4 excercises in neurobiology.pdf

7/29/2019 4 excercises in neurobiology.pdf

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Chapter 10Four Exercises in Neurobiology

Rollie SchaferDepartment of Biological SciencesNorth Texas State UniversityDenton, Texas 76203

Rollie Schafer received his doctorate at the University a t Coloradoin 1969, and has held teaching/research positions at New MexicoInstitute of Mining and Technology, the University of Michigan,and North Texas State University. He is presently Associate Pro-fessor of Biological Sciences and Physiology a t North Texas andalso serves as Assistant Graduate Dean for Research. W hile he wasa faculty member at the University of Michigan in Ann Arbor, heoccupied a laboratory and office next door to t hat of another neu-robiologist, Dr. B ruce Oakley. Oakley and Schafer taug ht th e lab-oratory course in neurobiology at Michigan and combined forcesto produce the laboratory manual, Experimental Neurobiology, ofwhich a portion is reprinted here. At North Texas, Dr. Schaferteaches neurobiology n the undergraduate, graduate, and medica1schools, and conducts research in the chemistry of the sense ofsmell.

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198 Neurobiology ExercisesThe four exercises which follow are reprinted from the laboratory textExperimental Neurobiology by Bruce Oakley and Rollie Schafer (Universityof Michigan Press, Ann Arbor, 1978, 367 pp.). Th e four exercises have beenchosen to exemplify several of the different areas covered in the manual: anelectrophysiological experiment on sensory reception (6.5); a quantitativestudy 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 experiencein neurobiology with empha sis in electrophysiological techniques. I t m ay a lsobe used effectively as a source book for sequenced independent projects. Thelevel of sophistication (or difficulty) of the experiments is graded so t h a t aninstructor can offer the laboratory for students of different levels and back-grounds. The selec tion of experiments (33 in all) is diverse enough to permitchoosing either a c ellular, organismic, or behavioral approach to complementthe instructors own lecture treatment. Introductory chapters cover basic ter-minology, practical use of electrophysiological equipment, and mam malianbrain anatomy and neuroanatomical terminology. The section on neuroanat-omy, Neuroanatomy: Dissection of the Sheep Brain, has been reprinted sep-arately by the University of Michigan Press for use in basic anatomy andphysiology courses.Each experiment in the man ual was selected with three criteria in m ind.An experiment ought to; (1) work in student hands; (2) utilize no highlyspecialized electrophysiological equipmen t; an d (3) have a balance of technicaland conceptual content. Some of the exercises are simple enough to be doneby beginning biology students, and the rest can be carried out by advancedundergraduates with little assistance from the instructional staff. Ma ny of theexercises are best done in two laboratory periods: a first period devoted tosetting up and learning procedures, followed by a second period devoted toproducing meaningful data . An estimate of the amount of time required islisted within each experiment.Laboratories-especially those which use more than very simple tech-niques-are a notorious time sink. Th e m anu al is designed to conserve pre-paratory and instructional time in several ways: (1) The instructions in allexercises are detailed an d complete. Minim al instructional assistance is re-quired to get students to the data-taking stage. (2) Potential problems arenoted in the Pitfalls and Suggestions and N otes for the Ins tructor sectionsof each experiment. (3 ) Each experiment has a detailed list of materials whichcan be used as a checklist and basis for purchases. (4) The information inchapter 10, Laboratory Organization, will aid the instructor to initiate anew 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 participa ntsat the second AB LE conference.


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Neurobiology Exercises 199

Contents

Introduction xiCHAPTER 1 . Electrical Principles and Electro-physiological Terminology 1

I . I Practical Electrophysiology 2I. 2 Review of Electrical Terms andPrinciples 3

CHAPTER 2. Survey of ElectrophysiologicalInstrumentation 92 . 1 Th e Electrophysiological Setup 102.2 Cables, Connectors, and Adapters 122. 3 Cathode Ray Oscilloscope 162. 4 60 Hz Interference 242. 5 Electronic Stimulator 252. 6 Preamplifier 292 .7 Audio Amplifier and Loudspeaker 342.8 Electrodes and Nerve Cha mbers 362. 9 Oscilloscope Cam era 402 . O Physiological Polygraph 42

CHAPTER 3. Neuroanatomy 453 I Cellular Neuroanatom y and Histological

Techniques 463 .2 Dissection of the Sheep Brain 49

CHAPTER 4. Cellular Potentials and Bio-electric Activity 754.1 Active Transport of Sodium 76

Frog Skin4. 2 Compound Action Potential 85

Frog Sciatic Nerve

4. 3 Compound Action Potential and SingleUnits 97Limulus Leg Nerve

4.4 Giant Nerve Fibers 104Earthworm Nerve Cord

4.5 The Membrane Potential 11 2lntracellular Recording from Frog Sartor iusMuscle Fibers

4.6 Volume Conduction 121The Potential Field Generated by a NerveImpulse

CHAPTER 5. Synaptic Transmission andEffector Processes 1295 .I Coelenterate Nerve Net 130

Luminescent Waves in the Hydroid5.2 Neuromuscular Synapse 138

Frog Sciatic-Gastrocnemius JunctionCardiac Muscle and Its NeuralRegulation 147Turtle HeartSmooth Muscle Regulation of BloodFlow 155Microcirculation in Amph ibians, Fish. andInsects

5 . 3

5.4

5. 5 Chromatophores 16 0Neurohumoral Control in Arthropods

CHAPTER 6 . Receptor P rocesses 1656 . 1 Visual Reception 266

Frog Electroretinogram (ERG)


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200 Neurobiology ExercisesCONTENTS

6 . 2 Mechanoreception 17 4Tactile Receptors on the Insect Leg

6. 3 Olfactory Reception 18 0Frog Electro-olfactogram (EOG)

6 . 4 Proprioception 187Frog Muscle Spindle

6. 5 Skin Receptive Fields 192Frog Cutaneo us ReceptorsGerbil Cochlear Microphonics

6 .6 Audition 196

CHAPTER 7.Central Processes 203 7 .1 Cockroach Ventral Nerve Cord 204

Spontaneous an d Elicited Responses7 . 2 The Rat Motor Cortex 212

Somatotopic Representation7 . 3 Spinal Reflexes 218

Effects of Surgical Abla tion of HigherNervous Centers in the Frog

7 . 4 Stretch Reflex 224Myographic Recording from the H uma n LegSomatosensory Representation in th e RatMedulla 232Electrophysiological Activity in SpinalTrigeminal, Cuneate, and Gracile Nuclei

7 . 5

7 . 6 Frog Optic Tect um 239Retinal Processing of Visual Form

7 . 7 Neural Regulation of Weight 2477 . 8 Chronic Stimulation of the Rat Brain 251

Lesions in the Rat Hypotha lamusSelf-stimulation of the Septal Nuclei a ndLateral Hypothalamic Areas

7 . 9 Chronic Recording from the Rat Brain 25 6Hippocampal Theta Rhythm

CHAPTER 8. Behavior 2618 .1 Animal Locomotion 26 2

Descriptive Analysis of Simple BehaviorSensory and Central Mechanisms

8. 2 Insect Flight 266

8 .3 Electrolocation and Electrocommunica-tion 272Electrical Signals and Behavior of WeaklyElectric Fish

8.4 Feeding Behavior 2818.5 Conditioned Taste Aversion in the Rat 287

One-Trial Learning o f LiCl Avoidance8 . 6 Sexual Behavior 291

Taste Reception in Flies

Responses of Cockroaches to Pheromones8 .7 Sensitive Plant 30 0

Nastic Responses and Conducted ActionPotentials in Mimosa pudica

CHAPTER 9. Surgery and Stereotaxic Tech-niques 3079 .1 Surgical Instrume nts and Micromanipula-9 . 2 Pithing the Frog 31 19. 3 Small Mammal Surgery 3139 . 4 Stereotaxic Procedures 318

tors 308

CHAPTER 10. Laboratory Organization 32510 .1 Setting Up the Laboratory 32610 .2 Electronic and Other Major Equipment 32 81 0 . 3 Electrode and Equipment Construction 33 410.4 Suppliers 3411 0 . 5 Basic Laboratory Supplies 3471 0 . 6 Animals 3491 0 . 7 Perfusion Solutions, Drugs, and Neuroactive

Agents 352Index 35 9


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6.5 Skin Receptive FieldsFrog Cuta neou s Receptors

IntroductionRationaleThe im mediate cause of a behavioral response isusually a n environmental stimulus which is de-tected by exteroceptors. Stimuli from the viscera,muscles, and joints are detected by subsurface en-teroceptors. In this exercise we will examine t he re-sponse properties and innervation patterns of tactilereceptors in frog skin.

Receptors which terminate in amphibian skin arecapable of responding t o touch, warmt h, cold, in-tense stimulation, s uch as a pinch, and chemical ir -ritants. Man y receptors are specialized to respond to

Line illustration is adapted from Jacobson and Baker, 1969

only one of these forms of stimula tion, includ ingreceptors which respond only to intense (nocicep-tive) stimulation. As is evident from commo n ex-perience, animals are often quite adept at localizinga cutaneous stimulus. An irritating insect may hequickly removed by a swipe of a leg. Exercise 7 3demonstrates that much of the neuronal circuitryresponsible for cutaneou s localization resides withinthe spinal cord. The receptors role in c utaneouslocalization can he studied by recording from thoseaxons which innervate the skin.

At first the problem seems elementary. The mostobvious arrangement would be a wiring diagramwith each point on the skin connected by its ownunique axon to that location in a central mot orswitchboard which, whe n sufficiently activated,would initiate th e correct wiping response In thejargon of neurobiolo gy, this woul d entail a soma to-topic representation with direct reflex connections.This may be in part correct, but it is also an over-simplification which neglects important c om-plexities. For examp le: [ I ) Axons have overlappingreceptive fields (skin regions innervated by morethan one axon]. ( 2 )The behavioral responsethreshold is lowered by stimulation elsewhere onthe body. (3) Would an animal stimulated simul-taneously at tw o widely separated points wipe half-way between? (4 ) n development how do the out-growing axons find their correct terminatio ns! As isfrequently the case, what seems patently simple isactually quite sophisticated. We will not at tempt toexplore these issues in this experiment, but willmake a beginning by examini ng the manner inwhich touch fibers respond and innervate the frogskin.Time to complete the exercise: On e 3-hour labora-tory period.

