Farhang 1

Parsa Farhang
NBIO 301: AC
Michael Kennedy
March 17th, 2019

Lab 5: Crayfish Slow Flexor Muscle and the Third Nerve

Introduction:

When people think of the nervous system, it is usually centered around the neurons and

synapses of the brain. This thought neglects the importance of neurons in the peripheral

nervous system, particularly those found at the neuromuscular junction (NMJ). Thus, it is of

paramount importance to not only gain an understanding of the relationship between muscles

and the neurons they’re synapsed with, but also the hands on skills/tools necessary to obtain

electrophysiological data in this important domain of neuroscience. In order to achieve both,

one must employ the services of a model organism.

A model organism commonly used to study the NMJ is the crayfish (Procambarus

clarkii). The crayfish is an aquatic crustacean found worldwide. Its body is covered by a hard

exoskeleton and divided into two parts: the cephalothorax and the abdomen. The latter

portion, the abdomen, is comprised of six distinct segments that help make up the tail of the

animal (Powell). Deep within the ventral parts of the abdomen are the superficial flexor

muscles (SFM), which are comprised of easily visible cells (great for making intracellular

recordings). This class of muscle is innervated by the crayfish’s third nerve and differs from

vertebrate skeletal muscles in several notable ways. Firstly, the muscle cells do not generate

action potentials (APs), instead displaying a graded contraction relative to the amount of inputs

from the third nerve. Additionally, the SFMs, unlike many vertebrate muscles, are innervated

at multiple sites by several motor neurons (6 total axons). Lastly, five of the neurons involved
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in SFM innervation are excitatory while one, which uses GABA, is inhibitory. Thus, given the

aforementioned reasons, the NMJ of SFM synapsed with the third nerve in crayfish is an ideal

target for the acquisition of general knowledge about the NMJ as well as the general skills

necessary for working with the NMJ in future laboratory settings.

The goal for this lab was to, therefore, dissect a crayfish so that we could make

simultaneous extracellular recordings from the third nerve and intracellular recordings from the

SFM muscles it innervates. We then hoped to be able to use this setup in order record

spontaneous post synaptic potentials and then causally link them to the specific axons in the

third nerve. Additionally, we wanted to, again, measure post synaptic potentials, and maybe

even facilitation, except this time via the simulation of the third nerve.

Ultimately, the data acquired from this lab would help us become more knowledgeable

about some of the basic properties of the NMJ, as well as become comfortable/skilled enough

to conduct simultaneous extracellular recordings of nerves and intracellular recordings from

the living muscle cells they are synapsed with for our future research related endeavors.

Methods:

We followed the procedure outlined in the Neuroscience 301 Course Manual. Data for

the section “Time course of persistence of facilitation” was obtained from Hope Willis and

Makaeel Karim.

Results:

Section 1: Extracellular Recordings From the 3rd Nerve

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Once a functional third nerve (Nerve 1) was inserted into the suction electrode, we

immediately noticed spontaneous activity. There was some noise we could not suppress (1-2

mV in amplitude) but it did not inhibit us from observing the spontaneous firing of a couple

axons. One axon with an amplitude of about 6 mV, was continuously present in our

spontaneous recordings, and was firing at a frequency of ~ 4.3 Hz (see Figure 1). One larger

axon fired only once (amplitude of ~ 33 mV) soon after we got the third nerve into the suction

electrode but then ceased to fire again (we knew it was a large axon because the amplitude of

its spontaneous firing was large, therefore implying a large influx of ions that propagated

through the extracellular fluid. That is why we can distinguish axons by their AP amplitudes in

extracellular recordings).

