Interaction between facilitation and presynaptic inhibition at the crayfish neuromuscular junction
Department of Biology, Simon Fraser University, Burnaby, British Columbia, V5A 1S6, Canada
* Author for correspondence (e-mail: cddemill{at}sfu.ca) at present address: Department of Biology, University of Victoria, PO Box 3020, Station CSC, Victoria, British Columbia, V8W 3N5, Canada
Accepted 15 March 2005
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Summary |
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Presynaptic inhibition reduced Ca entry into presynaptic excitor terminal boutons (range 0-50%, mean ± S.E.M.=20±1%, N=122 terminals; 12 preparations) and reduced the EJP amplitude (range 30-70%, mean ± S.E.M.=51±2%, N=27 cells). The decline in the EJP was proportional to the reduction of Ca influx raised to the power of 2.8. Since presynaptic inhibition reduces the number of Ca channels opened by an action potential, our data suggest cooperativity between Ca channel microdomains to initiate vesicle fusion at this synapse.
The amount of inhibition of Ca influx into an excitor bouton was not correlated with either the distance to the closest inhibitor bouton or the main excitor branch, although slightly more inhibition was seen for excitor boutons on tertiary versus secondary branches. Unlike inhibitor axon stimulation, bath application of GABA caused inhibition of Ca influx that steadily increased from proximal to distal terminal boutons on a branch. We propose a model where presynaptic inhibition causes localized shunting of an actively propagated action potential in the vicinity of release sites, which can recover its amplitude outside the shunted region.
Key words: calcium imaging, contraction, GABA, Procambarus clarkii
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Introduction |
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In the present study, we investigated the interaction between
activity-dependent short-term enhancement (primarily facilitation, not
augmentation or post-tetanic potentiation) and presynaptic inhibition in the
excitor axon. It has been shown that facilitation develops during
co-activation of the excitor and inhibitor axons, is masked by presynaptic
inhibition and is unmasked within a few ms of the offset of inhibitor activity
provided the excitor continues to fire
(Atwood and Bittner, 1971;
Baxter and Bittner, 1980
;
Dudel and Kuffler, 1961
). We
found that although presynaptic inhibition can block muscle contraction during
co-activation of the excitor and inhibitor axons, it does so without
eliminating facilitation. The result of this preservation of facilitation is a
shortened time to initiate contraction after the offset of inhibition. We have
studied the mechanism for this differential effect on release and facilitation
by measuring the action potential-mediated Ca influx into excitor terminals at
proximal to distal locations along transmitter-releasing axon branches in the
presence and absence of inhibitor axon activity. From the pattern of changes
of Ca influx during inhibition, we conclude that, although action potential
amplitudes are attenuated, their propagation is maintained into small distal
branches during inhibition.
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Materials and methods |
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Force transduction and electrophysiology
For force transduction experiments, the leg was attached to the bottom of a
Plexiglas recording chamber using periphery wax and cyanoacrylate glue,
leaving the dactylopodite free to move vertically over a ledge
(Fig. 2A). Force measurements
were obtained using a force-displacement transducer (model FT03; Grass
Instruments). The dactylopodite was attached to the force transducer with #9-0
surgical silk (Deknatel). The force transducer was mounted to a manipulator
that was used to raise the transducer above the claw until the thread was taut
and the dactylopodite was held level with the propodite in a neutral position,
being neither open nor closed. The signal from the force transducer was
amplified (low level D.C. Amplifier, 7P122; Grass Instruments, West Warwick,
RI, USA), filtered between 35 Hz and 2 kHz and digitized at a rate of 2
kHz.
The excitatory and inhibitory postsynaptic junction potentials (EJPs and
IJPs, respectively) were recorded with sharp (10-15 M) electrodes
filled with 3 mol l-1 KCl. Muscle fibres selected for recording
were primarily fibres termed `intermediate' by Delaney et al.
(1991
). These are located
distal and lateral to the tight midline proximal bundle, near the major Y
branch of the motor axons, and have unfacilitated EJPs in the range of 0.2-1
mV. Postsynaptic potentials were recorded with a high-impedance head stage
amplifier (IR283; Neurodata, Branford, CT, USA), amplified a total of
1000-fold, filtered between 0.05 Hz and 1 kHz and digitally sampled at 4
kHz.
