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.

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
<ol>
	<li>Select an active animal by offering it seaweed and confirming that bite intervals are no greater than between 3 and 5 s.</li>
	<li>Anesthetize the animal with 0.333 molar magnesium chloride (see Table 1) by injecting near the head with an 18 G needle on a 60 mL syringe so that the highest concentration of anesthetic will be around the buccal mass.
	<ol>
		<li>Take care to penetrate both the outer epithelium and the inner tissue layer with the needle. Ensure that the injection is roughly under the rhinophore, halfway between the rhinophore and the foot, and the needle should enter obliquely, pointing in the direction of the jaws.</li>
	</ol>
	</li>
	<li>After 10 min, gently attempt to insert a pin into the gill and rhinophore, verifying that these do not retract, to ensure sufficient anesthetization.</li>
	<li>Ensure that the lips and jaw of the slug are relaxed so that the odontophore can be exposed. 
	NOTE: The wrinkling on the lips of Figure 2A indicates that the animal&#8217;s lips and jaw are not sufficiently relaxed for the surgical procedure to be performed without damage. The smooth, relaxed lips of Figure 2B indicate that the jaws are fully relaxed.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60030/60030fig2.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/60030/60030fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>If an animal&#8217;s lips are not relaxed, inject an additional 30 mL of magnesium chloride and wait another 5 min. If this does not result in lip relaxation, return them to an isolated container with good water flow to allow them to recover (see step 4) and proceed with a different animal.</li>
</ol>
2. Exposing the Radular Surface
<ol>
	<li>Position the slug so that the head hangs downward, allowing the buccal mass to settle against the jaws.</li>
	<li>Apply pressure with the thumb and forefinger to push the buccal mass toward the jaws, holding the buccal mass in place.</li>
	<li>Rotate the jaws 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. (Figure 3).</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60030/60030fig3.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/60030/60030fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>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. 
	CAUTION: This pressure does risk greater damage to the animal.</li>
	<li>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 (Figure 4). Ensure that no more than half of the walls of the odontophore is exposed.</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60030/60030fig4.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/60030/60030fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
NOTE: A full eversion of the odontophore will cause major muscle damage from which the animals are very slow to recover.
3. Surgical Incisions
<ol>
	<li>Once the radular surface is fully exposed, arrange the slug under a dissection scope for the surgery.
	<ol>
		<li>Alternatively, use a wide rubber band and a third hand to stabilize the jaws and radular surface for the surgery, especially while learning. This, however, adds time and increased tissue damage to the procedure, which makes it less ideal over the long term.</li>
	</ol>
	</li>
	<li>Place the radular surface so that the cleft side faces the investigator.</li>
	<li>Gently grasp the radular surface, near the radular base, so that a horizontal fold is formed perpendicular to the anatomical crease. Use fine scissors to cut through this fold, making an incision along the anatomical crease (Figure 5).</li>
</ol>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60030/60030fig5.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/60030/60030fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Extend this initial incision to 3-5 cm to allow access to the interior of the buccal mass.</li>
	<li>Adjust light so that it points directly back through this incision.</li>
	<li>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. (Figure 6)</li>
</ol>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60030/60030fig6.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/60030/60030fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="7">
	<li>Reach into through the incision, grasp both strands of I7, and pull them up through the incision, where as much as the muscle can be cut away as is practical (Figure 7).</li>
</ol>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60030/60030fig7.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/60030/60030fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
NOTE: With practice, it is usually more effective to locate I7 by feel than by sight.
4. Post-operative Care
<ol>
	<li>After lesions have been performed, grasp the anterior tentacles, and push down on the radular surface to return the slug to its original configuration.</li>
	<li>Place post-surgical animals in a protected environment with good water flow. Increased oxygenation speeds the recovery. Ensure that the animals are alert and responsive on the day after surgery. If this is not the case, it can be assumed that they will not recover. 
	NOTE: Animals will usually begin to feed on the first or second day after surgery. Even animals that are having trouble biting should be offered seaweed, as it is our anecdotal observation that an animal&#8217;s recovery is improved by its attempts to eat.</li>
</ol>
5. For Sub-radular Fiber Lesion
<ol>
	<li>Follow the steps from 1.1 through 3.5</li>
	<li>Insert the tip of a small straight scalpel blade (#11 or similar) through the incision with the sharp edge angled upwards. Gently scrape the fine muscular fibers from the underside of the radular surface. (Figure 8).</li>
</ol>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/60030/60030fig8.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/60030/60030fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Return to step 4.1.</li>
</ol>

