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

Summary

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques^1,2,3,4. 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.

Abstract

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques^1,2,3,4. 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 innervation^4,6,7. 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 pools^10,11,12 in Aplysia or in other animal systems^2,3,13,14.

Protocol

1. Preparation of Recording Dish

1. During the force transducer experiments, the buccal ganglia, cerebral ganglion, and buccal mass are placed in a round Pyrex dish that is specialized for force studies.
2. To induce ingestive-like patterns in the experiments, we need to apply the non-hydrolyzable cholinergic agonist carbachol to the cerebral ganglion¹⁵. To avoid direct contact from carbachol onto the buccal ganglia and buccal mass, separate chambers are needed to isolate the cerebral ganglion from the buccal ganglia and the buccal mass (Figure 1).
3. Since the buccal mass is much thicker than the buccal ganglia, they....

Results

Figures 4 and 5 show typical results used to identify two I1/I3 motor neurons. Figure 4 shows the soma recordings of a large motor neuron, B3, during egestive-like and ingestive-like patterns (Figures 4C, 4D). The one-for-one corresponding spikes on the soma channel and the ipsilateral BN2 channel (Figure 4E) show that the specificity of B3 soma recording was maintained during patterns. B3 fires during the middle-to-late retraction phase.......

Discussion

In animals with large identified neurons, such as mollusks (for example, Lymnaea, Helix, and Aplysia), analysis of motor pools is typically done using intracellular recording^1,2,3,4. In this protocol, we describe a process for uniquely identifying the motor neurons for a motor pool using an extracellular technique. We used the force measurements as an illustration of this process. One could also use EMG to measure muscle innervations. Briefly, to do so, the protocol needs to be .......

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: 10-500 Hz for the I2 nerve/muscle; 300-500 Hz for all the other nerves
Pulsemaster Multi-Channel Stimulator	World Precision Instruments	A300
Stimulus Isolator	World Precision Instruments	A360
AxoGraph X	AxoGraph Scientific		Software for recordings
Gold Connector Pins	Bulgin	SA3148/1
Gold Connector Sockets	Bulgin	SA3149/1
Sylgard 184 Silicone Elastomer	Dow Corning
100 x 15 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
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
Kimwipes	Kimberly-Clark	34155