After, observing spontaneous activity, we then decided to stimulate tail hairs, via a Q-

tip, on the ipsilateral and contralateral sides of our recording and observe the resulting changes

in activity (note that the nerve fell out of the electrode so we had to suck it back in. Once it was

back in I noticed another axon firing spontaneous action potentials of roughly 10 mV in

amplitude at 4 Hz in frequency). We first stimulated from the ipsilateral side and noticed the

firing of two distinct axons, one with an amplitude around 10 mV (axon 1.1)( the spontaneous

axon) and one with an amplitude around 37 mV (axon 2.1) (see Figures 2a and 2b). Both axons

had a phasic component to their response. Shortly after stimulation, the larger axon (axon 2.1)

began firing from a frequency of 0 Hz to a frequency of 7 Hz, yet, after 1 second, ceased firing

and did not begin firing again even when the stimuli ceased. The smaller axon (axon 1.1), on the

other hand, increased in frequency from ~4 Hz pre stimuli to ~50 Hz post stimuli and then

slowly declined back to the tonic baseline characterized by the 4 Hz seen during spontaneous
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activity. Additionally, once the stimuli ended, unlike axon 2.1, there was yet another, albeit

brief, phasic response of APs firing at ~50Hz for axon 1.1. Next we decided to repeat the same

experiment except stimulate the contralateral hairs (again the nerve fell out of the electrode

and when we sucked it back in there was a new spontaneous axon firing at an amplitude of ~30

mV and a frequency of ~3 Hz). When stimulating the contralateral side three distinct axons can

be noticed, the spontaneous axon (axon 1.2), another axon firing APs with an amplitude of ~ 51

mV (axon 2.2) and another axon firing APs with an amplitude of ~ 25 mV (axon 3.2)(see Figure

3a and 3b). All display phasic responses. Axon 1.2 fires at a frequency of ~55-65 Hz

immediately following stimulation but then declines in frequency rapidly after 1.5 seconds

(then fires in a tonic manner). Axons 2.2 and 3.2, also displayed phasic characteristics. Upon

stimulation they went from a frequency of 0 Hz to firing at a frequency of 7 Hz. After 1.5

seconds both rapidly decline in frequency and cease. Other than the tonic nature of axon 1.2

another key difference between the axons is that axon 1.2 and 3.2 briefly fires in a phasic

manner again following the end of stimulation whereas axon 2.2 does not. Thus stimulation of

both the ipsilateral and contralateral sides cause the phasic increased frequency of

spontaneous axons upon stimulation commencement and termination (fires in a tonic manner

before, after, and between those two points). Additionally, new axons, not present during

spontaneous activity, fire in a phasic manner upon stimulation, do not display any tonic

properties and, depending on the axon, will show phasic properties upon termination of

stimulus (larger axons appear less likely to do this).

Section 2: Simultaneous Extracellular Recording From Third Nerve and Intracellular Recording

From SFM Cell

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We then wanted to study post synaptic potentials in SFMs generated by the stimulation

of the third nerve (Nerve 2) with the hopes of garnering a greater understanding of synaptic

transmission at the NMJ. Again, as a reminder, the SFM muscles are different from vertebrate

skeletal muscles in the sense that they do not fire APs, instead an increase in post synaptic

potentials gradually increases the force of contraction of that muscle. Anyways, we got the

third nerve into the suction electrode and soon after impaled an SFM muscle cell (Cell 1). We

knew the former occurred due to spontaneous APs (~ 8 - 10mV in amplitude) firing at a

frequency of ~21 Hz, and that the latter occurred because of the rapid drop of membrane

potential from -14 mV to ~-65 mV, which is the resting potential of an SFM cell (see Figure 4).

There was still spontaneous activity observed extracellularly from the third nerve (similar to

Section 1) but now we could simultaneously compare the spikes in the nerve to EPSPs (~4 mV in

amplitude) in the SFM it was synapsed with. I isolated one of these spike/EPSP pairings in

Figure 5 (my LabChart software would unfortunately not allow me to combine the channel

recordings). In this specific instance there is a ~.01 sec delay between the spike in the third

nerve and the EPSP peak in the SMF cell, which is likely due to the time it takes for the

transmitter to be released from the nerve, then travel through the synaptic cleft to bind to the

transmitter receptors and then finally trigger a response. The RC properties of a cell also likely

play a role, as the membrane capacitance creates a charging curve that therefore creates a

delay between the beginning of a stimuli and the EPSP peak. We then impaled another muscle

cell (Cell 2) synapsed with that same third nerve and noticed corresponding EPSPs to those

same spontaneous spikes of 8-10 mV in amplitude (see Figure 6). Given the particular nerve
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spikes of this third nerve (Nerve 2) generates EPSPs of similar waveform in both cells, it leads

me to believe that a given nerve spike will generate a very similar EPSP in different SFM cells.

