Spatial aspects of GDNF functions revealed in a compartmentalized microfluidic neuromuscular co-culture system

Abstract

Bidirectional molecular communication between the motoneuron and the muscle is vital for neuromuscular junction (NMJ) formation and maintenance. The molecular mechanisms underlying such communication are of keen interest and could provide new targets for intervention in motoneuron disease. Here, we developed a microfluidic platform with motoneuron cell bodies on one side and muscle cells on the other, connected by motor axons extending through microgrooves to form functional NMJs. Using this system, we were able to differentiate between the proximal and distal effects of oxidative stress and glial-derived neurotrophic factor (GDNF), demonstrating a dying-back degeneration and retrograde transmission of pro-survival signaling, respectively. Furthermore, we show that GDNF acts differently on motoneuron axons versus soma, promoting axonal growth and innervation only when applied locally to axons. Finally, we track for the first time the retrograde transport of secreted GDNF from muscle to neuron. Thus, our data suggests spatially distinct effects of GDNF – facilitating growth and muscle innervation at axon terminals and survival pathways in the soma.

On-chip, multisite extracellular and intracellular recordings from primary cultured skeletal myotubes

“…One plausible reason why MEA technology has not been adopted for skeletal muscle research and diagnostics is because progress in the culturing technologies of skeletal myotubes and NMJ from rodents and human is relatively recent151617181920. To date, only two original studies have been published on the use of substrate integrated planar MEA to electrophysiologically record from cultured skeletal myotubes2122.…”

“…It is important to note that currently in vitro preclinical drug screening as well as characterizations of skeletal muscle development and pathophysiology rely mainly on imaging of muscle contraction dynamics and associated alterations in their free intracellular calcium concentrations ([Ca 2+ ] i )15182023242526. The monitoring of muscle contraction dynamics or [Ca 2+ ] i imaging cannot provide the information obtained by electrophysiological recordings since [Ca 2+ ] i imaging reflects the integral of complex processes of Ca 2+ influx through voltage gated calcium channels, voltage dependent or independent release of Ca 2+ from intracellular stores, and the removal dynamics of Ca 2+ by a very large number of mechanisms.…”

A system for studying mechanisms of neuromuscular junction development and maintenance

“…We believe that such a method could allow both (1) growth of all the necessary neuronal cells (neurons and glial cells) and (2) the natural dynamics of paracrine secretion of trophic factors important for synaptogenesis to occur, as suggested in the recent work of Zahavi et al (2015).…”

“…To overcome these problems, different in vitro coculture systems have been set up in which motor neuron and skeletal muscle are grown together in order to recapitulate the formation and eventual disruption of the NMJ. To date, co-culture methods established from various species have been described, including mouse (Morimoto et al, 2013;Zahavi et al, 2015), rat (Das et al, 2010;Southam et al, 2013), Xenopus (Lu et al, 1996;Peng et al, 2003) and chick (Frank and Fischbach, 1979), and also heterologous co-cultures built from motor neuron and muscle cells obtained from different species, such as rat-human , mouse-human (Son et al, 2011) and mouse-chick (Soundararajan et al, 2007). However, these co-culture methods resulted in the formation of immature myofibers (thin muscle fiber, with centrally localized nuclei and no transversal triads) with immature sarcomeric structures (Das et al, 2007(Das et al, , 2009Southam et al, 2013).…”

Microfluidic Models of Neuromuscular Junctions

“…This device claims to be the first in monitoring in situ chemical synaptic transmission under physiologically relevant conditions. 15 Tong et al 16 & Zahavi et al 17 have achieved the bidirectional communication between motor neurons and muscle cells for the formation and healthy maintenance of neuromuscular junction. 16,17 The devices mentioned above hold great potential in high throughput drug screening, therapy design, conducting physiological and mechanistic studies, replacing animal models, soft-robotic actuators 18 etc..…”