Testing Monosynaptic Connections Using Tetrodotoxin and Tetrodotoxin-Resistant Sodium Channels

Begin with an electrophysiology setup containing an immobilized transgenic fly with an exposed brain.

Position a recording micropipette near a postsynaptic neuron connected to a presynaptic neuron co-expressing tetrodotoxin-resistant sodium channels and red light-sensitive channels, and an interneuron with tetrodotoxin-sensitive sodium channels.

Apply mild suction to the micropipette to form a tight seal with the neuronal membrane.

Then, apply a strong suction to disrupt the membrane.

Maintain a constant cell potential. Introduce tetrodotoxin, which blocks sodium channels in the interneuron, preventing signal transmission from the interneuron to the postsynaptic neuron.

However, the tetrodotoxin-resistant sodium channels remain unaffected, allowing sodium ions to enter the presynaptic neuron.

Illuminate the fly with the red-light pulses.

This stimulates the light-sensitive channels, allowing cations to influx and activate the presynaptic neuron.

This presynaptic neuron directly transmits a signal to the postsynaptic neuron without involving the interneuron, and the resulting electrical activity confirms the monosynaptic connection.