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Denervation of insect muscle: A comparative study of the changes in L -Glutamate sensitivity on locust retractor unguis and extensor tibiae muscle
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,2in,ropha~rr,a.,,loy~ Vol. IS. pp. 201. 208 0 Pergamon Press Ltd 1979. Printed in Great Britain
DENERVATION OF INSECT A COMPARATIVE STUDY CHANGES IN L-GLUTAMATE ON LOCUST RETRACTOR AND EXTENSOR TIBIAE
MUSCLE: OF THE SENSITIVITY UNGUIS MUSCLE
K. A. F. GRATION, R. B. CLARK and P. N. R. USHERWOOD Department
of Zoology, University of Nottingham, Nottingham NC7 ZRD, England (Accepted
University
Park.
2 August 1978)
Summary-The sensitivity of the extrajunctional membrane of the locust retractor unguis (R.U.) muscle to microiontophoretic application of L-glutamate was compared with that of the extensor tibiae (E.T.) muscle. The distribution of extrajunctional D-receptors for L-glutamate on R.U. muscle was more uniform than on E.T. muscle. Chronic denervation (at 30°C) caused an increase in the mean extrajunctional D-sensitivity of both muscles, complete development of which occurred 12-14 days post-denervation for R.U. muscle and l&12 days post-denervation for E.T. muscle. The maximum mean extrajunctional glutamate D-sensitivity of denervated R.U. fibres was about 20 times greater than the sensitivity of innervated control fibres, while the increase for denervated E.T. fibres was about 6-fold. For both muscles the increase in glutamate sensitivity was only partially accounted for by an increase in the input resistance of the fibres after denervation. The extrajunctional D-receptors on denervated R.U. muscle were uniformly distributed, but on ET. fibres there was considerable variation in local glutamate sensitivity. Between 2(r28 days post-denervation the extrajunctional sensitivity of both muscles declined at a time when muscle atrophy was evident. The reversal potential of extrajunctional glutamate responses of denervated muscle, determined using voltage-clamp, was +3.5 k 2.6mV (mean + SD., n = S), similar to the reversal potential of junctional glutamate responses of both innervated and denervated fibres. ’
I,-Glutamate, the putative transmitter at excitatory synapses on muscles of many insect species (Usherwood and Machili, 1968; Usherwood, 1976), also activates receptors on the extrajunctional muscle membrane of the extensor tibiae (E.T.) muscle of nymphal z.nd adult locusts. The fibres of this muscle have two pharmacologically distinct populations of extrajunctional glutamate receptors (denoted D- and H-receptors): activation of D-receptors mediates a transient increase in Na+ and K+ permeability, while H-receptor activation mediates an increased Cl- permeability (Lea and Usherwood, 1973; Cull-Candy, 1976). There i:; growing evidence that extrajunctional glutamate rzceptors might also occur on some vertebrate central ceurones and that like those of the locust E.T. muscle t ley might have significantly different physiological and pharmacological properties from their junctional counterparts on these cells (see review by Usherwood, 1978). It is difficult to judge from the fragmentary evidence available for both vertebrate and invertebrate excitable systems whether the presence of extrajunctional receptors on cells innervated by glutamatergic neurones is a widespread phenomenon. More information on this point is obviously required if effective pharmacological studies are to be made on these sysKey words: L-glutamate vation.
receptors,
insect muscle,
terns. There are indications that extrajunctional receptors are present on locust muscles other than the E.T. muscle, and on skeletal muscles of other insects, but their presence here has not been convincingly demonstrated. The orthopteran retractor unguis (R.U.) muscle falls into this category and since this muscle has been developed and extensively used for assaying pharmacological agents (Usherwood and Machili, 1968; McDonald, Farley and March, 1972; Clements and May, 1974; Walther and Rathmayer, 1974; Usherwood and Cull-Candy, 1975) it seemed an appropriate starting point in determining whether extrajunctional receptors are a common feature of insect skeletal muscles. It will be shown that the extrajunctional membrane of R.U. muscle fibres has similar pharmacological properties to the extrajunctional membrane of E.T. fibres. However, the distribution of extrajunctional receptors is much more uniform in R.U. muscle than in E.T. muscle, a difference which is maintamed following denervation. METHODS The
results
described
in this
paper
were
obtained
E.T. muscles of the metathoracic leg of the locust, Schistocerca gregaria (Usherwood and Machili, 1968). The .muscles were exposed by removing cuticle on the ventral side of the femur followed by dissection of the overlying flexor tibiae from
dener-
201
the R.U.
and
202
K. A. F.
GRATION.
R. B.
CLARK
muscle. The E.T. muscle provided support for the R.U. muscle during insertion of microelectrodes, which greatly facilitated voltage-clamp studies of the R.U. muscle fibres. The R.U. muscle is innervated by metathoracic nerve Sb which supplies it with two large excitatory motor axons (A, and A,) (Rees and Usherwood. 1972a). The muscle comprises two bundles of muscle fibres: one contains large diameter (5~80pm) “white” fibres and is innervated by axon A,, the other contains smaller diameter (20-40pm) “red” fibres and is innervated by axon AZ. Some fibres also receive endings from inhibitory neurones (C. Walther, personal communication). The R.U. muscle fibres are multiterminally innervated but the distal regions of the white fibres (i.e. IO?:, of the total fibre length, which is _ 1 cm) are devoid of nerve endings. These nerve-free regions are very suitable for investigating extrajunctional glutamate sensitivity since diffusion of iontophoretically-applied L-glutamate from extrajunctional sites of application to junctional glutamate receptors is obviated. The E.T. muscle receives endings for “fast” and “slow” excitatory motorneurones and an inhibitory motorneurone but ventral fibres in the mid-region of this muscle are exclusively innervated by the “fast” excitatory axon (Usherwood and Grundfest, 1965). The studies described in this paper were restricted to these fibres (see also Clark. Gration and Usherwood, 1978). The R. U. and E.T. muscle were denervated on one side of the animal by sectioning nerve 5 of the metathoracic ganglion using an aseptic procedure (Usherwood, 1963a). Operated animals were isolated in individual cages at 30°C. Denervated and contralateral control muscles were prepared side-by-side for physiological and pharmacological studies in the same perfusion bath. Preparations were continuously superfused with either standard saline, which contained NaCI 180, KC1 10, CaCl, 2. NaZHPO, 6, NaH2P04 4mM. pH 6.8 or Cl--free saline: NaCH,S04 180, KCH3S04 10. (C,H,.COO),Ca 2. NaH2P04 4, Na,HP04 6 mM. pH 6.8. High resistance (100-300 M0 in standard saline) microelectrodes filled with 0.1 M Na L-glutamate (pH 8) were used for the iontophoresis of glutamate. Backing currents in the range 3320nA were required to obtain optimum glutamate responses. Intracellular recordings were made with 2 M tripotassium citrate microelectrodes (5-I 5 MQ), using conventional electrophysiological techniques. A twoelectrode clamp circuit was used for voltage-clamp experiments. RESULTS
In standard saline iontophoresis onto the extrajunctional membrane
of L-glutamate at the distal end
and P. N. R.
