Effects of stimulation and neuromuscular drugs on vertebrate and invertebrate glycolytic muscles

Abstract

Muscle is necessary for locomotion in both vertebrate and invertebrate species. Variations in

how muscles of each of these groups function are beneficial for the specific animal. This study

was composed of three individual tests and sought to determine how vertebrate and invertebrate

muscles respond to three groups of variables and how their responses compare across muscle

types (vertebrate and invertebrate). Experiments consisted of measuring muscle contraction force

at increasing stimulation voltage, increasing stimulation frequency and stimulating muscles after

they have been rinsed in neuromuscular chemicals. Results did not support what is known about

vertebrate and invertebrate muscles which may be attributable to errors in the experimental

procedure, or because isolated muscles were not tested fast enough.

Introduction

Skeletal muscle is an important tissue type for both vertebrate and invertebrate animals

because it allows for locomotion and other movements. Muscle fibers create movement when the

individual muscle fibers, or sarcomeres, shorten. Sarcomeres are composed of a thick filament

made from myosin and a thin filament made from actin. The head of the myosin will bind to the

actin and pull the actin towards it via a power stroke resulting in the shortening of the sarcomere,

which translates into a contraction of the muscle.

The signal to contract is sent to the muscle by a combination of electrical and chemical

messengers. The nervous system sends an electrical signal to the neuromuscular junction, which

then uses acetylcholine as a chemical messenger. This message is received, and causes a second

electrical signal to be sent to the muscle fibers in order for them to contract. After the muscle

contracts, there is a refractory period in which the muscle is still above its resting voltage

potential.

Unlike action potentials, if a muscle receives a signal to contract before it has finished the

first contraction, it will still respond to the signal by contracting before it reaches its resting

voltage potential. This is called summation because the overlapping signals affect the muscle in

an additive fashion. Tetanus is an extreme form of summation in which the signals to the muscle

are so close together that the muscle remains at a high voltage, continuously contracting state in

which it cannot physically contract any faster. If signals to the muscle remain once the muscle

has reached tetanus, the muscle will eventually fatigue meaning the force of the contraction will

begin to decrease.

Vertebrate muscles differ from invertebrate muscles in ways that affect how each muscle

type will respond to chemical and electrical signals (Moyer and Schulte 2008). Motor neurons of

vertebrate muscles utilize an excitatory system in which acetylcholine is the only

neurotransmitter released at the neuromuscular junction. Thus, there is no inhibitory control over

muscle contraction, only excitation. Invertebrate muscles, however, are controlled by both

excitation and inhibition chemicals (Araque et al.1994). Acetylcholine acts in invertebrate

muscles to exciting the muscle cells, and serotonin acts by inhibiting muscle contraction.

Additionally, because vertebrate muscles contain motor neutrons that innervate many nerve

fibers, they have the ability to recruit muscle fibers when the muscle stimulation is stronger

(Beltman et al. 2004). Invertebrate muscles are not able to recruit muscle fibers through this

process (Katz 1949). A stronger stimulation may still be able to cause a stronger contraction in

invertebrate muscles, though, because a stronger signal can propagate further into a muscle

tissue, thus causing more muscle fibers to participate in the contraction.

This study sought to determine how vertebrate and invertebrate muscles differ in their

contraction force when stimulated at increasing voltages, how fast they can contract and for how

long until they fatigue, and how contraction strength is affected by neuromuscular chemicals.

The muscles were tested by varying the strength of an electrical stimulus, the time interval

between two stimuli, and the chemicals present. Based on the similarities and differences in

vertebrate and invertebrate muscles, I hypothesized that both invertebrate and vertebrate muscles

would increase contraction force in response to increases in voltage; however there would be

more of an effect for invertebrate. Additionally, I predicted that the vertebrate muscle would

respond to increases in stimulation frequency by going through the following phases:

summation, tetanty and fatigue. The invertebrate muscle should not go through these phases and

should produce contractions that increase in force as frequency increases until the muscle is

fatigued. Lastly, the vertebrate muscle would respond to having potassium solution applied to the

sciatic nerve but no change in contraction strength was expected from solutions being applied

directly to the muscle. The invertebrate muscle would increase contraction force in response to

phenoxybenzamine and decrease contraction force in response to serotonin relative to the

control.

