Introduction:

In this lab, we used both intracellular and extracellular recordings to examine the
neuromuscular junction of the invertebrate crayfish. The neuromuscular junction is where the
motor neuron innervates the muscle cell and causes muscular contraction with enough
stimulation. Here, we investigate the superficial third nerve, which innervates the slow flexor
muscle (SFM). The third nerve appears as a pale, whitish thread, located just posterior to the
ganglion. It travels laterally out to the fibers of the SFM. The SFM is located ventrally in each
abdominal segment and functions to hold the tail in a semi-curled position. The SFM is unique
because it is innervated by several (up to six) neurons, while typical vertebrate muscle cells
receive input from only one motor neuron. We distinguish these neurons through extracellular
recording of the third nerve. Five of these axons use the excitatory neurotransmitter, glutamate,
while one axon uses the inhibitory neurotransmitter, GABA. This again distinguishes the SFM
from typical vertebrate muscle cells, which only use excitatory transmission.
We surgically exposed the third nerve and SFM and applied a suction electrode to the
nerve for extracellular recording. We then inserted a microelectrode to record the muscle cells
intracellularly. Thus, we could correlate activity in motor neurons with responses in the muscle
cell.
The crayfish neuromuscular junction provides a tool to study synaptic transmission. Our
goal was to examine: 1) the properties of spontaneous activity in the third nerve, 2) evoked postsynaptic potentials (PSPs) in the SFM, 3) the response of the SFM to high frequency stimuli,
giving evidence for two possible neuroplasticity phenomena: postsynaptic facilitation and posttetanic potentiation.

Methods:
We followed the methods outlined in the Neurobiology 301, Winter 2014 course manual,
pages 159-176. Instead of crushing the third nerve to stop spontaneous activity, we applied a
hyperpolarizing current so as to not disturb our recordings.
Results:
An eight-second sample of extracellular recording from the third nerve is shown in
Figure 1a. There are three different size action potentials from the recording. These correspond to
the three different sized axons that innervate the third nerve. One fires at an extremely high rate
(40 Hz), another fires less frequently (20-25 Hz), and the last axon fires quite infrequently (4-6
Hz). These three axons are distinguished using the spike discriminator function shown in Figure
1b, and their corresponding rates of firing are quantified in Figure 1c. Notice as the axon size
increases, the rate of firing decreases.
We then use a microelectrode to record intracellularly from the SFM. Figure 2 shows a
brief sample of spontaneous activity from the third nerve and the corresponding activity in the
SFM. There are clearly two different sized amplitude action potentials from the third nerve (7
mV and 10 mV, respectively), but it is difficult to distinguish the size of the EPSPs. There are
two different size EPSPs. However, they vary by less than 0.05 mV. It appears as though the
smaller EPSP does correlate to the smaller amplitude action potential, while the larger EPSP
correlates to the larger amplitude action potential. However, this is not conclusive because the
rapid rate of spontaneous activity tended to obscure any possible correlations. Furthermore, some
action potentials did not cause any EPSPs. In Figure 2, there are thirteen different action

potentials, but only 4 resulting EPSPs. Figure 2b shows a possible correlation between an action
potential and its corresponding EPSP. There was a 9 ms delay between the action potential and
the EPSP.
During sensory stimulation, we observed a slight increase in third nerve firing rates, as
shown in Figure 3. The frequency increased by about 5 Hz, but we did not see any new axons
recruited. Thus, pre-existing axons increased their firing frequency. The corresponding trace
from the SFM appeared to slightly increase in frequency, but it is difficult to quantify because of
the already high rate of EPSP firing. Furthermore, the EPSP trace fluctuated after we tickled the
tail. This is likely not due to increased sensory stimulation, but because the microelectrode
shifted position. The SFM trace shows two 40 mV amplitude blips in the membrane potential,
which is definitely not from an EPSP.
After we silenced the third nerve using hyperpolarizing current, we used the pulse
simulator to deliver brief depolarizing currents into the third nerve. These were delivered at
frequencies of 1 Hz, 10 Hz, 20 Hz, and 50 Hz as seen in Figures 4-7. The stimulus frequencies of
up to 10 Hz 20 Hz mimic the natural frequencies of spontaneous activity in the nerve.
However, frequencies above 20 Hz are higher than anything observed in spontaneous activity of
the nerve. Thus, EPSP amplitudes from isolated stimuli (lower frequency) would best predict
EPSP amplitudes during spontaneous activity. There is no evidence of facilitation at lower
frequencies, which mimics the nature of spontaneous activity.
At around frequencies of 10 Hz and beyond, we began to see facilitation. Facilitation is
an increase of the amplitude of the EPSP at higher frequencies of firing. Note, however, that we
did not see summation until stimulating the nerve at 50 Hz. Summation is the increase in

