1The Otto Loewi Minerva Center for Cellular and Molecular Neurobiology, Department of Neurobiology, The Hebrew University, Jerusalem, Israel; and 2Department of Anaesthesia and Intensive Care, The University of Adelaide, Adelaide, South Australia 5005, Australia
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ABSTRACT |
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Parnas, Itzchak, Grigory Rashkovan, Jennifer Ong, and David I. B. Kerr. Tonic activation of presynaptic GABAB receptors in the opener neuromuscular junction of crayfish. 1184-1191 Release of excitatory transmitter from boutons on crayfish nerve terminals was inhibited by (R,S)-baclofen, an agonist at GABAB receptors. Baclofen had no postsynaptic actions as it reduced quantal content without affecting quantal amplitude. The effect of baclofen increased with concentration producing 18% inhibition at 10 µM; EC50, 50% inhibition at 30 µM; maximal inhibition, 85% at 100 µM and higher. There was no desensitization, even with 200 or 320 µM baclofen. Phaclofen, an antagonist at GABAB receptors, competitively antagonized the inhibitory action of baclofen (KD = 50 µM, equivalent to a pA2 = 4.3 ± 0.1). Phaclofen on its own at concentrations below 200 µM had no effect on release, whereas at 200 µM phaclofen itself increased the control level of release by 60%, as did 2-hydroxy-saclofen (200 µM), another antagonist at GABAB receptors. This increase was evidently due to antagonism of a persistent level of GABA in the synaptic cleft, since the effect was abolished by destruction of the presynaptic inhibitory fiber, using intra-axonal pronase. We conclude that presynaptic GABAB receptors, with a pharmacological profile similar to that of mammalian GABAB receptors, are involved in the control of transmitter release at the crayfish neuromuscular junction.
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INTRODUCTION |
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Receptors for the inhibitory transmitter GABA
(-aminobutyric acid) can be divided into ionotropic and metabotropic
subtypes. The ionotropic GABAA and GABAC
receptors, with an integral picrotoxin-sensitive chloride channel, are
members of the ligand-gated ion channel super family of receptors that
includes glycine, nicotinic, and serotonin (5-HT3)
receptors. The metabotropic GABAB receptors belong to the
larger super family of heptahelical transmembrane receptors that are
G-protein-linked to a variety of cellular effectors, including calcium
and potassium channels, which they regulate (Kerr and Ong
1995
). GABAB receptors are picrotoxin insensitive but respond selectively to baclofen as an agonist and are antagonized by phaclofen, the phosphonic analogue of baclofen, as well as by the
related sulfonic derivative 2-hydroxy-saclofen (Kerr et al.
1987
, 1988
). Baclofen has no actions at either
GABAA or GABAC receptors.
There is now increasing evidence for both invertebrate and vertebrate
presynaptic GABAB receptors. Using baclofen as an agonist, Miwa et al. (1990) found a pertussis toxin-sensitive
potassium-dependent hyperpolarization of the excitatory axon at the
lobster neuromuscular junction. Blundon and Bittner
(1992)
showed that baclofen depresses the amplitude of the
action potentials in crayfish excitatory axons. These actions suggest
that GABAB receptors are located on the excitatory axon
itself and may be involved in presynaptic inhibition by affecting the
amplitude of the excitatory action potential. However, using a
macropatch electrode, to give focal depolarization of individual
release boutons, Fischer and Parnas (1996a
,b
)
demonstrated the presence of both GABAA and
GABAB receptors on one and the same release bouton at the
crayfish opener muscle. When using such localized depolarization,
activation of GABAB receptors affects the release machinery
by a mechanism other than reduction of the amplitude of the action
potential in the excitatory axon.
In the present study we further characterize the GABAB receptors in the crayfish neuromuscular junction. We found considerable resemblance in the pharmacological properties of the mammalian and crayfish receptors. In addition, the simpler anatomic organization of the crayfish preparation enabled us to unravel a presynaptic tonic inhibitory effect on release, exerted through GABAB receptors by the small concentration of GABA normally present in the synaptic cleft.
