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Motor Nerve Tracing: Morphology of a Small Motor System

Introduction

If you have not already done so, read Appendix A, Crayfish Neuromuscular Preparation, for background. In Lab 2, Nerve Recording, you used extracellular recording to hypothesize the number of motor neurons innervating the superficial flexor muscle. In this lab, you will test that hypothesis with a staining technique that reveals the number and position of the motor neurons in the central nervous system.

Dissection

In this dissection, you will remove the ventral nerve cord with two adjacent ganglia and both third nerves from the most anterior ganglion. If you are careful, you should be able to obtain at least two such preparations from each crayfish tail.

Processing

Development

Because the next few steps must be done a day or two after the dissection, your instructor will probably do them. You will continue the procedure at the Evaluation step. Figure 3.2 illustrates processing in flowchart form. You should wear gloves for the procedures described below.

Fixation

Wear gloves and do these steps under a fume hood. Dispose of waste solutions in the containers provided under the hood.

Evaluation

Look at your preparation under the microscope.

Intensification

Wear gloves and do these steps under a fume hood. Dispose of waste solutions in the containers provided under the hood.

Clearing

Wear gloves and do these steps under a fume hood. Dispose of waste solutions in the containers provided under the hood.

Further Exploration

Try filling both third nerves in a segment to determine whether the same motor neurons innervate both superficial flexor muscles in that segment or whether the set of motor neurons are duplicated to innervate the other side. To see the position and structure of motor neurons involved in other motor patterns, such as tail flips, swimmeret movements, and tail fan movement, stain the deep branch of nerve 3 (Mittenthal and Wine, 1978), nerve 1, nerve 2, or any of the many nerves that leave the last abdominal ganglion. Nickel can be used instead of or in addition to cobalt in this process (Quicke and Brace, 1978). Try setting up a backfill with one nerve in a pool of cobalt chloride and another nerve in a pool of nickel chloride. You can distinguish the cells filled from the two nerves by their colors (cobalt-filled is red-brown, while nickel-filled is blue-black).

Lab Cleanup

After the dissection is finished and the ganglia are in the filling chamber, put the crayfish tail in the freezer along with the frozen heads, rinse the dissecting dish with fresh water, and clean up any spilled saline or Vaseline. During processing, dispose of chemical wastes in the containers provided in the fume hood.

Questions

  1. View the nerve cord under the microscope and determine how many motor neurons were stained. How many axons can you see in the nerve? How many cell bodies can you see in the ganglia? Make a sketch of the nerve cord, noting the positions of neuronal cell bodies relative to the midline of the ganglion and any neuronal processes you can see. Where would you expect synaptic input onto the motor neurons (such as input from sensory pathways) to occur?
  2. If you did Lab 2, Motor Nerve Recording, how does the count of stained cell bodies compare with your estimate of axon number based on the neural recordings? How do you account for any discrepancy? Is the number of cell bodies in the ganglion where nerve 3 originates the same as the number of axons in nerve 3?
  3. Compare the morphologies of the motor neurons innervating the SF muscle with neuronal morphologies illucidated by various other methods. Consider how morphology facilitates function. For examples, see Lichtman et al. (2008), Mulloney and Hall ( 2000), Murphy (2001), Purali (2005), and Purves et al. (2012).

References