Lastly, we were told during lab to not conduct the sensory stimulation part of the

simultaneous recording part of the lab given that our crayfish tail was contracting and would

potentially repeatedly knock out our intracellular electrode. I imagine that with Q-tip

stimulation we would see additional axons , as well as spontaneous ones, fire at a higher

frequency. Thus I believe that we would see a diversity of EPSPs (larger EPSPs corresponding to

larger axons – due to an increase in transmitter release from larger axons) at a higher frequency

and therefore see summation.

Section 3: Hardware Stimulation of the Third Nerve and the Corresponding Intracellular

Recording from an SFM Cell

Next we decided to use the hardware stimulator, attached to the suction electrode, to

manually stimulate a given third nerve (Nerve 3) and measure the corresponding response in

the SFM cell. To do this we set up the stimulator to trigger third nerve action potentials of ~4

mV at varying frequencies of 5 Hz, 10 Hz, 20 Hz and 50 Hz (The lab manual stated we use 2 Hz

instead of 5 Hz, but Professor Moody had us use 5 Hz when aiding us in the data collection

process). We then measured the response in the SFM cell (Cell 3) and displayed the data in

figures 7(a)-10 (a). As the frequency increased, we also noticed the increase in amplitude for

subsequent action potentials in a given trial. We therefore plotted graphs of the facilitation

indices for each frequency trial (see figures 7(b) – 10(b)). This is a plot of each EPSP amplitude

divided by the first EPSP of a trial relative to time. We saw that the facilitation index for EPSPs

increased with time within a given trial and also increased with a given frequency, meaning the
 Farhang 7

each subsequent EPSP amplitude tended to be larger than its predecessor. For lower

frequencies (5 Hz), for example, the facilitation index reached a maximum value of ~2.5

whereas that of higher frequencies (50 Hz) reached nearly 8. Additionally, some more data

that supports this observation is the fact that the average EPSP amplitude was 3.043 mV for 5

Hz, 4.12 mV for 10Hz, 5.233 mV for 20 Hz and 5.808 mV for 50 Hz. These observations are

likely due to facilitation and I will elaborate in the Discussion section of the lab.

Section 4: Time Course of Persistence of Facilitation:

This last exercise of the lab I was not able to obtain data in the allotted lab section so we

therefore got data from Hope Willis and Mekaeel Karim’s group (we did not receive their raw

LabChart data, just data point in an Excel spreadsheet, therefore we could not create raw data

figures). They, while maintaining the simultaneous intracellular/extracellular recording

apparatus, applied a constant test stimuli of 2 Hz (at whatever voltage they determined triggers

EPSPs consistently) via the hardware stimulator. They then interrupted this with high

frequency bursts of 5 Hz, 10 Hz, 20 Hz, and 40 Hz, and then measured the resulting EPSPs

during and after the burst. Then using the data, facilitation index plots were created, and show

that larger frequency bursts garnered larger max facilitation indices than their smaller

frequency counter parts (1.8 for 5 Hz vs. 3.6 for 40 Hz) (see Figures 11-14). Additionally, larger

frequency bursts (40 Hz) caused the SFM to take longer to return to the baseline EPSP

amplitude when compared to the 5 Hz bursts (over 30 seconds vs 10 seconds). The

mechanisms underlying these observations will be touched on in the Discussion section.

Discussion:
 Farhang 8

Through the experimentation of this lab I gained a lot more skills in regards to making

intracellular/extracellular recordings and now even have experience doing both simultaneously.

Additionally, this lab gave a much greater understanding of basic neuroscience principles

especially in relation to synaptic transmission at the neuromuscular junction.

First, in Section 1, we solidified our understanding of extracellular recordings which we

first conducted in the Cockroach Lab. We did this by, again, obtaining spontaneous

extracellular recordings of a nerve followed by extracellular recordings in the presence of

sensory stimulation (see figures 2a – 3b). Once again, we saw different axons firing in differing

phasic/tonic manners (for example, in each of our sensory stimulation trials the spontaneous

axon would fire in both phasic and tonic manners whereas larger axons, stimulated only by

sensory stimulation, only fired in a phasic manner).