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METHODS |
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Preparation
The opener neuromuscular system of the crayfish
Procambarus clarkii was used (Fischer and Parnas
1996a,b
). Animals were purchased from Atachafalaya Biological
Supply (Raceland) and kept in aquaria with circulating filtered fresh
water. The crayfish were fed with fish fillets twice a week. The first
walking leg was removed by autotomy and the opener muscle exposed as
described previously (Dudel and Kuffler 1961
;
Fischer and Parnas 1996a
). The preparation was held by
small springs in a chamber (3 × 2 cm) with shallow walls (4 mm).
The chamber was placed on the stage of a Zeiss upright microscope
(Axioscop FS). Modified Van-Harreveld solution in the chamber was
circulated through a cooling device using a peristaltic pump (Gilson
Minipuls 3), to keep the temperature at 12°C, and contained the
following (in mM): 220 NaCl, 5.4 KCl, 13.5 CaCl2, 2.5 MgCl2, and 10 tris (hydroxymethyl) aminomethane-maleate; pH was adjusted to 7.4 by adding NaOH. TTX (2 · 10
7M) was
added to block sodium excitability.
Stimulation and recording
To visualize single release boutons, an objective (Acroplan
×40/0.75 W) with 1.8-mm working distance was used, requiring
horizontal positioning of the macropatch electrode (Ravin et al.
1997). Macropatch electrodes (Dudel 1981
,
1983
) with a long shaft were pulled on the DMZ-Universal
puller (Zeitz-Instruments, Munich, Germany). The tip (8 µm) was
slightly bent to allow positioning of the macropatch electrode over a
single release bouton in the small space between the objective and the
preparation. In the different experiments, the seal resistance varied
between 200 and 250 k
, but it was constant throughout each of the
experiments. The bouton was depolarized by constant negative current
pulses (0.7 ms,
0.7 µA at a rate of 2 Hz) (Dudel
1981
, 1983
). At the temperature used (12°C),
quanta appeared after the stimulus artifact (Fig.
1), and single quanta events could be
detected and counted. Traces were digitized using a neurodata
(Neuro-Recorder DR-484) A/D converter at 50 kHz, and stored on video
cassettes. In parallel, the data were transferred to a Pentium computer
(Philips 90 MHz) using the Labview (AT-MIO-16F-5, NI-DAQ 4.9.0 driver
software) interface. The number of quantal events was counted for a
given number of pulses (usually 120, 1 min). Dividing this number by
the number of pulses gave the quantal content.
|
Experimental procedure
First, we established the control quantal content by counting the number of quanta for several sets of stimuli. When the quantal content stabilized, we changed the circulation fluid to one containing either an agonist or an antagonist of GABAB receptors, or both. The time required for a complete change of solution in the chamber was estimated to be 1.1 min, using a calibrated test solution containing Coomassie Blue. Because the crayfish presynaptic GABAB receptors did not show desensitization to agonists, this period required for a drug to reach its final concentration had no effect on the final steady level of the quantal content.
Concentration-response curves for the agonist baclofen were constructed
in the presence and absence of the antagonist phaclofen. As a measure
of agonist potency, the half-maximally effective concentration
(EC50) was the concentration of the agonist required to
produce 50% depression of release, estimated from the
concentration-response curve. Potency of the antagonist phaclofen was
obtained from the rightward shift of the baclofen
concentration-response curve in its presence, using the Gaddum-Schild
relationship CR = 1 + [B]/KD; rearranging, converting to logs, and averaging, gives an estimate of
antagonist potency (pA2 = log (CR 1)
log [B]), the
concentration ratio (CR) being derived from the shift of the
concentration-response curve in the presence of each concentration of
the antagonist [B] (see Barlow et al. 1997
). By
definition, the pA2 of an antagonist is the concentration
causing a two-fold rightward shift of the agonist
concentration-response curve. The pA2 for phaclofen was also estimated from the inhibition curve for increasing concentrations of phaclofen against the response to a fixed concentration of baclofen
(30 µM, close to the EC50), based on the method described by Lazareno and Birdsall (1993)
.
