Novel Mechanism for Presynaptic Inhibition: GABAA Receptors Affect the Release Machinery

I. Parnas, G. Rashkovan, R. Ravin, and Y. Fischer

The Otto Loewi Minerva Center for Cellular and Molecular Neurobiology, Department of Neurobiology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Parnas, I., G. Rashkovan, R. Ravin, and Y. Fischer. Novel Mechanism for Presynaptic Inhibition: GABAA Receptors Affect the Release Machinery. J. Neurophysiol. 84: 1240-1246, 2000. Presynaptic inhibition is produced by increasing Cl- conductance, resulting in an action potential of a smaller amplitude at the excitatory axon terminals. This, in turn, reduces Ca2+ entry to produce a smaller release. For this mechanism to operate, the "inhibitory" effect of shunting should last during the arrival of the "excitatory" action potential to its terminals, and to achieve that, the inhibitory action potential should precede the excitatory action potential. Using the crayfish neuromuscular preparation which is innervated by one excitatory axon and one inhibitory axon, we found, at 12°C, prominent presynaptic inhibition when the inhibitory action potential followed the excitatory action potential by 1, and even 2, ms. The presynaptic excitatory action potential and the excitatory nerve terminal current (ENTC) were not altered, and Ca2+ imaging at single release boutons showed that this "late" presynaptic inhibition did not result from a reduction in Ca2+ entry. Since 50 µM picrotoxin blocked this late component of presynaptic inhibition, we suggest that gamma -aminobutyric acid-A (GABAA) receptors reduce transmitter release also by a mechanism other than affecting Ca2+ entry.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Presynaptic inhibition is a powerful mechanism regulating release of neurotransmitter (see review in MacDermott et al. 1999). It is well accepted that rapid presynaptic inhibition is achieved by activation of presynaptic fast gamma -aminobutyric acid-A (GABAA) receptors, which increase Cl- conductance (Dudel and Kuffler 1961; Fuchs and Getting 1980; Takeuchi and Takeuchi 1966). The increase in Cl- conductance shunts the presynaptic membrane, resulting in shortening of the space constant of axon terminals. Thus,in cases of inexcitable terminals, the amplitude of the passively spreading action potential is reduced (Dudel 1963). The smaller depolarization results in a smaller influx of Ca2+, and release is reduced. These general mechanisms were found to operate in invertebrates such as crustaceans (Atwood and Wojtowicz 1986; Baxter and Bittner 1981; Dudel 1963; Dudel and Kuffler 1961) or in Aplysia (Kretz et al. 1986), as well as in vertebrates, including in the spinal cord and in the CNS of mammals (Clements et al. 1987; see review by Nicoll and Alger 1979). Thus, presynaptic inhibition affects release, indirectly, by reducing Ca2+ entry.

For presynaptic inhibition to be fully effective in reducing the space constant, the GABAA receptors should be located all along the axon terminal. Indeed, in the crayfish axoaxonic inhibitory synapses were found along the excitatory axon and especially near branch points (Atwood 1976; Atwood and Morin 1970). Such branch points have a low safety factor for conduction of action potentials and increase in Cl- conductance causes a conduction block (Parnas 1972; Parnas and Segev 1979; Segev 1990). The shunting effect, produced by increasing Cl- conductance, should last when the "excitatory" action potential spreads along its terminals. Indeed, it was found that presynaptic inhibition occurs only when the "inhibitory" action potential precedes the arrival of the excitatory action potential to its terminals (Dudel and Kuffler 1961).

