Four Exercises in Neurobiology
Department of Biological Sciences North Texas State University Denton, Texas 76203
Rollie Schafer received his doctorate at the University at Colorado in 1969, and has held teaching/research positions at New Mexico Institute of Mining and Technology, the University of Michigan, and North Texas State University. He is presently Associate Professor of Biological Sciences and Physiology at North Texas and also serves as Assistant Graduate Dean for Research. While he was a faculty member at the University of Michigan in Ann Arbor, he occupied a laboratory and office next door to that of another neurobiologist, Dr. Bruce Oakley. Oakley and Schafer taught the laboratory course in neurobiology at Michigan and combined forces to produce the laboratory manual, Experimental Neurobiology, of which a portion is reprinted here. At North Texas, Dr. Schafer teaches neurobiology in the undergraduate, graduate, and medica1 schools, and conducts research in the chemistry of the sense of smell.
The four exercises which follow are reprinted from the laboratory text Experimental Neurobiology by Bruce Oakley and Rollie Schafer (University of Michigan Press, Ann Arbor, 1978, 367 pp.). The four exercises have been chosen to exemplify several of the different areas covered in the manual: an electrophysiological experiment on sensory reception (6.5); a quantitative study of human reflexes (7.4); and comparative studies of locomotion (8.2) and simple responses to stimulation (8.7). The manual is intended to provide an introductory laboratory experience in neurobiology with emphasis in electrophysiological techniques. It may also be used effectively as a source book for sequenced independent projects. The level of sophistication (or difficulty) of the experiments is graded so that an instructor can offer the laboratory for students of different levels and backgrounds. The selection of experiments (33 in all) is diverse enough to permit choosing either a cellular, organismic, or behavioral approach to complement the instructor’s own lecture treatment. Introductory chapters cover basic terminology, practical use of electrophysiological equipment, and mammalian brain anatomy and neuroanatomical terminology. The section on neuroanatomy, Neuroanatomy: Dissection o f the Sheep Brain, has been reprinted separately by the University of Michigan Press for use in basic anatomy and physiology courses. Each experiment in the manual was selected with three criteria in mind. An experiment ought to; (1) work in student hands; (2) utilize no highly specialized electrophysiologicalequipment; and (3) have a balance of technical and conceptual content. Some of the exercises are simple enough to be done by beginning biology students, and the rest can be carried out by advanced undergraduates with little assistance from the instructional staff. Many of the exercises are best done in two laboratory periods: a first period devoted to setting up and learning procedures, followed by a second period devoted to producing meaningful data. An estimate of the amount of time required is listed within each experiment. Laboratories-especially those which use more than very simple techniques-are a notorious time sink. The manual is designed to conserve preparatory and instructional time in several ways: (1) The instructions in all exercises are detailed and complete. Minimal instructional assistance is required to get students to the data-taking stage. (2) Potential problems are noted in the “Pitfalls and Suggestions” and “Notes for the Instructor” sections of each experiment. (3) Each experiment has a detailed list of materials which can be used as a checklist and basis for purchases. (4) The information in chapter 10, “Laboratory Organization,” will aid the instructor to initiate a new laboratory or to reactivate it annually. The table of contents of the full manual is given on the next pages, followed by four representative exercises which were performed by participants at the second ABLE conference.
2.4 Smooth Muscle Regulation of Blood Flow 155 Microcirculation in Amphibians. 1 Visual Reception
Frog Skin 4.9
2 .5 Electronic Stimulator 25 Preamplifier 29 2.I
Turtle Heart 5.4 60 Hz Interference 24 2.3
CHAPTER 3.5 The Membrane Potential 112 lntracellular Recording from Frog Sartorius Muscle Fibers 4.1
2. Survey of Electrophysiological Instrumentation 9
Introduction xi CHAPTER 1. IO
The Electrophysiological Setup 10
4. Synaptic Transmission and Effector Processes 129
5 .7 Audio Amplifier and Loudspeaker 34 2.5
CHAPTER 4. Connectors. Neuroanatomy 45
Cellular Neuroanatomy and Histological Techniques 46 3 .3
Compound Action Potential and Single Units 97 Limulus Leg Nerve
2.8 Electrodes and Nerve Chambers 36
2. 2 Dissection of the Sheep Brain 49
3 .2 Compound Action Potential 85 Frog Sciatic Nerve
Frog Electroretinogram (ERG)
Insects Chromatophores 160 Neurohumoral Control in Arthropods
Active Transport of Sodium 76
CHAPTER 6 . Receptor Processes 165
6 .4 Giant Nerve Fibers
Practical Electrophysiology 2 Review of Electrical Terms and Principles 3
Earthworm Nerve Cord
I.6 Volume Conduction 121 The Potential Field Generated by a Nerve Impulse
Cables. Electrical Principles and Electrophysiological Terminology 1
4. and Adapters 12 Cathode Ray Oscilloscope 16 2.2
Coelenterate Nerve Net 130 Luminescent Waves in the Hydroid Neuromuscular Synapse 138 Frog Sciatic-Gastrocnemius Junction Cardiac Muscle and Its Neural Regulation 147
Oscilloscope Camera 40 Physiological Polygraph 42
5. Cellular Potentials and Bioelectric Activity 75
8. 2 The Rat Motor Cortex 212 Somatotopic Representation 7.8
Setting Up the Laboratory 326 Electronic and Other Major Equipment 328 Electrode and Equipment Construction Suppliers 341 Basic Laboratory Supplies 347 Animals
Mechanoreception 174 Tactile Receptors on the Insect Leg Olfactory Reception 180 Frog Electro-olfactogram (EOG) Proprioception 187 Frog Muscle Spindle Skin Receptive Fields 192 Frog Cutaneous Receptors Audition 196 Gerbil Cochlear Microphonics
6.7 7.5 10.2
Animal Locomotion 262 Descriptive Analysis of Simple Behavior Insect Flight 266 Sensory and Central Mechanisms
Electrolocation and Electrocommunication 272 Electrical Signals and Behavior of Weakly
8.6 10. Cuneate.5
Electric Fish Feeding Behavior 281 Taste Reception in Flies Conditioned Taste Aversion in the Rat 287 One-Trial Learning of LiCl Avoidance Sexual Behavior 291 Responses of Cockroaches to Pheromones Sensitive Plant 300 Nastic Responses and Conducted Action Potentials in Mimosa pudica
8. Surgery and Stereotaxic Techniques 307
CHAPTER 7. 9 Chronic Recording from the Rat Brain 256 Hippocampal Theta Rhythm
CHAPTER 9 .3 9.6 8. 6 Frog Optic Tectum 239 Retinal Processing of Visual Form
8. Drugs.4 7. Central Processes 203
Surgical Instruments and Micromanipulators 308 Pithing the Frog 31 1 Small Mammal Surgery 313 Stereotaxic Procedures 318
Stretch Reflex 224 Myographic Recording from the Human Leg Somatosensory Representation in the Rat Medulla 232 Electrophysiological Activity in Spinal
CHAPTER 10. and Gracile Nuclei 7 .3 6. Laboratory Organization 325
Cockroach Ventral Nerve Cord 204
Spontaneous and Elicited Responses 7 .1 9.3
10.4 6.3 Spinal Reflexes 218 Effects of Surgical Ablation of Higher Nervous Centers in the Frog
Trigeminal. and Neuroactive Agents 352
Lateral Hypothalamic Areas 7 .4
Neural Regulation of Weight 247 Lesions in the Rat Hypothalamus Chronic Stimulation of the Rat Brain 251 Self-stimulation of the Septal Nuclei and
10.2 9.5 6.7
and joints are detected by subsurface enteroceptors.Neurobiology Exercises
6. (2)The behavioral response threshold is lowered by stimulation elsewhere on the body. when sufficiently activated. and chemical irritants. would initiate the correct wiping response In the jargon of neurobiology. such as a pinch. Time to complete the exercise: One 3-hour laboratory period. intense stimulation. cold. At first the problem seems elementary. including receptors which respond only to intense (nociceptive) stimulation. this would entail a somatotopic representation with direct reflex connections.
Receptors which terminate in amphibian skin are capable of responding t o touch. An irritating insect may he quickly removed by a swipe of a leg. (3) Would an animal stimulated simultaneously at two widely separated points wipe halfway between? ( 4 )In development how do the outgrowing axons find their correct terminations! As is frequently the case. Many receptors are specialized to respond to
Line illustration is adapted from Jacobson and Baker. but will make a beginning by examining the manner in which touch fibers respond and innervate the frog skin. We will not attempt to explore these issues in this experiment.
Rationale The immediate cause of a behavioral response is usually an environmental stimulus which is detected by exteroceptors. Copyright © 1978 by the University of Michigan. 1969
From Bruce Oakley and Rollie Schafer. No part of this material may be used or reproduced in any manner whatsoever without written permission of the publisher. The most obvious arrangement would be a wiring diagram with each point on the skin connected by its own unique axon to that location in a central “motor switchboard” which. what seems patently simple is actually quite sophisticated. warmth. As is evident from common experience. Stimuli from the viscera.5 Skin Receptive Fields Frog Cutaneous Receptors
only one of these forms of stimulation. muscles. The receptor’s role in cutaneous localization can he studied by recording from those axons which innervate the skin. EXPERIMENTAL NEUROBIOLOGY: A LABORATORYMANUAL. Reprinted here by permission of the University of Michigan Press. All rights reserved. Exercise 7 3 demonstrates that much of the neuronal circuitry responsible for cutaneous localization resides within the spinal cord. but it is also an oversimplification which neglects important complexities. In this exercise we will examine the response properties and innervation patterns of tactile receptors in frog skin.
