evoked potentials in cat extraocular muscle -...

Evoked potentials in cat extraocularmuscle

Pinhas Nemet and James E. Miller

Cat extraocular muscle consisted of a peripheral portion ivith red histochemical properties anda central part with white muscle findings. Recordings were obtained from the two regions byextracellular electrodes. Third nerve stimulation produced action potentials which were pro-longed monophasic units (slow units) along the edge, and short-duration, biphasic spikes(fast units) in the center of the muscle. Third nucleus stimulation induced an immediate fasteye movement and an electro7nyographic pattern similar to III nerve stimulation. Vestibidarcomplex stimidation brought about a sloio movement that did not begin until several secondsafter excitation. The motor unit activity consisted of slow units throughout the muscle whichbegan at the start of stimulation. Intravenous succinylcholine produced fast and intermediatespike activity from all layers but very few slow units. Evoked fast eye movement appearedto be accompanied by fast and slow action potentials while slow vestibular movement wasassociated with only slow units. The motor unit pattern appeared to correlate with the histo-chemical organization and the type of movement.

JL hysiologic studies in extraocular muscleof the cat have shown the presence of dif-ferent types of muscle fibers. Two investi-gators1' - believed that they had identifiedslow fibers by intracellular recording. Thisfinding was not confirmed by anothergroup3 who were only able to demonstratetwitch fibers in the superior rectus and in-ferior oblique of the cat. The latter dividedthe cells into "slow multi-innervated twitchfibers" located near the outer and inner sur-face, and fast fibers in the central portionof the muscle.

Histologic studies have also indicated that

From the Department of Ophthalmology, AlbanyMedical College, Albany, N. Y.

This investigation was supported in part by GrantsNB-04936, NB-05794, and NB-05521 from theNational Institute of Neurological Diseases andBlindness of the National Institutes of Health,United States Public Health Service.

extraocular muscles were not homogeneousthroughout but contained a layer of smallcells around the border and larger cells inthe center.4-0 Muscle cells in the peripheryof guinea pig eye muscle4 had the morpho-logic characteristics of slow fibers. In themonkey5 cells with the histochemical prop-erties of red fibers were located principallyaround the outer portion of the muscle andthose having the features of white fiberswere predominantly in the central part.Electron micrographs indicated that themarginal cells did not contain an M line5

and in this respect resembled the slow fibersof the frog.7 They also had a poorly devel-oped sarcoplasmic reticulum in the A bandregion5 and by light microscopy would beclassified as felderstruktur fibers.

Since past electromyographic studieshave not taken into account the heterogene-ity of extraocular muscle, an attempt wasmade to differentiate between the outer red

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Evoked potentials in extraocidar muscle 593

and inner white portions of the muscle ofthe cat with the use of extracellular elec-trodes. Eye movements were produced bystimulating selected portions of the brain.Fast movements were obtained from IIInucleus excitation and slow movement fromvestibular complex or adjacent reticularformation. The effect of III nerve stimula-tion and succinylcholine was also evaluated.

MethodStimulation. Twenty-four adult cats weighing

2 to 3 kilograms were anesthetized with intra-peritoneal sodium pentobarbital (35 mg. per kilo-gram), supplemented by intravenous injection. Thecats were placed in a Kopf stereotaxic apparatusand portions of the temporal bone, the roof, andthe lateral wall of the orbit on one or both sideswere removed. One or more of the eye muscles(MR, SR, IR, 10, LR) were held at resting lengthby grasping the tendon of insertion with an im-mobile muscle clamp.

In 13 cats the dura was opened and the tem-poral lobe elevated. The III nerve was identifiedand stimulated through a pair of platinum elec-trodes by a Grass S4 stimulator and a 4B stimulusisolation unit. Cathodal stimulus parameters variedfrom 0.5 to 10 v. with the use of frequencies ashigh as 600 per second. A pulse duration of 0.1millisecond was utilized in the majority of experi-ments.

In the second group of 11 cats, insulated mono-polar 22 gauge needles were introduced into thenucleus of the III nerve and the vestibular com-plex or adjacent reticular formation by stereotaxiccoordinates.8 These areas were stimulated withsingle and repetitive pulses at frequencies of 1 to300 per second and voltages of 0.5 to 8 v.

