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 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.
1. Preparation of Solutions
2. Preparation of Recording Dish
3. Electrode Preparation
4. Animal Dissection and Buccal Mass Preparation
5. Hook Electrode Attachment
6. Positioning the Ganglion and Thinning the Sheath
7. Stimulation and Recording
At this time in the United States, invertebrate animals do not require formal approval by an institutional animal use and care committee. However, we have ensured that all treatments of Aplysia minimize harm and suffering to the animal, and that all dissections are done while the animal is fully anesthetized.
When an extracellular electrode is positioned above a neuron’s soma and used to stimulate the neuron, a one-for-one correspondence between spikes on the soma channel and on the nerve(s) the neuron projects to can be observed (Figure 6, left panel, stimulation of identified neuron B9). The soma channel (top channel) is set to stimulating mode when the current is applied (time 1 in the figure), and is then quickly switched to recording mode (time 2). By maintaining the position of the electrode, the activity of the neuron can be recorded during motor programs (Figure 6, right panel), along with activity from the I2 muscle, radular nerve, and bilateral buccal nerves 2 and 3.
Figure 7 shows video images taken from a series of swallowing patterns induced by application of carbachol to the cerebral ganglion and the subsequent placement of a seaweed strip into the grasper during carbachol-induced biting. In 7A, the radula is protracted to grasp the seaweed, and in 7B, the radula has retracted, pulling seaweed into the mouth. Videos and electrophysiological recordings can be directly synchronized to each other; the still images in Figure 7 correspond to times A and B indicated in Figure 6‘s right panel.
Figure 1. Schematic of overall setup for experiment. The main image shows an overhead view. The cerebral ganglion and buccal ganglia are pinned to Sylgard. The cerebral ganglion is in an isolated chamber so that carbachol can be applied to it. The cerebral-buccal connectives pass through a notch in the Sylgard, which is sealed with vacuum grease. The buccal mass, in the front chamber, is suspended by the buccal nerves from its posterior and by a silk suture at its anterior edge. An inset shows a side view of the setup, including an extracellular electrode positioned above the buccal ganglion. The entire structure within the dish is constructed from Sylgard. Dimensions indicated within the inset are: a. Length of notch connecting cerebral and buccal chambers, 0.5 cm. b. Width of buccal ganglia platform, 0.5 cm. c. Height of buccal mass chamber from bottom to buccal ganglia platform, 1.7 cm. Note that the height of the buccal mass chamber can be adjusted, depending on the size of the buccal mass, by placing a small platform made of Sylgard underneath the buccal mass.
Figure 2. Schematic of the Aplysia feeding apparatus, the buccal mass, showing the locations of key structures, including buccal nerves (BN) 1, 2, and 3, the radular nerve (RN), and the I2 muscle, and the attachment points for hook electrodes. The radular nerve projects ventrally from the rostral surface of the buccal ganglia and goes beneath the surface fibers of the I2 muscle. Buccal nerve 2 trifurcates into branches a, b, and c before going beneath the I1 muscle at the lateral groove. Branch a is the first branch to separate from the main trunk, and is adjacent to buccal nerve 3. The nomenclature of branches a, b, and c was used by Warman and Chiel5. Branches a, b, and c correspond to branches 3, 2, and 1, respectively, in the nomenclature of Nargeot et al.9 Furthermore, the RN, BN1, BN2, and BN3 correspond to nerves 1, 6, 5, and 4, respectively, in the nomenclature of Kandel10 and Scott et al.11
Figure 3. Attachment of hook electrode to nerve. The nerve is placed within the hook and lifted, and superglue is applied to the nerve and hook. The glue must completely cover the hook, and must not cover the end of the reference wire.
Figure 4. Setup of buccal ganglia, showing the arrangement of pins to position and secure the ganglia. Pins must be placed in the sheath surrounding the nerves and ganglia, being careful not to damage nerves or neurons. The left ganglion is shown as rotated forward to provide access to neurons that are proximate to the back chamber. To accomplish this rotation, excess sheath from the CBC is pulled forward and pinned between buccal nerves 2 and 3. Note that the caudal surface of the buccal ganglion (which is rounded, rather than flat) is schematically shown in the figure.
Figure 5. Positioning of extracellular electrodes over neuron somata in the buccal ganglion. Glass electrode tips should be gently pressed, using manipulators, into the thinned sheath above target cell bodies. Two electrodes can be simultaneously positioned above different cells, as pictured.
