Microfluidics of small-population neurons allows for a precise quantification of the peripheral axonal growth state

Abstract

Neurons are morphologically the most complex cell types and are characterized by a significant degree of axonal autonomy as well as having efficient means of communication between axons and neuronal cell bodies. For studying the response to axonal injury, compartmentalized microfluidic chambers (MFCs) have become the method of choice because they allow for the selective treatment of axons, independently of the soma, in a highly controllable and reproducible manner. A major disadvantage of these devices is the relatively large number of neurons needed for seeding, which makes them impractical to use with small-population neurons, such as sensory neurons of the mouse. Here, we describe a simple approach of seeding and culturing neurons in MFCs that allows for a dramatic reduction of neurons required to 10,000 neurons per device. This technique facilitates efficient experiments with small-population neurons in compartmentalized MFCs. We used this experimental setup to determine the intrinsic axonal growth state of adult mouse sensory neurons derived from dorsal root ganglia (DRG) and even trigeminal ganglia (TG). In combination with a newly developed linear Sholl analysis tool, we have examined the axonal growth responses of DRG and TG neurons to various cocktails of neurotrophins, glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) and leptin. Precise quantification of axonal outgrowth revealed specific differences in the potency of each combination to promote axonal regeneration and to switch neurons into an intrinsic axonal growth state. This novel experimental setup opens the way to practicable microfluidic analyses of neurons that have previously been largely neglected simply due to insufficient numbers, including sensory neurons, sympathetic neurons and motor neurons.