During vestibular complex stimulation, slow eyemovements were observed by inspection and rec-ords were taken with the globe both fixed andmobile. Third nucleus stimulation produced fastmovements of the globe. Displacements were ofsmall amplitude and in the upward and nasaldirection. Most of the recordings were obtainedin an essentially isometric state.

Recording. Recordings were obtained with a31 gauge needle containing two insulated stainlesssteel wires. The electrode was placed in the orbitalaspect (outer fibers), in the global portion (innerfibers), and/or within the center of the muscle.In 18 cats simultaneous recordings from globaland orbital aspects were obtained. In 2 cats themuscles were removed from the globe and theinsertion elevated. The electrode was placed inthe global or the orbital aspect and inserted per-pendicular to the muscle at increments of 0.1 mm.Responses were obtained at every level of pene-

tration until the needle had passed completelythrough the muscle.

The electrode was connected to a combinationof a Tektronix FM-122 preamplifier with a timeconstant of 1 second and a Honeywell differentialpreamplifier providing a total gain of x2,000. Oneto three channels were used for recording. Themuscle response and the stimulus were viewed ona 4 channel Tektronix RM-561A oscilloscope andsimultaneously recorded on a Honeywell 8100FM tape recorder. After the experiment, movingstrip photographs were taken with a Grass C4camera at paper speeds up to 1,000 mm. persecond.

Succinylcholine (10 to 125 Mg per kilogram)was injected by vein in 7 cats to produce musclecontraction. The larger quantities were injectedat tlie end of the experiment.

In 5 cats an electrolytic lesion was madethrough the stimulating electrode in the III nu-cleus, the vestibular complex, or reticular forma-tion. The lesions were obtained with a directcurrent of 30 Ma. for 20 seconds. The locationof the lesions was verified in histologic sectionsstained with methylene blue.

1 mV:

msec

Fig. 1. Stimulation of III nerve adjacent to pons;I stimulation, Calibration for time base and am-plitude is the same for all figures. A, Stimulation:5 v., frequency of 10 per second. Fast motor unitsof different latency recorded from inner portionof medial rectus. B, Stimulation; 4.5 v., frequencyof 100 per second. Recording from inferior rectus.Upper trace: fast action potentials in global as-pect. Lower trace: slower units in orbital (outer)aspect.


594 Nemet and Millet Investigative OphthalmologyOctober 1968

I \

Fig. 2. Stimulation of III nerve. Frequency of 10 per second. Recordings from one areaadjacent to the globe. Lower stimulating voltage produced a single prolonged motor unit;higher voltage induced motor units of short duration. A, Stimulation: 4 v. Slower singlemotor unit. B and C, Stimulation: 4.5 and 5 v., respectively. Faster response recorded.

Fig. 3. A, Fast units from inner portion of superi-or rectus obtained by stimulation of III nucleus(4.5 v., 50 per second). B, Motor units of longerduration recorded from outer layer of medialrectus (3.5 v., 100 per second). This was charac-teristic of vestibular complex stimulation, Similarunits were recorded from all layers of the muscle.

Results

Stimulation of III nerve. Recordings fromthe global and central or orbital parts of themuscle showed two different types of motorunits (Fig. 1). The first type (Fig. 1, A)had a biphasic or polyphasic spike form(fast unit). Duration was 0.2 to 0.5 milli-second and amplitudes measured 50 juv to1.5 mv. The faster motor units were foundin the global portion and the center of themuscle and were recorded from the outeraspect in only two experiments. The secondtype of motor unit (Fig. 1, B, lower trace)was monophasic, longer in duration (0.5 to3 milliseconds), and usually lower in am-plitude (50 to 700 fiv). The latter responsewas recorded mainly from the outer partof the muscle and was considered to rep-resent slow units.

The latency of both types was 1 millisec-ond in response to the lowest stimulating

voltage. Higher voltages produced manymotor units (2 to 14) in the central andglobal portions of the muscle for each stim-ulus, with a delay of 1 millisecond for thefirst motor unit followed by a train of actionpotentials extending over 5 milliseconds.Thus there was a distribution of latenciesindicating a gradation of nerve and muscleconduction velocities.

