electrophysiological identification of two types of fibres in rat

J. Phyeiol. (1979), 290, pp. 453-465 453With 7 text-figurewPrinted in Great Britain

ELECTROPHYSIOLOGICAL IDENTIFICATION OF TWO TYPES OFFIBRES IN RAT EXTRAOCULAR MUSCLES

BY D. J. CHIARANDINI AND E. STEFANI*From the Department of Ophthalmology, New York Univer8ity Medical Center,

New York, N.Y. 10016, U.S.A.

(Received 26 June 1978)

SUMMARY

1. The synaptic potentials and electrical properties of rat inferior rectus muscleswere examined in vitro.

2. In most fibres the spontaneous synaptic activity consisted of typical miniatureend-plate potentials which had a normal distribution of amplitudes and ratheruniform time courses. Suprathreshold and maximal nerve stimulation evokedunitary end-plate potentials (e.p.p.s). The synaptic activity of these fibres could berecorded only in the innervation zone of the muscle. These fibres were identified asbeing focally innervated.

3. Focally innervated fibres gave action potentials upon direct and indirectstimulation. They had an effective resistance (Reff) of 1'62 + 022 MQ (mean + S.E.,twenty-two fibres) and a time constant (Tm) of 3-8 + 04 msec (twenty-one fibres).Voltage-current curves in control saline were linear between membrane potentials of-50 to -140mV.

4. In a small number of fibres the spontaneous synaptic activity consisted ofminiature small-nerve junction potentials which had a skewed distribution ofamplitudes with predominance of smaller voltages and time courses with a widerange of variation. Nerve stimulation evoked composite small-nerve junctionpotentials (s.j.p.s) which could be resolved into unitary components by varying thestrength of stimulation. S.j.p.s had a higher threshold than e.p.p.s. Synapticpotentials could be recorded outside the innervation zone, at various sites alongthe muscle length. These fibres were recognized as being multiply innervated withpolyneuronal innervation.

5. Multiply innervated fibres lacked action potentials, had a large Reff of 6-0 +1-1 MD (six fibres) and a prolonged Tm of 29-8 + 4*8 msec. Reff show a moderatedecrease to hyperpolarization and a rather large decrease to depolarization whichdenote, respectively, the presence of anomalous and delayed rectification.

6. It is concluded that rat extraocular muscles contain at least two populations ofmuscle fibres that in terms of synaptic activity and electrical properties are com-parable to twitch fibres of other mammalian muscles and to slow or tonic fibres ofamphibians.

* Permanent address: E. Stefani, Departmento de Fisiologia, Centro de Investigacionesdel IPN, Apartado Postai 14-740, Mexico DF 14, Mexico.

0022-3751/79/3330-0574 $01.50 © 1979 The Physiological Society

D. J. CHIARANDINI AND E. STEFANI

INTRODUCTION

The synaptic activity and some of the electrical properties of extraocular muscleshave been studied previously in cat and rabbit (Hess & Pilar, 1963; Matyushkin,1964; Bach-y-Rita & Ito, 1966; Lennerstrand, 1974). Based on the characteristics ofthe synaptic potentials, Hess & Pilar (1963) and Matyushkin (1964) concluded thatextraocular muscles contain primarily two types of fibres: focally and multiplyinnervated. The first type exhibits action potentials and is equivalent to twitchfibres of other vertebrate muscles. The second type lacks action potentials and isthus comparable to slow or tonic fibres of amphibians. This fibre type is not found inother muscles of mammals with the exception of the ear muscles (Fernand & Hess,1969) and oesophagus musculature (Floyd, 1973).Bach-y-Rita & Ito (1966) and Lennerstrand (1974) have reported that in cat

extraocular muscles a large number of fibres are multiply innervated and have actionpotentials. The existence of such fibre type has been questioned on the basis that thecriteria used for their identification were not specific enough (Pilar, 1967; Barmack,Bell & Rence, 1971; Browne, 1976). Nevertheless, a very small number of fibres thatare multiply innervated and capable of firing action potentials has been identifiedelectrophysiologically in cat (Hess & Pilar, 1963) and in rabbit extraocular muscles,in a combined histological and electrophysiological study (Ozawa, Cheng, Davidowitz& Breinin, 1969).The present study was undertaken to characterize the synaptic activity and