From Bruce Oakley and R ollie Schafer, EXPERIMENTAL NEUROBIOLOGY: A LABORA-T OR Y M A NUA L . Copyright 97 8 by the University of M ichigan. All rights reserved. No partof this material may be used or reproduced in any manner whatsoever without written permissionof the publisher. Reprinted here by permission of the University of Michigan Press.


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202 Neurobiology Exercises

ProcedureElectronic instrumentationMount a pair of silver wire recording electrodes on amicromanipulatoror flexible rod and connect theirleads to the preamplifier input. Connect the lead of athird [ground! electrode to the ground terminal onthe preamplifier inputConnect the preamplifier output to th e verticalamplifier input of the oscilloscope. Connec t anaudio amplifier and loudspeaker to the accessoryoutput on th e hack of the oscillosc ope, or if no ac-cessory output is available, split the preamplifieroutput so that it drives both th e audio amplifier andthe oscilloscope. Use th e following initial settingson your equipment.Preamplifier. Input in differe ntial mode if possible;gain of 100 -1, 00 0x; band-pass filters (if available !set at about 30-100 Hz [low frequency filter] and3 kHz [high frequency filter).Oscilloscope. Time base at 10msec/div; verticalsensitivity at 0.1-0.01 V/div; trigger set on externalinput a nd th e triggering level control on automaticor free run.

CUT (THROUGH SKIN)

Fig. 6.5-1.Incision of the dorsal skin to expose the cutane-ous nerves for recording. Cut through the skin on threesides as indica ted, hut d o not elevate the f lap until r e adyto record. (Adapted rom Jacobson and Baker, 1969.)

Calibration. Adjust the preamplifier and oscillo-scope vertical gain to give a total syst em gain ofapproximately100uV/div. Check wi th th e pream-plifier calibrator if available.Audio amplifier. Set the tone control to midrangeand adjust the gain unt il a slight hum is heard. Oncerecording starts, readjust the gain so that action po-tentials are clearly audible but not offensive to thosearound you.

Leave all AC-powered equipment on while otherarrangement s are completed. Make any additionalground connections necessary to minim ize 60 Hzpickup. Remove any unnec essary AC-poweredequipment from the vicinity.Operative procedureDouble pith a large frog (see page 3 1I) .With scissorscut through the skin as shown in figure 6.5-1 to pro-duce the outline of a flap in the dorsal skin. D o no tpull the flop up yet; simply free the skin along thecut edges. The flap should be a large one, extendingfrom the level of th e cloaca almost to the tympanicmembranes. The longitudinal cu t should be lateralto t he derma l plica (a gold or brown longitudinalridge of glandular ac cumu lati ons on each side of th etrunk).

THREAD AND HOOELEVATE FLAP

NERVE SLIGHTLYSTRETCHED BETWEENFLAPOF SKIN AND BODY WALL

Fig. 6.5-2. Recording arrangement .The dorsal flapof skinis elevated enough to expose a cutaneous nerve. Then therecording electrodes are touched to the nerve. The nerveshouldbestretched only enough to accommodate elec-trode placement. After the first nerve is examined, it issevered and the skin flap elevated, if necessary, to allowrecording from a second nerve, and so on.


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Pin the animal to a frog board. Slightly elevate theskin along the longitudinal edge of the ski n flap andcarefully cut through the connective tissue attach-ments along the dermal plica. Do not lift the skinfurther.

Attach a bent pin or small fishhook to two 30 cmlength s of thre ad. Hook the free corners of th e skinflap so that th e thread can be used to elevate the flap(fig.6 . 5 - 2 ) .With Plasticine attac h the free part of t hethreads to a horizontal bar above th e frog. Nowslowly pull the threads and lift the flap while watch-ing for cutaneous nerves. The nerves pass from thebody wall at the midline (along the spine) out to t hedorsal skin, and look like white threads.

the individual nerves on to your sketch. A precisemap can be produced if enough care is taken withthe sketch and the stimulation. Also note on yoursketch the positio n of each nerves exit point fromthe dorsal musculature and its entry point on theskin. The use of colored pencils or different symbolswill minimize confusion from overlapping receptivefields.mapped, retract the electrode and cut the nerve asclose to the skin as possible. Elevate the skin a bitmore and go on to the next nerve. In this way acomple te map of th e dorsal skin innerva tion can beproduced. Keep the following questions in mind asyou proceed. Does each nerve innervate a uniquepatch of skin ? Can impulses be evoked by stimuliother than tou ching or scratching? What is thechara cterist ic shape of cutan eous receptive fields on

After each nerves receptive field has been

Do not stretch any of the nerves.Elevate th e edge of t he flap with t he hook and

thread t o expose enough of the most accessible nervefor recording. Connect the ground electrode to one ofthe front limbs. Record from the nerve by position-ing the paired electrodes vertically against th e nerve.Adjust the preamplifier to get a signal. Stimulate th eexternal surface of t he ski n by touching it near th esite where the nerve enters the skin. Gently rub theskin until action potentials are elicited by the stimu-lation. You should be able to discern th e borders ofthe receptive field of th e touc h fibers by carefulstimulation.

Keep the nerves moist with amphibian per-fusion fluid or mineral oil saturated w ithperfusion fluid. Periodically moisten theoutside of the skin with perfusion fluid.

Mapping receptive fieldsBy positioning t he sk in at different angles, and re-cording from different nerves you can map therecep tive fields of a numbe r of nerves. Each nerve isactua lly composed of many axons, not just one.Therefore, you should appreciate that these are ac-tually co mposite rece ptive fields of several touc hfibers firing simultaneously. Sketch an ou tline of thefrog and it s pattern of pigmentation. [A 1:1 repre-sentation is often helpful. Take measurement s witha sma ll plastic ruler.) Trace the receptive fields of

the frogs back? Where does the nerve contact theskin ? Are any areas completely devoid of innerva-tion? Is there bilateral symmetry between t he rightand left sides? Do any of the receptive fields crossthe dorsal midline or are they restricted to one side?Can this pattern be related to th e patterns seen inscratch reflexes [experiment 7.3)?

Consult other stud ents and compare your resultswith theirs. What similarities and differences arethere? If time permits you can conduct a second re-cording session with another frog, or perhaps with afrog or toad of a differ ent spe cies.Pitfalls an d Suggestions1. Dissect carefully.2. Pay close attentio n to th e geometry and neatnessof your setu p. If you have an awkward a rrange ment,it may be difficult to see during stimulatio n or tomove from one nerve to th e next. Less data will becollected.3. Drying and stretching the nerves are the majorpitfalls to avoid.4. When you stim ulate with a glass probe, you mayintroduce 60 Hz interference. Ground your free handto the cage or clip lead. Do not stimula te with ametal probe. It will produce artifacts.5 . Consider th e significance of receptive field overlap6. Wiping reflexes indicate t hat a dult frogs can accu-rately localize a point of irrita tion on th e skin. Cor-rect wiring for localization is established in de-


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velopment either by cutaneous axons innervatingthe correct patch of skin or by chemi cal influencefrom the patch of skin which dic tate appropriatecentral con nections for its axon s (lacobson andBaker, 1969).The alternatives and mechanisms arestill debated. damage the skin.

may need some suggestions on layout.2 The experiment can be used a s a takeoff point forexperiments on reflex properties and cutan eoustemperature receptors.3 Do not use TMS t o anesthetize the frogs; it may

MaterialsMaterials needed at each stationOscilloscopePreamplifierAudio amplifier and loudspeakerElectrical leads and connector s (1 set]MicromanipulatorPair of recording e lectrodes [se e page 336)Ground electrode [made rom a burnished insect pinFrog board and p ins2 glass tools for stimulating t he ski nSet of dissecting tools (furnished by students)Ringstand with horizontal barMaterials available in the laboratoryOscilloscope camera a nd filmDissecting microscopeExtra cables and con nectorsExtra ringstandsMedicine droppers and beakersToothpicks for stimulatingFine threadInsect pinsTackiwax or Plasticine (modelingclay]Fishhooks (no.14 or 16, short shank to min imizeweight]Solutions:

Amphibian perfusion fluid (SO ml/station)Mineral oil saturated with perfusion fluid (50 ml/station; 0.5 ml perfusion fluid in 100ml mineraloil]

with a lead soldered to it)

AnimalsFrogs or toa ds, Rana pip i en s or other species (Order1 frog/station/laboratory period .] Keep th e frogs ina cool place and provide the m with dripping water

Notes for the Instructor

Selected ReferencesIggo,A. 1974. Cutaneous receptors. In The peripheral

nervous system, ed J I Hubbard Plenum Press, NewYork and London, pp 347-404.1973 Somatosensory system Handbook of sen-sory physiology, vol II Springer, Berlin.Lynn, B 1975 Somatosensory receptors and their CNSconnections. In Annual review of physiology, ed. J H.Comroe, Jr. Annual Reviews, Inc., Palo Alto, Calif.,pp. 105-27.Rose, J. E., and Mountcastle, V B. 1959. Touch and kines-thesis. In Handbook of physiology. I. Neurophysiology,vol. 1, ed J Field American Physiol. Soc., Washington,D C., pp. 387-429.

RESEARCH REPORTSAdrian, E. D ;Cattell, McK ;and Hoagland, H. 1931 Sen-sory discharges in single cutaneous nerve fibers.I Physiol. 72: 377-91Baker, R. E., and Jacobson, M 1970. Development of re-flexes from skin grafts m Rana pipiens: Influences ofsize and position of the grafts. Dev. Biol 22: 476-94Cattel l, McK., and Hoagland, H. 1931. Response of tactilereceptors to intermittent stimulation. . Physiol. 72:392-404.Catton, W. T. 1958. Some properties of frog skinmechanoreceptors.I Physiol. 141: 305-22.Hogg, B M. 1935. Slow impulses from the cutaneousnerves of the frog. . Physiol. 84: 250-58.lacobson, M., and Baker, R. E. 1969 Development of neu-ronal connections with skin grafts in frogs: Behavioraland electrophysiological studies J Comp. Neurol. 137:121-37.Lowenstein, W. R 1956. Excitation and changes in adapta-tion by stretch of mechanoreceptors. J Physiol. 133:588-602.Silvey, G.E.; Gulley, R. L., and Davidoff,R. A. 1974. Thefrog dorsal column nucleus. Brain R e s . 73: 421-37Zotterman, Y. 1939. Touch, pain, and tickling. An elec-trophysiological investigation of cutaneous sensorynerves. J. Physiol. 95: 1-28.