The most frequent response to III nervestimulation was of the faster type in theglobal portion and the slower type in theorbital aspect (Fig. 1, B). Occasionally theslower type response was obtained in theglobal or central part with low stimulatingvoltage. An increase in stimulus strengthproduced a fast response which obscuredthe slower one (Fig. 2). In the orbital por-tion the slower motor units were observedwith both low and higher voltage.

Stimulation of III nucleus. Stimulation ofthe III nucleus gave essentially the sameresponse as III nerve stimulation, withslower motor units in the orbital aspectand faster units in the center and globalportions (Fig. 3, A). Fast responses weresometimes recorded from the orbital layers.

Stimulation of vestibular complex. Stimu-lation in the region of the vestibular com-plex caused slow eye movements. Motorunits of the slower type (Fig. 3, B) wererecorded from the orbital and global as-pects and the belly of the muscle. Stimula-tion was often accompanied by action po-tentials occurring at irregular intervalswhich were not directly related to stimulusfrequency. Spontaneous slow units often ap-peared after stimulation was stopped. Sloweye movements did not occur until several


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Evoked 'potentials in extraocidar muscle 595

A B C

Fig. 4. Spontaneous activity. A, Fast units, B, Intermediate units. C, Slower action potentials.

Fig. 5. Comparison of spontaneous activity following administration of succinylcholine andvestibular stimulation. Upper trace: outer layer of muscle. Lower trace: inner layer of muscle.A, Spontaneous activity after 20 fig succinylcholine intravenously. B, 250 /*g succinylcholineintravenously in same cat. C, Spontaneous activity following vestibular nucleus stimulationwith 4 v., frequency of 300 per second. Same spontaneous activity followed different fre-quencies.

Fig. 6. Cross sections of cat superior rectus x30, prepared to demonstrate «GPD and succinicdehydrogenase. The top of the figure is the outer portion of the muscle; the lower part isthe center of the muscle. The central portion contains a mixture of cells varying in activity.A, Histochemical preparation for «GPD. The cells nearest the periphery had the least activityand the majority of the central fibers were darker indicating greater reaction. B, Succinic de-hydrogenase. The outer portion was more reactive than the center.


596 Nemet and Miller Investigative OphthalmologyOctober 1968

seconds of stimulation had elapsed, whilemotor unit activity began with the firststimulus. This was in contrast to the fasteye movement which appeared almost im-mediately after stimulating the III nucleus.

Spontaneous activity. Spontaneous activ-ity was recorded from the global and orbit-al aspects of the muscle. This activity ap-peared to be peripheral since it was stillpresent when the III nerve was severed atthe brain stem. The form of the spontane-ous motor units was usually a biphasicspike wave extending over 0.2 to 0.5 milli-second. The amplitude for all types rangedfrom 50 /w to 1.2 mv., and the durationfrom 0.2 to 2.5 milliseconds (Fig. 4).

The intravenous administration of suc-cinylcholine (10 to 125 fxg per kilogram)was followed by activity occurring in boththe inside and outside of the muscle with aduration of 0.2 to 1.5 milliseconds and anamplitude of 50 /xv to 1.3 mv. (Fig. 5, Aand B). Motor units were usually biphasicspikes and the activity lasted from 3 to 15seconds.

Discussion

Muscle fibers that are considered slow inphysiologic terms have been identified inlower vertebrates and invertebrates. Thesecells undergo local partial depolarizationadjacent to the motor end plate and do notpropagate an action potential. It is neces-sary to record the restricted membrane al-terations by intracellular electrodes. How-ever, a group of slow fibers capable ofpropagating an action potential have beenidentified in birds. The response was mono-phasic, prolonged, and graded when mea-sured by extracellular electrodes.9"11 Intra-cellular action potentials12 and motorunits13 of similar configuration were ob-tained from rabbit and cat extraocularmuscle following nerve stimulation andlabyrinthine excitation. It was also pro-posed that the slow multi-innervatedtwitch fibers of eye muscle were similar toavian slow fibers.3

In addition to the variation of action po-tentials among slow fibers, alterations in

the form of twitch potentials were observedbetween red and white cat skeletal muscle.The rise time to peak was prolonged in redtonus fibers and the amplitude was lesswhen compared with white phasic fibers.14

The resting potential was lower and theaction potential more prolonged in red so-leus muscle of the rat than in the whiteextensor digitorum longus.15 Red twitch fi-bers more closely resembed slow fibers intheir response to acetylcholine applied tothe surface membrane.16 Depolarization de-veloped outside of the motor end-platezone, provided higher concentrations of thecholinergic agent were used. In contrast,white fibers did not respond outside of themotor end-plate region.