baseline voltage of each EPSP compared to the previous EPSP. In Figure 8, we plotted the
facilitation indices as a function of time for each stimulus frequency. We repeated each stimulus
frequency four times and averaged the amplitude of each EPSP after the initial EPSP for all
trials. We divide these values by the first (control) EPSP after the beginning of stimulation to
calculate a facilitation index. There was no facilitation at 1 Hz. At 10 Hz, the facilitation index
increased linearly over time. Interestingly, the 20 Hz facilitation index increased slower than the
50 Hz facilitation index but reached an overall slightly greater final value. This may have just
been due to discrepancies in EPSP measurement, since different sized EPSPs fired during the
stimulation. The final one or two EPSPs that I measured for the 20 Hz may have just been
abnormally higher than expected, simply due to random variation. An average of all the EPSP
amplitudes would give a more accurate facilitation index value.
Finally, we examined the time course of stimulation by using a function generator to fire
a steady 2 Hz pulse to the third nerve. Thus, even after the initial 5 Hz, 10 Hz, 20 Hz, and 40 Hz
stimulation ceases, the 2 Hz test pulse remains. We examined how long it takes for the SFM
EPSPs to return to its control (before stimulation) EPSP amplitude after the final stimulus occurs.
This corresponds with a return of facilitation index to a value of 1. The results are plotted in
Figure 9. The facilitation indices are calculated by measuring EPSP amplitude from the test pulse
at specific time intervals after the final stimulus was injected. The figure shows that facilitation
persists for longer times at higher stimulus frequencies. In other words, it takes longer for the
EPSP amplitude to return to its control amplitude at higher frequencies.
Discussion:

This lab allowed us to observe the spontaneous firing activity of the third nerve, the
effects of stimulating the third nerve at various frequencies, the response of the SFM, and the
properties of facilitation. After we entered the SFM, we found that the amplitude of spontaneous
activity from the third nerve correlated somewhat with the amplitude of EPSPs from the SFM.
Overall, however, a large nerve spike does not necessarily guarantee a large EPSP. There are two
reasons that explain this. First, the microelectrode could simply be located far away from the
synapse at which the axon synapses with the SFM. Voltage will decay as it travels this distance,
leading to an unreliable EPSP waveform that might be smaller than expected. Second, the SFM
we record from may be synapsed by the one axon that uses GABA as a neurotransmitter. This
would contribute inhibitory rather than excitatory signals, depressing the size of the overall
EPSP.
However, not all spikes correlated to an EPSP, as shown in Figure 2. This is because each
SFM cell is innervated by a different number of axons. Although we distinguished three different
axons in the third nerve, we only saw one or two different EPSP waveforms in the SFM. Thus,
even though the third nerve may generate an action potential, the particular axon that fires may
not connect directly to the SFM cell we record from. As a result, the EPSP does not show up on
the intracellular recording trace. Likewise, if we move to another muscle cell, the EPSP
distribution would likely change because different axons innervate the new muscle cell.
Furthermore, a large amplitude spike does not necessarily have to correlate with a large
amplitude EPSP. This is because the axon synapsing on to the particular muscle cell we were
recording from could have been located far away from the microelectrode. In this case, the
voltage may decay by the time it reaches the microelectrode, resulting in an abnormally small
EPSP.

There is a delay between the third nerve spike and the resulting EPSP because it takes
time for the current to travel down the axons innervating the SFM. This time course is dictated
by the properties of the third nerve axons, including the length constant (lambda). The longer
than expected delay (9 ms, as opposed to the stated 5 ms in the lab manual) shown in Figure 2b
can be explained by the relationship between the membrane and internal resistance of the axons
in question. The conduction velocity is smaller than a typical axon, perhaps caused by a smaller
membrane resistance or larger internal resistance. Both these factors decrease the length
constant, thus decreasing the conduction velocity. More obviously, the length of the axon itself
could have been longer, increasing the amount of time it takes to conduct the voltage. Finally, the
placement of the microelectrode could have influenced the time delay. The voltage has to travel a
distance once synapsing at the SFM; the farther away the microelectrode is, the longer the delay.
During high-frequency stimulation of the third nerve as shown in Figures 4-7, we begin
to observe facilitation in the SFM. That is, the amplitude of the EPSP increases in size in
response to a single spike from the third nerve. This should be distinguished from summation,
which is an increase in the baseline voltage at which each EPSP begins compared to the previous
EPSP. This should also be distinguished from post-tetanic potentiation, which would result in a
consistent increase in EPSP amplitude over minutes or hours due to enhanced neurotransmitter
release. We did not see this phenomenon; our EPSPs returned to their baseline within seconds.
Furthermore, we did not observe summation until applying 50 Hz frequency, which is unusual.
In contrast, we began to see facilitation at the 10 Hz pulse frequency. The lack of summation
may be due to an abnormally small muscle cell, or a muscle cell with unusual membrane
properties. An abnormally small cell or a cell with unusual membrane properties can decrease the
capacitance of the lipid bilayer, thus decreasing the time constant, tau. If the time constant