Recently, we found both GABAA and gamma -aminobutyric acid-B (GABAB) receptors at the same release bouton (Fischer and Parnas 1996a,b). Such GABAA receptors are expected to have no effect on the amplitude of the action potential at that same bouton (Segev 1990) but can still affect the space constant of the axon terminal and thus the amplitude of the passively spreading action potential at more distant boutons. However, we (Fischer and Parnas 1996a,b) found that activation of GABAA receptors under experimental conditions in which release is insensitive to changes in membrane conductance (Dudel 1981, 1983) reduced release from single boutons. Thus, presynaptic inhibition obtained under such conditions cannot be explained by a shunting mechanism. We (Fischer and Parnas 1996b), therefore, suggested that GABAA receptors located on release boutons reduce release by directly affecting the release machinery or by reducing Ca2+ entry by an effect (direct or indirect) on the voltage-activated Ca2+channels. In the present article, we further explore this possibility.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The opener neuromuscular junction of the crayfish Procambarus clarkii was used. The opener muscle with its single-excitatory axon and single-inhibitory axon was prepared as described in Fischer and Parnas (1996a,b). The preparation was mounted on a stage of a Zeiss upright microscope (Axioscope) with a long distance (1.8 mm) working objective ×40 (Parnas et al. 1999). Temperature was kept at 12 ± 1°C.

Stimulation and recordings

Each of the nerve bundles, one containing the excitatory axon, the other containing the inhibitory axon, was stimulated with brief pulses of 0.2 ms at a rate of 5 Hz. Alternating stimulation was such that in one trace only the excitatory axon was stimulated, and in the following trace, both axons were stimulated. Such alternating stimulation allowed us to obtain the control responses (excitatory axon alone) and experimental responses (excitatory-inhibitory stimulation) over the same period of time (Fischer and Parnas 1996a,b). The interval between the excitatory and inhibitory pulses varied between -5 ms (inhibitory axon preceding) to +4 ms (stimulation of the inhibitory axons was delivered after stimulation of the excitatory axon). Since the interval between the stimuli may not result with the same interval in the presynaptic terminal, we actually measured the time interval between the excitatory nerve terminal current (ENTC) and the inhibitory nerve terminal current (INTC) (see Fig. 1, arrowheads).



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Fig. 1. Samples of recordings. Excitatory axon stimulated alone (left); excitatory and inhibitory stimulation (right). The excitatory nerve terminal currents (ENTCs) are marked by filled arrowheads. The INTCs are marked by open arrowheads. The inhibitory nerve terminal currents (INTCs) appear 1 ms after the ENTCs. The quantal events are marked by asterisks. Right traces were selected to show a smaller quantal content with presynaptic inhibition.

Single quanta events were recorded with a macropatch electrode (Dudel 1981) as described in Parnas et al. (1999). At 12°C, the release of quanta is not very synchronized, and the number of quanta and delay to each quantum could be determined. The quantal content and synaptic delay histograms (Katz and Miledi 1965) were established as described in Parnas et al. (1999) and Ravin et al. (1999a,b).

Ca2+ imaging

Ca2+ imaging from single-release boutons was performed using fura-2 as described in detail by Ravin et al. 1997. Stimulation rate of the excitatory and inhibitory axons was 50 Hz.

Drugs and chemicals

Picrotoxin and gamma -aminobutyric acid (GABA) were purchased from Sigma. 2-OH-saclofen was obtained from Dr. D. Kerr, Department of Anesthesia and Intensive Care, The University of Adelaide, Adelaide, Australia. Fura-2 was purchased from Molecular Probes.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Timing experiments

We have monitored release from individual boutons, in response to excitatory test action potentials and checked for presynaptic effects of precisely timed inhibitory action potentials.

Figure 1 shows samples of recordings obtained with alternating stimulation. Accordingly, in one trace, the excitatory axon was stimulated alone, and in the following trace, the excitatory and inhibitory axons were stimulated. Even though the pulses were given in an alternating manner, the traces shown in Fig. 1 were grouped such that the responses to excitatory stimulation alone are shown on the left side, and those obtained with both excitatory-inhibitory stimulation are shown on the right. On the right, the traces were selected to show a smaller quantal content with the presynaptic inhibition (see also Fig. 2). In each trace, the stimulus artifact produced by stimulation of the excitatory axon appears first. The macropatch electrode recorded the ENTC, marked by the filled arrowheads. The upper traces show a failure in release; the other traces show one and two quanta (marked by asterisks). Because of the proximity of the excitatory and inhibitory release boutons (Atwood and Morin 1970), the macropatch electrode also recorded the INTC, marked by empty arrowheads. As seen, the INTC appears 1 ms after the ENTC. Due to the small driving force for Cl- ions, inhibitory quanta are too small to be detected by the macropatch electrode.