The time to onset of contraction in response to 20 Hz stimulation of the excitor was measured under conditions where the excitor was stimulated following a period of quiescence and compared with a paired sample t-test to that seen after co-activating the excitor and inhibitor axons. For co-activation, the inhibitor axon was stimulated in bursts of four action potentials at a frequency of 100 Hz, and the excitor axons were stimulated at 20 Hz with the final inhibitor stimulus in the burst preceding each excitor stimulus by 2-3 ms. The excitor and inhibitor axons were co-activated for 1.25 s (25 excitor stimuli at 20 Hz). The inhibitor to excitor stimulus delay was adjusted to produce maximal inhibition and was determined at the beginning of each experiment. Five to 10 trials were averaged for each condition with each animal.
Enhancement (facilitation and augmentation) was calculated by dividing the amplitude of the second EJP by each successive EJP.
Calcium imaging
To measure the effect of inhibition on Ca influx, a different stimulation
paradigm from that used for the muscle contraction experiments was employed to
avoid muscle movement; three excitor stimuli were delivered at 50 Hz and
compared with excitor stimuli delivered together with inhibitor activation
(Fig. 3). Studies of
presynaptic inhibition are typically not conducted at high stimulation rates
to avoid muscle movement (Govind et al.,
1995), but we wanted to study presynaptic inhibition at a more
physiologically relevant frequency for the muscle so we used a short train of
three stimuli. Ca influx was measured in terminal boutons located on
intermediate fibres in the vicinity of the main Y branch of the axon.
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The excitor axon was penetrated distal to the main Y branch with a sharp
electrode, containing 15 mmol l-1 membrane-impermeable Calcium
Green-1 (Molecular Probes, Eugene OR, USA), 300 mmol l-1 KCl and
1.0 mmol l-1 Hepes with a final resistance of approximately 40
M. The axon was filled with Calcium Green-1 by applying 5-10 nA of
negative, continuous current to a total of approximately 250 nA
min-1. Terminals on the surface of the muscle in the intermediate
fibre region were imaged using a custom-made epifluorescence microscope with a
60x, NA 0.9 water-immersion lens (Leica, Richmond Hill, ON, Canada). The
preparation was illuminated using a 75 W Xe arc lamp coupled to a switching
monocrometer (Polychrome II; T.I.L.L. Photonics, Pleasanton, CA, USA) to
select excitation wavelengths. A CCD camera (Imago PCO, Madison, WI, USA) and
image acquisition system (T.I.L.L. Photonics) were used to record images of
fluorescent transients controlled and synchronized to the electrophysiological
recordings. The image of the terminal bouton was binned on-chip prior to
readout by a factor of four in the vertical and horizontal dimensions. To
visualize the position of inhibitory terminal boutons relative to excitor
boutons at the end of the experiment, the inhibitor axon was filled with a
pipette containing 1 mmol l-1 Alexa 568 (Molecular Probes, Eugene,
OR, USA) dissolved in distilled water using approximately 300 nA
min-1 of 10 nA, negative continuous current. Green excitor and red
inhibitor terminals were simultaneously imaged using a dual FITC/Texas Red
dichroic mirror, a 510-550 nm bandpass and 600 nm long-pass emission filter
(51006; Chroma Optical, Rockingham, VT, USA) with sequential 488 and 564 nm
excitation. For most experiments, the change in fluorescence in the terminal
boutons was measured while stimulating the excitor axon alone three times at
50 Hz or with the excitor stimulation preceded by inhibitor stimuli at 100 Hz.
The stimulation protocol was as follows: four inhibitor stimuli, an excitor,
two inhibitor stimuli, an excitor and finally two more inhibitor stimuli and
an excitor (for a total of eight inhibitor stimuli and three excitor stimuli).
Each trial comprised 55 sequential 20-ms exposure time images (each image
required an additional 8-11 ms to readout for a total frame time of 28-31 ms,
depending on the size and position of the CCD readout region). Twenty to 40
trials were averaged, with alternating excitor stimulation and excitor plus
inhibitor stimulation. A 10-s delay was incorporated between each stimulation
trial to prevent a build-up of short-term synaptic plasticity. Several
terminals were imaged at a time, typically within a 60x60 µm field of
view. During the experiments, the inhibitor axon was monitored by penetrating
it with a sharp electrode and recording the resulting action potentials to
ensure faithful stimulation of the inhibitor axon.
Fluorescence transients were analyzed, using TILLVisionTM software, by
drawing a polygonal region of interest (ROI) around a varicosity from which an
average level of fluorescence intensity for the pixels enclosed by the ROI was
calculated for each frame. The same ROI was used for the excitor alone
condition (control) and the excitor plus inhibitor (inhibited) condition.
