J. Physiol. (1979), 290, pp. 551-568 With 8 text-ftgurew Printed in Great Britain
551
DESENSITIZATION OF GLUTAMATE RECEPTORS ON INNERVATED AND DENERVATED LOCUST MUSCLE FIBRES
BY R. B. CLARK, K. A. F. GRATION AND P. N. R. USHERWOOD From the Department of Zoology, University of Nottingham, Nottingham NG7 2RD
(Received 12 December 1977) SUMMARY
1. Depolarizations to L-glutamate, applied locally by microionophoresis to the extrajunctional membrane of locust extensor tibiae muscle fibres and measured either in current clamp or voltage clamp, increased in amplitude for equivalent doses of glutamate following chronic denervation of the muscle. 2. A two-pulse method was used to examine recovery from desensitization of junctional and extrajunctional receptors. A 'response ratio', i.e. the amplitude of response to the second (test) of a pair of glutamate pulses over the response to the first (control), was determined as a function of the time interval between the pulses. The 'response ratio' for extrajunctional depolarizations of innervated fibres increased exponentially with pulse interval, with a time constant of 15-6 + 4-7 sec (n = 11). Recovery of extrajunctional receptor populations from desensitization was accelerated after denervation. The recovery kinetics for responses from fibres 6-22 days after denervation were generally described by two exponential terms, with time constants in the range 0.5-10 sec which were inversely related to the glutamate sensitivity of the extrajunctional membrane. For junctional receptors on both innervated and denervated fibres the recovery kinetics were described by a single exponential with a time constant of 0.2-1 sec. 3. The results suggest that the increased extrajunctional glutamate sensitivity which occurs after denervation results from the 'appearance' of glutamate receptors with properties similar to those found at the post-junctional membrane on locust muscle fibres. INTRODUCTION
Motor innervation of vertebrate skeletal muscle maintains a large gradient of acetylcholine (ACh) receptors between the neuromuscular junction and the extrajunctional membrane, the sensitivity of the muscle fibre to ACh, applied locally by microionophoresis, falling by a factor of 10-100 within a few micrometers of the end-plate region (Peper & McMahan, 1972; Kuffler & Yoshikami, 1975). Following chronic denervation the extrajunctional membrane develops a 'supersensitivity' to ACh (Axelsson & Thesleff, 1959; Miledi, 1960; Albuquerque & McIssac, 1970) which results from its incorporation of newly synthesized receptors (Fambrough, Devreotes & Card, 1977). In locusts, where L-glutamate is the most likely candidate for the transmitter at excitatory junctions on skeletal muscle fibres (Beranek & Miller, 0022-3751/79/3210-0176 $01.50 © 1979 The Physiological Society
R. B. CLARK AND OTHERS 1968; Usherwood & Machili, 1968; Anwyl & Usherwood, 1974; Anwyl, 1977), the muscles react to denervation in an analogous fashion to vertebrate skeletal muscle (Usherwood, 1969; Cull-Candy, 1975, 1978; Mathers & Usherwood, 1978). The extrajunctional membrane of locust muscle normally exhibits a low sensitivity to glutamate. Jonophoresis of L-glutamate onto this region evokes a biphasic response (depolarization followed by hyperpolarization) which results from the simultaneous activation of two pharmacologically distinct populations of extrajunctional receptors, designated D- and H-receptors (Cull-Candy & Usherwood, 1973; Usherwood & Cull-Candy, 1974). Activation of D-receptors mediates a permeability increase for Na+ and probably K+ (depolarization); H-receptors increase Cl- permeability (hyperpolarization) (Lea & Usherwood, 1973; Cull-Candy, 1976). After denervation the depolarizing component of the extrajunctional glutamate response increases in amplitude for a fixed glutamate concentration (Usherwood, 1969; Cull-Candy, 1975, 1978), while the hyperpolarizing component apparently remains unaltered (CullCandy, 1975, 1978). The relationship between the extrajunctional receptors which appear after denervation and those found on the extrajunctional membrane of innervated fibres is unclear. In the present experiments response characteristics, in particular recovery from desensitization, of junctional and extrajunctional glutamate depolarizations were compared before and after denervation. The results suggest that denervation-induced supersensitivity to glutamate results from the incorporation into the extrajunctional membrane of receptors with properties similar to those of receptors occurring on the postjunctional membrane. A brief account of some of the results presented in this paper has appeared elsewhere (Clark, Gration & Usherwood, 1978). 552
METHODS
All of the experiments described in this paper were performed on the extensor tibiae muscle of the metathoracic leg of adult female locusts (Schitocerca gregaria). The right leg was denervated by sectioning nerve 5 of the metathoracic ganglion using the aseptic procedure of Usherwood (1963a). Risk of post-operative infection was reduced by sealing the wound in the cuticle with cyanoacrylate adhesive. Operated animals were isolated in individual cages at 30 ± 1 'C and fed daily on fresh green grass. After removing both the ventral and dorsal aspects of the cuticle, the femur was mounted ventral side uppermost in a Perspex chamber and the extensor muscle exposed by dissection of the overlying flexor tibiae and retractor unguis muscles. In those experiments where the contralateral unoperated leg served as the control, it was mounted in the chamber alongside the denervated leg and dissected in an identical manner. The extensor tibiae muscle of Schistocerca is innervated by 'fast' and 'slow' excitatory neurones and a single inhibitory neurone (Usherwood & Grundfest, 1965). The former runs in metathoracic nerve 5, the latter in metathoracic nerve 3b. Transection of nerve 5 completely denervates those fibres in the mid-region of the extensor tibiae muscle with exclusively 'fast' excitatory inputs. Some bundles of fibres at the distal end of the femur are innervated by both 'fast' and 'slow' excitatory inputs. In these fibres, miniature excitatory post-synaptic potentials (min. e.p.s.p.s) persisted after section of nerve 5 and contractions could be elicited from them by stimulating their 'slow' excitatory inputs. The results described below were obtained from fibres in about the 8th to 15th bundles of the extensor muscle, counting from the distal end of the femur, i.e. from those fibres with exclusively 'fast' neurone innervation. Muscle preparations were made during superfusion with a saline slightly modified after Usherwood & Grundfest (1965). This standard saline contained NaCl, 180; KCl, 10; NaH2PO4, 4; Na2HPO., 6; CaCl2, 2 mM (pH 6-8). Most of the experiments, however, were performed in a Cl0-free saline in order to almost eliminate the hyperpolarizing component of the extrajunctional
