Lab 5: The crayfish slow flexor muscle and the

third nerve
Author: Mackenzie Andrews

INTRODUCTION
The neuromuscular junction is the site where a motor neuron innervates a muscle cell.

Under normal conditions, an action potential in the motor neuron will release neurotransmitter

into the neuromuscular junction. This release of neurotransmitter will result in a post synaptic

potential in the muscle cell. With enough excitatory input, the muscle cell will contract.

In slow muscles, the cells do not generate action potentials. Instead, the contraction of the

muscle is graded with the amount of depolarization caused by the synaptic inputs from the motor

neuron. This depolarization is dependant on the size of the EPSP and frequency at which inputs

are received. The size of an EPSP is dependant on many factors such as number of vesicles

released by presynaptic cell, resting membrane resistance, receptor properties, voltage gated

channel properties, among others.

The frequency at which inputs are received can also affect the amount of depolarization

of the cell. At high frequency stimulus, two phenomenon occur to increase the magnitude of the

postsynaptic cell: summation and facilitation. Summation occurs due to the RC properties of the

postsynaptic membrane. The capacitance of the membrane causes the response to outlast the

stimulus, therefore, if another stimulus comes before the cell has discharged back to rest, the two

responses will summate and cause a larger depolarization. Facilitation is a phenomenon that

occurs when high frequency inputs cause each successive input to generate a larger EPSP.

Facilitation is believed to be one of the major the basis for learning.

 The crayfish is a model organism for studying synaptic transmission due to their large

size, accessibility, and simple muscle cell organization. The superficial flexor muscles (SFMs)

are slow muscle cells on the ventral abdominal segments innervated by the third nerve. The third

nerve contains six axons, five of which are excitatory, glutamatergic neurons, one of which is an

inhibitory GABAergic neuron.

In this lab, we used the crayfish to examine synaptic transmission by taking extracellular

recordings from the third nerve while simultaneously recording intercellularly from an SFM cell.

We recorded the response of the third nerve to a touch stimulus, observed postsynaptic potentials

in the muscle cell as a result of spontaneous activity in the third nerve and touch stimulus, and

examined the effect of stimulus frequency on synaptic facilitation.

METHODS
All methods used were as given in the protocol in the ​Neurobiology 301 Course Manual

(Winter Quarter, 2017) ​with the exception that we did not have to silence the third nerve do to

the fact that our EPSPs were already small enough to observe facilitation.

RESULTS
Extracellular Recordings of the Third Nerve

A crayfish was dissected to expose a section of SFM and a third nerve. A micropipet was

used to suck up the third nerve and take extracellular recordings of the nerve activity. Figure 1A

shows the spontaneous activity of the third nerve.

 During spontaneous activity, four separate axons could be distinguished. The spike

discriminator function in LabChart was used to differentiate between the four populations of

spikes, then the rate meter function was used to find the average frequency of firing for each

distinguishable axon (Figure 1B). The largest spikes, of amplitude around 7 mV, had an average

firing frequency was 3.4 Hz (first panel of figure 1B). The second largest spikes, of amplitude

around 2.5 mV, had an average firing frequency of 2.5 Hz (first panel of figure 1B). The second

to smallest spikes had an amplitude of 2.2 mV and a firing frequency of 4.8 Hz (last panel of

Figure 1B). Finally, the smallest spikes had an average firing frequency of 8.65 Hz and

amplitude of 1.7 mV.

Third Nerve Response to Tickle Stimulation

 We then stimulated the tail and carapace of the crayfish using the wooden end of a q-tip.

We began by stimulating the ipsilateral side of the tail, then the contralateral side of the tail, and

finally the side of the carapace directly adjacent to the nerve we were recording from (Figure 2A,

B, and C, respectively).

We then used the spike discriminator function to distinguish between the firing patterns

of the different axons being recorded from. The rate meter function was then used to quantify the

response of each axon to the different stimuli. Figure 2D shows the response of four

distinguishable axons to the ipsilateral stimulation. The first panel shows the response of the

largest axon which was not active prior to or following the stimulus (Axon 1). During the

stimulation, this axon reached a firing frequency of 6 Hz. The second panel shows the response

of an axon that Increased in firing frequency during the stimulation (Axon 2). This axon
increased from a frequency of 10 Hz to 52 Hz over the course of the stimulation. The firing

frequency then returned back to baseline 3 seconds after the end of the stimulation. The third

panel shows the response of an axon that immediately decreased in firing frequency during the

stimulation from a frequency of 20 Hz to a frequency of 5 Hz then increased in firing frequency

after the end of the stimulus (Axon 3). The final panel shows the response of an axon that

gradually decreased in firing frequency over the course of the stimulation from a frequency of 10

Hz to a frequency of 1.8 Hz (Axon 4). The axon quickly resumed its baseline firing rate after the

end of the stimulus.

