This article describes an electrophysiological setup to study neuronal connections in transgenic flies. The focus is on the interaction between presynaptic and postsynaptic neurons, utilizing tetrodotoxin and light-sensitive 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.
At the rig, immediately start perfusing the preparation with recording saline bubbled with carbogen. Use a gravity-fed saline solution reservoir placed above the microscope stage. To collect the waste saline, use a vacuum line in a two-liter flask.
Next, load the patch pipette with four microliters of internal recording solution, and attach the pipette to a micromanipulator. Then, zero the amplifier's offset and set the amplifier to deliver test pulses. Then, adjust IR oblique illumination in order to obtain a clear view of the brain.
Now, while applying positive pressure to the pipette, approach the cell with the pipette. When contact with the cell is made, release the positive pressure to seal the pipette to the membrane. Then, set the holding potential of the membrane to -60 millivolts, and the pipette should form a gigaohm seal with the cell membrane. The holding current should drop to a low sub-picoamp level.
To obtain constant gigaohm seals, make sure to keep your pipette clean by keeping positive pressure, and avoid hitting tissue when approaching your cells.
To proceed, set the amplifier to deliver test pulses from -50 to -60 millivolts. The test pulses will reveal a large capacitative transient representing the current necessary to charge the patch pipette. To remove capacitative transients, adjust the capacity compensation knobs on the amplifier.
Now, apply brief pulses of negative pressure to rupture the cell membrane and obtain the whole-cell configuration. A large increase in the capacitative transients indicates success. A small increase in the holding current should also be observed. Then, set the amplifier to current clamp mode and adjust the cell's potential to between -50 and -60 millivolts.
The holding potential may be adjusted to emphasize the excitatory or inhibitory connections accordingly. Once a connection is verified, set up the saline perfusion for recirculation of the saline. Set the pumps so the saline level in the bath stays stable.
Then, add enough tetrodotoxin to the system for a final concentration of one micromolar. This should stop the spiking activity of the patched neuron. If not, add more tetrodotoxin to the system. Now, apply brief pulses of red light to activate what is likely to be a monosynaptic connection.
繁體中文
