Optogenetic control of selective neural activity in multiple freely moving Drosophila adults

Abstract

We present an automated laser tracking and optogenetic manipulation system (ALTOMS) for studying social memory in fruit flies (Drosophila melanogaster). ALTOMS comprises an intelligent central control module for high-speed fly behavior analysis and feedback laser scanning (∼40 frames per second) for targeting two lasers (a 473-nm blue laser and a 593.5-nm yellow laser) independently on any specified body parts of two freely moving Drosophila adults. By using ALTOMS to monitor and compute the locations, orientations, wing postures, and relative distance between two flies in real time and using high-intensity laser irradiation as an aversive stimulus, this laser tracking system can be used for an operant conditioning assay in which a courting male quickly learns and forms a long-lasting memory to stay away from a freely moving virgin female. With the equipped lasers, channelrhodopsin-2 and/or halorhodopsin expressed in selected neurons can be triggered on the basis of interactive behaviors between two flies. Given its capacity for optogenetic manipulation to transiently and independently activate/inactivate selective neurons, ALTOMS offers opportunities to systematically map brain circuits that orchestrate specific Drosophila behaviors.operant learning | restraining order | restraining conditioning S ocial interactions are an important part of human life because they help us learn how to behave in a society. However, the mechanisms by which the neuron circuitry controls and modifies our behavior on the basis of previous experiences of interactions with others remain unclear. Drosophila courtship conditioning has been widely used for studying how genes and brain circuits control and modify a specific type of social interaction (1-6). In this behavioral assay, individual male fruit flies learn to suppress their courtship activity after several hours of exposure to an unreceptive female. Specific cuticular pheromones, such as 9-pentacosene, have been shown to potentially serve as conditioned stimuli (4, 7-9). Visual inputs act as conditioned stimuli for courtship through modulation of Ca 2+ /calmodulin-dependent protein kinase activity in the brain circuitry (4). An aversive male pheromone, cis-vaccenyl acetate, which is transferred to the female during copulation, may act as a punishment so that the rejected male forms a generalized memory that suppresses its subsequent courtship behavior (10). However, little is known about where and how the neural activities that represent the antecedent conditions and aversive consequence are associated in the brain-knowledge that is crucial for understanding courtship memory and decision making-because controlling female rejection behaviors (11) and acutely manipulating target neurons in courting males during social interaction are difficult. Here, we present an automated laser tracking system for real-time analysis and perturbation of social interactions between two freely moving adult flies that is equipped with highenergy laser irradiation as a controllable punishment source ...

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

“…From these exciting results, we realize that non-invasive, versatile, economical behavioral machines will help scientists screen behavior-related neuron circuits. In the present work, we improved our previous automated laser tracking and optogenetic manipulation system (ALTOMS) [10]. First, we found that ventral irradiation requires reduced laser intensity compared to dorsal irradiation.…”

“…In ventral irradiation ALTOMS, we attempted to use an economical long-pass filter (Thorlab FGL610, 610 nm long-pass color filter) to replace the two notch filters (Kaiser HNF-473.0, HNF-593.5) used in the first-generation ALTOMS [10]. Figure 2(D) displays the image of the CCD camera with the notch filters.…”

“…The ChR2 protein does not function properly in flies of the control batch [4]. For the jumping reflex assay, the dorsal anterior head and ventral posterior head of each fly were targeted with a 473-nm laser and illuminated for 10 cycles of 3-s on-and-off intervals [10]. For the backward reflex assay, backward rates were determined during 15 cycles of 1-s on/9-s off 593.5-nm laser irradiation on the thorax of VT50660-Gal4 > UAS-ReaChR at different laser energies and irradiation directions.…”

“…To understand the causal relationship between neural circuits and behavioral phenotypes, neuroscientists have created many behavioral assays using implanted optogenetic tools that monitor excitatory and/or inhibitory signals at the millisecond scale to record specific behavioral responses of fruit fly larvae [7,8], fruit fly adults [9], and one specific adult fly among multiple flies [10]. From these exciting results, we realize that non-invasive, versatile, economical behavioral machines will help scientists screen behavior-related neuron circuits.…”

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].…”