DESENSITIZATION OF LOCUST GLUTAMATE RECEPTORS
553 glutamate response (Cull-Candy, 1976) viz: NaCH3SO4, 180; KCH3SO4, 10; NaH2PO4, 4; Na2HPO4, 6; Ca propionate, 2 mm (pH 6-8). Preparations were equilibrated in the CL--free saline for 1-2 h before experiments were begun. All experiments were done at a room temperature of about 20 'C. High-resistance micro-electrodes were used for ionophoresis of L-glutamate, in order to minimize desensitization of the extrajunctional receptors (Cull-Candy, 1976). Electrodes containing one or two fine glass fibres, filled with 0-2 or 1-0 M L-glutamate at pH 8, had d.c. resistances of 100-300 Mil measured in Cl--free saline. To obtain stable responses to repeated doses of glutamate, braking currents in the range of 2-30 nA were applied to the electrodes. A 500 MK resistor was placed in series with the glutamate electrode to minimize changes in the ionophoresis currents due to occasional fluctuations in electrode resistance. Glutamate 'dose' is expressed in terms of the total net charge (mean current x pulse duration) passed through the electrode during the pulse. Glutamate doses ranged from 0 to 10 nC, with pulse durations from 1 to 20 msec. The 'sensitivity' of the muscle fibre membrane to ionophoretically applied glutamate was expressed in terms of the peak response produced per unit glutamate dose (i.e. mV/nC; see Miledi, 1960). Because the relation between response and applied dose is supralinear, however, the sensitivity value obtained depends on the point on the dose-response curve at which the measurement is made, and sensitivity values obtained using small glutamate doses will be lower than those made with larger doses. In these experiments comparisons between 'operated' and 'normal' fibres were made using a constant glutamate dose. Intracellular recording electrodes (5-15 MO) were filled with either 2 M-tri-K citrate (pH 7) or 3 M-KC1. For voltage-clamping, a conventional two-electrode clamp technique was used (Anwyl, 1977). The voltage and current electrodes were impaled in the fibres about 100 ,sm apart, with the glutamate electrode between them. The clamp current was filtered by a low-pass active filter with a bandwidth of 0-1 kHz before being displayed on an oscilloscope. -
RESULTS
Extrajunctional sensitivity to L-glutamate Ionophoresis of L-glutamate on to the extrajunctional membrane of locust muscle bathed in standard saline usually evoked a small biphasic response, a depolarization ('D-response') of up to 1 mV in amplitude and time to peak of 30-80 msec, followed by a more prolonged hyperpolarization ('H-response') of similar size (Cull-Candy, 1976). Superposition of the two components distorted the D-response, making accurate measurements of its amplitude and rise time difficult. This distortion was effectively eliminated by abolishing the H-response with Cl-free saline. There was considerable variation in the extrajunctional L-glutamate sensitivity of fibres from different control muscles, although the distribution of glutamate sensitivity on a given fibre was relatively uniform (Fig. 1A). The extrajunctional D-sensitivity of five fibres (from five different control muscles) with resting potentials in the range -50 to -56 mV, was 0-26 + 0-17 mV/nC (mean + S.D.). For 3-5 days after transaction of nerve 5, min. e.p.s.p.s were observed in many of the fibres, together with occasional 'giant' spontaneous potentials several millivolts in amplitude, which are characteristic of axotomized locust muscle with degenerating nerve terminals (Usherwood, 1963b, 1973). In these fibres the sensitivity and distribution of extrajunctional glutamate responses were not significantly different from those of the control fibres. Extrajunctional glutamate sensitivity increased significantly from 5 to 8 days after denervation (Fig. 1 B), by which time min. e.p.s.p.s had completely disappeared from all of the fibres examined. (In less extensive studies Mathers & Usherwood (1978) observed increased glutamate sensitivity only after 7-9 days after section of the
R. B. CLARK AND OTHERS nerve.) By 10-12 days the increased glutamate sensitivity had completely developed, when the mean (± S.E.) extrajunctional sensitivity (1.7 + 0-06 mV/nC) was approximately 5-6 times greater than that of control fibres. The glutamate sensitivity declined after prolonged denervation, and by 28 days, the longest period examined in this study, it had fallen by approximately twofold compared to the maximum sensitivity. It is noteworthy that by this time atrophy of the denervated muscle was obvious. 554
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Fig. 1. A, comparison of the distribution of extrajunctional glutamate sensitivity
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innervated and denervated locust muscle fibres. The glutamate electrode was moved in 10 jmn steps along the exposed surface of the fibre. Sensitivity at each point wvas measured in terms of the peak depolarization per unit glutamate dose (mV/nC) elicited by a 10 msec pulse of 18 nC. The depolarizations had times to peak of 30-60 msec. Note that the sensitivity scale is logarithmic. Open circles (o), innervated control fibre, resting potential, - 56 mV. Filled circles (@), fibre denervated for 21 days, resting potential, -51 mV. The mean glutamate sensitivity (+ S.D.) was 0-28 + 0-09 mV/nC for the control fibre and lI 0 ± i 63 mV/nC for the denervated fibre. B, changes in extrajunctional glutamate sensitivity and membrane potential following denervation. Glutamate sensitivity (a) was determined from maps similar to that in A. Each point is the mean + S.E. of mean of at least sixty values of glutamate sensitivity from maps of three fibres of two different preparations. Values are not corrected for the input resistance of the fibres. Membrane potential (0); each point is the mean +±s.E. of between three to eighteen determinations from two different preparations. The mean membrane potential (- 54.8 ± 2-3 mV, n = 18) of control fibres was significantly greater (P lnC) were generally required to evoke responses of an easily