This article describes a method for stimulating neuronal cultures using a pulsed electric field while monitoring neuronal activity through fluorescence imaging. The approach enables the observation of action potentials and calcium influx in response to electrical stimulation.
Take a coverslip containing a 1D patterned neuronal culture.
The neurons are loaded with a calcium indicator dye, which fluoresces upon calcium binding.
Transfer the coverslip to a fluorescence microscope imaging chamber containing an extracellular recording solution.
Assemble a holder with two parallel wire electrodes separated by a distance.
Invert the holder and position it above the neuronal culture with the electrodes immersed in the recording solution.
Connect the electrodes to an amplifier. Apply a non-DC pulsed bipolar square wave to generate a uniform uni-directional electric field across the neurons without causing injury.
The generated electric field excites the neurons, triggering an action potential.
The action potential travels along the axon, activating the voltage-gated calcium channels and causing calcium influx into the neuron terminal. Further, neurotransmitters are released into the synapse, facilitating synaptic transmission.
Following calcium ion influx, these ions bind to the intracellular dye and fluoresce, indicating neuronal activity in response to electrical stimulation.
Prior to an electric stimulation, place the seated coverslip into an imaging chamber. To stimulate the cells with an electric field of a constant direction, use two parallel electrodes separated by 11 millimeters and located at opposite sides of the culture plated on the 13-millimeter coverslips. Then, assemble the culture imaging chamber and matching electrode holder such that the wire electrodes are immersed in the extracellular media and positioned one millimeter above the culture.
Then, plug the single electrode pair to the operational amplifier that amplifies the signal from the generator. Apply a single pulse of bipolar square wave with a 50% duty cycle and amplitude of 22 volts. Use a bipolar wave with no DC component to avoid electrolysis that may occur at the electrodes. Apply the pulse lasting from 10 microseconds to four milliseconds to ensure effective stimulation, but prevent cell injury.
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