J. Physiol. (1976), 255, pp. 449-464 With 6 text-flguree Printed in Great Britain
449
TWO TYPES OF EXTRAJUNCTIONAL L-GLUTAMATE RECEPTORS IN LOCUST MUSCLE FIBRES
BY S. G. CULL-CANDY* From the Department of Zoology, the Univer8ity, Glasgow G12 8QQ
(Received 27 May 1975) SUMMARY
1. L-glutamate applied iontophoretically to the extrajunctional membrane of locust muscle produced a biphasic response, depolarization followed byhyperpolarization (i.e. DH-response). Applying L-glutamate and DL-ibotenate from multibarrel micropipettes allowed comparison of their extrajunctional responses. While glutamate produced a two component response, ibotenate produced a single component H-response. 2. The equilibrium values for the H-responses to L-glutamate and DLibotenate applied at the same extrajunctional site were very similar. The equilibrium value was 59-5 + 5-4 mV indicating an increased C1- conductance. The H-response was reversed and abolished in Cl- free medium. Picrotoxin 10-3 M selectively blocked the H-component of the DHresponse in a reversible manner. 3. The possibility that the D- and H-responses arose from the activation of two distinct types of extrajunctional glutamate receptors was investigated. Desensitization ofthe glutamate H-response by ibotenate and vice versa indicated the presence of an extrajunctional H-receptor sensitive to glutamate and ibotenate and an extrajunctional D-receptor sensitive to glutamate and insensitive to ibotenate. The junctional depolarizing response to glutamate was insensitive to ibotenate. 4. The presence of junctionally occurring H-receptors could not be discounted, although, if present, they were not measurably activated by the excitatory transmitter. 5. Double logarithmic plots (coulomb dose v8. response) for the actions of glutamate and ibotenate on H-receptors had values of 0-75, indicating that both drugs act on the same receptors with similar mechanisms. The value for the action of glutamate on the D-receptors was 1-5. 6. While the extrajunctional D-receptors show analogies to the extrajunctional ACh receptors in vertebrate muscle, the significance of the extrajunctional H-receptors remains speculative. * Present address: Department of Biophysics, University College London, Gower Street, London WCIE 6BT. 15-2
450
S. G. CULL-CANDY INTRODUCTION
In vertebrate skeletal muscle fibres acetylcholine (ACh) receptors occur both at the neuromuscular junction and in the extrajunctional membrane (Miledi, 1960; Katz & Miledi, 1964; Feltz & Mallart, 1971). In general the extrajunctional ACh receptors are restricted to the region immediately surrounding the junction, but in slow contracting muscles of the rat a low level of ACh sensitivity covers the entire muscle surface (Miledi & Zelena, 1966). In crustaceans and insects, L-glutamate (or a closely related substance) is the suggested transmitter at excitatory neuromuscular junctions (see Takeuchi & Takeuchi, 1964; Usherwood & Cull-Candy, 1975) which occur along the multiply innervated muscle fibres. In the present study glutamate and the glutamate analogue, ibotenate were iontophoretically applied to the locust muscle membrane. By using these two substances it was possible to distinguish two pharmacologically distinct populations of extrajunctional glutamate receptors. METRODS
All results described in this paper were obtained from the extensor tibiae muscle of the metathoracic leg of the locust, Schistocerca gregaria. The muscle was exposed by the removal of the cuticle covering the ventral aspect of the femur followed by dissection of the flexor tibiae and retractor unguis muscles (Hoyle, 1955). The large trachea which overlies the extensor muscle was cut away along one side of the muscle, to leave behind the fine tracheal branches which run close to the bundles of extensor muscle fibres. These branches were useful as reference points when the glutamate sensitivity of the muscle fibres was mapped since occasionally their apposition to the outer face of the muscle fibres or more often their intersection with the clefts between fibres marked the position of sites highly sensitive to L-glutamate. It has previously been demonstrated in other insect muscles that glutamate sensitive sites occur mainly but not exclusively in the clefts between fibres and that these coincide with junctional sites (Berdnek & Miller, 1968; Usherwood & Machili, 1968). The advantage of using the extensor tibiae muscle is that sites highly sensitive to glutamate can be found consistently on the exposed outer surface of the fibres; this allowed greatly improved examination of the properties and topography of junctional glutamate receptors. Furthermore, as the fibres of the extensor tibiae muscle are relatively large (ca. 100 #sm diam.) and have a high proportion of extrajunctional to junctional membrane, they were very suitable for investigating the glutamate sensitivity in the extrajunctional muscle membrane. Although a few experiments were performed on muscle fibres which received both excitatory and inhibitory innervation, as judged by the presence of spontaneous excitatory and inhibitory miniature potentials, most of this investigation was on fibres which received only excitatory innervation. Modified saline with an increased concentration of NaCl was used in most experiments (Usherwood & Grundfest, 1965). This saline contained NaCl, 200; KCl, 10; NaH2PO4, 4; Na2HPO4, 6; CaCl2, 2 mM at pH 6-8. Preparations were perfused with this saline for 2 hr before experimentation, to allow equilibration. In some experiments Na+ ions were replaced by choline to obtain a low Na+ (10 mM) saline, further
TWO TYPES OF EXTRA JUNCTIONAL RECEPTOR
451
substitution of the phosphate buffer with Hepes buffer (Sigma) produced Na+ free saline. Cl- free saline contained: NaMeSO4, 200; KMeSO4, 10; NaH2P04, 4; Na2HPO4, 6; Ca propionate, 2 mM at pH 6-8. Single and double barrel micropipettes when drawn contained central fibres of soft Pyrex glass (Tasaki, Tsukahara, Ito, Wayner & Yu, 1968; Peper & McMahan, 1972). Injection of drug into the shank just before use produced micropipettes with resistances between 150 and 300 Mn (measured in locust saline). The following drugs were used: 1 or 04- M-Na L-glutamate and Na D-glutamate, 041 m-Na DL-ibotenate dissolved in distilled water and adjusted to pH 7 with NaOH. At pH 7 glutamic and ibotenic acids are mainly anionic (Johnston, Curtis, de Groat & Duggan, 1968). 1 M y-amino-n-butyric acid (GABA) was dissolved in distilled water and adjusted to pH 4 with HCO (Takeuchi & Takeuchi, 1965). For the investigation of extrajunctional responses to L-glutamate or DL-ibotenate the drug-pipette required braking currents between 4 x 10-8 and 6 x 10-9 A to obtain optimum responses. A similar braking current was usually used to investigate the junctional responses to L-glutamate, although it was possible, with high resistance pipettes, to obtain fast (rise times less than 50 ms) and repeatable responses at these sites with no braking current (even though some diffusion of glutamate did occur from the tips of high resistance glutamate pipettes). Extrajunctional glutamate receptors were desensitized by the background leakage of drug which occurs from conventional micropipettes. It was therefore essential to use high resistance (> 150 MI) micropipettes and to allow a 60 s recovery period between pulses to obtain repeatable responses at a single site. For the investigation of extrajunctional receptors it was necessary that the iontophoretic pipette should be in close proximity to the muscle fibre membrane; therefore during passage of current the pipette was allowed to enter the muscle fibre and was then withdrawn the minimum distance necessary for it to leave the fibre. Penetration of the membrane was marked by the appearance of an intracellular electrotonic potential. This technique proved particularly useful for the examination of the tips of double barrel pipettes, the use of which usually required both tips should be at exactly the same distance from the receptor sites under investigation. The tips of double barrel pipettes which appeared symmetrical under a water immersion objective did not always penetrate the muscle fibre simultaneously. These were rejected. Intracellular recording electrodes were filled with 2 M potassium citrate acidified to pH 6 with citric acid and had resistance of 10-20 MQ. K citrate electrodes were also used to displace membrane potential of extensor tibiae muscle fibres. This avoided the injection of Cl- into the fibres during the injection of negative current and in addition these electrodes showed little tendency to block (Fatt, 1961 a). The current electrodes were lowered on to a fibre impaled by a recording electrode and inserted nearby by the passage of a short pulse of negative current through the electrode.