Changes in fluorescence (F/F) were calculated by
subtracting the average resting fluorescence (Fr) of the
terminal, calculated from the 10 frames prior to the stimulus, from the
fluorescence at time t (F) and correcting for background
fluorescence using the equation
[(F-Fr)/(Fr-Fb)]
where Fb is the background fluorescence including camera
offset, measured from a region of muscle adjacent to the terminals. The
fluorescence transient resulting from stimulating the excitor alone was then
compared with the excitor plus inhibitor transient to determine the reduction
in Ca entry into terminals caused by presynaptic inhibition. The fractional
change in fluorescence produced by three action potentials was
5%,
consistent with previous estimates for changes of about 10 nmol l-1
Ca per action potential (Delaney et al.,
1991
). Changes of this magnitude from resting Ca are small enough
that fluorescence changes should be linearly related to Ca concentration
within the accuracy of our measurements. To verify this, we confirmed for each
experiment that the fluorescence change resulting from six action potentials
at 50 Hz was virtually twice that produced by three action potentials,
allowing reductions in fluorescence transients during inhibition to be
directly equated to reductions in Ca influx without needing to convert
fluorescence into an estimate of Ca concentration.
As the majority of imaging and electrophysiological experiments were conducted separately, it was necessary to conduct several experiments where terminals were imaged and the resulting EJPs in corresponding muscle fibres were recorded concomitantly. Similarity between the two data sets indicated that presynaptic and postsynaptic data could be pooled from different experiments for comparison.
The amplitude of the fluorescence transient of the inhibited condition was compared with that of the control, non-inhibited, condition for each terminal bouton with a paired t-test. Postsynaptically, the amplitudes of the EJPs were also compared with a paired t-test. Experiments in which both electrophysiology and imaging were performed were compared with those with either imaging or electrophysiology alone using t-tests.
In order to test whether failure along the length of a branch or at branch points would occur with increased inhibition, the imaging and electrophysiological experiments described above were repeated with a longer period of stimulation of the inhibitor axon. Twenty inhibitor stimuli at 100 Hz were delivered before the first excitor stimulus for a total of 24 inhibitor stimuli. This was to ensure that the inhibitor was fully facilitated before excitation.
To test the effect of global activation of GABA receptors on Ca influx, the
preparation was exposed to bath application of 10-50 µmol l-1
GABA for at least 10 min before recording began. The excitor action potential
was recorded with an intracellular electrode in the main axon trunk distal to
the Y branch to ensure faithful following of the proximal axon to stimuli.
Action potential propagation, or lack thereof, into small distal branches and
terminals was inferred from Ca imaging. After examining the effect of GABA on
Ca influx in terminals, the preparation was rinsed with Ringer (modified Van
Harreveld's solution; Van Harreveld,
1936) and a final control recording was taken. Most experiments
measured the change in Ca when it had plateaued between 4 and 8 s of
continuous stimulation at 5 Hz. The change in fluorescence was
20-40% at
this time, which was sufficiently large enough to measure without averaging
trials and had the advantage that the muscle did not contract. Two experiments
were conducted with the standard protocol used to test presynaptic inhibition:
three excitor stimuli at 50 Hz.
Anatomical analysis of presynaptic inhibition
Images of the excitor axon (Calcium Green-1) were overlaid on images of the
inhibitor axon (Alexa 568), and the following measurements were made: distance
of excitor terminal boutons from the nearest branch, distance of excitor
boutons from the nearest inhibitor bouton and whether the bouton was on a
secondary, tertiary or quarternary branch
(Fig. 1). The amount of
inhibition at each terminal bouton was then compared with these anatomical
measures by correlation analysis and paired sample t-tests to observe
possible trends.
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Results |
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Intracellular recordings were obtained from individual muscle fibres in the
proximal region during stimulation sufficient to produce contraction, and an
example of data from one typical fibre is depicted in
Fig. 2C, with data from six
preparations summarized in Fig.