. For example: [ I ) Axons have overlapping receptive fields (skin regions innervated by more than one axon]. This may be in part correct. animals are often quite adept at localizing a cutaneous stimulus.
Do not pull the flop up yet. to allow recording from a second nerve. Input in differential mode if possible.)
Fig. 6.The dorsal flap of skin is elevated enough to expose a cutaneous nerve. or if no accessory output is available. Recording arrangement. extending from the level of the cloaca almost to the tympanic membranes.5-1.
THREAD AND HOO ELEVATE FLAP
CUT (THROUGH SKIN)
NERVE SLIGHTLY STRETCHED BETWEEN FLAP OF SKIN AND BODY WALL
Fig. simply free the skin along the cut edges.000x. trigger set on external input and the triggering level control on automatic or free run. band-pass filters (if available! set at about 30-100 Hz [low frequency filter] and 3 kHz [high frequency filter). it is severed and the skin flap elevated.hut do not elevate the flap until ready to record.
.5-2. and so on. vertical sensitivity at 0. The flap should be a large one. Leave all AC-powered equipment on while other arrangements are completed. Connect an audio amplifier and loudspeaker to the accessory output on the hack of the oscilloscope.1-0. Preamplifier. Remove any unnecessary AC-powered equipment from the vicinity.
Calibration. The nerve should be stretched only enough to accommodateelectrode placement. Once recording starts. Audio amplifier. Use the following initial settings on your equipment. (Adaptedfrom Jacobson and Baker. Incision of the dorsal skin to expose the cutaneous nerves for recording. With scissors cut through the skin as shown in figure 6.5-1 to produce the outline of a flap in the dorsal skin. 6. split the preamplifier output so that it drives both the audio amplifier and the oscilloscope. After the first nerve is examined. Operative procedure Double pith a large frog (see page 3 1 I). Connect the lead of a third [ground! electrode to the ground terminal on the preamplifier input Connect the preamplifier output to the vertical amplifier input of the oscilloscope. readjust the gain so that action potentials are clearly audible but not offensive to those around you. Cut through the skin on three sides as indicated. Make any additional ground connections necessary to minimize 60 Hz pickup.01 V/div. Then the recording electrodes are touched to the nerve. Adjust the preamplifier and oscilloscope vertical gain to give a total system gain of approximately 100 uV/div. Time base at 10 msec/div.202
Electronic instrumentation Mount a pair of silver wire recording electrodes on a micromanipulator or flexible rod and connect their leads to the preamplifier input. 1969. Set the tone control to midrange and adjust the gain until a slight hum is heard. if necessary. The longitudinal cut should be lateral to the dermal plica (a gold or brown longitudinal ridge of glandular accumulations on each side of the trunk). Check with the preamplifier calibrator if available. Oscilloscope. gain of 100-1.
retract the electrode and cut the nerve as close to the skin as possible. it may be difficult to see during stimulation or to move from one nerve to the next. Consider the significance of receptive field overlap 6. Connect the ground electrode to one of the front limbs. not just one. Ground your free hand to the cage or clip lead. and look like white threads. Keep the nerves moist with amphibian perfusion fluid or mineral oil saturated with perfusion fluid. Take measurements with a small plastic ruler.Neurobiology Exercises
Pin the animal to a frog board. It will produce artifacts. Hook the free corners of the skin flap so that the thread can be used to elevate the flap (fig. Slightly elevate the skin along the longitudinal edge of the skin flap and carefully cut through the connective tissue attachments along the dermal plica. Keep the following questions in mind as you proceed. Drying and stretching the nerves are the major pitfalls to avoid. If you have an awkward arrangement.) Trace the receptive fields of
. Periodically moisten the outside of the skin with perfusion fluid. 3.3)? Consult other students and compare your results with theirs. Sketch an outline of the frog and its pattern of pigmentation.
Elevate the edge of the flap with the hook and thread to expose enough of the most accessible nerve for recording. What similarities and differences are there? If time permits you can conduct a second recording session with another frog. 5 .
the individual nerves onto your sketch. 5 . You should be able to discern the borders of the receptive field of the touch fibers by careful stimulation. Do not lift the skin further. Elevate the skin a bit more and go on to the next nerve. Less data will be collected. you should appreciate that these are actually composite receptive fields of several touch fibers firing simultaneously. The nerves pass from the body wall at the midline (along the spine) out to the dorsal skin.
Do not stretch any of the nerves. and recording from different nerves you can map the receptive fields of a number of nerves. you may introduce 60 Hz interference. [A 1:1 representation is often helpful. Plasticine attach the free part of the threads to a horizontal bar above the frog. A precise map can be produced if enough care is taken with the sketch and the stimulation. Does each nerve innervate a unique patch of skin? Can impulses be evoked by stimuli other than touching or scratching? What is the characteristic shape of cutaneous receptive fields on the frog’s back? Where does the nerve contact the skin? Are any areas completely devoid of innervation? Is there bilateral symmetry between the right and left sides? Do any of the receptive fields cross the dorsal midline or are they restricted to one side? Can this pattern be related to the patterns seen in scratch reflexes [experiment 7. Dissect carefully.2 ) With . Therefore. Gently rub the skin until action potentials are elicited by the stimulation. 2. In this way a complete map of the dorsal skin innervation can be produced. Each nerve is actually composed of many axons. Stimulate the external surface of the skin by touching it near the site where the nerve enters the skin. The use of colored pencils or different symbols will minimize confusion from overlapping receptive fields. Adjust the preamplifier to get a signal. Record from the nerve by positioning the paired electrodes vertically against the nerve.
Pitfalls and Suggestions
1. Correct wiring for localization is established in de-
Mapping receptive fields By positioning the skin at different angles. After each nerve‘s receptive field has been mapped. When you stimulate with a glass probe. Attach a bent pin or small fishhook to two 30 cm lengths of thread. Also note on your sketch the position of each nerve’s exit point from the dorsal musculature and its entry point on the skin. or perhaps with a frog or toad of a different species. Do not stimulate with a metal probe. Now slowly pull the threads and lift the flap while watching for cutaneous nerves.6 . Pay close attention to the geometry and neatness of your setup. 4. Wiping reflexes indicate that adult frogs can accurately localize a point of irritation on the skin.
Physiol. Jr. Rose. 1939. 0.204
velopment either by cutaneous axons innervating the correct patch of skin or by chemical influence from the patch of skin which dictate appropriate central connections for its axons (lacobson and Baker. E. E. ed. Inc. In The peripheral nervous system. M. Hogg. B 1975 Somatosensory receptors and their CNS connections.. Palo Alto. 1974. Lynn.
Comroe. 387-429. pain. V B. 133: 588-602. Biol 22: 476-94 Cattell. Physiol. J. The frog dorsal column nucleus. and tickling. L. J. vol. Touch. Excitation and changes in adaptation by stretch of mechanoreceptors. Berlin. short shank t o minimize weight] Solutions: Amphibian perfusion fluid (SO ml/station) Mineral oil saturated with perfusion fluid (50 ml/ station. Lowenstein. 137: 121-37. J. and Baker.] Keep the frogs in a cool place and provide them with dripping water
Iggo. Catton. W.
may need some suggestions on layout. H. Some properties of frog skin mechanoreceptors. B. I Physiol. I. An electrophysiological investigation of cutaneous sensory nerves. D . J H. Cutaneous receptors..
Materials needed at each station Oscilloscope Preamplifier Audio amplifier and loudspeaker Electrical leads and connectors ( 1 set] Micromanipulator Pair of recording electrodes [see page 336) Ground electrode [madefrom a burnished insect pin with a lead soldered t o it) Frog board and pins 2 glass tools for stimulating the skin Set of dissecting tools (furnished by students) Ringstand with horizontal bar Materials available in the laboratory Oscilloscope camera and film Dissecting microscope Extra cables and connectors Extra ringstands Medicine droppers and beakers Toothpicks for stimulating Fine thread Insect pins Tackiwax or Plasticine (modeling clay] Fishhooks (no. I Physiol. ed J I Hubbard Plenum Press. M 1970. 1974. Silvey.R. vol II Springer.. 14 or 16. Touch and kinesthesis. Development of reflexes from skin grafts m Rana pipiens: Influences of size and position of the grafts. and Hoagland. Calif. R 1956. it may damage the skin. J Physiol. Slow impulses from the cutaneous nerves of the frog. E. W. 1931 Sensory discharges in single cutaneous nerve fibers. R. RESEARCH REPORTS Adrian. and Davidoff. 1. 1931. 1969). R. Neurol. E. McK .. ed J Field American Physiol. The alternatives and mechanisms are still debated. G. McK. R. Gulley. Rana pipiens or other species (Order 1 frog/station/laboratory period. 141: 305-22. 2 The experiment can be used as a takeoff point for experiments on reflex properties and cutaneous temperature receptors.