Intracellular injection of the proteolytic enzyme pronase
In some preparations, it was desired to have an opener muscle
innervated only by the excitatory axon. This was achieved by intra-axonal injection of the proteolytic enzyme pronase, as described by Parnas and Bowling (1977) for leech neurons, and by
Dudel and Parnas (1987)
for lobster axons. The pronase
diffuses into the very small terminals to dissolve them without any
overt damage to nearby neurons (Bowling et al. 1978
).
The injection solution contained 0.4% sulforhodamine B (RBI) 0.5%
protease type 14 (Sigma) 200 mM KCl, and the pH was adjusted to 7.4 by
adding NaOH. Beveled electrodes were inserted into one of the main
secondary branches of the inhibitor fiber, and the protease solution
was injected by pressure using a Pico-Injector (Medical System,
PLI-100). The preparation was then incubated for at least 2 h
after which the action of the agonist or antagonist was tested on
quantal content.
Electron microscopy
To ensure that the inhibitory axon was dissolved, we studied the
ultrastructure of the neuromuscular junction as described by
Atwood and Morin (1970) and found that incubation with
intra-axonal pronase for 2 h was sufficient to destroy the
inhibitory terminals (not shown).
Statistical evaluation
In each experiment the quantal content was established for a series of groups of pulses (usually 120 pulses). The range of fluctuation of the quantal content in the controls or after treatment is given together with the average and the standard deviation (SD). Significance was estimated using the paired two-tailed t-test. For comparison of results between groups of experiments, we used the unpaired two-tailed t-test. The level of significance has been set at P = 0.01. Theoretical curves were fitted to the experimental data points (Figs. 2A, 4, and 5) using the Prism computer program: sigmoidal dose response equation (variable slope).
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Drugs
(R,S)-Baclofen (RBI) was used as a GABAB receptor
agonist. The GABAB receptor antagonists, phaclofen and
2-hydroxy-saclofen (Kerr et al. 1987,
1988
) were synthesized by Professor R. H. Prager and his colleagues (The Flinders University of South Australia, Australia). 3-Aminopropylphosphonic acid (3-APPA) was purchased from Sigma.
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RESULTS |
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Lack of effect of baclofen on quantum size
Baclofen had no effect on quantum size. Figure 1 shows samples of recordings in a control (A), in the presence of 30 µM baclofen (B), and after washing (C). Traces were selected to show a failure of release, a single quantum, and two or three quanta (the onset of quanta is marked by asterisks). Note that even though quanta varied in amplitude, the smaller quanta could be easily distinguished from the noise level. The average amplitude of 200 consecutive single quanta (without any selection) was the same in controls as in the presence of 30 µM baclofen, which reduced the quantal content by 50% (Fig. 1D).
Baclofen reduced release in a concentration-dependent manner
Application of baclofen led to a reduction in the evoked quantal content. The lowest detectable threshold for this effect was 7-10 µM (14-18% reduction); effects of baclofen at lower concentrations could not be detected, because of small fluctuations in quantal content seen in controls. Upon switching from the control solution to baclofen-containing medium, it took some minutes for the full depressant effect on transmitter release to appear (Figs. 2 and 3), lower concentrations (<50 µM) requiring at least an additional 5 min after complete exchange of the medium in the chamber. The action of baclofen on release was concentration dependent, with an EC50 of 30 µM and a maximum reduction of 87% achieved at 100-120 µM (B 100 µM; Fig. 2A). This same level of reduction was seen at 200 µM baclofen (B 200 µM; Fig. 2B), and higher concentrations of baclofen, even up to 320 µM, never completely abolished release. There was no evidence of desensitization with prolonged application of baclofen (>20 min, Fig. 2B; see also Fig. 3), which enabled cumulative concentration-response curves to be constructed.
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Partial agonist/antagonist properties have been described for the
congener 3-aminopropylphosphonic acid (3-APPA), from which phaclofen is
derived (Chiefari et al. 1987; Kerr et al.
1989
). Such partial agonist properties were also found here, at
the crayfish opener muscle (Fig. 2C). In four preparations,
application of 3-APPA (300 µM) reduced transmitter release by an
average of 35 ± 8% (mean ± SD), whereas 100 µM or 200 µM 3-APPA had no effect on release. In combination with 30 µM
baclofen, which reduced release by 76%, the effect of 300 µM 3-APPA
did not add. Instead, in its continued presence, 3-APPA significantly
reduced the response to baclofen by an average of 35%, in keeping with
a partial agonist/antagonist action of 3-APPA at this receptor. After a
15-min wash out, 30 µM baclofen was again fully effective in reducing
release (Fig. 2C).
