Richard Wade-Martins

In our brains, different neurons make appropriate connections; however, there remain few in vitro models of such circuits. We use an open microfluidic approach to build and study neuronal circuits in vitro in ways that fit easily into existing bio-medical workflows. Dumbbell-shaped circuits are built in minutes in standard Petri dishes; the aqueous phase is confined by fluid walls – interfaces between cell-growth medium and an immiscible fluorocarbon, FC40. Conditions are established that ensure post-mitotic neurons derived from human induced pluripotent stem cells (iPSCs) plated in one chamber of a dumbbell remain where deposited. After seeding cortical neurons on one side, axons grow through the connecting conduit to ramify amongst striatal neurons on the other – an arrangement mimicking unidirectional cortico-striatal connectivity. We also develop a moderate-throughput non-contact axotomy assay. Cortical axons in conduits are severed by a media jet; then, brain-derived neurotrophic factor and striatal neurons in distal chambers promote axon regeneration. As additional conduits and chambers are easily added, this opens up the possibility of mimicking complex neuronal networks, and screening drugs for their effects on connectivity.

Understanding medium spiny neuron (MSN) physiology is essential to understand motor impairments in Parkinson’s disease (PD) given the architecture of the basal ganglia. Here, we developed a custom three-chambered microfluidic platform and established a cortico-striato-nigral microcircuit partially recapitulating the striatal presynaptic landscape in vitro using induced pluripotent stem cell (iPSC)-derived neurons. We found that, cortical glutamatergic projections facilitated MSN synaptic activity, and dopaminergic transmission enhanced maturation of MSNs in vitro. Replacement of wild-type iPSC-derived dopamine neurons (iPSC-DaNs) in the striatal microcircuit with those carrying the PD-related GBA-N370S mutation led to a depolarisation of resting membrane potential and an increase in rheobase in iPSC-MSNs, as well as a reduction in both voltage-gated sodium and potassium currents. Such deficits were resolved in late microcircuit cultures, and could be reversed in younger cultures with antagonism of protein kinase A activity in iPSC-MSNs. Taken together, our results highlight the unique utility of modelling striatal neurons in a modular physiological circuit to reveal mechanistic insights into GBA1 mutations in PD.