Figure 6. Example AxoGraph recordings. Channels are shown for one extracellular soma electrode (top channel) and (in sequence from the top) for hook electrodes on the I2 muscle, radular nerve, left buccal nerve 2, left buccal nerve 3, right buccal nerve 2, and right buccal nerve 3. At left, identification of a neuron by extracellular stimulation. The soma channel is used to apply a stimulating current (time 1, note stimulation artifact in all channels other than the soma channel, which is not recording), and is then switched to recording mode (time 2). Note the one-for-one correspondence between spikes on the soma channel and the BN2-L, BN3-L, and BN3-R channels. At right, recording of two cycles of feeding programs as the buccal mass swallows a strip of seaweed. Note that the activity of the neuron previously stimulated can be visualized on the soma channel (these spikes are highlighted by red bars above the soma channel). This is identified neuron B9 in the right buccal ganglion. Times A and B correspond to video frames shown in Figure 7. Click here to view larger figure.
Figure 7. Example video images corresponding to the recording shown in Figure 6. The suspended buccal mass is shown in a front view (left) and side view (right, a mirror positioned adjacent to the dish). A strip of seaweed has been placed in the mouth and the radula is in the process of swallowing the seaweed. A. The radula is protracted to grasp the seaweed. B. The radula has retracted, pulling seaweed into the mouth. Note that A and B correspond to the same times labeled A and B in Figure 6.
Previous work has characterized Aplysia motor programs in the intact animal and in reduced preparations, such as isolated ganglia. In the intact animal, although recordings of individual neurons have been obtained5, such experiments are very difficult, and electrodes cannot be moved from neuron to neuron during feeding. In isolated ganglia, the feeding movements induced by neural activity cannot be observed. The suspended buccal mass preparation bridges the gap between these two extremes.
Other semi-intact preparations have been used in previous studies, but the suspended buccal mass preparation has significant advantages. In one semi-intact preparation, the lips and buccal mass were still attached, but the preparation was too reduced for full feeding movements to be observed 14. A feeding head preparation15,16 allowed observation of feeding movements in vitro. However, this feeding head preparation was more complicated to set up than the suspended buccal mass described here, and only provided access to individual neurons in the cerebral ganglion, not in the buccal ganglion. There is an advantage to the feeding head preparation, in that the presence of the lips and anterior tentacles allowed the generation of biting patterns through a natural stimulus, application of seaweed. Although it would make the experiment more difficult, it may be possible to combine the preparations, leaving the lips attached to the buccal mass while also pinning the buccal ganglia for access to individual neurons.
Feeding patterns in Aplysia can be classified as ingestive or egestive based on the timing of activity on the radular nerve, which closes the food grasper, relative to the protraction and retraction phases of motor programs. If radular nerve activity occurs at the same time as the retraction phase, patterns are ingestive, because the grasper is closing and retracting as it attempts to pull food into the buccal cavity. If radular nerve activity occurs during the protraction phase, patterns are egestive, because the grasper is closing and protracting to push material out of the buccal cavity2.
Ingestive patterns can be divided into at least two distinct sub-types: biting, attempts to grasp food, and swallowing, transport of already grasped food1,2. However, most in vitro work (e.g. 14,17,18) has focused only on the ingestion/egestion dichotomy, not attempting to distinguish biting from swallowing. Two studies16,19 have classified in vitro ingestive patterns in biting-like and swallowing-like groups. In the first of these studies, the patterns were observed in isolated ganglia; if the associated feeding movements of the buccal mass could be observed, this would strengthen the argument that these patterns represent the biting and swallowing seen in vivo. In the second study, the patterns were observed in a feeding head preparation, so patterns were classified based on movements observed, but no recordings of buccal ganglion neurons were obtained.
The suspended buccal mass preparation provides a way to observe all three types of behaviors – biting, swallowing, and rejection – while simultaneously recording activity from key buccal nerves and muscles, and both stimulating and recording from individual neurons. The extracellular neuron technique6 is another key advance in this preparation, because the strong feeding-like movements elicited would almost certainly dislodge an intracellular electrode. With extracellular recordings of individual neurons, the timing of these neurons’ activity during motor programs can be ascertained when this would be difficult with nerve recordings alone, because many different units are firing simultaneously.
