Spatiotemporally precise optogenetic activation of sensory neurons in freely walking Drosophila.

Previous work has characterized how walking Drosophila coordinate the movements of individual limbs (DeAngelis et al., 2019). To understand the circuit basis of this coordination, one must characterize how sensory feedback from each limb affects walking behavior. However, it has remained difficult to manipulate neural activity in individual limbs of freely moving animals. Here, we demonstrate a simple method for optogenetic stimulation with body side-, body segment-, and limb-specificity that does not require real-time tracking. Instead, we activate at random, precise locations in time and space and use post hoc analysis to determine behavioral responses to specific activations. Using this method, we have characterized limb coordination and walking behavior in response to transient activation of mechanosensitive bristle neurons and sweet-sensing chemoreceptor neurons. Our findings reveal that activating these neurons has opposite effects on turning, and that activations in different limbs and body regions produce distinct behaviors.