measurable amplitude (i.e. > 1 mV) the question arose whether the time course of recovery measured from the two-pulse method reflected properties of the ionophoretic electrode rather than those of the glutamate responses. Although the net charge (mean current x pulse duration) passed through the electrode was identical for both doses, it is possible that the first dose altered the concentration of glutamate in the electrode tip, thereby changing the quantity of glutamate released by the second. This was checked in several experiments using a double barrelled glutamate electrode, one barrel delivering the 'control' dose and the other the 'test'. If the doses delivered by the two barrels were adjusted to give equal response amplitudes, desensitization measured by the response ratio was the same whether a pair of doses was delivered by a single barrel, or whether one dose was delivered from each of the two barrels. This was true even for the smallest time intervals (approximately 0- 1 see) used in the experiments. A problem with double-barrelled electrodes is that it cannot be guaranteed that doses delivered from the two barrels cover the same area of membrane, and hence the same receptor population, whereas this is unlikely to be a problem when the doses are delivered by a single barrel. There is the additional problem of barrel interactions with multiple-barrelled electrodes. The exponential recovery time constant seemed to decrease as the glutamate sensitivity of the extrajunctional membrane areas increased. There was indeed a good correlation between these values, as shown in Fig. 8. Extrajunctional Dresponses of innervated fibres associated with membrane areas of low glutamate sensitivity (0- 1-0-3 mV/nC), had correspondingly long recovery time constants (10-20 see). Extrajunctional responses of denervated fibres were associated with membrane areas of greater sensitivity, ranging from approximately 0-3 to about 3 mV/nC, and the recovery times measured in two-pulse experiments examined over
R. B. CLARK AND OTHERS 564 this range decreased as the sensitivity of the glutamate activated site increased. In those cases where the recovery had two discernible phases, both recovery times were plotted at the same sensitivity (Fig. 8). With few exceptions, the 'fast' and 'slow' recovery time constants fell into one of two distinct groups, both of which showed an inverse correlation between sensitivity and recovery time. 100
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Vp/Q (mV/nC) Fig. 8. Correlation between recovery time constant (tR) and coulombic sensitivity (V1/Q). In cases where the recovery had two phases, both time constants were plotted at the same coulombic sensitivity. Normal D-responses, filled triangles (A). Extrajunctional responses of fibres 6-22 days after denervation, filled circles (@), 'fast' recovery time and open circles (0), 'slow' recovery time. Junctional responses, filled squares (*).
Desensitization recovery kinetics for junctional glutamate receptors Junctional glutamate responses, which generally are associated with localized membrane areas of sensitivities in the range of 10-100 mV/nC (Beranek & Miller, 1968; Usherwood & Machili, 1968), are reduced in amplitude by dose repetition only when glutamate doses are applied at rates greater than about 1 or 2 per second. It was of interest to see how the desensitization recovery time constants for junctional responses compared with those of the extrajunctional responses. Recovery times in two-pulse experiments were determined for junctional responses of both control and denervated fibres, in a similar manner to that described above for extrajunctional responses. The recovery time course for junctions was described by a single exponential. For junctional areas with glutamate sensitivites in the range 10- 100 mV/nC, recovery times ranged from 0-2 to 1 sec. It is noteworthy that the extrapolation of extrajunctional recovery times to higher sensitivites appears to converge on the region of recovery times for junctions (Fig. 8). This suggests that as the extrajunctional glutamate sensitivity, and presumably the density of glutamate receptors, increases, the recovery from desensitization becomes more 'junction-like' in its characteristics.
DESENSITIZATION OF LOCUST GLUTAMATE RECEPTORS
565
DISCUSSION
The increased extrajunctional sensitivity to ACh which develops on vertebrate skeletal muscle following denervation results from the production of new ACh receptors which are incorporated into the extrajunctional membrane (Fambrough, Devreotes & Card, 1977). There is a good correlation between the sensitivity to ionophoretically applied ACh on the denervated rat diaphragm and the population density of extrajunctional receptors determined by the binding of 1251-labelled acbungarotoxin (Hartzell & Fambrough, 1972; Fambrough, 1974). The increased extrajunctional sensitivity to glutamate of denervated locust muscle presumably arises from an increased population density of glutamate receptors, but unfortunately an independent measure of receptor density does not seem possible at present as no substance is yet available with highly specific and relatively irreversible glutamate receptor binding properties. The increase in extrajunctional glutamate sensitivity of the locust extensor tibiae muscle following nerve section is small compared with the increase in ACh sensitivity of many denervated vertebrate muscles. For denervated rat diaphragm the population density of extrajunctional ACh receptors is up to a factor of several hundred greater than for innervated muscle (Hartzell & Fambrough, 1972; Fambrough, 1974). It is possible that neurotrophic influences in locust muscle are much less important in regulating receptor numbers and distribution than is the case in vertebrate muscle. This may be a common feature of arthropod skeletal muscle as denervated crayfish muscle fails to show any significant increase in extrajunctional glutamate sensitivity, even several months after any visible signs of functional synapses have disappeared (Frank, 1974). Junctional glutamate receptors on locust muscle mediate a membrane permeability increase for Na+ and probably K+ (Anwyl & Usherwood, 1974; Anwyl, 1977). The similar reversal potentials of junctional responses and extrajunctional responses of denervated muscle implies that the ionic selectivities (Na+: K+ permeability ratio) of the ionophores gated by the junctional and extrajunctional glutamate receptors are probably