<em>In vivo</em> Imaging of Intact <em>Drosophila</em> Larvae at Sub-cellular Resolution

Abstract

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution [1][2][3] . While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described 4,5,6,7,8 , the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm 1 , (2) a high survival rate of > 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days 2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter 1 in stable transgenic lines and the exon trap line FasII-GFP , the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

The Kinesin-3, Unc-104 Regulates Dendrite Morphogenesis and Synaptic Development in Drosophila

“…Next, larvae were mounted in a chamber originally designed for in vivo imaging and imaged within 15 min on a Zeiss LSM 710 confocal microscope. Details on the construction of the chamber and larvae mounting have been previously described (Fuger et al 2007; Zhang et al 2010). …”

Methods for studying planar cell polarity

“…The prepupa was paralyzed using levamisole. Similar methods can be used for larvae that need to be paralyzed or anesthetized, and are therefore studied in closed mounting arrangement with such agents added (Liu et al, 2012; Zhang et al, 2010). …”

Examining molecular determinants underlying heterogeneity of synaptic release probability using optical quantal imaging

“…However, this is not technically feasible due to the small size of AZs and the rapid locomotion that larvae undergo, preventing generation of P r maps in intact moving animals. Instead, we employed an alternative approach to repeatedly image the same NMJ at muscle 26 directly through the cuticle of intact larvae during anesthesia (Andlauer and Sigrist, 2012; Fouquet et al, 2009; Füger et al, 2007; Rasse et al, 2005; Zhang et al, 2010). Using this technique, we found that anesthesia eliminated action potential induced release and the associated GCaMP signals, preventing direct P r measurements in anesthetized larvae.…”