The invertebrate muscles tested were a glycolytic crab claw muscle and a glycolytic shrimp

tail muscle. The vertebrate muscle tested was a glycoltic frog gastrocnemius. It is important to

note that all three of these muscles are the same type of muscle; however they do vary in size

which can affect how the muscle responds to stimulation.

Methods

Invertebrate set up

Stimulator electrodes were inserted into an isolated crab claw and connected to PowerLab

in order to electrically stimulate the crab muscle. One end of a string was tied to the superior side

of the crab claw and the other was tied to a force transducer which was also connected to

PowerLab. The force transducer was used to detect and record the claw’s contraction force.

The shrimp tail muscle was used in the same set up as the crab claw. The most proximal

portion of the tail was pinned to the dissection platform and the string was tied to a hook, which

was placed in the most distal end of the tail.

Vertebrate Set up

A string was tied around the distal tendon (plantar tendon) of the gastrocnemius of an

isolated, skinned frog leg. The tendon was then severed below the string and the muscle was

strung perpendicular to the leg from the same force transducer used in the invertebrate

experiments. Using a plastic probe, the sciatic nerve was pulled partially from the leg and draped

over the stimulator electrodes. The force transducer and stimulator electrodes were both

connected to PowerLab in the same set up used for the invertebrate experiments.

Test 1: Contraction strength

Beginning at 0.2V, the muscle connected to PowerLab stimulated every 10 seconds for a

total of five times per voltage level. The stimulation voltage was increased by 0.2V until a

maximum contraction rate was reached. Data was collected from voltage levels that caused a

measurable contraction force in the muscle.

Test 2: Summation

The stimulator electrodes were used to stimulate the muscle using 2V of electricity at

increasing frequencies. The frequency of stimulation was increased until the muscle was

fatigued, which was determined as the point at which the muscle was no longer contracting. Data

was collected at frequencies that correlated with summation, tetany and fatigue.

Test 3: neuromuscular drug effects

For the control treatment and each drug test, the muscle its self was first rinsed in the

drug or control solution, then stimulated using 2V from the stimulator electrodes. Ten

stimulation trials were performed for every drug or control tested. For the invertebrate muscle,

the crab claw and shrimp tail were both tested. Serotonin and phenoxybenzamine were used as

the test drugs and marine Ringer’s solution was used as the control. For the vertebrate muscle,

tetraethylammonium and potassium were used as the test drugs and Ringer’s solution was used at

the control. In addition to rinsing the vertebrate muscle, the sciatic nerve was also tested with all

three solutions.

Statistical analysis

All data was collected in measures of Volts and converted to milligrams by measuring the

number of Volts produced when a known amount of force was applied to the force transducer.

Microsoft Excel was used for the statistical analysis and Two-tailed, repeated measures T-tests

were used to test for significance.

Results

Test 1: Contraction strength

The amount of force exerted by the frog gastrocnemius was higher when stimulated at

0.8Volts (X= 8.73+/- 0.17mg) then when stimulated at 1.0Volt (X= 7.33+/-1.17mg) (P=0.003)

(Figure 1) and was lower when stimulated at 1.8Volts (X=7.61+/- 0.26mg) then when stimulated

at 2.0Volts (X=7.93 +/-0.62mg) (P=0.002) (Figure 1). There was no linear correlation between

the force of the vertebrate muscle contraction and the voltage with which it was stimulated.

The amount of force exerted by the crab claw was higher when stimulated at 4.0Volts

(.43+/-0.055mg) than when it was stimulated at 3.0Volts (.147 +/- 0.045mg) (P=0.01) (Figure 2)

and higher when stimulated at 10.0Volts (1.07 +/-0.12mg) than when stimulated at 4.0Volts

(.43+/-0.055mg) (P=0.002) (Figure 2).

Test 2: Summation

The vertebrate muscle muscle contraction strength was significantly higher during tetany

(0.135+/- 0.0001mg) than during the initial summation (X=0.053+/-0.03mg) (P=0.05) (Figure 3).

The c ontraction strength during tetany (0.135+/- 0.0001mg) was significanlty higher than

during fatigue (X=0.096+/- 0.0007mg) (P>0.0001) (Figure 3).

The muscle contration force of the crab claw when stimulated at 1.1Hz (X=0.34 +/-

0.026mg) was lower than when the frequency was 0.8Hz (X=0.38+/- 0.021mg) (P<0.0001)

(Figure 4). The contraction force decreased significantly when the stimulation frequency was

increased from 1.1Hz to 1.7Hz(X=0.119+/- 0.015mg) (P<0.0001) (Figure 4), and the contraction

force decreased even more when the frequency was changed from 1.7Hz to 2.0Hz (X=0.078+/-

0.008mg) (P= 0.021) (Figure 4).