electrical properties of rat extraocular muscles using the inferior rectus muscle 'invitro', a preparation used previously to study some of the contractile properties ofextraocular muscles (Chiarandini, 1976). The global layer of the muscle (Mayr, 1971)was explored and two types of fibres were recognized. Most of the fibres were focallyinnervated and had synaptic activity and electrical properties comparable to thoseof twitch fibres of other mammalian muscles. A smaller number of muscle cells weremultiply innervated with polyneuronal innervation. In various respects these fibresare akin to slow or tonic fibres of amphibian muscles.

METHODS

Experiments were made on inferior rectus muscles of Wistar rats weighing 125-175 g. Theanimals were anaesthetized in most cases with Na thiamylal and occasionally with ether. Noapparent difference was observed between the two procedures and the barbiturate anaesthesiawas preferred. The muscle was dissected attached with its proximal end to a fragment of thepresphenoid bone and its distal end to the sclera.

Conventional techniques for intracellular recording and current injection were used. Recordingmicropipettes were filled with 3 M-KCl, had a resistance of 40-50 MQ and tip potentials of -5 mVor less. Although these micropipettes gave rather noisy records they were preferred to thosewith lower resistance which usually damaged the fibres, most likely because of the small celldiameter (10-40 ,um (Mayr, 1971)). Current injecting micropipettes were filled with 2 M-Kcitrate. The micropipettes were placed 50-200 ,um apart. Rectangular pulses of current witha duration of 30-125 msec were used to stimulate directly the fibres and to measure the effectiveresistance between the inside and the outside of the cells (Fatt & Katz, 1951). In most fibresdirect current was also applied to polarize the fibres to membrane potentials of -70 to -80 mV.The injected current was measured with an operational amplifier in the ammeter configuration,which was connected to the experimental chamber via a Ag-AgCl wire. Most of the studied

454

ELECTRICAL PROPERTIES OF EXTRAOCULAR MUSCLES 455

fibres were located on the superficial layers of the global region of the muscle. They were impaledeither in the region where the nerve enters and branches (innervation zone) or in the distal thirdof the muscle.

Synaptic potentials and action potentials were evoked by stimulating the nerve to the inferiorrectus muscle with 0 3 msec pulses via a suction electrode connected to the stimulus isolationunit of a stimulator. Stimulation voltages were adjusted as required. Extracellular stimulationof the fibres was carried out with a pair of nichrome wires connected to an isolation unit andplaced near the muscle surface.The muscles were bathed with a saline containing (mM): NaCl, 136; KCl, 5; CaCl1, 10; MgSO4,

1 2; glucose, 11; and imidazole sulphate 5. The relatively high Ca concentration was used toimprove the condition of the tissue. Isotonic Ca solution was prepared by replacing all the NaClof the saline by CaCl2 and adding sucrose to maintain the osmolarity. The pH of the bathingsolution was 7-35. The solution was oxygenated constantly and flowed at a rate of about 2 ml./min through the chamber. Experiments were conducted at room temperature (21-25 00). Underthese conditions the synaptic activity remained normal for several hours.

RESULTS

Two distinct populations of muscle cells were distinguished in the inferior rectusmuscle of the rat by their synaptic activities and electrical properties. One of thepopulations comprised a very small proportion of the muscle fibres which werecharacterized by their lack of action potentials, their slow membrane time constant(range 18-50 msec) and the presence of multifocal synaptic activity. The otherpopulation included most ofthe muscle cells. These had overshooting action potentials,a fast membrane time constant (range 1-4-8-3 msec) and focal synaptic activity.