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Fig. 2. Presynaptic inhibition obtained when the inhibitory axon was stimulated at different intervals from the stimulation of excitatory axon (taken as zero time). Average results of 7 experiments. Delays were measured between the ENTC and INTC. For each interval, the quantal content is given as a percent of control. For each point, the quantal content was obtained by giving 1500 pulses. Vertical bars: SD.

Figure 2 shows the level of presynaptic inhibition (at 12°C) when the inhibitory stimulus was given at different intervals from the excitatory stimulus. Each point represents the average result of seven experiments. The control quantal content, obtained when the excitatory axon was stimulated alone, was taken as 100%. At intervals of -5 and -4 ms between the INTC and the ENTC (inhibitory stimulation preceded excitatory stimulation), the level of presynaptic inhibition was about 6% (an insignificant change, P > 0.55, unpaired t-test). At -3 ms, presynaptic inhibition reduced the quantal content by 19 ± 2.8% (mean ± SD); maximal presynaptic inhibition was obtained at -2 ms (36 ± 11%). When the ENTC and INTC coincided (zero interval), inhibition was still 29 ± 9.5%. Surprisingly, presynaptic inhibition was quite prominent when stimulation of the inhibitory axon followed the stimulation of the excitatory axon by 1 and even 2 ms. The values of inhibition were 21 ± 5% and 12 ± 6%, respectively (P = <0.0001 and <0.04, respectively, unpaired t-test). Thus, it is clear that at 12°C activation of GABA receptors 1 or 2 ms after the arrival of the action potential to the excitatory release bouton was still effective in reducing excitatory release (denoted throughout as "late" presynaptic inhibition). This finding suggests an extremely fast action of the GABA receptors on release probably by a mechanism which is not related to changes in Ca2+ entry.

Late presynaptic inhibition is blocked by picrotoxin and not by 2-hydroxy-saclofen

Picrotoxin blocks presynaptic inhibition produced by activation of GABAA receptors (Takeuchi and Takeuchi 1969). The effect of presynaptic inhibition produced by inhibitory impulses preceding the excitatory impulses is blocked by picrotoxin (Takeuchi and Takeuchi 1969). We tested for effects of picrotoxin on the late component of presynaptic inhibition. The results depicted in Fig. 3A were obtained when the INTC followed the ENTC by +1 ms, producing presynaptic inhibition of about 20%. Superfusion of 50 µM picrotoxin, a concentration known to block GABAA and not GABAC receptors (Johnston 1996), blocked the late component of presynaptic inhibition and reappeared after washing. 2-hydroxy-saclofen, a specific antagonist of GABAB receptors (Kerr et al. 1988; Parnas et al. 1999), did not block the late component of presynaptic inhibition (Fig. 3B). We conclude that similar to the classical presynaptic inhibition, the late component of presynaptic inhibition is also produced by activation of GABAA receptors.



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Fig. 3. Effect of picrotoxin and 2-hydroxy-saclofen on the late presynaptic inhibition. Interval between ENTC and INTC, +1 ms. Each point represents the quantal content obtained for 256 pulses. A: the first 4 points show that presynaptic inhibition was about 20%. Superfusion for 16 min, with a saline solution containing 50 µM picrotoxin, blocked the late presynaptic inhibitory effect. Washing of picrotoxin for additional 16 min restored the late presynaptic inhibition. B: a different preparation, same experimental conditions as in A, but the preparation was superfused with 2-hydroxy-saclofen. 2-hydroxy-saclofen did not block the late presynaptic inhibition.