identical. It is possible that the junctional and extrajunctional ionophores may differ in other properties, such as the elementary conductance and mean open time, as is the case for ACh receptor ionophores in vertebrate muscles (Katz & Miledi, 1972; Dreyer, Walther & Peper, 1976; Neher & Sakmann, 1976). These results from locust muscle show some similarity to those from vertebrate skeletal muscle. For both cat (Axelsson & Thesleff, 1959) and frog (Trautmann & ZilberGachelin, 1976; see also Neher & Sakmann, 1976) muscles, the reversal potential of ACh responses is unchanged by denervation. The reversal potential of extrajunctional glutamate responses of innervated locust muscle is uncertain at present because of the small magnitude of the D-currents and the possible influence of residual H-currents in Cl-free saline. It should be noted, however, that junctional and extrajunctional ACh reversal potentials of innervated frog skeletal muscle, determined by voltage-clamp, are identical (Mallart, Dreyer & Peper, 1976). Interpreting the time constants obtained from the two-pulse recovery experiments in terms of the rates of recovery of glutamate receptors from desensitization is extremely difficult, due to the nature of the ionophoretic method of glutamate
R. B. CLARK AND OTHERS application. The local concentration of released glutamate near the muscle membrane is governed by diffusion (see Peper, Dryer & Muller, 1976). It is greatest on those areas of membrane nearest the focus of release (i.e. micropipette tip) and declines rapidly as the distance from the source is increased. It follows that the fraction of receptors activated (and desensitized), per unit of membrane surface, will be greatest on those areas nearest the micro-electrode tip. Therefore, although in a two-pulse experiment the 'control' dose may be applied to a receptor population with a uniform density of glutamate receptors may influence the amplitude of the 'test' response, and receptors has been reduced by desensitization, the reduction being greatest on those areas of membrane nearest the focus of release. Such alterations in the local population density of glutamate receptors may influence the amplitude of the test response, and hence the rates of recovery determined from the two-pulse experiments may contain a contribution from a time-dependent change in the distribution of receptors available for activation as well as from the rate of recovery of the glutamate receptors from desensitization and it seems difficult to separate the two. This complication may explain in part the correlation between the glutamate sensitivity of the membrane and the rate of recovery from desensitization measured in two-pulse experiments (Fig. 8) as alteration of local receptor population density is likely to be least on membrane areas of greatest glutamate sensitivity (i.e. receptor density). The effect is also likely to be smaller for a receptor population with a highly localized distribution, thus the junctional recovery times are probably closer to the 'true' recovery rate. It is difficult to see how this problem can be circumvented with the ionophoretic technique in its present form. The basis of the two phase recovery from desensitization observed at many that extrajunctional areas on denervated muscle is not clear. It is tempting to suggesttypes the response results from the activation of a mixture of two (or more) receptor with different recovery characteristics, but there is no convincing evidence to support this. In view of the non-uniform distribution of glutamate sensitivity seen on many of the denervated fibres, responses may often be obtained from areas with a nonuniform receptor density. In such cases the recovery from desensitization might be expected to depend on the relative contribution to the response from 'patches' of different receptor density.
5666
Our thanks are due to Dr R. Ramsey for construction of the voltage-clamp apparatus and for valuable technical advice. This research was supported by a grant from the Science Research
Council.
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E. X. & McIssAc, R. J. (1970). Fast and slow mammalian muscles after denerALBUQu-ERQUE, vation. Expl Neurol. 26, 183-202. R. (1977). Permeability of the post-synaptic membrane of an excitatory glutamate ANDwY, synapse to sodium and potassium. J. Physiol. 273, 367-388. R. & UsHmUwooD, P. N. R. (1974). Voltage clamp studies of glutamate synapse. AwwyL, Nature, Lond. 252, 591-593. J. & THiSLIEF, S. (1959). A study of supersensitivity in denervated mammalian AxELSSON, skeletal muscle. J. Physiol. 147, 178-193. R. & MILLER, P. L. (1968). The action of iontophoretically applied glutamate on BERANEK, insect muscle fibres. J. exp. Biol. 49, 83-93. CLARK, R. B., GRATION, K. A. F. & USHERWOOD, P. N. R. (1978). Denervation-induced changes in extrajunctional glutamate responses of insect muscle. J. Physiol. 276, 75P.
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CULL-CANDY, S. G. (1975). Effect of denervation and local damage on extrajunctional L-glutamate receptors in locust muscle. Nature, Lond. 258, 530-531. CULL-CANDY, S. G. (1976). Two types of extrajunctional L-glutamate receptors on locust muscle fibres. J. Physiol. 255, 449-464. CULL-CANDY, S. G. (1978). Glutamate sensitivity and distribution of receptors along normal and denervated locust muscle fibres. J. Physiol. 276, 165-181. CULL-CANDY, S. G. & USHERWOOD, P. N. R. (1973). Two populations of L-glutamate receptors on locust muscle fibres. Nature, Lond. 246, 62-64. DAOUD, M. A. R. & USHERWOOD, P. N. R. (1978). Desensitization and potentiation during glutamate application to locust skeletal muscle. Comp. Biochem. Physiol. 59C, 105-110. DREYER, F., WALTHER, C. & PEPER, K. (1976). Junctional and extrajunctional acetylcholine receptors in normal and denervated frog muscle fibres. Noise analysis experiments with different agonists. Pflugers Arch. 366, 1-9. DUDEL, J. (1 975). Potentiation and desensitization after glutamate induced postsynaptic currents at the crayfish neuromuscular junction. Pflugers Arch. 356, 317-327. FAMBROUGH, D. M. (1974). Acetylcholine receptors. Revised estimates of extrajunctional density in denervated rat diaphragm. J. yen. Physiol. 64, 468-472. FAMBROUGH, D. M., DEVREOTES, P. N. & CARD, D. J. (1977). The synthesis and degradation
