Lab 5.

1
Objectives - after completing this lab you should be able to:
1) explain why we can use the frog gastrocnemius muscle to study skeletal muscle
contraction
2) perform the dissection to prepare the nerve-muscle preparation
) describe a basic muscle twitch and the physiological events that cause it or occur
during it
!) describe the effect of varying stimulus intensities
5) describe the effect of varying stimulus fre"uencies
#) differentiate between subthreshold$ threshold$ maximal and supermaximal stimuli
%) interpret data collected from frog muscle contractions
&) describe the process and result of pithing
') describe the function and composition of (ingers solution
BACKGRO!":
) skeletal muscle cell will contract only when ade"uately stimulated. *he
basic response of skeletal muscle to ade"uate stimulation is the muscle twitch. *he
twitch is modified by changing the intensity +volts) or fre"uency +pulses per second$
pps) of stimulation or by changing the condition of the muscle tissue +temperature$ ,
2

level$ ion concentration).
-n a whole muscle$ weak stimuli excite only those motor units which are most
sensitive. .ore intense stimuli excite more motor units. *hus$ as the intensity of the
stimulus is increased$ the strength of contraction increases. /trength of contraction
is also increased by increasing the fre"uency of stimulation so that the muscle
cannot fully relax before it contracts again. *hese phenomena are explained further
in the textbook.
0e are using the gastrocnemius muscle of a frog to observe skeletal muscle
contraction because even though the frog is an amphibian$ its skeletal muscle tissue
works the same way as mammalian muscle. *he main difference is that mammalian
tissue re"uires a high level of oxygen and glucose and a controlled temperature
environment. *he frog1s muscle will work at room temperature because its en2ymes
are not as temperature-dependent as mammalian en2ymes.