Later, in Sections 2, we impaled an SFM cell with an intracellular electrode and soon

after began recording simultaneous muscle responses to third nerve stimulation via the

hardware stimulator. We did notice an association between distinct nerve spikes and particular

EPSP waveforms during simultaneous recording of spontaneous activity of the third nerve

synapsed with an SFM. Larger nerve spikes in the third nerve corresponded to larger EPSPs in

the SFM cell. This is likely due to a larger depolarization of the third nerve which lead to a

greater influx of calcium and therefore a greater release of transmitter. This larger transmitter

release would likely result in a larger inward current into the SFM cell and therefore a larger

EPSP. Additionally, we had to impale a couple of cells during this simultaneous recording

portion of the lab and therefore saw the resulting EPSPs in the SFM resulting from the same

spontaneous activity of a third nerve. As stated in the result section, we did see very similar
 Farhang 9

EPSPs (roughly the same amplitude) generated in different SFM cells resulting from the same

spontaneous nerve spikes in the third nerve.

Using the same simultaneous recording apparatus, in Section 3 we stimulated the third

nerve with a hardware stimulator and observed the resulting EPSPs in the SFM cell we were

recording from. Our stimulus frequencies ranged from 5 – 50 Hz whereas the spontaneous

frequency was ~21 Hz. Furthermore, those EPSPs resulting in isolation (essentially the first

EPSP of a given trial) were not as good at predicting the EPSP amplitude of spontaneous activity

when compared to those resulting from higher frequency bursts, given that the frequency of

spontaneous action potentials was in the range of some of the higher burst frequencies. This

notion is supported by the fact that EPSPs resulting from high frequency bursts had an

amplitude of 4 – 7 mV (similar to the ~6-10mV amplitude of EPSPS from spontaneous activity)

whereas isolated EPSPs had an amplitude of ~1.7 mV. This is likely due to the high frequency

EPSPs displaying facilitation through the residual calcium hypothesis (no spike broadening

because we controlled the width of the action potentials generated in the pre synaptic third

nerve via the hardware stimulator).

Lastly, in Section 4, we observed the time course of facilitation in a crayfish third nerve

(using the same mechanical setup as Section 3). As seen in the facilitation plots of figures 11-

14, it takes time for the effects of facilitation to leave after a high frequency burst. This, as

mentioned earlier, is likely due to the residual calcium hypothesis which essentially states that

subsequent action potentials in a high frequency burst increase in amplitude due to residual

calcium in the presynaptic terminal (the calcium pump cannot pump out the previous action

potentials calcium fast enough). This means that the next action potential will have more
 Farhang 10

calcium, trigger more vesicular release, and therefore a larger post synaptic response. In line

with this theory, the effects of facilitation lasted longer during higher frequency bursts, again,

likely due to the residual calcium theory (less time to pump out presynaptic calcium from the

last action potential during a high frequency burst, therefore leaving more calcium in the

terminal for the next action potential to summate on). Thus, it appears that the recovery time

of EPSP amplitude does in fact depend on the burst frequency.

All in all, this was a perfect lab to end the quarter on as it helped build/solidify lecture

knowledge and laboratory techniques that we have been exposed to over the past 10 weeks.

Bibliography:

1. Powell, Kevin. “Crayfish Dissection.” Biology Junction,

https://www.biologyjunction.com/crayfish_dissection.htm. Accessed March 4th 2019.

