Inhibiton and gamma-aminobutyric acid-induced conductance on locust (Schistocerca gregaria) extensor tibiae muscle fibres

0022-1910/87 $3.00+0.00 Pergamon Journals Ltd

J. heel Physiol. Vol. 33, No. 3, pp. 183-189, 1987 Printed in Great Britain

INHIBITON AND GAMMA-AMINOBUTYRIC ACID-INDUCED CONDUCTANCE ON LOCUST (SCHISTOCERCA GREGARIA) EXTENSOR TIBIAE MUSCLE FIBRES RODERICK H. SCOTT* and IAN R. DUCE Department of Zoology, Nottingham University, University Park, Nottingham NG7 2RD, England (Received

27 March 1986; revised 15th May 1986)

Ahetraet-I. Increases in membrane conductance (g,,,) were induced by GABA in distal bundles 32, 33 and 34 of extensor tibiae muscles of the locust (Schbtocerca gregaria). 2. Bath application of GABA (10e5-5 x 10eJ M) induced reductions in muscle fibre space constant (A). 3. GABA (5 x 1O-3 M) induced additional membrane conductance of 2.21 f 0.03 x 10m6S/mm, 0.38 f 0.03 x 10e6 S/mm and 0.29 k 0.06 x 10m6S/mm on muscle bundles 34, 33 and 32 respectively. The greater sensitivity of muscle fibres in bundle 34 to GABA is due at least in part to a larger number of GABA receptors on bundle 34 muscle fibres. 4. The decrement of electrotonic potentials in the presence of GABA were measured over distances of both half fibre length and whole fibre length. Good agreement was obtained between changes in space constant produced by GABA using half fibre length and whole fibre length data. 5. By taking into account changes in space constant induced by GABA it was possible to demonstrate that presynaptic GABA receptors were involved in the inhibition of slow excitatory postsynaptic potentials by GABA. 6. “Slow” excitatory postsynaptic potentials recorded under current clamp were inhibited in a dose-dependent manner by GABA. This inhibition was not dependent on muscle-fibre GABA sensitivity and could not be completely accounted for by GABA-induced changes in the cable properties of the muscle fibres. Key Word Index: Locust muscle, gamma-aminobutyric

INTRODUCI’ION The amino acid gamma-aminobutyric acid (GABA) is one of the major inhibitory neurotransmitters in both invertebrates and vertebrates. The inhibitory actions of GABA are not confined to the central

nervous system. GABA has been found to have wide spread inhibitory action on arthropod muscles. Peripheral inhibition mediated by GABA has been studied extensively in crustacean neuromuscular preparations (Kravitz et al., 1963; Takeuchi and Takeuchi, 1965; Constanti and Smart, 1982). Inhibition is also found in skeletal muscles of insects where GABA is the suggested neurotransmitter (Usherwood and Grundfest, 1965; Hoyle, 1978). Only around l&20% of fibres in the locust extensor tibiae muscle bundles receive inhibitory innervation (Usherwood and Grundfest, 1965). At these inhibitory synapses, the sensitivity of GABA responses and IPSPs to picrotoxin (Usherwood and Grundfest, 1965), and similar reversal potentials for GABA responses and inhibitory postsynaptic potentials (IPSPs) (Usherwood and Cull-Candy, 1975), provide evidence that GABA is likely to be the inhibitory neurotransmitter. *Correspondence to: Dr Roderick H. Scott, Department of Pharmacology, St George’s Hospital Medical School, Cranmer Terrace, London SW17 ORE, England. 183