1. The experiment requires careful attention to thegeometry of th e setup in recording. The students


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7.4 Stretch ReflexMyographic Recording from t h e H u m a n Leg

IntroductionRationaleExperimentat ion with t he vertebrate stretch reflex isan excellent introducti on to the subject of motorcontrol. Consider, for example, the sequence ofevents which occurs when a person jumps to thefloor from a low stool. The extenso r muscles of t helegs are stretched on landing. This also lengthens themuscle spindles, i.e., specialized stretch receptorsarranged in parallel with th e muscle fibers. Themuscle spindle discharge is conveyed to the centralnervous syst em via fast-conducting A-alpha fiberswhich ente r the spinal cord through the dorsal rootsand synapse in the anterior horn with motor neuronsof th e same extensor muscle. The m otor neuronstrigger contraction (fig. 7.4-1), thereby completingthe reflex arc and oppo sing the force of landi ng.

Stretch receptors, which occ ur in all skeletal mus-cles, play an especially important role in antigravityreflexes and aid in maintaining muscle tone (con-stant slight contraction).Th e importance of th estretch receptors is underscored by th e fact that ap-proximatel y 40% of the axon s in nerves inne rvat ingskeletal muscl es are sensory fibers, primarily frommuscle spindles. Of t he mot or fibers in su ch nerves,about one-third are A-gamma axons innervating theintrafusal mu scle fibers of the muscle spindles (Fo ramore comple te discussion of stretch reflex functionsand muscle spindle morphology consult a physiol-ogy or neuroscience text.)The stretch reflex [also known as the myotatic,tendon, or knee-jerk reflex) is unusual because it ismonosynaptic, i.e., sensory axons from th e musclespindles synapse directly wit h motor neuron s, thereare no interneurons (fig. 7.4-1).However, this directpathway is on ly part of the story. Branches of th esensory axons also spread to adjacent spinal seg-

SENSORYNEURON

ENDNGS

Fig. 7.4-1. A two-neuron stretch reflex. This kind of path-way is involved in the ankle-jerk and knee-jerk reflexes.Stretch of the muscle, induced experimentally by strikingthe tendon, results in excitation of the stretch receptorThe receptor, in turn, excites spinal motor neurons whichproduce muscle contraction. Although not shown here,input from stretch receptors also passes to adjacent spinalsegments and ascends to the brain.

From Bruce Oakley and Rollie Schafer, EXPERIM ENTAL NEUROBIOLOGY: A LABORA-T OR Y M A NUA L . Copyright1978 by the University of Michigan .All rights reserved. N o partof this m aterial may be used or reproduced in any manner whatsoever without written permissionof the publisher. Reprinted here by permission of t he University of Michigan Press.


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STRETCH REF LEX 7.4ments and to the brain. In turn, motor neu rons re-ceive facilitatory and inhibitory inp uts from manysources. Motor neuron activity at a ny given momentin time depends on the summation of all excitatoryand inhibitory inputs, including those of the musclespindles.In combination with other tests, stretch reflexesare employed in clinical neurology. Stretch reflexesmay be accentuated, reduced, or abolished by injuryor disease, but specific diagnoses can be mad e onlyafter applying a number of tests. Clinicians distin-guish three types of reflexes:superficial reflexes(suc h as the c orneal reflex), visceral reflexes (such asbladder emptying), and deep tendon reflexes (such asthe patellar and Achilles tendon reflexes which willbe studied in th is exercise)

Probably the most intri guing aspect of hum an re-flexes is th e progression of change s which ta kesplace during normal d evelopment. A number ofnewborn reflexes are present at birth: grasping,sucking, rooting, etc. The newborn reflexes subsideas developme nt proceeds and the brains inhibitoryinfluence increases. New reflexes also appear, be-come modified or inhibited, and disappear. In a veryreal sense the developing human passes through aquadrupedal stage (with its attendant reflexes) o nthe way to becoming a bipedal animal. In adulthood,disease and injury may cause the reappearance ofreflexes of an earlier stage of development Forexample, stroking the outer margin of a newborninfants foot will elicit a spreading of the toes andexten sion of th e big toe. This reflex (Babinskis ign]reappears in an adult w ho has a damaged or diseasedcorticospinal sy stem and indicates an abno rmal de-crease in cerebral inhibiti on of spinal reflex activity .The Babinski sign may also be seen during the earlystages of reco very from spinal dama ge. We suggestthat interested students examine an illustratedmanual of reflex testin g (e.g., Fiorentino, 1972, 1973)or a discussion of clini cal analysis (e.g., Curtis,Jacobson, and Marcus, 1972) in order to appreciatebetter the extent and roles of reflex activity i nhuman posture, locomotion, and development.Time to complete the exercise: One 3-hour labora-tory period. Additional tim e or a second period maybe necessary to comple te the last two optional exer-cises.

ProcedureStretch reflexes can be elicited experimentally bystriking the tendon of a muscle or by electricallystimulating it s nerve. We will use both meth ods ofstimul ation and record the electrical response of th emuscle, rather than t he mechanic al response, to in-crease the accuracy of latency measurements.

TARSAL BONE

Fig. 7.4-2. Extensors and flexors of the human knee andankle joints. Stretch reflexes are elicited by striking thepatellar tendon or the Achilles tendon. The femoral neweand spinal segments L2-L4 are involved in the patellartendon reflex. The tibial newe ( a branchof the sciatic)and spinal segments L5 and S i, chiefly the latter, are In-volved in the Achilles endon reflex. The rectus femorisand vastus la teralis are part of the quadriceps muscle.


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7.4 STRETCH R EF LEXElectromyographic recording and reflex timeObtain a rubber percussion hamm er. Seat the sub-ject on th e edge of a sturd y table so that the thighsare well supported and the legs are hanging freelyTap the Achilles tendon with the hammer. Th eAchilles tendon is located above the heel and con-nects the g astrocnemius muscle to the tarsal hone ofthe foot (fig.7.4-2).A few trials should produce aconsistent reflexive contraction of the gastroc-nemius. The downward movement of the foot isplanta r flexion. The opposite, upward motion is dor-siflexion. Which mu scles are involved in plantarflexion and dorsiflexion of the ank le joint ?

Place t wo surface electrodes 8-10 cm apart overthe belly of the gastro cnemius muscle (actually thegastrocnemius-soleus muscle complex). Place aground electrode on the upper part of the ankl e ofth e same leg. Use a light coating of electrically co n-ducting electrode paste between the s kin and theelectrodes. Use shielde d cables to connect the re-cording electrodes to the input of your oscilloscope.Use differential input if available; otherwise con-nect one of the recording leads to the ground termi-nal on the oscilloscope. Connect the ground elec-trode to the ground terminal on the oscilloscope orto the shield of the recording electrode leads [fig .7.4-3).

BATTERY BO X PERCUSSION HAMMERWITH INERTIA ACTIVATEDMICROSWITCHI

Fig. 7.4-3. Arrangement for recording from the gastroc-nemius muscle . Striking the Achilles tendon with thepercussion hammer elicits the stret ch reflex and starts t heoscilloscope sweep. The electrica l activity of the muscle isdisplayedon the oscilloscope. t is important to use differ-ential amplificationand shielded cables.

Ask the subject to move the foot up and down,alternating plantar flexion and dorsiflexion. Set thehorizontal sweep to auto matic with a sweep speed of0 5 sec/div Switch to AC coupling on the verticalamplifier and set the hand-pass filter, if present, to-3dB at 10 Hz [lower cutoff) and 3 kHz [uppercutoff). ncrease the vertical gain to about 1.0 mV/divor more, until a response is seen every time the sub-ject contracts the gastrocnem ius muscle. The re-sponse is an electromyogram or E M G . The elec-tromyogram is the summate d electrical activity (ac-tion pote ntials) of the thou sand s of musc le fiberswhich make up the gastrocnemius. If 60 Hz pickupis a problem, rearrange the recording and groundleads to minimize it .

Use a percussion hamm er with an inertia-acti-vated switch. Connect one cable from the switch tothe battery and another from the switch to t he hori-zontal trigger input of the oscillosco pe. Conne ctanother cable from the outpu t of the battery box tothe ground terminal. Swit ch the trigger mode switchto external triggering. Adjust the triggering levelcontrol to trigger the oscilloscope sweep only whenthe hamm er is lightly tapped against a solid object.Set the sweep speed to 10 msecidiv.Tap the Achilles tendon to elicit the stretch reflexwhile recording the electromyogram. Readjust thesweep speed to obtain th e best display. Measure thereflex time by determining the period between thebeginning of the sweep (when the hamm er struc kthe tendon) and the onset of electrical activity in themuscle Measure the reflex time i n 10 trials, usingthe same tapping force Is the reflex time constant?What is the average reflex time ?

Perform additional tests, using differing amo unt sof force. Does the reflex time chang e? Why or whynot ? Measure the distance between the muscle belly(widest part of the muscle) and the presu med site ofthe sen sory-motor synapse in the spinal cord. As-sume that synaptic transmission takes about0.5 msec, hut remember that even though thestretch reflex is labeled a monosynaptic reflex, thepathway measured with this technique includes theneuromuscul ar synapse as well. Calculate the meanconductio n rate in the nerves mak ing up the reflexpathway. How would you design an anim al experi-ment to measure th e reflex time more precisely!


