Using Light-Sheet Microscopy to Understand Evoked Motor Sequence Generation

Abstract

Motor sequences are critical elements of everyday behavior, but how they are produced by central neural circuits is poorly understood. The complexity and scale of the circuitry involved makes motor sequence generation very difficult to study at the cellular level in large, mammalian brains. However, the neural circuits of the fruitfly also drive complex motor sequences, and are small enough to investigate at a brain-wide scale via emerging methods in microscopy. A critical behavioral sequence for the fruitfly, called ecdysis, is required for molting at each developmental stage and consists of three serially executed, stereotyped behavioral programs at the pupal stage. The neural circuit controlling the pupal ecdysis sequence includes approximately 300 peptidergic neurons that express the Ecdysis Triggering Hormone receptor (ETHR) and are activated by peripheral release of Ecdysis Triggering Hormone (ETH). The activation of these neurons leads directly to the three phases of associated motor neuron activity that mediate the ecdysis sequence, and existing data indicate that specific subpopulations are required for each behavioral phase of ecdysis. However, the identities of the individual neurons that control each behavioral phase remain largely unknown, as do the mechanisms by which they regulate motor output. To achieve a detailed cellular-level understanding of the ecdysis circuit, we have built a light-sheet microscope that is capable of imaging the Drosophila pupal CNS rapidly at high resolution. We are currently using calcium biosensors to monitor the neural activity in ETHR-expressing neurons and motor neurons of excised brains in response to ETH. Single-cell activity imaging on the light-sheet microscope confirms that individual neurons respond to ETH with varying onset times and distinct activity profiles that suggest the generation of fictive ecdysis behavior. Analysis of these data, and data collected from other populations of neurons, are being used to generate a predictive model of the circuit underlying the ecdysis sequence with the general goal of understanding how nervous systems orchestrate complex motor sequences in response to input stimuli.