2D. In the control portion of the sample trace
(Fig 2C, top), an initial rapid
build-up of the EJP amplitude occurred for the first 10-12 stimuli followed by
a small slower increase in EJP amplitude. The rapid build-up of EJP amplitude
is characteristic of facilitation, while the slower increase may include a
component of augmentation. With inhibition, the EJP amplitude was greatly
reduced, but increased with each successive stimulus. The first EJP produced
50 ms after the offset of inhibitor activity showed a marked increase
(48%; paired t-test, P<0.001, N=6
preparations), achieving an amplitude greater than those of EJPs that were
coincident with the onset of contraction when no preceding period of
inhibition was applied. Although, the first EJP following the cessation of
inhibition did not immediately reach the same amplitude that it would have
attained in the absence of preceding inhibitor activity, it rapidly achieved
full amplitude with further stimulation.
Imaging and electrophysiology
We investigated the effect of presynaptic inhibition on Ca entry into
excitor terminal boutons with short trains of three stimuli at 50 Hz. For
technical reasons, the majority of imaging and electrophysiological
experiments were conducted separately. To allow comparison of pre- and
postsynaptic data between experiments, several experiments were conducted
where terminal boutons were imaged and the resulting EJPs in muscle fibres
were recorded at the same time. In this series of experiments with both
imaging and electrophysiology, the Ca transient was reduced by 18±2%
(mean ± S.E.M., N=60
terminals from four preparations). In imaging-only experiments, the Ca
transient was reduced by 21±2% (mean ±
S.E.M., N=62 terminals from eight
preparations). Thus, overall, co-activation of the inhibitor axon reduced Ca
entry into terminals by 20±1% (mean ±
S.E.M., range 0-50%, paired t-test,
P<0.001, N=122 terminals from 12 preparations;
Fig. 3A). One experiment with
typical fluorescence intensities and fractional fluorescence changes was
analyzed in detail to determine the accuracy of an estimate of the percent
inhibition for a particular terminal. Where the inhibition was greater than
20%, a significant difference between the control and the inhibited
fluorescence transients was observed with only five trials averaged (paired
t-test, P<0.05). With an increased number of trials
(20-30), we were able to observe a significant difference between the control
and the inhibited fluorescence transients of 6% (paired t-test,
P<0.05).
Data from one muscle cell (Fig. 3B) illustrates a reduction in the amplitude of the EJPs caused by the inhibitor. Normalized and pooled data for 27 proximal cells from 18 experiments (Fig. 3C), revealed inhibition for the first, second and third EJPs of 42±4%, 51±4% and 59±2% (mean ± S.E.M.), respectively. The amplitudes of the first, second and third inhibited EJPs were significantly smaller than the respective control conditions (paired t-test, P<0.001, N=27 cells). The slight increase in percent inhibition with each subsequent stimulus was significant (paired t-test, P<0.05) and raises the question of whether or not the inhibitor was fully facilitated for the first excitor stimulus, which is addressed below. As most experiments were done with just electrophysiology, results from experiments with both imaging and electrophysiology were compared and found not to be significantly different. In experiments where imaging and electrophysiology were combined, the EJP was inhibited 51±8%, 55±6% and 59±6% for the first, second and third stimuli, respectively (mean ± S.E.M., N=5 cells from four preparations) versus 40±4%, 50±5% and 60±3% (mean ± S.E.M., N=22 cells from 14 preparations) for experiments without imaging.
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Anatomical effects on presynaptic inhibition
Images of the excitor and inhibitor axons were overlaid in order to analyze
the effects of anatomy on inhibition. Variation in the amount of inhibition at
different terminal boutons was seen (Fig.
4A). However, evidence for a consistent trend of increasing
inhibition from proximal to distal along long branches of terminal boutons was
never observed (Fig. 4B). We
did find evidence for slightly more inhibition in terminal boutons on
quaternary branches (24±2%, N=36 terminals) versus
those on tertiary branches (18±1%, N=86 terminals, two-sample
t-test, P<0.05; Fig.
4C). The distance separating an excitor bouton from the nearest
inhibitor bouton was compared with the amount of inhibition observed, and no
correlation was detected (r2=0.001, N=95
terminals). Similarly, we found no correlation between the distance of the
excitor bouton to the nearest main branch and the amount of inhibition of the
Ca transient (r2=0.04, N=92 terminals). Failure
of propagation of the action potential did not seem to occur because we always
observed Ca entry into all terminal boutons, even at the end of long (>150
µm) branches. A limitation of this analysis was that the exact location of
axo-axonal synapses within large boutons or along thin axons was not known.
Others have observed with electronmicrographic studies that excitor and
inhibitor terminal boutons are sometimes closely associated, without any
axo-axonal synapses, while sometimes excitor terminals received numerous
axo-axonal synapses from several different converging inhibitor terminals
(Atwood et al., 1984).