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STRETCH R E F L E X 7.4Patellar tendon (knee-jerk) reflexSeat the subject on a sturdy table with t he thighswell supported and the legs swinging freely. Place apair of electrodes about 10 cm apart on the quad-riceps muscles on the front of the thi gh (fig 7.4-2).Transfer the ground electrode to the kne e. Feel theposition of the patellar tendon just ben eath thekneecap. Place one hand on the patella and use theother hand with the hammer to gently strike thetendon to elicit th e stretch reflex. Observe the elec-tromyogram. Measure the reflex time in 10 trialsand compare this average with t he average obtainedfor the Achilles tendon reflex What are the possiblesources of differenc e?Study the effects of varying the amount of tensionin the muscle by voluntary contraction on the partof the subject Can the reflex be inhibited or en-hanced in this man ner? Ask the subject to lock thefingers of his tw o hands, one against th e other, infront of his chest and pull outward at the m omen tthe ha mmer strikes the patellar tendon [Jendrassik'smaneuver]. Is the reflex facilitated by this method ?Does isometric contraction elsewhere facilitate thereflex? Speculate on the mec hanism of enhan ce-ment. What synaptic inputs influence spinal motorneurons other than the excitatory input of thestretch receptors?Electrical stimulation of the Achilles tend on reflex[at the instructor's option]The stretch reflex can also he elicited by direct elec-trical stimulation of afferent sensory fibers. In hu-mans this is often referred to as th e H-reflex. We willstimulate the tibial nerve through the skin at theknee, which will produce a reflexive contraction ofthe gastrocnemius muscle.Recording orrongement. Place recording electrodeson the gastro cnemius muscle and a ground electrodeon the foot as in figure 7.4-3.Adjust the vertical gainuntil a response is seen as before. Connect a trigger-ing lead from the trigger output on th e electronicstimulator to th e trigger input of the oscilloscopetime base. Connec t a ground lead between the os-cilloscope and stimulator. Set the sweep speed to10msec/div and switch the t ime base to externaltriggering. Reduce the sti mulato r voltage to its low-est possible value. Actuate the stimula tor and adjustthe triggering level control so that a single sweep is

triggered for each stimulus pulse. Turn the st imu -lator output off.Stimulating arrang ement. Tape a plate electrodejust above the front of th e subject's knee, usin g athin film of electrode paste between the s kin and theelectrode. Connect this electrode to th e positiveoutpu t of the s timula tor with an appropriate lead.Obtain a hall electrode with an insulated h andle.Connect this stimulating electrode to the negativeoutput of the stimulator, and coat th e hall with athi n film of electro de paste.Set the stimulator to give monophasic squarewave stimuli at 1 pulseisec and 0.6 msec duration.The subject should grasp the insul ated part of thestimulating electrode and apply the ball to the hol-low at the hack of the knee in t he midline [t he pop-liteal fossa]. The tibial nerve passes through th eknee beneath this point. Stimulate at 1 pulseisecwhile gradually increasing the s timulus voltage.

The subject should control both th e stimu-lus voltage an d the site of stimulation.

Fig. 7.4-4. Responseof the gastrocnemiusmuscle to directelectrical stimulation of the tibial nerve. The peak in theupper trace is a stimulus marker which indicates the ap-plicationof a 0.5 msec stimulus to the tibial nerve. Thelower trace shows he stimulus artifact I );the response ofthe muscle to direct stimulation of the motor neurons inthe tibial nerve (2);nd the reflexive responseof the mus-cle elicited hy stimulating the sensory fibersof the tibialnerve (3).pproximately 30 msec elapsed hetween thestimulus and the onset of the reflexive response.


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7.4 STRETCH REFLEXProbe in different areas with the stimulating elec-trode while gradually increasing the voltage to30-40 V and watch ing the oscilloscope screen for areflexive response. The subject should expect t o feeldiscomfort as the stimulat ing current passesthrough the s kin. The first wave on the oscilloscopescreen will be the stimulu s artifact. A second wavewill follow very closely or he combined with thestimulu s artifact. Th is is the response of the muscleto direct stimulation of the motor fibers in the tibialnerve and i s not a reflexive response. Look for a thirdwave with a latency of 25-35 msec (fig. 7.4-4). Thi swill he the reflexive response initiated by stimula -tion of the sensory fibers of the tibial nerve. Readjustthe gain and swe ep speed to obtain the best display.In some subjects t he ampli tude of th e reflexiveresponse will he larger than the response to directstimula tion. Are the magnitudes of the direct andreflexive responses linearly related to t he stim ulusvoltage? Why might the reflexive response disappearat stimu lus voltages strong enough to stimulat e themotor axons?5 pulsesisec. Does the response dimin ish? At fre-quencies above 5 pulses/sec the response may de-cline. This indicates that t he motor neurons whichhave reflexiv ely discharged are in a period of ine x-citahility. Such inexcitability is caused by pre- andpostsynaptic factors, including recurrent inhibitionof th e motor neurons. Can you think of any otherreasons for motor neuron unresponsiveness? Hint:What happens to muscle spindle discharge duringmuscle contraction?response by (1) ontracting t he gastrocnemiusslightly and (2 )contracting the antagonists of th egastrocnemiu s to produce dorsiflexion of the foot.Does antagonist activity increa se or decrease thereflexive response? What wa s the effect of synergis-tic activity? Illustrate your c onclusions by drawing anerve-muscle wiring diagram summarizing theprobable factors at work in this system.Reciprocal action of antagonistic muscles(at he instructors option)Electromyography will he used to observe the actionof two antagonist ic muscl es of th e ankle joint-thegastrocnemi us and the tibialis anterior. Contractionof the g astrocnemius produces plantar flexion of the

Stimul ate with increasing frequency u p to

Attem pt to change the ampli tude of the reflexive

foot, wh ereas contraction of the tibialis anterior re-sults in dorsiflexion A dual-trace oscilloscope or apolygraph will he necessary to record the electricalactivity of both muscles simultaneously.Recording arrang ement. Place two recording elec-trodes over the belly of the gastrocnemius muscleand a ground electrode just above the ank le joint, asin figure 7.4-3. Connect these electrodes to one ofthe vertical amplifier inputs of one chan nel of adual-trace oscilloscope using shielded cables. Usethe differentia! mode and A C coupling.

A second pair of recording electrodes must heplaced over the anterior tibialis muscle, located justlateral to the tibia in t he upper part of the lower leg.Feel the subjects tibia (shinb one) t the front of thelower leg. Place your fingers 2 cm lateral to the mar-gin of the tibia about 8-12 c m below the kneecap.Ask the subject to point th e toes inward (supinatingor inverting th e foot] and dorsiflect the foot. Youshould he able to feel and see the contraction of theanterior tibialis muscle beneath th e skin. Place apair of recording electrodes over the belly of the an-terior tibialis muscle and connect the m to th e sec-

Fig. 7.4-5. Responses of the gastrocnemiusmuscle (uppert r ace )and the anterior tibialis muscle ( l o w e r race) in asubject standing erect and rocking back and forth. Notethe reciprocal firingof the two antagonistic muscles. Thegastrocnemiuscontracts as the subject leans forward, andthe tibia lis anterior contracts as the subject leans back-ward. The upper trace is unfiltered, while the lower traceis limited to a band-pass of 0.1-10 kHz to limit noise andAC pickup.


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210 Neurobiology ExercisesSTRETCH REF LEX 7.4

ond cha nnel of the dual-trace oscilloscope usingshielded cables. Use differential mode an d AC c ou-pling.Set the sweep speed of t he tim e base to 1sec/divand th e triggering mode to automatic. Adjust the

gain of both vertical amplifiers to about 1mV/div. Iffrequency filters are available on the vertical am-plifiers, limit the band-pass filter to lower the noiselevel (e.g., lower -3dB cutoff at 10Hz and the upper-3dB cutoff at 3 kHz). If 60 Hz AC pickup is a prob-lem, rearrange the leads, check the ground connec-tion to t he ankle, and make sure that electrode pastewas used wit h every electrode. Ask the subject toalternate plantar flexion of t he foot with dorsiflex-ion. Adjust the gain to get the best display of th eelectromyo gram of each muscle.Observation of reciprocal action . The subjectshould stand erect, placing more weight on the legwith the recording electrodes than on the other leg.When the subject rocks backward and forward, al-ternating contraction of the gastrocnemiu s and an-terior tibialis muscles should be apparent (fig.7.4-5).Notice that although one muscle may predominate,the othe r muscle may also be somewhat active. Askthe subject to stand on one foot. Co-contraction of

IMBALANCE ,FORWARDRETURN TOMEANPOSTURE

antagonists as observed here is necessary to stabilizejoints against twisting and slipping. Joint stabiliza-tion is particularly im portan t in leg and posturalmuscles which contribute to the bipedal locomotionin the hum an. Th e joints are mechanica lly stabilizedwhen motionless. Stretch reflexes help to maintai nbalance [fig. 7 .4 - 6 ) .Can yo u detect muscle activityin a relaxed standing posture?Within the l imits of cable length and th e introduc-tion of movement artifacts you can explore the ac-tion of the ankl e flexors. What happens, for example ,when t he subject stretches upward or squats? Whatother locomot ory activities require the coordinationof antagonists? How are such activities related to th estretc h reflex studied in th e first three sections ofthis exercise?Multiple-joint and multiple-action muscles(at he instructor's option)It is easy to record from the biceps and triceps mus-cles of th e upper arm. These antagonistic musclesare involved in a number of different movementsand involve more than one joint. Explore their func-tions using electromyography. What is the effect ofloading (increased resistance] on the electromyo-gram!

Fig. 7.4-6. Balance maintenance and the stretch reflex. Animbalance forward activates the spindles of the gastroc-nemius and soleus muscles. Their activation is relayed tothe motor neurons, and the muscles contract and balanceis restored.


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7.4 STRETCH REFLEXPitfalls and Suggestions1. In this exercise an electronic stimula tor is used tostimulate a huma n subject. Take care to stimulateonly the leg. If the subject touches the ball electrodewith the hand, a current will pass between the handand the other stimulating electrode on the leg, a cir-cuit which includes the torso and the heart.2. Prior study of the stre tch reflex and reciprocalinnervation in a textbook of physiology or neu-roscience will make t he exercise more rewarding.3. After the experiments are completed, wash all

Materials available in the laboratoryTape measureOscilloscope camera and filmElectrode pasteMasking tape [used to attach surface electrodes; do

not use adhesive tape which is painful to pull off )Extra cables and connectors1spare percussion hamme r with inertia-activatedswitchNotes for the Instructor

electrode paste off th e legs and electrodes. Electrodepaste left on the skin may cause itching. 1, Safety and subjectmou nt conce rn of the instru ctor. Before the labora-

should be the para-MaterialsMaterials needed a t each stationOscilloscope or polygraph (A dual-tra ce oscilloscope

or polygraph is needed to perform the section onthe reciprocal acti on of antagonistic mu scles.)Preamplifie r (opt iona l f the oscilloscope vertical

amplifier sensitiv ity goes to 1.0 mv/div)5 surface electrodes with at taching straps [plate elec-

trodes, such as Harvard no. 321 or Grass no. E4).Set of con nectors a nd cables (Shielded coaxial cablesare mandatory to suppress 60 Hz AC pickup.)Ball-tipped stimulating electrode w ith insulatedhandle. (Thi s s most easily made up from a phoneplug. Solder a single conductor insulated lead tothe tab attac hed to the phone plug tip, and roundthe tip to a ball with a file. See fig. 2.2-8E for anillustration of a phone plug.)

