Miniature end-plate potentials in the frog muscle spindle

Brain Research Bulletin, Vol. 3, pp. 161-165.

Printed in the U.S.A.

Miniature End-Plate Potentials in the Frog Muscle Spindle F. ITO, Y. ITO, N. FUJITSUKA

AND M. MATSUURA

Department of Physiology, Nagoya University School of Medicine, Nagoya 466, Japan (Received 11 October 1977) ITO, F., Y. ITO, N. FUJITSUKA AND M. MATSUURA.kkature end-plate potentials in the frog muscle spindle. BRAIN. RES. BULL. 3(2) 161-165, 1978. - Spontaneous mixture end-plate potentials (m.e.p.p.s) were recorded from the intrafusal muscle fibers in the muscle spindles isolated from the frog sartorius and semitendinosus muscles by means of microelectrode (mtracellularly) and across a Vaseline gap made transversely on the intrafusal bundle (extracellularly). The latter method was available to record simultaneously the m.e.p.p.s and the afferent terminal activities, of which the latter was easily distinguishable from the former by concomitant occurrence with the axonal responses. Simple type spindles in the sartorius muscle have a restricted end-plate region along the intrafusal bundle on either side of the capsule, while complex type spindles in the semitendinosus muscle are innervated by plural regions which are found on both sides of the capsule in most preparations. Application of 2~ hypertonic Ringer’s solutions with NaCl or sucrose induced an increase in the rate of the m.e.p.p.s preceding a decrease in the rate of afferent discharges from the spindle. Frog muscle spindle

M.e.p.p.

Vaseline gap method

Intrafusal muscle fiber

inverted microscope. The ends of the preparation was tied to the tip of a pair of rods, which was attached on a differential electromagnetic puller. The general situation of the isolated spindle in the Ringer’s pool was monitored with a binocular microscope at magnification of 6- 16 x , while the distance from the sensory terminal in the capsule to the boundary between the Vaseline and the Ringer’s pool was measured with the inverted microscope at a magnification of 100 X. Potential differences across the IFM and axon gaps were recorded through Ringer-agar bridges immersed in the Ringer’s pools; each bridge was in turn connected to calomel electrodes led to two differential high input-hnpedance amplifiers (cf. [ 71). In all records made by the Vaseline gap method, negativity of the spindle pool (P4) relative to the axon pool (Pl) or the polar pools (P2 and P3) was displayed as an upward deflection. Both the potential differences displayed on a dual beam oscilloscope were simultaneously photographed on running film. The interval times between successive spikes were measured from the film with the aid of calibration. Some of the data were coded into a paper tape which was provided for constructing interval histograms by means of a computer (FACOM 270-20). The normal Ringer’s solution used had the following composition (mM): NaCl 116; KC1 2.5; CaC12 1.8; Naz HP04 2.15 and NaH2P04 0.85. The hypertonic solution was prepared by adding 115 mM of NaCl or 230 mM of sucrose to normal Ringer’s solution. The tonicities of these solutions were two times of that of the normal Ringer’s solution [2]. The pH of these solution was in the range from 6.6 to 7.2. No significant effects of pH change of Ringer’s solution at this range on responses of the muscle spindle have been observed [ 6 ] .

potentials and the membrane properties of intrafusal muscle fibers in the frog muscle spindle have extensively been studied [3,9], but spontaneous miniature end-plate potentials (m.e.p.p.s) have not been recorded from the intrafusal muscle fibers. In the present study, the m.e.p.p.s were recorded with intrace~ular micropipette from intrafusal muscle fiber and also by Vaseline gap method from intrafusal bundle. The latter method is available for simultaneous activities in end-plates along the plural intrafusal musde fibers in a spindle. The recorded m.e.p.p.s, however, were often interm~gled with the sensory terminal responses [7], because the sensory terminal has been found even at 10 pm in the vicinity of the end-plate region of the frog spindle with electron microscopic technique [ 8]_ Those two kinds of the spontaneous activities should be distinguished from each other.