We have observed that feeding movements in the suspended buccal mass appear qualitatively similar to those seen in vivo. Importantly, stimuli used to generate biting and rejection patterns (carbachol and buccal nerve 2 branch a stimulation, respectively) have been used in more reduced preparations, and our finding that the stimuli generate physiological movements lends credibility to their use in other settings. By contrast, to generate swallowing patterns, we combined an artificial stimulus (carbachol) with a natural food stimulus (a seaweed strip) which could not be applied to isolated ganglia. We have also observed that the neural patterns in the suspended buccal mass appear to be more similar to in vivo patterns than are patterns obtained in more reduced preparations, suggesting sensory feedback may be important in shaping the patterns from the CPG. For example, motor programs in isolated ganglia are typically several times longer in duration than motor programs in intact animals, whereas motor programs in the suspended buccal mass are typically much closer in duration to motor programs in intact animals (unpublished data). Another advantage of the suspended buccal mass is that simultaneous side and front views of the feeding apparatus can be obtained. The front view provides a similar view of the mouth to that which would be seen in vivo, while allowing better observation of jaw muscle contraction. The side view allows observation of buccal muscle contractions and the forward and backward movement of the grasper underneath the muscles, making it easier to understand biomechanical changes during the different behaviors.
In this preparation, we have recorded from up to five nerves (radular nerve and bilateral buccal nerves 2 and 3) and the I2 muscle simultaneously, and placed up to two extracellular electrodes over identified nerve cells in the ganglion. It would be possible to record from additional nerves and/or muscles, if the experimenter desired. It would also be possible to stimulate and record from neurons in the cerebral ganglion, which could give insight into whether stimulation of cerebral buccal interneurons, a common technique17,19, induces motor programs similar to in vivo behavior. Although doing so increases the technical difficulty of the preparation, perfusion of the buccal artery15 could allow the preparation to generate stronger and longer-lasting behaviors (unpublished observations).
The authors have nothing to disclose.
This research was supported by NIH grant NS047073 and NSF grant DMS1010434.
Name | Company | Catalog Number | Comments |
Sodium chloride | Fisher Scientific | S671 | Biological, Certified |
Potassium chloride | Fisher Scientific | P217 | Certified ACS |
Magnesium chloride hexahydrate | Acros Organics | 19753 | 99% |
Magnesium sulfate heptahydrate | Fisher Scientific | M63 | Certified ACS |
Calcium chloride dihydrate | Fisher Scientifc | C79 | Certified ACS |
Glucose (dextrose) | Sigma-Aldrich | G7528 | BioXtra |
MOPS buffer | Acros Organics | 17263 | 99% |
Carbachol | Acros Organics | 10824 | 99% |
Sodium hydroxide | Fisher Scientific | SS255 | Certified |
Hydrochloric acid | Fisher Scientific | SA49 | Certified |
Single-barreled capillary glass | A-M Systems | 6150 | |
Flaming-Brown micropipette puller model P-80/PC | Sutter Instruments | Filament used: FT345B | |
Enamel coated stainless steel wire | California Fine Wire | 0.001D, coating h | |
Household Silicone II Glue | GE | ||
Duro Quick-Gel superglue | Henkel corp. | ||
A-M Systems model 1700 amplifier | A-M Systems | Filter settings: 300-500 Hz nerves,10-500 Hz I2 muscle | |
Pulsemaster Multi-Channel Stimulator | World Precision Instruments | A300 | |
Stimulus Isolator | World Precision Instruments | A360 | |
AxoGraph X | AxoGraph Scientific | ||
Veeder-Root Totalizing Counter | Danaher | C342-0562 | |
Gold Connector Pins | Bulgin | SA3148/1 | |
Gold Connector Sockets | Bulgin | SA3149/1 | |
Sylgard 184 Silicone Elastomer | Dow Corning | ||
100 x 50 mm Crystalizing Dish | Pyrex | ||
High Vacuum Grease | Dow Corning | ||
Pipet Tips | Fisher Scientific | 21-375D | |
Minutien Pins | Fine Science Tools | 26002-10 | |
Modeling Clay | Sargent Art | 22-4400 | |
Silk Sutures | Ethicon | K89OH | |
Whisper Air Pump | Tetra | 77849 | |
Aquarium Tubing | Eheim | 7783 | 12/16 mm |
Elite Airstone | Hagen | A962 | |
Vannas Spring Scissors | Fine Science Tools | 15000-08 | |
Dumont #5 Fine Forceps | Fine Science Tools | 11254-20 | |
Yaki Sushi Nori Seaweed | Rhee Bros | ||
Kimwipes | Kimberly-Clark | 34155 |