of acetylcholine receptors. In Synapses, ed. COTTRELL, G. A. & USHERWOOD, P. N. R., pp. 202-236. Glasgow: Blackie. FAMBROUGH, D. M. & HARTZELL, H. C. (1 972). Acetylcholine receptors: number and distribution at neuromuscular junctions in rat diaphragm. Science, N.Y. 176, 189-191. FRANK, E. (1 974). The sensitivity to glutamate of denervated muscles of the crayfish. J. Phy8iol. 242, 371-382. HARTZELL, H. C. & FAMBROUGH, D. M. (1972). Acetylcholine receptors. Distribution and extrajunctional density in rat diaphragm after denervation correlated with acetylcholine sensitivity. J. gen. Physiol. 60, 248-262. JAN, L. Y. & JAN, Y. N. (1976). L-glutamate as an excitatory transmitter at the Drosophila larval neuromuscular junction. J. Physiol. 262, 215--236. KATZ, B. & MImDi, R. (1972). The statistical nature of the acetylcholine potential and its molecular components. J. Physiol. 224, 665-699. KATZ, B. & THESLEFF, S. (1957). A study of the desensitizationn' produced by acetylcholine at the motor end-plate. J. Physiol. 138, 63-80. KUFFLER, S. W. & YOSHIKAMI, D. (1975). The distribution of acetylcholine sensitivity at the post-synaptic membrane of vertebrate skeletal twitch muscles: Iontophoretic mapping in the micron range. J. Physiol. 244, 703-730. LEA, T. J. & USHERWOOD, P. N. R. (1973). Effect of ibotenic acid on chloride permeability of insect muscle fibres. Comp. gen. Pharmac. 4, 351-363. MAGAZANIK, L. G. & VYSKOCIL, F. (1975). The effect of temperature on desensitization kinetics at the post-synaptic membrane of the frog muscle fibre. J. Physiol. 249, 285-300. MALLART, A., DREYER, F. & PEPER, K. (1976). Current-voltage relation and reversal potential at junctional and extrajunctional ACh receptors of the frog neuromuscular junction. Pfluigers Arch. 362, 43-47. MATHERS, D. A. & USHERWTOOD, P. N. R. (1978). The sensitivity of locust skeletal muscle fibres to L-glutamate following denervation and local injury. Comp. Biochem. Physiol. 60C, 7-10. M1LEDI, R. (1960). The acetylcholine sensitivity of frog muscle fibres after complete or partial denervation. J. Physiol. 151, 1-23. NEHER, E. & SAKMANN, B. (1976). Noise analysis of drug induced voltage clamp currents in denervated frog muscle fibres. J. Physiol. 258, 705-729. PEPER, K., DREYER, F. & MULLER, K. D. (1976). Analysis of cooperativity of drug-receptor interaction by quantitative iontophoresis at frog motor end plates. Cold Spring Harb. Symp. quant. Biol. 60, 187-192. PEPER, K. & McMAHAN, U. J. (1972). Distribution of acetylcholine receptors in the vicinity of nerve terminals on skeletal muscle of the frog. Proc. R. Soc. B 181, 431-440. TAKEUCHI, A. & TAKEUCHI, N. (1964). The effect of crayfish muscle of iontophoretically applied glutamate. J. Physiol. 170, 296-317. TRAUTMANN, A. & ZILBER-GACHELIN, N. F. (1976). Further investigations on the effect of denervation and pH on the conductance change at the neuromuscular junction of the frog. Pflugerm Arch. 364, 53-58.
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USHERWOOD. P. N. R. (1963a). Response of insect muscle to denervation. I. Resting potential changes. J. Insect Physiol. 9, 247-255. USHERWOOD, P. N. R. (1 963b). Response of insect muscle to denervation. II. Changes in neuromuscular transmission. J. Insect Physiol. 9, 811-825. USHERWOOD, P. N. R. (1969). Glutamate sensitivity of denervated insect muscle fibres. Nature, Lond. 223, 411-413. USHERWVOOD, P. N. R. (I 973). Release of transmitter from degenerating locust motoneurones. J. exp. Biol. 59, 1-16. USHERWOOD, P. N. R. & CULL-CANDY, S. G. (1974). Distribution of glutamate sensitivity on insect inmuscle fibres. Neuropharmacology 13, 455-461. USHERWOOD, P. N. R. & GRUNDFEST, H. (1965). Peripheral inhibition in skeletal muscle of insects. J. Neurophysiol. 28, 497-518. USHERWOOD, P. N. R. & MACHILM, P. (1968). Pharmacological properties of excitatory neuromuscular synapses in the locust. J. exp. Biol. 49, 341-361.
551
DESENSITIZATION OF GLUTAMATE RECEPTORS ON INNERVATED AND DENERVATED LOCUST MUSCLE FIBRES
BY R. B. CLARK, K. A. F. GRATION AND P. N. R. USHERWOOD From the Department of Zoology, University of Nottingham, Nottingham NG7 2RD
(Received 12 December 1977) SUMMARY
1. Depolarizations to L-glutamate, applied locally by microionophoresis to the extrajunctional membrane of locust extensor tibiae muscle fibres and measured either in current clamp or voltage clamp, increased in amplitude for equivalent doses of glutamate following chronic denervation of the muscle. 2. A two-pulse method was used to examine recovery from desensitization of junctional and extrajunctional receptors. A 'response ratio', i.e. the amplitude of response to the second (test) of a pair of glutamate pulses over the response to the first (control), was determined as a function of the time interval between the pulses. The 'response ratio' for extrajunctional depolarizations of innervated fibres increased exponentially with pulse interval, with a time constant of 15-6 + 4-7 sec (n = 11). Recovery of extrajunctional receptor populations from desensitization was accelerated after denervation. The recovery kinetics for responses from fibres 6-22 days after denervation were generally described by two exponential terms, with time constants in the range 0.5-10 sec which were inversely related to the glutamate sensitivity of the extrajunctional membrane. For junctional receptors on both innervated and denervated fibres the recovery kinetics were described by a single exponential with a time constant of 0.2-1 sec. 3. The results suggest that the increased extrajunctional glutamate sensitivity which occurs after denervation results from the 'appearance' of glutamate receptors with properties similar to those found at the post-junctional membrane on locust muscle fibres. INTRODUCTION
Motor innervation of vertebrate skeletal muscle maintains a large gradient of acetylcholine (ACh) receptors between the neuromuscular junction and the extrajunctional membrane, the sensitivity of the muscle fibre to ACh, applied locally by microionophoresis, falling by a factor of 10-100 within a few micrometers of the end-plate region (Peper & McMahan, 1972; Kuffler & Yoshikami, 1975). Following chronic denervation the extrajunctional membrane develops a 'supersensitivity' to ACh (Axelsson & Thesleff, 1959; Miledi, 1960; Albuquerque & McIssac, 1970) which results from its incorporation of newly synthesized receptors (Fambrough, Devreotes & Card, 1977). In locusts, where L-glutamate is the most likely candidate for the transmitter at excitatory junctions on skeletal muscle fibres (Beranek & Miller, 0022-3751/79/3210-0176 $01.50 © 1979 The Physiological Society