Percussion hammerPercussion ham mer with inertia-activated switch.[Make this up by adding a spring-loaded micro-switch, e.g., Microswitch Co. series 31 1SM withroller lever, to the uppe r part of the hand le. Tapethe switc h onto the handle after attaching a two-conductor cable to the switch. Weight the ar m onthe sw itch wi th a blob of solder. Arrange theswitch to be activated by the inertia of the solderwhen th e ha mme r is struck against a solid object.)

tory period, check all electrical equipment. EachAC-powered unit should be provided with a properlygrounded three-prong power cord. Do not useequipment wit h any hint of malfunction. The in-term itte nt square-wave output of the electronicstimulator is relatively innocuous, but AC line cur-rent is very dangerous, and every precaution shouldbe taken to prevent exposure to it. Remove all unes-sential AC-powered equipment and metal objects.Do not use metal tables. Subjects should not groundthemselves by touching a grounded cage or wirewith their hands. Ground the leg only, as directed.2. The stim ulating electrodes will sting, especiallythe cathode. Discomfort can be minimize d by usingenough electrode paste and a smooth ball-shapedcathode. A pointed cathode will produce a high cur-rent density and attendant irritation. Current den-sity is inversely proportional to electrode area.Long-term stimulation will lead to hyperemia atboth electrodes, but more at the cathode. Bony andfatty areas should be avoided because the y presentmore resistance a nd increase discomfort.3. Time permitting, a motion picture of huma n re-flexes is a useful adjunct.4. We suggest that a polygraph not be used. Sincehigh paper speeds are necessary, much wastage will5. The position suggested for the Achilles tendonreflex (legshanging freely) s not ideal. There are twoalternative positions: (1) the subject kneel s on a pad-ded table with the knee joint at a 90" angle and thefeet hanging over the edge of the table; and (2 ) hesubject reclines on his side on a padded table wit hone leg straight and the othe r leg bent a t the kne eand lying across the straight leg. In the second posi-

occur.Battery box (Make this up by placing a 1.5 Vflashlight cell in a minibox and adding binding

posts and/or banana jacks. Th e battery is necessaryto provide a triggering voltage which is actuatedby the percussion ham mer switch. If a battery isused without enclosing it in a minibox, solder theleads securely to the battery termi nals.)


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212 Neurobiology ExercisesSTR ETC H REFLEX 7.4

tion the tendon of the crossed leg is struck. Bothpositions require a padded surface and more roomthan ma y be available in many labs.6. Your st uden ts may occasionally observe muscleaction potentials (MAPS) between EM G responses.Thes e are active, singlemotor units, not single mus-cle cells. The EM G is the async hronous discharge ofpotentials from multiple motor units.Selected ReferencesChusid, J. G.. and McDonald, J. J. 1973. Correlative neu-roanatomy, 15th ed. Lange Med Pub., Los Altos, Calif.Curtis, B. A,; Jacobson. S.; and Marcus, E. M 1972. Anintroduction to the neurosciences. W. B. Saunders,Philadelphia.Fiorentino, M.R. 1972. Normal an d abnormal develop-ment: The influenceof primitive reflexes on motor de-velopment. Charles C Thomas, Springfield, I l l . (Well-Illustrated, showing normal newborn reflexes and aber-rant patterns of development.)1973 Reflex testing methods forevaluating C.N.S.development. Charles C Thomas, Springfield, Ill.(Well-illustrated,showing reflex patterns in children.]Ochs, S. 1965. Elements ojneurophysiology. John Wileyand Sons, New York.Willis, W. D., Jr., and Grossman, R. G. 1977. Medical neu-robiology: Neuroanatomical and neurophysiologicalprinciples basic to clinical neuroscience. C. V. Mosby,St. Louis.


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Neurobiology Exercises 21 38.2 Insect FlightSensory and Cent ra l Mech anisms

IntroductionRationaleThe ability to sustain flight has evolved in three liv-inggroups of animals: insects, birds, and hats. Flightis the most com plex form of lo comotion and re-quires an advanced neuromuscu lar system for coor-dination. The evolutionary success of flying animalscomes largely from exploiting the aerial environ-ment.

Insects are particularly favored in the evolution offlight. Their small size leads to a large ratio of sur-face area to mass. Th e power-to-weight ratio in in-sects is enhanced by the air tubes or trachea whichgo directly to the tissues and thu s eliminate th e needfor a complex and heavy circulatory system. Theexoskeleton provides a strong and light structural

material, and th e skeletal veins, air-filled tubeswhich support the me mbranous wings, would heonly slightly stronger even if they were made of alight metal like t itaniu m. Finally, streamlining ofthe body is unimportant to most flying insects sincethey are light and fl y at low speed. Birds in generalhave had to evolve streamlined shapes, hut insectshave been free to develop other morphological adap-tations with out the overriding necessity to stream-line the body.Cockroaches, th e subject of thi s exercise, repre-sent a group of relatively unspecia lized fliers. In fact,immatu re cockroaches (nymphs or larvae) do nothave wings, and in some species the adults are wing-less, or only the male adults have wings. Cock-roaches have changed little since they evolved dur-ing the Carboniferous period, before the appearanceof flowe ring plants. Th e specializ ed fliers, bees, flies,and moths, evolved later during the Mesozoicperiod. Bee flight muscles, for example, are highlyspecialized with den se arrays of contractile proteinsand numerous mitochondria, whereas in cock-roaches the flight muscles are not too different fromleg muscles (fig.8.2-1). We will use cockroaches be-cause they are large, hardy, and easily obtained andwill maintain a regular and sustained flight.Time to complete th e exercise: One 3-hour labora-tory period.ProcedureObtain an adult mal e cockroach [see experiment 8.6for sexing cockroaches).Attach a wooden applicatorstick to the pro notum [the shieldlike area behind thehead) using melted wax. Hold the insect still whilethe wax solidifies. After the wax hardens, carefullyseparate the cockroach from its grip on your handwithou t breaking the wax bond. Suspend the

From Bruce Oakley and Rollie Schafer, EXPERIM ENTAL NEUROBIOLOGY: A LABORA-T OR Y M A NUA L . Copyright 1978 by the U niversity of Michigan. All rights reserved.No partof this material may be used or reproduced in any manner whatsoever without written permissionof the publisher. Reprinted here by permission of the University of Michigan Press.


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INSECT F LIG H T 8.2mounted cockroach in air by attaching theapplicator stick to a horizontal rod fixed to aringstand. Give the cockroach a small st yrofoam ballto grip with the feet while it hangs in the air. Simi-larly prepare three or four more males.Normal flightSuspend a cockroach in front of a small fan which isplugged into a n autotransformer. Use a swivel clampfor mounting so that th e flying attitu de may be var-ied. The fan will act as a simple wind tunnel . Adjusta stroboscopic lamp to shine on th e cockroach, butdo not turn it on yet.

A

The flashing of a stroboscope will triggerepileptic seizures in some people. If you feelodd, ill at ease, or dizzy during the courseof the experiment, leave the vicinity of theflashing light.

Turn th e fan on and adjust the autotransformerto subject the insect t o a low velocity breeze(1-2 misec 2-4 mph, if you can estima te it).Now,remove the Styrofoam ball from the cockroachsgrip. using care to avoid pulling th e insect off th eapplicator stick. At this point the insect may beginto fly. If not, give it from 30 seconds to 1 minutebefore doing any thing more.After a period of time with no flight, attempt totrigger flying with a slight increase i n wind velocityand by touching or lightly pinching the tip of theabdo men. Let it grip one end of a glass rod, whichyou then w ithdraw. If this does not initia te flight,discard the animal and get another.

When flying is obtained, turn the room lights offand the stroboscope on. Adjust th e stroboscope fre-quen cy In the range of 20-40 flashesisec until th ewing motion appears nearly stopped.

CAUTION: The fan blades may appear to bestill or only slowly moving. This is an illu-sion! The y are rapidly moving and are dan-gerous!

Fig. 8.2-1.Wing movements in insect flight. These aresketches of wing movements taken from high-speed cinephotographs.The progressionof wing movements readsfrom right to left. The upper surface of the wing is whiteand the lower surfaceblack. A. The locust beats its foreand hindwings slightly out of phase.B. Beetle. C. Thebutterfly beats its fore- and hindwings exactly in phase. D.The dragonfly beats the fore- and bindwings exactly out ofphase to neutralize torque. The wing tips of the forewingsare marked with block do ts . [Adapted from Nachtigall,1974.)

Determine t he frequency of wing beat of theforewings by finding the fastest flashing rate wh ichproduces the appearance of com pletely stopped mo-tion. (If the strobe light is not calibrated, you willnot be able to measure th e frequency unless youdrive the strobe with a stimulator.)

Observe the movements of the fore- and hind-wings by adjusting the stroboscope to mak e it appearthat they are flapping very slowly. Do they beat to-gether or ou t of phase? If out of phase, how far out ofphase? [See ig. 8.2-2.)How steady is th e rate of beat-ing? Do you suppose the rate of beating wouldchange if the wings were lightened by clipping themshorter? Do not test this now.

Does the angle of incidence (angle of attack )of th ewings change during their reciprocating motion?