Notes for the Instructor
1. J. pp.. and Jacobson. T. 73: 421-37 Zotterman. and Hoagland.. lacobson. The students
. 84: 250-58. D C. 3 Do not use TMS t o anesthetize the frogs. pp 347-404. In Annual review of physiology. E. H. 105-27. 1959.5 ml perfusion fluid in 100 ml mineral oil] Animals Frogs or toads.A. Neurophysiology. Cattell. New York and London. Annual Reviews. Response o f tactile receptors to intermittent stimulation. Brain Res.. 72: 392-404. Soc. Physiol. Y. In Handbook of physiology.. 72: 377-91 Baker. 1973 Somatosensory system Handbook of sensory physiology. M. 1935. 95: 1-28.. Dev.. 1958. The experiment requires careful attention t o the geometry of the setup in recording. pp. 1969 Development of neuronal connections with skin grafts in frogs: Behavioral and electrophysiological studies J Comp. A. Washington. and Mountcastle.
primarily from muscle spindles. there are no interneurons (fig. sensory axons from the muscle spindles synapse directly with motor neurons. The extensor muscles of the legs are stretched on landing. 7. in turn. Copyright © 1978 by the University of Michigan. or knee-jerk reflex) is unusual because it is monosynaptic.4-1)..However. which occur in all skeletal muscles. No part of this material may be used or reproduced in any manner whatsoever without written permission of the publisher. input from stretch receptors also passes to adjacent spinal segments and ascends to the brain. 7. excites spinal motor neurons which produce muscle contraction.. the sequence of events which occurs when a person jumps t o the floor from a low stool. All rights reserved.Neurobiology Exercises
7. Although not shown here. about one-third are A-gamma axons innervating the intrafusal muscle fibers of the muscle spindles (Fora more complete discussion of stretch reflex functions and muscle spindle morphology consult a physiology or neuroscience text. results in excitation of the stretch receptor The receptor.
. for example. A two-neuron stretch reflex. Stretch o f the muscle. Consider.) The stretch reflex [also known as the myotatic.e. 7. Reprinted here by permission of the University of Michigan Press. play an especially important role in antigravity reflexes and aid in maintaining muscle tone (constant slight contraction). EXPERIMENTAL NEUROBIOLOGY: A LABORATORY MANUAL. induced experimentally by striking the tendon.
From Bruce Oakley and Rollie Schafer. The motor neurons trigger contraction (fig. The importance of the stretch receptors is underscored by the fact that approximately 40% of the axons in nerves innervating skeletal muscles are sensory fibers. specialized stretch receptors arranged in parallel with the muscle fibers. The muscle spindle discharge is conveyed to the central nervous system via fast-conducting A-alpha fibers which enter the spinal cord through the dorsal roots and synapse in the anterior horn with motor neurons of the same extensor muscle.4 Stretch Reflex Myographic Recording from the Human Leg
Stretch receptors. i.
Fig. Of the motor fibers in such nerves. This kind of pathway is involved in the ankle-jerk and knee-jerk reflexes. Branches of the sensory axons also spread to adjacent spinal seg-
Rationale Experimentation with the vertebrate stretch reflex is an excellent introduction to the subject of motor control. this direct pathway is only part of the story. This also lengthens the muscle spindles.e. thereby completing the reflex arc and opposing the force of landing. i.4-1). tendon.4-1.
This reflex (Babinski‘ssign] reappears in an adult who has a damaged or diseased corticospinal system and indicates an abnormal decrease in cerebral inhibition of spinal reflex activity. and deep tendon reflexes (such as the patellar and Achilles tendon reflexes which will be studied in this exercise) Probably the most intriguing aspect of human reflexes is the progression of changes which takes place during normal development. chiefly the latter. In a very real sense the developing human passes through a quadrupedal stage (with its attendant reflexes) on the way to becoming a bipedal animal. sucking. Motor neuron activity at any given moment in time depends on the summation of all excitatory and inhibitory inputs. In turn. Fiorentino. Curtis. become modified or inhibited. 1972) in order to appreciate better the extent and roles of reflex activity in human posture. motor neurons receive facilitatory and inhibitory inputs from many sources. including those of the muscle spindles.g. The tibial newe (a branch of the sciatic) and spinal segments L5 and S i . are Involved in the Achilles tendon reflex. The Babinski sign may also be seen during the early stages of recovery from spinal damage. We suggest that interested students examine an illustrated manual of reflex testing (e. The femoral newe and spinal segments L2-L4 are involved in the patellar tendon reflex.
Stretch reflexes can be elicited experimentally by striking the tendon of a muscle or by electrically stimulating its nerve. locomotion. Clinicians distinguish three types of reflexes: superficial reflexes (such as the corneal reflex). Stretch reflexes may be accentuated.
Time to complete the exercise: One 3-hour laboratory period. 1972. We will use both methods of stimulation and record the electrical response o f the muscle. Stretch reflexes are elicited by striking the patellar tendon or the Achilles tendon. but specific diagnoses can be made only after applying a number of tests. to increase the accuracy of latency measurements. disease and injury may cause the reappearance of reflexes of an earlier stage of development For example. and development. stroking the outer margin of a newborn infant’s foot will elicit a spreading of the toes and extension of the big toe. reduced. rather than the mechanical response.
. and disappear. A number of newborn reflexes are present at birth: grasping. New reflexes also appear. rooting. visceral reflexes (such as bladder emptying). etc. The newborn reflexes subside as development proceeds and the brain’s inhibitory influence increases. stretch reflexes are employed in clinical neurology. 1973) or a discussion of clinical analysis (e.g. Jacobson..4
ments and to the brain..4-2. The rectus femoris and vastus lateralis are part of the quadriceps muscle. or abolished by injury or disease. and Marcus. In adulthood. Additional time or a second period may be necessary t o complete the last two optional exercises.206
7. 7. Extensors and flexors of the human knee and ankle joints. In combination with other tests.
Readjust the sweep speed to obtain the best display. Tap the Achilles tendon to elicit the stretch reflex while recording the electromyogram.4-3. 7. otherwise connect one of the recording leads to the ground terminal on the oscilloscope. The downward movement of the foot is plantar flexion.Increase the vertical gain to about 1. The Achilles tendon is located above the heel and connects the gastrocnemius muscle to the tarsal hone of the foot (fig. The electrical activity of the muscle is displayed on the oscilloscope. alternating plantar flexion and dorsiflexion.4-2). rearrange the recording and ground leads to minimize it. The electromyogram is the summated electrical activity (action potentials) of the thousands of muscle fibers which make up the gastrocnemius. Does the reflex time change? Why or why not? Measure the distance between the muscle belly (widest part of the muscle) and the presumed site of the sensory-motor synapse in the spinal cord. Connect one cable from the switch to the battery and another from the switch to the horizontal trigger input of the oscilloscope. Seat the subject on the edge of a sturdy table so that the thighs are well supported and the legs are hanging freely Tap the Achilles tendon with the hammer. Arrangement for recording from the gastrocnemius muscle.
BATTERY BOX PERCUSSION HAMMER WITH INERTIA ACTIVATED MICROSWITCH
Ask the subject to move the foot up and down. The opposite. Place a ground electrode on the upper part of the ankle of the same leg.4
STRETCH R E F L E X
Electromyographic recording and reflex time Obtain a rubber percussion hammer. Connect another cable from the output of the battery box to the ground terminal. hut remember that even though the stretch reflex is labeled a monosynaptic reflex. Use a light coating of electrically conducting electrode paste between the skin and the electrodes.A few trials should produce a consistent reflexive contraction of the gastrocnemius. upward motion is dorsiflexion.
7. Measure the reflex time by determining the period between the beginning of the sweep (when the hammer struck the tendon) and the onset of electrical activity in the muscle Measure the reflex time in 10 trials.Neurobiology Exercises
7. Assume that synaptic transmission takes about 0. The response is an electromyogram or EMG. Striking the Achilles tendon with the percussion hammer elicits the stretch reflex and starts the oscilloscope sweep. How would you design an animal experiment to measure the reflex time more precisely!
Fig. Use differential input if available. Use shielded cables to connect the recording electrodes to the input of your oscilloscope. Connect the ground electrode to the ground terminal on the oscilloscope or to the shield of the recording electrode leads [fig. Set the horizontal sweep to automatic with a sweep speed of 0 5 sec/div Switch to AC coupling on the vertical amplifier and set the hand-pass filter. Which muscles are involved in plantar flexion and dorsiflexion of the ankle joint? Place two surface electrodes 8-10 cm apart over the belly of the gastrocnemius muscle (actually the gastrocnemius-soleus muscle complex).
. 7. If 60 Hz pickup is a problem. using differing amounts of force. I t is important to use differential amplificationand shielded cables. until a response is seen every time the subject contracts the gastrocnemius muscle. the pathway measured with this technique includes the neuromuscular synapse as well. to -3dB at 10 Hz [lower cutoff) and 3 kHz [upper cutoff). Calculate the mean conduction rate in the nerves making up the reflex pathway. if present.4-3). Use a percussion hammer with an inertia-activated switch. Set the sweep speed to 10 msecidiv. Switch the trigger mode switch to external triggering.5 msec. using the same tapping force Is the reflex time constant? What is the average reflex time? Perform additional tests. Adjust the triggering level control to trigger the oscilloscope sweep only when the hammer is lightly tapped against a solid object.0 mV/div or more.
Place a pair of electrodes about 10 cm apart on the quadriceps muscles on the front of the thigh (fig 7. Obtain a hall electrode with an insulated handle. Set the sweep speed to 10 msec/div and switch the time base to external triggering.4-4. Feel the position of the patellar tendon just beneath the kneecap.4
Patellar tendon (knee-jerk) reflex Seat the subject on a sturdy table with the thighs well supported and the legs swinging freely. which will produce a reflexive contraction of the gastrocnemius muscle.