Multiply innervated fibresSpontaneous synaptic potentials. The recording of spontaneous synaptic activity

provided a direct and unequivocal means to recognize the presence of multifocalinnervation in this fibre population. The synaptic activity could be recorded in varioussites along the muscle's length and it consisted of potentials with a markedly widerange of rise- and half-decay times. These potentials are equivalent to the spontaneoussynaptic potentials of frog slow fibres (Burke, 1957) and will be referred to asminiature small-nerve junction potentials (min.s.j.p.s) following the nomenclatureused by Hess & Pilar (1963) in the case of cat extraocular muscles. As in frog slowfibres, the considerable variability in the time course of min.s.j.p.s is determined bythe different distances between the site of origin of the miniature potentials and therecording electrode (Burke, 1957).The characteristics of the min.s.j.p.s were studied in detail in several fibres. The

frequency of occurrence ofthese potentials varied from 0 9 to 3*1 sec1. The amplitudesranged from 0 3 to 5 4 mV and had a skewed distribution with a marked predominanceof smaller amplitudes (Fig. 1, right). A considerable number of min.s.j.p.s were verysmall and difficult to distinguish from the base line noise and, therefore, some un-certainty is present in the values given below. An outstanding property of themin.s.j.p.s was the above mentioned variation of their time courses. Potentials withfast and slow time courses occurred in an intermingled fashion in every fibre (Fig. 1,left). There was no clear relationship between rise time and amplitude of the potentials(Fig. 2). Min.s.j.p.s with very similar rise times but with very different amplitudeswere commonly observed. The over-all variation of rise and half-decay times in all

456 D. J. CHIARANDINI AND E. STEFANI

fibres was markedly wide. It ranged, respectively, from 2*5 to 90*0 msec and from2*5 to 80*0 msec. Even when considering individual fibres the variation of bothparameters was still rather wide. For instance, in two fibres the rise time rangedfrom 2*5 to 32*5 msec and from 10 to 75 msec while the half-decay time, in the samefibres, ranged from 3*5 to 17 msec and from 15 to 80 msec.Evoked synaptic potentials. Further evidence for multiple innervation in fibres

which exhibited min.s.j.p.s was obtained from experiments carried out with nerve-muscle preparations. The application of above threshold stimuli to the nerve of theinferior rectus muscle evoked composite small-nerve junction potentials (s.j.p.s)

150 r

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0

._

._

E

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100 -

50

0 1 2 4mV

5

Fig. 1. Spontaneous synaptic activity in a multiply innervated fibre. Left: continuousrecording showing several min.s.j.p.s with various amplitudes and time courses. Right:distribution of min.s.j.p. amplitudes in the same fibre. The distribution of amplitudesis skewed with a predominance of smaller min.s.j.p.s.

5 -

4 _F

3

E2

0

! I* .

4 ei20 40

msec

T

60 80

Fig. 2. Relationship between the amplitudes and rise times of min.s.j.p.s recorded fromone multiply innervated fibre. Ordinate: amplitude of min.s.j.p.s. Abscissa: rise time ofmin.s.j.p.s. There is no clear relationship between both parameters.

.~~ .o.-.-

-glw-

1 00 msec

I

.

I

i ilj

-h-l ,


which could be separated into components with different thresholds and time coursesby varying the strength of stimulation to the nerve.A composite s.j.p. evoked by maximal nerve stimulation is shown in Fig. 3. In

the upper record it can be seen that the stimulus artifact is closely followed by a fastinward going deflection which corresponds to the extracellularly recorded actionpotential fired by neighbouring focally innervated muscle fibres. This, in turn, is

Fig. 3. Compound s.j.p. in a multiply innervated fibre. Records obtained with twodifferent sweep speeds. Supramaximal stimulation of the nerve evoked a s.j.p. withtwo components which had different rise times. The downward deflection recordeda few msec before the beginning of the s.j.p., seen more prominently in the top record,corresponds to the action potential fired by adjacent focally innervated fibres recordedextracellularly.

followed by the two components of the synaptic potential of the impaled cell whichhave different rise times. The lower record shows the same s.j.p. at slower sweepspeed. The observation that the action potential of the neighbouring fibres occursa few msec before the synaptic potential indicates that motor axons eliciting thecomponents of the s.j.p.s have a lower conduction velocity than axons to the focallyinnervated fibres. No attempt was made to measure the conduction velocity of theaxons because the site of stimulation could not be determined precisely enough towarrant such calculation. However, when s.j.p.s and end-plate potentials wererecorded in the same preparation it was found that the latencies of the former wereabout 5 times those of the latter. Furthermore, the motor axons to multiply inner-vated fibres had a higher threshold than the axons to focally innervated fibres. Inthree cells the threshold of the s.j.p.s was 2-4, 4-2 and 5*0 V, 3-6 times higher thanthe threshold of end-plate potentials measured in the course of the same experiments.