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8.2 INSECT F LIG H TSome insects, such as the bee, change the angle ofincidence t o achieve lift on the upstroke as well asthe downstroke. Is this true of the cockroach? Canyou detect changes in posture and wing attitudewhen the insect corrects for the roll, yaw, and pitchthat you induce by turning the insect [fig. 8.2-3)?After observing, give the Styrofoam ball back to thecockroach to termina te the flight and rest the insect.Turn the strobe light off.ExperimentsAs evident from the questions just asked, there aremany aspects t o winged flight. Take a few minut esto read over the following and plan a set of experi-ments. Three main areas of investigation are availa-ble with th e tools you have at hand.AerodynamicsThe questions in Normal flight dealt primarilywith the mechanical aspects of flying, and may havesuggested experiments to do. It ought to be possible

0.5 ONE CYCLE0 20 40 60

TIME, STARTING WHEN THE HINDWINGSARE MIDWAY UP (MSEC)

Fig. 8.2-2. Standard wing stroke of the locust Schistocercogregaria. The typical wing posi tions shown above thecurves were drawn from photographs. The d o t t ed l i n es onthe forewings are thin filaments glued there to ind icatethe twisting of the wing plane. Th e wing tips are markedby black d o t s . Th e two sine waves represent the up-and-down strokes of the fore- and hindwings. (Adapted fromWeis-Fogh, 1956b.)

to analyze the flight response into subco mponents.move ment s of the fore- and hindwin gs; position ofthe body, legs, and antennae; action of a single wing;or the effe cts of chang es in the body posit ion anddirection of the wind stream. If you have read dis-cussions of animal aerodynamics, such as those hyPringle ( 1957], Pennycuick (1972), or Nachtigall( 1974),you may be able to ask mor e informed ques-tions This preparation is strongly encouraged ifmore than one laboratory period is available.Sensory mechanismsInsects apparently depend on the simultaneous acti-vation of several kinds of sensory receptors to in-itiate and maintain flight. The most completeanalyses of the sensory mechanisms of insect flighthave come from the locust and bee. With modifica-tions, these observations also apply to cockroaches.Flight is induced in a numbe r of in sects by theremoval of support. Receptors on the legs thu s con-tribute to the initiation of flight. You may observethe effec ts of supp ort and lack thereof by repeatedlyremoving the Styrofoam ball from the grasp of thecockroach. Is this sufficient to sustain flight in theabsence of wind! Ho w may seconds is flight sus-tained wi tho ut wind! What is the effect of repeatedtrials? What is the influence of changing the timeinterval between trials? How many legs must betouching a surface to inhibit flight? You may con-sider amputating legs or parts of legs to elimina tesensory input, but remember that the acute traumamay confound the results of your experiments. De-spite this, amputation experiments often work wellin insects and other arthropods.

Receptors on the head and ante nnae may also playa role in flight. For example, stim ula tio n of groups oftactile hairs on the head of the locust will result insustained flight if support is simultaneou sly re-moved. Examine th e head of a cockroach wi th amagnifying glass or dissecting microscope. Are hairsvisible which might serve a similar purpose? Thereceptors on the bead can be selectively stimulat edby a fine jet of air from a Pasteur pipette co nnec tedvia rubber tubing to an air line. Alternatively, sen-sory input from tactile hairs can be eliminated bycovering them with grease, e.g., silicone stopcockgrease, or extra-fast drying modeling cem ent.

The influence of t he anten nae on flight is moredifficult to test. Amp utation of th e antenn ae of


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Fig. 8.2-3 Roll, yaw, and pitch in a flying ocust (Adaptedfrom Weis-Fogh, 1956b )

cockroaches usually leads to a general depression ofactivity; hence it is probably a relatively poor ex-perimental approach On the other hand, since manyof the proprioc eptors of the an tenn ae are located inthe first three basal segments, it would be possible toinactivate them by immobilizing the bases of theantennae with cement. This will not, however,eliminate tactile input from the antenna e becausetactile hairs are found on every segmenton the wings. In the locust, the stimulation of airmoving across the wings is sufficient to main tainflight once it has begun, but it is an insufficientstimulus t o initiate flight. Does a similar mecha-nism operate in the cockroach? You may want to usethe fine jet of air to explore this problem. You mayalso consider amput atio n of the wing s and observa-tion of other componen ts of the flight posture. Useof larvae (ny mph s)witho ut wings may be useful toobserve postural responses in cockroaches witho utwings.Finally, the possible influence of the caudal cercilocated at th e tip of the abd omen can be evaluatedThey are an important mechanoreceptive input (seeexperiment 7.1).The cerci can be stimulated wi th anair jet or they can be amputat ed.

Tactile receptors and proprioceptors are also found

Central mechanismsComplete reflexive pathways for walking are oftenpresent in th e thoracic ganglia of insec ts like cock-roaches. For example, a male praying ma ntis whic hhas been beheaded will continue circular walkingmovements for days. This particular example is ex-treme and related to t he sexual behavior of t he man-tis, but it illustrates the degree of autonomy possiblein individual ganglia You may want to ask whetherthe thr ee thoracic ganglia of the cock roach contai n acomp lete set of neural cir cuits for flying.

Tests of the quest ion of autonomy will have torely on ablation (surgical destruction) of the twomajor areas of the cock roach brain: th e supra- andthe subesophageal ganglia (see fig. 7.1-2, page 2 0 5 ) .The objective will he to remove any influence theymight have on the thoracic ganglia. The supra-esophageal ganglion can be destroyed by inserting ahot needle into the head capsule between the com -pound eyes and antennae. Use a blunt needle (abo ut1 mm diameter)which is nearly red hot.

Although preliminary observations are possible,one or more hou rs may be required for a stable re-covery st ate. Plan your ex periments accordingly.You should prepare several insects since some abla-tions may be faulty.

The supra- and subesophageal ganglia may be to-tally removed simply by beheading the insect. Lessrecovery time will be needed after beheading. If ex-cessive bleeding takes place, l igh t ly cauterize thewound with a hot needle.The abdo minal ganglia also influence the activityof the thoracic ganglia Therefore, you may want tosever the nerve cord between the a bdomen andthorax in a few insects. This can be done by cutting asmall, transverse slit in the ventral abdomen at t hefirst or second segment (sternit e)behind the thorax.After making the slit, insert the opened tips of a finepair of scissors into t he opening and snip. Since thenerve cord is adjacent to th e sternite , you will cutthrough it. Do not insert the scissors too far or youwill also cut the gut. Cutting the nerve cord at thissite will eliminate all afferent input from the abdo-men, including the sensory input from the cerci.Pitfalls and Suggest ions1. Turn the strobe lamp off when it is not in use.Flashing strobe lamps may trigger epileptiform sei-


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zures in some people, as noted previously, and long-term exposure will make most persons develop aheadache. Do not look directly into the lamp at anytime.2. Be ready to make observations and take data w henthe insect begins flying. Terminate flight by replac-ing the Styrofoam hall whenever you are not maki ngobservations. Avoid fatiguing the insect as this willaffect your results.3. The wax is more likely to hold the anim al if t hewax is curved aroun d the lateral edges of th e tergum .MaterialsMaterials needed at each station2 ringstands5 short rods, about 0.32 x 20 cm (114x 8 inches]5 right-angle clampsSwivel clampFan [A small, plastic-blade fan is safest, e.g., Rotron

Muffin Fan available from electronics suppliers.]Autotransforme r [AC rheostat for controlling fan

speed; low amperage capacity is sufficient.)Stroboscopic lamp [A photic sti mulato r can be used;e.g., Grass Instrument Co. PS22 or PS33.)5 Styrofoam halls, about 2-3 cm in diameterBunsen burner or soldering iron for melting waxGlass rod with fire-polished endsPithing needle and 1 pair of fine scissors [furnishe d

by students]Materials available in the laboratoryApplicator sticksWax (e.g., in order of preference, S. S. White CrownSticky Wax, Surgident Periphery Wax, Cenco

Universal Red Wax]and rods

Extra right-angle clamps, swivel clamps, ringstands,Extra Styrofoam ballsBeakers or other containers for melting waxPasteur pipettes or glass tubing pulled out to

1-2 mm tipsRubber tubing, approxima tely 7 mm (114 inch) nsidediameterSilicone stopcock grease [a viscous grease, such asDow Corning silicone stop cock grease] or fast-

drying modeling cemen t (e.g., Testors extra-fastdrying cement for wood models available fromhobby shops)

Fine camel hair paintbrushes or bristle s from a largerMagnifying glassDissecting microscope and illuminatorAnimalsPeriplaneta americana are available commercially

paintbrush, for stimulating the anal cerci

from Carolina Biological Supply. (Order 7- 8cockroaches/station.] Oth er species, such asLeucophaea maderae, may also be used as long asthe ad ults have wings. In some parts of the c oun-try, wild cockroaches can be trapped. Trappingmay be advantageous because wild cockroachesare often more vigorous fliers than laboratory-reared cockroaches. Flying grasshoppers or beetlescan be used if they are available. Place the roachesin a plastic wastebasket or a similar containerwith the u pper walls smeared with pet roleum jelly(Vaseline] o prevent escape. Provide the roacheswit h vertically sta nding pieces of cardboard toclimb upon. A sponge wetted with water everyfew days will supply sufficient water. Feed labchow or dry dog food. Supplement with apples ororanges periodically if th e roaches are kept for along period.

Notes for the Instructor1. The most difficulty will he encount ered in fixingthe insects to applicator sticks with wax. The bondbetween the cockroach and the stick is usually bro-ken by students who hastily try to pull the insect'sgrip loose by pulling on the stick. Pull gently, freeingone leg at a time.2 Caution the st udent s not to look directly in thelight. Also caution th em to avoid the fan blades,which ma y appear slowly moving or stationary inthe flashing light. Do not use the strobe light in thepresence of an epileptic.3. Check the instruction ma nual for the strobe lampto determine if there is any suggested limitation oncontinuous operation. Some units require periodicresting.Selected ReferencesNachtigall,W 1974. Insects in flight.A glimpse behindthe scenes in biophysical esearch. McGraw-Hill,NewYork


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INSECT FLIGHT 8.2Pennycuick, C 1972. Animal f l ight . The Institute of Biol-ogy's Studies in Biology, no. 33. Edward Arnold PubLtd., LondonPringle, . W. S . 1957. Insect flight. Cambridge UniversityPress, Cambridge

1968. Comparative physiology of the flight motorAdv. Insect Phyciol 5: 163-227.Soc 7 Halsted (Wiley), New York.Rainey, R C. 1976 Insect flight Symp. Royal Entomol.Wells, M 1968.Lower animals McGraw-Hill, New York.Weis-Fogh, T. 1956a The flight of locusts. Sci A m 194

Wilson, D. M. 1968a. The flight control system of theIocust. So. m . 218: R3-90.1968b.The nervous control of insect flight andrelated behavior Adv Insect Phyciol. 5: 289-338

116-24.