acid, conductance

In addition to mediating postsynaptic inhibitory neurotransmission GABA has been suggested to be involved in presynaptic inhibition. The amplitude of excitatory postsynaptic potentials (EPSPs) in crayfish muscle was reduced by GABA in a manner that could not be accounted for by reductions in postsynaptic muscle-membrane input resistance (Dude1 and Kuffler, 1961). In insect preparations GABA has been shown to reduce the frequency and amplitude of spontaneous miniature excitatory postsynaptic potentials (Kerkut and Walker, 1967). Morphological evidence has also indicated possible presynaptic inhibition. Fine structural studies occasionally reveal axo-axonic contacts between motor nerve fibres (Osborne, 1975). Takeuchi and Takeuchi (1967) found that the space constant of the crayfish muscle was long compared to the muscle length and the input conductance depended on the muscle length: space constant ratio L/A as well as the membrane conductance. In this study cable properties of distal muscle fibres in the locust extensor tibiae have been considered when investigating their varying sensitivity to GABA. These data have then been used to determine whether GABA has presynaptic as well as postsynaptic actions, and have revealed new evidence supporting presynaptic inhibition mediated by GABA. Some of the results have been presented in preliminary form (Duce and Scott, 1983).

184

RODERICKH. SCOTTand IAN R. DUCE MATERIALS AND METHODS

Adult locusts (Schistocerca gregaria) from a colony maintained in the Zoology Department of Nottingham University were used in this study. The metathoracic leg was cut from the locust at the joint between the coxa and thorax and pinned out on Sylgard in a perfusion bath. The preparation was perfused at a rate of 3 ml/min with phosphatebuffered locust saline pH 6.8, containing (mM) 180 NaCl, 10 KCl, 6 Na,HPO,, 4 NaH2P0,, 2 CaCl, (Clark er af., 1979). The extensor tibiae muscle was exposed by dissecting away the overlying flexor muscles and main tracheae (Hoyle, 1955). Microelectrodes with resistances of 10-20 MO, filled with 1 M potassium citrate were used. A recording electrode was inserted into the middle of a muscle fibre. Two current injecting electrodes were inserted into the muscle fibre, one within .50pM of the recording electrode, the other at a distance of half the muscle fibre length from the recording electrode. Constant hyperpolarizing current pulses of 10-20 nA in amplitude, 200-4OOmS in width and at a frequency of 0.1 Hz were injected through either of the two electrodes. The electrotonic potentials VO,(injection of current within 50 pm of the recording electrode) and V, (injection of current at a distance of half the muscle fibre length from the recording electrode) were measured after equilibration before, during and after perfusion of GABA (Fig. 1). Conductance changes induced by GABA in terms of membrane conductance (S/mm) were calculated considering changes in space constant occurring over half the length of the muscle fibre (Orkand, 1962; Takeuchi and Takeuchi, 1967). The space constant I. is defined as the distance from the current injecting electrode over which the electrotonic potential decays to 37% of its maximal value in an infinite cable. In

5x 10-3M

“r “0

“x

GABA

“0

“x

Fig. 1. The change in conductance induced by bath application of 5 x IO-’ M GABA, to muscle bundle 34. V, and V, indicate hyperpolarizing electrotonic potentials (downward deflections), induced by injecting hyperpolarizing current pulses at (V,) a distance of half fibre length and (V,) within SOpm of the recording electrode.

a “short” cable the potential decay does not reach 37% of the maximal value as I is larger than the length of the muscle fibre (Orkand, 1962). In this study the potential decayed along half the fibre length to between 80 and 90% of the maximum, so the fibres were treated as short cables with infinite resistance at both ends. The space constant was calculated using: L VU -= cash Y A VX where V, = electrotonic potential produced by current I being injected within 50,~~rn of the recording electrode (mV); V, = electrotonic potential produced by current I being injected at a distance of half the muscle length from the recording electrode (mV); L = muscle length (mm); I. = space constant (mm). Membrane conductance (g,) (S/mm) was calculated using: 1 I