USHERW~D
of “white” R.U. muscle hbres evoked biphasic responses, which consisted of a depolarization preceding a hyperpolarization. Replacement of standard saline by Cl--free saline resulted in the rapid reversal and eventual virtual abolition of the hyperpolarizing component. There is a small residual current which possibly arises from a permeability increase for methylsulphate ions {Lea and Usherwood, 1973) although contribution of K + ions to the hyperpolarizing current has not been ruled out. The transient depolarizations that remained were of a similar amplitude to the extrajunctional D-response recorded from E.T. fibres in Cl--free saline (Cull-Candy, 1976; Clark er al., 1978). Slight displacement of the L-glutamate electrode had little effect on either the amplitude or rise time of these responses, indicating a non-focal distribution of receptors. Mapping the extrajunctional sensitivity to glutamate on R.U. muscle fibres revealed a low but essentially uniform L-glutamate sensitivity (0.14.5 mV/nC) over the extrajunctional membrane but with high levels of sensitivity (up to 1.4 mV/nC) at the muscle-apodeme junction. The distribution of extrajunctional glutamate sensitivity over R.U. white fibres was more uniform than over E.T. fibres from the same leg and was more consistent between fibres. To compare the relative distribution of extrajunctional sensitivity for R.U. and E.T. muscles the coefficient of variation of glutamate sensitivity (i.e. S.D./mean sensitivity) was determined on areas of both R.U. and ET. fibres. The glutamate sensitivity of R.U. fibres was measured on extrajunctional areas well away from the muscle-apodeme junction (>4#pm; Fig. 1). For R.U. fibres the coefhcient of variation was 0.22 _t 0.04 (mean It SD., n = 12) while for E.T. fibres a value of 0.60 + 0.21 (n = 14) was obtained. The difference between the mean values of the coefftcients for the two muscles was highly significant (P < 0.001. Student’s r-test). The relationship between glutamate dose and response amplitude for the interaction of L-glutamate with extrajun~tional D-receptors on R.U. muscle fibres was measured at the resting potential (approximately -65 mV in Cl--free saline). The data confirm the non-focal distribution of these receptors, since both the amplitude and rise time of the glutamate potentials increased with dose. This is similar to the interaction between acetylcholine (ACh) and extrajunctional receptors on frog muscle fibres (Feltz and Mallart, 1971). The limiting slope of double Iogarithmic dose-amplitude plots (Fig. 2) was 1.48 I: 0.2 (mean & S.D., n = 6). which is approximately the same as that for the interaction of L-glutamate with extrajunctional D-receptors on E.T. muscle fibres (Cull-Candy, 1976: Clark et al., 1978). Extrajunctional D-receptors on R.U. muscle are readily desensitized. Recovery from desensitization was examined by varying the time interval between identical glutamate doses delivered from a single high resistance glutamate micropipette and measuring the ratio of the amplitude of the second (“test”) response
203
Denervated insect muscle
Fig. 1. The distribution of extrajunctional glutamate sensit vity on a locust R.U. fibre (resting potential -63 mV). The glutamate electrode was moved in 1Opm steps along the centre of the muscle fibre and the sensitivity at each point measured in terms of the peak depolarization per unit glutamate dose (mV/nC) produced by a constant dose of 2 nC (IO msec). The upper points (@).show the distribution of glutamate sensitivity in the region of the muscleapodeme junction: the lower points (0) show the distribution on an area of extrajunctional membrane starting 4IOprn from the apodeme. Note that the glutamate sensitivity scale is logarithmic. The recording electrode was n:ver more than 400pm from any extrajunctional site to which glutamate was applied. This is unlikely to cause any significant error in estimating the local glutamate sensitivity, as the length constant of R.U. fibres is several mm.
EPSP’s were almost completely absent from R.U. fibres and the extrajunctional glutamate sensitivity of these fibres had increased by approximately 3-fold compared with control levels (Fig. 4) although at this time the sensitivity of ET. fibres had not significantly changed and min. EPSP’s were present in most fibres. By 5-7 days, min. EPSP’s had disappeared from E.T. fibres and their extrajunctional sensitivity had increased. The difference between the time required for the appearance of extrajunctiona) supersensitivity on R.U. and E.T. fibres can probably be attributed, in part, to different rates of axon terminal degeneration (Usherwood, 1963a, b; Rees and Usherwood, 1972a). Complete development of extrajunctional supersensitivity took from 12 to 14 days on R.U. fibres and from 10 to 12 days on the E.T. fibres (Fig. 4). The mean (& S.D.) maximum extrajuncrional glutamate sensitivity of R.U.. and E.T. fibres was 3.4 + 0.8mV/nC (n = 60) and 1.7 + 1.1 mV/nC (n = IOO), res~ctively. Examples of the distributions of giutamate sensitivity over R.U. and E.T. fibres from a muscle denervated for 7 days are compared in Figure 5. The distribution of glutamate sensitivity over the R.U. fibre was uniform and there was little variation in this parameter between this fibre and other fibres in the muscle. However, the distribution of glutamate sensitivity over the E.T. fibre at this time was very
to that of the first (“control”) (Clark et a/., 1978). For pulse separations of less than 1-2 set the test response was barely detectabIe. The response ratio increased with increasing time interval until at separations in tl+e range 40-50 set the test and control responses had identical amplitudes (Fig. 3a). The kinetics of recovery fram desensitization (Fig. 3b) were found to be best described by an equation containing a single exponential term, i.e. identical to that describing the recovery from desensitization of D-responses of E.T. muscle fibres (Clark ef al., 1978). The exponential time constants of recovery ranged from 10.5 to 22sec (mean rt SD., 12 F 3 set, n = 7), being very similar to those measured from the curves for recovery from desensitization of D-responses on E.T. muscle fibres. Comparison of pharmacological properties of denerwted R.U. and ET. muscle fibres The extrajunctional glutamate sensitivity of chronically denervated R.U. and E.T. muscles was investigs.ted for up to 28 days after denervation (Fig. 4). Botween I and 3 days post-denervation miniature excitatory postsynaptic potentials (min. EPSP’s} were observed in most fibres of the R.U. and E.T. muscles. The magnitude and dist~butjon of the extrajunctiona1 glutamate sensitivity of these fibres were not significantly different from those of fibres of the contralaterai control mu&es. After 4 days denervation, min.
40-J 7’‘llo0 Dose
(nC)
Fig. 2. Upper graph. Relationship between extrajunctional glutamate potential amplitude (V,) and coulombic dose (nC), on double log co-ordinates. The limiting slope (solid line) of the dose-response relationship is 1.55. Line drawn by eye. Lower graph. Relationship between time to response peak (r,) and cou~ombic dose, on double log co-ordinates. The time to peak was taken from the onset of the glutamate pulse. The slope of the solid line is 0.25. Line drawn by eye.
204
K. A. F. GRATION, R. B. CLARK
and P. N. R.