RESEARCH REPORTSAltman, J . S. 1974. Changes in thc flight motor pattemduring the developmentof the Australian plague locust,Ch o r t o i ce t es terminifera. J . Comp . Phys iol. 9 7: 127-42.Camhi, M. 1970. Sensory control of abdomen posture inflying locusts. I . E xp Biol 52: 533-38Gettrup, E. 1965.Sensory mechanisms in locomotion:The campaniform sensilla of the insect wing and theirfunction during flight. Cold Spring Harbor Symp.Quan t . Biol. 30: 615-22.Jensen, M. 1956. Biology and physics of locust flight.Ill. The aerodynamicsof locust flight. Philos. Trans B.239: 51 1-52.Kutsch, W. 1974. The influence of the wing sense organson the flight motor pattern in maturing adult locusrs.J. Comp. Phyciol. RR: 413-24.Weis-Fogh, T. 1956b. Biology and physics of Iocusr flight.II . Flight performance of the desert locust. Phil. Trans.B. 239: 459-510.IV. Notes on sensory mechanisms in locust flight. Phil.Trans B. 239:553-84Weis-Fogh,T., and Jensen, M. 1956.Biology and physics oflocust Right. I. Basic principles in insect flight. A Crit-ical review. Phil. Trans. B. 239: 415-58.Wendlcr, G. 1974. The influence of proprioceptive feed-back on locust flight co-ordination. 1. Comp Physiol.RR : 173-200.

Wilson, D. M. 1961. The centra l nervous control of flightin a locust. J. Exp . Biol. 38: 471-90.Wilson, D. M.,and Weis-Fogh, T. 1962. Patterned activityof co-ordinated motor units, studied in flying locusts.J. E x p . Biol. 39 . 643-67.

1956c. Biology and physics of locust flight .


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8.7 SensitivePlantNa stic Responses and Conducted Ac tion Potentials inM i m o s a pudica

IntroductionRationaleBacteria, protozoa, plants, and animals often sharesimilar adaptive mechanisms. Photoreactivity, forexample, is a common thread running through allphylogenetic levels. Sensitivity to mechanicalstimuli is also widespread. While all plants are me-chanically sensitive in that their growth patterns aredeeply influenced by physical contacts with wind,water, gravity, and ot her forces and objects in th eenviro nment, only a few plants are capable of rapidmovement when stimulated. This ability is as-sociated with carnivorous (e.g., Venus flytrap) orprotective (e.g., Mimosa ) adaptations.

Mimosa pudica is a member of the pea family,Leguminosae. Many memb ers of thi s family areequipped with organs called pulvini at t he bases of

the leaflets, leaves, and petioles. The pulvini containosmotically active effector cells which cause foldingof the leaflet pairs and drooping of th e petiole.Mimosa pudica, which folds its leaflets in th e darkor when disturbed (fig.8.7-1), is appropriatelynamed: Mimosa is from the Greek for mimic, andpudica from Latin for modest or bashful. The pla nthides its leaves when disturbed. Because the move-ments of Mimosa pudica leaves and stems are inde-pendent of th e direction of th e mechanical stimu lus,they have been termed nasti c responses. This termdistinguishes such movements from tropic re-sponses w hich proceed in a direction determined bythe dir ectio n of t he sti mul us. Folding of leafl ets inresponse to mechanical disturbance is termed se i s -monasty.

The rapid nastic response of Mimosa pudi ca ismediated through interc onnect ed pathways of excit-able cells in the leaves, petioles, and stems (fig.8.7-2).A given pathw ay is comprise d of a ser ies ofcells arranged end to end with low resistance path-ways between them. The conductile pathways carrypropagated action potentials, and are thu s analogousto nerve fibers in animals although conduction i nplant cells is generally much slower.In the pulvin i at the base of leaflets, leaves, andpetiol es are a class of effector cells who se turgorpressure holds these st ructur es unfurled and erect(fig.8.7-1). A disturbance, such as brushing the handagainst a lea f, is sufficient t o excite conductile cellsin the leaves. Excitation is conducted to the pulvinareffector cells, triggering in succession a massiveefflux of pot assium, a rapid outflow of water, a nd adrop in turgor pressure. The leaflets fold and thepetioles droop. This nastic response is aided byanoth er class of cells arranged as antag onists, whic hgain potassium and water to increase turgor pres-sure.

From Bruce Oakley and Rollie Schafer, EXPERIMENT AL NEUROBIOLOGY: A LABORA-T O R Y M A N U A L .Copyright 978 by the University of Mich igan. All rights reserved.No partof this material may beused or reproduced in any manner whatsoever without written perm issionof the publisher. Reprinted here by permission of th e University of Michigan Press.


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SENSITIVE PLANT 8.7We will explore two aspects of th e rapid nastic

response. First, mechanical sti mulati on will be usedto examine th e nature of t he response and its deter-mining factors. Second, you will record conducte dpotentials from t he petioles, stems, and leaves.Time to complete the exercise: One 3-hour labora-tory period A second session or a longer laboratoryperiod would be useful since the plant requires10-15 minute s to recover between stimulations.ProcedureMimosn pudica is literally a very sensitive plant. Becareful throughout the experiment not to acciden-tally bump theplant or j a r t s table. Moreover, eachtime the plant is stimulated it will take 10-15 m in-ute s to recover. Therefore, for each test tha t followsstimula te fresh portions of the leaf or new leaves.Some patience will be required since even in fullyrecovered and responsive leaflets there may be a

LEAFLETS

Fig. 8.7-1. The nastic response of Mimosopudlca. A touchto the leaf results in foldingof the paired leaflets anddrooping of the petiole. The effectororgans (pulvini) t thebases of leaflets and petioles arenot visible.

delay of several seconds between stim ulus an d re-sponse, depending on the mann er of stimulation andother factors. Examine figure 8.7-1 to learn themeaning of the te rms leaf, leaflets, petiole, and ste mbefore proceeding further.Exploring Mimosas behaviorGently touch one of the leaflets at a leaf tip wi th athin glass rod or a Pasteur pipette. Can you mak eonly those leaflets in the vicinity of the touch fold?Note th at a sufficient stimulus at the tip of the leafwill initia te a wave of leaflet folding toward the baseof th e leaf.Pinching stimul i. Use a pair of forceps with rubber-guarded jaws to st imula te by pinching. First, pinc hthe leaf near its tip.

Pinch no harder than necessary to produce aresponse.

Can waves of leaflet folding be readily elicited bypic hin g? In spite of the difficulty in giving a con-sistent pinch w ith ha nd-held forceps, can you getsome idea of th e force required to get a co nductedwave of leaf folding! Ho w muc h consist ency of re-sponse is there from leaf to leaf?

Notice that often a wave of leaflet folding willproceed to the base of the leaf, but will not elicitdrooping of the petiol e. What is the result of elicitinga wave of leaflet foldin g in a second leaf on the sa mepetiole after the leaflets of the first leaf ha ve folded?We can describe the result of th is experi ment as aform of facilita tion.

Pinch a leaflet near t he base of a leaf. Does out-ward [centrifugal)propagation of leaflet folding oc-cur? Does propagation occur in both directions whe na leaflet between the leaf tip and base is pinched?Does centrifugal propagation into th e leaves occurwhen the petiole is pinched? Finally, pinch o ne ofthe petioles or stems near t he base of the plant.Watch for a few minutes.Brushing stimuli. Pinching s a somewhat unnaturalstimulu s. Try to elicit the same kinds of responsesby brushing a fine glass rod or Pasteur pipette alongthe axis of one of the leaves. Does sensitivity varyaccording to the extent or direction of the bru shingmoveme nt? Does recovery take more or less timethan w hen th e leaf was pinched!


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8.7 S E N S I T I V E PLANTDamaging stimuli. Use a pair of clean, sha rp surgicalscissors to cut 2-3 mm off the ti p of a distal leaflet.Do t his very carefully to prevent jostling and induc-ing leaflet folding through mech anical stimulation.Watch carefully, allowing ample time to observe theeffects of the damage. Does damage always elicit awave of leaflet folding? In wha t respects are th emoveme nts after damage different from those afterbrushing or pinching stimuli ? Finally, ask yourself ifyou can clearly distinguish in this ex periment be-tween hormonal or electrical propagation of t he re-sponses along the leaf and petiole.Further behavioral experimentsHere are some suggestions for other experimentsusing simple observational techniques.1. Measure the conducti on velocity in leaves andpetioles. Use cutting as a stimulu s, stopwatches formeasu ring, and the pulvinar responses of leaflet andpetiole bases as time marks. Is conduction velocityconstant along a leaf? Compare centripetal [inward)with centrifugal [outward) onduction velocities.2 Examine the facilitation of the drooping responseof th e petiole when two or four leaves which branchfrom the sam e point are stimulated in succession.3 . Examine the effect of co ntinuous wind on respon-siveness. Does the plant become more or less re-sponsive after a fan hasge ntly bl own on it for 20-30minutes? Use brushing stimuli to test.4. Examin e the effects of various levels of ill umi na-tion on nastic responses. (There are both slow andrapid nastic responses; you have been observingrapid responses.) What happens to the pla nt after 5,10 ,20 , and 30 minutes in the dark?Recording ProcedureConducted electrical potentials can he measured byinserting fine wire electrodes into a petiole, stem, orleaf. Since they are much slower than nerve actionpotentials, these potentials can be recorded equallywell on a polygraph or oscilloscope. Wire electrodesin the s tems will also pick up the electrical spikesproduced by rapid ionic fluxes in the pulvini and bymechanical moveme nt of a stem when it droops.Recording setupTo keep the leads short, mount the preamplifier near

the M i m o s a plant at the same height as the stemsfrom which you intend t o record. Connect the o ut-put of the preamplifier to the input of an oscillo-scope or a physiological polygraph [fig.9.7-3). Turnon your equipment and use the following initial set-tings.Preamplifier. Input in differential mode if possible,gain of 10x; band-pass filters (i f available] set atabout 0.1 Hz [low frequency filter) and 1 kHz (highfrequency filter)Oscilloscope or Polygraph.speed at 1 sec/div or 1 cm/sec; sensitivity at50 mV/div or SO mV/cm; trigger set on automatic orfree run.Calibration. Adjust the preamplifie r and oscillo-scope or polygraph gain to give a total syste m gain ofapproximately 5 mV/div or 5 mV/cm. Check wit hthe preamplifier calibrator if available.