R. B. CLARK AND OTHERS 1968; Usherwood & Machili, 1968; Anwyl & Usherwood, 1974; Anwyl, 1977), the muscles react to denervation in an analogous fashion to vertebrate skeletal muscle (Usherwood, 1969; Cull-Candy, 1975, 1978; Mathers & Usherwood, 1978). The extrajunctional membrane of locust muscle normally exhibits a low sensitivity to glutamate. Jonophoresis of L-glutamate onto this region evokes a biphasic response (depolarization followed by hyperpolarization) which results from the simultaneous activation of two pharmacologically distinct populations of extrajunctional receptors, designated D- and H-receptors (Cull-Candy & Usherwood, 1973; Usherwood & Cull-Candy, 1974). Activation of D-receptors mediates a permeability increase for Na+ and probably K+ (depolarization); H-receptors increase Cl- permeability (hyperpolarization) (Lea & Usherwood, 1973; Cull-Candy, 1976). After denervation the depolarizing component of the extrajunctional glutamate response increases in amplitude for a fixed glutamate concentration (Usherwood, 1969; Cull-Candy, 1975, 1978), while the hyperpolarizing component apparently remains unaltered (CullCandy, 1975, 1978). The relationship between the extrajunctional receptors which appear after denervation and those found on the extrajunctional membrane of innervated fibres is unclear. In the present experiments response characteristics, in particular recovery from desensitization, of junctional and extrajunctional glutamate depolarizations were compared before and after denervation. The results suggest that denervation-induced supersensitivity to glutamate results from the incorporation into the extrajunctional membrane of receptors with properties similar to those of receptors occurring on the postjunctional membrane. A brief account of some of the results presented in this paper has appeared elsewhere (Clark, Gration & Usherwood, 1978). 552
METHODS
All of the experiments described in this paper were performed on the extensor tibiae muscle of the metathoracic leg of adult female locusts (Schitocerca gregaria). The right leg was denervated by sectioning nerve 5 of the metathoracic ganglion using the aseptic procedure of Usherwood (1963a). Risk of post-operative infection was reduced by sealing the wound in the cuticle with cyanoacrylate adhesive. Operated animals were isolated in individual cages at 30 ± 1 'C and fed daily on fresh green grass. After removing both the ventral and dorsal aspects of the cuticle, the femur was mounted ventral side uppermost in a Perspex chamber and the extensor muscle exposed by dissection of the overlying flexor tibiae and retractor unguis muscles. In those experiments where the contralateral unoperated leg served as the control, it was mounted in the chamber alongside the denervated leg and dissected in an identical manner. The extensor tibiae muscle of Schistocerca is innervated by 'fast' and 'slow' excitatory neurones and a single inhibitory neurone (Usherwood & Grundfest, 1965). The former runs in metathoracic nerve 5, the latter in metathoracic nerve 3b. Transection of nerve 5 completely denervates those fibres in the mid-region of the extensor tibiae muscle with exclusively 'fast' excitatory inputs. Some bundles of fibres at the distal end of the femur are innervated by both 'fast' and 'slow' excitatory inputs. In these fibres, miniature excitatory post-synaptic potentials (min. e.p.s.p.s) persisted after section of nerve 5 and contractions could be elicited from them by stimulating their 'slow' excitatory inputs. The results described below were obtained from fibres in about the 8th to 15th bundles of the extensor muscle, counting from the distal end of the femur, i.e. from those fibres with exclusively 'fast' neurone innervation. Muscle preparations were made during superfusion with a saline slightly modified after Usherwood & Grundfest (1965). This standard saline contained NaCl, 180; KCl, 10; NaH2PO4, 4; Na2HPO., 6; CaCl2, 2 mM (pH 6-8). Most of the experiments, however, were performed in a Cl0-free saline in order to almost eliminate the hyperpolarizing component of the extrajunctional
DESENSITIZATION OF LOCUST GLUTAMATE RECEPTORS
553 glutamate response (Cull-Candy, 1976) viz: NaCH3SO4, 180; KCH3SO4, 10; NaH2PO4, 4; Na2HPO4, 6; Ca propionate, 2 mm (pH 6-8). Preparations were equilibrated in the CL--free saline for 1-2 h before experiments were begun. All experiments were done at a room temperature of about 20 'C. High-resistance micro-electrodes were used for ionophoresis of L-glutamate, in order to minimize desensitization of the extrajunctional receptors (Cull-Candy, 1976). Electrodes containing one or two fine glass fibres, filled with 0-2 or 1-0 M L-glutamate at pH 8, had d.c. resistances of 100-300 Mil measured in Cl--free saline. To obtain stable responses to repeated doses of glutamate, braking currents in the range of 2-30 nA were applied to the electrodes. A 500 MK resistor was placed in series with the glutamate electrode to minimize changes in the ionophoresis currents due to occasional fluctuations in electrode resistance. Glutamate 'dose' is expressed in terms of the total net charge (mean current x pulse duration) passed through the electrode during the pulse. Glutamate doses ranged from 0 to 10 nC, with pulse durations from 1 to 20 msec. The 'sensitivity' of the muscle fibre membrane to ionophoretically applied glutamate was expressed in terms of the peak response produced per unit glutamate dose (i.e. mV/nC; see Miledi, 1960). Because the relation between response and applied dose is supralinear, however, the sensitivity value obtained depends on the point on the dose-response curve at which the measurement is made, and sensitivity values obtained using small glutamate doses will be lower than those made with larger doses. In these experiments comparisons between 'operated' and 'normal' fibres were made using a constant glutamate dose. Intracellular recording electrodes (5-15 MO) were filled with either 2 M-tri-K citrate (pH 7) or 3 M-KC1. For voltage-clamping, a conventional two-electrode clamp technique was used (Anwyl, 1977). The voltage and current electrodes were impaled in the fibres about 100 ,sm apart, with the glutamate electrode between them. The clamp current was filtered by a low-pass active filter with a bandwidth of 0-1 kHz before being displayed on an oscilloscope. -
RESULTS