USHERWOOD
(A) ImV
I
I ,I ?I
2,
-,
I I 0
I IO Days
after
I
20
I 1 1 30
deneivation
Fig. 4. Comparison of the extrajunctional glutamate sensitivities of locust retractor unguis (of and extensor tibiae (0) fibres, denervated for different time periods (G28 days). Each datum point is the mean +S.D. of at least 60 sensitivity measurements from 3 fibres of 2 different muscles.
)
I
zi
0
Pulse
interval
SO (set)
Fig. 3. (A) D-responses evoked by two successive glutamate doses, applied iontophoreticaily to the same area of extrajunctional membrane. The pulse separation, indicated in the left-hand column, was varied between 540 sec. Resting potential of muscle fibre -63.5 mV; control response amplitude 1.6 mV; glutamate dose 5 nC. Note the miniature excitatory post-synaptic potentials in most of the records. (B) Examination of the desensitization recovery kinetics of extrajunctional glutamate responses, using a two-pulse technique (see text for explanation). The “response ratio”, R, i.e. the amplitude of the second (test) response over the amplitude of the first (control), is plotted as a function of the time interval between the glutamate pulses. The time course of recovery from desensitization is fitted to an exponential equation (solid line) with a time constant of 13.9sec. Resting potential -62mV; control response amplitude 1.4 mV; glutamate dose 4 nC.
non-uniform and showed only localized areas of increased glutamate sensitivity. In some cases the mean extrajunctional sensitivities of denervated and contralateral control E.T. fibres were not significantly different. On the other hand, denervated R.U. fibres always had a greater glutamate sensitivity than fibres of the contralateral control muscle. These differences in the relative distributions of glutamate sensitivity on R.U. and E.T. fibres were measured in terms of the coefficient of variation of the sensitivity distribution (Fig. 5). The coefficient for 3.5 denervated R.U. fibres (6-18 days after denervation) was 0.32 + 0.1 (mean f SD.) while that for 38 ET. fibres over a similar denervation period was 0.76 & 0.36. The difference between the coefficients was found to be highly significant (P < 0.001, Student’s t-test). Between 20-28 days after denervation, the extrajunctional sensitivity of R.U. and E.T. muscle declined (Fig. 4). In R.U. fibres sensitivity decreased &fold, to a level comparable to that of the 6 day-denervated muscle, while that for E.T. fibres a a-fold decrease was observed. Former junctional areas on muscles denervated for more than 20 days were easily distinguished from extrajunctiobal areas by their significantly higher sensitivity to glutamate. As in normal muscles their sites were generally located in the clefts between fibres (Usherwood and Machili, 1968). The sensitivity of these areas was similar to that of junctions on control fibres, i.e. lO-lOOmV/nC. Measurement of extrajunctional glutamate sensitivity in terms of the membrane depolarization per
205
Denervated insect muscle
01
I
0
I
50 Distance
I
I
I
100
150
200
( ,um 1
Fig. 5. Comparison of the distribution of extrajunctional glutamate sensitivity on 7 days denervated locust retractor unguis and extensor tibiae fibres. Solid circles (a), R.U. fibre, resting potential -57.6 mV. Open circles (o), E.T. fibre, resting potential - 51.3 mV. The mean glutamate sensitivity (*SD.) was 1.42 + 0.5 mV/nC for the R.U. fibre, 0.78 & 0.5 mV/nC for the E.T. fibre and the respective coefficients of variation of the glutamate sensitivities were 0.35 and 0.64. The small difference in resting potential accounts for only IO?< of the difference in glutamate sensitivity since the reversal potential of the glutamate response is near zero (See Fig. 6B).
unit glutamate dose depends partly on the electrical properties of the muscle membrane (Albuquerque and M&sac, 1970). Following denervation the input resi:;tance of R.U. and E.T. fibres, measured by current injection through a second intracellular microelectrade (resistance 5-10 MR), increased from 565 + 196 kR (mean 2 SD., 58 observations on IO muscles) to 943 + 266 kQ (30 observations on 7 muscles) 620 days after denervation) and from 260 & 56 kR (52 observations on 8 muscles) to 497 t_ 91 ki2 (43 observations on 8 muscles) over a similar denervation period, respectively. Note that the increased input reGtance of the denervated fibres was not sufficient to account for the observed increase in sensitivity to glutamate (Fig. 4). A comparison of the extrajunctional glutamate currents of innervated and denervated muscle hbres under voltage clamp supports this conclusion. The inward currents associated with extrajunctional glutamate responses of denervated R.U. fibres were much larger (maximum recorded amplitude of 28 nA, at a membrane potential of - 60 mV) than the extrajunctional currents from innervated fibres (Fig. 6a). A similar difference in the magnitude of extrajunctional currents has previously been observed for E.T. fibres (Clark et al., 1978). The relationship between peak current and membrane potential for extrajunctional responses of denervated R.U. fibres was approximately linear (Fig. 6) and the reversal potential for fibres 8-14 days post-denervation, determined from a linear regression over mem-
brane potentials in the range + 10 to -6OmV, was + 3.5 k 2.6 mV (data from 8 fibres of 6 preparations). The reversal potential of glutamate currents recorded from former junctional areas on 8-14 day denervated fibres was + I.7 + 2.6 mV (data from 7 fibres of 4 preparations), a value which was not significantly different from that of junctions on control muscles, i.e. -3.7 & 2.9 mV (n = 6). The reversal potential of extrajunctional glutamate currents of innervated muscle fibres could not be determined reliably using voltage clamp. A slow outward current, whose amplitude increased as the membrane potential was reduced, was often superimposed on the inward current generated by activation of extrajunctional D-receptors. The outward current possibly resulted from activation of extrajunctional H-receptors. the current being carried primarily by CH,SO; ions (Lea and Usherwood, 1973) or poss i biy K+ rather than Cl-. In one experiment the glutamate electrode was replaced by one fihed with 0.1 M DLibotenic acid, which activates only the H-receptors on the extrajunctional membrane (Lea and Usherwood, 1973; Cull-Candy, 1976). When DL-ibotenate was ejected onto extrajunctional membranes of E.T. and R.U. fibres in Cl--free saline, an outward current was generated whose time course was similar to that seen with glutamate. The amplitude of this current was up to 2-4nA at membrane potentials near zero. The inward current decreased in amplitude as membrane potential was reduced and at about -2OmV,