Effect of inhibitory stimulation on the delay histogram of excitatory release

Previous experiments (Arechiga et al. 1990; Ravin et al. 1997) measuring synaptic delay histograms at the opener neuromuscular system of crayfish showed that, at 12°C, release starts at most 1 ms after the beginning of the depolarizing pulse, reaches its peak about 2-3 ms later, and terminates within 4-5 ms. It is obvious that inhibitory action potentials appearing 1 ms after the arrival of the excitatory action potential can only block the release during the later part of the delay histogram.

The results depicted in Fig. 4A show one example that this was indeed the case. Alternating stimulation was given, and synaptic delay histograms were constructed for stimulation of the excitatory axon alone, and when the inhibitory axon was also stimulated, such that the interval between the ENTC and INTC was +1 ms (see oscilloscope trace in Fig. 4A).



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Fig. 4. A: effect of presynaptic inhibition on time course of excitatory release. The oscilloscope trace shows that the interval between the negative peaks of the ENTC and INTC was +1 ms. The delay to the beginning of each quantum was measured from the negative peak of the ENTC (taken as zero time). 1500 pulses were given when the excitor axon was stimulated alone (histogram, solid line) and when the 2 axons were stimulated (dashed line). B: percent block of excitatory release as a function of time. Vertical bars: SD. For further details, see text.

When the excitatory axon was stimulated alone (Fig. 4A, solid line), release started about 1 ms after the negative peak of the ENTC. Maximal release was obtained at about 2 ms, and release stopped at about 4 ms. When stimulation of the excitatory axon was followed by stimulation of the inhibitory axon (Fig. 4A, dashed line), excitatory release followed the same kinetics for a period of about 0.5 ms. At later times, the number of quanta released per time bin was smaller than in the control. In 12 such experiments, we calculated percent inhibition, in time, when the inhibitory INTC appeared 1 ms after the ENTC. The results depicted in Fig. 4B show the average inhibition obtained, in time, after the negative peak of the ENTC. Since the INTC appeared 1 ms after the ENTC, it took some time until presynaptic inhibition became apparent. On average, the inhibitory effect became apparent about 0.75 ms after the INTC, and thereafter the percent of inhibition was between 25-33%, creeping up as time elapsed. The differences between percent inhibition for the first and last points was not significant (P = 0.28 unpaired t-test). Thus, it seems that inhibitory impulses given at +1 ms, once they started to reduced the quantal content, the effect was about the same throughout the entire delay histogram.

Although synaptic delay histograms for inhibitory release were never measured, release of inhibitory quanta is also very fast, as found by Dudel (1977), who measured inhibitory synaptic currents in crayfish and found that the minimal delay at 13.5°C is <0.5 ms. Thus, it can be assumed that at 12°C release of quanta from the inhibitory axon terminals starts also with a minimal delay of 0.5 ms (or less) after the INTC. Since presynaptic inhibitory effects were detected already at 0.75 ms after the negative peak of INTC, we conclude that the presynaptic effect of GABA on excitatory release must be extremely fast (about 0.2 ms after initial GABA release). In other words, the "early" inhibitory quanta reduced the release of the late excitatory quanta, and this relation was kept throughout the delay histogram with slightly stronger inhibition toward the end of the histogram. This probably results due to a cumulative effect of inhibitory quanta released at early and somewhat later times, but still before the release of the late excitatory quanta.

