Imaging Neural Activity in Intact, Semirestrained Drosophila Larvae

The Drosophila larval nerve cord, which is the equivalent of the vertebrate spinal cord, houses the circuits required to process somatosensory stimuli (e.g., tactile, temperature, vibration, and self-movement) and generate the patterned muscle contractions underlying movement and behavior. Within this complex structure reside many cell types and cellular processes, making it difficult to experimentally access, when compared to peripheral parts of the nervous system (i.e., primary sensory neuron dendrites, motor neuron axons and synapses, and muscles). Additionally, the neurons in the larval nerve cord have small cell bodies, precluding traditional electrophysiological approaches. As such, the function of neurons in the nerve cord is less well studied than other parts of the nervous system, severely limiting our understanding of how larvae process sensory information and generate movement. Ca²⁺-sensitive fluorescent proteins enable the study of neuronal activity in live, genetically tractable animals, even those with small neuronal cell bodies. In addition, live imaging of neurons within the nerve cord in whole, intact animals is possible because larvae are translucent, and the use of intact animals allows for the peripheral sensory neuron circuits to remain intact. Ca²⁺-sensitive fluorescent proteins increase their fluorescence when voltage-gated Ca²⁺ channels are opened in depolarized neurons. Here, we describe an assay where a Ca²⁺-sensitive fluorescent protein (GCaMP6m) is expressed under the control of a GAL4 driver in a subset of neurons that reside in a circuit for vibration sensation. External vibration (sound) stimulates sensory neurons that activate the cells expressing the Ca²⁺-sensitive fluorescent protein. Visualization of the calcium-induced fluorescent signal with microscopy allows for quantification of neuronal activity.