decreases, then much higher frequencies are required to cause summation. This is because the
voltage caused by one EPSP will decay more quickly; therefore, the cell requires much higher
frequencies to summate, or increase the baseline of the membrane potential over time.
Facilitation is a type of short-term plasticity that increases synaptic strength between the
third nerve and SFM. There are two main mechanisms that explain this phenomenon, both of
which are pre-synaptic. The first is the residual calcium hypothesis. Calcium enters the
presynaptic terminal after stimulating the nerve. At higher frequency stimulation, there is not
enough time for calcium to exit the presynaptic terminal before the next stimulus arrives, so we
get summation of calcium concentration. Therefore, more neurotransmitter (in this case,
glutamate) is released because of increased probability of vesicle exocytosis. This leads to an
increase in EPSP amplitude. The second hypothesis is based on spike broadening. This involves
the voltage-gated potassium channels, which have a certain inactivation period. At high
frequencies, this inactivation accumulates, so we get less outward V-K current to repolarize the
cell at higher frequencies. Thus, the action potential broadens at higher frequencies. This means
there is more time for calcium to enter the cell through V-Ca channels. Increased calcium that
enters per spike will then cause more neurotransmitter release. The conclusion is the same from
both mechanisms the amount of neurotransmitter released per spike at higher frequencies is
higher than the baseline neurotransmitter released at low frequency action potentials, translating
to greater amplitude EPSP.
These conclusions explain why higher frequency stimulation led to greater facilitation
indices. At the highest frequency (Figure 7), there is the shortest amount of time for calcium to
leave the cell, leading to the greatest calcium summation. In contrast, there may be the most
spike broadening, leading to the greatest amount of calcium entry. There is the most residual

calcium leftover in the presynaptic terminal, which takes the longest time to remove. This leads
to an increase in the time course of facilitation, as depicted in Figure 9.
Overall, we successfully observed spontaneous activity in the third nerve and
corresponding EPSPs in the SFM. By artificially stimulating the third nerve, we saw evidence of
facilitation and summation, but not potentiation.
Facilitation is simply one way a synapse can increase in strength. The facilitation process
increases the amount of neurotransmitter released between cells, increasing the postsynaptic cell
response. Facilitation is merely a short-term example of plasticity. On the long term scale,
plasticity has profound implications in areas of memory and learning. Simultaneous firing of
neurons when we learn new skills leads to increased synaptic strength. Amazingly, this occurs in
much the same manner as in crayfish neuromuscular junction. This increased synaptic strength
allows us to store information in an extremely complex manner. The third nerve and SFM is one
of the most basic models that we can investigate, and it already shows a significant capacity to
learn through facilitation. There are millions of other neurons in our bodies that have this
potential to increase firing efficiency through use and experience. Thus, every time we learn a
new concept in school or learn a new sport, synaptic connections between neurons radically
change, forever reshaping the way our brain functions.

Figure 1: Third Nerve Spontaneous Activity. We used a suction electrode to do extracellular

recordings from the third nerve. The above figure shows spontaneous activity generated by three
different sized axons in the third nerve. These axons are distinguished by their action potential
waveform and amplitude. From this figure, it is evident that there are at least two different axons
firing at high frequencies, one approximately reaching a maximum voltage of 8 mV and the other
staying at fairly low voltages, around 2-3 mV.

Figure 1b: Spike Histogram of Spontaneous Third Nerve. Using the spike discriminator function
on LabChart revealed three spontaneously firing axons, as opposed to two, as I initially thought. The
three distinct clusters of spike amplitude and waveform correlate to three distinct axons because
these characteristics are unique and remain constant for each axon. There were three distinct clusters
of spike amplitudes, but there was also quite a few outliers scattered in between. This may just be due
to random noise addition and subtraction from the overall spike amplitude.