Antagonism of baclofen by phaclofen, a GABAB receptor antagonist
Phaclofen is a competitive, surmountable, and reversible
antagonist of baclofen (Kerr et al. 1987). At the
crayfish presynaptic GABAB receptor, it was also an
effective antagonist as seen in Fig. 3. The control average quantal
content was 0.15 ± 0.01; in the presence of baclofen (50 µM),
this was reduced to 0.02 ± 0.01, recovering to 0.12 ± 0.01 after wash out. Phaclofen (100 µM) did not affect basal release
(0.14 ± 0.01), but in its continued presence, the response to 50 µM baclofen was virtually blocked (release 0.13 ± 0.01). Upon
wash out, the original basal level of stimulated release was restored,
whereupon another application of 50 µM baclofen again depressed
release to the same extent as previously (0.02 ± 0.01),
indicating reversibility of the antagonism. In a similar experiment,
phaclofen (100 µM) antagonized the action of baclofen (100 µM) by
25%. The average quantal content in the control was 0.36 ± 0.03. In the presence of 100 µM baclofen, this declined to 0.09 ± 0.02 (75% inhibition), whereas in the combined presence of phaclofen
and baclofen, the quantal content was 0.17 ± 0.01, a 21% change
from the level with baclofen alone (highly significant P < 0.01).
To establish the properties of phaclofen as an antagonist at GABAB receptors in this preparation, we measured the effects of different concentrations of phaclofen on the concentration-response curve for the presynaptic action of baclofen. Figure 4 shows that the typical concentration-response curve for baclofen was shifted to the right, in a parallel manner, in the presence of 60 µM phaclofen, indicative of surmountable, competitive antagonism. Interestingly, as can be seen, the concentration-response curve for baclofen, over the lower concentration range 10-30 µM, was altered in the presence of phaclofen (60 µM, Fig. 4). From the control concentration-response curve, the threshold response to baclofen was found to be a minimal 18% depression of release at 10 µM. However, in the presence of phaclofen (60 µM), which shifted the curve to the right, the minimum detectable response induced by 10 µM baclofen became a 5% depression. After wash out of phaclofen, the control concentration-response curve to baclofen was reestablished, although the response to 10 µM baclofen remained near 10%, rather than the original 18% depression. Using the rightward shifts of the baclofen concentration-response curve due to various concentrations of phaclofen and applying the Gaddum-Schild relationship (see METHODS), a mean value of pA2 = 4.3 ± 0.1 was found for its antagonist action at the crayfish GABAB receptor.
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In a further six experiments, we examined the concentration-dependent
displacement of baclofen from the GABAB receptor by the
antagonist. Three different concentrations of phaclofen (15, 30, and 60 µM) were used against the response to 30 µM baclofen, which is
close to the EC50 for reduction in release; Fig.
5 shows the resultant
concentration-dependent reduction of the baclofen response (taking the
control response to 30 µM baclofen alone as 100%). Phaclofen at 15 µM had no effect on the baclofen response, but at 30 and 60 µM,
phaclofen reduced the effect of baclofen by 24 ± 12.1% and
82 ± 9.75%, respectively. Using these results, an
EC50 value of 45 µM was calculated for phaclofen to
reduce the response to 30 µM baclofen by half; applying the method of Lazareno and Birdsall (1993), these results again
yielded an estimated pA2 of 4.3 ± 0.1 for phaclofen
as an antagonist at the crayfish GABAB receptor. Each of
these estimates was based on steady-state analysis with prolonged
exposure to the agents, which overcomes any problem of limitation of
drug penetration at the macropatch electrode.
