The Otto Loewi Minerva Center for Cellular and Molecular Neurobiology, Department of Neurobiology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
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ABSTRACT |
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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
-aminobutyric
acid-A (GABAA) receptors reduce transmitter release also by
a mechanism other than affecting Ca2+ entry.
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INTRODUCTION |
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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
-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
-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.
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METHODS |
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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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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 -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.
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RESULTS |
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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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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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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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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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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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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.
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DISCUSSION |
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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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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.
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REFERENCES |
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