Optical Recording of Visually Evoked Activity in the Drosophila Central Nervous System

Drosophila has become a powerful experimental animal for the analysis of neuronal circuits and computations underlying innate behavior. In Drosophila , perturbational genetics is currently combined with the direct recording of neural activity in the CNS and eventually the quantitative analysis of behavior. Any deviation of the recorded response from the normal response is indicative of the functional role of the manipulated neurons and mechanisms in a specific computation or behavior. In these experiments, strong correlation is established by directly recording the membrane potential with electrodes (whole cell recording from the soma) or by optical recording changes in the concentration of intracellular calcium. In addition to recordings from the soma, optical measurements provide access to subcellular compartments as well as large ensembles of visual interneurons. Furthermore, optical recordings are not limited by the small size of the cell body, and neurons located deep inside the brain can be analyzed by using two-photon laser scanning microscopy (2PLSM). The latter aspect is of particular importance, as studying vision in flies requires that the large compound eyes covering almost the entire head of the fly remain fully intact. However, optical imaging of sensory processing in the fly visual system comes along with inherent difficulties of the approach: Fluorescence excitation causes blinding of the fly and photons from the visual stimulus enter the detection pathway and corrupt the recorded signals. In this chapter, I describe a method and guidelines suitable to bypass these problems. Genetic targeting of a population of visual interneurons is used to express a genetically encoded fluorescent indicator for intracellular calcium (GECI). The GECI molecules are expressed in the soma as well as all subcellular compartments. Thus, the requirement of dye application is overcome, and ultimately, a functionally homogeneous population of neurons can be analyzed with high spatial resolution. Fluorescence of the GECI is excited and recorded using in vivo 2PLSM that helps to prevent direct excitation of photoreceptors by laser light. Optical recordings are performed during visual stimulation and sensory processing of the fly. By separating fluorescence recording and visual stimulus presentation in time, even most subtle changes in GECI fluorescence are captured, while the visual stimulus is excluded from the recorded fluorescence signal.