Synaptic Plasticity: Short-Term Changes at the Neuromuscular Junction

Introduction

If you have not already done so, read Appendix A, Crayfish Neuromuscular Preparation, for background. In this lab exercise, you will stimulate the motor neurons innervating the superficial flexor (SF) muscle of the crayfish abdomen while recording the resulting synaptic potentials in muscle fibers. Your main goal will be to demonstrate and quantify some types of synaptic plasticity seen at these neuromuscular synapses. Synaptic plasticity, the change in the strength of synaptic communication between neurons, is thought to be one of the major mechanisms of learning and memory (Byrne et al., 2009; Purves et al., 2012). You should see three forms of intrinsic, use-dependent synaptic plasticity: two-pulse facilitation, post-tetanic potentiation (PTP), and synaptic depression. These are changes in synaptic strength resulting from the synapse’s history of activity (Purves et al., 2012; Zucker et al., 2009).

Dissection

The dissection is the same as for Lab 5, Synaptic Connectivity. You can go back to that lab or watch Video 6.1, Dissection, Recording, and Stimulation, for the entire procedure. Use any segment except the first one, which is damaged from the initial cut.

Recording

Prepare for an extracellular recording of nerve 3 on one side and an intracellular recording of the superficial flexor muscle on the same side as in Lab 5, Synaptic Connectivity. However, instead of recording from the nerve, you will stimulate it. Before starting to record, 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.

• Figure 6.1 shows the setup for stimulation and recording. The suction electrode is connected to a stimulus isolation unit (SIU) for stimulation of nerve 3. If it were connected to an AC amplifier you could record spontaneous extracellular action potentials from the spontaneously firing motor neurons in this nerve.
• Video 6.2, Cutting Nerve 3. Cut nerve 3 near the ganglion. Locate the cut end of the nerve and suck it into the tip of the suction electrode (suck saline up to the internal wire first).
• Next, record intracellularly from a SF muscle fiber on the same side as the nerve you are stimulating (see Lab 4, Muscle Resting Potential, for recording methods). Start by recording from fibers in the medial half of the muscle. Start with the oscilloscope set 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 resting potential (at least −50 mV), set this channel to AC coupling, decrease the time scale (5 to 20 ms/div), and slow the vertical scale to 20 to 50 mV/div (corresponding to 2 to 5 mV/div in reality). You will not see spontaneous EPSPs with the nerve cut. (You may see very small EPSPs, or “minis”, which represent spontaneous release of transmitter.)
• Video 6.3, Stimulating Nerve 3. Set the stimulator to a low voltage (around 5.0 V) and short duration (about 2 ms to start with), and set it to repeat a stimulus about once every 10 seconds (see Figure C.4 for stimulator settings). Slowly increase the voltage until it causes a single EPSP in the muscle. Once you can elicit an EPSP, decrease the stimulus duration until the EPSP fails. Balance the stimulus voltage and duration to elicit an EPSP with the shortest stimulus possible (aim for a duration of 0.5 ms). If your stimulus evokes more than one EPSP, reduce the voltage.
• If you cannot elicit an EPSP in this muscle fiber, check that the suction electrode is still in contact with the nerve and that the nerve’s connection with the muscle is intact. If it seems to be alright, try recording in a different location. Remember that you need to record near a synaptic junction to see EPSPs. If you have not been able to stimulate any EPSPs after recording from several muscle fibers in different locations, try releasing the nerve from the suction electrode and sucking it in again at a point closer to the muscle.

Experiments

Two-Pulse Facilitation

Once you can reliably stimulate an EPSP in a muscle fiber, set the stimulator to produce a short train of pulses (150 ms interval for 1 s). Note the increase in EPSP size with each stimulus pulse. Now quantify facilitation by setting the stimulator to produce a pair of pulses 5 s apart. Measure the amplitude of each EPSP evoked by these pulses. Gradually reduce the interval between pulses and measure the EPSP amplitudes in each case (Video 6.4, Eliciting Facilitation). Calculate a facilitation index from the ratio of amplitudes (F = (EPSP₂−EPSP₁)/EPSP₁, or F = EPSP₂/EPSP₁−1) at each interval (see the end of Video 6.4, Eliciting Facilitation). First, reduce the interval in 1 s increments down to 1 s. Next, try intervals of 600, 400, 200, and 100 ms, followed by 80, 60, 40, and 20 ms. The range of intervals that gives good facilitation may vary from one muscle fiber to the next; concentrate your measurements on the interval range that is of most interest. When collecting data, wait at least 5 s after each stimulus pair to allow the synapse to return to its normal, non-facilitated state. Be sure to collect an entire data set from one muscle fiber. If possible, use the same fiber for the next set of experiments as well.