Electrical properties. The resting potential (Erv) measured initially after the pene-tration with the recording micro-electrode was - 63-5 + 4*8 mV (mean + s.E. of eightfibres). In most cases, however, the Erp declined gradually up to 15-20 mV in thefollowing 10-15 min. To study the electrical properties at more physiological levelsof polarization, inward constant current was applied through the second intracellularmicropipette to repolarize the fibres to a membrane potential of -70 to -80 mV.

Fig. 4A shows depolarizing responses to outward current pulses. This cell had an

458 D. J. CHIARANDINI AND E. STEFANIinitial resting potential of -70 mV and was repolarized to -75 mV. The first andsmallest pulse failed to evoke a response but subsequent larger pulses evoked 'local'oscillatory responses, which became faster with stronger pulses. Action potentialscould not be evoked in any of the eight fibres studied, even though the cells weredepolarized to near 0 mV. For comparison, an action potential evoked by intracellularstimulation in a focally innervated fibre is shown in Fig. 4C.

A C20 mV 120 mV

10 nA1-0msec _10nA

B D 2 msec

10mV20 msec

| 10 nA -5 msec

Fig. 4. Comparison of the electrical properties of multiply and focally innervated fibres.A, Multiply innervated fibre which had an initial Erp of -70 mV and was polarized to-75 mV. Depolarizing pulses of increasing intensity failed to induce an action potentialbut evoked a 'local' response which was followed by an oscillation of the membranepotential. This response should be compared with the action potential induced bysimilar pulses in a focally innervated fibre (C). B, multiply innervated fibre which hadan initial Erp of -60 mV and was polarized to -80 mV. The records demonstrate thecharacteristically high Reff and long Tm of multiply innervated fibres, which in this fibreare approximately 4-3 MfQ and 28 msec. These records should be compared with thoseshown in D which were obtained in focally innervated fibre at a sweep speed 4 timesfaster.

Fig. 4B shows voltage responses to inward pulses of current in another multiplyinnervated fibre. From these experiments voltage-current curves were obtained andone of them is shown in Fig. 5A. The effective resistance (Reff) in six of these fibres,at a membrane potential close to -70 mV, was 6-0 + 11 MO. In control saline thevoltage-current relationship for membrane potentials between -50 and -90 mVwas approximately linear (Fig. 5A filled circles) but below -90 mV it showed asmall decrease in resistance to hyperpolarization or 'anomalous rectification' (Katz,1949) and a rather large decrease in resistance to depolarization above -50 mV,which can be explained by the K-delayed rectifier. Fig. 5A also shows the effects ofisotonic Ca solution on the voltage-current relationship (open circles). As in frog slowor tonic muscle fibres an elevated Ca concentration in the bathing saline produceda marked increase of the membrane resistance (Stefani & Steinbach, 1969). The timeconstant of the cell membrane, Tm) was 29-8 + 4-8 msec (six fibres) in normal saline.


This value was obtained assuming that the fibres behave electrically as infinite cables(i.e. rm was measured as the time to reach 84% of the steady voltage change).

The high effective resistance and relatively short length of the fibres, about 10 mm, raise thepossibility that the fibres may behave as short cables. In this case the above given value of %mwould be over-estimated since the percentage of the final value of the voltage deflexion at which%m should be measured is less than 84% and depends on the ratio between the length of the fibresand their space constant (Stefani & Steinbach, 1969). To establish whether the infinite or the

AB-10

> -30 -40 - PE

,I | | S I II . I

-30 -20 -10 10 20 30 -60 -40 -20 20 40' -90 nA ,i-100 nA

o -110 -120

/ -130 -140

>o -150 --160

Fig. 5. Voltage-current relationships in normal (*) and isotonic Ca salines (0) A,multiply innervated fibre which had an initial Erp of -60 mV and was polarized to-70 mV. The resistance was relatively constant for membrane potentials between -50and -90 mV but decreased at more depolarized and hyperpolarized levels of potential.R,,f measured at -70 mV was about 4-0 MCI and it was increased by the isotonic Casaline to about 11-0 MI). B, focally innervated fibre with an Erp of -72 mV which waspolarized to -80 mV. The voltage-current relationship was linear in normal saline.The isotonic Ca saline increased slightly Rdf measured at -80 mV from 1-4 to 1-8 MC.