Fig. Tape a plate electrode just above the front of the subject's knee. The tibial nerve passes through the knee beneath this point.208
Stimulating arrangement. We will stimulate the tibial nerve through the skin at the knee. Set the stimulator to give monophasic square wave stimuli at 1 pulseisec and 0. Observe the electromyogram. Reduce the stimulator voltage to its lowest possible value. What synaptic inputs influence spinal motor neurons other than the excitatory input of the stretch receptors? Electrical stimulation of the Achilles tendon reflex [at the instructor's option] The stretch reflex can also he elicited by direct electrical stimulation of afferent sensory fibers. Is the reflex facilitated by this method? Does isometric contraction elsewhere facilitate the reflex? Speculate on the mechanism of enhancement.6 msec duration. Connect this electrode to the positive output of the stimulator with an appropriate lead. using a thin film of electrode paste between the skin and the electrode.
The subject should control both the stimulus voltage and the site of stimulation. Connect a ground lead between the oscilloscope and stimulator. Place one hand on the patella and use the other hand with the hammer to gently strike the tendon to elicit the stretch reflex. in front of his chest and pull outward at the moment the hammer strikes the patellar tendon [Jendrassik's maneuver]. Connect a triggering lead from the trigger output on the electronic stimulator to the trigger input of the oscilloscope time base. 7. Turn the stimulator output off. Response of the gastrocnemiusmuscle to direct electrical stimulation of the tibial nerve. Transfer the ground electrode to the knee. Approximately 30 msec elapsed hetween the stimulus and the onset of the reflexive response.4-2). The peak in the upper trace is a stimulus marker which indicates the application of a 0. Actuate the stimulator and adjust the triggering level control so that a single sweep is
triggered for each stimulus pulse. Adjust the vertical gain until a response is seen as before. The lower trace shows the stimulus artifact ( I ). In humans this is often referred to as the H-reflex.4-3. one against the other. Measure the reflex time in 10 trials and compare this average with the average obtained for the Achilles tendon reflex What are the possible sources of difference? Study the effects of varying the amount of tension in the muscle by voluntary contraction on the part of the subject Can the reflex be inhibited or enhanced in this manner? Ask the subject to lock the fingers of his two hands. Place recording electrodes on the gastrocnemius muscle and a ground electrode on the foot as in figure 7. the response of the muscle to direct stimulation of the motor neurons in the tibial nerve (2). and the reflexive response of the muscle elicited hy stimulating the sensory fibers of the tibial nerve (3). Recording orrongement. Connect this stimulating electrode to the negative output of the stimulator. Stimulate at 1 pulseisec while gradually increasing the stimulus voltage. The subject should grasp the insulated part of the stimulating electrode and apply the ball to the hollow at the hack of the knee in the midline [the popliteal fossa].
.5 msec stimulus to the tibial nerve. and coat the hall with a thin film of electrode paste.
You should he able to feel and see the contraction of the anterior tibialis muscle beneath the skin. and the tibialis anterior contracts as the subject leans backward. Recording arrangement. 7. Use the differentia! mode and AC coupling. Readjust the gain and sweep speed to obtain the best display. This will he the reflexive response initiated by stimulation of the sensory fibers of the tibial nerve. whereas contraction of the tibialis anterior results in dorsiflexion A dual-trace oscilloscope or a polygraph will he necessary to record the electrical activity of both muscles simultaneously.Neurobiology Exercises
7. The subject should expect to feel discomfort as the stimulating current passes through the skin. located just lateral to the tibia in the upper part of the lower leg. This indicates that the motor neurons which have reflexively discharged are in a period of inexcitahility. Look for a third wave with a latency of 25-35 msec (fig. Such inexcitability is caused by pre. Reciprocal action of antagonistic muscles (at the instructor’s option) Electromyography will he used to observe the action of two antagonistic muscles of the ankle joint-the gastrocnemius and the tibialis anterior.4
Probe in different areas with the stimulating electrode while gradually increasing the voltage to 30-40 V and watching the oscilloscope screen for a reflexive response. 7. In some subjects the amplitude of the reflexive response will he larger than the response to direct stimulation. Can you think of any other reasons for motor neuron unresponsiveness? Hint: What happens to muscle spindle discharge during muscle contraction? Attempt to change the amplitude of the reflexive response by (1)contracting the gastrocnemius slightly and (2)contracting the antagonists of the gastrocnemius to produce dorsiflexion of the foot. Are the magnitudes of the direct and reflexive responses linearly related to the stimulus voltage? Why might the reflexive response disappear at stimulus voltages strong enough to stimulate the motor axons? Stimulate with increasing frequency up to 5 pulsesisec.1-10 kHz to limit noise and AC pickup. Place your fingers 2 cm lateral to the margin of the tibia about 8-12 cm below the kneecap. A second pair of recording electrodes must he placed over the anterior tibialis muscle.and postsynaptic factors. Does antagonist activity increase or decrease the reflexive response? What was the effect of synergistic activity? Illustrate your conclusions by drawing a nerve-muscle wiring diagram summarizing the probable factors at work in this system. Connect these electrodes to one of the vertical amplifier inputs of one channel of a dual-trace oscilloscope using shielded cables.
. Responses of the gastrocnemiusmuscle (upper t r a c e ) and the anterior tibialis muscle (lower trace) in a subject standing erect and rocking back and forth.4-4). Note the reciprocal firing of the two antagonisticmuscles.4-5. The upper trace is unfiltered. Place a pair of recording electrodes over the belly of the anterior tibialis muscle and connect them to the sec-
Fig. as in figure 7. A second wave will follow very closely or he combined with the stimulus artifact. This is the response of the muscle to direct stimulation of the motor fibers in the tibial nerve and is not a reflexive response. including recurrent inhibition of the motor neurons. The first wave on the oscilloscope screen will be the stimulus artifact. Contraction of the gastrocnemius produces plantar flexion of the
foot. Feel the subject’s tibia (shinbone)at the front of the lower leg.4-3. Ask the subject to point the toes inward (supinating or inverting the foot] and dorsiflect the foot. Place two recording electrodes over the belly of the gastrocnemius muscle and a ground electrode just above the ankle joint. Does the response diminish? At frequencies above 5 pulses/sec the response may decline. The gastrocnemiuscontracts as the subject leans forward. while the lower trace is limited to a band-pass of 0.
ond channel of the dual-trace oscilloscope using shielded cables. What is the effect of loading (increased resistance] on the electromyogram!
Observation of reciprocal action. Joint stabilization is particularly important in leg and postural muscles which contribute to the bipedal locomotion in the human.. Ask the subject to alternate plantar flexion of the foot with dorsiflexion. Notice that although one muscle may predominate. Adjust the gain of both vertical amplifiers to about 1 mV/div. Ask the subject t o stand on one foot. 7. An imbalance forward activates the spindles of the gastrocnemius and soleus muscles. Stretch reflexes help to maintain balance [fig. 7. Use differential mode and AC coupling. Adjust the gain to get the best display of the electromyogram of each muscle. lower -3dB cutoff at 10 Hz and the upper -3dB cutoff at 3 kHz).
antagonists as observed here is necessary to stabilize joints against twisting and slipping. These antagonistic muscles are involved in a number of different movements and involve more than one joint. The subject should stand erect. If frequency filters are available on the vertical amplifiers. check the ground connection to the ankle.4-5). alternating contraction of the gastrocnemius and anterior tibialis muscles should be apparent (fig. and the muscles contract and balance is restored. If 60 Hz AC pickup is a problem. Set the sweep speed of the time base to 1 sec/div and the triggering mode t o automatic. Balance maintenance and the stretch reflex. limit the band-pass filter to lower the noise level (e. the other muscle may also be somewhat active.
Fig. rearrange the leads. placing more weight on the leg with the recording electrodes than on the other leg. Explore their functions using electromyography. Their activation is relayed to the motor neurons. Co-contraction of
RETURN TO MEANPOSTURE
. when the subject stretches upward or squats? What other locomotory activities require the coordination of antagonists? How are such activities related to the stretch reflex studied in the first three sections of this exercise? Multiple-joint and multiple-action muscles (at the instructor's option) It is easy to record from the biceps and triceps muscles of the upper arm. The joints are mechanically stabilized when motionless. for example. What happens. When the subject rocks backward and forward.Can you detect muscle activity in a relaxed standing posture? Within the limits of cable length and the introduction of movement artifacts you can explore the action of the ankle flexors. and make sure that electrode paste was used with every electrode.
If the subject touches the ball electrode with the hand. but AC line current is very dangerous. Set of connectors and cables (Shielded coaxial cables are mandatory to suppress 60 Hz AC pickup..4
Pitfalls and Suggestions
1. 2. Since high paper speeds are necessary. Arrange the switch to be activated by the inertia of the solder when the hammer is struck against a solid object. [Make this up by adding a spring-loaded microswitch. Each AC-powered unit should be provided with a properly grounded three-prong power cord.g. Ground the leg only. Solder a single conductor insulated lead to the tab attached to the phone plug tip. series 31 1SM with roller lever. a motion picture of human reflexes is a useful adjunct. Electrode paste left on the skin may cause itching. Prior study of the stretch reflex and reciprocal innervation in a textbook of physiology or neuroscience will make the exercise more rewarding. We suggest that a polygraph not be used. The stimulating electrodes will sting. 4. E4). much wastage will occur. 5. The intermittent square-wave output of the electronic stimulator is relatively innocuous. 321 or Grass no. Bony and fatty areas should be avoided because they present more resistance and increase discomfort. a current will pass between the hand and the other stimulating electrode on the leg.) Preamplifier (optional if the oscilloscope vertical amplifier sensitivity goes to 1. Remove all unessential AC-powered equipment and metal objects. A pointed cathode will produce a high current density and attendant irritation. In the second posi-
Materials needed at each station Oscilloscope or polygraph (A dual-trace oscilloscope or polygraph is needed to perform the section on the reciprocal action of antagonistic muscles. The position suggested for the Achilles tendon reflex (legshanging freely) is not ideal. If a battery is used without enclosing it in a minibox.Neurobiology Exercises
7. wash all electrode paste off the legs and electrodes. check all electrical equipment. Do not use equipment with any hint of malfunction. to the upper part of the handle. 3. Discomfort can be minimized by using enough electrode paste and a smooth ball-shaped cathode. Safety and subject mount concern of the instructor. a circuit which includes the torso and the heart.