g,=2Vcoth0

L 1

where I = current injected (nA) (Cole and Curtis, 1938; Hodgkin and Rushton, 1946; Weidmann, 1952; Orkand, 1962; Takeuchi and Takeuchi, 1967). The decay in electrotonic potential along the whole length of the fibre was also measured. The recording electrode was positioned as close as possible to the central apodeme and the current injecting electrode V, was positioned within 50f~rn of the recording electrode. The second current injecting electrode V, was inserted into the same muscle fibre close to the end where the muscle attached to the exoskeleton. The distance between V. and VL was close to the whole length of the muscle fibre. Slow EPSPs were evoked by stimulating nerve 3b either antidromically or orthodromically using a suction electrode (Hoyle, 1955, 1978; Usherwood and Grundfest, 1965). The stimulus applied to evoke EPSPs and contractions was l-4 V in amplitude with a width of 0.4ms and a frequency of 0.1 Hz. Contractions were abolished by titrating MgClz into the perfusing saline; concentrations between 10 and 20 mM were required to stop contractions. In studies on presynaptic actions of GABA, slow EPSPs were recorded from distal muscle bundles 34 and 32 as well as muscle bundles 21 to 26. Muscle bundle 34 and 32 receive inhibitory and slow excitatory innervation only; bundles 21-26 do not receive inhibitory innervation but certain fibres receive slow excitatory innervation in addition to innervation from the fast excitor motoneurone (Hoyle, 1978). Slow events were identified by their time course, approx. 80 ms compared with less than 10ms for fast EPSPs. Once contractions had stopped the muscle fibres were impaled with two microelectrodes, one for recording the potentials and a second intracellular electrode for injecting DC current to counteract changes in membrane potential induced by application of GABA. RESULTS

Conductance measurements

Current pulses between + I5 and - 50 nA were injected and the resulting changes in membrane potential were plotted (Fig. 2). The current-voltage relationships revealed that no anomalous rectification

GABA-induced conductance on locust muscle 20

1

5

nA -48

-36

-24

185

t

-12

-40

\-

mV

+--•--Imb34

I

--60

I 10-5

-I-

I

I

10-e

GABA

60

Fig. 2. Current-voltage relationships of a muscle fibre in muscle bundle 34, in the presence and absence of lo-’ M GABA. Control (saline) mean electrotonic potential + SE (n = 3, 1 series before and 2 after GABA application). V, = current injected at a distance of half the fibre length from the recording electrode; V, =current injected at a distance of less than 5Orm from the recording electrode.

occurred in the locust extensor tibiae muscle fibres (Fig. 2). This finding was in agreement with previous studies on insect muscle fibres (Werman et al., 1961; Belton and Grundfest, 1962). Muscle bundles 34, 33 and 32 varied in size and space constant in normal locust saline. The space constants of the muscle fibres were considerably greater than the length of the muscle (Table 1) and did not change significantly in the presence of 10e5 M GABA. The space constant decreased when increasing concentrations of GABA were applied (Fig. 3). An increase in GABA concentration from 10s5 to 5 x 10e3 M caused reductions in the space constant of 49.4, 55.5 and 32.2% for fibres in muscle bundles 34, 33 and 32 respectively. In the case of fibres in muscle bundle 34 at GABA concentrations greater than 5 x lo-’ M the space constant decreased to less than the length of the muscle fibre length. The mean values for the resting membrane conductance for fibres in muscle 34 were significantly different from those of fibres from bundles 33 and 32 (34, g, = 0.34 x low6 S/mm; 33 and 32 g,,, = 0.19 x 10e6 S/mm). When GABA was applied no desensitization was found to occur as the GABA-induced change in conductance equilibrated and did not decline in the continued presence of GABA (Fig. 1). Fibres in muscle bundle 32 and 33 had similar sensitivities to GABA. GABA (5 x 10e3 M) induced an eight times greater change in membrane conductance in muscle bundle 34 than in bundles 33 and 32 (Fig. 4). In all three muscle bundles, the receptor population started to saturate with doses greater than

I

10-3

10-Z

(Ml

Fig. 3. The effect of GABA on the space constant of fibres in muscle bundles 34, 33, 32. Graph of space constant (A) plotted against log GABA concentration + SEM (n = 6).

