Here we present a protocol for minimally invasive surgical lesioning of muscles intrinsic to the feeding apparatus of the marine mollusk Aplysia californica to understand the roles of these muscles during feeding behavior.
Aplysia californica is a model system for studying the neural control of learning and behavior. This animal has a semi-open circulatory system, making it possible to access many of its internal structures without causing any significant damage. Many manipulations can be easily performed both in vivo and in vitro, making it a highly tractable model for the analysis of behavior and neural circuitry. To better understand the functions of muscles within the feeding grasper, we have developed a technique for lesioning them without opening the main body cavity of the animal or damaging the outer layers of the feeding organ (i.e., the buccal mass). In this technique, the grasper is partially everted, allowing direct access to the musculature. This procedure allows animals to recover quickly and reliably. This has made it possible to lesion the I7 muscles and sub-radular fibers, allowing us to show that both muscles significantly contribute to the opening in vivo.
The feeding system of Aplysia californica has a long history of use as a model system for understanding learning and memory1, motivated behaviors2,3, and the interaction between behavior, biomechanics and neural control during feeding4. It has highly accessible neural circuitry, with a relatively small number of large, identifiable neurons. The animal has a semi-open circulatory system, making it possible to access many of its internal structures without causing significant damage. It is also robust to many manipulations both in vivo and in vitro, making it a highly tractable model for the analysis of behavior and neural circuitry.
To understand the neural patterns that give rise to feeding behaviors, it is important to describe the underlying mechanics of the soft structure that makes up the feeding organ, the buccal mass4. While there has been work done to characterize the outer muscles that make up the buccal mass5,6, the inner muscles of the underlying structure within the buccal mass that controls the surface of the grasper, the odontophore, have been largely inaccessible to in vivo experimentation. Although there have been in vitro studies on the functions of some of these muscles7,8, the lack of direct access to these muscles has made it difficult to study their role in intact, behaving animals.
Most techniques for electrode implantation or lesions in Aplysia or similar molluscan species require that the body wall be opened9,10,11,12. Opening the body wall causes epithelial injury, and the incision must be securely sealed to prevent hemolymph escape. Even more serious difficulties are posed when attempting to reach the inner muscles of the grasper of Aplysia (muscles underlying the radular surface or within the odontophore): having entered through the main body cavity, one must then go through some portion of the muscular wall of the buccal mass to gain access to the interior structures (Figure 1A). This accumulated injury and difficulty of access has made the approach through conventional means problematic because animals do not recover well from these surgeries (of animals with full eversions, only 17% regained any feeding ability, N = 12. Around 85% of non-everted animals regained the ability to feed, N = 84).
The I7 muscle, which has been characterized as a radular opener8, is deep inside the odontophore itself, further complicating access. It stretches between the base of the radular stalk (Figure 1C) and the underside of the radular surface, through a lumen in the odontophore (Figure 1C). On three sides of the I7 muscles are walls of muscle, and the fourth wall consists of the radular stalk. For the purposes of a biomechanical study, major impairment to any of these structures would compromise the normal function of the feeding apparatus. We developed a novel approach of working the odontophore out through the jaws, and conducting the surgery through an incision to the thin, cartilaginous radular surface, that made it possible to lesion the I7 muscle, as well as newly-described fine muscle fibers that run just beneath the radular surface, which we refer to as the sub-radular fibers (Figure 1C).
Figure 1: Anatomical Overview. (A) Location of the buccal mass within Aplysia. (B) External anatomy of odontophore. The surface of radula and radular sac are yellow; muscles composing the odontophore are shown in red, based on their actual colors. (C) Sagittal section of odontophore, showing the location of sub-radular fibers (curved pink line) and I7 muscle (straight pink line). Cross section of the I6 muscle is shown in dark red. Please click here to view a larger version of this figure.
Aplysia are invertebrates and thus not subjected to IAUC approval. To minimize discomfort to animals, ensure that they are fully anesthetized before applying the surgical techniques described below.
1. Animal Selection and Anesthetization
Figure 2: Tension and Relaxation in Anesthetized Aplysia Mouths. (A) Aplysia showing a high degree of muscle tension around the lips. This correlates with jaw tension and contraindicates proceeding with the surgery. (B) Aplysia with relaxed lips, showing the inside of jaws (light grey). Colors again correspond to those observed in the animal. Please click here to view a larger version of this figure.
2. Exposing the Radular Surface
Figure 3: Supporting the Buccal Mass Against the Inside of the Jaws. Fingers support the buccal mass that has been pushed up against the inside edge of the jaws until the tip of the prow can be seen. Please click here to view a larger version of this figure.
Figure 4: Partial Eversion of the Odontophore. The radular surface is fully exposed, but the sides of the odontophore are not uncovered, making this only a partial eversion. Further eversion will likely result in damage to the animal. Please click here to view a larger version of this figure.
NOTE: A full eversion of the odontophore will cause major muscle damage from which the animals are very slow to recover.
3. Surgical Incisions
Figure 5: Location of Incision to the Radular Surface. (A) Radular surface, with an incision. (B) Radular surface with circles showing where the strands of the bilateral I7 muscle attach; dotted lines show the location of the descending muscles underneath the radular surface. Please click here to view a larger version of this figure.
Figure 6: Location of I7 through the Radular Surface Incision. Looking through the incision, both strands of I7 can be seen between the inner surfaces of I4. Please click here to view a larger version of this figure.