Extrajunctional sensitivity to L-glutamate Ionophoresis of L-glutamate on to the extrajunctional membrane of locust muscle bathed in standard saline usually evoked a small biphasic response, a depolarization ('D-response') of up to 1 mV in amplitude and time to peak of 30-80 msec, followed by a more prolonged hyperpolarization ('H-response') of similar size (Cull-Candy, 1976). Superposition of the two components distorted the D-response, making accurate measurements of its amplitude and rise time difficult. This distortion was effectively eliminated by abolishing the H-response with Cl-free saline. There was considerable variation in the extrajunctional L-glutamate sensitivity of fibres from different control muscles, although the distribution of glutamate sensitivity on a given fibre was relatively uniform (Fig. 1A). The extrajunctional D-sensitivity of five fibres (from five different control muscles) with resting potentials in the range -50 to -56 mV, was 0-26 + 0-17 mV/nC (mean + S.D.). For 3-5 days after transaction of nerve 5, min. e.p.s.p.s were observed in many of the fibres, together with occasional 'giant' spontaneous potentials several millivolts in amplitude, which are characteristic of axotomized locust muscle with degenerating nerve terminals (Usherwood, 1963b, 1973). In these fibres the sensitivity and distribution of extrajunctional glutamate responses were not significantly different from those of the control fibres. Extrajunctional glutamate sensitivity increased significantly from 5 to 8 days after denervation (Fig. 1 B), by which time min. e.p.s.p.s had completely disappeared from all of the fibres examined. (In less extensive studies Mathers & Usherwood (1978) observed increased glutamate sensitivity only after 7-9 days after section of the
R. B. CLARK AND OTHERS nerve.) By 10-12 days the increased glutamate sensitivity had completely developed, when the mean (± S.E.) extrajunctional sensitivity (1.7 + 0-06 mV/nC) was approximately 5-6 times greater than that of control fibres. The glutamate sensitivity declined after prolonged denervation, and by 28 days, the longest period examined in this study, it had fallen by approximately twofold compared to the maximum sensitivity. It is noteworthy that by this time atrophy of the denervated muscle was obvious. 554
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innervated and denervated locust muscle fibres. The glutamate electrode was moved in 10 jmn steps along the exposed surface of the fibre. Sensitivity at each point wvas measured in terms of the peak depolarization per unit glutamate dose (mV/nC) elicited by a 10 msec pulse of 18 nC. The depolarizations had times to peak of 30-60 msec. Note that the sensitivity scale is logarithmic. Open circles (o), innervated control fibre, resting potential, - 56 mV. Filled circles (@), fibre denervated for 21 days, resting potential, -51 mV. The mean glutamate sensitivity (+ S.D.) was 0-28 + 0-09 mV/nC for the control fibre and lI 0 ± i 63 mV/nC for the denervated fibre. B, changes in extrajunctional glutamate sensitivity and membrane potential following denervation. Glutamate sensitivity (a) was determined from maps similar to that in A. Each point is the mean + S.E. of mean of at least sixty values of glutamate sensitivity from maps of three fibres of two different preparations. Values are not corrected for the input resistance of the fibres. Membrane potential (0); each point is the mean +±s.E. of between three to eighteen determinations from two different preparations. The mean membrane potential (- 54.8 ± 2-3 mV, n = 18) of control fibres was significantly greater (P lnC) were generally required to evoke responses of an easily measurable amplitude (i.e. > 1 mV) the question arose whether the time course of recovery measured from the two-pulse method reflected properties of the ionophoretic electrode rather than those of the glutamate responses. Although the net charge (mean current x pulse duration) passed through the electrode was identical for both doses, it is possible that the first dose altered the concentration of glutamate in the electrode tip, thereby changing the quantity of glutamate released by the second. This was checked in several experiments using a double barrelled glutamate electrode, one barrel delivering the 'control' dose and the other the 'test'. If the doses delivered by the two barrels were adjusted to give equal response amplitudes, desensitization measured by the response ratio was the same whether a pair of doses was delivered by a single barrel, or whether one dose was delivered from each of the two barrels. This was true even for the smallest time intervals (approximately 0- 1 see) used in the experiments. A problem with double-barrelled electrodes is that it cannot be guaranteed that doses delivered from the two barrels cover the same area of membrane, and hence the same receptor population, whereas this is unlikely to be a problem when the doses are delivered by a single barrel. There is the additional problem of barrel interactions with multiple-barrelled electrodes. The exponential recovery time constant seemed to decrease as the glutamate sensitivity of the extrajunctional membrane areas increased. There was indeed a good correlation between these values, as shown in Fig. 8. Extrajunctional Dresponses of innervated fibres associated with membrane areas of low glutamate sensitivity (0- 1-0-3 mV/nC), had correspondingly long recovery time constants (10-20 see). Extrajunctional responses of denervated fibres were associated with membrane areas of greater sensitivity, ranging from approximately 0-3 to about 3 mV/nC, and the recovery times measured in two-pulse experiments examined over
R. B. CLARK AND OTHERS 564 this range decreased as the sensitivity of the glutamate activated site increased. In those cases where the recovery had two discernible phases, both recovery times were plotted at the same sensitivity (Fig. 8). With few exceptions, the 'fast' and 'slow' recovery time constants fell into one of two distinct groups, both of which showed an inverse correlation between sensitivity and recovery time. 100
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Vp/Q (mV/nC) Fig. 8. Correlation between recovery time constant (tR) and coulombic sensitivity (V1/Q). In cases where the recovery had two phases, both time constants were plotted at the same coulombic sensitivity. Normal D-responses, filled triangles (A). Extrajunctional responses of fibres 6-22 days after denervation, filled circles (@), 'fast' recovery time and open circles (0), 'slow' recovery time. Junctional responses, filled squares (*).