Materials available in the laboratory Tape measure Oscilloscope camera and film Electrode paste Masking tape [used to attach surface electrodes. e. See fig. Subjects should not ground themselves by touching a grounded cage or wire with their hands. especially the cathode. Take care to stimulate only the leg.)
. solder the leads securely to the battery terminals. In this exercise an electronic stimulator is used to stimulate a human subject. Current density is inversely proportional to electrode area. After the experiments are completed. but more at the cathode.2-8E for an illustration of a phone plug. Before the laboratory period. The battery is necessary to provide a triggering voltage which is actuated by the percussion hammer switch.) Ball-tipped stimulating electrode with insulated handle. Tape the switch onto the handle after attaching a twoconductor cable to the switch. Do not use metal tables. (This is most easily made up from a phone plug. 2. and (2)the subject reclines on his side on a padded table with one leg straight and the other leg bent at the knee and lying across the straight leg.5 V flashlight cell in a minibox and adding binding posts and/or banana jacks. and round the tip to a ball with a file.0 mv/div) 5 surface electrodes with attaching straps [plate electrodes. There are two alternative positions: ( 1) the subject kneels on a padded table with the knee joint at a 90" angle and the feet hanging over the edge of the table. Weight the arm on the switch with a blob of solder. do not use adhesive tape which is painful to pull off ) Extra cables and connectors 1 spare percussion hammer with inertia-activated switch
Notes for the Instructor
should be the para1. and every precaution should be taken to prevent exposure to it. 2. such as Harvard no.) Battery box (Make this up by placing a 1. Long-term stimulation will lead to hyperemia at both electrodes.) Percussion hammer Percussion hammer with inertia-activated switch. Microswitch Co. as directed. 3. Time permitting.
..showing reflex patterns in children.. Charles C Thomas.] Ochs. Willis. 6. J. W. M 1972. V. John Wiley and Sons. A. D. and McDonald.212
7. Philadelphia. development.4
tion the tendon of the crossed leg is struck. 1965. Lange Med Pub. Saunders.. Louis. Ill. Correlative neuroanatomy. (WellIllustrated. Jacobson. W. 1972.N. Jr. Springfield. The EMG is the asynchronous discharge of potentials from multiple motor units. not single muscle cells. Springfield.. S. showing normal newborn reflexes and aberrant patterns of development. An introduction to the neurosciences. Ill. Curtis. St. Your students may occasionally observe muscle action potentials (MAPS) between EMG responses. (Well-illustrated. G. G. These are active. Normal and abnormal development: The influence of primitive reflexes on motor development. Mosby. and Marcus. 1973. New York. M. B. Calif. B.
Chusid. C. 15th ed.S. Los Altos. single motor units.) 1973 Reflex testing methods forevaluating C.. R. Medical neurobiology: Neuroanatomical and neurophysiological principles basic to clinical neuroscience. J.. S. Fiorentino. Both positions require a padded surface and more room than may be available in many labs. R. Charles C Thomas. 1977. Elements ojneurophysiology. and Grossman.
carefully separate the cockroach from its grip on your hand without breaking the wax bond. The power-to-weight ratio in insects is enhanced by the air tubes or trachea which go directly to the tissues and thus eliminate the need for a complex and heavy circulatory system. flies. air-filled tubes which support the membranous wings.
Obtain an adult male cockroach [see experiment 8.2 Insect Flight Sensory and Central Mechanisms
Rationale The ability to sustain flight has evolved in three livinggroups of animals: insects. The exoskeleton provides a strong and light structural
material. The evolutionary success of flying animals comes largely from exploiting the aerial environment. Hold the insect still while the wax solidifies. Insects are particularly favored in the evolution of flight. Reprinted here by permission of the University of Michigan Press. Flight is the most complex form of locomotion and requires an advanced neuromuscular system for coordination. 8. We will use cockroaches because they are large. the subject of this exercise.
. immature cockroaches (nymphs or larvae) do not have wings.2-1). and the skeletal veins. Attach a wooden applicator stick to the pronotum [the shieldlike area behind the head) using melted wax. and in some species the adults are wingless. Suspend the
From Bruce Oakley and Rollie Schafer. Time to complete the exercise: One 3-hour laboratory period. and hats. No part of this material may be used or reproduced in any manner whatsoever without written permission of the publisher. are highly specialized with dense arrays of contractile proteins and numerous mitochondria. evolved later during the Mesozoic period. In fact. bees.6 for sexing cockroaches). Copyright © 1978 by the University of Michigan. represent a group of relatively unspecialized fliers. and easily obtained and will maintain a regular and sustained flight. The specialized fliers. birds. whereas in cockroaches the flight muscles are not too different from leg muscles (fig. streamlining of the body is unimportant to most flying insects since they are light and fly at low speed. Finally.Neurobiology Exercises
8. Cockroaches have changed little since they evolved during the Carboniferous period. hardy. Their small size leads to a large ratio of surface area to mass. After the wax hardens. or only the male adults have wings. for example. Birds in general have had to evolve streamlined shapes. and moths. hut insects have been free to develop other morphological adaptations without the overriding necessity to streamline the body. All rights reserved. EXPERIMENTAL NEUROBIOLOGY: A LABORATORY MANUAL. Cockroaches. before the appearance of flowering plants. would he only slightly stronger even if they were made of a light metal like titanium. Bee flight muscles.
Now. The upper surface of the wing is white and the lower surface black. B. Wing movements in insect flight. This is an illusion! They are rapidly moving and are dangerous! Determine the frequency of wing beat of the forewings by finding the fastest flashing rate which produces the appearance of completely stopped motion. 8. f time with no flight. discard the animal and get another. using care to avoid pulling the insect o applicator stick. When flying is obtained. Do they beat together or out of phase? If out of phase. give it from 30 seconds t o 1 minute before doing anything more. Similarly prepare three or four more males.and hindwings by adjusting the stroboscope to make it appear that they are flapping very slowly. you will not be able t o measure the frequency unless you drive the strobe with a stimulator.
CAUTION: The fan blades may appear to be still or only slowly moving.)
.) Observe the movements of the fore.) ing? Do you suppose the rate of beating would change if the wings were lightened by clipping them shorter? Do not test this now. Turn the fan on and adjust the autotransformer to subject the insect to a low velocity breeze (1-2 misec 2-4 mph. turn the room lights off and the stroboscope on. but do not turn it on yet.2
mounted cockroach in air by attaching the applicator stick to a horizontal rod fixed to a ringstand. how far out of How steady is the rate of beatphase? [See fig.and bindwings exactly out of phase to neutralize torque. or dizzy during the course of the experiment. leave the vicinity of the flashing light. At this point the insect may begin to fly. The butterfly beats its fore. These are sketches of wing movements taken from high-speed cine photographs. Give the cockroach a small styrofoam ball to grip with the feet while it hangs in the air. The dragonflybeats the fore. Normal flight Suspend a cockroach in front of a small fan which is plugged into an autotransformer.2-1. Does the angle of incidence (angle of attack) of the wings change during their reciprocating motion?
Fig. attempt to After a period o trigger flying with a slight increase in wind velocity and by touching or lightly pinching the tip of the abdomen. Adjust the stroboscope frequency In the range of 20-40 flashesisec until the wing motion appears nearly stopped. The fan will act as a simple wind tunnel. Let it grip one end of a glass rod. which you then withdraw. If not. If you feel “odd.The progression of wing movements reads from right to left. [Adapted from Nachtigall. A. remove the Styrofoam ball from the cockroach’s f f the grip. If this does not initiate flight. Use a swivel clamp for mounting so that the flying attitude may be varied. The wing tips of the forewings are marked with block dots. C. if you can estimate it).” ill at ease.2-2.
The flashing of a stroboscope will trigger epileptic seizures in some people. The locust beats its fore and hindwings slightly out of phase. Adjust a stroboscopic lamp to shine on the cockroach. (If the strobe light is not calibrated.214
8. 8.and hindwings exactly in phase. D. Beetle.
Amputation of the antennae of
As evident from the questions just asked.
