Chemically induced synaptic activity between mixed primary hippocampal co-cultures in a microfluidic system

Abstract

Primary neuronal cultures are an invaluable in vitro tool for examining the fundamental physiological changes that occur in diseases of the central nervous system. In this work, we have used a microfluidic device to grow twin cultures of primary hippocampal neuronal/glia cells which are synaptically connected but environmentally isolated. Immunocytochemical staining, for β-III-Tubulin and synaptophysin, indicated that the two neuronal populations were physically connected and that synapses were present. By dispensing predefined volumes of fluids into the device inlets, one culture was chemically stimulated and the consequent increase in neuronal activity in the opposing culture was monitored using calcium imaging. To optimise the experimental procedures, we validated a numerical model that estimates the concentration distribution of substances under dynamic fluidic conditions, proposing that no cross contamination of chemical stimuli occurred during the experiments. Calcium imaging and local chemical stimulation were used to confirm synaptic connectivity between the cultures. Chemical stimulation of one population, using KCl or glutamate, resulted in a significant increase of calcium events in both neurons and astrocytes of the connected population. The integration of the system and techniques described here presents a novel methodology for probing the functional synaptic connectivity between mixed primary hippocampal co-cultures, creating an in vitro testing platform for the high-throughput investigation of synaptic activity modulation either by novel compounds or in in vitro disease models.

In Vitro Microfluidic Models for Neurodegenerative Disorders

“…Neurons and glial cells in each chamber grow within the microchannels where they physically interact and form synapses. The function of neuron–glia synapse was confirmed by calcium imaging . Yang et al developed a co‐culture CMS combined with electrodes to study activity‐dependent myelination.…”

Quantitative propagation of assembled human Tau from Alzheimer's disease brain in microfluidic neuronal cultures

“…Typically, to facilitate neuronal culture, microfluidic devices are fabricated using gas-permeable silicone elastomer polydimethylsiloxane (PDMS). The most common device layout used to create synaptically connected but environmentally isolated neuronal populations ( 18 , 19 ) comprises two microfluidic culture compartments where neuronal cells grow processes and subsequently become connected by an array of microchannels through which axonal growth is guided; whether axons or dendrites can reach the opposite culture compartment depends on the length and shape of the microchannels ( 17 , 20 , 21 ). This structure enables axons from one culture compartment to form synaptic connections with dendrites from the other compartment.…”

High-throughput platforms for the screening of new therapeutic targets for neurodegenerative diseases

“…The authors isolated distal axons from cell bodies in a microfluidic device and concluded that the most susceptible part of the neuron to excitotoxicity is the distal axon [65]. More recently, Roberston et al [66] developed a high-throughput platform to monitor neuronal synaptic communication for drug screening purposes. The authors have combined a microfluidic system with a calcium-imaging array to study primary hippocampal cultured networks.…”

“…Scanning electron microscopy (SEM) images of the 3 μm gaps and apical and basolateral fluidic chamber. Reproduced, with permission, from[66].Version: Postprint (identical content as published paper) This is a self-archived document from i3S -Instituto de Investigação e Inovação em Saúde in the University of Porto Open Repository For Open Access to more of our publications, please visit http://repositorio-aberto.up.pt/ A01/00…”