Lab 5 Report

7/21/2019 Lab 5 Report

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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 (S!". The third nerve appears as a pale, whitish thread, located just posterior to the

ganglion. It travels laterally out to the fibers of the S!. The S! is located ventrally in each

abdominal segment and functions to hold the tail in a semi#curled position. The S! is uni$ue

because it is innervated by several (up to six" neurons, while typical vertebrate muscle cells

receive input from only one motor neuron. %e distinguish these neurons through extracellular

recording of the third nerve. ive of these axons use the excitatory neurotransmitter, glutamate,

while one axon uses the inhibitory neurotransmitter, &''. This again distinguishes the S!

from typical vertebrate muscle cells, which only use excitatory transmission.

%e surgically exposed the third nerve and S! and applied a suction electrode to the

nerve for extracellular recording. %e 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. )ur

goal was to examine* +" the properties of spontaneous activity in the third nerve, " evo-ed post#

synaptic potentials (Ss" in the S!, /" the response of the S! to high fre$uency stimuli,

giving evidence for two possible neuroplasticity phenomena* postsynaptic facilitation and post#

tetanic potentiation.



Methods:

%e followed the methods outlined in the 0eurobiology /1+, %inter 1+2 course manual,

pages +34#+56. Instead of crushing the third nerve to stop spontaneous activity, we applied a

hyperpolari7ing current so as to not disturb our recordings.

Results:

'n eight#second sample of extracellular recording from the third nerve is shown in

igure +a. There are three different si7e action potentials from the recording. These correspond to

the three different si7ed axons that innervate the third nerve. )ne fires at an extremely high rate

(21 H7", another fires less fre$uently (1#3 H7", and the last axon fires $uite infre$uently (2#6

H7". These three axons are distinguished using the spi-e discriminator function shown in igure

+b, and their corresponding rates of firing are $uantified in igure +c. 0otice as the axon si7e

increases, the rate of firing decreases.

%e then use a microelectrode to record intracellularly from the S!. igure shows a

brief sample of spontaneous activity from the third nerve and the corresponding activity in the

S!. There are clearly two different si7ed amplitude action potentials from the third nerve (5

m8 and +1 m8, respectively", but it is difficult to distinguish the si7e of the 9Ss. There are

two different si7e 9Ss. However, they vary by less than 1.13 m8. It appears as though the

smaller 9S does correlate to the smaller amplitude action potential, while the larger 9S

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. urthermore, some

action potentials did not cause any 9Ss. In igure , there are thirteen different action



potentials, but only 2 resulting 9Ss. igure b shows a possible correlation between an action

potential and its corresponding 9S. There was a 4 ms delay between the action potential and

the 9S.

:uring sensory stimulation, we observed a slight increase in third nerve firing rates, as

shown in igure /. The fre$uency increased by about 3 H7, but we did not see any new axons

recruited. Thus, pre#existing axons increased their firing fre$uency. The corresponding trace

from the S! appeared to slightly increase in fre$uency, but it is difficult to $uantify because of

the already high rate of 9S firing. urthermore, the 9S trace fluctuated after we tic-led the

tail. This is li-ely not due to increased sensory stimulation, but because the microelectrode

shifted position. The S! trace shows two 21 m8 amplitude blips in the membrane potential,

which is definitely not from an 9S.

'fter we silenced the third nerve using hyperpolari7ing current, we used the pulse

simulator to deliver brief depolari7ing currents into the third nerve. These were delivered at

fre$uencies of + H7, +1 H7, 1 H7, and 31 H7 as seen in igures 2#5. The stimulus fre$uencies of

up to +1 H7 ; 1 H7 mimic the natural fre$uencies of spontaneous activity in the nerve.

However, fre$uencies above 1 H7 are higher than anything observed in spontaneous activity of

the nerve. Thus, 9S amplitudes from isolated stimuli (lower fre$uency" would best predict

9S amplitudes during spontaneous activity. There is no evidence of facilitation at lower

fre$uencies, which mimics the nature of spontaneous activity.