Figure 2E shows the rate meter representation of the firing pattern response to the

contralateral tickle. All of the axons exhibited a similar response as they did to the ipsilateral

tickle. Axon 1 began fired at a rate of 10 Hz during this stimulus. Axon 2 increased in firing to a

frequency to 67 Hz then returned to baseline levels 2 seconds after the stimulus. Axon 3

immediately dropped from a frequency of 60 Hz to a frequency of 2 Hz during the stimulus then

returned to its baseline frequency 2 seconds post stimulus. Finally, Axon 4 initially declined in

firing rate but then began to restore firing rate to 40% of baseline by the end of the stimulation.

The frequency response of the axons to the carapace tickle is shown in figure 2F. Axon 1

did not respond to the carapace tickle so there was no rate meter representation for this axon.

Axon 2 responded similarly with a ramped up frequency over the course of the stimulation

reaching a maximum firing frequency of 18 Hz. Axon 3 decreased in firing frequency, similar to

the tail tickles, however the decrease was more gradual and only slowed to a frequency of 6 Hz.

Finally, Axon 4 initially had an increase in firing frequency (from 3 to 6 Hz) then returned to

baseline for the rest of the stimulus.

Intracellular Recordings from the Superficial Flexor Muscle

After getting extracellular recordings of the third nerve action potentials, a glass

microelectrode filled with KCl solution was inserted into one of the SFM cells to get intracellular

recordings of the post synaptic potentials in the muscle cell. Figure 3 shows an example of the
response of the muscle cell to the spontaneous activity of the third nerve (this recording was

taken during the second week of lab but is not the same animal used for the rest of the report).

The muscle cell had two distinct responses resulting from the activity of two of the axons in the

third nerve. One of the responses was an EPSP of magnitude 0.6 mV which resulted from the

firing of one of the excitatory axons which had a recorded extracellular action potential

amplitude of 7 mV. The second response was an IPSP of magnitude -1.4 mV which resulted

from the single inhibitory axon in the third nerve. This axon had a recorded firing amplitude of

7.5 mV. Both postsynaptic responses resulted approximately 3 ms after the action potential.

Third Nerve and SFM Response to Tickle Stimulation

After getting a stable intracellular recording, the carapace of the crayfish was

gently tickled to observe the response of the third nerve and muscle cell (Figure 4,

the data for this figure was gathered by Nick Tolley). During rest, one group of

action potentials had an average recorded amplitude of 4 mV which resulted in an

average EPSP amplitude of 1.3 mV. Another group of action potentials had an

average amplitude of 6.8 mV resulting in an EPSP amplitude of 0.78 mV. During

stimulation, no additional axons began firing, but the frequency of firing increased,
causing a summation of the postsynaptic responses. This summation resulted in an

increase in membrane potential and what appears to be a partial muscle

contraction.

Postsynaptic Response to Varying Frequency Stimulus

After observing the response of the muscle cell to action potentials generated by the third

nerve, we stopped recording from the nerve and switched to stimulating. Figure 5 shows the

intracellular recordings of the muscle cell response to four different frequencies of 1.5 mV

stimulation. Figure 5A shows the response of the muscle cell to a 2 Hz signal. The size of the

EPSPs generated increased from a baseline of 0.51 mV to a maximum of 0.74 mV over the

course of the 3.5 second stimulus. We next stimulated with a 10 Hz signal (Figure 5B). At 10 Hz,

the muscle cell EPSPs increased in amplitude from 0.45 mV to 1.1 mV over the course of the

signal. At 20 Hz, the EPSPs increased in amplitude from 0.5 mV to 1.8 mV (Figure 5C). Finally,

at a stimulus frequency of 50 Hz, the EPSPs increased in amplitude from 0.52 mV to 2.3 mV

(Figure 5D). Baseline membrane potential also increased from -71.5 mV to -70.1 mV.
Facilitation Index of Varying Frequency Stimulus

The increase in EPSP amplitude observed in the previous signals is a result of short term

facilitation. The amount of facilitation observed can be quantified by dividing a given EPSP by

the baseline EPSP amplitude (the size of the first EPSP recorded during the stimulus), yielding

the facilitation index. Figure 6 shows the facilitation index of each successive EPSP during the 2,

10, and 20 Hz stimuli (the 50 Hz stimuli facilitation indexes were not calculated due to the fact

that summation also occurred which would add an additional variable to the facilitation

mechanism).

