テトロドトキシン耐性ナトリウム チャネルを用いたショウジョウバエのシナプス接続を調べる

The overall goal of this technique is to identify monosynaptic connections electrophysiologically in drosophila using tetrodotoxin resistant sodium channels. The main advantages of this technique is that it is straightforward and can reveal the chemical nature of synapses. It is also suitable for the study of volume or bulk transmission.

Generally, in videos new to this method, we struggle because of the difficulty of the dissection. By simply understanding the method, however, the logic is easily transferable to other model systems such as mice. For the recordings, have patch pipettes for the resistance near seven megaohms.

Also, have external recording saline with a pH of 7.3 or less aerated continuously with carbogen containing 95%oxygen and 5%carbon dioxide. For the subjects, have a fly stock expressing csChrimson, propagating on food containing 0.2 millimolar of all trans-Retinal. For the recordings, select one to three-day-old adults.

Load them into a glass scintillation vial and immobilize it using one minute exposure to ice-cold conditions. Then, lower the light level on the dissecting scope and coarse forceps to load an anesthetized fly into the custom-made recording chamber. Secure the fly to the chamber by surrounding with wax or UV curable epoxy.

Position it dorsal side up with the foil roughly halfway up the thorax. The key to obtaining a great preparation is having the hole cut in the foil to be just the right size, so the fly is not squeezed nor able to fall through. Illuminate the preparation with optical fiber lights from either side and keep them as dim as possible.

Next, cover the preparation with four to six drops of recording saline. Next, sharpen the tungsten wire or filament for the dissection. Apply about 20 volts of AC current to a potassium nitrate-saturated solution and repeatedly dip the tip of the wire very quickly into the solution.

After 20 dips, the tip will be sharp. Now, adjust the lights to provide just enough visibility to use the sharpened wire to expose the brain by removing the cuticle, followed by the trachea and fat bodies. To access the antennal lobes, use a bent tungsten wire to gently tuck the antenna under the foil shield.

Then use sharp forceps to gently tear away the glial sheathing covering the area of interest. Now transfer the preparation to the electrophysiology rig to make recordings. 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 and a 2 liter flask. Next, load the patch pipette with 4 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 minus 60 millivolts. And the pipette should form a gigaohm seal with the cell membrane.

The holding current should drop to as low sub-picoamp level. To obtain constant gigaohm seals, make sure to keep your pipette clean by keeping positive pressure and avoid hitting tissue or approaching your cells. To proceed, set the amplifier to deliver test pulses from minus 50 to minus 60 millivolts.

The test pulses will reveal a large capacitative transient representing the current necessary to charge the patch pipette. To remove capacitative transience, 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 transience 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 minus 50 and minus 60 millivolts. In preparation, configure the data acquisition system as indicated in the tech's protocol. The system requires a high-powered red LED positioned beneath the preparation, positioned to illuminate the fly at 0.238 milliwatts per square millimeter.

Now, activate the presynaptic neurons optogenetically, using pulsed red light in csChrimson expression. Other methods can also be used. Before applying tetrodotoxin, a synaptic connection should be visible in response to a 40 millisecond light pulse.

No response means a connection is unlikely. The holding potential may be adjusted to emphasize the excitatory or inhibitory connections, accordingly. Once a connection is verified, set up the saline profusion for recirculation at the saline.

Set the pump 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. Then, proceed with adding pharmacological antagonists to the recirculating saline to test the chemical nature of the synaptic connections.

Transgenic expression of the tetrodotoxin in sensitive sodium channel notchback rescues excitability in neurons and results in a large plateau potential. Interneurons in the antennal lobe respond to odors. However, excitatory post-synaptic currents are not easily resolved at the soma.

So, do the responses arise directly from olfactory receptor neuron input? Using tERPs reveals that the olfactory receptor neurons make direct connections with the local interneurons. Stimulation of the serotonergic neurons in the antennal lobe results in a mixture of excitation and inhibition in local interneurons.

Application of methysergide, a broad 5HT-receptor antagonist, blocked a slow hyperpolarization, but had no effect on the depolarization, while mecamylamine blocks the fast depolarization. This suggests that the stimulation is cholinergic. tERPs was used to resolve extrasynaptic volume transmission that occurs with either GABA or neuromodulators.

In one example, application of tetrodotoxin did not suppress an inhibitory signal in the local interneuron. This inhibition was blocked using the serotonin antagonist, methysergide, suggesting a direct signal from a specific serotonergic neuron. After watching this video, you should have a good understanding of how to obtain whole cell recordings from drosophila neurons and assess their conductivity using tERPs.

While attempting this procedure, it's important to remember to be patient and practice the difficult dissection repeatedly. Once mastered, this technique can be done in about an hour if it is performed properly. Following this procedure, other methods like pharmacology can be performed in order to answer additional questions like the nature of the chemical synapse.

Don't forget that working with tetrodotoxin can be extremely hazardous and precautions, such as wearing gloves and working with small volumes, should always be taken while performing these procedures.