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Over antagonism at high antagonist concentrations
Although 100 µM phaclofen had no effect on the resting level of evoked release, phaclofen at 200 µM increased the level of evoked release, even in the presence of 100 µM baclofen. Such an increase of release above the previous basal control indicates that 200 µM phaclofen had removed an underlying tonic inhibitory action of GABA at the synapse ("over-antagonism"). In a typical experiment, 100 µM baclofen reduced the control level of release (0.15 ± 0.014) by 69%, after which phaclofen (200 µM) was added in the continued presence of baclofen without washing. Even though baclofen (100 µM) was still present, the quantal content recovered to above its original control level, to an average of 0.23 ± 0.01, an increase of 153% (significant, P < 0.01). After washing, the quantal content declined to its initial level (0.15 ± 0.014), and a further application of 100 µM baclofen again inhibited release, as in the beginning of the experiment (not shown). This effect was not seen when using lower concentrations of phaclofen.
In Fig. 6, different concentrations of phaclofen (50, 100, and 200 µM) were added after 100 µM baclofen (B 100 µM) had produced its maximal effect (76% depression of release). With baclofen (100 µM) still present, addition of the lowest concentration of phaclofen (P 50 µM) had only a very slight effect, whereas phaclofen at 100 µM (P 100 µM) produced partial recovery, to 50% of control release. However, in the presence of 200 µM phaclofen (P 200 µM), the quantal content rose to a peak level of 78% over and above the initial control level; washing then restored release to the basal level, whereupon adding 100 µM baclofen once again effectively reduced release. In all experiments (n = 5) where 100 µM phaclofen was used, there was no increase in the basal level of evoked release, whereas in all experiments with 200 µM phaclofen (n = 4), evoked release increased above the basal level, on average by 62 ± 8%.
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When phaclofen (200 µM) was added alone in the absence of baclofen, there was again an increase in the basal level of evoked release. In four experiments, 200 µM phaclofen significantly increased release, to an average of 167 ± 24% (Fig. 7A, P = 0.01) of the control. Also, we tested for any similar effect of high concentrations of the GABAB receptor antagonist 2-OH-saclofen (Fig. 7A); at 100 µM 2-OH-saclofen had no effect on the basal level of evoked release (average 99.3 ± 3.7%; n = 4; nonsignificant, P = 0.7), whereas 200 µM 2-OH-saclofen significantly increased the basal level of evoked release to 144 ± 20.3% (n = 4, P = 0.01).
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Effect of phaclofen and 2-OH-saclofen on preparations without an inhibitory axon, following intra-axonal pronase treatment
An increase in the basal level of release after addition of
phaclofen or 2-OH-saclofen could have resulted from one or both of the
following mechanisms: phaclofen or 2-OH-saclofen may, at high
concentrations, affect release directly, or they may act by the removal
of a tonic inhibitory effect of GABA present in the synaptic cleft, as
found for example for the postsynaptic membrane of the neuromuscular
junction of the crab (Parnas et al. 1975). One way to
distinguish between these two mechanisms is to test for effects of
phaclofen or 2-OH-saclofen, on the opener neuromuscular system, after
removal of the inhibitory axon by intracellular injection of the
proteolytic enzyme pronase (Bowling et al. 1978
;
Parnas and Bowling 1977
). Removal of the inhibitory axon
presumably leaves the presynaptic release bouton without inhibitory
innervation, and therefore without an inhibitory synaptic cleft
containing GABA. If the effect of the antagonists is to increase
release directly, we would expect high antagonist concentrations to
still increase the release by an action at the excitatory boutons. If,
on the other hand, the effect is indirect, because of removal of tonic
inhibition, then we expect the effect to disappear. Comparison of Fig.
7A with Fig. 7B shows that the latter indeed was
the case; in preparations with the inhibitory axon removed, 200 µM
phaclofen or 200 µM 2-OH-saclofen no longer had any effect on the
basal level of release, in contrast to preparations with intact
inhibitory input. The average percentage of the quantal content in
comparison with the control was 101 ± 10.8% SD (4 experiments,
insignificant change, P = 0.2). If there was any
leakage of pronase from the damaged inhibitory terminals into the
synaptic cleft, it had no effect on the GABAB receptors of
the excitatory terminal because baclofen at 100 µM was still
effective in reducing release (Fig. 7B), as in normal
noninjected preparations.