K. A. F. GRATION.R. B.
206
CLARK
(A)
n
(b)
*4
and P. N. R. USHERW~~
the D-current (inward) and the outward H-current were equal and opposite. In some preparations the H- and D-currents had similar time-courses and could not be distinguished during glutamate iontophoresis. As a result “apparent” reversal values for D-responses in Cl-free saline of about - 20 mV were obtained. However, application of ibotenate to these fibres demonstrated unequivocally the presence of an outward current presumably equal in amplitude but opposite in polarity to the inward D-current. Since the extrajunctional inward currents recorded from denervated fibres were considerably larger than those of innervated fibres the accompanying outward H-current did not greatly influence their reversal values. Figure 7 shows examples of the time course of recovery from desensitization, measured using the twopulse method, for extrajunctional glutamate responses recorded from denervated R.U. muscles. The kinetics of recovery from desensitization were similar to those of extrajunctional responses of innervated fibres but the recovery time constants were generally much smaller. The time constant was smaller (i.e. recovery from desensitization was more rapid) for responses from membrane areas of higher glutamate sensitivity. Many of the extrajunctional responses from denervated muscle had recovery kinetics consisting of “fast” and “slow” phases of recovery. The time course of recovery from desensitization in these cases was best described by an equation containing two exponential terms (Fig. 7). DIfCUSSlON
Fig. 6. {A) Comparison of the extrajunctional glutamate currents from an innervated (b) and a 16 day denervated (c) locust retractor unguis fibre, under voltage clamp. Both fibres were clamped at a membrane potential of - 60 mV. The glutamate dose was 2 nC (trace a). Current calibration is 2nA for the response from innervated fibre, IOnA for the denervated. Note miniature excitatory postsynaptic currents on trace (b). (B) Relationship between membrane potential (E,) and peak clamp current (I,) of extrajunctional gfutamate responses from three denervated R.U. fibres. Inward clamp currents are shown positive. The solid lines are linear regression fits to the data. The reversal potentials, determined from the regression lines are +9 mV (0). $8.5 mV (0). and f3.1 mV (m).
The properties of extrajunctional glutamate responses of the innervated locust R.U. muscle are in most respects identical to those which can be recorded from E.T. muscles, Although H-receptors have been observed on fibres of the locust retractor unguis (Usherwood, unpublished) and anterior coxal adductor muscles (Lea and Usherwood, 1973), the presence of D-receptors on muscles of this insect other than the E.T. muscle has not hitherto been reported. The high glutamate sensitivity observed near the muscle-apodeme junction of R.U. fibres is analogous to the high ACh sensitivity of myotendinous junctions of some vertebrate skeletal muscles (Miledi and Zelena, 1966; Albuquerque and M&sac, 1970). A similar high glutamate sensitivity occurs near the muscle-apodeme junction of E.T. fibres (Cull-Candy, 1978) although the region of high sensitivity appears to be more restricted than for the R.U. muscle-apodeme junction. The increase in extrajunctional glutamate sensitivity of R.U. fibres which follows denervation (Usherwood, 1969) is quaiitatively similar to that which occurs on denervated E.T. fibres (Cull-Candy, 1975; 1978; Clark er al., 1978). The distribution of extrajunctional glutamate sensitivity on denervated locust
207
Denervated insect muscle
and changes are observed in the muscle cytoarchitecture (Rees and Usherwood, 1972b). Possibly, protein synthesis is impaired at this stage, causing a decline in the turn-over of extrajunctional receptors and a consequent reduction in receptor density. A consistent finding was that the sensitivity of former junctional areas bad not declined even when muscle atrophy was very obvious. This suggests that the density of glutamate receptors on the former post-synaptic membrane remains unchanged after denervation. A similar conclusion has been reached by Hartzell and Fambrough (1972) for ACh receptors on the denervated rat diaphragm. The similar reversal potentials of the junctional currents and the extrajunctional currents on denervated muscle suggests that the apparent increase in
IIP
*n 0
IO
Pulse Interval(set
20 )
Fig. 7. Desensitization recovery kinetics of extrajunctional glutamate responses of denervated R.U. fibres. The response ratio, R (see text and Fig. 3 for explanation), is plotted as a function of the time interval between successivt: glutamate doses. Open circles (o), data from a 4 day denervated fibre, resting potential -57.8 mV. Control response amplitude 1.5 mV, glutamate dose 3.0 nC. The solid line is a single exponential of time constant 7.5 sec. Solid circles (e), data from a 6 day denervated fibre, resting potential -49.6 mV. Control response amplitude 2.6 mV. glutamate dose 2.8 nC. The solid line is a double exponential of time constants 0.45 set and 4.2 sec.
ET. muscle is similar to the heterogeneous distribution of extrajunctional ACh sensitivity on 3-day denervated rat diaphragm (Hartzell and Fambrough, 1972) and soleus/extensor digitorum muscles (Albuquerque and M&sac, 1970) with the receptors tending to occur in “patches”. The distribution on the locust R.U. muscle is more like the homogeneous distribution of ACh sensitivity on the rat muscles 10 days post-denervation. The difference between the distribution of glutamate sensitivity on both innervated and denervated R.U. and E.T. muscles may indicate a true difference in the density of extrajunctional glutamate receptors on these muscles, but unfortunately an independent measure of glutamate receptor densitf, is, as vet, unavailable. ‘The decline in extrajunctional glutamate sensitivity of both R.U. and E.T. fibres which occurs about 20 days post-denervation is analogous to the decrease in the extrajunctional ACh sensitivitv of rat soleus and extensor digitorum longus muscles after prolonged denervation (Albuquerque and M&sac, 1970), which Hartzell and Fambrough (1972) have suggested is due, in part, to selective loss of ACh receptors from the membrane. It may be significant that the fall in glutamate sensitivity of denervated R.U. and E.T. muscle occurs at a time when muscle atrophy is very obvious. By 20-24 days post-denervation, the crosssectional area of R.U. muscle decreases by about 65%
glutamate receptor density which occurs after denervation results from the “appearance” of receptors which are similar, if not identical, to those occurring at the junctions. The junctional receptors mediate a permeability increase for Na+ and probably K+ and Ca” (Anwyl and Usherwood. 1975; Anwyl, 1977) and it seems likely from the reversal potentiaf that the Na+/K’ permeability ratio for the extrajunctionat receptors on denervated muscle is identical to that for junctional receptors. The reversal potential of D-responses of innervated muscle is unclear at present for the reasons discussed earlier. However, voltage clamp studies on vertebrate muscle (e.g. frog: Mallart, Dreyer and Peper, 1976) have shown that the reversal potentials of junctional and extrajunctional ACh responses are identical.
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K. A. F. GRATION, R. B. CLARK and P. N. R. USHERW~~D
Hartzell, H. C. and 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. Lea. T. J. and Usherwood, P. N. R. (1973). Effect of ibotenic acid on chloride permeability in insect muscle fibres. Comp. gen. Pharmac. 4: 351-363. Mallart, A., Dreyer, F. and Peper, K. (1976). Current-voltage relation and reversal potential at junctional and extrajunctional ACh-receptors of the frog neuromuscular junction. Pj7iigers Arch. ges. Physiol. 362: 4347. McDonald. T. J., Farley, R. D. and March, R. B. (1972). Pharmacological profile of the excitatory neuromuscular synapses of the insect retractor unguis muscle. Comp. gen. Pharmac. 3: 327-338. Miledi, R. and Zelena, J. (1966). Sensitivity to acetylcholine in rat slow muscle. Nature, Lond. 210: 855-856. Rees, D. and Usherwood, P. N. R. (1972a). Fine structure of normal and degenerating motor axons and nervemuscle synapses in the locust Schisrocerca gregaria. Comp. Biochem. Physiol. 43A: 85-101. Rees, D. and Usherwood, P. N. R. (1972b). Effects of denervation on the ultrastructure of insect muscle. J. Cell Sci. IO: 667-682. Usherwood, P. N. R. (1963a). Response of insect muscle to denervation. I. Resting potential changes. J. insect Physioi. 9: 247-255.