RESULTS
Comparison of the extrajunctional responses to glutamate and ibotenate Glutamate locally applied to locust excitatory junctional receptors causes a depolarization of the membrane potential (Beranek & Miller, 1968; Usherwood & Machili, 1968). When applied from a high resistance micropipette to the extrajunctional membrane of a muscle at the resting potential, glutamate produces either a small biphasic (depolarization followed by hyperpolarization) or hyperpolarizing response (Cull-Candy & Usherwood, 1973). Jbotenate, an isoxazole which shows certain structural
452 S. G. CULL-CANDY similarities to glutamate, has been reported to activate glutamate receptors in spinal interneurones (Johnston et al. 1968), Renshaw cells (Johnston, Curtis, Davies & McCulloch, 1974), central neurones (Shinozaki & Konishi, 1970) and snail neurones (Walker, Woodruff & Kerkut, 1971). The application of ibotenate to locust muscle fibres via the bathing medium causes an increase in Cl- conductance of the muscle fibre (Lea & Usherwood, 1973). A
L
-glutamate
B
-60
-60
-635
-63-5
-67 S
-675S
-70
~
DL-ibotenate -NNW
-70
Fig. 1. Comparison of the extrajunctional responses to L-glutamate and DL-ibotenate applied iontophoretically from a twin barrel micropipette to the same region of extrajunctional membrane. Note that since the fibres are multiply innervated spontaneous miniature excitatory potentials are recorded from junctional and extrajunctional regions in these and in subsequent records. A, at the resting potential, -60 mV, glutamate produced a DH-response, the H-component of which was annulled at - 63-5 mV when a D-component alone was seen. The H-component reversed at membrane potentials greater than - 63-5 mV so that a two-component (DH) depolarization occurred at - 67-5 and at -70 mV. The D-component increased in amplitude as the membrane potential increased from -60 to -70 mV. B, at the resting potential, -60 mV, ibotenate produced a pure H-response which was annulled at - 63-5 mV, no net ionic movement occurring at this level, in comparison with the glutamate induced Dresponse at - 63-5 mV. The H-response reversed at membrane potentials greater than - 63-5 mV; depolarizing H-responses which increased in amplitude with increased membrane potential occurred at - 67-5 and -70 mV. Calibration: 1 mV and 200 ms.
Comparisons were made of the extrajunctional responses to glutamate and ibotenate by the iontophoretic application of these drugs to the same extrajunctional site from twin barrel pipettes which contained glutamate in one barrel and ibotenate in the other (see Fig. 1). Glutamate produced a biphasic response, depolarization followed by a hyperpolarization, while ibotenate produced only a hyperpolarizing response at the resting potential.
453 TWO TYPES OF EXTRA JUNCTIONAL RECEPTOR Hyperpolarization of the membrane by current injection abolished the hyperpolarizing response to glutamate and ibotenate at the same membrane potential level (Fig. 1). At this membrane potential a depolarizing response to glutamate was clearly present in fibres which had produced biphasic responses to glutamate at their resting potential. Further hyperpolarization of the membrane caused the second component of the biphasic response to glutamate and the single component of the response to ibotenate to reappear as a depolarization.
Equilibrium potential for the H-response The equilibrium potential for the H-response could be accurately determined in those fibres where the D-response was small or absent. Values were also obtained in fibres which normally produced DHresponses to glutamate either by removal of the ions which generated the D-component or by the use of ibotenate. Values for EH-glutamate and EH-ibotenate obtained from single muscle fibres by using either double barrel pipettes, glutamate in one barrel and ibotenate in the other, or two single barrel pipettes, were not measurably different. The mean value (± S.D.) obtained was - 59-5 + 5.4 mV (for twelve fibres investigated simultaneously with glutamate and ibotenate) when the resting potential value was - 53-6 + 36 mV. This is compatible with the idea that the H-response was generated by an increased Cl- permeability, and is similar to figures for the equilibrium value for GABA action (-60 to -70 mV), at inhibitory junctional receptors in locust muscle fibres, which also causes an increased Cl- permeability (Usherwood & Grundfest, 1965). Very few fibres (less than 1 %) produced a depolarizing H-response at the resting potential and when present it was usually associated with fibre damage. Chloride involvement in the H-response Cl- in the bathing medium was substituted by the impermeant methyl sulphate anion (see Methods). The driving force on Cl-, across the muscle fibre membrane, would be expected to rapidly reverse and so result in the reversal and eventual abolition of a Cl- mediated event as Cl- leaks from the fibres (see Fatt, 1961 b; Usherwood & Grundfest, 1965). Fig. 2 shows a typical experiment in which the H-response to glutamate had reversed 4 or 5 min after Cl--free saline entered the bath. The now depolarizing H-response increased in size as the Cl- was removed from the bath and extracellular spaces. Over a period of 1-2 hr, which presumably represented the time for most of the Cl- to leave the fibre, the depolarizing H-response declined in amplitude and finally disappeared completely. Displacement of the muscle fibre membrane potential by depolarizing or hyperpolarizing current injection failed to restore the H-response.
S. G. CULL-CANDY 454 Reapplication of normal saline to preparations treated with C1- free saline caused the reappearance of the H-response in all cases and a very marked increase in amplitude of response in those preparations where glutamate still produced small H-responses in Cl- free saline. Similar results were obtained with ibotenate. ControlI' Cl free-* 3 min-
4 min--
-
---
5 min-.
60 min
70 min
--
80 min
Cl
>
I Fig. 2.. The effect of Cl--free saline on the extrajunctional response to glutamate. In normal saline the muscle fibre produced a DH-response (the D-component being only just visible). 5 min after Cl- removal the Hresponse reversed so that a two component (DH) depolarization was present. At 70 min, the H-response had disappeared leaving the small Dcomponent. In this fibre a small hyperpolarizing H-response, following the D-component, was present at 80 min. Addition of CO- ions to the saline results in the rapid reappearance of an H-response of a slightly larger amplitude than before Cl- removal. The slight change in time course may be due to reposition of the glutamate micropipette. Calibration: 1 mV and 200 ms.
455 TWO TYPES OF EXTRAJUNCTIONAL RECEPTOR In some preparations (approximately 30 %) a very small hyperpolarizing H-response reappeared at the resting potential in muscle fibres soaked for several hours in Cl- free saline (see Fig. 2); this was probably the result of a glutamate induced conductance increase, in the membrane, to MeSO4 , since ibotenate is known to have such an effect (Lea & Usherwood, 1973). The action of picrotoxin on the H-response Locust extensor muscles exposed to picrotoxin 0-3 M were investigated with double barrel pipettes which contained glutamate and ibotenate (eleven fibres). This permitted simultaneous investigation of the action of picrotoxin on the glutamate and ibotenate response. The treatment of fibres in picrotoxin for 20 min completely blocked the H-response to ibotenate and the H-component of the DH-response to glutamate. With lower concentrations of picrotoxin (i.e. 104 M) only a partial block of the Hresponse was observed. Reappearance of the H-response on washing took approximately 20 min. These results provide additional evidence that the H-response is Cl- mediated.