Effects of presynaptic inhibition on the excitatory action potential

One of the mechanisms explaining presynaptic inhibition is the reduction of the amplitude of the excitatory action potential (Dudel 1963, 1965a,b). This was shown by comparing the action potentials in the preterminal of the excitatory axon when stimulated alone and with properly timed inhibitory stimulation or after addition of GABA to the bathing solution (Baxter and Bittner 1991). We repeated such experiments with inhibitory stimulation given at different delays from the excitatory action potential. The intracellular electrode recorded excitatory action potential from one of the secondary branches of the excitatory axon. The axon branch was impaled about 20 µm from a release site, as verified by recordings with a macropatch electrode positioned as close as possible to the recording microelectrode, as done by Wojtowicz et al. (1987). When the inhibitory action potential preceded the excitatory action potential by 2 ms, the latter became slightly smaller (Fig. 5A and see also Fig. 1 of Baxter and Bittner 1991). When the inhibitory INTC appeared 1 ms after the ENTC, the shape of the excitatory action potential at the preterminal remained exactly the same. The amplitude of the action potential and the long decay phase shows perfect superposition (Fig. 5B). As found by others (Atwood 1976; Baxter and Bittner 1991; Fuchs and Getting 1980), when GABA was added to the circulating solution, the reduction of the excitatory action potential was much more pronounced (Fig. 5C, dashed line). The GABA effect was reversible on washing (Fig. 5C, open dots).



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Fig. 5. Effects of presynaptic inhibition on the amplitude of the excitatory action potential. The action potential was recorded intracellularly from the preterminal of the excitatory axon 20 µm away from a release site. The solid lines in A-C show the action potential when the excitatory axon was stimulated alone. A, dashed line: the amplitude of the excitatory action potential was slightly reduced when the inhibitory axon was stimulated with -2 ms delay. B, dashed line: when the inhibitory axon was stimulated with +1 ms delay there was no change in the excitatory action potential. C: 30 µM GABA produced a marked reduction in the amplitude of the excitatory action potential (dashed line) with recovery after washing (dots).

We conclude that late presynaptic inhibition did not result from changes in the shape of the excitatory presynaptic action potential (at least as detected in the preterminal 20 µm away from a release site, see also Dudel 1963). Since the INTC appeared after the ENTC, there was no change in the amplitude or shape of the ENTC (not shown). Even though such results suggest that Ca2+ entry into the excitatory release boutons may not be affected by the late presynaptic inhibition, nevertheless, it is still possible that activation of the GABAA receptors reduces (directly or indirectly) the entry of Ca2+ through the voltage-dependent Ca2+ channels (Kretz et al. 1986).

Effects of inhibitory stimulation on Ca2+ entry

Ravin et al. (1997) developed a technique for Ca2+ imaging from single-release boutons depolarized by a macropatch electrode. Ravin et al. (1997, 1999a,b) showed that even though the Ca2+ imaging technique detects the average level of Ca2+ in the bouton, this level is lower when Ca2+ entry is reduced, for example, by increasing the extracellular Mg2+ concentration. Ravin et al. (1997, 1999a,b) demonstrated that this technique is sufficiently sensitive to detect small changes in Ca2+ entry. They also showed that the slope relating log release to log [Ca2+]i is only slightly larger than 1. We used the same imaging technique, but unlike Ravin et al. (1999a,b), who depolarized single-release boutons, we stimulated the excitatory axon alone or in combination with inhibitory stimulation.

Figure 6A shows profiles of Ca2+ accumulation when the excitatory axon was stimulated alone (solid line) and when the INTC preceded the ENTC by 2 ms. With inhibitory stimulation, Ca2+ accumulation was reduced by about 4% (dotted line), recovering to the control level when inhibitory stimulation was stopped (Fig. 6A, dots). The results presented in Fig. 6A were actually from the experiment that showed the largest reduction in Ca2+ accumulation. In four additional experiments, the reduction in Ca2+ accumulation was even smaller than 4%. Figure 6B shows Ca2+ accumulation curves when the delay between the ENTC and INTC was +1 ms. The three curves, control (solid line), with late presynaptic inhibition (dashed line), and after recovery (dots),are actually the same. Similar results were obtained in four additional experiments. We conclude that a reduction in Ca2+ entry does not account for the late component of presynaptic inhibition. In contrast, when 30 µM GABA was added, the reduction in Ca2+ accumulation was much more pronounced (Fig. 6C). The insert shows the control ENTC and its smaller amplitude in the presence of 30 µM GABA. Thus, the reduction in Ca2+ entry resulted from smaller depolarization of the presynaptic terminal.