Time base or paper

Push a metal rod into the soil and connect the rodto a grounding point on the preamplifier input. Useheavy surgical scissors or good quality shears to cuttwo 20 cm lengths of fine stainless steel wire. Cutthrough th e wire at a 45-degree angle to produce asharp point. Avoid bending the wires; once theybend, they stay bent. Attach each wire to one of theinputs on the preamplifier.Choose a healthy looking and accessible pair ofleaves on the M i m o s a plant. Gri p one of th e elec-trode wires betwe en the jaws of a smooth-surface dpair of forceps about 3-5 mm from the cut end of thewire. Carefully insert the tip of this wire through th estem, several centimeters below th e branch point ofthe petiole from which you intend to record (fig.8.7-3).Use the same technique t o insert the secondrecording electrode into the petiole a bout midwaybetw een the base of the leaf and the base of thepetiole. Insert both electrodes at angles which do notput too much mechanical stress on the stem or thepetiole.Recording propagated potentialsAllow 10-15 minute s for the plant to recover.Meanwhile, make sure all adjustments and connec-tions are complete and calibrate the system gain.When the leaflets unfold, you may begin. Turn onyour preamplifier and recheck everything beforestimulating.


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S E N S I T I V E P L A N T 8.7

A

NONGLANDULARHAIRS

C

UPRAISED LEAFLETS

B CROSS SECTION OF PETIOLE D CROSS SECTION OF LEAF AXIS(RACHILLA)AND TWO PULVINULESFig. 8.7-2 Cross sections of a petiole ( A and B 42x) andleaf axis (C and D 60x) f the sensit lve plant Mimosap u d i ca . The conducted potentials which course throughthe stemlike tissue probably travel in the phloem and theprotoxylem (differentiating xylem) Thc cells in theseregions have resting membrane potentials of about- 160 mV , whereas cells in other regions (e.g., paren-chymal have resting potentials around -50 mV The leaf-

lets fold upward upon stimulation because turgor pressurerapidly increases in the abaxial epithelial cells of the pul-vinules while it decreases in the adaxial cells Th is is ac-complished by the active movement of potassium ions(followed by the osmotic flow of water) from the adaxialcells to the abaxial cells (Scanningelectron micrographscourtesy of P. Dayanandan.)


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NeurobiologyExercises 223

Fig. 8.7-3. Recording arrangement .Stainless steel wires areinserted through the stem an d petiole for recording thepassage of conducted potentials

For initial observations use the cutting method ofstimul ation by carefully snipping2-3 mm off the tipof one of the distal leaflets Observe both the activityof the plant and the recording trace. One personshould watc h th e plant, describing its responsesaloud, while the other watches t he recording trace.You should see a large potential change 1 second ormore after the wave of leaflet folding reaches thebase of the leaf. This is a propagated potential in th epetiole passing the first recording electrode. Youmay also pick up a spikelike potential as the pul-vinus at the base of t he petiole reacts (fig.8.7-4). naddition, you may see a third potential change as apropagated potential passes th e second recordingelectrode in the stem. You will have to allow timefor recovery after each response.

After obtaining responses and noting their generalform using the previous recording arrangement, shiftthe recording electrode in the stem to the midd le of aleaf. Now, you can record the passage of potentialsin the leaf and in the petiole. This arrangement isparticularly useful for studying the time courses offacilitory effects and recovery.

Suggested experimen ts with electrical conduct ion1. Characterize the magnitude, waveform, and timerelationships of the recorded potential changes. Arethe potentials all-or-none, or graded! The potentialsrecorded with your gross electrodes are, like thecompound action potential of a nerve, the summedpotentials of many cells2. Measure conduction velocity in the petiole elec-trically. Move the tw o recording electrodes to a newpart of the plant, this time placing both electrodes ina petiole--one near th e base of the petiole an d one atthe distal end of the petiole. Stimulate by snippingdistal leaflets as before. Record the time interval be-twee n the peaks of the rapid upward and downwarddeflections of the trace produced as the conductedpotential passes the first and second electrodes.Measure the distance between the two electrodesand compute th e conduct ion velocity. Remember,mm/msec m/sec3 . Examine the effectsof temperature and illumina-tion on conduction. Th is problem requires somethought t o separate the effects of light and heat.4. Examine the effects of electrical stimula tion usingan electronic stimula tor and another set of wirestimulating electrodes. For example, you might at -tempt to block the conduc ted potentials by usingDC current to hyperpolarize a region between thesite of stim ulu s and the site of recording. Whatwould this tell you about the nature of the con-ducted potential!

5 Sec

Fig. 8.7-4. Potentials rrcorded with the arrangementshown in figure 8.7-3. At A, thc conducted potentialpasses the recording electrode in the petiole The rapidpotential change at B is associated with the response of thepulvinus at the base of the petiole. The waveforms of bothpotential changes have been d istorted byAC recording.The propagated potential would appear as a fully mono-phasic wave in DC ecording.


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Pitfalls and Suggestions1.You should meet no special difficulties other thanwaiting for a response to occur and waiting for theplant to recover betwee n stimulations. Use this tim eto think about the design of yourexpenments. If youdo not wait long enough you will get only partialresponses.2. Be sure to check your recording setup carefullybefore stimulating the plant (e.g., is the preamplifieron?).3. Be careful around other experimenters. Disturbinga plant wastes time and could ruin an experiment.MaterialsMaterials needed at each stationOscilloscope or polygraph (A polygraph IS preferable

since it produces a continuous, permanent record.)Preamplifier (Alternatively, one can plug the elec-trode wires directly into the input of an oscillo-scope or polygraph. The adva ntages of using apreamplifier are that the electrode wires may bekept quite short, minimizing 60 Hz interference,and th e preamplifier band-pass filters may beused.]Cables and connectors (1 et ]Electrode wire (T he wire must be stiff enough topenetrate the M i m o s a stems. Tungsten wire ofabout 40 gauge is good, as is stainless steel kymo-graph pen cleaning wire, e.g., Harvard Ink Clean-ing Wire, no. 808. Silver and copper wire are muchless suitable because they are too limp. Be sure toremove the insulation present on some wires orthe electrodes will not work.]Metal rod and clip lead for ground connection to thesoilFine glass rods or Pasteur pipettes

Forceps with rubber-protected laws or fine rubbertubing slipped over the lawsSurgical scissors, fine and of good qualityFlat-jawed forceps for gripping electrode wireSmall metric rulerMaterials available in the laboratoryOscilloscope camera and filmElectronic stimulatorsSmall fan and rheostat to control it s speedStopwatches

Dissecting microscopeExtra electrode wireHeavy surgical scissors or good quality shears forcuttin g electrode wire (Most wire-cutting pliers

will not cut the wire without bending it.)Desk lamps for strong illuminationCardboard boxes for light shadesRingstandsPlantsM i m o s a pudica (Have2-10 plants available/station.]Occasionally, M i m o s a p u d i c a plants may be

found in plant specialty stores or obtained from ahobbyist. Otherwise they will have to be startedfrom seed at least 2, and preferably 4, month s inadvance. Young plants will produce two leaves perpetiole (fig. 8.7-1) and older plants will producefour. Seeds may be obtained locally on occasion(ask for "sensitive plants"] but i t is usually easierto order the m directly from a supplier (e.g., Caro-lina Biological Supply, no. LB 600).Lightly nickthe seeds with sandpaper before planting. Placeseveral seeds atop damp potting soil in a smallflowerpot. Cover the seeds with a very thin layerof soil, sprinkle with water, and place the potin a covered container until th e seeds sprout(1 to 2 weeks at roo m temperature). Keep theseedlings out of direct sunlight until they havereached a height of about 5 cm. At this point, placethe m in strong light. The use of a 40-watt fluores-cent grow lamp on a ti mer is strongly suggested.Place the lamp 30-50 cm above the plants andsupply them with 16 hour s of ligh t per day. Keepthe soil moist with frequent, but not excessive,watering. Consult a local plant supplier orbotanist if problems are encountered.

Notes for the Instructor1. Ask students to treat the plants with reverence. Ifthey approach the exercise too casually, much tim ewill be wasted with accidental stimulations andfalse starts. A drooped petiole may require 10-30minute s recovery time. Leaflets should be at least75% unfolded before retest. Obviou sly, if mo replants are available, more experimen ts can be run ina given time period.2. Several sets of electrodes can be connecte d to thesame preamplifier through an EKG lead selector orsimilar switching device.


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3. A sensitive force transducer may be used t o recordthe mechanical activity of the plant [petioledroop-ing/ while simultaneously recording the electricalactivity.Selected ReferencesBose, J. C. 1906 Plant response as a means ofphysiologi-cal investigation. Longmans, Green, and Co., LondonBunning, E. 1959. Die seismonastschen Reaktionen InHandbuch der Pflanzenphysiologie [Encyclopedia ofplant physiology), vol. 17 1), ed. W Ruhland. Springer-Verlag, Berlin, pp 184-238Higinbotham, N. 1973. Electropotentials of plant cells.Annu. Rev. Plant Physiol. 24: 25-46Sibaoka, T 1969. Physiology of rapid movements inhigher plants Annu. Rev Plant Physiol 2 0 165-84.Umrath, K. l959a. Der Erregungvorgang. n Handbuch derPflanzenphysiologie [Encyclopedia of plant physiology),

vol. 17(1), ed. W. Ruhland. Springer-Verlag, Berlin, pp24-110.1959b.Mogliche Mechanismen von Krummungs-bewegungen. In Handbuch der Pflanzenphysiologie[Encyclopedia ofplant physiology],vol. 17 (1), ed.

W. Ruhland. Springer-Verlag, Berlin, pp 11 -18RESEARCH REPORTSFondeville, J. C., Schneider, M J .; Borthwick, H A ;andHendncks, S . B 1967. Photocontrol of Mimosa pudicaleaf movement. Planta 75: 228-38.Satter, R. L., and Galston, A . W. 1971 Potassium flux: Acommon featu re of Albizzia leaflet movement con-trolled by phytochrome or endogenous rhythm Science174: 518-20.Sibaoka, T. 1962. Excitable cells in Mimosa. Science 137:226.Toriyama, H., and Jaffe, M. J. 1972. Migration of calciumand its role in the regulation of seismonasty in themotor cell of Mimosa pudica L. Plant

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