Evidence for two population of extrajunctional receptors: specific action of ibotenate The demonstration of a biphasic potential response to glutamate, and a monophasic response to ibotenate when these drugs are applied to the extrajunctional membrane, raises the possibility that there are two types of extrajunctional glutamate receptors, one of which is activated by ibotenate. A classical property of receptors is their desensitization or refractoriness in the continued presence of an agonist (Katz & Thesleff, 1957). It follows that if a receptor is desensitized by an agonist, the agonist has activated the receptor. As ibotenate applied iontophoretically to an extrajunctional site on locust muscle produced a response which resembled the hyperpolarizing component of the response to glutamate, it was of interest to see if the hyperpolarizing component of the glutamate response could be desensitized with ibotenate or vice versa to determine whether these two drugs acted on the same receptor. The experiments (twenty fibres, different preparations) were performed by the iontophoretic application of ibotenate and glutamate from two single micropipettes or, more commonly, from double barrel micropipettes one barrel being filled with glutamate and the other with ibotenate, to receptors in the extrajunctional membrane (see Fig. 3). Ibotenate was applied either by current passage or by the removal of the positive braking current from one barrel which allowed anions to diffuse outwards. The
456
S. G. CULL-CANDY hyperpolarizing response to glutamate was desensitized by ibotenate and vice versa, demonstrating that the extrajunctional glutamate receptor, which altered membrane permeability to produce a hyperpolarization, is also ibotenate sensitive (Fig. 3). Further, these experiments showed that the extrajunctional depolarizing response produced by glutamate remained unaffected by ibotenate sufficient to totally abolish the hyperpolarizing response. DL -ibotenate B
A
I
L -glutamate
__________L
-l___________
D
C
r
-r
I
Fig. 3. Selective desensitization by DL-ibotenate of the H-component of the extrajunctional DH-response to L-glutamate when applying ibotenate and glutamate iontophoretically from a twin barrel micropipette to the same region of extrajunctional membrane. The drug ejection currents passing through the pipettes are monitored on the lower traces. A, an H-response to ibotenate. B, DH-response to glutamate 1 min later. C, after ejecting ibotenate at 5 s intervals for 30 s the H-response to ibotenate was abolished due to receptor desensitization. D, 5 s later glutamate generated a pure D-response, which in the absence of an H-response has increased in amplitude from B. Calibration: 1 mV and 200 ms, 10 nC.
Junctional receptors The iontophoretic application of L-glutamate to junctional receptors produced a transient depolarizing response. Depolarization of the membrane potential, by current injection, reversed this response and gave a direct value for its equilibrium potential (see Fig. 4). The mean value ( ± S.D.) obtained in normal saline was - 0-25 + 4-2 mV (for fourteen fibres) when the resting potential value was 55-0 + 2-1 mV. The mean value obtained in Na+-free saline was - 13-8 + 2-04 mV (for twelve fibres) when the resting potential value was 56-6 + 1-5 mV. Depolarization of the membrane potential by current injection revealed that the junctional response to glutamate on many occasions became biphasic as the membrane potential was reduced (Fig. 4). The biphasic junctional response was
457 TWO TYPES OF EXTRAJUNCTIONAL RECEPTOR investigated in more detail, to determine if its H-component was due to the activation of H-receptors and to determine whether the junctional glutamate receptors, which mediate a depolarization, were sensitive to locally applied ibotenate. In some experiments low Na+ or Na+-free saline was used. This reduced the size of the depolarizing junctional response and made the H-response more noticeable, even at membrane potentials close to the resting potential. L-glutamate
DL-ibotenate
A -15
-15-
-20 -\>
-20--
-25 -40
=
-
-2
5-
-40
Fig. 4. A comparison at a range of membrane potentials of the action of L-glutamate (A) and DL-ibotenate (B) when applied iontophoretically from a twin barrel micropipette to a single junctional site. These experiments were performed in low Na+ saline to reduce the proportion of depolarizing to hyperpolarizing responses. At -40 mV glutamate generated a junctional depolarization. At -25 and -20 mV the junctional response became biphasic. The first phase, which is the typical excitatory junctional response to glutamate, was annulled at -15 mV, and reversed at -5 mV. The H-component generated by both ibotenate and glutamate was close to its equilibrium value at -40 mV in this fibre, and increased in amplitude as the muscle membrane potential was reduced (membrane depolarized). Note the similarity between the time course and equilibrium potential of H-responses generated by both glutamate and ibotenate. Calibration: 1 mV and 200 ms.
Using double barrel pipettes, one barrel of which was filled with glutamate and the other with ibotenate, it was possible to locate glutamate sensitive junctional regions and to compare the action of ibotenate and glutamate on the same receptors (Fig. 4). In the experiment illustrated in the Figure, at the resting potential glutamate produced a depolarization, while no response to ibotenate was seen. When the membrane was depolarized, by current injection, a hyperpolarizing component of the response to glutamate and a hyperpolarizing response to ibotenate became evident, demonstrating that the response to glutamate consisted of two components, while the response to ibotenate was a single component. That
458 S. G. CULL-CANDY ibotenate and glutamate acted on the same receptors to generate the Hresponse at the junctional region was shown by desensitizing the hyperpolarizing component of the response to glutamate, with ibotenate, and vice versa. As was the case for extrajunctional D-receptors, junctional receptors which mediate a depolarization were unaffected by locally applied ibotenate. The origin of the H-response at the junction is unclear since it is likely that the iontophoretic application of glutamate to the junction might activate some extrajunctionally occurring H-receptors (Cull-Candy & Usherwood, 1973). It is thus not possible to tell from iontophoretic mapping whether H-receptors exist in the junctional membrane.
Does the transmitter activate H-receptors? To determine if the H-receptors are measurably involved in synaptic transmission, e.p.s.p.s were reversed by depolarizing a fibre via current injection, both in normal and in low Na+ saline (see Fig. 5). Involvement +10 -
0-
-10
-
'I! m
-20-
-30-
-40--
Fig. 5. Reversal of excitatory post-synaptic potentials (e.p.s.p.s) in low Na+ (10 mM) saline. A fast depolarizing event occurred between membrane potentials of -40 and -15 mV. Between - 15 and - 10 mV, the e.p.s.p. reversed in a biphasic manner (the muscle fibre having multiple innervation), a hyperpolarization preceding a depolarization. At 0 mV and more positive membrane potentials a pure fast hyperpolarizing e.p.s.p. occurred. The rise time of the e.p.s.p. was reduced at lower membrane potentials. No H-component was visible. Calibration: 10 mV and 20 ms.
459 TWO TYPES OF EXTRAJUNCTIONAL RECEPTOR of the H-receptors in synaptic transmission would be expected to produce a biphasic junctional response to the transmitter similar to that observed in the extrajunctional membrane on iontophoretic application of glutamate. Close to the reversal level of the e.p.s.p. an initial hyperpolarization was followed by a depolarization as the response reversed (see Fig. 5). This would be expected in a situation where transmitter was not released simultaneously along the multiply innervated locust muscle fibre and responses at proximal junctions were reversed first (cf. Burke & Ginsborg 1956). No H-component was present, which indicated that the transmitter either did not reach or did not significantly activate H-receptors.
'Coulomb dose'-response relationships for extrajunctional receptors The peak amplitude of both the extrajunctional D- and H-responses was related to the coulomb dose of glutamate or ibotenate released from the micropipette. Coulomb dose-response relationships were investigated independently for the two types of response (i.e. in each case in the absence of the other response). Intervals of 120 s were allowed between successive iontophoretic applications to prevent desensitization of receptors. Drugs were applied from single barrel or double barrel micropipettes. Both techniques gave similar results. H-receptors. Coulomb dose-response relations of ibotenate and glutamate on the H-receptors were obtained over a wide range of membrane potentials (ca. -50 to - t- mV) to reduce inaccuracy caused by the small driving force on the iouE involved in the H-response. For the action of glutamate on H-receptors the mean value ( + S.D.) of the limiting slope of a double logarithmic plot was 0-75 + 0-09 (for six fibres in different preparations). For the action of ibotenate on H-receptors the mean value was 0-76 + 0-08 (for nine fibres in different preparations). The similarity of these values gives support to the concept that ibotenate and glutamate were acting on the same receptor to produce the H-response, and that the mechanisms underlying the interaction of both drugs with the H-receptor may be very similar or identical (see Fig. 6). D-receptors. Coulomb dose-response relationship of glutamate on the D-receptors were obtained at the resting potential (approximately -60 mV) in a Cl--free saline, which abolished the H-component. For the action of glutamate on D-receptors the mean value ( ± S.D.) of the limiting slope of a double logarithmic plot was 1-5 + 0-06 (for ten fibres in different preparations). This approaches the value of 2-0 obtained for junctional glutamate receptors in locust muscle (Walther & Usherwood, 1972) and of 2 3 in crayfish muscle (Takeuchi & Takeuchi, 1964) and suggests a similarity between the junctional glutamate receptors and the extrajunctional
S. G. CULL-CANDY
460 A
5"
1-0_
1*0
E
; 0-5_5, -
0.1
10
5
I
i-glutamate (nC)
40 8
2-0
, 10 E
ff
05
I
n-1 v,I. 1
I
I
I
I
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5
I I I
10
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20
DL-ibotenate (nC)
Fig. 6. For description
see
opposite
page.