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DISCUSSION |
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Although Kaupmann et al. (1997) could not detect
any invertebrate GABAB receptor protein in an insect
(Drosophila) or a nematode (Hemonchus),
nevertheless the present results have demonstrated inhibitory
GABAB receptors on presynaptic terminals in a crustacean (Procambarus). From their agonist and antagonist profiles,
using known agents selective for mammalian GABAB receptors,
these presynaptic receptors at the crayfish opener muscle are typical
GABAB receptors, their pharmacology being very similar to
that found in the mammalian CNS. The crayfish GABAB
receptors are activated by GABA itself, as well as by the agonist
baclofen, and both these agonists are blocked by 2-OH-saclofen.
Moreover, complete block of the inhibitory actions of GABA requires the
combined application of picrotoxin and 2-OH-saclofen, which are
antagonists at GABAA and GABAB receptors, respectively (Fischer and Parnas 1996a
,b
), so that each
of these receptor types must be present in this preparation.
In the present study, activation of the presynaptic GABAB
receptors with baclofen gave a depression of excitatory transmitter release at the crayfish opener, and this effect was blocked by the
specific GABAB receptor antagonist phaclofen. In addition, we have confirmed that the more potent antagonist 2-OH-saclofen is
effective against baclofen at the crayfish GABAB receptors, as was originally shown by Fischer and Parnas (1996a).
In particular, we have established the concentration-dependent
depression of excitatory transmitter release by baclofen at the
crayfish opener neuromuscular system, with an EC50 value of
30 µM and a maximum depression of 87% at 150 µM. We made two
different estimates of potency for phaclofen as an antagonist at the
crustacean GABAB receptors, both of which gave a mean value
of pA2 = 4.3, close to the pA2 value of 4.0 previously obtained for phaclofen in the mammal (Kerr et al.
1990
). In addition, we found that the phosphonic analogue of
GABA, 3-APPA, itself partly reduced transmitter release, yet attenuated
the action of baclofen when coapplied. Such actions are consistent with
partial agonist/antagonist properties of 3-APPA at these crustacean
GABAB receptors, as also found in the rat brain
(Drew et al. 1990
; Kerr et al. 1989
).
Thus, in relation to the agents examined so far (GABA, baclofen,
3-APPA, phaclofen and 2-OH-saclofen), the pharmacology of the
crustacean GABAB receptor closely resembles that of its
mammalian counterpart. We did not attempt to use the more recent,
potent agonists or antagonists for GABAB receptors, based
on P-substituted phosphinic analogues of GABA (Froestl and
Mickel 1997
), because many of these have been found to have
significant affinity for GABAC receptors, which crustacean
GABAA receptors closely resemble (Johnston
1997
).
In all experiments, we found that there was a delay of some minutes
before the full depressant action of baclofen was exerted on
transmitter release, and recovery was slow after wash out, suggesting
that this baclofen action is mediated through a G-protein rather than
through a rapidly acting channel-linked mechanism, although we have no
formal proof for this. In general, G-protein-coupled inhibitory
receptors, including GABAB receptors, are blocked by pertussis toxin (Thalmann 1987), as was originally shown
by Miwa et al. (1990)
, who found that the conductance
change induced by baclofen in crustacean motor fibers was abolished by
treatment with pertussis toxin. It could be argued that the delayed
onset of the depressant action of baclofen in our preparation is
somehow related to its slow diffusion from the fluid in the perfusion chamber to the receptors beneath the macropatch electrode; but we
discount this because the seal resistance at the macropatch electrode
is low (~200 K
), so that materials in solution can easily diffuse
below the rim of the electrode to reach the receptors. Instead, the
evidence is more consistent with the notion that the GABAB
receptors involved are G-protein coupled to some mechanism controlling
excitatory transmitter release. Indeed, such pertussis toxin-sensitive
G-proteins, likely involved, are highly conserved across vertebrates
and invertebrates as common transducing elements linking a variety of
ligands to intracellular effectors (Simon et al. 1991
).
For instance, in the crustacean neuromuscular junction, different
presynaptic receptors are coupled to G-proteins, including glutamate
(Miwa et al. 1987
), serotonin (Dixon and Atwood
1989
), and GABA (Miwa et al. 1990
). With the
presynaptic GABAB receptors in the crayfish, the ultimate
action is a longer lasting and possibly a tonic reduction in excitatory
transmitter release.