Histochemical preparations of cat extra-ocular muscle indicated that the outer por-tion consisted of cells resembling red fibers(Fig. 6) since this layer had more activityfor succinic dehydrogenase and less foralpha glycerophosphate dehydrogenase(aGPD).17 The center and global surfacehad the histochemical properties of whitemuscle with the predominating cells havinga greater reaction for «GPD and less forsuccinic dehydrogenase. Also included inthe inner portion were cells varying fromred to white. Thus, within the muscle therewere sporadic fibers which resembled thered cells located about the periphery. Ifthe red cells are capable of propagating anaction potential, it would be expected thatthe response would be of long duration,similar to avian slow muscle. Althoughslower motor units with prolonged actionpotentials would predominate around theouter portion of the muscle, they wouldalso occur in the inner portion because ofthe presence of red fibers. Action poten-tials of shorter duration would be locatedprincipally in the inner portion of the mus-cle where the white fibers are concentrated.A response pattern similar to the histo-chemical picture was obtained with intra-cellular electrodes.3 Slow multi-innervatedfibers were present on the orbital and glo-bal margins of the muscle while the fastergroup was located in the center.


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Evoked potentials in extraocular muscle 597

Since the present study utilized extracel-lular electrodes, only fibers that propagatedan action potential were recorded. How-ever, again the topographical arrangementof the action potentials obtained by the IIInerve and III nucleus stimulation were sim-ilar to past results obtained by intracellularrecording and were compatible with thehistochemical studies. In addition, with ves-tibular complex or reticular formation stim-ulation, motor units of prolonged durationwere consistently present about the periph-ery, but were also distributed throughoutthe muscle.

It can also be observed (Fig. 1 A, 2, C,3, A) that stimulation of the III nucleus ornerve initiated a series of motor units withvarying latencies. The first motor unit wasobserved 1 millisecond after stimulationwhile the last occurred 5 to 6 millisecondslater. This signifies that an evoked fastmovement is produced by motor units withfast, intermediate, and slower conductingvelocities, and presumably varying contrac-tile properties. At the same time, the slowermotor units were occurring in the peripheryof the muscle. Thus fast movement ap-peared to be a mixture of slow, interme-diate, and fast components while the slowervestibular-induced movement was accom-panied solely by units low in amplitude andprolonged in duration. Vestibular move-ment also appeared to be graded in that itdid not occur until several seconds of stimu-lation had elapsed.

The succinylcholine experiments indi-cated that the faster motor units, not theslow group, were activated by this agentsince the contracture was accompanied byaction potentials of short and intermediateduration. Furthermore, it was often possi-ble to stimulate the vestibular or reticularformation while the induced contractionwas still in effect and obtain the mono-phasic slower units.

Another indicator of a spectrum of motorunits was spontaneous activity. Some motorunits were of very short duration (Fig. 4,A), others intermediate (Fig. 4, B), andthe long action potential noted with vestib-

ular stimulation was also observed (Fig.4,C).

In two III nerve experiments, fast re-sponses were obtained from the orbitalaspect and were occasionally observed afterIII nucleus stimulation. These exceptionsmay have been due to the limitations ofthe recording system. The diameter of therecording electrode was approximately thewidth of the fine cell layer. It was difficultto maintain the needle in or adjacent tothis fine layer, and the contraction mayhave resulted in the passage of the needleinto the more interior part of the muscle.

Recordings from the central aspect oc-casionally gave slower responses for lowstimulating voltage and faster response forhigher voltage (Fig. 2). Fibers having thesame enzymatic reactions as the outer fiberswere foimd throughout the muscle (Fig. 6).The recording electrode may have beennear red fibers that were excited by a lowlevel of stimulation. As the amplitude ofstimulation was increased, the predomi-nating white fiber responses would thenoverride the initial response. A low thresh-old for response implies that the slow motorunits are supplied by heavily myelinatednerves. This correlates with past histologicdeterminations of eye muscle showing thatmultiple motor end plates of en grappeconfiguration were often derived from thickmedullated nerves.18'20

The present study does not indicatewhether nonpropagating slow fibers arefound in extraocular muscle. It does illus-trate, however, that it is possible to produceaction potentials of different configurationby stimulating regions of the brain thatwill induce slow or fast eye movement. Itwas also shown that the variations in actionpotentials coincides with the histologiccharacteristics of the muscle. Potentials oflonger duration were more prevalent in thered portion while the white component wasmore complex since it contained a varietyof motor units varying in duration and la-tency.