TWO TYPES OF EXTRAJUNCTIONAL RECEPTOR 4-0
461
C
2-0
0~~~~~~~~
oU
0-
1
5 10 L-glutamate (nC)
20
Fig. 6. A, B, double log plot of the amplitude of the H-response generated by L-glutamate (A) and DL-ibotenate (B) against the coulomb strength of the drug ejection currents. Each graph shows the result of three separate experiments. The membrane potential of the fibre under investigation was, in some experiments, displaced from the resting level. Glutamate limiting slope values, with fibre membrane potentials in parentheses were filled circles 0-69 (-50 mV), open circles 0-74 (-52 mV) and triangles 0-88 (-52 mY). Ibotenate limiting slope values, with fibre membrane potentials were, filled circles 0-70 (-75 mY), open circles 0-75 (-80 mY) and triangles 0-98 (-53 mY). C, double log plot of the amplitude of the D-response generated by L-glutamate against the coulombic strength of the drug ejection currents. The graph shows the result of three separate experiments with limiting slope values, filled circles 1-48, open circles 1-42 and triangles 1-48. All slopes were obtained at the resting potential of the muscle fibre under investigation.
D-receptors. The lower value for the slope of the extrajunctional Dreceptors may be due to the ease with which these receptors can be desensitized when compared to junctional receptors (Cull-Candy & Usherwood, 1973). DISCUSSION
On the basis of pharmacological evidence two populations of extrajunctionally occurring glutamate receptors (D- and H-receptors) exist in locust muscle fibres. The simultaneous activation, by glutamate, of these mixed receptor types generates a biphasic response, depolarization followed
462 S. 0. CULL-CANDY by a hyperpolarization. As the D-response to glutamate remained when the H-component of the biphasic response had been desensitized by ibotenate, it was concluded that the depolarizing and hyperpolarizing phases of the extrajunctional response resulted from the activation of two types of receptors: D-receptors which were activated by glutamate but were insensitive to ibotenate, and H-receptor8 which were activated by both glutamate and ibotenate. Certain locust muscle fibres receive an inhibitory innervation, the transmitter being GABA (Usherwood & Grundfest, 1965). H-receptors occurred in fibres both with and without inhibitory innervation (as judged by the presence of spontaneous miniature inhibitory potentials) and were not detectably sensitive to locally -applied GABA. This indicates that they are not simply extrajunctional GABAreceptors with a sensitivity to glutamate, despite the similarity in the ionic conductance changes which underly the H-response to glutamate and the inhibitory junctional response to GABA. A more complex relationship between H-receptors and GABA-receptors or inhibitory innervation remains possible. D-receptors, however, showed similarities to junctional glutamate receptors in terms of the ionic conductance changes mediated. The lack of action of ibotenate on junctional glutamate receptors (Lea & Usherwood, 1973; Cull-Candy & Usherwood, 1973) was confirmed by the iontophoretic application of ibotenate from double barrel micropipettes, which contained ibotenate, and glutamate to locate junctional sites. The selective excitation of H-receptors by ibotenate from amongst a mixture of D- and H-glutamate receptors demonstrated that the extrajunctional D-receptors are also insensitive to ibotenate and in this respect are pharmacologically more akin to the junctional glutamate receptors. Additional evidence that the D- and H-receptors are of two distinct types is provided by the observed variation in the proportion of Dresponse to H-response seen in different muscle preparations (Cull-Candy & Usherwood, 1973), and by the difference in characteristics of the coulomb dose-response curves relating to the actions of glutamate and ibotenate on these receptors. The slope of the coulomb dose-response relationship (on a log-log plot) for glutamate action on extrajunctional D-receptors approaches the values previously found for the action of glutamate on junctional receptors of locust (Walther & Usherwood, 1972; S. G. CullCandy, unpublished) and of crayfish (Takeuchi & Takeuchi, 1964). The slope values for coulomb dose-response curves of ibotenate and glutamate acting on H-receptors are in agreement with the idea that the two agonists both act on a single type of receptor. There is an increasing volume of evidence that excitatory glutamate receptors in arthropod muscles show pharmacological differences from those in central neurones (see Takeuchi & Takeuchi, 1964; Usherwood &
463 TWO TYPES OF EXTRAJUNCTIONAL RECEPTOR Cull-Candy, 1975; Curtis & Watkins, 1960; Krnjevic6 & Phillis, 1963). The lack of action of ibotenate on locust excitatory receptors while it excites central neurones (Johnston et al. 1968) apparently supports the idea of such a difference, but considerable caution is necessary as more than one type of glutamate receptor may exist on central neurones (Johnston et al. 1974) as presently described for locust muscle. It was not possible to determine whether or not H-receptors occur in the junctional membrane. It does seem that H-receptors are not mneaaurably activated by the transmitter. This confirms experiments where the H-receptors were mapped and is predictable on the basis of the respective sensitivities of the receptor types. Thus, as H-receptors are about 200 times less sensitive than the excitatory junctional receptors, a 10 mV e.p.s.p. would be accompanied by a 50 ,uV H-response if H-receptors occurred junctionally. While in certain properties the glutamate sensitive D-receptors may be analogous to the extrajunctional ACh receptors in vertebrate muscle, the significance of the H-receptors is unclear. I wish to express thanks to Professor P. N. R. Usherwood for much invaluable discussion and encouragement. I also gratefully acknowledge Dr R. J. Dowson, Miss B. P. Fulton and Professor R. Miledi for invaluable discussions at various stages in this work, and am indebted to Professor S. Thesleff for constructive criticism of the manuscript. A gift of ibotenic acid was kindly supplied by Professor C. H. Eugster.
REFERENCES BERANEK, R. & MILT.x.R, P. L. (1968). The action of iontophoretically applied glutamate on insect muscle fibres. J. exp. Biol. 49, 83-93. BuExE, W. & GINSBORG, B. L. (1956). The action of the neuromuscular transmitter on the slow fibre membrane. J. Phy8iol. 132, 599-610. CULL-CANDY, S. G. & USHERWOOD, P. N. R. (1973). Two populations of L-glutamate receptors on locust muscle fibres. Nature, New Biol. 246, 62-64. CuRTs, D. R. & WATKINS, J. C. (1960). The excitation and depression of spinal neurones by structurally related amino acids. J. Neurochem. 6, 117-141. FATT, P. (1961 a). Intracellular microelectrodes. Meth. med. 9, 381-404. FATT, P. (1961 b). The change in membrane permeability during the inldbitory process. In Nervou8 Inhibition, ed. FLOREY E., pp. 87-91. Oxford: Pergamon. FELTZ, A. & MALLRT, A. (1971). An analysis of the acetylcholine responses of junctional and extrajunctional receptors of frog muscle fibres. J. Physiol. 218, 85-100. HoYLE, G. (1955). The anatomy and innervation of locust skeletal muscle. Proc. R. Soc. Lond. B 143, 281-292. JOHNSTON, G. A. R., CuRTis, D. R., DAVIES, J. & McCuLLocH, R. M. (1974). Spinal interneurone excitation by conformationally restricted analogues of L-glutamic acid. Nature, Lond. 248, 804-805. JOHNSTON, G. A. R., CURTIS, D. R., DE GROAT, W. C. & DUGGAN, A. W. (1968). Central actions of ibotenic acid and muscimol. Biochem. Pharmac. 17, 2488-2489.