Figure 1c: Rate Meter of Spontaneous Firing Third Nerve. The rate meter function in LabChart
quantifies the spike histogram from figure 1b. Note that the bin size is set to one second, so the y-axis
is essentially frequency (Hz). The leftmost plot shows the lowest frequency firing rate (approximately
4-6 counts/bin), but correlates to the axon with the highest amplitude action potential. There was also
the most variation in the firing pattern of this axon. The middle plot shows another axon that fired at a
higher frequency (~ 20-25 counts/bin). This axon showed more consistency over time in firing pattern.
Finally, the rightmost graph shows an axon firing at extremely high frequencies (40-45 counts/bin).
The firing pattern of this axon was the most steady.

Figure 2: Third Nerve and SFM Traces. The top trace shows a voltage trace from extracellular
recording of the third nerve. The bottom trace shows a voltage trace from intracellular recording of the
SFM. Note the two distinct waveform amplitudes in this section of recording. The EPSPs from the SFM
were approximately .7 mV in amplitude, as we expected. The amplitude of the spontaneous action

potential was approximately 10 mV, smaller than a typical action potential waveform as expected
(extracellular recording).

Figure 2b: Superposed Third Nerve and SFM Trace. A zoom-in of one spontaneous action
potential and its corresponding EPSP. Note that the y-axis does not have a consistent voltage for the
third nerve and muscle; the relative amplitudes are not to scale. The action potential amplitude was
approximately 6 mV and the EPSP amplitude was approximately .5 mV. This graph primarily shows the
latency of onset of EPSP the delay was approximately 9 ms. This delay varied as we examined each
action potential and its corresponding EPSP.

Figure 3: Response of Third Nerve to Tail Tickling, Ipsilateral. The marking in the figure above
shows the time at which we began stimulating the crayfish tail with the wooden end of a Q-tip. All the
spontaneous activity amplitude remained around 0.02 0.04 mV. However, there was an increase in
the firing rate of the axons. By using the rate meter, we found that the rate of firing increased from
approximately 8-10 Hz to 13-16 Hz within three seconds after stimulation. Note that the EPSP trace
showed large blips, most likely because the microelectrode moved. There do not appear to be any
significant changes in the EPSP trace compared to baseline rates of firing.

Figure 4: 1 Hz Stimulus Application to Third Nerve. We used the pulse simulator to deliver a 1
Hz pulse to the third nerve, shown in the top trace. The bottom trace shows the response of the SFM.
We stopped spontaneous activity from the third nerve using a hyperpolarizing current. The SFM shows
relatively consistent amplitude of EPSPs, all at approximately 0.5 mV. There is no change in overall
membrane potential.

Figure 5: 10 Hz Stimulus Application to Third Nerve. A 10 Hz pulse delivered to the third nerve
(top trace) results in the response shown in the bottom trace. The EPSPs increase from approximately .
32 mV to approximately 2.38 mV. The membrane potential does not increase throughout the
stimulation.

Figure 6: 20 Hz Stimulus Application to Third Nerve. A 20 Hz pulse delivered to the third nerve
(top trace) results in the response shown in the bottom trace. The EPSPs increase from approximately .
73 mV to approximately 3.56 mV. The membrane potential grew slightly more positive (0.32 mV)
throughout the stimulation.

Figure 7: 50 Hz Stimulus Application to Third Nerve. A 50 Hz pulse delivered to the third nerve
(top trace) results in the response shown in the bottom trace. The EPSPs increase from approximately
1.29 mV to approximately 5.91 mV. The membrane potential grew much more positive than the
previous runs (~ 4 mV).

Figure 8: Facilitation at Crayfish Neuromuscular Junction. Facilitation indices were averaged

over four separate runs at each frequency. This was done by finding the amplitude of the EPSP from
the SFM after stimulation began and dividing that number by the amplitude of the control (first) EPSP
observed. The facilitation index increased as the applied frequency increased.

Figure 9: Time Course of Persistence of Facilitation. An underlying function generator applied a

repetitive pulse that allowed us to observe the persistence of facilitation after the initial burst of
stimulation (5 Hz, 10 Hz, 20 Hz, or 40 Hz) was applied. Unfortunately, we did not leave the recording
device active for a long time after the end of stimulus for the 5 Hz and 10 Hz stimuli. As a result, we
only received 2-3 seconds worth of data. However, we made sure that the EPSPs did return to baseline
before stopping the recording. As frequency of applied stimulus increases, there is a longer
persistence of facilitation. It takes longer for the EPSPs to return to its original (pre-stimulus) value at

higher frequencies. Note that the facilitation index reaches a maximum for the 10 Hz, 20 Hz, and 40
Hz pulses quickly after stimulus is released, then slowly decay over several seconds.