During the 2 Hz stimulus, the facilitation index reached a maximum value of 1.76 over

the course of 3 seconds (Figure 6A). During the 10 Hz stimulus, the facilitation index reached a
mean maximum value of 2.25 over the course of 1.5 seconds (Figure 6B). After 1.5 seconds, the

facilitation index remained relatively constant, without further increase. The 20 Hz stimulus

showed similar behavior as the 10 Hz stimulus, reaching a mean maximum facilitation index of

3 after 0.6 seconds, then leveling off (Figure 6C).

Facilitation Decay Post High Frequency Stimulus

After a high frequency stimulus, the short term facilitation observed will decay back to a

baseline level. To observe facilitation decay, a 1 Hz square wave was delivered to the muscle cell

via a function generator to generate a 1 msec stimulus of amplitude 1.5 mV. A burst of a 20 Hz

stimulus was then delivered to the cell to cause facilitation. At the end of the burst, the cell was

allowed to relax back to its baseline EPSP level (Figure 7A).

 The facilitation index was calculated for the EPSPs both during a post stimulus (Figure

7B). Over the course of the 1.5 second burst, the facilitation index reached a maximum of 2.3.

Following the burst, the facilitation index decayed back to 1 over the course of 5.5 seconds.

DISCUSSION
In order to sense environmental information and respond appropriately, organisms must

be able to encode a wide range of stimuli. In this lab, we observed multiple ways in which the

crayfish neuromuscular junction is able to vary its information response to varying stimuli.

The fact that the third nerve carries 6 axons allows for each axon to respond differently to

a given stimulus. As observed in Figure 2, each axon being recorded from responded differently

to a given stimulus. Some of the axons increased in their firing rate, while others decreased in

their firing rate. The axons that decrease in firing frequency could be fast adapting axons.

Adaptation allows for an organism to encode change in stimulus as opposed to the absolute

magnitude of stimulation. This type of frequency response is crucial to be able to differentiate

between benign versus potentially threatening environmental cues.

 The third nerve also contains two distinct types of neurons, 5 excitatory and 1 inhibitory.

Figure 3 shows the effect of an inhibitory neuron on the muscle cell. The firing of the inhibitory

neuron causes a hyperpolarizing IPSP in the postsynaptic muscle cell. The hyperpolarizing signal

causes the muscle cell to be less prone to contraction. Inhibitory signals are crucial for bimodal

movement. In the crayfish, the SFM holds the tail in a curled position, however, in order for the

animal to swim quickly, the SFM must relax to allow the deep phasic flexors and extensors to

rapidly move the tail. In the human, all skeletal muscle comes in pairs where one of the muscles

must relax for the ther to contract. For example, in order for the bicep to contract, inhibitory

signals but be sent to the tricep to keep it relaxed during the bicep contraction.

Figure 3 also shows the correlation (or lack thereof) between neuronal activity and

postsynaptic response. Four separate axons can be observed in the extracellular trace from the

third nerve, however only 2 distinct postsynaptic response are observed. In addition, the size of

the action potential does not determine the size of the EPSP. The fact that not every axon

generates a postsynaptic response suggests that not every axon synapses on the particular muscle

cell being recorded from. Therefor, axons that generate a response in the muscle cell observed

may not generate a response in surrounding muscle cells.

The lack of proportionality of action potential amplitude and postsynaptic response

magnitude can be attributed to the fact we were recording extracellularly. While recording

extracellularly, the increased proximity and size of the axon will generate a larger signal. In

addition, the actual strength of the synaptic connection may be greater meaning that a smaller

action potential could still yield a large EPSP.

The muscle cells themselves are also capable of differential response to varying stimulus

from the neurons. Through facilitation, muscle cells are able to respond in proportionally greater

magnitudes to high intensity stimulus than to low intensity stimulus. As observed in Figure 5,

higher frequency stimulation results in increasingly greater EPSPs. Facilitation also includes a

temporal component, so longer lasting high frequency burst will result in progressively larger
postsynaptic responses. The frequencies observed in the facilitation experiments were generally

larger than firing frequencies that occur during spontaneous activity. During spontaneous

activity, the firing frequencies of axons were on the order of 2 to 9 Hz (Figure 1). Therefore, the

high amount of facilitation observed during the 10, 20, and 50 Hz stimuli is not indicative of

EPSP amplitudes during spontaneous activity.

During this lab, we observed the activity of a single nerve containing 6 axons synapsed to

a single muscle cell. Even in this relatively simple example of synaptic transmission, a large

amount of variability in information encoding and response could be observed. On the scale of

an entire organism, this variability allows for the complexity in perception and behavior that is

observed on the macroscopic scale.