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KATZ, B. & MILEDI, R. (1964). Further observations on the distribution of acetylcholine-reactive sites in skeletal muscle. J. Phyaiol. 170, 379-388. KATZ, B. & THESLEFF, S. (1957). A study of the 'desensitization' produced by acetylcholine at the motor endplate. J. Physiol. 138, 60-80. KRNJEVI6, K. & PHiLLIs, J. W. (1963). Iontophoretic studies of neurones in the mammalian cerebral cortex, J. Physiol. 165, 274-304. LEA, T. J. & USHERWOOD, P. N. R. (1973). The site of action of ibotenic acid and the identification of two populations of glutamate receptors on insect muscle fibres. Coamp. gen. Pharmac. 4, 333-350. MILEDI, R. (1960). Junctional and extra-junctional acetylcholine receptors in skeletal muscle fibres. J. Physiol. 151, 24-30. MILEDI, R. & ZELENA, J. (1966). Sensitivity to acetylcholine in rat slow muscle. Nature, Lond. 210, 855-856. 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. SHINOZAKI, H. & KoNismI, S. (1970). Actions of several anthelmintics and insecticides on rat cortical neurones. Brain Res. 24, 368-371. TAKEUCHI, A. & TAREumI, N. (1964). The effect on crayfish muscle of iontophoretically applied glutamate. J. Physiol. 170, 296-317. TAK UcHI, A. & TAKEUCHI, N. (1965). Localized action of gamma-aminobutyric acid on the crayfish muscle. J. Physiol. 177, 225-238. TASAKI, K., TSUKAHARA, Y., ITO, S., WAYNER, M. J. & YU, W. Y. (1968). A simple, direct and rapid method for filling microelectrodes. Physiol. & Behav. 3, 1009-1 01 0. USHERWOOD, P. N. R. & CULL-CANDY, S. G. (1975). Pharmacology of somatic nervemuscle synapses. In Insect Muscle, ed. USHERWOOD, P. N. R. London: Academic Press. USHERWOOD, P. N. R. & GRUNDFEST, H. (1965). Peripheral inhibition in skeletal muscle of insects. J. Neurophysiol. 28, 497-518. USHERWOOD, P. N. R. & MACHILI, P. (1968). Pharmacological properties of excitatory neuromuscular synapses in the locust. J. exp. Biol. 49, 341-361. WALKER, R. J., WOODRUFF, G. N. & KERKUT, G. A. (1971). The effect of ibotenic acid and muscimol on single neurones of the snail, Helix asper8a. Comp. gen. Pharmac. 2, 168-174. WALTHER, C. & USHERWOOD, P. N. R. (1972). Characterization of glutamate receptors at the locust excitatory neuromuscular junction. Verh. dt. zool. Ges. 65, 309-312.
449
TWO TYPES OF EXTRAJUNCTIONAL L-GLUTAMATE RECEPTORS IN LOCUST MUSCLE FIBRES
BY S. G. CULL-CANDY* From the Department of Zoology, the Univer8ity, Glasgow G12 8QQ
(Received 27 May 1975) SUMMARY
1. L-glutamate applied iontophoretically to the extrajunctional membrane of locust muscle produced a biphasic response, depolarization followed byhyperpolarization (i.e. DH-response). Applying L-glutamate and DL-ibotenate from multibarrel micropipettes allowed comparison of their extrajunctional responses. While glutamate produced a two component response, ibotenate produced a single component H-response. 2. The equilibrium values for the H-responses to L-glutamate and DLibotenate applied at the same extrajunctional site were very similar. The equilibrium value was 59-5 + 5-4 mV indicating an increased C1- conductance. The H-response was reversed and abolished in Cl- free medium. Picrotoxin 10-3 M selectively blocked the H-component of the DHresponse in a reversible manner. 3. The possibility that the D- and H-responses arose from the activation of two distinct types of extrajunctional glutamate receptors was investigated. Desensitization ofthe glutamate H-response by ibotenate and vice versa indicated the presence of an extrajunctional H-receptor sensitive to glutamate and ibotenate and an extrajunctional D-receptor sensitive to glutamate and insensitive to ibotenate. The junctional depolarizing response to glutamate was insensitive to ibotenate. 4. The presence of junctionally occurring H-receptors could not be discounted, although, if present, they were not measurably activated by the excitatory transmitter. 5. Double logarithmic plots (coulomb dose v8. response) for the actions of glutamate and ibotenate on H-receptors had values of 0-75, indicating that both drugs act on the same receptors with similar mechanisms. The value for the action of glutamate on the D-receptors was 1-5. 6. While the extrajunctional D-receptors show analogies to the extrajunctional ACh receptors in vertebrate muscle, the significance of the extrajunctional H-receptors remains speculative. * Present address: Department of Biophysics, University College London, Gower Street, London WCIE 6BT. 15-2
450
S. G. CULL-CANDY INTRODUCTION
In vertebrate skeletal muscle fibres acetylcholine (ACh) receptors occur both at the neuromuscular junction and in the extrajunctional membrane (Miledi, 1960; Katz & Miledi, 1964; Feltz & Mallart, 1971). In general the extrajunctional ACh receptors are restricted to the region immediately surrounding the junction, but in slow contracting muscles of the rat a low level of ACh sensitivity covers the entire muscle surface (Miledi & Zelena, 1966). In crustaceans and insects, L-glutamate (or a closely related substance) is the suggested transmitter at excitatory neuromuscular junctions (see Takeuchi & Takeuchi, 1964; Usherwood & Cull-Candy, 1975) which occur along the multiply innervated muscle fibres. In the present study glutamate and the glutamate analogue, ibotenate were iontophoretically applied to the locust muscle membrane. By using these two substances it was possible to distinguish two pharmacologically distinct populations of extrajunctional glutamate receptors. METRODS
All results described in this paper were obtained from the extensor tibiae muscle of the metathoracic leg of the locust, Schistocerca gregaria. The muscle was exposed by the removal of the cuticle covering the ventral aspect of the femur followed by dissection of the flexor tibiae and retractor unguis muscles (Hoyle, 1955). The large trachea which overlies the extensor muscle was cut away along one side of the muscle, to leave behind the fine tracheal branches which run close to the bundles of extensor muscle fibres. These branches were useful as reference points when the glutamate sensitivity of the muscle fibres was mapped since occasionally their apposition to the outer face of the muscle fibres or more often their intersection with the clefts between fibres marked the position of sites highly sensitive to L-glutamate. It has previously been demonstrated in other insect muscles that glutamate sensitive sites occur mainly but not exclusively in the clefts between fibres and that these coincide with junctional sites (Berdnek & Miller, 1968; Usherwood & Machili, 1968). The advantage of using the extensor tibiae muscle is that sites highly sensitive to glutamate can be found consistently on the exposed outer surface of the fibres; this allowed greatly improved examination of the properties and topography of junctional glutamate receptors. Furthermore, as the fibres of the extensor tibiae muscle are relatively large (ca. 100 #sm diam.) and have a high proportion of extrajunctional to junctional membrane, they were very suitable for investigating the glutamate sensitivity in the extrajunctional muscle membrane. Although a few experiments were performed on muscle fibres which received both excitatory and inhibitory innervation, as judged by the presence of spontaneous excitatory and inhibitory miniature potentials, most of this investigation was on fibres which received only excitatory innervation. Modified saline with an increased concentration of NaCl was used in most experiments (Usherwood & Grundfest, 1965). This saline contained NaCl, 200; KCl, 10; NaH2PO4, 4; Na2HPO4, 6; CaCl2, 2 mM at pH 6-8. Preparations were perfused with this saline for 2 hr before experimentation, to allow equilibration. In some experiments Na+ ions were replaced by choline to obtain a low Na+ (10 mM) saline, further
TWO TYPES OF EXTRA JUNCTIONAL RECEPTOR
451