Test 3: neuromuscular drug effects

Neuromusclular drugs were applied first to the muscle then to the sciatic nerve of the

vertabrate muscle. When the neuromusclular drugs were applied directly to the muscle, the

contraction force of the muscle rinsed in ringers solution (the control) (X=13.5 +/- .005mg) was

significantly lower than when it was rinsed with tetraethylammonium (X=15.3 +/- 2.12mg) (P=

0.005) but the difference between tetraethylammonium and potassium was not significantly

different. When neuromuscular drugs were applied to the sciatic nerve, the contraction force of

the muscle was significantly higher when the nerve was rinsed with potassium (8.8 +/- 0.46mg)

than when it was rinsed with tetraethylammonium (0.009 +/- 0.0003mg) (P<0.0001).

Neuromuscular drugs were applied directly to the muscles of both the crab claw and a

shrimp tail in the invertebrate muscle tests. When serotonin (X=461+/- 46.8mg) was applied to

the crab claw muscle, the contraction strength was significantly higher than when

phenoxybenzamine (X=351+/- 48.6) was applied (P= 0.002). There was no significant difference

betwee the control and the treatments. When marine Ringer’s solution (control) ( X=97.9 +/-

7.0mg) was applied to the shrimp tail muscle, the contrction strength was significantly higher

than when serotonin (X=82.7 +/- 6.04mg) was applied (P<0.0001). There was no significant

difference between the serotonine and phenoxybenzamine treatments.

Figure 1:Vertebrate muscle stimulated at increasing voltages. The difference between 0.8V and 1.0V was significant ( P= 0.003), and the difference between 1.8V and 2.0V was significant (P= 0.002) ( Two-tailed, repeatd measures T-test, DF=39).

Figure 2: Invertebrate muscle stimulated at increasing voltages. The difference between 3.0V and 4.0V was significant (P=0.01) and the differnece between 4.0V and 10V was significant (P=0.002) ( Two-tailed, repeatd measures T-test, DF=8).

Figure 3: The average vertebrate muscle contraction strength when stimulated at increasing frequencies until fatigued. The difference between summation and tetany was significant (P= 0.05) and the differnece between tetany and fatigue was significant (P<0.0001) (Two-tailed, repeatd measures T-test, DF=9).

Figure 4: The average invertebrate muscle contraction strength when stimulated at increasing frequencies until fatigued. The difference between 0.8Hz and 1.1Hz was significant (P<0.0001), difference between 1.1Hz and 1.7Hz was significant (P<0.0001) and the differnece between 1.7Hz and 2.0Hz was significant (P= 0.021) (Two-tailed, repeatd measures T-test, DF=19).

Figure 5: The average vertebrate muscle contraction strenth when drugs were applied directly to the muscle. The difference between the control and tetraethylammonium was significant (P= 0.005) but the difference between tetraethylammonium and potassium was not (Two-tailed, repeatd measures T-test, DF=14) (Note: error bars too small to depict).

Figure 6: The average vertebrate muscle contraction strenth when drugs were applied directly to the sciatic nerve. The difference between tetraethylammonium and potassium was significant (P<0.0001) (Two-tailed, repeatd measures T-test, DF=14) (Note: error bars too small to depict).

Figure 7: The average invertebrate muscle contraction strenth of a crab claw when drugs were applied directly to the muscle. The difference between serotonin and phenoxybenzamine was significant (P= 0.002) (Two-tailed, repeatd measures T-test, DF=29).

Figure 8: The average invertebrate muscle contraction strenth of a shrimp when drugs were applied directly to the muscle. The difference between the control and serotonin was significant (P<0.0001) (Two-tailed, repeatd measures T-test, DF=29).

Discussion

Test 1: Contraction strength

Based on the type of vertebrate muscle and the nature of the muscle innervations, it would be

expected that motor neuron recruitment would lead to an increase in the number of muscle fibers

stimulated with each increasing voltage, thus contraction force should have been positively

correlated with stimulation voltage (Belanger 2005). However, the expected results were not

obtained. In contrast, muscle contraction force was initially high before it decreased, and at a

certain point then began to increase. This portion of the study should be repeated to ensure the

muscle is not fatigued before the next set of stimulations in the set, and that there is no disruption

during the experiment; it should be ran from beginning to end without any breaks to ensure they

equipment remains calibrated and the muscle remains moist. Due to a fire drill during the

experiment, these conditions were not able to be met.

