Partial Treatment of In Vivo Single Axons by Mounting a Microfluidic Device Directly

Abstract

Mixing happens slowly under flow conditions with a low Reynolds number when viscous effect is dominant. This allows multi-channel perfusion to flow laminarly even without any physical separations. Furthermore, the individual flow width, and therefore position, is governed by the respective volumetric flow rate, enabling flow focusing. This has been used in a variety of biological context, including single cells partial treatment studies called PARTCELL. PDMS is the most common material for making microfluidics devices. Typically a slab of PDMS with channels patterned is plasma-treated such that it can form bonds with a glass slide to seal the channels. Suspendable samples (usually dissociated cells) are subsequently flown into the channels and let adhere to the glass slide before an experiment commences. This process severely limits the control over sample placement and sample selection. Here, we propose a novel direct mounting method that enables sample placement and preparation to precede device sealing, thus extending the use of partial treatment to study small living organisms. The device was used to treat single axons partially in their proximal segments with Y-27632, a known axonal tension disruptor through inhibiting the ROCK pathway. Such treatment allowed us to reduce the tension level in the axons being perfused. Once tension was reduced, we observed simultaneously a reduction in vesicle density at the distal presynaptic terminal (PT) of the axons. Switching off the Y-27632 flow can reverse this process and led to the restoration of vesicles at the PTs. Note that the distal PTs were not subjected to any treatment (drug flow only at proximal segments), thus suggesting that tension along the axons directly correlates with the vesicle density at the PTs. Such fundamental experiments cannot be performed without using the direct mounting method.