A Minimally Invasive Lesion Technique for Muscles Intrinsic to the Odontophore of Aplysia californica

The feeding system of Aplysia has been long used as a model system for the study of motor control and soft body biomechanics. However, the muscles deep inside of the feeding grasper have been inaccessible to in vivo manipulation. This surgery allows us to access these muscles in otherwise intact behaving animals.

Not only does this allow us in vivo access for the first time, but the technique involves a single incision to a single part of a slug, which means that the damage to the other regions are relatively small. The best preparation is to do careful dissections of the buccal mass and thoroughly acquaint oneself with the anatomy. Select an active animal by offering it seaweed and confirming that bite intervals are no greater than five seconds.

10 minutes after anesthesia, gently attempt to insert a pin into the gill and rhinophore, verifying that these do not retract to ensure sufficient anesthetization. Ensure that the lips of the slug are smooth, indicating the jaws are fully relaxed. In this way, odontophore is exposed.

In the case where wrinkling appears on the lips, the animal's lips and jaw are not sufficiently relaxed for the surgical procedure. Refer to the manuscript for tips on inducing lip relaxation. Position the slug so that the head hangs downward, allowing the buccal mass to settle against the jaws.

Apply pressure with the thumb and forefinger to push the buccal mass toward the jaws, holding the buccal mass in place. Rotate the jaw so that they are visible. At the same time, maintain the pressure on the buccal mass so that the prow of the buccal mass is visible through the jaws.

Gently work the tips of the blunt forceps into the cleft of the odontophore and use them to lever the radular surface through the jaws. If the jaws are not sufficiently relaxed, use the forceps to gently grasp the edge of the cleft to assist this process. Once the surface is exposed, work the jaws clear of the anterior portion of the radular surface all the way around the perimeter.

This makes the odontophore less likely to retract. Ensure that no more than half of the walls of the odontophore is exposed. Once the radular surface is fully exposed, arrange the slug under a dissection microscope for the surgery.

For scientists with less experience, use a wide rubber band and a third hand to stabilize the jaws and radular surface for the surgery. Position the radular surface so that the cleft side faces the investigator. Gently grasp the radular surface near the radular base so that a horizontal fold is created perpendicular to the anatomical crease.

Use fine scissors to cut through this fold, making an incision along the anatomical crease. Extend this initial incision to three to five centimeters to allow access to the interior of the buccal mass. Adjust light so that it points directly back through this incision.

Part the edges of the incision so that the back of the lumen of the odontophore and the thin vertical strands of the I7 muscle are visible. Reach into the incision. Grasp both strands of I7 and pull them up through the incision, where as much of the muscle can be cut away as is practical.

After lesions have been performed, grasp the anterior tentacles and push them down on the radular surface to return the slug to its original configuration. Place post-surgical animals in a protected environment with good water flow. Ensure that the animals are alert and responsive on the day after surgery.

On the first or second day after surgery, animals begin to feed. To perform sub-radular fiber lesion after surgical incision, insert the tip of a small, straight scalpel blade through the incision with the sharp edge angled upwards. Gently scrape the fine muscular fibers from the underside of the radular surface.

Then, perform post-operative care as previously. In this anatomical study, sham lesions had no significant effect on the width of the opening at the peak of biting. Whereas both I7 and sub-radular fibrous lesions did significantly reduce bite width.

The most important thing to remember is that the lips and jaws of the animal are fully relaxed before attempting to push the grasper out through the jaws. Most likely, both I7 and the sub-radular fibers are multifunctional muscles and we've only identified certain aspects of the behavior to which they contribute. In addition, this technique of protracting the odontophore gives access to whole portions of the anatomy that were previously inaccessible.

Besides opening up anatomical regions and soft body biomechanics, we hope that this might serve as a foundation for other investigators who are working with soft structures that might be otherwise difficult to investigate.