In optogenetics, neuron cells are manipulated in vivo by light (often laser light) to help in understanding the way the brain and nervous system work, including at high levels such as in social and behavioral studies. One important technique in optogenetics is the use of genetically modified animals, which can allow specific neural circuits to be better targeted by optical probing.
A group of researchers from National Tsing Hua University and Hsuan Chuang University, both in Hsinchu, Taiwan, Academia Sinica (Nankang Taipei, Taiwan), and the University of California, San Diego (La Jolla) has been developing and improving an optogenetic setup for studying memory in the common fruit fly (Drosophila Melanogaster) called the automated laser-tracking and optogenetic-manipulation system (ALTOMS). The noninvasive system can simultaneously deliver heat “punishment” (serving as an incentive for the fly to alter its behavior) as well as simultaneously activate channelrhodopsin-2 (ChR2) and a red-shifted variant of channelrhodopsin (ReaChR), which serve to activate or inhibit specific neurons. The ALTOMS also includes a real-time image-analyzing system to track the flies.
The researchers have shown experimentally that ALTOMS is capable of contributing to experiments on Drosophila behavior; for example, they anticipate that the system could be used to map memory circuits in the Drosophila brain.
Three-color system
The system has three wavelengths: 1064 nm for inflicting heat punishment (earlier setups had used a blue laser, which, because it was visible to the flies, created conflicting reactions in the flies) and 473 and 593 nm to activate ChR2 and ReaChR. The beams are directed at the flies using galvanometer mirrors and, optionally, a digital micromirror device (DMD), with the DMD giving the setup the ability to produce arbitrary illumination patterns for neuronal triggering. The researchers also replaced an earlier set of delicate notch filters with a more robust long-pass filter.
The setup contains four subsystems (see figure): the arena itself (the 20-mm-diameter x 3-mm-high chamber in which the fly is experimented on); the image-capture module (ICM), which contains white LED illumination and a CCD camera; the laser-scanning module (LSM); and the intelligent central-control module (ICCM), which analyzes the video from the ICM and calculates how to target the fly (or flies) based on the fly positions, orientations, and wing-extension angles. The researchers also developed an easy-to-use graphical user interface (GUI).
The cuticle of the fruit fly, through which the triggering light must pass, is nonuniform, so the researchers measured its optical transmission at 473 nm in six places: dorsal (backside) anterior head, dorsal thorax, dorsal abdomen, ventral (front-side) abdomen, ventral posterior head, and proboscis. The ventral side of the fly proved to be the best area for laser illumination due to its higher optical transmission and greater concentration of neurons.
Experiments showed that the real-time tracking and analysis system had a very low error rate of below 0.0071%. Fly orientation was detected at an accuracy of 94%. To test restraining conditioning, a wild virgin female fly and a male fly (both genetically modified for neuronal triggering by light) were placed in the arena. The male fly was hit with a punishment beam if it lingered at a distance of 3.5 mm or less from the female for longer than 2 s. Testing confirmed that the flies could be trained against their natural behavior.
For jumping and backward-reflex tests, a control group of flies was fed a diet that inactivated the ChR2 protein; the experimental group ate food that kept the ChR2 functional. Jumping was tested with 473 nm light in 10 cycles of 3 s on-and-off intervals aimed at the fly’s head, while the backward reflex was tested using 593 nm light aimed at the fly’s thorax.
The experiments showed that jumping starts being triggered at a 3 mW/mm2 light intensity and saturates at a 21 mW/mm2 intensity. In addition, only “giant fiber” neurons triggered their jumping. Backward reflex tests showed results consistent with the fly’s anatomy and neuron placements.
“The major improvements of the second-generation ALTOMS system are that the irradiation direction is changed to the ventral side and an IR laser is inserted because we want to create a less perturbative ALTOMS system,” says Yen-Yin Lin, an assistant research fellow at National Tsing Hua University. “Our next focus will be the development of a new arena for a new behavior assay. In our current demonstration, this system can noninvasively trigger ChR2, NpHR [another rhodopsin-enabled protein], ReaChR, and invisible punishment, so the design of the arena is flexible. As a result, we will try to create different arena designs.” The group will also begin to systematically study various fruit fly behaviors using ALTOMS.
REFERENCE
1. Y.-Y. Lin et al., Biomed. Opt. Express 6(2) (Jan. 2015); http://dx.doi.org/10.1364/BOE.6.000514.