lo-‘M GABA. Complete saturation was not achieved in 5 x lO-3 M GABA and higher concentration were not applied owing to damaging effects on the muscle membrane. Hill plots, of log dose GABA against log Y/(1-Y), (where Y is the membrane conductance normalized with respect to Y,,,, at 5 x 10m3M GABA, for any given dose of GABA) were prepared for each muscle bundle. Hill coefficients were calculated from the slopes of the Hill plots. The Hill coefficients for muscle bundles 33 and 32 were 1.73 + 0.13 and 1.74 &-0.12 (mean f SE, n = 5) and were significantly lower (Mann-Whitney U test, P < 0.05) than the Hill coefficient for fibres in muscle bundle 34, (2.09 + 0.09 mean f SE, n = 5). These results suggested that the mean number of bound GABA molecules required to elicit one unit response was greater for GABA receptors on bundle 34 than on bundles 33 and 32. The non-integral values, greater than 1, for Hill coefficients implied that positive cooperativity in GABA binding was involved in this system. The decrement in electrotonic potential over half the fibre length was too small to give an indication of the maximal possible decrement of response. In order to obtain this information electrodes were placed at either end of the muscle fibres and the decay of potential was measured along the whole fibre length (Table 2). 24 “lb34 20

Table 1. The muscle fibre lengths and space constant values for different muscle bundles Muscle bundle

Mean length (mm It SE)

Mean space constant

34 33

1.37 f 0.04 1.47 f 0.03 1.72f 0.04

2.29 k 0.07 4.79 5 0.16 4.43 * 0.14

32

(I

mm * SE)

n

58 41 46

GABA

(Ml

Fig. 4. Graph changes in membrane conductance Agg, induced by GABA bath applied to fibres (n = 6) of distal muscle bundles 34, 33 and 32.

RODERICK

186

H. Scorr and

IAN

R.

DUCE

Table 2. Percentage decay of electrotonic potentials recorded in muscle bundles 34 and 32 in varying concentrations of GABA Muscle

GABA

[Ml

bundle

n

5 x 10-l 5x10 4 0 5x10 3 5 x IO ~4

34 34 34 32 32 32

6 6 IO 6 5 IO

0

mean % reduction in potential along half the fibre length 50.5 + 35.1 * 19.5 * 16.2 k 13.8 + 10.2 *

12.4 14.4 4.5 3.2 15.9 7.2

n

mean % reduction in potential along the whole libre length

8 4 32 2 3 IO

82.6 f 3.1 53.0 f I I. I 31.2fll.l 29 19.7 14.0 f 3.7

The space constants calculated from the half fibre length data predicted that in the presence of 5 x low3 M GABA the decrement of response will be to less than 37% of maximum along the whole-fibre length in muscle fibres of bundle 34 but not 32. Figure 5 shows that this is in fact the case and illustrates the good correlation obtained between experiments measuring decrement of response over whole and half fibre length. The preparation was considerably less stable when intracellular electrodes were placed at both ends of muscle fibres so complete does-response curves were not generated.

not account for the reductions in slow EPSP amplitude observed. The 30% reduction in the slow EPSP amplitude seen in fast muscle bundles must be entirely due to a presynaptic action since there are no postsynaptic GABA receptors and GABA does not affect the cable properties of the muscle fibre membrane. The amplitudes of fast EPSPs recorded from fibre in muscle bundles 21-26 were not reduced by bath application of 5 x low3 M GABA.

