The overall goal of these methods is to study gustatory coding in a simple animal mandi-casecsta by recording the activity of multiple receptor neurons in vivo, while delivering and monitoring precisely timed pulses of tastants. These methods can help answer key questions about gustation, such as how tasters are encoded by populations of gustatory receptor neurons. The main advantage of this technique is that it allows in vivo recordings for multiple gustatory neurons before, during, and after precisely delivered tastant stimuli.

Individuals new to this method might find it challenging because the dissection procedure is intricate. Because of this, we hope that the visual demonstration of the method will be helpful. These techniques were developed by Sam Writer when he was a graduate student in my lab, along with Alejandra, Kui Sun will demonstrate these procedures.

Three days after eclosion, chose a moth of either sex that has a generally healthy appearance. The wings should be fully extended, and the proboscis and antennae should be well formed and intact. In a fume hood, insert the selected moth into a restraining tube until the head is exposed.

Then, immobilize the moth by inserting tissue paper into the other end of the tube. Now, using pressurized air delivered from a syringe, remove the hair from the exposed head. Then, place the tube on a clay platform in a Petri dish.

Orient the head dorsal side up. Next, protect the antennae and the proboscis. First, make covers for each structure from 200 microliter pipette tips cut into half centimeter lengths.

To manipulate the proboscis, prepare a hook from a piece of wire. Extend the proboscis into one of the covers until the most proximal part of the proboscis is protected. Secure the tube to the dorsal part of the head capsule using firm batik wax.

Next, carefully sheet the antennae and secure the tubes with batik wax. Now, stabilize the brain by cutting the buckle compressor muscle. Under the dissection microscope, use microdissection tools to open the head capsule by making a small cut just below the proboscis.

To confirm the muscle is cut, offer sucrose solution to the distal two thirds of the proboscis, and for five minutes make sure that the proboscis does not extend. Then, close the head capsule using a layer of melted batik wax. To perfuse the brain with saline, build a wax cup around the ventral side of the head.

Using an electric waxer, start building a wax cup along the front of the head moving towards the back. Keep the proboscis and antennae tubes inside the cup. Continue building the cup outward and upward until it reaches the level of the eyes.

Using forceps, take the labial palps and attach them to the wax cup by using melted wax. Next, seal any openings in the wax cup and tubes using a thing layer on binary epoxy. Mix the epoxy using a toothpick.

First, cover the outer and inner surfaces of the cup. Second, fill the part of the restraining tube surrounding the neck so the head is held more firmly. After 20 minutes, check the cup for leaks.

To begin the brain surgery, first cut off the labial palps. To open the ventral side of the head capsule, cut the cuticle along the back and front of the head and around the eyes. Then, cut along the back of the head and make more cuts along the front of the head near the proboscis.

Then, gently lift the flap of cuticle with forceps to expose the brain. Now, perform the most critical step, exposing the SEZ and maxillary nerves. Slowly and carefully remove fat tissue and the trachea.

Rinse the brain frequently with fresh saline. Be very careful, because it is very easy to crush the maxillary nerves. When the SEZ and the maxillary nerves are finally visible, they will be covered by a thin sheath.

To remove this sheath, drain any excess saline and then weaken the sheath by applying a 10%collagenase dispase solution. After soaking for five minutes, rinse out the solution several times with saline. Then, after several rinses with saline gently remove the sheath using superfine microdissection forceps.

Lastly, secure a saline drip line into the perfusion cup. This section describes how to build and use a tastants delivery system. The system consists of a testing tube with several openings.

The openings provide entrance for the animal's proboscis, the tastants manifold, a color sensor, and they allow for the flor through of water. To build this system, first prepare a testing tube for the proboscis. Make a proboscis sized hole using a soldering iron.

Then, build a screened in section within the tube to hold the proboscis in place. Cut five millimeters above the hole, place the mesh there with wax, and reconnect the tubes using wax and epoxy. Use a pressurized perfusion system to deliver the tastants.

Join as many tastant tubes as needed to a manifold. To connect the tastant output tube, make a hole about one centimeter above the mesh, then secure the output tube to it with epoxy. In each tastant, include an artificial dye to be detected by a color sensor.

Attach a color sensor to the apparatus at a hole one half centimeter about the mesh, and secure it there using epoxy. Use a peristaltic pump to deliver distilled water into the system via silicon tubing. Collect the waste water with a large container.

Use a pneumatic pump to deliver the tastants with puffs of air. Now, begin pumping clean water through the tubing, and deliver pulses of tastant to test the color sensor. Now, load the distill two thirds of the moth proboscis into the testing tube.

Using forceps, extend the proboscis and gently push it into the hole. Then, seal the proboscis in place with soft dental wax and a layer of epoxy. Now, connect the saline perfusion line and set the perfusion flow.

Then, immerse the ground electrode of the twisted wire tetrode into the saline bath. Then, using a micromanipulator, position the tetrode next to the maxillary nerve, and slowly advance it into the nerve. After detecting spiking signals, let the preparation stabilize for at least 10 minutes before taking recordings.

Recordings were made from gustatory receptor neurons before, during, and after tastant delivery using the described protocol. The onset and offset of a one second pulse of each tastant was monitored by the color sensor. The tastants elicited spikes that were detected by each of the four channels.

Within the spike trains, individual action potentials could be observed. Spike sorting software was used to assign these spikes to unique neurons. The response of three different gustatory receptor neurons to six tastants delivered in sequence reveals different levels of baseline activity and selectivity to the tastants.

Also, responses had different timing. Some are locked to the duration of the stimulus, while others outlast the stimulus. Intracellular recordings could also be made from gustatory receptor axons using conventional sharp glass electrodes.

The intracellular recordings were very similar to responses observed using the described tetrode technique, but the tetrode recordings were easier to make, longer lasting, and more robust. After watching this video, you should be able to expose the maxillary nerves and the sub-esophagal zone in mandi-casecsta, and be able to build a tastant delivery system with precise temporal control. While performing this protocol, wear laboratory safety gloves, and a face mask because the fine powdery scaled shed by mandi-casecsta can be allergenic.

Once mastered, this procedure can be completed in three and a half hours. Following this procedure, simultaneous recordings from multiple brain areas can also be performed to answer additional questions such as, how gustatory receptor neuron responses to tastants are transformed when they are transmitted to the postsynaptic target neurons.