The technical assistance of Mrs. Edith Patnode


598 Nemet and Miller In vestigative OphthalmologyOctober 1968

and Mrs. Lois Leebrick is gratefully acknowl-edged.

REFERENCES1. Hess, A., and Pilar, G.: Slow fibres in the

extraocular muscles of the cat, J. Physiol.169: 780, 1963.

2. Pilar, G.: Further study of the electrical andmechanical responses of slow fibers in catextraocular muscle, J. Gen. Physiol. 50: 9,1967.

3. Bach-y-Rita, P., and Ito, F.: In vivo studieson fast and slow muscle fibers in cat extra-ocular muscles, J. Gen. Physiol. 49: 1177,1966.

4. Hess, A.: The structure of slow and fastextrafusal muscle fibers in the extraocularmuscles and their nerve endings in guineapigs, J. Cell. & Comp. Physiol. 58: 63, 1961.

5. Miller, J. E.: Cellular organization of rhesusextraocular muscle, INVEST. OPHTH. 6: 18,1967.

6. Peachey, L. D.: Fine structure of two fibertypes in cat extraocular muscles, J. Cell. Biol.31: 84A, 1966.

7. Peachey, L. D., and Huxley, A. F.: Structuralidentification of twitch and slow striated mus-cle fibers of the frog, J. Cell. Biol. 13: 177,1962.

8. Snider, R. S., and Niemer, W. T.: A stereo-taxic atlas of the cat brain, Chicago, 1964,The University of Chicago Press.

9. Cinsborg, B. L., and Mackay, B.: The latis-simus dorsi muscles of the chick, J. Physiol.153: 19P, 1960.

10. Ginsborg, B. L., and Mackay, B.: A histo-chemical demonstration of two types of motorinnervation in avian skeletal muscle, Bibliot.Anat. 2: 174, 1961.

11. Zelena, J., Vyklicky, L., and Jirmanova, I.:Motor end-plates in fast and slow muscles ofthe chick after cross-union of their nerves,Nature 214: 1010, 1967.

12. Matyushkin, D. P.: Motor systems in theoculomotor apparatus of higher animals, Fed.Proc. (Trans. Suppl.) 23: T1103, 1964.

13. Baichenko, P. I., Matyushkin, D. P., andSuvorov, V. V.: Participation of fast andtonic oculomotor systems in stretch reflexesand labyrinthine reflexes of extraocular mus-cles, Neurosc. Trans. 3: 350, 1967-68.

14. Buller, A. J., Lewis, D. M., and Ridge, R.M. A. P.: Some electrical characteristics offast twitch and slow twitch skeletal musclefibres in the cat, J. Physiol. 180: 29P, 1965.

15. Yonemura, K.: Resting and action potentialsin red and white muscles of the rat, Jap. J.Physiol. 17: 708, 1967.

16. Miledi, R., and Zelena, J.: Sensitivity toacetylcholine in rat slow muscle, Nature 210:855, 1966.

17. Pearse, A. G. E.: Direct relationship of phos-phorylase and mitochondrial alpha-glycero-phosphate dehydrogenase activity in skeletalmuscle, Nature 191: 504, 1961.

18. Hines, M.: Studies on the innervation ofskeletal muscle. III. Innervation of the ex-trinsic eye muscles of the rabbit, Am. J.Anat. 47: 1, 1931.

19. Feindel, W., Hinshaw, J. R., and Weddell,G.: The pattern of motor innervation in mam-malian striated muscle, J. Anat. 86: 35, 1952.

20. Garven, H. S. D.: The nerve-endings in thepanniculus carnosus of the hedgehog, withspecial reference to the symphathetic inner-vation of striated muscle, Brain 48: 380,1925.


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