Aerodynamics The questions in “Normal flight” dealt primarily with the mechanical aspects of flying. If you have read discussions of animal aerodynamics. action of a single wing. Flight is induced in a number of insects by the removal of support. these observations also apply to cockroaches. The wing tips are marked by black dots. give the Styrofoam ball back to the cockroach to terminate the flight and rest the insect. you may be able to ask more informed questions This preparation is strongly encouraged if more than one laboratory period is available. and pitch that you induce by turning the insect [fig. sensory input from tactile hairs can be eliminated by covering them with grease.Neurobiology Exercises
8. or Nachtigall ( 1974). STARTING WHEN THE HINDWINGS ARE MIDWAY UP (MSEC)
.. change the angle of incidence t o achieve lift on the upstroke as well as the downstroke.and hindwings.g. Standard wing stroke of the locust Schistocerco gregaria. 8. 8. e.2
Some insects. You may observe the effects of support and lack thereof by repeatedly removing the Styrofoam ball from the grasp of the cockroach. Receptors on the head and antennae may also play a role in flight. Three main areas of investigation are available with the tools you have at hand. Take a few minutes to read over the following and plan a set of experiments. Receptors on the legs thus contribute to the initiation of flight. Pennycuick (1972). Is this sufficient to sustain flight in the absence of wind! How may seconds is flight sustained without wind! What is the effect of repeated trials? What is the influence of changing the time interval between trials? How many legs must be touching a surface to inhibit flight? You may consider amputating legs or parts of legs to eliminate sensory input. Sensory mechanisms Insects apparently depend on the simultaneous activation of several kinds of sensory receptors to initiate and maintain flight. For example. 1956b.2-3)? After observing. and may have suggested experiments to do. amputation experiments often work well in insects and other arthropods. Turn the strobe light off. Despite this. but remember that the acute trauma may confound the results of your experiments. legs. or extra-fast drying modeling cement. position of the body. The influence of the antennae on flight is more difficult t o test. The dotted lines on the forewings are thin filaments glued there to indicate the twisting of the wing plane. Alternatively.and hindwings. silicone stopcock grease. movements of the fore. It ought to be possible
0. and antennae. such as those hy Pringle ( 1957]. The typical wing positions shown above the curves were drawn from photographs. stimulation of groups of tactile hairs on the head of the locust will result in sustained flight if support is simultaneously removed.
to analyze the flight response into subcomponents. Are hairs visible which might serve a similar purpose? The receptors on the bead can be selectively stimulated by a fine jet of air from a Pasteur pipette connected via rubber tubing to an air line. such as the bee. (Adapted from Weis-Fogh. or the effects of changes in the body position and direction of the wind stream. Is this true of the cockroach? Can you detect changes in posture and wing attitude when the insect corrects for the roll. The most complete analyses of the sensory mechanisms of insect flight have come from the locust and bee. With modifications.5
TIME.2-2. Examine the head of a cockroach with a magnifying glass or dissecting microscope. The two sine waves represent the up-anddown strokes of the fore. there are many aspects t o winged flight.
The supraesophageal ganglion can be destroyed by inserting a hot needle into the head capsule between the compound eyes and antennae. one or more hours may be required for a stable recovery state. yaw. but it illustrates the degree of autonomy possible in individual ganglia You may want to ask whether the three thoracic ganglia of the cockroach contain a complete set of neural circuits for flying. Do not insert the scissors too far or you will also cut the gut. In the locust. lightly cauterize the wound with a hot needle. You may also consider amputation of the wings and observation of other components of the flight posture. insert the opened tips of a fine pair of scissors into the opening and snip. including the sensory input from the cerci. Does a similar mechanism operate in the cockroach? You may want to use the fine jet of air to explore this problem. hence it is probably a relatively poor experimental approach On the other hand. You should prepare several insects since some ablations may be faulty. Tests of the question of autonomy will have to rely on ablation (surgical destruction) of the two major areas of the cockroach brain: the supra. The objective will he to remove any influence they might have on the thoracic ganglia.and subesophageal ganglia may be totally removed simply by beheading the insect. This can be done by cutting a small. since many of the proprioceptors of the antennae are located in the first three basal segments. Use a blunt needle (about 1 m m diameter) which is nearly red hot. Plan your experiments accordingly. Since the nerve cord is adjacent to the sternite.216
Fig.1-2. you will cut through it. Cutting the nerve cord at this site will eliminate all afferent input from the abdomen. it would be possible to inactivate them by immobilizing the bases of the antennae with cement. but it is an insufficient stimulus t o initiate flight. and pitch in a flyinglocust (Adapted from Weis-Fogh. a male praying mantis which has been beheaded will continue circular walking movements for days. Finally. Flashing strobe lamps may trigger epileptiform sei-
. transverse slit in the ventral abdomen at the first or second segment (sternite)behind the thorax. however.1).and the subesophageal ganglia (see fig. 7.
Pitfalls and Suggestions
1. For example. eliminate tactile input from the antennae because tactile hairs are found on every segment Tactile receptors and proprioceptors are also found on the wings. If excessive bleeding takes place. Less recovery time will be needed after beheading. Use of larvae (nymphs)without wings may be useful to observe postural responses in cockroaches without wings. This will not. The cerci can be stimulated with an air jet or they can be amputated. This particular example is extreme and related to the sexual behavior of the mantis. Turn the strobe lamp off when it is not in use. 1956b )
cockroaches usually leads to a general depression of activity.2-3 Roll. Although preliminary observations are possible. page 205). the stimulation of air moving across the wings is sufficient to maintain flight once it has begun. you may want t o sever the nerve cord between the abdomen and thorax in a few insects. The abdominal ganglia also influence the activity of the thoracic ganglia Therefore.
Central mechanisms Complete reflexive pathways for walking are often present in the thoracic ganglia of insects like cockroaches. the possible influence of the caudal cerci located at the tip of the abdomen can be evaluated They are an important mechanoreceptive input (see experiment 7. The supra. After making the slit. 8.
g.. Testors extra-fast drying cement for wood models available from hobby shops)
Notes for the Instructor
1. e.] Autotransformer [AC rheostat for controlling fan speed. Also caution them to avoid the fan blades. PS22 or PS33. for stimulating the anal cerci Magnifying glass Dissecting microscope and illuminator Animals Periplaneta americana are available commercially from Carolina Biological Supply.
Fine camel hair paintbrushes or bristles from a larger paintbrush.Neurobiology Exercises
zures in some people.g. 2 Caution the students not to look directly in the light. 3. which may appear slowly moving or stationary in the flashing light. Cenco Universal Red Wax] Extra right-angle clamps. Grass Instrument Co.New
. about 2-3 cm in diameter Bunsen burner or soldering iron for melting wax Glass rod with fire-polished ends Pithing needle and 1 pair of fine scissors [furnished by students] Materials available in the laboratory Applicator sticks Wax (e. ringstands. Trapping may be advantageous because wild cockroaches are often more vigorous fliers than laboratoryreared cockroaches. S. Insects in flight. S. The bond between the cockroach and the stick is usually broken by students who hastily try to pull the insect's grip loose by pulling on the stick. (Order 7-8 cockroaches/station. wild cockroaches can be trapped. such as Leucophaea maderae. Place the roaches in a plastic wastebasket or a similar container with the upper walls smeared with petroleum jelly (Vaseline] to prevent escape. in order of preference. Pull gently.) Stroboscopic lamp [A photic stimulator can be used. and longterm exposure will make most persons develop a headache. A sponge wetted with water every few days will supply sufficient water. White Crown Sticky Wax. as noted previously. may also be used as long as the adults have wings. Do not look directly into the lamp at any time.g. Avoid fatiguing the insect as this will affect your results.g. Provide the roaches with vertically standing pieces of cardboard to climb upon. Do not use the strobe light in the presence of an epileptic. The wax is more likely to hold the animal if the wax is curved around the lateral edges of the tergum. Feed lab chow or dry dog food. low amperage capacity is sufficient.. Some units require periodic resting.
Materials needed at each station 2 ringstands 5 short rods. swivel clamps.) 5 Styrofoam halls. Flying grasshoppers or beetles can be used if they are available.. plastic-blade fan is safest. 3. approximately 7 m m (114 inch) inside diameter Silicone stopcock grease [a viscous grease. about 0. such as Dow Corning silicone stopcock grease] or fastdrying modeling cement (e. 2. Terminate flight by replacing the Styrofoam hall whenever you are not making observations.
Nachtigall. W 1974. freeing one leg at a time.. Be ready to make observations and take data when the insect begins flying. A glimpse behind the scenes in biophysicalresearch. Supplement with apples or oranges periodically if the roaches are kept for a long period. and rods Extra Styrofoam balls Beakers or other containers for melting wax Pasteur pipettes or glass tubing pulled out to 1-2 m m tips Rubber tubing. Check the instruction manual for the strobe lamp to determine if there is any suggested limitation on continuous operation. Surgident Periphery Wax.32 x 20 cm (114 x 8 inches] 5 right-angle clamps Swivel clamp Fan [A small. McGraw-Hill. The most difficulty will he encountered in fixing the insects to applicator sticks with wax. Rotron Muffin Fan available from electronics suppliers.] Other species. In some parts of the country. e.
B. The Institute of Biology's Studies in Biology.. J M. 1974. Biology and physics of locust flight. S . 1956c.A m . Kutsch. 1957. M. 1968a. 1956a The flight of locusts. 1965. G.The influence of proprioceptive feedback on locust flight co-ordination. M. Wells. Changes in thc flight motor pattem during the development of the Australian plague locust. Comparative physiology of the flight motor
Adv. D. 1956. Biology and physics of locust flight. 38: 471-90. R C. Notes on sensory mechanisms in locust flight. I. T. 218: R3-90. J. 1956b. 239: 459-510. 33. Trans B. Basic principles in insect flight. 1976 Insect flight Symp. Jensen. Weis-Fogh. Comp. E. So. Comp Physiol.Biology and physics of locust Right.. W. A Critical review. Sensory mechanisms in locomotion: The campaniform sensilla of the insect wing and their function during flight. I. S.218
8. Physiol. M. J. 239: 553-84 Weis-Fogh. D. 1974. T. Wendlcr. J. The flight control system of the Iocust. 1956. and Weis-Fogh. Quant. studied in flying locusts. Chortoicetes terminifera. Trans. The aerodynamics of locust flight.