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To test the possibility that the inhibitor was not fully facilitated when four stimuli were applied prior to the onset of excitor stimulation, as in the imaging and electrophysiological experiments, 20 inhibitor stimuli were delivered to more fully develop inhibitor facilitation. A slightly greater reduction of Ca influx was observed with an increased number of inhibitory stimuli (paired t-test, P<0.05, N=14 terminals from two preparations). For these experiments, inhibition stimulated in the standard manner ranged from 8 to 26% (13±2.3%, mean ± S.E.M.), while more inhibitor stimuli preceding excitor activation caused a 13-35% (22±1.8%, mean ± S.E.M.) reduction in Ca influx (Fig. 5A). Faithful propagation of an action potential to terminals was inferred from the fact that we always observed a Ca transient in the excitor terminals. Thus, although there was greater inhibition of Ca entry with increased inhibitor stimulation, neither increasing inhibition along the length of branches nor branch point failure were observed. Even with more sustained trains of inhibition, the action potential was still able to propagate to distal terminals.
Although 20 action potentials are sufficient for inhibitor facilitation to
substantially reach a plateau, there was still 10% greater inhibition of
the second and third EJPs compared with the first (paired t-test,
P<0.05, 10 cells from three preparations;
Fig. 5B). Inhibition mediated
by GABAB receptors has been reported
(Parnas et al., 1999
) but we
could find no inhibitory effect of baclofen (30-180 µmol l-1;
N=4) on excitatory transmission, and baclofen did not occlude
inhibition of the EJP by the inhibitor axon nor remove the reduced build-up of
facilitation during short trains. Therefore, our data suggest that
GABAA-mediated presynaptic inhibition has a small inhibitory effect
on facilitation of the excitor, which is not expected from its minor reduction
of Ca influx.
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Discussion |
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Presynaptic inhibition at the crayfish neuromuscular junction can cause
relaxation of the muscle in the face of ongoing excitor axon activity. In this
instance, relaxation is produced by the addition of inhibitor axon activity
rather than the reduction of excitor axon firing. Added inhibitor axon
activity during sustained excitor axon firing is not entirely wasteful since
processes that facilitate release are maintained and can be expressed
immediately after the inhibition is removed when the inhibitor is silenced.
Thus, although the muscle is relaxed when the inhibitor is co-activated with
the excitor, the time to onset of contraction is shortened if the excitor
continues to be activated after a period of excitor and inhibitor
co-activation, as predicted by Atwood and Wojtowicz
(1986). This phenomenon could
be behaviourally significant for motor control in the crayfish because it
increases the kinetics of contraction. In central synapses in other nervous
systems, such a mechanism could be used to regulate the latency, and thus the
timing, of information transfer at specific gating points and could underlie
some priming or attentional phenomena.
With co-activation of the inhibitor axon, we measured that a moderate (20%
average) reduction in Ca entry into presynaptic excitor axon terminal boutons
is associated with a 51% reduction in the amplitude of the postsynaptic EJP.
This is consistent with a relationship between Ca entry and transmitter
release proportional to approximately the third power. Presynaptic inhibition
most likely reduces Ca entry into excitor motor terminals by decreasing the
amplitude of the action potential by 10-20 mV
(Baxter and Bittner, 1981),
thus activating fewer voltage-gated Ca channels. A non-linear relationship
between a change in presynaptic Ca entry and neurotransmitter release, with a
reduced number of open Ca channels, indicates a requirement for overlapping Ca
microdomains to elicit neurotransmitter release
(Zucker, 1999
). Thus, this
synapse only requires a small reduction in Ca entry to have a large effect on
neurotransmitter release.
Although short-term or `paired-pulse' facilitation at the crayfish
neuromuscular junction and other synapses requires Ca influx
(Katz and Miledi, 1968), it is
not as sensitive to reduced Ca as release itself
(Magleby, 1987
;
Zucker and Regehr, 2002
). We
observed a 50% reduction in release. If this were due to loss of Ca influx
(and thus transmitter release) in half of the terminals with little or no
reduction in the other half then we would expect to see a 50% reduction in
facilitation. We observed an average 20% reduction of Ca influx in all
terminals, which, based on the other studies cited, is consistent with our
observation that facilitation is minimally affected. Thus, the model, where
most or all terminals continue to experience near normal Ca influx during
inhibition, is supported by our measurements that reveal a moderate reduction
of Ca influx into most terminals with no evidence for complete failure of
influx into any terminals during inhibition.
