The overall goal of this procedure is to isolate individual sensory neurons of plegia in short-term culture. For patch clamp studies, this is accomplished by first dissecting the ganglia containing sensory cells from an anesthetized animal. The second step is to digest the ganglia in enzyme solution overnight.
Next, the sensory neurons of interest are dissected from the digested ganglia. The final step is the dissociation of the prepared culture dishes for adhering to the culture substrate overnight. Ultimately, voltage clamping is used to study the voltage and agonist gated ion currents expressed in these neurons.
The main advantage of this technique over existing methods, such as in situ cell recording, is that isolating the neurons allows the use of a single electrode voltage clamping technique. This method can help identify key unknowns in the field of aple neurobiology, such as the identity of ion currents activated by neurotransmitters implicated in learning and memory. Generally, individuals new to this method will struggle due to the sensitivity of the neurons to enzymatic digestion and dispersal onto culture dishes.
We first had the idea for this method when we realized that certain glutamatergic currents important in learning and memory would benefit from the cell isolation that this method provides. Demonstrating the procedure will be graduate students, Andy Kesel and Justin Greer. Begin this procedure by placing an anesthetized animal ventral side down in the dissection tray.
Pin the animal through the edges of the body wall and para poal borders, but avoid the tail and head to expose the dorsal surface of the genital groove. Next, rinse the dorsal surface in slow running cold tap water. Then apply a light stream of 70%isopropyl alcohol from a squeeze bottle along the genital groove and over the top of the head.
To make the initial incision tautly hold a fold of tissue to the left of the base of the genital groove with the rib nose forceps while snipping a shallow hole all the way through the smooth flat area of body wall just to the right of the genital groove with a pair of fine angled scissors. Then expand the incision anteriorly to a point between the ROF fours while pulling upward with the lower blade to avoid nicking the gut. Next, use clean forceps and fine scissors to remove the head ganglia by severing the point where the nerves forming the head ganglia turn into a ring around the esophagus in order to remove pleural petal and cerebral ganglia as a group.
After that, remove the buccal ganglion that adheres to the ventral side of the esophagus. To isolate the ganglia of interest, trim the head ganglia apart, leaving a length of connective attached to each ganglion of interest that is equal to at least the diameter of the ganglion. This will retard the enzyme from over digesting the cells of interest in the next step.
Then rinse each target ganglion twice in dishes of artificial sea water with penicillin streptomycin. If targeting the PVC cells, leave the right pleural hemi ganglion attached to the right petal hemi ganglion, and similarly, leave the left plural hemi ganglion attached to the left petal hemi ganglion. Then place the rinsed ganglia in enzyme solution and attach the cap tightly.
Subsequently, place the tube on its side on a rotary shaker and set at slow speed for 13 to 15 hours overnight at approximately 23 degrees Celsius. In this procedure, fill the center of the 35 millimeter dishes that were PDL coated and UV sterilized with approximately 0.5 millimeters of artificial seawater and penicillin streptomycin to create an island of media inside. To prepare the microdissection of digested ganglia, pour the enzyme solution containing the digested pleural pedal and buccal ganglia into the dissection dish.
Next, place the dish on the stage of the microscope against a black surface under the reflected light. Then using the attached connective tissue pinned down each pleural petal hemi ganglion dorsal side up the tissues will be soft and intolerant of stretching. Then isolate the PVC cells from a pleural hemi ganglion using two pairs of forceps while holding a fine dissection pin with one of them as a probe.
Use the probe to detach these cells from the petal, plural, connective. Then let the cell cluster fall to the bottom of the dissection dish. Transfer the cell cluster to a prepared 35 millimeter culture dish by gently sucking up the cluster into the tip of a slightly fire polished disposable glass pasture pipette.
Then dispense it slowly into the media island in a culture dish, being careful to avoid introducing air bubbles into the pipette. Repeat the isolation of the PVC cluster of the other hemi ganglion and place it into a separate culture dish. Here is a photo micrograph of the V-shaped dark orange PVC cell cluster of the right pleural hemi ganglion.
To dissect the BSC neurons. First pin out the buccal ganglion with the ventral side up with care to spare. The cells of interest subsequently isolate the BSC cells using two pairs of forceps while holding a fine dissection pin with one of them as a probe.
Next, use the probe to detach these cells from the buccal ganglion. Then let the cells fall to the bottom of the dissection dish. This photo micrograph shows the position of the glutamatergic cells of the buccal ganglion.
The BSC neurons are identifiable by their dark orange color. The isolation of PVC and BSC neurons produces four culture dishes containing neuron clusters. Two dishes containing BSC clusters and two dishes containing PVC clusters.
Discard the rest of the ganglia and set aside the 100 millimeter dissection dish to dissociate the cell clusters. Place a culture dish containing cells on the stage of the low power microscope and remove the cover. Gently flick the bottom of the culture dish adjacent to the cell cluster.
With a fine dissection pin held in forceps five to 10 flicks may be needed to completely dissociate the cells. Then repeat with other culture dishes. Next, place the culture dishes containing media islands with the dissociated cells in a large container that permits air circulation, such as a 150 by 25 millimeter round plastic Petri dish.
And place this dish in an incubator set to 17 degrees Celsius. Afterward, place an open dish of water as a source of humidity in the chamber and leave them undisturbed overnight to allow the cells to adhere to the polylysine coating in the center of the dish. On the next day, gently flood the dishes with 2.5 milliliters of artificial seawater and penicillin streptomycin, examine and count the cells using an inverted cell culture microscope.
Here are live BSC neurons after one day in culture, the BSC cells are generally larger than PVC cells at 40 microns. Shown here is a live PVC neuron after one week in culture showing process outgrowth. Here is an excitatory current carried by sodium ions in response to a pulse of AL glutamate, and here is a current in response to D aspartate.
The currents activated by NMDA are generally smaller in amplitude than those activated by the other agonists. While attempting this procedure, it's important to remember to not damage the neurons during dissociation. One advantage of this technique is that it creates two cultures from each ganglion, so that one may be used as a match control to the experimental culture.
This technique can be used to create primary cultures of neurons from plegia or other marine invertebrates. After watching this video, you should have a good understanding of how to isolate sensory neurons of plegia by enzymatic digestion of the ganglia, followed by careful dissociation of the cells of interest.