't around fre$uencies of +1 H7 and beyond, we began to see facilitation. acilitation is

an increase of the amplitude of the 9S at higher fre$uencies of firing. 0ote, however, that we

did not see summation until stimulating the nerve at 31 H7. Summation is the increase in



baseline voltage of each 9S compared to the previous 9S. In igure <, we plotted the

facilitation indices as a function of time for each stimulus fre$uency. %e repeated each stimulus

fre$uency four times and averaged the amplitude of each 9S after the initial 9S for all

trials. %e divide these values by the first (control" 9S after the beginning of stimulation to

calculate a facilitation index. There was no facilitation at + H7. 't +1 H7, the facilitation index

increased linearly over time. Interestingly, the 1 H7 facilitation index increased slower than the

31 H7 facilitation index but reached an overall slightly greater final value. This may have just

been due to discrepancies in 9S measurement, since different si7ed 9Ss fired during the

stimulation. The final one or two 9Ss that I measured for the 1 H7 may have just been

abnormally higher than expected, simply due to random variation. 'n average of all the 9S

amplitudes would give a more accurate facilitation index value.

inally, we examined the time course of stimulation by using a function generator to fire

a steady H7 pulse to the third nerve. Thus, even after the initial 3 H7, +1 H7, 1 H7, and 21 H7

stimulation ceases, the H7 test pulse remains. %e examined how long it ta-es for the S!

9Ss to return to its control (before stimulation" 9S amplitude after the final stimulus occurs.

This corresponds with a return of facilitation index to a value of +. The results are plotted in

igure 4. The facilitation indices are calculated by measuring 9S 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 fre$uencies. In other words, it ta-es longer for the

9S amplitude to return to its control amplitude at higher fre$uencies.

Discussion:



This lab allowed us to observe the spontaneous firing activity of the third nerve, the

effects of stimulating the third nerve at various fre$uencies, the response of the S!, and the

properties of facilitation. 'fter we entered the S!, we found that the amplitude of spontaneous

activity from the third nerve correlated somewhat with the amplitude of 9Ss from the S!.

)verall, however, a large nerve spi-e does not necessarily guarantee a large 9S. There are two

reasons that explain this. irst, the microelectrode could simply be located far away from the

synapse at which the axon synapses with the S!. 8oltage will decay as it travels this distance,

leading to an unreliable 9S waveform that might be smaller than expected. Second, the S!

we record from may be synapsed by the one axon that uses &'' as a neurotransmitter. This

would contribute inhibitory rather than excitatory signals, depressing the si7e of the overall

9S.

However, not all spi-es correlated to an 9S, as shown in igure . This is because each

S! cell is innervated by a different number of axons. 'lthough we distinguished three different

axons in the third nerve, we only saw one or two different 9S waveforms in the S!. Thus,

even though the third nerve may generate an action potential, the particular axon that fires may

not connect directly to the S! cell we record from. 's a result, the 9S does not show up on

the intracellular recording trace. =i-ewise, if we move to another muscle cell, the 9S

distribution would li-ely change because different axons innervate the new muscle cell.

urthermore, a large amplitude spi-e does not necessarily have to correlate with a large

amplitude 9S. 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

9S.



There is a delay between the third nerve spi-e and the resulting 9S because it ta-es

time for the current to travel down the axons innervating the S!. This time course is dictated

by the properties of the third nerve axons, including the length constant (lambda". The longer

than expected delay (4 ms, as opposed to the stated 3 ms in the lab manual" shown in igure b

can be explained by the relationship between the membrane and internal resistance of the axons

in $uestion. The conduction velocity is smaller than a >typical? axon, perhaps caused by a smaller

membrane resistance or larger internal resistance. oth these factors decrease the length

constant, thus decreasing the conduction velocity. !ore obviously, the length of the axon itself

could have been longer, increasing the amount of time it ta-es to conduct the voltage. inally, the

placement of the microelectrode could have influenced the time delay. The voltage has to travel a

distance once synapsing at the S!@ the farther away the microelectrode is, the longer the delay.