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Figures:

Spontaneous Activity

Figure 1. Spontaneous Activity. Extracellular recordings from a crayfish third nerve (Nerve 1) via
a suction electrode. The y axis is measuring membrane potential in mV and the x axis is
measuring time in seconds. The arrow marks the point in which the third nerve was successfully
placed into the suction electrode. Notice the continuous presence of spontaneous action
potentials of ~5 mV firing at ~4.3 Hz.
 Farhang 12

Ipsilateral Stimulation of Crayfish Tail

Figure 2a. Ipsilateral Stimulation of Crayfish Tail. Extracellular recordings from a crayfish third
nerve (Nerve 1), via a suction electrode, during contralateral stimulation of its tail through
touch. The y axis is measuring membrane potential in mV and the x axis is measuring time in
seconds. Two axons fired during stimulation: the spontaneous axon with an amplitude of ~10
mV (axon 1.1), and another axon firing APs with an amplitude of ~ 37 mV (axon 2.1). The left
most arrow indicates start of stimulation and the right most arrow indicates the end of
stimulation. Notice the differing phasic vs. tonic properties of the axons.
 Farhang 13

Distinguishing Axons During Ipsilateral Stimulation of Crayfish Tail

Figure 2b. Distinguishing Axons During Ipsilateral Stimulation of Crayfish Tail. This is a
histogram representing action potential data in Figure 2a. The x-axis represents the amplitude
of a given action potential and the y-axis represents the number of times this action potential
was observed during the first two seconds following stimulation. Notice the thwo distinct
groups of action potentials corresponding to axon 1.1 and 2.1 in Figure 2a
 Farhang 14

Contralateral Stimulation of Crayfish Tail

Figure 3a. Contralateral Stimulation of Crayfish Tail. Extracellular recordings from a crayfish
third nerve (Nerve 1), via a suction electrode, during contralateral stimulation of its tail through
touch. The y axis is measuring membrane potential in mV and the x axis is measuring time in
seconds. Three axons fired during stimulation: the spontaneous axon with an amplitude of ~30
mV (axon 1.2), another axon firing APs with an amplitude of ~ 47 mV (axon 2.2) and another
firing APs with an amplitude of ~25 mV (axon 3.2). The left most arrow indicates start of
stimulation and the right most arrow indicates the end of stimulation. Notice the differing
phasic vs. tonic properties of the axons.
 Farhang 15

Distinguishing Axons During Contralateral Stimulation of Crayfish Tail

Figure 3b. Distinguishing Axons During Contralateral Stimulation of Crayfish Tail. This is a
histogram representing action potential data in Figure 3a. The x-axis represents the amplitude
of a given action potential and the y-axis represents the number of times this action potential
was observed during the first two seconds following stimulation. Notice the three distinct
groups of action potentials corresponding to axon 1.2, 2.2, and 3.2 in Figure 3a
 Farhang 16

Simulataneous Recordings of Third Nerve and SFM Cell

Figure 4. Simultaneous Recordings of Third Nerve and SFM Cell. This is a simultaneous
extracellular recording of a crayfish third nerve (Nerve 2), via a suction electrode, and an
intracellular recording of the SFM muscle cell it is synapsed with (Cell 1). The top graph
measures the extracellular voltage of the third nerve in mV on the y-axis. The bottom graph
measures intracellular voltage of the SFM cell on the y-axis. Both graphs share a common x-
axis which measures time in seconds. The arrow represents when we got our intracellular
electrode into the SFM cell. Notice the little EPSPs generated, once the electrode is fully in the
cell.
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EPSP in SFM Cell

Figure 5. EPSP in SFM Cell. This data was obtained during a simultaneous extracellular
recording of a crayfish third nerve (Nerve 2), via a suction electrode, and an intracellular
recording of the SFM muscle cell it is synapsed with (Cell 1). The graph represents an EPSP of ~4
mV in amplitude in the SFM cell of a crayfish. Y-axis measure voltage in mV and the x-axis
measures time. The arrow indicates the point in which in action potential of ~10 mV in the third
nerve was fired as determined by our simultaneous extracellular recordings (I could not combine
the data from the channels due to my lack of access to the appropriate software). Notice the
time delay between the action potential firing and the EPSP reaching its peak voltage.
 Farhang 18

Simulataneous Recordings of Third Nerve and SFM Cell

Figure 6. Simultaneous Recordings of Third Nerve and SFM Cell. This is a simultaneous
extracellular recording of a crayfish third nerve (Nerve 2), via a suction electrode, and an
intracellular recording of the SFM muscle cell it is synapsed with (Cell 2). The top graph
measures the extracellular voltage of the spontaneous activity of the third nerve in mV on the y-
axis. The bottom graph measures intracellular voltage of the SFM cell on the y-axis. Both
graphs share a common x-axis which measures time in seconds. Notice the little EPSPs
generated as well as the spontaneous action potentials of ~10mV from the third nerve.
 Farhang 19