Posttetanic Potentiation

In this experiment, you will examine the effect of a tetanic stimulus on PSP amplitude. To do so, you will first elicit a single EPSP (EPSP₁), followed by a 20 Hz (50 ms interval) tetanus for 5 s, followed by a final single pulse to elicit a test EPSP (EPSP₂) at varying times after the train. For each delay after the tetanus, calculate a facilitation index (EPSP₂/EPSP₁−1). Try a range of delays from 1 to 10 s. Wait at least 20 s between trials to allow the synapse to return to its non-potentiated state.

If you do not have software that has been set up to produce this type of stimulus, you can follow this procedure with an electronic stimulator. (Names of settings vary with different stimulators; ask your instructor for help.)

1. Set the repetition rate to 20 Hz (or period of 50 ms).
2. Start acquiring data on the computer, setting a duration of 30 s.
3. Set the mode to single pulse and manually trigger a single pulse.
4. Quickly switch to train (or repeat) mode and hold the trigger switch down for 5 s.
5. Quickly switch back to single-pulse mode and manually trigger a single pulse after the desired delay. The delay will not be precise, but you can measure the actual delay afterward.

On your computer, expand the EPSP trace and scroll through the portion that occurred during the tetanic stimulation. What happens to EPSP amplitude during this time? You will probably see an initial increase in amplitude (due to facilitation), followed by some decrease, followed by amplitude fluctuations. The decrease is synaptic depression, while the fluctuations reflect the interactions between facilitation and depression.

Once you have a complete set of data for a 5 s tetanus, repeat this experiment with a longer or shorter tetanus duration. What effect does this have on the magnitude and duration of posttetanic potentiation?

Comparisons

After you have complete sets of facilitation and posttetanic potentiation data from one synapse, move to another muscle fiber and repeat both experiments. Do the two types of facilitation vary quantitatively between muscle fibers? Do any synapses not show facilitation or PTP?

Further Exploration

You have now seen the changes of synaptic strength that result from patterns of nerve activity. Neuromodulators can also bring about dramatic changes in the strength of synaptic transmission. Apply neuromodulators such as dopamine and serotonin and examine their effects on synaptic strength and plasticity (Djokaj et al., 2001; Miller et al., 1985; Glusman and Kravitz, 1982).

Long-term potentiation (LTP) and long-term depression (LDP) are other forms of use-dependent plasticity that maintain altered synaptic transmission much longer than PTP (Byrne et al., 2009). Experiment with the ability of the crayfish neuromuscular junction to produce LTP by using longer trains of repeated stimuli (over 10 s) at varying stimulus frequencies. Watch out for muscle contractions that could dislodge the electrode during long-duration high-frequency stimulation.

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. From your two-pulse experiments, plot facilitation index vs. stimulus interval. Describe the time course of facilitation. Fit an exponential curve to your data (F_t = F₀ e^−t/τ, where F_t is the facilitation index at interval t). If any of your facilitation indices are below 0, you must omit them from the analysis. What do the parameters of this exponential equation indicate about the time course of facilitation? If you did this experiment with more than one synapse, how did your results vary?
2. From your posttetanic potentiation experiments, plot facilitation index vs. delay after tetanus. Fit an exponential curve to these data. How do the time courses of two-pulse facilitation and posttetanic potentiation compare? If you did this experiment with more than one synapse, how did your results vary?
3. Describe what happened to EPSP amplitudes during the PTP stimulus train. Explain this variation in terms of the dynamic interaction between facilitation, PTP, and synaptic depression.
4. Briefly discuss presynaptic mechanisms that might account for facilitation, depression, and posttetanic potentiation (Xu-Friedman and Regehr, 2003; Zucker et al., 2009; Zucker and Regehr, 2002).
5. What postsynaptic mechanisms might account for these three forms of synaptic plasticity?
6. Design experiments that could distinguish between presynaptic and postsynaptic mechanisms of synaptic plasticity.

References

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