<ol>
	<li>Pinsker, H., Kupfermann, I., Castellucci, V., Kandel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Habituation+and+Dishabituation+of+the+GM-Withdrawal+Reflex+in+Aplysia.">Habituation and Dishabituation of the GM-Withdrawal Reflex in Aplysia.</a> Science. 167, 1740-1742 (1970).</li><li>Kupfermann, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+Behavior+in+Aplysia:+A+Simple+System+for+the+Study+of+Motivation.">Feeding Behavior in Aplysia: A Simple System for the Study of Motivation.</a> Behavioral Biology. 10, 1-26 (1974).</li><li>Susswein, A. J., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+as+a+regulator+of+behavior:+New+ideas+from+Aplysia+feeding.">Nitric oxide as a regulator of behavior: New ideas from Aplysia feeding.</a> Progress in Neurobiology. 97, 304-317 (2012).</li><li>Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aplysia+feeding+biomechanics.">Aplysia feeding biomechanics.</a> Scholarpedia. 2, 4165 (2007).</li><li>Neustadter, D. M., Drushel, R. F., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinematics+of+the+buccal+mass+during+swallowing+based+on+magnetic+resonance+imaging+in+intact,+behaving+Aplysia+californica.">Kinematics of the buccal mass during swallowing based on magnetic resonance imaging in intact, behaving Aplysia californica.</a> Journal of Experimental Biology. 205, 939-958 (2002).</li><li>Neustadter, D. M., Herman, R. L., Drushel, R. F., Chestek, D. W., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+kinematics+of+multifunctionality:+comparisons+of+biting+and+swallowing+in+Aplysia+californica.">The kinematics of multifunctionality: comparisons of biting and swallowing in Aplysia californica.</a> Journal of Experimental Biology. 210, 238-260 (2007).</li><li>Brezina, V., Evans, C. G., Weiss, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+membrane+ion+currents+of+a+model+molluscan+muscle,+the+accessory+radula+closer+muscle+of+Aplysia+california.+I.+Hyperpolarization-activated+currents.">Characterization of the membrane ion currents of a model molluscan muscle, the accessory radula closer muscle of Aplysia california. I. Hyperpolarization-activated currents.</a> Journal of Neurophysiology. 71, 2093-2112 (1994).</li><li>Evans, C. G., Rosen, S., Kupfermann, I., Weiss, K. R., Cropper, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+Radula+Opener+Neuromuscular+System+in+Aplysia.">Characterization of a Radula Opener Neuromuscular System in Aplysia.</a> Journal of Neurophysiology. 76 (2), 1267-1281 (1996).</li><li>Cullins, M. J., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrode+Fabrication+and+Implantation+in+Aplysia+californica+for+Multi-channel+Neural+and+Muscular+Recordings+in+Intact,+Freely+Behaving+Animals.">Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals.</a> Journal of Visualized Experiment. (40), 1791 (2010).</li><li>Dudek, F. E., Cobbs, J. S., Pinsker, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bag+cell+electrical+activity+underlying+spontaneous+egg+laying+in+freely+behaving+Aplysia+brasiliana.">Bag cell electrical activity underlying spontaneous egg laying in freely behaving Aplysia brasiliana.</a> Journal of Neurophysiology. 42, 804-817 (1979).</li><li>Hermann, P., Maat, A., Jansen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Neural+Control+of+Egg-Laying+Behaviour+in+the+Pond+Snail+Lymnaea+Stagnalis:+Motor+Control+of+Shell+Turning.">The Neural Control of Egg-Laying Behaviour in the Pond Snail Lymnaea Stagnalis: Motor Control of Shell Turning.</a> Journal of Experimental Biology. 197, 79-99 (1994).</li><li>Jansen, R. F., Pieneman, A. W., Ater Maat, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+Generation+in+the+Buccal+System+of+Freely+Behaving+Lymnaea+stagnalis.">Pattern Generation in the Buccal System of Freely Behaving Lymnaea stagnalis.</a> Journal of Neurophysiology. 82, 3378-3391 (1999).</li><li>Kupfermann, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociation+of+the+appetitive+and+consummatory+phases+of+feeding+behavior+in+Aplysia:+a+lesion+study.">Dissociation of the appetitive and consummatory phases of feeding behavior in Aplysia: a lesion study.</a> Behavioral Biology. 10, 89-97 (1974).</li><li>Scott, M. L., Kirk, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+consummatory+feeding+behavior+after+bilateral+lesions+of+the+cerebral-buccal+connectives+in+Aplysia+california.">Recovery of consummatory feeding behavior after bilateral lesions of the cerebral-buccal connectives in Aplysia california.</a> Brain Research. 585, 272-274 (1992).</li><li>de Boer, P. A., Jansen, R. F., ter Maat, A., van Straalen, N. M., Koene, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distinction+between+retractor+and+protractor+muscles+of+the+freshwater+snail&#8217;s+male+organ+has+no+physiological+basis.">The distinction between retractor and protractor muscles of the freshwater snail&#8217;s male organ has no physiological basis.</a> Journal of Experimental Biology. 213, 40-44 (2010).</li><li>Chiel, H. J., Weiss, K. R., Kupfermann, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+identified+histaminergic+neuron+modulates+feeding+motor+circuitry+in+Aplysia.">An identified histaminergic neuron modulates feeding motor circuitry in Aplysia.</a> Journal of Neuroscience. 6, 2427-2450 (1986).</li><li>Hurwitz, I., Neustadter, D., Morton, D. W., Chiel, H. J., Susswein, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+patterns+of+the+B31/B32+pattern+initiators+innervating+the+I2+muscle+of+the+buccal+mass+during+normal+feeding+movements+in+Aplysia+californica.">Activity patterns of the B31/B32 pattern initiators innervating the I2 muscle of the buccal mass during normal feeding movements in Aplysia californica.</a> Journal of Neurophysiology. 75, 1309-1326 (1996).</li><li>Kier, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diversity+of+hydrostatic+skeletons.">The diversity of hydrostatic skeletons.</a> Journal of Experimental Biology. 215, 1247-1257 (2012).</li></ol>