Muscle Response to Nerve Stimulation: 5 Hz

Figure 7 a. Muscle Response to Nerve Stimulation: 5 Hz. This is a simultaneous extracellular

recording of a crayfish third nerve (Nerve 3), via a suction electrode, and an intracellular
recording of the SFM muscle cell it is synapsed with (Cell 3). The third nerve is being stimulated
by a hardware stimulator at 5 Hz. The top graph measures the amplitude of pulses generated
from the hardware stimulator in mV on the y-axis. The bottom graph measures intracellular
voltage of the SFM cell on the y-axis. Both graphs share a common x-axis which measures time
in seconds. Notice the arrows marking the commencement of stimulation.
 Farhang 20

Muscle Response to Nerve Stimulation: 5 Hz

Facilitation Plot
3

2.5

1.5

0.5

0
0 0.5 1 1.5 2 2.5

Figure 7 b. Muscle Response to Nerve Stimulation: 5 Hz Facilitation Plot. This is a plot of the
facilitation indices of all of the EPSPs in Figure 7 a (divide each EPSP amplitude by the amplitude
of the first EPSP in the trial). The amplitude of the first EPSP was ~ 1.71 mV. The y-axis is
measuring the facilitation indices and the x-axis is time relative to the first EPSP of that trial.
Each dot represents an individual EPSP.
 Farhang 21

Muscle Response to Nerve Stimulation: 10 Hz

Figure 8 a. Muscle Response to Nerve Stimulation: 10 Hz . This is a simultaneous extracellular

recording of a crayfish third nerve (Nerve 3), via a suction electrode, and an intracellular
recording of the SFM muscle cell it is synapsed with (Cell 3). The third nerve is being stimulated
by a hardware stimulator at 10 Hz. The top graph measures the amplitude of pulses generated
from the hardware stimulator in mV on the y-axis. The bottom graph measures intracellular
voltage of the SFM cell on the y-axis. Both graphs share a common x-axis which measures time
in seconds. Notice the arrows marking the commencement of stimulation as well as the trend in
increasing amplitude for subsequent EPSPs as the trial progresses.
 Farhang 22

Muscle Response to Nerve Stimulation: 10 Hz Facilitation Plot

7

0
0 0.5 1 1.5 2 2.5 3 3.5

Figure 8 b. Muscle Response to Nerve Stimulation: 10 Hz Facilitation Plot. This is a plot of the
facilitation indices of all of the EPSPs in Figure 8 a (divide each EPSP amplitude by the amplitude
of the first EPSP in the trial). The amplitude of the first EPSP was ~ 1.29 mV. The y-axis is
measuring the facilitation indices and the x-axis is time relative to the first EPSP of that trial.
Each dot represents an individual EPSP.
 Farhang 23

Muscle Response to Nerve Stimulation: 20 Hz

Figure 9 a. Muscle Response to Nerve Stimulation: 20 Hz . This is a simultaneous extracellular

recording of a crayfish third nerve (Nerve 3), via a suction electrode, and an intracellular
recording of the SFM muscle cell it is synapsed with (Cell 3). The third nerve is being stimulated
by a hardware stimulator at 20 Hz. The top graph measures the amplitude of pulses generated
from the hardware stimulator in mV on the y-axis. The bottom graph measures intracellular
voltage of the SFM cell on the y-axis. Both graphs share a common x-axis which measures time
in seconds. Notice the arrows marking the commencement of stimulation as well as the trend in
increasing amplitude for subsequent EPSPs as the trial progresses.

