REFLEXES IN THE FROG
reflex.htm last updated 1 September 2009 at 1:54 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 "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 behaviors
require both the brain and spinal cord, which behaviors can be produced in the
absence of the brain, and which behaviors 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. These experiments allows students
to gain experience dissecting and stimulating peripheral nerves.
Before beginning the experiments, students will become familiar with the legal
and ethical implications of working with live animals in a laboratory (previous
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 will be instructed on the use of an electronic stimulator and pin
electrodes as well as how to pith a frog. The instructor will demostrate all
procedures without revealing the expected results.
Observe the following behaviors of a normal intact frog. Record detailed notes
(create a table for your observations).
Voluntary activity and equilibrium
Righting responses (place the frog on its back and watch it right itself;
may require several attempts)
Responses to mechanical, chemical, and electrical stimuli
touch the eye (cornea) with a blunt object,
lightly pinch the skin of the foot with a pair of forceps,
apply a pledget soaked in a weak acid (10% Acetic Acid) to
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)
apply a brief electrical shock to the foot using a hospital-grade
electronic stimulator. (Your instructor will demonstrate how to use
this piece of equipment. Stimulus parameters will be explained: 0.1
ms duration, 30 Hz, 10-30 Volts.)
"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
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. Your instructor will
explain and demonstrate. Record the time at which your
frog was pithed and proceed immediately to Experiment 2.
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 Dr. Davis or the 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 (the same stimulus parameters
used in Experiment 1). Observe the response. Repeat each minute until the leg
is raised in response to the stimulus (called a flexion reflex.) The interval
between successive stimulations should be no less than one minute. 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 number can be shared with the
class. The instructor will explain the physiological basis for spinal shock.
With the frog suspended from ringstand, use the electronic stimulator to apply
a short burst of weak tetanic stimuli to the toes of one foot. (A tetanic stimulus
will be described at the beginning of lab.) Increase the strength of the stimulus
at intervals of 30 seconds while watching for a bilateral response. What happens
when the palm of the hand is electrically stimulated with a weak tetanic stimulus?
A strong tetanic stimulus? How do these experiment illustrate the phenomenon
Once the flexion reflex has returned, remove the animal from the ringstand
and repeat the observations made on the intact frog (listed under Experiment
# 1). Make notes of your observations. Which of these behaviors are independent
of the brain?
until all groups have completed this series of experiments.
Your instructor will demonstrate 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 double-pithed animal?
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 Dr. Davis 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 by the instructor, 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 below the forelegs and one just above 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
for one second with a weak voltage 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?
By adjusting the stimulus intensity, is it possible to produce contractions
in muscles of both legs? What happens when you stimulate the muscles of the
leg directly with the electrodes? What do you conclude from these observations?
Are the movements the result of reflexes? Do the movements involve the participation
of sensory axons?
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.