophila behavior by two-photon excited red-activatable channelrhodopsin

“…For example, channelrhodopsin-2 (ChR2) is a light-gated ion channel that has already proven to be useful in the fruit fly [1][2][3][4][5]. The most efficient wavelength for activating ChR2 is 460 nm [6].…”

“…Because scattering effects are inversely proportional to the fourth power of the radiation wavelength, strong scattering and pigment absorption increase the power level required to activate ChR2 in vivo. Hence, even small improvements can facilitate in vivo experiments, such as optical transmission through the cuticles [4]. Longer wavelengths can be used to achieve deeper penetration in living animals and reduce scattering effects.…”

“…Red-activatable ChR (ReaChR), for example, which is optimally excited by light in the orange to red region (590-630 nm) [10,12], was developed to perform single-photon neuron activation in deep-tissue experiments. The feasibility of this approach has been experimentally demonstrated with the fruit fly [4,12]. On the other hand, a red-shifted cruxhalorhodopsin, Jaws, can perform single-photon neuron inhibition in deep-tissue experiments [13].…”