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Synaptic Connectivity: Nerve-Target Matching and Synaptic Integration

Introduction

If you have not already done so, read Appendix A, Crayfish Neuromuscular Preparation, for background. In this lab exercise, you will simultaneously record intracellularly from the superficial flexor (SF) muscle of the crayfish abdomen and extracellularly from ganglionic nerve 3, which innervates it. Your goals are to map the innervation pattern of motor neurons onto the muscle, describe excitatory postsynaptic potentials (EPSPs) in muscle fibers, matching EPSPs with the identifiable action potentials (APs) in the nerve that cause them, and document examples of synaptic integration. You may find it helpful to refer to your data from Lab 2, Nerve Recording, in which you determined the number of AP classes in nerve 3 and how nerve 3 activity changed with sensory stimulation.

You will see that individual muscle fibers are selectively innervated by some neurons, but not others, in that some action potentials in the nerve will not match with any EPSP in a given muscle fiber. You will also see evidence of polyneuronal innervation, in that most muscle fibers have EPSPs resulting from activity in more than one axon. Finally, you will examine some basic integrative features of the synapse, including temporal and spatial summation of EPSPs and IPSPs (Byrne, 2008; Nicholls et al., 2012; Purves, et al., 2012).

Dissection

The dissection is the same as for Lab 4, Resting Potential. Before starting, look at the methylene blue-stained specimen that was prepared in advance (it will resemble Figure A.2). This specimen shows nerve 3 leaving the ganglion, projecting to the superficial flexor muscle, and branching out over the surface of the muscle. At high magnification, you should be able to see single stained axons in nerve 3. Look closely at the nerve as it enters the muscle and note where it appears to terminate on the muscle. The fine branching ends of the nerves on the muscle are where synaptic contacts are made.

Recording

Figure 5.1 shows the setup for dual intracellular and extracellular recording. Video 5.2, Nerve-Muscle Recording, shows the recording procedure. Before starting to record from the muscle, replace the saline in the dissecting dish with fresh cold saline. Repeat this every half hour or so, but do not interrupt a good recording to do so.

Start with an extracellular recording of nerve 3 on one side (see Lab 2, Nerve Recording, for recording methods). If nerve 3 is not visible, apply Janus green for 15 s and then wash with saline. Be sure that the suction electrode placement will not prevent access to the muscle with the intracellular electrode. It is usually best to have the manipulator for the intracellular electrode on the same side as the muscle and the manipulator for the nerve on the opposite side. Rotate the preparation dish to get a good orientation. Next, record intracellularly from a muscle fiber on the same side as the nerve you are recording (see Lab 4, Resting Potential, for recording methods). If there are EPSPs in the fiber you are recording, they will appear as small (1 to 10 mV) deflections in the oscilloscope trace, as seen at the end of Video 5.2, Nerve-Muscle Recording.

Not every location from which you record on the superficial flexor will show EPSP activity. To see EPSPs, you must record near a synapse. Although each crayfish and abdominal segment is a little different, you may be able to direct your search by looking at a methylene blue-stained muscle and noting the location of fine nerve branches on the muscle. Generally, EPSPs can best be recorded in the middle of the muscle.

When starting muscle recording, set the oscilloscope channel with the intracellular recording to DC coupling with a fairly fast time scale (2 ms/div) and vertical scale of 0.1 V/div (your amplifier increases the signal 10×, so this corresponds to 10 mV/div of real voltage). Once you have a good muscle resting potential with obvious small depolarizing deflections on the oscilloscope, set this channel to AC coupling, slow the time scale (5 to 20 ms/div), and increase the vertical scale to 20 to 50 mV/div (corresponding to 2 to 5 mV in reality). Now the trace should appear more like that shown in Video 5.3, PSP Recording.

Experiments

EPSP Characteristics

Measure the waveform characteristics of some single EPSPs, including amplitude, rise time (from the initial positive deflection to the peak), and duration (measure at half the amplitude). Note the variation in amplitudes of the extracellular APs. Each extracellular action potential size class corresponds to a different axon in the nerve because each is caused by an axon of a different diameter. Larger diameter axons produce larger extracellular AP current densities than smaller diameter axons. Find an AP size class that always closely precedes a particular EPSP size. Does more than one size of AP correspond to EPSPs in this fiber? Measure the delay between several different AP size classes and the onset of the EPSPs that regularly follow them. Note that not every AP in the nerve can be matched to an EPSP in the muscle fiber. This is because not every axon in nerve 3 innervates every muscle fiber (selective innervation). If there is an obvious large AP that you cannot match with an EPSP, it may be from the inhibitory axon. Look for inhibitory postsynaptic potentials (IPSPs) or changes in the shapes of EPSPs after this axon fires. Are there any EPSPs that do not have an AP immediately preceding them? What does this mean?

Mapping Innervation

Draw a diagram of the muscle from which you are recording. Record from a variety of locations, marking them on your map. At each location, note the resting potential of the muscle fiber and which sizes of APs match EPSPs in the fiber. From this set of data, map the innervation of the muscle.

Reflex Activity

Changes in PSP activity in muscles due to sensory input are important because they lead to the behavioral response to sensory stimulation. While recording, gently prod the telson with a nonmetallic object (be careful not to disturb your muscle recording). The reflex activity evoked by this prodding will cause more AP activity in the nerve. Does it also activate a new size of AP in the nerve and a new EPSP in the muscle? Repeat this while recording from several different muscle fibers. Look for IPSPs during this reflex-evoked activity.

While eliciting reflex activity in the nerve and muscle, or when you have a spontaneously very active nerve and muscle, gather examples of temporal and spatial summation of EPSPs.

Further Exploration

You can follow up on questions raised by the above experiments in several ways:

Lab Cleanup

During the lab, be sure to immediately discard used glass electrodes in the appropriate container. After the lab, remove your last electrode from its holder and discard it as well. Clean up any spilled saline and rinse the ground electrode with distilled water. Expel saline from the suction electrode and rinse it with distilled water. Put the crayfish tail in the freezer along with the frozen heads and rinse the dissecting dish with fresh water.

Questions

  1. Why must the electrode be near a synaptic junction for PSPs to be recorded?
  2. Show your measurements of the waveforms of some EPSPs, including amplitude, rise time, and duration. Why do different AP classes often cause EPSPs of different amplitudes? Would you expect one AP class to elicit the same size PSP in different muscle fibers? Does the size of an AP correlate with the size of the PSP it causes? What would PSPs with different rise times tell you? Explain why you might see a PSP without an AP associated with it.
  3. Present your measurements of some intervals between APs in the nerve and the PSPs they cause in the muscle. Are the intervals shorter when the APs are larger? What physiological processes account for this interval?
  4. Present a map of the innervation of the superficial flexor muscle, marking all the sites from which you recorded and which AP size classes caused PSPs at each site. Are there medial-to-lateral differences in innervation of the muscle? What does selective innervation of particular muscle fibers by particular motor neurons suggest about the development of these synaptic connections?
  5. Show examples of what happened to nerve and PSP activity when you stimulated the tail.
  6. Present examples of the synaptic integration you observed.
  7. It is easy to find EPSPs in your recordings, but much harder to see evidence of inhibition. Why is this? Consider the ion conductances activated by excitatory and inhibitory innervation and explain why EPSPs are easier to record than IPSPs. How could the inhibitory axon affect EPSPs without producing a distinct hyperpolarizing IPSP? How could you enhance the amplitude of IPSPs to make them more visible in a recording?
  8. Briefly explain why the crayfish neuromuscular junction is a good model for human brain synapses while the mammalian neuromuscular junction is not.

References