:uring high#fre$uency stimulation of the third nerve as shown in igures 2#5, we begin

to observe facilitation in the S!. That is, the amplitude of the 9S increases in si7e in

response to a single spi-e from the third nerve. This should be distinguished from summation,

which is an increase in the baseline voltage at which each 9S begins compared to the previous

9S. This should also be distinguished from post#tetanic potentiation, which would result in a

consistent increase in 9S amplitude over minutes or hours due to enhanced neurotransmitter

release. %e did not see this phenomenon@ our 9Ss returned to their baseline within seconds.

urthermore, we did not observe summation until applying 31 H7 fre$uency, which is unusual.

In contrast, we began to see facilitation at the +1 H7 pulse fre$uency. The lac- of summation

may be due to an abnormally small muscle cell, or a muscle cell with unusual membrane

properties. 'n 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 fre$uencies are re$uired to cause summation. This is because the

voltage caused by one 9S will decay more $uic-ly@ therefore, the cell re$uires much higher

fre$uencies to summate, or increase the baseline of the membrane potential over time.

acilitation is a type of short#term plasticity that increases synaptic strength between the

third nerve and S!. There are two main mechanisms that explain this phenomenon, both of

which are pre#synaptic. The first is the residual calcium hypothesis. Aalcium enters the

presynaptic terminal after stimulating the nerve. 't higher fre$uency 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 9S amplitude. The second hypothesis is based on spi-e broadening. This involves

the voltage#gated potassium channels, which have a certain inactivation period. 't high

fre$uencies, this inactivation accumulates, so we get less outward 8#B current to repolari7e the

cell at higher fre$uencies. Thus, the action potential broadens at higher fre$uencies. This means

there is more time for calcium to enter the cell through 8#Aa channels. Increased calcium that

enters per spi-e will then cause more neurotransmitter release. The conclusion is the same from

both mechanisms ; the amount of neurotransmitter released per spi-e at higher fre$uencies is

higher than the baseline neurotransmitter released at low fre$uency action potentials, translating

to greater amplitude 9S.

These conclusions explain why higher fre$uency stimulation led to greater facilitation

indices. 't the highest fre$uency (igure 5", 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

spi-e broadening, leading to the greatest amount of calcium entry. There is the most residual



calcium leftover in the presynaptic terminal, which ta-es the longest time to remove. This leads

to an increase in the time course of facilitation, as depicted in igure 4.

)verall, we successfully observed spontaneous activity in the third nerve and

corresponding 9Ss in the S!. y artificially stimulating the third nerve, we saw evidence of

facilitation and summation, but not potentiation.

acilitation 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. acilitation is merely a short#term example of plasticity. )n the long term scale,

plasticity has profound implications in areas of memory and learning. Simultaneous firing of

neurons when we learn new s-ills leads to increased synaptic strength. 'ma7ingly, 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 S! 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 gure shows spontaneous activity generated by three

dierent sized axons in the third nerve. These axons are distinguished by their action potentialwaveform and amplitude. From this gure, it is evident that there are at least two dierent axonsring at high freuencies, one approximately reaching a maximum voltage of ! m" and the other

staying at fairly low voltages, around #$% m".



Figure 1b: Spike Histogram of Spontaneous Third Nerve. &sing the spi'e discriminator functionon (ab)hart revealed three spontaneously ring axons, as opposed to two, as * initially thought. The

three distinct clusters of spi'e amplitude and waveform correlate to three distinct axons becausethese characteristics are uniue and remain constant for each axon. There were three distinct clustersof spi'e amplitudes, but there was also uite a few outliers scattered in between. This may +ust be due

to random noise addition and subtraction from the overall spi'e amplitude.



Figure 1c: Rate eter of Spontaneous Firing Third Nerve. The rate meter function in (ab)hart uanties the spi'e histogram from gure b. -ote that the bin size is set to one second, so the y$axisis essentially freuency /z0. The leftmost plot shows the lowest freuency ring rate approximately 1$2 counts3bin0, but correlates to the axon with the highest amplitude action potential. There was alsothe most variation in the ring pattern of this axon. The middle plot shows another axon that red at ahigher freuency 4 #5$#6 counts3bin0. This axon showed more consistency over time in ring pattern.

