FlexiChip1.0: A simple device for in vivo imaging of sensory-motor activities in the Drosophila larval CNS
With the advent of many genetically encoded neurophysiological fluorescent sensors, it is desirable to develop methods to reliably record central nervous system (CNS) activity in intact specimens. In the Drosophila larva, the near transparent body-wall is ideal for conducting live-imaging of its CNS, but intrinsic motor activities hinder the stable recording of fluorescent signals. One major component that results in CNS movement involves the digging apparatus located in the anterior-end of the larva (also called the Cephalo-Pharyngeal Skeleton (CPS)). The CPS and associated muscles drive digging and feeding behaviors, and its attachment to the CNS capsule results in severe displacements of the CNS during live-imaging. To overcome this problem we designed a PDMS (PolyDiMethylSiloxane) chip that traps the CPS of the larva so that movement of the CNS from the anterior is reduced.
Figure panels above summarize the basic design and operation of the device. A. Key features in the chip are a main channel (arrow) that fits the larva, a clasping mechanism (perpendicular double-arrow) and side channels (arrowhead). The main channel is contoured to fit a mid or late 3rd instar larva, the auxillary channels can be used to keep the preparation moistened, or for the introduction of electrical or mechanical probes for body-wall stimulation. B. Bending the PDMS chip opens the clasp (double-headed arrow) so that the CPS area can be inserted into the gap (arrow in C), so that the larva’s ventral side is facing upwards, and then carefully releasing of the bending force to trap the CPS/anterior-end of the larva. This anchoring procedure negates or isolates its movement. D. Arrowhead in D indicates the approximate location of the CNS ventral cord (VC) that resides just below the ventral body-wall of the larva so that live-imaging can be carried out, often with almost no extraneous tissue obstruction. E. Schematic of the set up, cross-sectional view (not drawn to scale). Larval cartoon (L) is colored in blue. A cover-glass is placed on top of the larva before visualization of fluorescent activities in the VC. The larval posterior-end protrudes into a funnel shaped outer chamber that is open to ambient air, this allows respiration to continue through posterior spiracles during live-imaging.
