REFLEXES IN THE FROG
reflex.htm last updated 7 October 2019 at 3:47 pm
The basic unit of behavior is the reflex. Reflexes involve the excitation
of sensory receptors, conduction of electrical signals (action potentials) by
sensory neurons (sensory afferents) to the central nervous system (spinal cord
and brainstem) where, either directly or indirectly, motoneurons are activated.
Sufficiently excited motoneurons generate action potentials which travel along
motor axons to effector organs (muscles or glands). Sensory inputs are processed
within the CNS by interneurons such that appropriate responses are generated
by the effector organs. Although reflexes require part of the CNS, reflexes
do not require conscious thought processes.
In today's experiments, you will first examine behaviors in an intact frog.
Subsequently, the brain will be disrupted by a procedure called "single pithing"
and behaviors will be re-examined. Next, the spinal cord will be destroyed producing
what is called a "double-pithed" frog in which the behaviors will again be tested.
Students are to organize their observations so that comparisons can be made
between the results obtained in an intact, a single-pithed, and a double-pithed
frog. From these observations, students should be able to deduce which responses
require both the brain and spinal cord, which responses can be produced in the
absence of the brain, and which responses are expressed independently of both
the brain and spinal cord.
Upon the completion of our tests for reflexes, we will examine the effects
of stimulating branches of the sciatic nerve. This phase allows students to
gain experience dissecting and stimulating peripheral nerves.
Before beginning the experiments, students have become familiar with the legal
and ethical implications of working with live animals in a laboratory in aprevious
lab period. All of the experiments in this lab have been approved by the Wofford
College Institutional Animal Care and Use Committee which is ultimately responsible
for assuring the proper use of live animals on this campus.
Please read the instructions before beginning. If you are unsure about something,
ask for help from the instructor or lab assistant. Our supply of animals is
limited. You should have watched the videos on the use of an electronic stimulator
and pin electrodes, tying sutures, how to pith a frog and how to open the abdomen
and expose nerves for stimulation. For each of the experiments described below,
your instructor will demostrate the procedures without revealing the expected
Useful terms to describe movements: Adduction, abduction, flexion, extension.
Observe the following behaviors of a normal intact frog. Record detailed notes
on the datasheet you printed from the lab Moodle.
Voluntary activity and posture
Righting responses (Place the frog on its back and watch it right itself;
this may require several attempts.)
Responses to mechanical, chemical, and electrical stimuli
Mechanical # 1: touch the cornea with a blunt object (wooden handle
of a probe)
Mechanical # 2: lightly pinch the skin of the back foot with a pair
Chemical # 1-3: Apply a pledget soaked in a weak acid (10% Acetic
the skin near the midline on the dorsal surface (rinse with
near the anus (rinse with water afterwards)
to the dorsal surface of the thigh (rinse with water afterwards)
Electrical: Apply a brief electrical shock to the foot using a hospital-grade
electronic stimulator. The video shows to use this piece of equipment.
Stimulus parameters are: 0.1 ms duration, 30 pps, 10-30 Volts.
Mechanical # 4 : "Tickle" the skin with a sharp probe near the midline
on the dorsal surface and then near the anus
Swimming patterns in a tank of water :
observe fore- and hindlimb movments and patterns of coordination
observe responses to a silhouette of a bird passing over the tank
the frog's eyes are above the surface
the frog's eyes are below the surface
until all groups have completed this first round of experiments.
PITHING A FROG
The remaining experiments will be performed on pithed frogs. A single-pithed
frog is prepared by inserting a needle into the cranial cavity through the foramen
magnum and mechanically disrupting the brain. This renders the animal asensate
and is an approved method for euthanasia of amphibians. Pithing is explained
and demonstrated in the video you watched before coming to lab.
Record the time at which your frog was pithed and proceed immediately to Experiment
Note: Students may elect to pith their frog or not.
No one should feel pressured to pith. There will be other opportunities during
the semester for students to pith a frog. Students who pith a frog should do
so under the supervision of your lab instructor or a lab assistant.