. Sci A m 194 116-24. J. RR: 413-24. Royal Entomol. Patterned activity of co-ordinated motor units. Soc 7 Halsted (Wiley). T. Insect flight. Weis-Fogh. 239: 415-58. T. II. J. Animal flight. M. 30: 615-22.2
Pennycuick. 5: 289-338 RESEARCH REPORTS Altman. The nervous control of insect flight and related behavior Adv Insect Phyciol. C 1972. 1962. Philos. Edward Arnold Pub Ltd. 39. Cold Spring Harbor Symp.
Wilson. Biology and physics of Iocusr flight. Phil. D. Trans. Sensory control of abdomen posture in flying locusts. 643-67. Cambridge University Press. Trans B. Wilson. Ill. Cambridge 1968. no. IV. Phyciol. 239: 51 1-52. 97: 127-42. 1970. Biol. Comp. 1961. Wilson. 1968b. Phil. The influence of the wing sense organs on the flight motor pattern in maturing adult locusrs.. Flight performance of the desert locust. W. Exp. M. Lower animals McGraw-Hill. Biol. Insect Phyciol 5: 163-227. Exp Biol 52: 533-38 Gettrup. Phil. London Pringle. New York. New York. 1. Camhi. J. Exp. 1974. The central nervous control of flight in a locust. B.
Rainey. and Jensen. Biol. M 1968.
is appropriately named: Mimosa is from the Greek for mimic. This term distinguishes such movements from tropic responses which proceed in a direction determined by the direction of the stimulus. such as brushing the hand against a leaf.g.Neurobiology Exercises
8. Leguminosae. EXPERIMENTAL NEUROBIOLOGY: A LABORATORYMANUAL. Photoreactivity.7 Sensitive Plant Nastic Responses and Conducted Action Potentials in Mimosa pudica
the leaflets. leaves.7-2). is a common thread running through all phylogenetic levels.7-1). water. Reprinted here by permission of the University of Michigan Press. All rights reserved. and petioles are a class of effector cells whose turgor pressure holds these structures unfurled and erect (fig. and a drop in turgor pressure. a rapid outflow of water.8. 8. Venus’ flytrap) or protective (e. Mimosa pudica. gravity.. Excitation is conducted to the pulvinar effector cells. This ability is associated with carnivorous (e. and stems (fig. protozoa. A given pathway is comprised of a series of cells arranged end to end with low resistance pathways between them. The conductile pathways carry propagated action potentials.
. they have been termed nastic responses.7-1). The plant hides its leaves when disturbed. and pudica from Latin for modest or bashful. is sufficient to excite conductile cells in the leaves. leaves. The pulvini contain osmotically active effector cells which cause folding of the leaflet pairs and drooping of the petiole. A disturbance. Mimosa) adaptations. and petioles. This nastic response is aided by another class of cells arranged as antagonists. triggering in succession a massive efflux of potassium. plants. No part of this material may be used or reproduced in any manner whatsoever without written permission of the publisher. and other forces and objects in the environment.
Rationale Bacteria. The leaflets fold and the petioles droop. Many members of this family are equipped with organs called pulvini at the bases of
From Bruce Oakley and Rollie Schafer. Folding of leaflets in response to mechanical disturbance is termed seismonasty. 8. Mimosa pudica is a member of the pea family. Copyright © 1978 by the University of Michigan. petioles. The rapid nastic response of Mimosa pudica is mediated through interconnected pathways of excitable cells in the leaves. and animals often share similar adaptive mechanisms.g. and are thus analogous to nerve fibers in animals although conduction in plant cells is generally much slower. only a few plants are capable of rapid movement when stimulated. Sensitivity to mechanical stimuli is also widespread. Because the movements of Mimosa pudica leaves and stems are independent of the direction of the mechanical stimulus. While all plants are mechanically sensitive in that their growth patterns are deeply influenced by physical contacts with wind. In the pulvini at the base of leaflets. which folds its leaflets in the dark or when disturbed (fig.. for example. which gain potassium and water to increase turgor pressure.
. leaflets. Examine figure 8. and stem before proceeding further. Therefore. Some patience will be required since even in fully recovered and responsive leaflets there may be a
Fig. Can you make only those leaflets in the vicinity of the touch fold? Note that a sufficient stimulus at the tip of the leaf will initiate a wave of leaflet folding toward the base of the leaf.7-1.220
8. but will not elicit drooping of the petiole. Second. each time the plant is stimulated it will take 10-15 minutes t o recover. pinch the leaf near its tip. petiole. The nastic response o f Mimoso pudlca. Can waves of leaflet folding be readily elicited by piching? In spite of the difficulty in giving a consistent pinch with hand-held forceps. Pinch no harder than necessary to produce a response. and leaves. First. depending on the manner of stimulation and other factors. mechanical stimulation will be used to examine the nature of the response and its determining factors. Does outward [centrifugal)propagation of leaflet folding occur? Does propagation occur in both directions when a leaflet between the leaf tip and base is pinched? Does centrifugal propagation into the leaves occur when the petiole is pinched? Finally. Pinching stimuli. Does sensitivity vary according to the extent or direction of the brushing movement? Does recovery take more or less time than when the leaf was pinched!
Mimosn pudica is literally a very sensitive plant.7-1 t o learn the meaning of the terms leaf. Try to elicit the same kinds of responses by brushing a fine glass rod or Pasteur pipette along the axis of one of the leaves. Brushing stimuli. Use a pair of forceps with rubberguarded jaws to stimulate by pinching. Watch for a few minutes. First. A touch to the leaf results in foldingof the paired leaflets and drooping of the petiole.7
We will explore two aspects of the rapid nastic response. 8. Time t o complete the exercise: One 3-hour laboratory period A second session or a longer laboratory period would be useful since the plant requires 10-15 minutes to recover between stimulations. Pinch a leaflet near the base of a leaf. can you get some idea of the force required t o get a conducted wave of leaf folding! How much consistency of response is there from leaf to leaf? Notice that often a wave of leaflet folding will proceed to the base of the leaf. you will record conducted potentials from the petioles. stems. What is the result of eliciting a wave of leaflet folding in a second leaf on the same petiole after the leaflets of the first leaf have folded? We can describe the result of this experiment as a form of facilitation. Exploring Mimosa‘s behavior Gently touch one of the leaflets at a leaf tip with a thin glass rod or a Pasteur pipette. Pinching is a somewhat unnatural stimulus.
delay of several seconds between stimulus and response. pinch one of the petioles or stems near the base of the plant. for each test that follows stimulate fresh portions of the leaf or new leaves. Moreover. The effectororgans (pulvini)at the bases of leaflets and petioles are not visible. Be careful throughout the experiment not to accidentally bump theplant or jar its table.
Turn on your preamplifier and recheck everything before stimulating. they stay bent. When the leaflets unfold. Use heavy surgical scissors or good quality shears to cut two 20 cm lengths of fine stainless steel wire. Check with the preamplifier calibrator if available. you have been observing rapid responses. Since they are much slower than nerve action potentials.7
Damaging stimuli. Adjust the preamplifier and oscilloscope or polygraph gain to give a total system gain of approximately 5 mV/div or 5 mV/cm. Recording propagated potentials Allow 10-15 minutes for the plant to recover. Attach each wire to one of the inputs on the preamplifier.7-3). or leaf.
Conducted electrical potentials can he measured by inserting fine wire electrodes into a petiole. 4.Neurobiology Exercises
8. Watch carefully. Do this very carefully to prevent jostling and inducing leaflet folding through mechanical stimulation. Carefully insert the tip of this wire through the stem. stopwatches for measuring. band-pass filters (if available] set at about 0. Cut through the wire at a 45-degree angle to produce a sharp point. Connect the output of the preamplifier to the input of an oscilloscope or a physiological polygraph [fig. Recording setup To keep the leads short. (There are both slow and rapid nastic responses.Use the same technique to insert the second recording electrode into the petiole about midway between the base of the leaf and the base of the petiole. Use a pair of clean. Turn on your equipment and use the following initial settings. Meanwhile. you may begin.1 Hz [low frequency filter) and 1 kHz (high frequency filter) Oscilloscope or Polygraph. 10. and the pulvinar responses of leaflet and petiole bases as time marks. and 30 minutes in the dark?
the Mimosa plant at the same height as the stems from which you intend to record. 3 . Choose a healthy looking and accessible pair of leaves on the Mimosa plant. Does damage always elicit a wave of leaflet folding? In what respects are the movements after damage different from those after brushing or pinching stimuli? Finally. Avoid bending the wires.) What happens to the plant after 5. several centimeters below the branch point of the petiole from which you intend to record (fig. sharp surgical scissors to cut 2-3 m m off the tip of a distal leaflet. Is conduction velocity constant along a leaf? Compare centripetal [inward) with centrifugal [outward)conduction velocities. Time base or paper speed at 1 sec/div or 1 cm/sec. ask yourself if you can clearly distinguish in this experiment between hormonal or electrical propagation of the responses along the leaf and petiole. stem. make sure all adjustments and connections are complete and calibrate the system gain. sensitivity at 50 mV/div or SO mV/cm. 9. Measure the conduction velocity in leaves and petioles. gain of 10x. Calibration. Does the plant become more or less responsive after a fan hasgently blown on it for 20-30 minutes? Use brushing stimuli to test. allowing ample time to observe the effects of the damage. Insert both electrodes at angles which do not put too much mechanical stress on the stem or the petiole. Grip one of the electrode wires between the jaws of a smooth-surfaced pair of forceps about 3-5 m m from the cut end of the wire. Use cutting as a stimulus.20. Wire electrodes in the stems will also pick up the electrical spikes produced by rapid ionic fluxes in the pulvini and by mechanical movement of a stem when it droops. Further behavioral experiments Here are some suggestions for other experiments using simple observational techniques. Input in differential mode if possible. trigger set on automatic or free run. Preamplifier. mount the preamplifier near
. 8.7-3). Examine the effect of continuous wind on responsiveness. Examine the effects of various levels of illumination on nastic responses. these potentials can be recorded equally well on a polygraph or oscilloscope. once they bend. Push a metal rod into the soil and connect the rod to a grounding point on the preamplifier input. 2 Examine the facilitation of the drooping response of the petiole when two or four leaves which branch from the same point are stimulated in succession.