Muscle Response to Nerve Stimulation: 20 Hz

Facilitation Plot
7
6
5
4
3
2
1
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

Figure 9 b. Muscle Response to Nerve Stimulation: 20 Hz Facilitation Plot. This is a plot of the
facilitation indices of all of the EPSPs in Figure 9 a (divide each EPSP amplitude by the amplitude
of the first EPSP in the trial). The amplitude of the first EPSP was ~ 1.79 mV. The y-axis is
measuring the facilitation indices and the x-axis is time relative to the first EPSP of that trial.
Each dot represents an individual EPSP.
 Farhang 24

Muscle Response to Nerve Stimulation: 50 Hz

Figure 10 a. Muscle Response to Nerve Stimulation: 50 Hz . This is a simultaneous extracellular

recording of a crayfish third nerve (Nerve 3), via a suction electrode, and an intracellular
recording of the SFM muscle cell it is synapsed with (Cell 3). The third nerve is being stimulated
by a hardware stimulator at 50 Hz. The top graph measures the amplitude of pulses generated
from the hardware stimulator in mV on the y-axis. The bottom graph measures intracellular
voltage of the SFM cell on the y-axis. Both graphs share a common x-axis which measures time
in seconds. Notice the arrows marking the commencement of stimulation as well as the trend in
increasing amplitude for subsequent EPSPs as the trial progresses.
 Farhang 25

Muscle Response to Nerve Stimulation: 50 Hz

Facilitation Plot
8
7
6
5
4
3
2
1
0
0 0.2 0.4 0.6 0.8 1 1.2

Figure 10 b. Muscle Response to Nerve Stimulation: 50 Hz Facilitation Plot. This is a plot of the
facilitation indices of all of the EPSPs in Figure 10 a (divide each EPSP amplitude by the
amplitude of the first EPSP in the trial). The amplitude of the first EPSP was ~ 1.72 mV. The y-
axis is measuring the facilitation indices and the x-axis is time relative to the first EPSP of that
trial. Each dot represents an individual EPSP.

Facilitation Time Course: 5 Hz

2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0 2 4 6 8 10 12

Figure 11. Facilitation Time Course: 5 Hz. This is a plot of the facilitation indices of EPSPs in data
obtained by Hope Willis and Mekaeel Karim for the “Time course of persistence of facilitation”
portion of the lab. The amplitude of the first EPSP was ~2.17 mV. The y axis measures the
facilitation indices and the x axis is time relative to the first EPSP of that trial. Each dot
represents and individual EPSP. The arrow indicates the termination of bursting behavior
(subsequent EPSPs are from a 2 Hz baseline stimulation).
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Facilitation Time Course: 10 Hz

2.5

1.5

0.5

0
0 2 4 6 8 10 12
Figure 12. Facilitation Time Course: 10 Hz. This is a plot of the facilitation indices of EPSPs in
data obtained by Hope Willis and Mekaeel Karim for the “Time course of persistence of
facilitation” portion of the lab. The amplitude of the first EPSP was ~2.16 mV. The y axis
measures the facilitation indices and the x axis is time relative to the first EPSP of that trial.
Each dot represents and individual EPSP. The arrow indicates the termination of bursting
behavior (subsequent EPSPs are from a 2 Hz baseline stimulation).

Facilitation Time Course: 20 Hz

3

2.5

1.5

0.5

0
0 5 10 15 20 25

Figure 13. Facilitation Time Course: 20 Hz. This is a plot of the facilitation indices of EPSPs in
data obtained by Hope Willis and Mekaeel Karim for the “Time course of persistence of
facilitation” portion of the lab. The amplitude of the first EPSP was ~2.385 mV. The y axis
measures the facilitation indices and the x axis is time relative to the first EPSP of that trial.
Each dot represents and individual EPSP. The arrow indicates the termination of bursting
behavior (subsequent EPSPs are from a 2 Hz baseline stimulation).
 Farhang 27

Facilitation Time Course: 40 Hz

4
3.5
3
2.5
2
1.5
1
0.5
0
0 5 10 15 20 25 30 35

Figure 14. Facilitation Time Course: 40 Hz. This is a plot of the facilitation indices of EPSPs in
data obtained by Hope Willis and Mekaeel Karim for the “Time course of persistence of
facilitation” portion of the lab. The amplitude of the first EPSP was ~1.99 mV. The y axis
measures the facilitation indices and the x axis is time relative to the first EPSP of that trial.
Each dot represents and individual EPSP. The arrow indicates the termination of bursting
behavior (subsequent EPSPs are from a 2 Hz baseline stimulation).