Uncovering the neural encoding of behavior in the adult Drosophila motor system

One of the most important goals in neuroscience research has always been to understand how animals control their behavior. However, the long focus on the role of brain neurons in behavioral control might be missing the full story. In fact, brain-wide fluctuations in neural activity are highly correlated to the behavioral state, suggesting a closed-loop system where the behavioral command and the body state interact to interpret sensory inputs and dynamically guide action selection. However, to date, little is known about how and where information about the behavioral state is generated. In principle, information about the behavioral state is a relatively high-level encoding of the body state derived from the integration of signals from primary sensors and the local network. Local motor circuits residing in the spinal cord of mammals or the ventral nerve cord (VNC) of insects may give rise to the behavioral state and pass to the brain via brain-projecting interneurons---termed ascending neurons (ANs). However, what information ANs carry and where this information originates is still a mystery due to the technical challenges in measuring these signals in behaving animals. Therefore, it is critical to develop tools to track neuron identity and record neuronal activity during behavior to crack the functional network. In this work, a method is introduced for imaging the motor circuit in the VNC of Drosophila melanogaster. This preparation demonstrates how to retain fly limb function after dissection and allows simultaneous recording of a tethered fly's behavior and its neural activity in the VNC. Incorporating this methodology with other approaches can verify the conclusions from the experiments such as those manipulating neuronal excitability, and motivate further investigation into neuronal function in specific types of behavior. Since it has long been theorized that ANs are the cellular basis of the behavioral state information sent in the brain, the technique introduced in this work was designed to resolve questions relating to what type of information ANs convey. Here, a large-scale functional screen was performed to relate neural activity, behavior, and neuronal morphology for 247 genetically identified ANs in the adult fly. This is the first large-scale screen of neural activity in a behaving animal, and this work uncovered three fundamental features of AN populations. First, rather than low-level limb movements, ANs encode high-level behaviors like walking, grooming, and resting. Second, ANs project nearly exclusively to an integrative sensory center (AVLP) and an action selection hub (GNG). Remarkably, the encodings projected to these distinct areas reflect their potential roles in contextualizing sensory cues (AVLP) and guiding future actions (GNG). Third, the behavioral encodings of ANs are closely linked to their motor system patterning. In conclusion, this work provides an indispensable tool to study the motor system beyond the brain network by overcoming a major technical barrier in in-vivo recording of the VNC of a behaving fly. This tool was used to help resolve what is encoded by ANs as a higher-level behavioral state, suggesting that the primary cellular origin of the behavioral information that is used to modulate brain function comes from the VNC. Overall, the technique and the findings on AN encoding should inspire follow-up studies to uncover additional neural mechanisms used in adaptive behavior generation.