The conducted potentials which course through
the stemlike tissue probably travel in the phloem and the protoxylem (differentiating xylem) Thc cells in these regions have resting membrane potentials of about .g. whereas cells in other regions (e. parenchymal have resting potentials around -50 mV The leaf-
lets fold upward upon stimulation because turgor pressure rapidly increases in the abaxial epithelial cells of the pulvinules while i t decreases in the adaxial cells This is accomplished by the active movement of potassium ions (followed by the osmotic flow of water) from the adaxial cells to the abaxial cells (Scanningelectron micrographs courtesy of P..)
. Dayanandan.7-2 Cross sections of a petiole ( A and B 42x) and leaf axis (C and D 60x)of the sensitlve plant Mimosa pudica.222
SENSITIVE P L A N T
NONGLANDULAR HAIRS UPRAISED LEAFLETS
CROSS SECTION OF PETIOLE
D CROSS SECTION OF LEAF AXIS
(RACHILLA)AND TWO PULVINULES
Are the potentials all-or-none. The propagated potential would appear as a fully monophasic wave in D C recording. like the compound action potential of a nerve. or graded! The potentials recorded with your gross electrodes are. you can record the passage of potentials in the leaf and in the petiole. Examine the effects of electrical stimulation using an electronic stimulator and another set of wire stimulating electrodes. 8.7-3. shift the recording electrode in the stem to the middle of a leaf. Stimulate by snipping distal leaflets as before. Record the time interval between the peaks of the rapid upward and downward deflections of the trace produced as the conducted potential passes the first and second electrodes. What would this tell you about the nature of the conducted potential!
Fig. This arrangement is particularly useful for studying the time courses of facilitory effects and recovery. Move the two recording electrodes to a new part of the plant.7-4. Characterize the magnitude. this time placing both electrodes in a petiole--one near the base of the petiole and one at the distal end of the petiole. After obtaining responses and noting their general form using the previous recording arrangement.
Fig. You may also pick up a spikelike potential as the pulvinus at the base of the petiole reacts (fig. You will have to allow time for recovery after each response.In addition. For example.7-3. Measure conduction velocity in the petiole electrically. Potentials rrcorded with the arrangement shown in figure 8. Examine the effects of temperature and illumination on conduction. 4. the summed potentials of many cells 2. Remember. This is a propagated potential in the petiole passing the first recording electrode. and time relationships of the recorded potential changes. you might attempt to block the conducted potentials by using DC current to hyperpolarize a region between the site of stimulus and the site of recording. The waveforms of both potential changes have been distorted by A C recording.7-4). 8.
. Measure the distance between the two electrodes and compute the conduction velocity.Neurobiology Exercises
Suggested experiments with electrical conduction 1. waveform. you may see a third potential change as a propagated potential passes the second recording electrode in the stem.Stainless steel wires are inserted through the stem and petiole for recording the passage of conducted potentials
For initial observations use the cutting method of stimulation by carefully snipping 2-3 m m off the tip of one of the distal leaflets Observe both the activity of the plant and the recording trace. This problem requires some thought to separate the effects of light and heat. At A. Recordingarrangement. Now. while the other watches the recording trace. mm/msec m/sec 3 . thc conducted potential passes the recording electrode in the petiole The rapid potential change at B is associated with the response of the pulvinus at the base of the petiole. describing its responses aloud. You should see a large potential change 1 second or more after the wave of leaflet folding reaches the base of the leaf. One person should watch the plant. 8.
If you do not wait long enough you will get only partial responses. LB 600). At this point.
Materials needed at each station Oscilloscope or polygraph (A polygraph I S preferable since it produces a continuous.] Occasionally. Tungsten wire of about 40 gauge is good.] Metal rod and clip lead for ground connection to the soil Fine glass rods or Pasteur pipettes Forceps with rubber-protected laws or fine rubber tubing slipped over the laws Surgical scissors. Place the lamp 30-50 cm above the plants and supply them with 16 hours of light per day. Carolina Biological Supply. Be careful around other experimenters. M i m o s a p u d i c a plants may be found in plant specialty stores or obtained from a hobbyist.. more experiments can be run in a given time period. permanent record. Keep the seedlings out of direct sunlight until they have reached a height of about 5 cm.] Cables and connectors ( 1set] Electrode wire (The wire must be stiff enough to penetrate the M i m o s a stems. Use this time to think about the design of yourexpenments. Young plants will produce two leaves per petiole (fig. Leaflets should be at least 75% unfolded before retest. and place the pot in a covered container until the seeds sprout ( 1 to 2 weeks at room temperature). fine and of good quality Flat-jawed forceps for gripping electrode wire Small metric ruler Materials available in the laboratory Oscilloscope camera and film Electronic stimulators Small fan and rheostat to control its speed Stopwatches
Notes for the Instructor
1. Several sets of electrodes can be connected to the same preamplifier through an EKG lead selector or similar switching device. Seeds may be obtained locally on occasion (ask for "sensitive plants"] but it is usually easier to order them directly from a supplier (e. Be sure to remove the insulation present on some wires or the electrodes will not work. 2. as is stainless steel kymograph pen cleaning wire.224
Pitfalls and Suggestions
1. Ask students to treat the plants with reverence. Harvard Ink Cleaning Wire. sprinkle with water.7-1) and older plants will produce four. 2. one can plug the electrode wires directly into the input of an oscilloscope or polygraph. months in advance. 808. much time will be wasted with accidental stimulations and false starts. A drooped petiole may require 10-30 minutes recovery time. Consult a local plant supplier or botanist if problems are encountered. if more plants are available. Otherwise they will have to be started from seed at least 2. no. You should meet no special difficulties other than waiting for a response to occur and waiting for the plant to recover between stimulations. is the preamplifier on?). 3. Place several seeds atop damp potting soil in a small flowerpot.) Desk lamps for strong illumination Cardboard boxes for light shades Ringstands Plants M i m o s a pudica (Have 2-10 plants available/station.) Preamplifier (Alternatively. e.
Dissecting microscope Extra electrode wire Heavy surgical scissors or good quality shears for cutting electrode wire (Most wire-cutting pliers will not cut the wire without bending it.g.
. Obviously.Be sure to check your recording setup carefully before stimulating the plant (e. 8. Lightly nick the seeds with sandpaper before planting. Disturbing a plant wastes time and could ruin an experiment.g.. and preferably 4. The advantages of using a preamplifier are that the electrode wires may be kept quite short.. Silver and copper wire are much less suitable because they are too limp. Cover the seeds with a very thin layer of soil. If they approach the exercise too casually. no. watering.g. Keep the soil moist with frequent. and the preamplifier band-pass filters may be used. The use of a 40-watt fluorescent grow lamp on a timer is strongly suggested. place them in strong light. minimizing 60 Hz interference. but not excessive.
In Handbuch der Pflanzenphysiologie [Encyclopedia of plant physiology]. N. Photocontrol o f Mimosa pudica leaf movement. B 1967. vol.In Handbuch der Pflanzenphysiologie [Encyclopedia of plant physiology). Springer-Verlag. Science 137: 226. Ruhland. W. A.. Rev.
Bose. Green. L. 17(1). ed. W Ruhland. N e w Phvtol. Plant Physiol. Annu. Migration o f calcium and its role in the regulation of seismonasty in the motor cell of Mimosa pudica L. Mogliche Mechanismen von Krummungsbewegungen. R.. J. C. Schneider. Springer-Verlag. and Galston. and Hendncks. 1971 Potassium flux: A common feature of Albizzia leaflet movement controlled by phytochrome or endogenous rhythm Science 174: 518-20.Berlin. 1972. Excitable cells in Mimosa. SpringerVerlag. S . Planta 75: 228-38. Toriyama. Die seismonastschen Reaktionen In Handbuch der Pflanzenphysiologie [Encyclopedia of plant physiology). 1952. Berlin. W. 1959. 50: 357-82. ed. 24: 25-46 Sibaoka. l959a. pp 111-18 RESEARCH REPORTS Fondeville. Physiology of rapid movements in higher plants Annu. Ruhland. 17 (1). T. and Co. E. London Bunning. Rev Plant Physiol 2 0 165-84. H. Longmans. H A .. Satter. M. M. Borthwick. 1973. and Jaffe. Electropotentials o f plant cells. J. ed. 1962. vol. pp 184-238 Higinbotham.
3. Umrath. pp 24-110. 17 (1). Leaf movements in Mimosa pudica L.. W. 1906 Plant response as a means ofphysiological investigation.. K. Sibaoka. M J. Der Erregungvorgang. T 1969. Plant Physiol 49: 72-81 Weintrauh.Berlin.vol. 1959b. J. A sensitive force transducer may be used to record the mechanical activity of the plant [petioledrooping/ while simultaneously recording the electrical activity. C.