Optical Dissection of Neural Circuits Responsible for Drosophila Larval Locomotion with Halorhodopsin

Abstract

Halorhodopsin (NpHR), a light-driven microbial chloride pump, enables silencing of neuronal function with superb temporal and spatial resolution. Here, we generated a transgenic line of Drosophila that drives expression of NpHR under control of the Gal4/UAS system. Then, we used it to dissect the functional properties of neural circuits that regulate larval peristalsis, a continuous wave of muscular contraction from posterior to anterior segments. We first demonstrate the effectiveness of NpHR by showing that global and continuous NpHR-mediated optical inhibition of motor neurons or sensory feedback neurons induce the same behavioral responses in crawling larvae to those elicited when the function of these neurons are inhibited by Shibirets, namely complete paralyses or slowed locomotion, respectively. We then applied transient and/or focused light stimuli to inhibit the activity of motor neurons in a more temporally and spatially restricted manner and studied the effects of the optical inhibition on peristalsis. When a brief light stimulus (1–10 sec) was applied to a crawling larva, the wave of muscular contraction stopped transiently but resumed from the halted position when the light was turned off. Similarly, when a focused light stimulus was applied to inhibit motor neurons in one or a few segments which were about to be activated in a dissected larva undergoing fictive locomotion, the propagation of muscular constriction paused during the light stimulus but resumed from the halted position when the inhibition (>5 sec) was removed. These results suggest that (1) Firing of motor neurons at the forefront of the wave is required for the wave to proceed to more anterior segments, and (2) The information about the phase of the wave, namely which segment is active at a given time, can be memorized in the neural circuits for several seconds.

A Model of Larval Biomechanics Reveals Exploitable Passive Properties for Efficient Locomotion

“…Positive feedback may still be used to align ongoing neural motor control signals with the mechanics of the body and environment. This is consistent with the observation that waves of muscle activation travel slower in larvae which have been experimentally deprived of mechanosensory input [18,19].…”

Principles of designing interpretable optogenetic behavior experiments

“…Light may also distract the animal, perhaps eliciting increased locomotion (Godsil and Fanselow 2004) or encouraging the use of a visual distractor to mask the optogenetic stimulus (Huber et al 2008). Choosing wavelengths of light that the animal cannot see, or is less responsive to, may ameliorate this (Inada et al 2011;Klapoetke et al 2014), as can usage of animals that are engineered to be insensitive to light (Kocabas et al 2012).…”

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

“…Next, we evaluated the effectiveness of optogenetic inhibition of neural activity in living flies with pan-neuronal expression of NpHR, which is a yellow light-driven pump specific for chloride ions (22,(35)(36)(37). With continuous yellow laser irradiation of the head or thorax, we found that experimental flies fed with alltrans-retinal exhibited an anesthesia rate positively correlated with laser energy and that most flies fainted after 60 s or 400 s laser irradiation at 34 or 23 mW/mm 2 , respectively (Fig.…”

“…Recent advances in the optogenetic manipulation of neural activity at the millisecond time scale by using light-activated channelrhodopsin-2 (ChR2) excitation or halorhodopsin (NpHR) inhibition have made it possible to study how neural circuits control behavior (18)(19)(20)(21)(22)(23). Combining online image analysis and two lasers for acute punishment and optogenetic manipulation of selective neural activities, this automatic laser tracking and optogenetic manipulation system (ALTOMS) can precisely specify the timing of the purported associated events (antecedent conditions and response-dependent outcome), which could not be done in previous studies; this ability gives us better experimental control over courtship conditioning and an automated platform to systematically identify the neural circuits responsible for specific Drosophila behaviors.…”