Usherwood, P. N. R. (1963b). Response of insect muscle to denervation. II. Changes in neuromuscular transmission J. insect Physiol. 9: 81 l-825. Usherwood. P. N. R. (1969). Glutamate sensitivity of denervated insect muscle fibres. Nature, Lond. 223: 41 l-413. Usherwood, P. N. R. (1976). Amino acid receptors on insect muscle. In: Electrobiology of Nerve, Muscle and Synapse (Reuben, J. P., Purpura, D. P.. Bennet, M. V. L. and Kandel, E.. Eds), pp. 81-93. Raven Press, New York. Usherwood, P. N. R. (1978). Amino acids and synaptic transmission. In: Advances in Comparative Physiology Biochemistry (Lowenstein, 0. E., Ed.) Academic Press, New York. In press. Usherwood. P. N. R. and Cull-Candy, S. G. (1975). Pharmacology of somatic nerve-muscle synapses. In: Insect Muscle (Usherwood, P. N. R.. Ed.). .on. 207-280. Academic Press, London. Usherwood, P. N. R. and Grundfest, H. (1965). Peripheral inhibition in skeletal muscle of insects. J. Neurophysiol. 28: 497-518. Usherwood, P. N. R. and Machili, P. (1968). Pharmacological properties of excitatory neuromuscular synapses in the locust. J. exp. Biol. 49: 341-361. Walther, C. and Rathmayer, N. (1974). The effect of Habrobracon venom on excitatory neuromuscular transmission in insects. J. camp. Physiol. 89: 23-38.
,2in,ropha~rr,a.,,loy~ Vol. IS. pp. 201. 208 0 Pergamon Press Ltd 1979. Printed in Great Britain
DENERVATION OF INSECT A COMPARATIVE STUDY CHANGES IN L-GLUTAMATE ON LOCUST RETRACTOR AND EXTENSOR TIBIAE
MUSCLE: OF THE SENSITIVITY UNGUIS MUSCLE
K. A. F. GRATION, R. B. CLARK and P. N. R. USHERWOOD Department
of Zoology, University of Nottingham, Nottingham NC7 ZRD, England (Accepted
University
Park.
2 August 1978)
Summary-The sensitivity of the extrajunctional membrane of the locust retractor unguis (R.U.) muscle to microiontophoretic application of L-glutamate was compared with that of the extensor tibiae (E.T.) muscle. The distribution of extrajunctional D-receptors for L-glutamate on R.U. muscle was more uniform than on E.T. muscle. Chronic denervation (at 30°C) caused an increase in the mean extrajunctional D-sensitivity of both muscles, complete development of which occurred 12-14 days post-denervation for R.U. muscle and l&12 days post-denervation for E.T. muscle. The maximum mean extrajunctional glutamate D-sensitivity of denervated R.U. fibres was about 20 times greater than the sensitivity of innervated control fibres, while the increase for denervated E.T. fibres was about 6-fold. For both muscles the increase in glutamate sensitivity was only partially accounted for by an increase in the input resistance of the fibres after denervation. The extrajunctional D-receptors on denervated R.U. muscle were uniformly distributed, but on ET. fibres there was considerable variation in local glutamate sensitivity. Between 2(r28 days post-denervation the extrajunctional sensitivity of both muscles declined at a time when muscle atrophy was evident. The reversal potential of extrajunctional glutamate responses of denervated muscle, determined using voltage-clamp, was +3.5 k 2.6mV (mean + SD., n = S), similar to the reversal potential of junctional glutamate responses of both innervated and denervated fibres. ’
I,-Glutamate, the putative transmitter at excitatory synapses on muscles of many insect species (Usherwood and Machili, 1968; Usherwood, 1976), also activates receptors on the extrajunctional muscle membrane of the extensor tibiae (E.T.) muscle of nymphal z.nd adult locusts. The fibres of this muscle have two pharmacologically distinct populations of extrajunctional glutamate receptors (denoted D- and H-receptors): activation of D-receptors mediates a transient increase in Na+ and K+ permeability, while H-receptor activation mediates an increased Cl- permeability (Lea and Usherwood, 1973; Cull-Candy, 1976). There i:; growing evidence that extrajunctional glutamate rzceptors might also occur on some vertebrate central ceurones and that like those of the locust E.T. muscle t ley might have significantly different physiological and pharmacological properties from their junctional counterparts on these cells (see review by Usherwood, 1978). It is difficult to judge from the fragmentary evidence available for both vertebrate and invertebrate excitable systems whether the presence of extrajunctional receptors on cells innervated by glutamatergic neurones is a widespread phenomenon. More information on this point is obviously required if effective pharmacological studies are to be made on these sysKey words: L-glutamate vation.
receptors,
insect muscle,
terns. There are indications that extrajunctional receptors are present on locust muscles other than the E.T. muscle, and on skeletal muscles of other insects, but their presence here has not been convincingly demonstrated. The orthopteran retractor unguis (R.U.) muscle falls into this category and since this muscle has been developed and extensively used for assaying pharmacological agents (Usherwood and Machili, 1968; McDonald, Farley and March, 1972; Clements and May, 1974; Walther and Rathmayer, 1974; Usherwood and Cull-Candy, 1975) it seemed an appropriate starting point in determining whether extrajunctional receptors are a common feature of insect skeletal muscles. It will be shown that the extrajunctional membrane of R.U. muscle fibres has similar pharmacological properties to the extrajunctional membrane of E.T. fibres. However, the distribution of extrajunctional receptors is much more uniform in R.U. muscle than in E.T. muscle, a difference which is maintamed following denervation. METHODS The
results
described
in this
paper
were
obtained
E.T. muscles of the metathoracic leg of the locust, Schistocerca gregaria (Usherwood and Machili, 1968). The .muscles were exposed by removing cuticle on the ventral side of the femur followed by dissection of the overlying flexor tibiae from
dener-
201
the R.U.
and
202
K. A. F.
GRATION.