THE JUNCTIONAL

METHOD

The experiments were carried out with muscle spindles isolated from the sartorius and the ventral part of semitendinosus muscles of the frog (Rana nigromaculata). The isolated spindle receptor included an intact sensory axon which was also isolated for I- 2 mm near spindle capsule, but the motor axon was removed. The preparation was mounted in a Ringer’s pool in a glass chamber (P4 in the schematic diagram of experimental arrangement in Fig. 1). In the chamber, Vaseline embankments of 1 mm width were drawn in T or rr shape. The intrafusal muscle bundle was passed into another Ringer’s pools (P2 and/or P3 in Fig. 1) through embankments (IFM gap), while the axon was partitioned with the other embankment (axon gap). In the T shaped embankment, Ringer’s pools P4 and P3 were a common. The glass chamber was placed on a stage of an 161

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(A in Fig. 3) was drawn by excluding interposed sensory terminal responses, while that of sensory terminal responses was done by excluding interposed m.e.p.p.s (B in Fig. 3). with an aid of a computer from an admixed train of those two spontaneous activities. Complex type muscle spindles isolated from the semitendinosus muscles yielded both of the negatively and positively deflected m.e.p.p.s regardless of the relative position of the capsule to the IFM gap, as shown in Fig. 2, C and D, The results indicate that the m.e.p.p.s in the simple type spindle may originate at a restricted region along the intrafusal muscie bundle on either side of the capsule, but those in the complex type spindles at the plural sites along the bundle on both sides of the capsule in the most preparations.

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RESULTS

Miniature End-Plate Potentials Along the Intrafusal Muscle Fibers M.e.p.p.s were recorded intracellularly from an intrafusal muscle fiber (R4 in Fig. 1) or extracellularly from the intrafusal bundle across the Vaseline gap (R3 in Fig. I ). The intra- or extra-celIularly recorded m.e.p.p.s were often observable simultaneously with the axonal responses recorded across the axon gap (R 1 in Fig. 1). It, however, was difficult to record the intracellular m.e.p.p.s simultaneously with the IFM gap responses (extracellular m.e.p.p.s and sensory terminal responses); because the extracellular m.e.p.p.s could be recorded only when a restricted region between 10 and 200 pm from the sensory nerve terminal was placed underneath the IFM gap, and the insertion of microelectrode into the restricted region through the Vaseline was impossible. Spontaneous propagated spikes along the intrafusal muscle fiber were rarely observed by microelectrode inserted into an intrafusal fiber or across the IFM gap which was placed 1 mm apart from the restricted region (R2 in Fig. 1). As was shown in Fig. 2, the m.e.p.p.s recorded with the IFM gap were easily distinguishable from the sensory terminal responses, because the latter occurred simultaneously with responses recorded with the axon gap. When the spindle capsule was moved from the inside of the Vaseline embankment in the Ringer’s pool in common with the axon gap, a decrease in the amplitude of the negatively deflected m.e.p.p.s were observed in simple type muscle spindles isolated from the sartorius muscles (Fig. 2, A and B). Figure 3 shows individual interval histograms of the two kinds of spontaneous activities recorded with IFM gap from a spindle preparation. The interval histogram of m.e.p.p.s

Alternation Between Sensory Ter~inai M.E.P.P.S in Hypertonic Solutions

Responses

It has been well known that an mcreasc of tonicity in the Ringer’s solution were follo\;red by a reversible increase of discharge rate of m.e.p.p.s in the frog extrafusal muscle fibers [4] while it resulted in a reversible decrease in rate of spontaneous afferent discharges of the frog muscle spindles [6,12]. In Fig. 4, the 1n.e.p.p.s and the spontaneous af‘ferent discharges in an isolated sartorius spindle were concomitantly recorded before and during treatment of a Na(‘l hypertonic solution. After trzatr’nent of 2x NaCl hypertonic soiution into the termin: pool (P4), increasing the rate of m.e.p.p.s, decreasing the rate of the spontaneous afferent discharges (Fig. 4. B and (‘1. The spontaneous afferent discharges disappeared approximately 12 min after the treatment, while the m.e.p.p.s became often synchronized (Fig. 4, D). After the preparation was returned into the normal Ringer’s solution. the rate of m.c,p.p.s rapldly retained the normal value but the spontaneous afferent

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FIG. 2. Simultaneous record of miniature end-plate potentials and sensory terminal responses from a simple type spindle in sartorius muscle (A and B) and from a complex type in semitendinosus (C and D). Upper traces: afferent discharges recorded across axon gap. Lower traces: admixed responses recorded by IFM gap. A and C: the capsule of the spindles was located in the Vaseline embankment as was shown in the upper illustration. B and D: the capsule was just outside the

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discharges reappeared 10 min later and recovered to the normal rate with approximately 30 min. Changes in the rates of the spontaneous afferent discharges and the m.e.p.p.s before and during treatment of the hypertonic solution to a preparation were plotted in Fig. 5. The rate of m.e.p.p.s was started to increase shortly after the treatment and attained the maximum at 6 to 7 while that of afferent , discharges was decreased min, between 8 and 12 min after the treatment. The 2 x sucrose hypertonic solution resulted in essentially the same phenomena of alternation as the above. On the contrary, application of d-tubocurarine blocked the m.e.p.p.s but did not affect onto the sensory terminal responses.