substitution of the phosphate buffer with Hepes buffer (Sigma) produced Na+ free saline. Cl- free saline contained: NaMeSO4, 200; KMeSO4, 10; NaH2P04, 4; Na2HPO4, 6; Ca propionate, 2 mM at pH 6-8. Single and double barrel micropipettes when drawn contained central fibres of soft Pyrex glass (Tasaki, Tsukahara, Ito, Wayner & Yu, 1968; Peper & McMahan, 1972). Injection of drug into the shank just before use produced micropipettes with resistances between 150 and 300 Mn (measured in locust saline). The following drugs were used: 1 or 04- M-Na L-glutamate and Na D-glutamate, 041 m-Na DL-ibotenate dissolved in distilled water and adjusted to pH 7 with NaOH. At pH 7 glutamic and ibotenic acids are mainly anionic (Johnston, Curtis, de Groat & Duggan, 1968). 1 M y-amino-n-butyric acid (GABA) was dissolved in distilled water and adjusted to pH 4 with HCO (Takeuchi & Takeuchi, 1965). For the investigation of extrajunctional responses to L-glutamate or DL-ibotenate the drug-pipette required braking currents between 4 x 10-8 and 6 x 10-9 A to obtain optimum responses. A similar braking current was usually used to investigate the junctional responses to L-glutamate, although it was possible, with high resistance pipettes, to obtain fast (rise times less than 50 ms) and repeatable responses at these sites with no braking current (even though some diffusion of glutamate did occur from the tips of high resistance glutamate pipettes). Extrajunctional glutamate receptors were desensitized by the background leakage of drug which occurs from conventional micropipettes. It was therefore essential to use high resistance (> 150 MI) micropipettes and to allow a 60 s recovery period between pulses to obtain repeatable responses at a single site. For the investigation of extrajunctional receptors it was necessary that the iontophoretic pipette should be in close proximity to the muscle fibre membrane; therefore during passage of current the pipette was allowed to enter the muscle fibre and was then withdrawn the minimum distance necessary for it to leave the fibre. Penetration of the membrane was marked by the appearance of an intracellular electrotonic potential. This technique proved particularly useful for the examination of the tips of double barrel pipettes, the use of which usually required both tips should be at exactly the same distance from the receptor sites under investigation. The tips of double barrel pipettes which appeared symmetrical under a water immersion objective did not always penetrate the muscle fibre simultaneously. These were rejected. Intracellular recording electrodes were filled with 2 M potassium citrate acidified to pH 6 with citric acid and had resistance of 10-20 MQ. K citrate electrodes were also used to displace membrane potential of extensor tibiae muscle fibres. This avoided the injection of Cl- into the fibres during the injection of negative current and in addition these electrodes showed little tendency to block (Fatt, 1961 a). The current electrodes were lowered on to a fibre impaled by a recording electrode and inserted nearby by the passage of a short pulse of negative current through the electrode.
RESULTS
Comparison of the extrajunctional responses to glutamate and ibotenate Glutamate locally applied to locust excitatory junctional receptors causes a depolarization of the membrane potential (Beranek & Miller, 1968; Usherwood & Machili, 1968). When applied from a high resistance micropipette to the extrajunctional membrane of a muscle at the resting potential, glutamate produces either a small biphasic (depolarization followed by hyperpolarization) or hyperpolarizing response (Cull-Candy & Usherwood, 1973). Jbotenate, an isoxazole which shows certain structural
452 S. G. CULL-CANDY similarities to glutamate, has been reported to activate glutamate receptors in spinal interneurones (Johnston et al. 1968), Renshaw cells (Johnston, Curtis, Davies & McCulloch, 1974), central neurones (Shinozaki & Konishi, 1970) and snail neurones (Walker, Woodruff & Kerkut, 1971). The application of ibotenate to locust muscle fibres via the bathing medium causes an increase in Cl- conductance of the muscle fibre (Lea & Usherwood, 1973). A
L
-glutamate
B
-60
-60
-635
-63-5
-67 S
-675S
-70
~
DL-ibotenate -NNW
-70
Fig. 1. Comparison of the extrajunctional responses to L-glutamate and DL-ibotenate applied iontophoretically from a twin barrel micropipette to the same region of extrajunctional membrane. Note that since the fibres are multiply innervated spontaneous miniature excitatory potentials are recorded from junctional and extrajunctional regions in these and in subsequent records. A, at the resting potential, -60 mV, glutamate produced a DH-response, the H-component of which was annulled at - 63-5 mV when a D-component alone was seen. The H-component reversed at membrane potentials greater than - 63-5 mV so that a two-component (DH) depolarization occurred at - 67-5 and at -70 mV. The D-component increased in amplitude as the membrane potential increased from -60 to -70 mV. B, at the resting potential, -60 mV, ibotenate produced a pure H-response which was annulled at - 63-5 mV, no net ionic movement occurring at this level, in comparison with the glutamate induced Dresponse at - 63-5 mV. The H-response reversed at membrane potentials greater than - 63-5 mV; depolarizing H-responses which increased in amplitude with increased membrane potential occurred at - 67-5 and -70 mV. Calibration: 1 mV and 200 ms.
Comparisons were made of the extrajunctional responses to glutamate and ibotenate by the iontophoretic application of these drugs to the same extrajunctional site from twin barrel pipettes which contained glutamate in one barrel and ibotenate in the other (see Fig. 1). Glutamate produced a biphasic response, depolarization followed by a hyperpolarization, while ibotenate produced only a hyperpolarizing response at the resting potential.
453 TWO TYPES OF EXTRA JUNCTIONAL RECEPTOR Hyperpolarization of the membrane by current injection abolished the hyperpolarizing response to glutamate and ibotenate at the same membrane potential level (Fig. 1). At this membrane potential a depolarizing response to glutamate was clearly present in fibres which had produced biphasic responses to glutamate at their resting potential. Further hyperpolarization of the membrane caused the second component of the biphasic response to glutamate and the single component of the response to ibotenate to reappear as a depolarization.
Equilibrium potential for the H-response The equilibrium potential for the H-response could be accurately determined in those fibres where the D-response was small or absent. Values were also obtained in fibres which normally produced DHresponses to glutamate either by removal of the ions which generated the D-component or by the use of ibotenate. Values for EH-glutamate and EH-ibotenate obtained from single muscle fibres by using either double barrel pipettes, glutamate in one barrel and ibotenate in the other, or two single barrel pipettes, were not measurably different. The mean value (± S.D.) obtained was - 59-5 + 5.4 mV (for twelve fibres investigated simultaneously with glutamate and ibotenate) when the resting potential value was - 53-6 + 36 mV. This is compatible with the idea that the H-response was generated by an increased Cl- permeability, and is similar to figures for the equilibrium value for GABA action (-60 to -70 mV), at inhibitory junctional receptors in locust muscle fibres, which also causes an increased Cl- permeability (Usherwood & Grundfest, 1965). Very few fibres (less than 1 %) produced a depolarizing H-response at the resting potential and when present it was usually associated with fibre damage. Chloride involvement in the H-response Cl- in the bathing medium was substituted by the impermeant methyl sulphate anion (see Methods). The driving force on Cl-, across the muscle fibre membrane, would be expected to rapidly reverse and so result in the reversal and eventual abolition of a Cl- mediated event as Cl- leaks from the fibres (see Fatt, 1961 b; Usherwood & Grundfest, 1965). Fig. 2 shows a typical experiment in which the H-response to glutamate had reversed 4 or 5 min after Cl--free saline entered the bath. The now depolarizing H-response increased in size as the Cl- was removed from the bath and extracellular spaces. Over a period of 1-2 hr, which presumably represented the time for most of the Cl- to leave the fibre, the depolarizing H-response declined in amplitude and finally disappeared completely. Displacement of the muscle fibre membrane potential by depolarizing or hyperpolarizing current injection failed to restore the H-response.
S. G. CULL-CANDY 454 Reapplication of normal saline to preparations treated with C1- free saline caused the reappearance of the H-response in all cases and a very marked increase in amplitude of response in those preparations where glutamate still produced small H-responses in Cl- free saline. Similar results were obtained with ibotenate. ControlI' Cl free-* 3 min-
4 min--
-
---
5 min-.
