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.

INSTRUCTIONS

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.

Diagram of Frog Skeleton

EXPERIMENT 1.

Observe the following behaviors of a normal intact frog. Record detailed notes (create a table for your observations).

Wait 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.

EXPERIMENT 2

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.

EXPERIMENT 3

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 of divergence?

EXPERIMENT 4

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?

Wait until all groups have completed this series of experiments.

EXPERIMENT 5

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.

Wait until all groups have completed this series of experiments and all results to this point have been summarized.

EXPERIMENT 6

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 provided.

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.

At 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.