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Appendix A. Crayfish Neuromuscular Preparation

Introduction

The superficial flexor nerve-muscle system of the crayfish abdomen (tail) is an excellent model preparation in which to experimentally examine important concepts in neuroscience. For example, many properties of excitability, including action potential generation and conduction, resting potential generation and maintenance, synaptic transmission, and modulation and plasticity of the synapse, can be easily examined here.

The superficial flexor (SF) is one of the four main groups of muscles in each abdominal segment that control movements of the tail (Figure A.1; see Larimer and Moore, 2003; Atwood, 2008; Kennedy et al., 1966). The abdominal muscles are of two classes: extensors that straighten the tail and flexors that bend it. Extensors and flexors are subdivided into phasic muscles and tonic muscles. The phasic muscles are involved in rapid tail flips for escape. They are the deep flexors, which curl the tail (in a lobster, these are the big muscles that make up the bulk of a lobster dinner), and the deep extensors, which return the tail to its extended position after a tail flip. The tonic superficial flexor and extensor muscles control fine movements of the tail that maintain the position of the tail in space (posture). The SF is a thin muscle sheet on the ventral surface of each tail segment, immediately under the cuticle (skin). It is innervated by a small set of motor neurons whose axons travel in the superficial branch of nerve 3 of each tail ganglion (Figure A.2). The superficial branch of nerve 3 is purely motor, with no sensory axons. Thus, all of the action potentials recorded from this nerve are commands that set the contractile state of the muscle and consequently contributing to tail posture.

Skeletal muscle of arthropods, including crayfish, has a number of features that make it distinctly different from vertebrate skeletal muscle, especially mammalian skeletal muscle such as our own (Figure A.3):

  1. Arthropod muscles are innervated by relatively few excitatory motor neurons (sometimes only one).
  2. Arthropod motor neurons innervate each muscle fiber at multiple points (multiterminal innervation).
  3. More than one motor neuron may innervate one muscle fiber (polyneuronal innervation).
  4. Inhibitory motor neurons may innervate muscle fibers (and sometimes the terminals of the excitatory motor nerve endings).
  5. The tonic superficial flexor does not have “all-or-none” propagated action potentials, but instead has graded electrical responses dependent upon the level of the excitation and inhibition. The degree of depolarization determines the amount of Ca2+ that enters the cell through voltage-gated channels; the amount of Ca2+ entry in turn determines the strength of muscle contraction. Unlike the superficial flexor, fast phasic crayfish muscles may fire Ca2+-based action potentials.

Synaptic transmission from the motor neurons onto crayfish muscle, however, has features that make it remarkably similar to synapses in our brains (Figure A.4). As in human brains, glutamate is an excitatory transmitter and GABA is an inhibitory transmitter. In addition, crayfish neuromuscular synapses show the types of synaptic plasticity thought to be involved in vertebrate learning and memory and in altering the efficacy of central synaptic transmission in normal and pathological brain network function. The multiterminal, polyneuronal, and inhibitory innervation of crustacean muscle, the use of glutamate and GABA as transmitters, and the extensive synaptic plasticity make the crayfish neuromuscular junction a good simplified model for the complex mix of synaptic interactions that occur in our own brains.

Lab Exercises

In the neuromuscular preparation set of lab exercises, you will:

Dissections

The dissections for these lab exercises are similar. In each case, you will remove the tail from the crayfish and pin it ventral-side-up in a dish (Video A.1, Preparing Crayfish Abdomen), remove the swimmerets (Video A.2, Removing Swimmerets), and open the cuticle between two of the abdominal segments. For Lab 2, you will only expose the area around the ventral nerve cord. For Lab 4, you will only expose the superficial flexor muscle. For Labs 5 and 6, you will expose both the nerve cord and the muscle. Details of those dissections are shown in videos in each lab.

References

There is a rich body of literature on many different aspects of the superficial flexor muscle preparation, and there is still plenty of room for creativity in extending the published literature and exploring new research areas. The literature listed below includes background on superficial flexor motor innervation, patterns of motor activity and reflex actions, motor neuron morphology and central nervous system organization, development and maintenance of the motor innervation, synaptic physiology, central and peripheral neuromodulation, and sensory feedback to the postural motor programs, as well as a few references to guide you to more information on other neuromuscular systems of the tail. Specific supplemental references are given in each lab exercise.