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

Summary

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.

Abstract

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 programs^2,3,4, but it is very difficult to directly record from individual neurons⁵. Additionally, in vivo, ingestive programs can be further divided into bites and swallows^1,2, 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.

Protocol

1. Preparation of Solutions

1. To prepare magnesium chloride solution that is isotonic to the seawater in which the animals are kept (~ 1,000 millosmolar), mark a large jug at the level of the desired volume. Fill the jug with distilled water to approximately 80% of this level, and weigh out the appropriate amount of magnesium chloride hexahydrate to create a 333 mM solution in the final volume. Add the magnesium chloride to the jug, close the lid and shake vigorously until the magnesium chloride is fully dissolved, and then add distilled water until the final volume is reached.
2. To prepare Aplysia saline, again mark a large jug at the level....

Results

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.......

Discussion

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.

Materials

List of materials used in this article

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