R. B.
CLARK
muscle. The E.T. muscle provided support for the R.U. muscle during insertion of microelectrodes, which greatly facilitated voltage-clamp studies of the R.U. muscle fibres. The R.U. muscle is innervated by metathoracic nerve Sb which supplies it with two large excitatory motor axons (A, and A,) (Rees and Usherwood. 1972a). The muscle comprises two bundles of muscle fibres: one contains large diameter (5~80pm) “white” fibres and is innervated by axon A,, the other contains smaller diameter (20-40pm) “red” fibres and is innervated by axon AZ. Some fibres also receive endings from inhibitory neurones (C. Walther, personal communication). The R.U. muscle fibres are multiterminally innervated but the distal regions of the white fibres (i.e. IO?:, of the total fibre length, which is _ 1 cm) are devoid of nerve endings. These nerve-free regions are very suitable for investigating extrajunctional glutamate sensitivity since diffusion of iontophoretically-applied L-glutamate from extrajunctional sites of application to junctional glutamate receptors is obviated. The E.T. muscle receives endings for “fast” and “slow” excitatory motorneurones and an inhibitory motorneurone but ventral fibres in the mid-region of this muscle are exclusively innervated by the “fast” excitatory axon (Usherwood and Grundfest, 1965). The studies described in this paper were restricted to these fibres (see also Clark. Gration and Usherwood, 1978). The R. U. and E.T. muscle were denervated on one side of the animal by sectioning nerve 5 of the metathoracic ganglion using an aseptic procedure (Usherwood, 1963a). Operated animals were isolated in individual cages at 30°C. Denervated and contralateral control muscles were prepared side-by-side for physiological and pharmacological studies in the same perfusion bath. Preparations were continuously superfused with either standard saline, which contained NaCI 180, KC1 10, CaCl, 2. NaZHPO, 6, NaH2P04 4mM. pH 6.8 or Cl--free saline: NaCH,S04 180, KCH3S04 10. (C,H,.COO),Ca 2. NaH2P04 4, Na,HP04 6 mM. pH 6.8. High resistance (100-300 M0 in standard saline) microelectrodes filled with 0.1 M Na L-glutamate (pH 8) were used for the iontophoresis of glutamate. Backing currents in the range 3320nA were required to obtain optimum glutamate responses. Intracellular recordings were made with 2 M tripotassium citrate microelectrodes (5-I 5 MQ), using conventional electrophysiological techniques. A twoelectrode clamp circuit was used for voltage-clamp experiments. RESULTS
In standard saline iontophoresis onto the extrajunctional membrane
of L-glutamate at the distal end
and P. N. R.
USHERW~D
of “white” R.U. muscle hbres evoked biphasic responses, which consisted of a depolarization preceding a hyperpolarization. Replacement of standard saline by Cl--free saline resulted in the rapid reversal and eventual virtual abolition of the hyperpolarizing component. There is a small residual current which possibly arises from a permeability increase for methylsulphate ions {Lea and Usherwood, 1973) although contribution of K + ions to the hyperpolarizing current has not been ruled out. The transient depolarizations that remained were of a similar amplitude to the extrajunctional D-response recorded from E.T. fibres in Cl--free saline (Cull-Candy, 1976; Clark er al., 1978). Slight displacement of the L-glutamate electrode had little effect on either the amplitude or rise time of these responses, indicating a non-focal distribution of receptors. Mapping the extrajunctional sensitivity to glutamate on R.U. muscle fibres revealed a low but essentially uniform L-glutamate sensitivity (0.14.5 mV/nC) over the extrajunctional membrane but with high levels of sensitivity (up to 1.4 mV/nC) at the muscle-apodeme junction. The distribution of extrajunctional glutamate sensitivity over R.U. white fibres was more uniform than over E.T. fibres from the same leg and was more consistent between fibres. To compare the relative distribution of extrajunctional sensitivity for R.U. and E.T. muscles the coefficient of variation of glutamate sensitivity (i.e. S.D./mean sensitivity) was determined on areas of both R.U. and ET. fibres. The glutamate sensitivity of R.U. fibres was measured on extrajunctional areas well away from the muscle-apodeme junction (>4#pm; Fig. 1). For R.U. fibres the coefhcient of variation was 0.22 _t 0.04 (mean It SD., n = 12) while for E.T. fibres a value of 0.60 + 0.21 (n = 14) was obtained. The difference between the mean values of the coefftcients for the two muscles was highly significant (P < 0.001. Student’s r-test). The relationship between glutamate dose and response amplitude for the interaction of L-glutamate with extrajun~tional D-receptors on R.U. muscle fibres was measured at the resting potential (approximately -65 mV in Cl--free saline). The data confirm the non-focal distribution of these receptors, since both the amplitude and rise time of the glutamate potentials increased with dose. This is similar to the interaction between acetylcholine (ACh) and extrajunctional receptors on frog muscle fibres (Feltz and Mallart, 1971). The limiting slope of double Iogarithmic dose-amplitude plots (Fig. 2) was 1.48 I: 0.2 (mean & S.D., n = 6). which is approximately the same as that for the interaction of L-glutamate with extrajunctional D-receptors on E.T. muscle fibres (Cull-Candy, 1976: Clark et al., 1978). Extrajunctional D-receptors on R.U. muscle are readily desensitized. Recovery from desensitization was examined by varying the time interval between identical glutamate doses delivered from a single high resistance glutamate micropipette and measuring the ratio of the amplitude of the second (“test”) response
203
Denervated insect muscle
Fig. 1. The distribution of extrajunctional glutamate sensit vity on a locust R.U. fibre (resting potential -63 mV). The glutamate electrode was moved in 1Opm steps along the centre of the muscle fibre and the sensitivity at each point measured in terms of the peak depolarization per unit glutamate dose (mV/nC) produced by a constant dose of 2 nC (IO msec). The upper points (@).show the distribution of glutamate sensitivity in the region of the muscleapodeme junction: the lower points (0) show the distribution on an area of extrajunctional membrane starting 4IOprn from the apodeme. Note that the glutamate sensitivity scale is logarithmic. The recording electrode was n:ver more than 400pm from any extrajunctional site to which glutamate was applied. This is unlikely to cause any significant error in estimating the local glutamate sensitivity, as the length constant of R.U. fibres is several mm.
EPSP’s were almost completely absent from R.U. fibres and the extrajunctional glutamate sensitivity of these fibres had increased by approximately 3-fold compared with control levels (Fig. 4) although at this time the sensitivity of ET. fibres had not significantly changed and min. EPSP’s were present in most fibres. By 5-7 days, min. EPSP’s had disappeared from E.T. fibres and their extrajunctional sensitivity had increased. The difference between the time required for the appearance of extrajunctiona) supersensitivity on R.U. and E.T. fibres can probably be attributed, in part, to different rates of axon terminal degeneration (Usherwood, 1963a, b; Rees and Usherwood, 1972a). Complete development of extrajunctional supersensitivity took from 12 to 14 days on R.U. fibres and from 10 to 12 days on the E.T. fibres (Fig. 4). The mean (& S.D.) maximum extrajuncrional glutamate sensitivity of R.U.. and E.T. fibres was 3.4 + 0.8mV/nC (n = 60) and 1.7 + 1.1 mV/nC (n = IOO), res~ctively. Examples of the distributions of giutamate sensitivity over R.U. and E.T. fibres from a muscle denervated for 7 days are compared in Figure 5. The distribution of glutamate sensitivity over the R.U. fibre was uniform and there was little variation in this parameter between this fibre and other fibres in the muscle. However, the distribution of glutamate sensitivity over the E.T. fibre at this time was very
to that of the first (“control”) (Clark et a/., 1978). For pulse separations of less than 1-2 set the test response was barely detectabIe. The response ratio increased with increasing time interval until at separations in tl+e range 40-50 set the test and control responses had identical amplitudes (Fig. 3a). The kinetics of recovery fram desensitization (Fig. 3b) were found to be best described by an equation containing a single exponential term, i.e. identical to that describing the recovery from desensitization of D-responses of E.T. muscle fibres (Clark ef al., 1978). The exponential time constants of recovery ranged from 10.5 to 22sec (mean rt SD., 12 F 3 set, n = 7), being very similar to those measured from the curves for recovery from desensitization of D-responses on E.T. muscle fibres. Comparison of pharmacological properties of denerwted R.U. and ET. muscle fibres The extrajunctional glutamate sensitivity of chronically denervated R.U. and E.T. muscles was investigs.ted for up to 28 days after denervation (Fig. 4). Botween I and 3 days post-denervation miniature excitatory postsynaptic potentials (min. EPSP’s} were observed in most fibres of the R.U. and E.T. muscles. The magnitude and dist~butjon of the extrajunctiona1 glutamate sensitivity of these fibres were not significantly different from those of fibres of the contralaterai control mu&es. After 4 days denervation, min.