short cable model is more suitable for these fibres it is necessary to know their space constant, aparameter not measured in these experiments. The value of the space constant could have beenobtained from the effective resistance by applying cable equations. For this calculation, however,it would have been necessary to assume the fibre radius and specific myoplasmic resistivitywhich introduces large uncertainties in the computation (Katz, 1948) and makes the procedurerather questionable. This approach, therefore, was not pursued.

Focally innervated fibresSpontaneous synaptic potentials. Typical miniature end-plate potentials (min.-

e.p.p.s), as observed in amphibian and mammalian focally innervated fibres (Fatt &Katz, 1952; Liley, 1956) were recorded only when fibres were impaled in the inner-vation zone of the muscle, which lies approximately in the junction of the proximaland middle thirds of the muscle.

Figure 6 shows recordings of min.e.p.p.s in one focally innervated fibre and anhistogram representing the frequency distribution of their amplitudes. The meanfrequency of occurrence of min.e.p.p.s varied from cell to cell and ranged from 1-2 to11 -0 sec-'. The amplitude of min.e.p.p.s fluctuated from 0-3 to 2-5 mV and had anormal distribution (Fig. 6, right). The overall range of variation of the rise and half-decay times in all fibres was narrow, from 0-6 to 5-6 msec and from 0O8 to 4-4 msec,

D. J. CHIARANDINI AND E. STEFANI

respectively. In individual fibres these ranges were narrower. For instance, in twofibres the rise time varied from 0-8 to 2*4 msec and from 1.5 to 4 0 msec, while inthe same cells the half-decay time ranged from 0-8 to 2-5 msec and from 1P3 to3-4 msec.Evoked synaptic potentials. End-plate potentials (e.p.p.s) (Fatt & Katz, 1951) were

evoked by nerve stimulation in nerve-muscle preparations in which synaptic trans-mission was partially blocked with 1 0 to 2 0 x 10-6 M D-tubocurarine to prevent thefiring of action potentials by the fibres. E.p.p.s were recorded in the innervation zone.Their amplitude varied among different fibres from 1 to 10 mV. The rise and half-

150 -

100

E 50

10 msec 0 2

mV

Fig. 6. Spontaneous synaptic activity in a focally innervated fibre. Left: continuousrecording of min.e.p.ps which have a rather uniform time course. Right: distributionof min.e.p.ps amplitudes in the same fibre. In these fibres the distribution is normal.

decay times of the e.p.p.s had rather uniform values from 1t0 to 2-8 msec and from1-2 to 3-8 msec. The latency of the e.p.p.s, measured from the beginning of thestimulation artifact, ranged from 1 2 to 3'5 msec and their threshold ranged from0 4 to 0*8 V.

Fig. 7 shows superimposed e.p.p.s obtained with nerve stimulation slightly abovethreshold (upper record), and with stimulation 4 times threshold (lower record). Thestronger stimulation failed to evoked more components in the e.p.p. indicating thatthe fibre was innervated by a single motor axon. This observation was confirmed inmany other fibres, though in one case, an apparent second component appeared witheight times threshold stimulation. In this fibre the time course of the two componentswas identical which suggests a repetitive firing of the same nerve ending and nota composite e.p.p.

Electrical properties. The Erp measured immediately after the penetration of themicro-electrode was - 67-5 + 1*0 mV (mean + S.E., 153 fibres). To study the electricalproperties, the fibres were polarized routinely to a membrane potential of - 70 to- 80 mV passing direct current through the second micro-electrode. In twenty-twofibres Reff was 1 62 + 0-22 MIQ. These fibres gave action potentials after direct or

460


indirect stimulation. Fig. 4C shows an action potential evoked by depolarizing thefibre to about -50 mV with a pulse of outward current. In eight fibres which had anErp of - 69-6 + 2-5 mV, the action potential evoked by direct stimulation had an over-shoot of 26-5 + 2-8 mV and a duration of 0 70 + 0 03 msec, measured at 0 mV level.Tetrodotoxin (6 x 10-7 M) reversibly blocked the action potentials in few minutes.Fig. 4D shows voltage responses to hyperpolarizing pulses. Tm, the time taken bythe membrane potential to reach 84% of the full deflexion, was 3-8 + 0 4 msec intwenty-one fibres.