Suspend a single-pithed frog from a ringstand with a hook through the anterior
part of the lower jaw. Immediately test for a reflex by moderate electrical
stimulation of the plantar surface of the foot (stimulus parameters: 0.1 ms
duration, 30 pulses per second, 30 Volts applied for 1 second). Repeat the stimulus
at one minute intervals until you observe a response (typically a flexion of
the leg being stimulated.) Record the time (in minutes) for the flexion reflex
to return following pithing. The interval during which reflexes cannot be obtained
is called spinal shock. Determine the duration of spinal shock (in minutes)
so that you can share this information with the class. The instructor will explain
the physiological basis for spinal shock.
Part A: With the frog suspended from ringstand, use the electronic stimulator
to apply a tetanic stimuli (40 pulses per second) to the toes of one foot. (A
tetanic stimulus will be described at the beginning of lab.) Increase the length
of time the stimulus is applied to the toes at intervals of 30 seconds while
watching for a bilateral response.
Part B: Apply a tetanic stimulus for about 3 seconds to the palm of the hand?
What do you observe? Are other limbs involved? Does this experiment illustrate
the phenomenon of divergence?
Once the flexion reflex has returned and you have checked for bilateral responses
to prolonged stimuli, remove the animal from the ringstand and repeat the observations
made on the intact frog (listed under Experiment # 1). Make
notes of your observations on your data sheet. Which of these behaviors are
independent of the brain?
until all groups have completed this series of experiments.
The latter part of the video demonstrated how to "double pith" a
frog. Double-pithing completely destroys the spinal cord but leaves the peripheral
nervous system intact. Repeat all the observations listed in Experiment 1 on
your double-pithed frog. You will omit Experiments 3 and 4 above.
Make notes of your results so that you can compare each of the behaviors in
intact, single-, and double-pithed frogs. Which behaviors required a functional
spinal cord but do not require the brain? Can any behaviors be produced in a
Note: Again, students may elect to double-pith their
frog or not. No one should feel pressured . Students who double-pith should
do so under the supervision of your lab instructor or the lab assistant.
until all groups have completed this series of experiments and all results to
this point have been summarized.
Following the example provided in the video, open the abdomen of the frog
by making a longitudinal cut through the ventral body wall from the pelvic region
through the shoulder girdle. Make lateral incisions on each side, one just anterior
the forelegs and one just anterior to the hindlegs. Remove the viscera from
the abdominal cavity to expose the three spinal nerves that merge to form the
sciatic plexus of the sciatic nerve on each side. Using glass probes, lift up
each of the three nerves on the left side and place a loose ligature around
each one. Keep the nerve moistened with the Ringer's Solution
Place the most lateral of the spinal nerves of the left sciatic nerve on stimulating
electrodes and be sure the electrode does not touch any other tissue. Stimulate
at 4 pulses per second at 10 Volts so that contractions are observed in the
muscles of the left leg. Note which muscles contract and the type of movement
produced. Next, stimulate only the middle nerve while noting whether the pattern
of muscle contraction of leg movement. Finally, stimulate the most medial nerve
alone while watching for muscle contractions. Is the pattern of muscle contraction
the same for each nerve stimulated? Why or why not? What does this experiment
demonstrate regarding the trajectory of motor axons in the sciatic plexus?
Place one of the nerves on the electrode at stimlulate with 10 volts at 4
pulses per second. While keeping the pin electrodes in place, have a lab partner
move the decade knob so that a 40 pulse per second stimulus is delivered. Note
the change in the charatericstics of the muscle contraction. Switch back to
4 pulses per second.
What happens when you stimulate the muscles of the leg directly with the
electrodes? What do you conclude from these observations? Are theses movements
the result of reflexes? Do the movements involve the participation of sensory
In the remaining time, students may investigate the internal organs of a double-pithed
frog. As we will be conducting experiments in a subsequent lab involving the
heart, don't expect the instructor to divulge information related to the heart
during this lab period.
the end of all experiments, place the frog carcass in the ziplock bag offered
by the lab assistant. Clean all lab materials and leave the lab space neat.