Abstract
New in vitro technologies that assess neuronal excitability and the derived synaptic activity within a controlled microenvironment would be beneficial for the characterisation of compounds proposed to affect central nervous system (CNS) function. Here, a microfluidic system with computer controlled compound perfusion is presented that offers a novel methodology for the pharmacological profiling of CNS acting compounds based on calcium imaging readouts. Using this system, multiple applications of the excitatory amino acid glutamate (10 nM–1 mM) elicited reproducible and reversible transient increases in intracellular calcium, allowing the generation of a concentration response curve. In addition, the system allows pharmacological investigations to be performed as evidenced by application of glutamatergic receptor antagonists, reversibly inhibiting glutamate-induced increases in intracellular calcium. Importantly, repeated glutamate applications elicited significant increases in the synaptically driven activation of the adjacent, environmentally isolated neuronal network. Therefore, the proposed new methodology will enable neuropharmacological analysis of CNS active compounds whilst simultaneously determining their effect on synaptic connectivity.
Highlights
Stage drug development heavily relies upon in vitro cultures, with neurons grown on coverslips prior to drug screening
We present a microfluidic system that integrates computer-controlled perfusion of multiple compounds with Ca2+ imaging techniques to provide a platform for the characterisation of central nervous system (CNS) active compounds
Microfluidic cultures were stained for β-III-Tubulin, glial fibrillary acidic protein (GFAP) and synaptophysin revealing that only neuronal, but not astrocytic processes, were able to traverse across the microchannel array to the adjacent chamber and that synapses were formed both within and outside the microchannels
Summary
Stage drug development heavily relies upon in vitro cultures, with neurons grown on coverslips prior to drug screening. The use of microfluidic procedures to model in vitro pathological conditions has gradually increased over the past decade, due to the level of control and manipulation available over the cellular microenvironment and experimental conditions[14,15] These systems show potential for use as drug discovery platforms, taking advantage of engineering techniques to facilitate high-throughput pharmacological assays, either by device design or the incorporation of perfusion systems to wash on/off multiple compounds[16,17,18,19]. We present a microfluidic system that integrates computer-controlled perfusion of multiple compounds with Ca2+ imaging techniques to provide a platform for the characterisation of CNS active compounds The novelty of this approach is its ability to simultaneously detect direct responses within a primary hippocampal culture to repeated drug applications whilst monitoring the consequent synaptic activity in an adjacent, functionally connected but environmentally isolated hippocampal culture. The proposed platform provides a novel, miniaturised solution amenable to CNS drug discovery, offering the ability to simultaneously screen both the direct effects of compounds on cells, as well as how such drugs influence communication between synaptically connected cultures
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