Slow excitatory postsynaptic

The differences in the size and space constant for fibres in muscle bundles 34, 33 and 32 make it necessary to calculate conductance changes induced by GABA in terms of membrane conductance when comparing different muscle bundles. In the flexor tibialis muscle of the locust the length constant (space constant) has also been found to vary and ranged from 1.5 to 4.5mm (Brookes and Werman, 1980). Similar differences between fibres have been found in crustacean muscle fibres (1.8-3.1 mm; Constanti and Smart, 1982). Takeuchi and Takeuchi (1967) found the L/d ratio increased with increases in conductance induced by GABA, this was due to decreases in the space constant 1. In this study it was found that the value of 1 was reduced with increasing GABAinduced conductance changes. GABA (5 x 10m3M) when applied to distal muscle bundles induced additional membrane conductance which was 8 times greater in fibres in bundle 34 than either 33 or 32. This may be due at least in part to a greater number of GABA receptors being activated on bundle 34 muscle fibres. The differences in membrane conductance in a saline environment for the muscle fibres suggest that other mechanisms also may be involved, e.g. the differential GABA sensitivity seen may reflect varying internal chloride concentrations which could affect GABA induced conductance changes. Brookes and Werman (1973) found the limiting slope of the Hill plot for flexor tibialis muscle fibres gave a value close to 3 (2.8) considering GABA induced conductance changes. This value of 3 for the Hill coefficient is higher than the values of 2.09, 1.73 and 1.74 presented here and may relate to cumulative application of GABA leading to hysteresis, the log slope of 3.15 was reduced to 2.45 when decreasing concentrations were applied, (Werman, 1976). The partial substitution of propionate for chloride in the bathing saline (Brookes and Werman, 1980) and the variable resting membrane conductance (Brookes and Werman, 1973) were reported to have no influence on the limiting slope.

potentials

The slow EPSPs in muscle fibres with membrane potentials of between -52 and -67 mV had amplitudes of between 3 and 20 mV when recorded from muscle bundles 34, 32 and fibres in bundles 21-26. Bath application of GABA (5 x 10e5 to 5 x 10m3M) resulted in dose-dependent reductions in the amplitude of the slow EPSPs in all muscle fibres studied (Fig. 6). The application of GABA produced changes in the cable properties of the membranes of GABA sensitive muscle bundles, in bundles 34 and 32, but did not affect GABA-insensitive fibres in bundles 21-26. An indication of the maximal reduction in the slow EPSP amplitude resulting from postsynaptic effects of GABA was obtained by comparing the percentage reduction in electrotonic potential over half and whole fibre length under different conditions. The percentage reductions in electrotonic potentials measured over half the fibre length were normalized with respect to the decay in potential which occurred along the length of the fibre without GABA being present. The intracellular electrodes were inserted into the centre of the muscle fibre; under these conditions the maximal distance any nerve terminal was away from the recording electrode was half the fibre length. Data summarized in Table 2 shows the percentage reduction in potential that can occur along half the fibre length. A concentration of 5 x lo-‘M GABA applied to fibres of muscle bundle 32 induced an increase in conductance and increased the percentage decay of an electrotonic potential along half the length of the fibre from 10 to 16%. Since not all the nerve terminals are at the ends of the muscle fibres but spread along the length of the fibres the decay in electrotonic potential over half the fibre length represents an overestimation of the possible decrement in EPSP amplitude due to GABA altering cable properties of the muscle fibre. The reduction in slow EPSP amplitude induced by 5 x 10e3 M GABA applied to muscle bundle 32 was 49%. Clearly the postsynaptic action of GABA can-

DJSCUSSION

Fig. 5. Traces

IOmv

illustrating

1 mln

M GABA

mb32

15nA

- 50mV

IOnA

- - ,_ -

-

_.

. --

- _

- -.

- _ -

- .., ._

-.

the electrotonic potentials when current was injected within 50 pm (V,) of the recording electrode and at a distance approaching (VL) from the recording electrode. GABA (5 x lo-’ M) was applied to fibres of muscle bundles 34 and 32.

~xIO-~

1 min

Vo

5 x 1O-3 M GABA

VL

mb34

. - _I

- _

the whole fibre length

_.

188

RODERICK H. Scorr and IAN R. DUCE

32

‘OmV l-

IOOms

GABA

CM)

A

GAEIA 10-4

A_

5x10-4

-A.-

10-3

-L

5x10.’