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Fig. 6. Effect of presynaptic inhibition on Ca2+ accumulation in a single presynaptic bouton. A and B: same bouton. A: the interval between the ENTC and INTC was -2 ms. Solid line: control, stimulation of the excitatory axon at 50 Hz for 7 s. Dashed line: with inhibitory stimulation at -2 ms interval. Ca2+ accumulation declined by 4%. Dots: recovery when inhibitory stimulation stopped. The interval between each run was 7 min. B, same as in A, but the inhibitory axon was stimulated with a delay of +1 ms. C, another preparation, solid line: control. Dashed line: in the presence of 30 µM GABA. Dots: recovery after washing. The insert shows the ENTC in the control (solid line) and in the presence of 30 µM GABA (dashed line) with recovery after washing (dots). D, another preparation: effect of increasing [Mg2+]o on Ca2+ accumulation. Solid line: control in normal solution. Short dashes: in the presence of 5 mM [Mg2+]o. Filled dots: recovery after washing with normal solution. Long dashes: in the presence of 10 mM [Mg2+]o. Empty dots: recovery after washing with normal solution. Interval between runs, 7 min. For further details, see text.

Effect of increasing extracellular Mg2+ concentration

Even with the nonlinearity (power of 4) of the dependence of release on Ca2+ concentration (Dodge and Rahamimoff 1967; Ravin et al. 1997), a 4% reduction in Ca2+ accumulation (and assuming the same reduction in Ca2+ entry) obtained with presynaptic inhibition (at -2-ms interval) is too small to account for a reduction of 36% (on average) in quantal content (Ravin et al. 1999a). This result raised the possibility that even when the INTC preceded the ENTC by 2 ms, the presynaptic inhibition affected release by an additional mechanism, that is to say, in addition to reducing Ca2+ entry.

To test for such a possibility, we reduced Ca2+ accumulation (entry) by increasing the concentration of extracellular Mg2+. This treatment is expected to reduce the quantal content only as a result of the reduced Ca2+ entry. Indeed, a much larger reduction in Ca2+ accumulation was required to inhibit release to the extent (36%) obtained with presynaptic inhibition at -2 ms.

Figure 6D shows profiles of Ca2+ accumulation recorded from a single-release bouton that was depolarized directly (Ravin et al. 1997). In the control (solid line) in the presence of normal [Mg2+]o (2.5 mM), Ca2+ reached a level of 5 µM. [Recall that for the crayfish, the Kd of fura-2 is 850 nM Ca2+ (Ravin et al. 1997).] At 5 mM [Mg2+]o, it reached a level of 4.3 µM. After washing, there was a recovery to the initial control level (5.1 µM) where, on addition of 10 mM [Mg2+]o, the value declined to 3.9 µM, with recovery after washing to 5 µM. The quantal content in the control was 0.54 ± 0.2 SD (40 consecutive points, each of 380 pulses). At 5 and 10 mM [Mg2+]o, the respective quantal contents were 0.46 ± 0.17 and 0.33 ± 0.2. With washings, the quantal content recovered to values of 0.53 ± 0.2 and 0.52 ± 0.15, respectively. Thus, decline of 14% in Ca2+ accumulation, a much larger value than the 4% seen with presynaptic inhibition, reduced release by only 15%. A decline of 22% in Ca2+ accumulation was associated with a 39% inhibition. In five experiments, increasing [Mg2+]o to 5 and 10 mM reduced Ca2+ accumulation, on average, by 8.4 ± 5 and 18 ± 9%, respectively. The quantal content was reduced from an average of 0.54 ± 0.24 in the control to a value of 0.46 ± 0.17 in the presence of 5 mM [Mg2+]o (14% inhibition). After washing, the quantal content recovered to 0.53 ± 0.2 (98%). In the presence of 10 mM [Mg2+]o, the quantal content was 0.33 ± 0.2 (38%), and it recovered to a value of 0.52 ± 0.15 (98%). It should be recalled that with presynaptic inhibition (at -2 ms), a 4% reduction in Ca2+ accumulation was associated with a 36% inhibition of release, and with presynaptic inhibition at +1 ms, the quantal content was reduced by about 20% without any change in the Ca2+ profile.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present article, we show that presynaptic inhibition, operating by activation of GABAA receptors, is achieved by more than the classical mechanism found first for presynaptic inhibition by Dudel and Kuffler (1961), namely, the shunting effect which is produced by an increase in Cl- conductance (MacDermott et al. 1999). This conclusion is based on the following results: 1) Maximal presynaptic inhibition (about 35%) obtained with properly timed (-2 ms) inhibitory stimulation was associated with a reduction in presynaptic Ca2+ accumulation of only ~4%. A much larger reduction in Ca2+ accumulation was required to reduce release by 35% when [Mg2+]o was increased. Our results with increasing [Mg2+]o are similar to those reported by Ravin et al. (1999b). 2) Late presynaptic inhibition of about 20% was not associated with any reduction in Ca2+ influx. 3) Inhibition was seen when inhibitory stimulation was given after the excitatory impulse without having a major effect on the time course of release.