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By recording spontaneous m.e.p.p.s across a Vaseline gap on intrafusal bundle, the activities of the plural end-plates in the bundle were simultaneously observed. As the m.e.p.p.s in each end-plate is discharged at random [4], the histogram of interval length between successive spontaneous activities in an assembly of the end-plates might be exponential, as is shown in Fig. 3. In the frog muscle spindle, the motor innervation is derived collaterally from the two extrafusal motor systems [S] , the twitch motor system providing a collateral plate innervation for the large intrafusal muscle fibers, and the non-twitch system providing a collateral grape innervation for the smaller fibers. The present study indicates that the simple type spindles in sartorius muscle have a focal end-plate region as is so in the extrafusal twitch muscle fiber, while the complex type spindles in semitendinosus

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FIG. 4. Effects of 2~ hypertonic solution with NaCl on the sensory terminal responses and m.e.p.p.s of a simple type spindle. A: control. B, C and D: 5, 10 and 15 min after treatment of the hypertonic solution, respectively. Upper traces: -he afferent discharges recorded by axon gap. Lower traces: admixed records of sensory terminal responses and m.e.p.p.s by IFM gap.

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M.E.P.P.S IN FROG MUSCLE SPINDLE muscle are innervated multiply as well as the extrafusal slow muscle fiber [ 101. These results are in good agreement with histological evidence [ 1I. It was shown that the static and dynamic responses of the sensory ending are controlled by the large and the small motor fibers, respectively [ 111. The sartorius spindles may be controlled by the static sensitivity in the motor system, whereas the semitendinosus spindles are under dynamic sensitivity control. It was demonstrated in this study that the application of

hypertonic solutions to the preparation resulted in a decrease of spontaneous afferent discharge rate following an increase of the rate of m.e.p.p.s. This suggests that those two spontaneous activities may be modified by different mechanisms with each other in hypertonic solutions. ACKNOWLEDGEMENTS This study was supported by grants from the Ministry of Education (222110 and 287014).

REFERENCES 1. Barker, D. L’innervation motrice du muscle stri6 des vertebres. Actual. neurophysiol. 8: 23-71, 1968. 2. Caputo, C. Volume and twitch tension changes in single muscle fibers in hypertonic solution. J. gen. Physiol. 52: 793-809, 1968. 3. Eyzaguirre, C. Functional organization of neuromuscular spindle in toad. J. Neurophvsiol. 20: 523-592.1957. 4. Fatt, P. and B. Katz.- Spontaneous subthreshold activity at motor nerve endings. J. Physiol. 117: 109-128,1952. 5. Gray, E. G. The spindle and extrafusal innervation of a frog muscle.Proc. R. Sot. 146: 416-430,1957. 6. Ito, F. The behavior of frog muscle spindle in hyper and hypotonic solutions. Jap. J. Physiol. 20: 394-407, 1970. I. Ito, F. and Y. Komatsu. Sensory terminal responses of frog muscle spindle recorded across Vaseline gap onto intrafusal muscle flbre. PjliigersArch. ges. Physiol. 366: 25-30,1976.

8. Karlsson, U., E. Anderson-Cedergren and D. Ottoson. Cellular organization of the frog muscle spindle as revealed by serial sections for electronmicroscopy._. J. Ultrastruct.Res. 14: l-35, 1966. 9. Koketsu, K. and S. Nishi. An analysis of junctional potentials of intrafusal muscle fibres in frogs. J. Physiol. 139: 15-26, 1957. 10. Kuffler, S. W. and E. M. Vaughan Williams. Properties of the ‘slow’ skeletal muscle fibres of the froe. J. Phvsiol. 121: I_ 318-340,1953. 11. Matthews, P. B. C. and D. R. Westbury. Some effects of fast and slow motor fibres on muscle spindles of the frog. J. Physiol. 178: 178-192,1965. 12. Ottoson, D. The effect of osmotic pressure changes on the isolated muscle spindle. Acta physiol. stand. 64: 93-105, 1965. MU