The authors have nothing to disclose.

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.

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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60030/60030fig1.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/60030/60030fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>Blunt forceps</td>
			<td>Fine Science Tools</td>
			<td>11210-10</td>
			<td>2 pair</td>
		</tr>
		<tr>
			<td>Scalpel blade (#11)</td>
			<td>Fine Science Tools</td>
			<td>10011-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15024-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Webcam</td>
			<td>Logitech</td>
			<td>c920</td>
			<td>for recording data</td>
		</tr>
	</tbody>
</table>

a minimally invasive lesion technique for muscles intrinsic to the odontophore of aplysia californica

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.

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).
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/60030/60030fig9.jpg" /> 
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 &#60; 0.031 for the I7 lesion group, indicated with a single asterisk, or p &#60; 0.002 for the SRF lesion group, indicated by a double asterisk). <a href="https://www.jove.com/files/ftp_upload/60030/60030fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Body Weight</td>
			<td>Magnesium Chloride Dose</td>
		</tr>
		<tr>
			<td>&#60;200 g</td>
			<td>&#189; bodyweight</td>
		</tr>
		<tr>
			<td>200-350 g</td>
			<td>1/3 bodyweight</td>
		</tr>
		<tr>
			<td>350-450 g</td>
			<td>&#188; bodyweight</td>
		</tr>
	</tbody>
</table>
Table 1: Magnesium Chloride dosage by bodyweight.

Watch this Scientific Journal Video about A Minimally Invasive Lesion Technique for Muscles Intrinsic to the Odontophore of Aplysia californica at JoVE.com

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

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.

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

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 ...

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Neuroscience

5.9K Views.  Case Western Reserve University. 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.

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

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

A technique is described for implanting four in vivo electrodes to monitor the neuromuscular control of feeding behavior in Aplysia californica.

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal.  ...

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 &deg;C) that are natural for Aplysia, preparations are stable for long periods of time. 
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. 
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

