This article describes a method for studying synaptic connections between light-sensitive cortical neurons and iPSC-derived neurons using optogenetics. The approach involves whole-cell recordings to measure postsynaptic currents in response to light stimulation.
Place a coverslip containing a co-culture in a recording chamber perfused with an oxygenated extracellular solution.
The co-culture consists of astrocytes, rat cortical neurons expressing light-sensitive ion channels fused to a fluorescent protein, and iPSC-derived neurons expressing another fluorescent protein.
Using a confocal microscope, identify a fluorescent iPSC-derived neuron.
Advance a recording pipette filled with an intracellular solution toward the neuron to establish a whole-cell configuration.
Maintain a constant negative membrane potential for accurate current measurements.
Illuminate the co-culture with light to activate light-sensitive ion channels in the cortical neurons, thereby generating an action potential.
The action potential causes neurotransmitter release into the synaptic cleft.
If synaptically connected, the neurotransmitters bind to receptors on the patched iPSC-derived neuron, opening ion channels and generating postsynaptic currents.
Increased postsynaptic currents in iPSC-derived neurons following light stimulation confirm synaptic connections with cortical neurons.
Use a confocal microscope equipped with a 60x water-immersion lens. Perfuse the cells at room temperature in an external solution, bubbled constantly with 95% oxygen, 5% carbon dioxide. Use an internal solution to fill the recording micropipette.
To perform optogenetic stimulation, patch a tdTomato-positive cell in whole-cell mode and set the voltage clamp at negative 70 millivolts. With the whole-field mercury lamp and a 480-over-40 excitation filter, stimulate the whole field for 30 seconds. Record postsynaptic currents, or PSCs, of a patched cell induced by photostimulation of ChR2-expressing presynaptic cortical neurons.
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