For the invertebrate muscle, a clear positive correlation was found when the crab claw

was stimulated at increasing voltages. Based on Moyes and Schulte (2008), these results were to

be expected. When the muscle was stimulated with higher voltages, the signal was able to travel

further in the muscle which led to a contraction of greater force.

The vertebrate muscle was only stimulated up to 2.0Volts while the invertebrate muscle

was first stimulated at 3.0Volts. It is possible that the positive trend was not found in the frog

gastrocnemius muscle stimulation because the voltages used were not strong enough (Chaffee

and Light 1934). Overall, the muscle contraction force of the vertebrate muscle was much larger

than the invertebrate muscle. It is possible that this is due to differences in how each muscle is

innervated and because the muscles tested are not directly comparable (Loeb et al. 1987). The

gastrocnemius is important for jumping, thus is a long muscle whereas the crab claw is important

for gripping and thus, is a short muscle. A last possibility could be that even though the volts

were converted into milligrams for each set of experiments, the equipment used was not

calibrated to the same measurements or sensitivity since the two experiments were performed

two weeks apart.

Test 2: Summation

As shown in figure 3, the vertebrate muscle responded as expected to increased

stimulation frequency. The muscle contraction initially increased, then reached a plateau at a

high contraction force, then contraction force quickly decreased as the muscle fatigued. These

results are expected because when a vertebrate muscle is stimulated too frequently, the motor

neuron sending the message does not have time to repolarize.

When invertebrate muscles are stimulated at higher frequencies, the signals to the muscle

can lead to a graded response in the muscle which means that instead simply contracting more

frequently at a consistent force, like in vertebrate muscles, invertebrate muscles will respond to

the stimulus by contracting at a constant rate but with more force instead (Moyes and Schulte

2008). Although this is known to be the affect of increasing muscle stimulation, this is not was

the results from this experiment show. Figure 4 shows that muscle contraction strength decreases

with increasing stimulation frequency.

It is possible that the results obtained were due to errors in experimental set up and

protocol. Prior to data collection, the muscle had been stimulated by increasing voltages in Test 1

which may have prematurely began to fatigue the muscle because proper wait time between Test

1 and Test 2 was not allowed. If this was the case, the results would make sense because the

initial contractions are strong, and the muscle then the contraction force quickly decreases as

stimulation frequency is increased.

Test 3: neuromuscular drug effects

The vertebrate muscle was tested with neuromuscular drugs by directly rinsing the

muscle and sciatic nerve in separate experiments. Vertebrate muscles function by reacting to the

release of acetylcholine at the neuromuscular junction, and since tetraethylammonium and

specifically has no affect on the muscle itself, no change between test groups should be expected.

However, when the muscle was rinsed with tetraethylammonium, the contraction strength of the

muscle increased. Tetraethylammonium blocks potassium channels from opening which will

affect the motor neurons that travel to the muscle. If the potassium channels don’t open, then

potassium cannot rush into the neuron and the cell will not be able to repolarize because

potassium won’t be able to rush out of the neuron.

When the neuromuscular drugs were used directly on the sciatic nerve, the potassium

treatment lead to a large increase in muscle contraction force. This result should have been

expected because potassium is needed in order to for the nerve to function properly, so if there is

a high concentration of potassium available in the surrounding environment, the nerve will be

able to repolarize efficiently.

When the invertebrate crab had a higher contraction force when rinsed in serotonin than

when phenoxybenzamine which is the opposite of what would be predicted. Serotonin causes

invertebrate muscles to relax, thus the contraction should have been weaker than when it was

rinsed with phenoxybenzamine, which inhibits the action of serotonin. The inhibitory effect of

serotonin was seen in the shrimp tail experiment; when serotonin was used to rinse the muscle, it

had a decrease in contraction strength as compared to the control of marine Ringer’s solution.

It is likely that the neuromuscular drug experiments did not yield the expected results

because the muscles had been isolated and used for stimulation experiments for over an hour.

The shrimp tail was isolated less than ten minutes before it was used in experiments, which may

be why it is the only one that correctly responded to the drugs.

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