40-J 7’‘llo0 Dose
(nC)
Fig. 2. Upper graph. Relationship between extrajunctional glutamate potential amplitude (V,) and coulombic dose (nC), on double log co-ordinates. The limiting slope (solid line) of the dose-response relationship is 1.55. Line drawn by eye. Lower graph. Relationship between time to response peak (r,) and cou~ombic dose, on double log co-ordinates. The time to peak was taken from the onset of the glutamate pulse. The slope of the solid line is 0.25. Line drawn by eye.
204
K. A. F. GRATION, R. B. CLARK
and P. N. R.
USHERWOOD
(A) ImV
I
I ,I ?I
2,
-,
I I 0
I IO Days
after
I
20
I 1 1 30
deneivation
Fig. 4. Comparison of the extrajunctional glutamate sensitivities of locust retractor unguis (of and extensor tibiae (0) fibres, denervated for different time periods (G28 days). Each datum point is the mean +S.D. of at least 60 sensitivity measurements from 3 fibres of 2 different muscles.
)
I
zi
0
Pulse
interval
SO (set)
Fig. 3. (A) D-responses evoked by two successive glutamate doses, applied iontophoreticaily to the same area of extrajunctional membrane. The pulse separation, indicated in the left-hand column, was varied between 540 sec. Resting potential of muscle fibre -63.5 mV; control response amplitude 1.6 mV; glutamate dose 5 nC. Note the miniature excitatory post-synaptic potentials in most of the records. (B) Examination of the desensitization recovery kinetics of extrajunctional glutamate responses, using a two-pulse technique (see text for explanation). The “response ratio”, R, i.e. the amplitude of the second (test) response over the amplitude of the first (control), is plotted as a function of the time interval between the glutamate pulses. The time course of recovery from desensitization is fitted to an exponential equation (solid line) with a time constant of 13.9sec. Resting potential -62mV; control response amplitude 1.4 mV; glutamate dose 4 nC.
non-uniform and showed only localized areas of increased glutamate sensitivity. In some cases the mean extrajunctional sensitivities of denervated and contralateral control E.T. fibres were not significantly different. On the other hand, denervated R.U. fibres always had a greater glutamate sensitivity than fibres of the contralateral control muscle. These differences in the relative distributions of glutamate sensitivity on R.U. and E.T. fibres were measured in terms of the coefficient of variation of the sensitivity distribution (Fig. 5). The coefficient for 3.5 denervated R.U. fibres (6-18 days after denervation) was 0.32 + 0.1 (mean f SD.) while that for 38 ET. fibres over a similar denervation period was 0.76 & 0.36. The difference between the coefficients was found to be highly significant (P < 0.001, Student’s t-test). Between 20-28 days after denervation, the extrajunctional sensitivity of R.U. and E.T. muscle declined (Fig. 4). In R.U. fibres sensitivity decreased &fold, to a level comparable to that of the 6 day-denervated muscle, while that for E.T. fibres a a-fold decrease was observed. Former junctional areas on muscles denervated for more than 20 days were easily distinguished from extrajunctiobal areas by their significantly higher sensitivity to glutamate. As in normal muscles their sites were generally located in the clefts between fibres (Usherwood and Machili, 1968). The sensitivity of these areas was similar to that of junctions on control fibres, i.e. lO-lOOmV/nC. Measurement of extrajunctional glutamate sensitivity in terms of the membrane depolarization per
205
Denervated insect muscle
01
I
0
I
50 Distance
I
I
I
100
150
200
( ,um 1
Fig. 5. Comparison of the distribution of extrajunctional glutamate sensitivity on 7 days denervated locust retractor unguis and extensor tibiae fibres. Solid circles (a), R.U. fibre, resting potential -57.6 mV. Open circles (o), E.T. fibre, resting potential - 51.3 mV. The mean glutamate sensitivity (*SD.) was 1.42 + 0.5 mV/nC for the R.U. fibre, 0.78 & 0.5 mV/nC for the E.T. fibre and the respective coefficients of variation of the glutamate sensitivities were 0.35 and 0.64. The small difference in resting potential accounts for only IO?< of the difference in glutamate sensitivity since the reversal potential of the glutamate response is near zero (See Fig. 6B).
unit glutamate dose depends partly on the electrical properties of the muscle membrane (Albuquerque and M&sac, 1970). Following denervation the input resi:;tance of R.U. and E.T. fibres, measured by current injection through a second intracellular microelectrade (resistance 5-10 MR), increased from 565 + 196 kR (mean 2 SD., 58 observations on IO muscles) to 943 + 266 kQ (30 observations on 7 muscles) 620 days after denervation) and from 260 & 56 kR (52 observations on 8 muscles) to 497 t_ 91 ki2 (43 observations on 8 muscles) over a similar denervation period, respectively. Note that the increased input reGtance of the denervated fibres was not sufficient to account for the observed increase in sensitivity to glutamate (Fig. 4). A comparison of the extrajunctional glutamate currents of innervated and denervated muscle hbres under voltage clamp supports this conclusion. The inward currents associated with extrajunctional glutamate responses of denervated R.U. fibres were much larger (maximum recorded amplitude of 28 nA, at a membrane potential of - 60 mV) than the extrajunctional currents from innervated fibres (Fig. 6a). A similar difference in the magnitude of extrajunctional currents has previously been observed for E.T. fibres (Clark et al., 1978). The relationship between peak current and membrane potential for extrajunctional responses of denervated R.U. fibres was approximately linear (Fig. 6) and the reversal potential for fibres 8-14 days post-denervation, determined from a linear regression over mem-
brane potentials in the range + 10 to -6OmV, was + 3.5 k 2.6 mV (data from 8 fibres of 6 preparations). The reversal potential of glutamate currents recorded from former junctional areas on 8-14 day denervated fibres was + I.7 + 2.6 mV (data from 7 fibres of 4 preparations), a value which was not significantly different from that of junctions on control muscles, i.e. -3.7 & 2.9 mV (n = 6). The reversal potential of extrajunctional glutamate currents of innervated muscle fibres could not be determined reliably using voltage clamp. A slow outward current, whose amplitude increased as the membrane potential was reduced, was often superimposed on the inward current generated by activation of extrajunctional D-receptors. The outward current possibly resulted from activation of extrajunctional H-receptors. the current being carried primarily by CH,SO; ions (Lea and Usherwood, 1973) or poss i biy K+ rather than Cl-. In one experiment the glutamate electrode was replaced by one fihed with 0.1 M DLibotenic acid, which activates only the H-receptors on the extrajunctional membrane (Lea and Usherwood, 1973; Cull-Candy, 1976). When DL-ibotenate was ejected onto extrajunctional membranes of E.T. and R.U. fibres in Cl--free saline, an outward current was generated whose time course was similar to that seen with glutamate. The amplitude of this current was up to 2-4nA at membrane potentials near zero. The inward current decreased in amplitude as membrane potential was reduced and at about -2OmV,