3efore the frog is used it will be double pithed by the instructor. 4ithing is a
procedure in which the central nervous system of the frog is destroyed$ leaving the
peripheral nerves intact. ) dissecting needle is inserted into the cranium through
the foramen magnum and used to disrupt the brain tissue. )s soon as the needle
goes through the foramen magnum$ the spinal cord is severed and the frog feels no
pain +remember$ there are no sensory receptors in 56/ tissue). -n addition$
destruction of the cortex prevents perception of pain or other sensations via the
cranial nerves. 0hat has 7ust been described is called 8single-pithing9$ and it is not
sufficient for testing muscle responses. *he spinal cord must also be destroyed$
and a frog with both brain and spinal cord destroyed is 8double-pithed9. )t this point$
LAB 6: Skeletal Muscle Function
Lab 5.2
the frog is clinically dead$ but its tissues continue to be viable for minutes to hours$
depending on how well we take care of the frog.
)s soon as the frog1s muscle or nerve tissue is exposed$ it should be fre"uently
+once every 1 or 2 min) bathed with (inger1s solution$ an isotonic solution that has
approximately the same ion concentrations as does amphibian :5;. <ust a few drops
are enough to make sure the tissues don1t dry out and have enough of the right ions.
;rogs absorb oxygen through their .,-/* skin and through their lungs. 3ecause the
lungs stop working when we pith the frog$ we rely on the skin to allow oxygen to diffuse
into the blood. *he heart keeps working and circulating the blood through the skin and
muscle$ and supplies enough oxygen as long as the skin is kept moist +damp paper
towel).
#ROC$"R$%
). ;rog 6erve-.uscle 4reparation
:"uipment and materials=
Labscribe software>hardware
double-pithed frog
dissecting tray
dissecting kit
(ingers solution in dropper bottle
2 pieces thread$ 12? each
1. (efer to the diagram below. .ake an incision through the skin at the base of one
thigh +where it 7oins the trunk) and cut it all the way around.
2. /trip the skin down over the entire leg$ carefully freeing it from underlying tissues at
the knee. 4ull$ don1t cut.
. @eep exposed tissues moist with (ingers solutions at all time.
!. Ase blunt dissection to separate the muscles on the dorsal surface of the thigh to
expose the sciatic nerve. Bave the instructor cut the superficial fascia if it is in the way.
5. 3e careful not to touch the nerve with metal or your fingers$ and also not to stretch
it. Con1t pull up on it or twist it.
#. Asing a glass rod or the tip of a pipette$ carefully pull a thread under the nerve. *his
thread will be used to position the stimulating electrode in part C, 6,* *-: *B-/
*B(:)C.
%. -nsert a glass rod between the gastrocnemius muscle and the leg bones and run it
up and down to separate them.
&. *ie the other piece of thread firmly around the )chilles tendon +using a s"uare knot)$
close to the muscle.
Lab 5.
'. 5ut the )chilles tendon distal to the tie$ leaving at least 1>&? of the tendon distal to
the knot.
1D. Ase metal clips to secure frog to board in dissecting tray.
11. @eep the rest of the body covered with a wet paper towel.
3. )pparatus 4reparation
1. /tart Lab/cribe using icon on desktop.
2. /ettings menu$ Load$ group$ choose ahk11!$ ,@
. /ettings$ choose summation-tetanus$ ,@.
!. )ttach tendon to transducer as demonstrated by instructor.
5. 4ut stimulating electrode under nerve as demonstrated by instructor.
#. Bave instructor check setup before proceeding.
Lab 5.!
5. varying stimulus intensity to demonstrate recruitment
1. .ake sure preferences are set for a single 1D ms stimulus of amplitude DE. 5hange
if necessary.
2. *ype DE in the marks window$ click /tart on the screen$ then hit the enter key.
. 5hange stimulus amplitude to D.25 E and repeat step 2.
!. (epeat$ each time increasing stimulus voltage as indicated in the data chart.
5. /ave file to desktop as 8your name muscle9
#. /croll back to the first response or use the .arks window to find the first mark.
%. 5lick )utoscale.
&. 5lick the two cursor button.
'. 4lace one cursor at the beginning of the contraction and the other at the peak.
1D. .easure and record amplitude and duration of contraction and relaxation phases.
11. (epeat measurement for all other responses.
12. 4rint threshold and maximum response responses and label with your names and
the stimulus intensity if it isn1t already on the screen.
stimulus +E) amplitude duration of
contraction
duration of
relaxation
D.D25
D.D5D
D.D%5
D.1
D.15
D.2
D.25
D.
continue only if at this point the amplitude is still increasing
C. varying stimulus fre"uency to demonstrate twitch summation and tetanus
Lab 5.5
1. (inse muscle with (ingers.
2. :dit$ preferences$ count F D$ pps +fre"uency) 1 +this will change-see table below)
. -n the marks window$ enter 1pps$ click on start$ hit the enter key$ wait until the
muscle stops contracting and click on stop.
!. (ecord the response of the muscle at each stimulus fre"uency as one of the
follwoing= no summation, twitch summation, unfused tetanus, complete (fused)
tetanus
fre"uency of stimulation +B2) response
1
5
1D
15
2D
5L:)6A4=
1. frog and tissues go in 2ip lock bag on counter
2. rinse clips$ board and pan in plain water and put in drainer
. wash dissecting instruments in soapy water$ rinse$ dry and return to plastic box
!. rinse stimulating electrode prongs in dB
2
, and gently dry
5. close Lab/cribe and )dobe(eader$ shut down computer
Guestions
1. 0hat was the thresholdH
2. 0hat was the maximum stimulusH
. (ecruitment began at IIIE and ended at IIIE.
!. Bow long did contraction and relaxation take at the threshold stimulusH
contraction=
relaxation=
5. 0ave summation leads not only to a sustained muscle contraction$ but also an
increase in contraction strength :E:6 );*:( (:5(A-*.:6* /*,4/. *he
physiological basis for the increase in strength +not the sustained contraction) is=
Lab 5.#
#. 5aJJ is pumped out of a cardiac contractile cell via cotransporter that exchanges
5aJJ for 6aJ. +refer to how cotransporter systems work i.e. an active transport pump
is re"uired to set up the initial ion gradient) Bow would muscle activity be affected by a
higher than normal level of 6aJ in the cytosolH
a. muscle contraction would be impaired
b. muscle relaxation would be impaired
c. there would be no change in muscle activity
%. Cendrodotoxin blocks voltage-gated @ channels in the motor neuron axon terminal.
a. 0hat effect would dendrodotoxin have on repolari2ation of the axon terminalH
enhance$ prevent or impair$ or noneH
b. 0hat effect would dendrodotoxin have on the release of )5hH
enhance$ prevent or impair$ or noneH
c. 0hat effect would dendrodotoxin have on the duration of muscle contractionH
shorten$ lengthen$ or noneH
&. 4ithing destroys the frog1s IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII$ but leaves
the IIIIIIIIIIIIIIIIIIIIII intact.
'. -s the frog used in today1s lab single- or double-pithedH
1D. Bow long are the frog1s tissues viable after pithingH
11. 0hat are the characteristics of (inger1s solutionH
12. 0hat two functions does (inger1s solution performH
1. 0hat two factors allow oxygen to be supplied to a pithed frog1s tissuesH
1!. (ecruitment increases the strength of muscle contraction by adding more and
more IIIIIIIIIIIIIIIIIIIIIIIIIIIII. *his is done by increasing the stimulus
IIIIIIIIIIIIIIIIIIIIIIIIIIIIII.
Lab 5.%
15. Bow are the frog1s en2ymes different from mammalian en2ymesH
1#. Craw a muscle twitch and label the stimulus and the three phases. Label the x and
y axes.