Figure 7: Pulling the I7 Muscle Strand Through the Incision. The I7 muscle is highly elastic and can be pulled up through the incision for removal. Please click here to view a larger version of this figure.
NOTE: With practice, it is usually more effective to locate I7 by feel than by sight.
4. Post-operative Care
5. For Sub-radular Fiber Lesion
Figure 8: Lesioning the Subradular Fibers. The edge of the scalpel blade is angled upwards through the incision to the underside of the radular surface so that it can gently scrape away the sub-radular fibers. Please click here to view a larger version of this figure.
Previous work had suggested that the I7 muscle contributed to the opening of the grasper8. Our own anatomical studies suggested that the sub-radular fibers might also contribute to grasper opening. To test these hypotheses, animals were induced to generate bites both before and after receiving a surgical procedure. Sham animals were subjected to all the surgical steps, including the incision in the radular surface, but no internal muscles were removed. Animals subjected to an I7 lesion had both I7 muscles removed. Animals subjected to a sub-radular fiber lesion had ~25% of the sub-radular fibers removed immediately beneath the incision. Sham lesions had no significant effect on the width of the opening at the peak of biting, whereas both I7 and sub-radular fibers lesions did significantly reduce bite width (Figure 9).
Figure 9: Results of Lesions on Opening Width During Peak Biting. Data shown are the differences between the averaged normalized opening width of the radula before and after the surgical procedure for 5 animals in each of the 3 groups (sham, I7 lesion, or SRF lesion), with each animal serving as its own control. Averages were taken of 5 bites before, and 5 bites after the surgical procedure to determine the mean normalized difference. Opening width was the distance from the center of radula to the radular edge at the peak protraction, normalized by the distance from the inner surface of the radular base to the cleft-side edges of the radular surface. The differences are shown as the means plus or minus the standard deviation. After establishing that the difference data were normally distributed, the probability that the lesion had no effect was determined (i.e., the null hypothesis was tested that the effects of the surgical procedures would be zero, on average) by applying a paired t-test to each independent group. The data demonstrates that the sham lesion had no significant effect, whereas a lesion of the I7 muscles or a lesion of the sub-radular fibers did have a significant effect on radular opening (p < 0.031 for the I7 lesion group, indicated with a single asterisk, or p < 0.002 for the SRF lesion group, indicated by a double asterisk). Please click here to view a larger version of this figure.
Body Weight | Magnesium Chloride Dose |
<200 g | ½ bodyweight |
200-350 g | 1/3 bodyweight |
350-450 g | ¼ bodyweight |
Table 1: Magnesium Chloride dosage by bodyweight.
The most critical steps within the protocol are the need to ensure that the animal is fully anesthetized, and that the eversion of the buccal mass is just enough to access the underlying muscles. It may require some practice to perfect these steps, but once they are mastered, the yield from surgeries is likely to be greater than 85% of all experiments done. The most important way to properly modify and troubleshoot the protocol is to spend time doing dissections of the buccal mass so that the locations of the internal muscles are completely clear to the investigator. Because the suggested incision through the radular surface inevitably causes some damage to the underlying sub-radular fibers, it may be appropriate to modify the exact location of the incision to avoid specific regions of these fibers.
One limitation of the surgical technique is that it may have non-specific effects on feeding responses, such as the strength of protraction. One way to overcome this limitation is to have animals serve as their own controls. In addition, it is critical to have a sham lesion group which is subjected to the entire surgical protocol except for the removal of the specific muscle (i.e., I7 or the SRFs). By following these suggestions, an investigator will reduce the effects of variability between animals and have an intrinsic measure of the non-specific effects of surgery.
Previous work has used approaches through the body wall to lesion or record either from nerves13,14, or muscles15,16,17. In our laboratory, we have anecdotally observed that body wall incisions are often accompanied by a significant loss of hemolymph and thus of body volume. Animals often require several days to recover from this, and if the body wall lesion is not carefully sutured, animals may not recover. In addition, post-mortem examination of the animals reveals considerable scarring around the incision and a strong immune response (anecdotal observations). In contrast, animals show no loss of hemolymph or change in body volume after recovery from the protocol described here (based on observations in 96 animals).
Future applications of the technique may extend it to other muscles within the feeding apparatus of Aplysia, and to other animals. We have focused on the removal of the I7 muscle and sub-radular fibers. These same general surgical techniques also allow access to most of the other muscles of the odontophore. Some of these, such as the internal portion of the I5 muscle, are best accessed through the radular surface. Others, like the inner leaflets of I4, may be better reached through the exterior epithelium of the odontophore. We have made preliminary trials where an incision under the radular cleft of the partially everted odontophore allowed access for a sharpened hook to be inserted that could then be used to lesion another muscle within the odontophore, muscle I88. Because the surgical protocol described here does not open the main body cavity, no suturing is required.
The protocol that we have described may be of general interest to other investigators working on soft tissue structures that would otherwise be difficult to manipulate, e.g., the feeding apparatus of other mollusks. More generally, this protocol could suggest other novel surgical approaches to the analysis of soft structures such as tongues, trunks or tentacles18.
We would like to acknowledge the hard work that Sherry Niggel, Sisi Lu, and Joey Wu put into improving and validating these protocols. This work was supported by NSF Grant IOS 1754869.
Name | Company | Catalog Number | Comments |
Blunt forceps | Fine Science Tools | 11210-10 | 2 pair |
Scalpel blade (#11) | Fine Science Tools | 10011-00 | |
Spring scissors | Fine Science Tools | 15024-10 | |
Webcam | Logitech | c920 | for recording data |
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