K. A. F. GRATION.R. B.
206
CLARK
(A)
n
(b)
*4
and P. N. R. USHERW~~
the D-current (inward) and the outward H-current were equal and opposite. In some preparations the H- and D-currents had similar time-courses and could not be distinguished during glutamate iontophoresis. As a result “apparent” reversal values for D-responses in Cl-free saline of about - 20 mV were obtained. However, application of ibotenate to these fibres demonstrated unequivocally the presence of an outward current presumably equal in amplitude but opposite in polarity to the inward D-current. Since the extrajunctional inward currents recorded from denervated fibres were considerably larger than those of innervated fibres the accompanying outward H-current did not greatly influence their reversal values. Figure 7 shows examples of the time course of recovery from desensitization, measured using the twopulse method, for extrajunctional glutamate responses recorded from denervated R.U. muscles. The kinetics of recovery from desensitization were similar to those of extrajunctional responses of innervated fibres but the recovery time constants were generally much smaller. The time constant was smaller (i.e. recovery from desensitization was more rapid) for responses from membrane areas of higher glutamate sensitivity. Many of the extrajunctional responses from denervated muscle had recovery kinetics consisting of “fast” and “slow” phases of recovery. The time course of recovery from desensitization in these cases was best described by an equation containing two exponential terms (Fig. 7). DIfCUSSlON
Fig. 6. {A) Comparison of the extrajunctional glutamate currents from an innervated (b) and a 16 day denervated (c) locust retractor unguis fibre, under voltage clamp. Both fibres were clamped at a membrane potential of - 60 mV. The glutamate dose was 2 nC (trace a). Current calibration is 2nA for the response from innervated fibre, IOnA for the denervated. Note miniature excitatory postsynaptic currents on trace (b). (B) Relationship between membrane potential (E,) and peak clamp current (I,) of extrajunctional gfutamate responses from three denervated R.U. fibres. Inward clamp currents are shown positive. The solid lines are linear regression fits to the data. The reversal potentials, determined from the regression lines are +9 mV (0). $8.5 mV (0). and f3.1 mV (m).
The properties of extrajunctional glutamate responses of the innervated locust R.U. muscle are in most respects identical to those which can be recorded from E.T. muscles, Although H-receptors have been observed on fibres of the locust retractor unguis (Usherwood, unpublished) and anterior coxal adductor muscles (Lea and Usherwood, 1973), the presence of D-receptors on muscles of this insect other than the E.T. muscle has not hitherto been reported. The high glutamate sensitivity observed near the muscle-apodeme junction of R.U. fibres is analogous to the high ACh sensitivity of myotendinous junctions of some vertebrate skeletal muscles (Miledi and Zelena, 1966; Albuquerque and M&sac, 1970). A similar high glutamate sensitivity occurs near the muscle-apodeme junction of E.T. fibres (Cull-Candy, 1978) although the region of high sensitivity appears to be more restricted than for the R.U. muscle-apodeme junction. The increase in extrajunctional glutamate sensitivity of R.U. fibres which follows denervation (Usherwood, 1969) is quaiitatively similar to that which occurs on denervated E.T. fibres (Cull-Candy, 1975; 1978; Clark er al., 1978). The distribution of extrajunctional glutamate sensitivity on denervated locust
207
Denervated insect muscle
and changes are observed in the muscle cytoarchitecture (Rees and Usherwood, 1972b). Possibly, protein synthesis is impaired at this stage, causing a decline in the turn-over of extrajunctional receptors and a consequent reduction in receptor density. A consistent finding was that the sensitivity of former junctional areas bad not declined even when muscle atrophy was very obvious. This suggests that the density of glutamate receptors on the former post-synaptic membrane remains unchanged after denervation. A similar conclusion has been reached by Hartzell and Fambrough (1972) for ACh receptors on the denervated rat diaphragm. The similar reversal potentials of the junctional currents and the extrajunctional currents on denervated muscle suggests that the apparent increase in
IIP
*n 0
IO
Pulse Interval(set
20 )
Fig. 7. Desensitization recovery kinetics of extrajunctional glutamate responses of denervated R.U. fibres. The response ratio, R (see text and Fig. 3 for explanation), is plotted as a function of the time interval between successivt: glutamate doses. Open circles (o), data from a 4 day denervated fibre, resting potential -57.8 mV. Control response amplitude 1.5 mV, glutamate dose 3.0 nC. The solid line is a single exponential of time constant 7.5 sec. Solid circles (e), data from a 6 day denervated fibre, resting potential -49.6 mV. Control response amplitude 2.6 mV. glutamate dose 2.8 nC. The solid line is a double exponential of time constants 0.45 set and 4.2 sec.
ET. muscle is similar to the heterogeneous distribution of extrajunctional ACh sensitivity on 3-day denervated rat diaphragm (Hartzell and Fambrough, 1972) and soleus/extensor digitorum muscles (Albuquerque and M&sac, 1970) with the receptors tending to occur in “patches”. The distribution on the locust R.U. muscle is more like the homogeneous distribution of ACh sensitivity on the rat muscles 10 days post-denervation. The difference between the distribution of glutamate sensitivity on both innervated and denervated R.U. and E.T. muscles may indicate a true difference in the density of extrajunctional glutamate receptors on these muscles, but unfortunately an independent measure of glutamate receptor densitf, is, as vet, unavailable. ‘The decline in extrajunctional glutamate sensitivity of both R.U. and E.T. fibres which occurs about 20 days post-denervation is analogous to the decrease in the extrajunctional ACh sensitivitv of rat soleus and extensor digitorum longus muscles after prolonged denervation (Albuquerque and M&sac, 1970), which Hartzell and Fambrough (1972) have suggested is due, in part, to selective loss of ACh receptors from the membrane. It may be significant that the fall in glutamate sensitivity of denervated R.U. and E.T. muscle occurs at a time when muscle atrophy is very obvious. By 20-24 days post-denervation, the crosssectional area of R.U. muscle decreases by about 65%
glutamate receptor density which occurs after denervation results from the “appearance” of receptors which are similar, if not identical, to those occurring at the junctions. The junctional receptors mediate a permeability increase for Na+ and probably K+ and Ca” (Anwyl and Usherwood. 1975; Anwyl, 1977) and it seems likely from the reversal potentiaf that the Na+/K’ permeability ratio for the extrajunctionat receptors on denervated muscle is identical to that for junctional receptors. The reversal potential of D-responses of innervated muscle is unclear at present for the reasons discussed earlier. However, voltage clamp studies on vertebrate muscle (e.g. frog: Mallart, Dreyer and Peper, 1976) have shown that the reversal potentials of junctional and extrajunctional ACh responses are identical.
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