60 min
70 min
--
80 min
Cl
>
I Fig. 2.. The effect of Cl--free saline on the extrajunctional response to glutamate. In normal saline the muscle fibre produced a DH-response (the D-component being only just visible). 5 min after Cl- removal the Hresponse reversed so that a two component (DH) depolarization was present. At 70 min, the H-response had disappeared leaving the small Dcomponent. In this fibre a small hyperpolarizing H-response, following the D-component, was present at 80 min. Addition of CO- ions to the saline results in the rapid reappearance of an H-response of a slightly larger amplitude than before Cl- removal. The slight change in time course may be due to reposition of the glutamate micropipette. Calibration: 1 mV and 200 ms.
455 TWO TYPES OF EXTRAJUNCTIONAL RECEPTOR In some preparations (approximately 30 %) a very small hyperpolarizing H-response reappeared at the resting potential in muscle fibres soaked for several hours in Cl- free saline (see Fig. 2); this was probably the result of a glutamate induced conductance increase, in the membrane, to MeSO4 , since ibotenate is known to have such an effect (Lea & Usherwood, 1973). The action of picrotoxin on the H-response Locust extensor muscles exposed to picrotoxin 0-3 M were investigated with double barrel pipettes which contained glutamate and ibotenate (eleven fibres). This permitted simultaneous investigation of the action of picrotoxin on the glutamate and ibotenate response. The treatment of fibres in picrotoxin for 20 min completely blocked the H-response to ibotenate and the H-component of the DH-response to glutamate. With lower concentrations of picrotoxin (i.e. 104 M) only a partial block of the Hresponse was observed. Reappearance of the H-response on washing took approximately 20 min. These results provide additional evidence that the H-response is Cl- mediated.
Evidence for two population of extrajunctional receptors: specific action of ibotenate The demonstration of a biphasic potential response to glutamate, and a monophasic response to ibotenate when these drugs are applied to the extrajunctional membrane, raises the possibility that there are two types of extrajunctional glutamate receptors, one of which is activated by ibotenate. A classical property of receptors is their desensitization or refractoriness in the continued presence of an agonist (Katz & Thesleff, 1957). It follows that if a receptor is desensitized by an agonist, the agonist has activated the receptor. As ibotenate applied iontophoretically to an extrajunctional site on locust muscle produced a response which resembled the hyperpolarizing component of the response to glutamate, it was of interest to see if the hyperpolarizing component of the glutamate response could be desensitized with ibotenate or vice versa to determine whether these two drugs acted on the same receptor. The experiments (twenty fibres, different preparations) were performed by the iontophoretic application of ibotenate and glutamate from two single micropipettes or, more commonly, from double barrel micropipettes one barrel being filled with glutamate and the other with ibotenate, to receptors in the extrajunctional membrane (see Fig. 3). Ibotenate was applied either by current passage or by the removal of the positive braking current from one barrel which allowed anions to diffuse outwards. The
456
S. G. CULL-CANDY hyperpolarizing response to glutamate was desensitized by ibotenate and vice versa, demonstrating that the extrajunctional glutamate receptor, which altered membrane permeability to produce a hyperpolarization, is also ibotenate sensitive (Fig. 3). Further, these experiments showed that the extrajunctional depolarizing response produced by glutamate remained unaffected by ibotenate sufficient to totally abolish the hyperpolarizing response. DL -ibotenate B
A
I
L -glutamate
__________L
-l___________
D
C
r
-r
I
Fig. 3. Selective desensitization by DL-ibotenate of the H-component of the extrajunctional DH-response to L-glutamate when applying ibotenate and glutamate iontophoretically from a twin barrel micropipette to the same region of extrajunctional membrane. The drug ejection currents passing through the pipettes are monitored on the lower traces. A, an H-response to ibotenate. B, DH-response to glutamate 1 min later. C, after ejecting ibotenate at 5 s intervals for 30 s the H-response to ibotenate was abolished due to receptor desensitization. D, 5 s later glutamate generated a pure D-response, which in the absence of an H-response has increased in amplitude from B. Calibration: 1 mV and 200 ms, 10 nC.
Junctional receptors The iontophoretic application of L-glutamate to junctional receptors produced a transient depolarizing response. Depolarization of the membrane potential, by current injection, reversed this response and gave a direct value for its equilibrium potential (see Fig. 4). The mean value ( ± S.D.) obtained in normal saline was - 0-25 + 4-2 mV (for fourteen fibres) when the resting potential value was 55-0 + 2-1 mV. The mean value obtained in Na+-free saline was - 13-8 + 2-04 mV (for twelve fibres) when the resting potential value was 56-6 + 1-5 mV. Depolarization of the membrane potential by current injection revealed that the junctional response to glutamate on many occasions became biphasic as the membrane potential was reduced (Fig. 4). The biphasic junctional response was
457 TWO TYPES OF EXTRAJUNCTIONAL RECEPTOR investigated in more detail, to determine if its H-component was due to the activation of H-receptors and to determine whether the junctional glutamate receptors, which mediate a depolarization, were sensitive to locally applied ibotenate. In some experiments low Na+ or Na+-free saline was used. This reduced the size of the depolarizing junctional response and made the H-response more noticeable, even at membrane potentials close to the resting potential. L-glutamate
DL-ibotenate
A -15
-15-
-20 -\>
-20--
-25 -40
=
-
-2
5-
-40
Fig. 4. A comparison at a range of membrane potentials of the action of L-glutamate (A) and DL-ibotenate (B) when applied iontophoretically from a twin barrel micropipette to a single junctional site. These experiments were performed in low Na+ saline to reduce the proportion of depolarizing to hyperpolarizing responses. At -40 mV glutamate generated a junctional depolarization. At -25 and -20 mV the junctional response became biphasic. The first phase, which is the typical excitatory junctional response to glutamate, was annulled at -15 mV, and reversed at -5 mV. The H-component generated by both ibotenate and glutamate was close to its equilibrium value at -40 mV in this fibre, and increased in amplitude as the muscle membrane potential was reduced (membrane depolarized). Note the similarity between the time course and equilibrium potential of H-responses generated by both glutamate and ibotenate. Calibration: 1 mV and 200 ms.
Using double barrel pipettes, one barrel of which was filled with glutamate and the other with ibotenate, it was possible to locate glutamate sensitive junctional regions and to compare the action of ibotenate and glutamate on the same receptors (Fig. 4). In the experiment illustrated in the Figure, at the resting potential glutamate produced a depolarization, while no response to ibotenate was seen. When the membrane was depolarized, by current injection, a hyperpolarizing component of the response to glutamate and a hyperpolarizing response to ibotenate became evident, demonstrating that the response to glutamate consisted of two components, while the response to ibotenate was a single component. That
458 S. G. CULL-CANDY ibotenate and glutamate acted on the same receptors to generate the Hresponse at the junctional region was shown by desensitizing the hyperpolarizing component of the response to glutamate, with ibotenate, and vice versa. As was the case for extrajunctional D-receptors, junctional receptors which mediate a depolarization were unaffected by locally applied ibotenate. The origin of the H-response at the junction is unclear since it is likely that the iontophoretic application of glutamate to the junction might activate some extrajunctionally occurring H-receptors (Cull-Candy & Usherwood, 1973). It is thus not possible to tell from iontophoretic mapping whether H-receptors exist in the junctional membrane.
Does the transmitter activate H-receptors? To determine if the H-receptors are measurably involved in synaptic transmission, e.p.s.p.s were reversed by depolarizing a fibre via current injection, both in normal and in low Na+ saline (see Fig. 5). Involvement +10 -
0-
-10
-
'I! m
-20-
-30-
-40--
Fig. 5. Reversal of excitatory post-synaptic potentials (e.p.s.p.s) in low Na+ (10 mM) saline. A fast depolarizing event occurred between membrane potentials of -40 and -15 mV. Between - 15 and - 10 mV, the e.p.s.p. reversed in a biphasic manner (the muscle fibre having multiple innervation), a hyperpolarization preceding a depolarization. At 0 mV and more positive membrane potentials a pure fast hyperpolarizing e.p.s.p. occurred. The rise time of the e.p.s.p. was reduced at lower membrane potentials. No H-component was visible. Calibration: 10 mV and 20 ms.