Most published experiments on presynaptic inhibition have been performed at room temperature, and in the case of a mammalian tissue, even at higher temperatures. At room temperature, and certainly at higher temperatures, the time course of release is brief. Therefore, presynaptic inhibition could be demonstrated only when the "inhibitory potential" preceded the "excitatory potential." At 12°C, the time course of release is markedly slowed, enabling presynaptic inhibition to operate also when the inhibitory potential appeared after the excitatory potential. In this case, however, inhibition could act only on those excitatory quanta which were destined to be released toward the later part of the synaptic delay histogram. Such late presynaptic inhibition was not associated with any reduction in Ca2+ entry, probably because, at that time, the voltage-dependent Ca2+ channels activated by the excitatory stimulation already closed.

The effect of activation of the GABAA receptors located directly on the excitatory release bouton was extremely fast. Inhibition of release appeared 0.2-0.3 ms after the release of the first inhibitory quanta. These numbers are correct if we assume (at 12°C) a minimal delay of about 0.5 ms for release of the first quanta from the inhibitory terminals (Dudel 1977).

Such a rapid action with no concomitant reduction in Ca2+ influx raises the possibility that the GABAA receptors operate directly on the release machinery. Presynaptic inhibitory muscarinic receptors in brain synaptosomes were found to be directly linked to proteins of the release apparatus (SNARE, SNAP-25, syntaxin, Ilouz et al. 1999; Linial et al. 1997; Parnas et al. 2000), and activation of these receptors in the frog neuromuscular junction reduced release without any effect on the time course of release (Slutsky et al. 1999). It may be that a similar linkage also exists between GABAA receptors and core proteins of the exocytotic machinery.

Finally, it should be emphasized that the mechanisms involved in presynaptic inhibition are quite similar in crustaceans and mammals. The increase in Cl- conductance, the similar sensitivities to antagonists, the presence of presynaptic GABAA and GABAB receptors (the latter with similar responses to different agonists and antagonists suggest substantial structural conservation of these receptors). It is, thus, quite possible that, in vertebrates as well as in crustaceans, presynaptic inhibition produced by activation of GABAA receptors is achieved not only by a reduction in Ca2+ entry.


    ACKNOWLEDGMENTS

We thank H. Parnas for reading the manuscript and for helpful comments. We thank Dr. Kerr for the 2-OH-saclofen. I. Parnas is Greenfield Professor of Neurobiology.

This research was supported by Grant SFB 391 to Prof. J. Dudel, I. Parnas, and H. Parnas from the Deutsche Forschungsgemeinschaft, Germany. We are grateful to the Anna Lea Foundation for continuous support.

Present address of Y. Fischer: Brain Research Institute, University of Zurich, CH-8057 Zurich, Switzerland.


    FOOTNOTES

Address for reprint requests: I. Parnas, Dept. of Neurobiology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel (E-mail: ruthy{at}vms.huji.ac.il).

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 31 January 2000; accepted in final form 24 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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