*~~~~~~~~~~~~~~~~ /|5 mV

\ ~ /~ 1 msec

Fig. 7. Evoked synaptic release in a focally innervated fibre. Synaptic transmission waspartially blocked with D-tubocurarine. Upper record: several superimposed e.p.p.sevoked by threshold stimulation. Lower record: stimulation four times threshold failedto evoke further components in the e.p.p. indicating that the fibre is focally innervated.

In seventeen fibres the voltage-current characteristics of the cell membrane werestudied for a membrane potential range between - 160 and -40 mV. Fig. 5B showsthe results obtained in a fibre which had a spontaneous Erp of -72 mV. The voltage-current relationship in control saline (filled circles) was linear. This was a consistentfinding in all the fibres studied. In Fig. 5B it is also shown that in the same fibre theisotonic Ca saline increased Rerf only slightly. In one fibre in which the action potentialmechanism was inactivated with TTX, the voltage-current characteristic was studiedat membrane potential levels of from -70 to -10 mV. Reff decreased markedlywhen the membrane potential was less negative than -50 mV, indicating, asexpected, the presence of delayed rectification.

Correlation between action potentials and innervation. In another series of experi-ments it was attempted to establish what kind of synaptic activity was present inmuscle fibres capable of firing action potentials. It was found that from a total ofninety-eight fibres which responded with spikes to indirect stimulation, eighty-sevenexhibited min.e.p.p.s while eleven did not show any spontaneous synaptic potentials.Furthermore, in another series ofmuscles, in which synaptic transmission was partiallyblocked with D-tubocurarine, all of the eighteen fibres that responded with actionpotentials to direct stimulation showed typical e.p.p.s upon nerve stimulation.

D. J. CHIARANDINI AND E. STEFANIDISCUSSION

The present study demonstrates that in rat extraocular muscles there are twomain populations of muscles fibres with different synaptic activities and electricalproperties. One of them includes focally innervated fibres while the other comprisesmultiply innervated fibres.

Recently, Chiarandini (1976) studied tensions evoked in rat extraocular musclesby elevated K concentrations and found two kinds of contractile responses: phasicand tonic tensions. He concluded that these responses are due to the activation ofmuscle cells which have, respectively, the contractile characteristics of focallyinnervated or twitch fibres of amphibians and mammals and of multiply innervatedor tonic fibres of amphibians. The two types of fibres identified electrophysiologicallyin this study correspond, most likely, to the two types previously recognized by theircontractile properties.Most of the fibres analysed were focally innervated and had synaptic potentials

which were essentially identical to those of twitch fibres ofmammalian muscles (Boyd& Martin, 1956a, b; Liley, 1956) and of cat extraocular muscles (Hess & Pilar, 1963;Bach-y-Rita & Ito, 1966). The electrical properties of the focally innervated or twitchfibres of inferior rectus, namely, overshooting action potentials, brief Tm and a linearvoltage-current relationship, are characteristics common to other mammalian twitchfibres (Boyd & Martin, 1959; Kiyohara & Sato, 1967). The apparent lack of 'anoma-lous rectification' in focally innervated fibres of rat inferior rectus could be explainedby a large Cl conductance which could shunt any change in the K conductancebrought about by hyperpolarization of the cell. Twitch fibres of rat diaphragm areknown to have a large Cl conductance (Palade & Barchi, 1975).A minority of fibres exhibited spontaneous synaptic potentials which had a

relatively low frequency of occurrence, widely different time courses and a skeweddistribution of amplitudes. This synaptic activity denotes the presence of multipleinnervation in these fibres. The observation that the various components of the s.j.p.shave different thresholds indicates that the multiple innervation has a polyneuronalorigin. Motor axons to the multiply innervated fibres appear to have smaller dia-meters than the axons to the focally innervated fibres, as revealed by their relativelyhigher threshold and slower conduction velocity. In all these respects, the synapticactivity of these fibres is similar to that of multiply innervated fibres of frog andavian muscles (Burke, 1957; Ginsborg, 1960) and of cat extraocular muscles (Hess &Pilar, 1963).The examination of the electrical properties of tonic fibres of rat inferior rectus