Desensitization recovery kinetics for junctional glutamate receptors Junctional glutamate responses, which generally are associated with localized membrane areas of sensitivities in the range of 10-100 mV/nC (Beranek & Miller, 1968; Usherwood & Machili, 1968), are reduced in amplitude by dose repetition only when glutamate doses are applied at rates greater than about 1 or 2 per second. It was of interest to see how the desensitization recovery time constants for junctional responses compared with those of the extrajunctional responses. Recovery times in two-pulse experiments were determined for junctional responses of both control and denervated fibres, in a similar manner to that described above for extrajunctional responses. The recovery time course for junctions was described by a single exponential. For junctional areas with glutamate sensitivites in the range 10- 100 mV/nC, recovery times ranged from 0-2 to 1 sec. It is noteworthy that the extrapolation of extrajunctional recovery times to higher sensitivites appears to converge on the region of recovery times for junctions (Fig. 8). This suggests that as the extrajunctional glutamate sensitivity, and presumably the density of glutamate receptors, increases, the recovery from desensitization becomes more 'junction-like' in its characteristics.
DESENSITIZATION OF LOCUST GLUTAMATE RECEPTORS
565
DISCUSSION
The increased extrajunctional sensitivity to ACh which develops on vertebrate skeletal muscle following denervation results from the production of new ACh receptors which are incorporated into the extrajunctional membrane (Fambrough, Devreotes & Card, 1977). There is a good correlation between the sensitivity to ionophoretically applied ACh on the denervated rat diaphragm and the population density of extrajunctional receptors determined by the binding of 1251-labelled acbungarotoxin (Hartzell & Fambrough, 1972; Fambrough, 1974). The increased extrajunctional sensitivity to glutamate of denervated locust muscle presumably arises from an increased population density of glutamate receptors, but unfortunately an independent measure of receptor density does not seem possible at present as no substance is yet available with highly specific and relatively irreversible glutamate receptor binding properties. The increase in extrajunctional glutamate sensitivity of the locust extensor tibiae muscle following nerve section is small compared with the increase in ACh sensitivity of many denervated vertebrate muscles. For denervated rat diaphragm the population density of extrajunctional ACh receptors is up to a factor of several hundred greater than for innervated muscle (Hartzell & Fambrough, 1972; Fambrough, 1974). It is possible that neurotrophic influences in locust muscle are much less important in regulating receptor numbers and distribution than is the case in vertebrate muscle. This may be a common feature of arthropod skeletal muscle as denervated crayfish muscle fails to show any significant increase in extrajunctional glutamate sensitivity, even several months after any visible signs of functional synapses have disappeared (Frank, 1974). Junctional glutamate receptors on locust muscle mediate a membrane permeability increase for Na+ and probably K+ (Anwyl & Usherwood, 1974; Anwyl, 1977). The similar reversal potentials of junctional responses and extrajunctional responses of denervated muscle implies that the ionic selectivities (Na+: K+ permeability ratio) of the ionophores gated by the junctional and extrajunctional glutamate receptors are probably identical. It is possible that the junctional and extrajunctional ionophores may differ in other properties, such as the elementary conductance and mean open time, as is the case for ACh receptor ionophores in vertebrate muscles (Katz & Miledi, 1972; Dreyer, Walther & Peper, 1976; Neher & Sakmann, 1976). These results from locust muscle show some similarity to those from vertebrate skeletal muscle. For both cat (Axelsson & Thesleff, 1959) and frog (Trautmann & ZilberGachelin, 1976; see also Neher & Sakmann, 1976) muscles, the reversal potential of ACh responses is unchanged by denervation. The reversal potential of extrajunctional glutamate responses of innervated locust muscle is uncertain at present because of the small magnitude of the D-currents and the possible influence of residual H-currents in Cl-free saline. It should be noted, however, that junctional and extrajunctional ACh reversal potentials of innervated frog skeletal muscle, determined by voltage-clamp, are identical (Mallart, Dreyer & Peper, 1976). Interpreting the time constants obtained from the two-pulse recovery experiments in terms of the rates of recovery of glutamate receptors from desensitization is extremely difficult, due to the nature of the ionophoretic method of glutamate
R. B. CLARK AND OTHERS application. The local concentration of released glutamate near the muscle membrane is governed by diffusion (see Peper, Dryer & Muller, 1976). It is greatest on those areas of membrane nearest the focus of release (i.e. micropipette tip) and declines rapidly as the distance from the source is increased. It follows that the fraction of receptors activated (and desensitized), per unit of membrane surface, will be greatest on those areas nearest the micro-electrode tip. Therefore, although in a two-pulse experiment the 'control' dose may be applied to a receptor population with a uniform density of glutamate receptors may influence the amplitude of the 'test' response, and receptors has been reduced by desensitization, the reduction being greatest on those areas of membrane nearest the focus of release. Such alterations in the local population density of glutamate receptors may influence the amplitude of the test response, and hence the rates of recovery determined from the two-pulse experiments may contain a contribution from a time-dependent change in the distribution of receptors available for activation as well as from the rate of recovery of the glutamate receptors from desensitization and it seems difficult to separate the two. This complication may explain in part the correlation between the glutamate sensitivity of the membrane and the rate of recovery from desensitization measured in two-pulse experiments (Fig. 8) as alteration of local receptor population density is likely to be least on membrane areas of greatest glutamate sensitivity (i.e. receptor density). The effect is also likely to be smaller for a receptor population with a highly localized distribution, thus the junctional recovery times are probably closer to the 'true' recovery rate. It is difficult to see how this problem can be circumvented with the ionophoretic technique in its present form. The basis of the two phase recovery from desensitization observed at many that extrajunctional areas on denervated muscle is not clear. It is tempting to suggesttypes the response results from the activation of a mixture of two (or more) receptor with different recovery characteristics, but there is no convincing evidence to support this. In view of the non-uniform distribution of glutamate sensitivity seen on many of the denervated fibres, responses may often be obtained from areas with a nonuniform receptor density. In such cases the recovery from desensitization might be expected to depend on the relative contribution to the response from 'patches' of different receptor density.
5666
Our thanks are due to Dr R. Ramsey for construction of the voltage-clamp apparatus and for valuable technical advice. This research was supported by a grant from the Science Research
Council.
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