At higher concentrations, phaclofen removed a fraction of tonic
inhibition, resulting in a substantial reduction in the basal level of
inhibition ("over antagonism"). As seen in Figs. 6 and 7, 200 µM
phaclofen antagonized the depresssant action of 100 µM baclofen,
raising the quantal content to 78% over the initial basal control. A
similar increase in the basal level of release was seen when 200 µM
phaclofen was added alone. Likewise, 200 µM of 2-OH-saclofen, on its
own, significantly increased basal release (Fig. 7A). It
seems that a presynaptic tonic inhibitory action of GABA on release of
glutamate from excitatory terminals must be present at the crayfish
opener muscle system, acting on a high-affinity state of the
GABAB receptor and displaceable only by high concentrations
of phaclofen or 2-OH-saclofen. In the crayfish opener system, these
underlying levels of GABA were readily removed by destruction of the
inhibitory axon along with its terminals, using intra-axonal pronase,
as confirmed by electron microscopy studies (N. Feinstein and I. Parnas, unpublished observations). Here, such destruction enabled us to
uncover a new tonic presynaptic effect of the inhibitory transmitter
present at the synaptic cleft. The actual concentration of GABA in the
synaptic cleft was never determined, but it is estimated to be in the
submicromolar range, since Fischer and Parnas (1996a,b
)
found that a concentration of 2 µM GABA already produced some
detectable GABAB receptor-mediated presynaptic inhibition.
It should be noted that for GABA to act at such low concentrations to
produce tonic inhibition, two conditions must be met. The presynaptic
receptors must be of a high affinity to GABA, and they should not show
desensitization at these or even higher concentrations (Fischer
and Parnas 1996a
,b
). The tonic inhibitory effect is substantial
because its removal increased the basal level of release by ~50%,
which suggests that, in systems where presynaptic inhibition exists,
there is an additional securing mechanism to prevent release operating
as long as the excitatory axon is not activated. It is also interesting
to note that, when a smaller fraction of tonic inhibition was removed
by lower concentrations of phaclofen, we could detect a depressant
response to lower concentrations of baclofen in the presence of
phac-lofen. The latter not only shifted the baclofen
concentration-response curve to the right (Fig. 4), but also lowered
the minimal detectable response to baclofen, which evidently was
normally occluded by endogenous GABA in the absence of phaclofen.
Modulation of glutamate release through GABAB
receptor-activated G-proteins as seen here could involve a decreased
Ca2+ influx at the terminal or might be due to some action
at the release mechanism itself, independent of alterations in
K+ conductance as discussed recently by Zhang et al.
(1996). In this regard, the present method for studying
modulation of release offers several advantages, because the detection
of release does not depend on propagation of an action potential or on
its amplitude. The focal depolarization technique detects only effects
on the region below the macropatch electrode and thus provides direct access to the presynaptic receptors on excitatory boutons. The latter
raises the feasibility of directly examining the influence of
modulators such as GABA on intracellular Ca2+
concentration, and associated changes in transmitter release at single
release boutons, independently of alterations in K+
conductance, as was done recently by Parnas et al.
(1996)
and Ravin et al. (1997)
. Thus our present
technique may profitably be used for future characterization of the
receptors involved.
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ACKNOWLEDGMENTS |
---|
We are grateful to the Goldie Anna fund for continuous support. We thank Prof. R. H. Prager for the synthesis of phaclofen and 2-hydroxy-saclofen, and R. Ravin for continuous discussions and help. Professor I. Parnas is the Greenfield Professor of Neurobiology.
This work was supported by an SFB 391 grant from the Deutsche Forschungsgemeinschaft, Germany, to Drs. Dudel and Parnas. J. Ong was the recipient of an Australian Research Council Senior Research Fellowship.
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FOOTNOTES |
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Address for reprint requests: I. Parnas, The Otto Loewi Minerva Center for Cellular and Molecular Neurobiology, Dept. of Neurobiology, The Hebrew University, Jerusalem, Israel.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 September 1998; accepted in final form 3 November 1998.
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