459 TWO TYPES OF EXTRAJUNCTIONAL RECEPTOR of the H-receptors in synaptic transmission would be expected to produce a biphasic junctional response to the transmitter similar to that observed in the extrajunctional membrane on iontophoretic application of glutamate. Close to the reversal level of the e.p.s.p. an initial hyperpolarization was followed by a depolarization as the response reversed (see Fig. 5). This would be expected in a situation where transmitter was not released simultaneously along the multiply innervated locust muscle fibre and responses at proximal junctions were reversed first (cf. Burke & Ginsborg 1956). No H-component was present, which indicated that the transmitter either did not reach or did not significantly activate H-receptors.
'Coulomb dose'-response relationships for extrajunctional receptors The peak amplitude of both the extrajunctional D- and H-responses was related to the coulomb dose of glutamate or ibotenate released from the micropipette. Coulomb dose-response relationships were investigated independently for the two types of response (i.e. in each case in the absence of the other response). Intervals of 120 s were allowed between successive iontophoretic applications to prevent desensitization of receptors. Drugs were applied from single barrel or double barrel micropipettes. Both techniques gave similar results. H-receptors. Coulomb dose-response relations of ibotenate and glutamate on the H-receptors were obtained over a wide range of membrane potentials (ca. -50 to - t- mV) to reduce inaccuracy caused by the small driving force on the iouE involved in the H-response. For the action of glutamate on H-receptors the mean value ( + S.D.) of the limiting slope of a double logarithmic plot was 0-75 + 0-09 (for six fibres in different preparations). For the action of ibotenate on H-receptors the mean value was 0-76 + 0-08 (for nine fibres in different preparations). The similarity of these values gives support to the concept that ibotenate and glutamate were acting on the same receptor to produce the H-response, and that the mechanisms underlying the interaction of both drugs with the H-receptor may be very similar or identical (see Fig. 6). D-receptors. Coulomb dose-response relationship of glutamate on the D-receptors were obtained at the resting potential (approximately -60 mV) in a Cl--free saline, which abolished the H-component. For the action of glutamate on D-receptors the mean value ( ± S.D.) of the limiting slope of a double logarithmic plot was 1-5 + 0-06 (for ten fibres in different preparations). This approaches the value of 2-0 obtained for junctional glutamate receptors in locust muscle (Walther & Usherwood, 1972) and of 2 3 in crayfish muscle (Takeuchi & Takeuchi, 1964) and suggests a similarity between the junctional glutamate receptors and the extrajunctional
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TWO TYPES OF EXTRAJUNCTIONAL RECEPTOR 4-0
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Fig. 6. A, B, double log plot of the amplitude of the H-response generated by L-glutamate (A) and DL-ibotenate (B) against the coulomb strength of the drug ejection currents. Each graph shows the result of three separate experiments. The membrane potential of the fibre under investigation was, in some experiments, displaced from the resting level. Glutamate limiting slope values, with fibre membrane potentials in parentheses were filled circles 0-69 (-50 mV), open circles 0-74 (-52 mV) and triangles 0-88 (-52 mY). Ibotenate limiting slope values, with fibre membrane potentials were, filled circles 0-70 (-75 mY), open circles 0-75 (-80 mY) and triangles 0-98 (-53 mY). C, double log plot of the amplitude of the D-response generated by L-glutamate against the coulombic strength of the drug ejection currents. The graph shows the result of three separate experiments with limiting slope values, filled circles 1-48, open circles 1-42 and triangles 1-48. All slopes were obtained at the resting potential of the muscle fibre under investigation.
D-receptors. The lower value for the slope of the extrajunctional Dreceptors may be due to the ease with which these receptors can be desensitized when compared to junctional receptors (Cull-Candy & Usherwood, 1973). DISCUSSION
On the basis of pharmacological evidence two populations of extrajunctionally occurring glutamate receptors (D- and H-receptors) exist in locust muscle fibres. The simultaneous activation, by glutamate, of these mixed receptor types generates a biphasic response, depolarization followed
462 S. 0. CULL-CANDY by a hyperpolarization. As the D-response to glutamate remained when the H-component of the biphasic response had been desensitized by ibotenate, it was concluded that the depolarizing and hyperpolarizing phases of the extrajunctional response resulted from the activation of two types of receptors: D-receptors which were activated by glutamate but were insensitive to ibotenate, and H-receptor8 which were activated by both glutamate and ibotenate. Certain locust muscle fibres receive an inhibitory innervation, the transmitter being GABA (Usherwood & Grundfest, 1965). H-receptors occurred in fibres both with and without inhibitory innervation (as judged by the presence of spontaneous miniature inhibitory potentials) and were not detectably sensitive to locally -applied GABA. This indicates that they are not simply extrajunctional GABAreceptors with a sensitivity to glutamate, despite the similarity in the ionic conductance changes which underly the H-response to glutamate and the inhibitory junctional response to GABA. A more complex relationship between H-receptors and GABA-receptors or inhibitory innervation remains possible. D-receptors, however, showed similarities to junctional glutamate receptors in terms of the ionic conductance changes mediated. The lack of action of ibotenate on junctional glutamate receptors (Lea & Usherwood, 1973; Cull-Candy & Usherwood, 1973) was confirmed by the iontophoretic application of ibotenate from double barrel micropipettes, which contained ibotenate, and glutamate to locate junctional sites. The selective excitation of H-receptors by ibotenate from amongst a mixture of D- and H-glutamate receptors demonstrated that the extrajunctional D-receptors are also insensitive to ibotenate and in this respect are pharmacologically more akin to the junctional glutamate receptors. Additional evidence that the D- and H-receptors are of two distinct types is provided by the observed variation in the proportion of Dresponse to H-response seen in different muscle preparations (Cull-Candy & Usherwood, 1973), and by the difference in characteristics of the coulomb dose-response curves relating to the actions of glutamate and ibotenate on these receptors. The slope of the coulomb dose-response relationship (on a log-log plot) for glutamate action on extrajunctional D-receptors approaches the values previously found for the action of glutamate on junctional receptors of locust (Walther & Usherwood, 1972; S. G. CullCandy, unpublished) and of crayfish (Takeuchi & Takeuchi, 1964). The slope values for coulomb dose-response curves of ibotenate and glutamate acting on H-receptors are in agreement with the idea that the two agonists both act on a single type of receptor. There is an increasing volume of evidence that excitatory glutamate receptors in arthropod muscles show pharmacological differences from those in central neurones (see Takeuchi & Takeuchi, 1964; Usherwood &
463 TWO TYPES OF EXTRAJUNCTIONAL RECEPTOR Cull-Candy, 1975; Curtis & Watkins, 1960; Krnjevic6 & Phillis, 1963). The lack of action of ibotenate on locust excitatory receptors while it excites central neurones (Johnston et al. 1968) apparently supports the idea of such a difference, but considerable caution is necessary as more than one type of glutamate receptor may exist on central neurones (Johnston et al. 1974) as presently described for locust muscle. It was not possible to determine whether or not H-receptors occur in the junctional membrane. It does seem that H-receptors are not mneaaurably activated by the transmitter. This confirms experiments where the H-receptors were mapped and is predictable on the basis of the respective sensitivities of the receptor types. Thus, as H-receptors are about 200 times less sensitive than the excitatory junctional receptors, a 10 mV e.p.s.p. would be accompanied by a 50 ,uV H-response if H-receptors occurred junctionally. While in certain properties the glutamate sensitive D-receptors may be analogous to the extrajunctional ACh receptors in vertebrate muscle, the significance of the H-receptors is unclear. I wish to express thanks to Professor P. N. R. Usherwood for much invaluable discussion and encouragement. I also gratefully acknowledge Dr R. J. Dowson, Miss B. P. Fulton and Professor R. Miledi for invaluable discussions at various stages in this work, and am indebted to Professor S. Thesleff for constructive criticism of the manuscript. A gift of ibotenic acid was kindly supplied by Professor C. H. Eugster.
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