M

Fig. 6. (a) Graph of percentage reduction in slow EPSP amplitude plotted against log GABA concentration. GABA induced a dose-dependent reduction in EPSP amplitude. Bach point shows the mean + SE of 6 values. When applied to fibres in muscle bundles 34,32 and fast muscle bundles. (b) Traces of slow EPSPs recorded from a fibre in muscle bundle 32 with a membrane potential of - 50 mV. The amplitude of the slow EPSP is reduced by GABA (10e4-5 x IO-’ M).

Postsynaptic GABA receptor-GABA molecule stochiometry studied in crustacean preparations has yielded Hill coefficients of between 1 and 4 (Nistri and Constanti, 1979). Therefore there seems to bc some variability in the lower limit for the number of GABA molecules required to activate one unit response from a receptor ion channel complex. The differences in Hill coefficient seen for the muscle bundles in this work may reflect varying ratios in population densities of GABA junctional and GABA extrajunctional receptors in fibres from different bundles. Cull-Candy and Miledi (1981) recorded small diffuse ionophoretic GABA responses and have suggested that extrajunctional GABA receptors exist in this preparation. Junctional acetylcholine receptors on frog muscle have been shown to have slightly higher Hill coefficient for acetylcholine n = 2.7 and nicotine n = 1.7 compared with their extrajunctional counterparts (acetylcholine, n = 2.0 and nicotine, n = 1.5; Peper et al., 1975). Cull-Candy (1976) has shown differences in glutamate receptor Hill coefficients in insect muscle, junctional D receptors have an n value of 1.5 and extrajunctional H receptors have an n value of 0.75, although in this case the differences may be accounted for by extrajunction glutamate H receptors desensitizing faster. Cooperativity, whereby the binding of one molecule to a receptor affects the binding of subsequent molecules has been found in this study. Cooperativity in GABA binding to locust muscle has been previously reported by Brookes er al. (1973) and has been shown for the action of GABA on crustacean muscle (Takeuchi and Takeuchi, 1967, 1969; Feltz, 1971; Constanti, 1977). The reductions in the amplitude of slow EPSPs induced by bath application of GABA cannot be completely accounted for by changes in decrement of electrotonic potentials over half the muscle fibre length. GABA was also found to have an inhibitory effect on slow EPSPs evoked in muscle fibres which were insensitive to GABA and did not receive inhibitory innervation. The likelihood of GABA acting postsynaptically at the glutamate receptor ion channel complex seems remote, because fast excitatory

responses also mediated by glutamate were found in this study and previously (Usherwood and Grundfest, 1965), not to be attenuated by GABA. These data suggested that GABA acted presynaptically to reduce glutamate release from slow excitatory neuromuscular junctions. What is particularly interesting is that this presynaptic inhibitory action of GABA was independent of postsynaptic inhibitory innervation of a particular muscle fibre. These findings support the suggestion that all slow excitatory neuromuscular junctions on the locust extensor tibiae muscles have functional GABA receptors on their surfaces (Usherwood and Cull-Candy, 1975). The involvement of GABA receptors (1) mediating presynaptic inhibitioin on slow excitatory terminals and (2) mediating postsynaptic inhibition may be important in neural control of locust extensor tibiae muscles. Inhibitory control may therefore be effected at slow excitatory terminals and not be dependent only on postsynaptic inhibitory innervation, which is restricted to a few muscle fibres in proximal and distal muscle bundles (Usherwood and Grundfest, 1965; Hoyle, 1978). The integration of slow excitatory and inhibitory responses on locust muscles is believed to be involved in control of postural movement. Locust skeletal muscles also receive innervation from octopaminergic neurones. Activity in these neurones increases transmitter release from slow excitatory motoneurones and increases cyclic AMP in muscle fibres (Evans, 1981, 1984). A number of systems thus interact to give rise to fine control of locust skeletal muscle movement. Acknowledgements-We

thank the SERC for financial support and Professor P. N. R. Usherwood, Dr R. L. Ramsey and Professor H. Shinozaki for suggestions and encouragement.

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189

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