Multifunctionality, the ability of one peripheral structure to generate multiple, distinct behaviors1, allows animals to rapidly adapt their behaviors to changing environments. The marine mollusk Aplysia californica provides a tractable system for the study of multifunctionality. During feeding, Aplysia generates several distinct types of behaviors using the same feeding apparatus, the buccal mass. The ganglia that control these behaviors contain a number of large, identified neurons that are accessible to electrophysiological study. The activity of these neurons has been described in motor programs that can be divided into two types, ingestive and egestive programs, based on the timing of neural activity that closes the food grasper relative to the neural activity that protracts or retracts the grasper2. However, in isolated ganglia, the muscle movements that would produce these behaviors are absent, making it harder to be certain whether the motor programs observed are correlates of real behaviors. In vivo, nerve and muscle recordings have been obtained corresponding to feeding programs2,3,4, but it is very difficult to directly record from individual neurons5. Additionally, in vivo, ingestive programs can be further divided into bites and swallows1,2, a distinction that is difficult to make in most previously described in vitro preparations.
The suspended buccal mass preparation (Figure 1) bridges the gap between isolated ganglia and intact animals. In this preparation, ingestive behaviors - including both biting and swallowing - and egestive behaviors (rejection) can be elicited, at the same time as individual neurons can be recorded from and stimulated using extracellular electrodes6. The feeding movements associated with these different behaviors can be recorded, quantified, and related directly to the motor programs. The motor programs in the suspended buccal mass preparation appear to be more similar to those observed in vivo than are motor programs elicited in isolated ganglia. Thus, the motor programs in this preparation can be more directly related to in vivo behavior; at the same time, individual neurons are more accessible to recording and stimulation than in intact animals. Additionally, as an intermediate step between isolated ganglia and intact animals, findings from the suspended buccal mass can aid in interpretation of data obtained in both more reduced and more intact settings. The suspended buccal mass preparation is a useful tool for characterizing the neural control of multifunctionality in Aplysia.

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

We describe a technique to extracellularly record and stimulate from nerves, muscles, and individual identified neurons in vitro while eliciting and observing different types of feeding behaviors in the feeding apparatus of Aplysia.

Multifunctionality, the ability of one peripheral  structure to generate multiple, distinct behaviors1, allows animals  to rapidly adapt their behaviors ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the development of several animal models of these neuromuscular disorders has provided the scientific community with different therapeutic scenarios aimed at delaying or reversing the progression of these conditions. By taking advantage of the retrograde machinery of neurons, one of these approaches has been to target skeletal muscles in order to shuttle therapeutic genes into corresponding spinal cord motor neurons. Although once promising, the success of such gene delivery approach has been hampered by the sub-optimal number of transduced motor neurons it has so far shown to yield. Motor end plates (MEPs) are highly specialized regions on the skeletal musculature that are in direct synaptic contact to the spinal cord &#945; motor neurons. In this regard, it is important to note that, so far, the efforts to retrogradely transfer genes into motor neurons were made without reference to the location of the MEP region in the targeted muscles. Here, we describe a simple protocol 1) to reveal the exact location of the MEPs on the surface of skeletal muscles and 2) to use this information to guide the intramuscular delivery and subsequent optimal retrograde transport of retrograde tracers into motor neurons. We hope to utilize the results from these tracing experiments in further studies into investigating retrograde transport of therapeutic genes to spinal cord motor neurons through the targeting of MEPs.

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy

Stereological methods are designed to describe quantitative parameters without making assumptions about size, shape, orientation and distribution of cells or structures. These methods have been revolutionary for quantitative analysis of the mammalian brain, in which volumetric cell populations are too high to count manually, and stereology is now the technique of choice whenever estimates of three-dimensional quantities need to be extracted from measurements on two-dimensional sections. All stereological methods are in principle unbiased; however, they rely on proper knowledge about the structure of interest and the characteristics of the tissue. Stereology is based on Systematic Uniformly Random Sampling (SURS), with adjustment of sampling to the most efficient level in respect to precision, providing reliable, quantitative information about the whole structure of interest. Here we present the optical fractionator in conjunction with BrdU immunohistochemistry to estimate the production and survival of newly-formed neurons in the granule cell layer (including the sub-granular zone) of the rat hippocampus following electroconvulsive stimulation, which is among the most potent stimulators of neurogenesis. The optical fractionator technique is designed to provide estimates of the total number of cells from thick sections sampled from the full structure. Thick sections provide the opportunity to observe cells in their full 3-D extent and thus, allow for easy and robust cell classification based on morphological criteria. When correctly implemented, the sensitivity and efficiency of the optical fractionator provides accurate estimates with a fixed and predetermined precision.

Here, we present a stereological method, the optical fractionator, used to quantify the formation of new neurons, and their survival, in the rat hippocampus following electroconvulsive stimulation. When correctly implemented, the sensitivity and efficiency of stereological methods ensures accurate estimates with a fixed and predetermined precision.

Stereological methods are designed to describe quantitative parameters without making assumptions about size, shape, orientation and distribution of cells ...