Assessing Neural Connection Formation in a Co-Culture using Electrophysiological Recordings

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