demonstrated that large depolarizations do not evoke action potentials but 'local'oscillatory responses. Previous studies (Hess & Pilar, 1963; Matyushkin, 1964) hadsuggested the absence of action potentials in tonic fibres of eye muscles but in theseearlier experiments the cells had low resting potentials which raised the possibilitythat the lack of spikes was due to an inactivation of the action potential mechanism(Adrian & Marshall, 1977). Since in the present study the fibres were routinelypolarized to a membrane potential of -70 to -80 mV prior to testing their abilityto give action potentials, it can be concluded that the absence of action potentialsis a genuine property of these fibres and not a consequence of cathodal depressionof the cells.

462

ELECTRICAL PROPERTIES OF EXTRAOCULAR MUSCLES

The initial Erp of multiply innervated fibres was only few millivolts less negativethan in focally innervated fibres but it declined considerably with time. This gradualfall can be attributed to an increase of the shunt around the micro-electrode, mostlikely due to the development of cell damage at the impalement site. The attenuatinginfluence of such a shunt on Erp should be noticeable in these muscle cells becausethev have a relativeely high Reir. Stefani & Steinbach (1969) and Hodgkin & Nakajima(1972) have discussed the effects of micro-electrode shunt on Erp in the case of frogmuscle fibres.

Rert of multiply innervated or tonic fibres is large, about four times that of focallyinnervated or twitch fibres. Two factors may contribute to this difference: theirrelatively smaller diameter (15-25,um, Mayr, 1971) and, possibly, a higher specificmembrane resistance. Studies on the repriming of tensions evoked in these musclefibres by elevated KCl concentrations suggest that tonic fibres are highly imperme-able to Cl (Chiarandini, 1976). This suggestion has been confirmed recently by directmeasurements of Reff in the presence and absence of Cl (Bondi & Chiarandini, 1978).The long Tm of these fibres then could be a consequence of a large specific membraneresistance. Elevated [Ca]o greatly increased Reff in multiply innervated fibres, mostlikely by reducing their resting K conductance. This large increase in resistance isin keeping with the existence of a negligible Cl conductance in these fibres.

Although synaptic activity was examined in over one hundred fibres with actionpotentials, none of these fibres was recognized as being multiply innervated. Thereare at least two possible explanations for this failure to identify fibres equivalent tothose described in cat eye muscles by Bach-y-Rita & Ito (1966) and Lennerstrand(1974). First, in the present study the type of innervation of the fibres was identifieddirectly by the features of the synaptic activity while the mentioned authors basedtheir identification on other criteria. As held by Pilar (1967), Barmack, Bell &Rence (1971) and Browne (1976) it is possible that these criteria were not specificfor the recognition of multiple innervation. Secondly, it is conceivable that the cellularpopulation of the muscle regions explored in the above-mentioned studies and in thepresent analysis were different. Extraocular muscles have five to six fibre populationsin two layers: orbital and global, each one with specific cellular populations (forreview see Peachey, 1971; Chiarandini & Davidowitz, 1979. The present study wasconfined to the most superficial fibres of the global region of the muscle while theother studies included muscle cells of both layers.

It is noteworthy to mention that in the global region of rat extraocular musclesMayr (1971) has shown the existence of multiply innervated fibres which are morpho-logically comparable to tonic fibres of amphibian muscles. Similarly, this studydemonstrates that multiply innervated fibres of frog muscles and rat extraocularmuscles have physiological properties in common. Both types of fibres lack actionpotentials, have a high Reit and a large Tm. Moreover, they receive polyneuronalinnervation and their synaptic activities are characterized by long-lasting spontaneousand evoked synaptic potentials with widely different time courses.

The authors wish to thank Dr Ardith Bondi for her comments and Ms K. Rhee for herassistance. This study was supported by grant EY 01297 from 1T.S.P.H.S. and partially by theKirby Eye Surgery Fund.

463

464 D. J. CHIARANDINI AND E. STEFANI

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