Finally, the rightmost graph shows an axon ring at extremely high freuencies 15$16 counts3bin0.The ring pattern of this axon was the most steady.

Figure ! : Third Nerve and SF 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 the7F8. -ote the two distinct waveform amplitudes in this section of recording. The 9:7:s from the 7F8were approximately .; m" in amplitude, as we expected. The amplitude of the spontaneous action



potential was approximately 5 m", smaller than a typical action potential waveform as expectedextracellular recording0.

Figure !b: Superposed Third Nerve and SF Trace. < zoom$in of one spontaneous action

potential and its corresponding 9:7:. -ote 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 2 m" and the 9:7: amplitude was approximately .6 m". This graph primarily shows the

latency of onset of 9:7: > the delay was approximately ? ms. This delay varied as we examined each

action potential and its corresponding 9:7:.



Figure ": Response of Third Nerve to Tai# Tick#ing$ %psi#atera#. The mar'ing in the gure above

shows the time at which we began stimulating the craysh tail with the wooden end of a @$tip. <ll the

spontaneous activity amplitude remained around 5.5# > 5.51 m". /owever, there was an increase in

the ring rate of the axons. Ay using the rate meter, we found that the rate of ring increased from

approximately !$5 /z to %$2 /z within three seconds after stimulation. -ote that the 9:7: trace

showed large blips, most li'ely because the microelectrode moved. There do not appear to be any

signicant changes in the 9:7: trace compared to baseline rates of ring.



Figure &: 1 H' Stimu#us App#ication to Third Nerve. We used the pulse simulator to deliver a

/z pulse to the third nerve, shown in the top trace. The bottom trace shows the response of the 7F8.

We stopped spontaneous activity from the third nerve using a hyperpolarizing current. The 7F8 shows

relatively consistent amplitude of 9:7:s, all at approximately 5.6 m". There is no change in overall

membrane potential.

Figure (: 1) H' Stimu#us App#ication to Third Nerve. < 5 /z pulse delivered to the third nerve

top trace0 results in the response shown in the bottom trace. The 9:7:s increase from approximately .

%# m" to approximately #.%! m". The membrane potential does not increase throughout thestimulation.



Figure *: !) H' Stimu#us App#ication to Third Nerve. < #5 /z pulse delivered to the third nerve

top trace0 results in the response shown in the bottom trace. The 9:7:s increase from approximately .

;% m" to approximately %.62 m". The membrane potential grew slightly more positive 5.%# m"0

throughout the stimulation.

Figure +: () H' Stimu#us App#ication to Third Nerve. < 65 /z pulse delivered to the third nervetop trace0 results in the response shown in the bottom trace. The 9:7:s increase from approximately

.#? m" to approximately 6.? m". The membrane potential grew much more positive than the previous runs 4 1 m"0.



Figure ,: Faci#itation at -raysh Neuromuscu#ar /unction. Facilitation indices were averagedover four separate runs at each freuency. This was done by nding the amplitude of the 9:7: from

the 7F8 after stimulation began and dividing that number by the amplitude of the control rst0 9:7:observed. The facilitation index increased as the applied freuency increased.

Figure 0: Time -ourse of ersistence of Faci#itation. <n underlying function generator applied arepetitive pulse that allowed us to observe the persistence of facilitation after the initial burst of

stimulation 6 /z, 5 /z, #5 /z, or 15 /z0 was applied. &nfortunately, we did not leave the recordingdevice active for a long time after the end of stimulus for the 6 /z and 5 /z stimuli. <s a result, we

only received #$% seconds worth of data. /owever, we made sure that the 9:7:s did return to baselinebefore stopping the recording. <s freuency of applied stimulus increases, there is a longer

persistence of facilitation. *t ta'es longer for the 9:7:s to return to its original pre$stimulus0 value at



higher freuencies. -ote that the facilitation index reaches a maximum for the 5 /z, #5 /z, and 15/z pulses uic'ly after stimulus is released, then slowly decay over several seconds.

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Lab 5 Report