The amount of inhibition at a given terminal could potentially depend on
the morphology of the excitor and inhibitor axons and the physical
relationship between their terminals
(Atwood and Wojtowicz, 1986).
We measured inhibition in a large number of terminals (122) over many
locations (18) to test for morphological trends. Although the amount of
inhibition at individual terminal boutons varied, trends of increasing
inhibition along a branch were not observed. Slightly greater inhibition was
observed at terminal boutons on quaternary branches, as would be predicted by
anatomical models (Atwood et al.,
1984
), but complete blockade of the action potential was never
observed. Furthermore, although increasing the number of inhibitor axon action
potentials that preceded the excitor action potentials (from 4 to 20) did
result in slightly greater inhibition, the action potential still appeared to
propagate actively, as evidenced by a relatively consistent reduction of Ca
influx along branches. Therefore, we propose that the action potential
propagates actively in the excitor motor axon and that presynaptic inhibition
reduces the amplitude of the action potential at specific terminals. The
action potential would then recover between the electrically shunted regions,
resulting from inhibitory axo-axonal synapse activity. This hypothesis is
supported by anatomical data where inhibitory axo-axonal synapses are
typically found on the excitor axon a few microns proximal to sites of
excitatory transmitter release (Atwood and
Morin, 1970
; Jahromi and
Atwood, 1974
).
The build-up of facilitation is essential to elicit contraction of the opener muscle in the crayfish neuromuscular junction. Because it was observed that the majority of facilitation was maintained during inhibition, it follows that an actively propagated action potential must reach the majority of the terminals. If presynaptic inhibition worked by blocking the action potential at branch points or constrictions, thus electrically isolating entire branches of the motor axon, facilitation would be blocked in groups of terminals.
Although the predominant effect of presynaptic inhibition is to reduce
release while preserving facilitation, we saw some evidence that facilitation
was slightly inhibited. During sustained 20 Hz trains, the first one or two
EJPs stimulated after cessation of inhibition were not as large as they would
have been had the inhibition been absent
(Fig. 2D). In addition, we
observed that with three high-frequency (50 Hz) stimuli, the build-up of
paired-pulse facilitation was slightly reduced
(Fig. 3D). This contrasts to
the results of Baxter and Bittner
(1981), who reported no
decrease in facilitation during inhibition. The stimulation paradigm used in
our experiments differed from their study in that they stimulated with a short
train (11 excitor stimuli at 100 Hz) while we used a longer, lower-frequency
train (25 excitor stimuli at 20 Hz). An explanation for the difference between
the results of our study and their work may be that during inhibition we had
some loss of augmentation, which may have normally contributed during the
longer stimulus trains we used. Some loss of augmentation during presynaptic
inhibition is expected because it is dependent on the build-up of free Ca
concentration in these terminals (Delaney
and Tank, 1994
), which is reduced. However, a reduction of
augmentation would not apply for our three-stimulus/50 Hz paradigm, where we
clearly saw a small reduction of facilitation. Another explanation could be
activation of a GABAB receptor. GABAB receptors have
been indicated to inhibit transmitter release from crayfish excitor motor
terminals (Fischer and Parnas,
1996a
,b
;
Parnas et al., 1999
). The
apparent persistence of inhibition for a few hundred milliseconds after the
offset of inhibitor axon firing (Fig.
2D) would be consistent with the decay of a
GABAB-mediated contribution to inhibition. However, we were unable
to observe any inhibitory effect of high concentrations of baclofen on
excitatory transmission, nor did we observe occlusion of the inhibitor
axon-mediated inhibition by baclofen. Perhaps changes in the distribution of
Ca in the active zone associated with the reduction in the number or duration
of Ca channel openings caused by the reduced action potential amplitude have a
small inhibitory effect on facilitation.
Bath application of GABA produced increasing amounts of inhibition of Ca
influx along a branch in several preparations. In some instances, with 10-30
µmol l-1 GABA, the inhibition between proximal and distal
terminals ranged from 0 to >60%. Increasing GABA to 40 or 50 µmol
l-1 blocked action potentials entirely. A similar result was seen
in preliminary work by Tank and Delaney
(1988). However, inhibition
induced by stimulation of the inhibitor axon did not produce this result. This
indicates that bath application of GABA affects the excitor motor axon in a
different way than presynaptic inhibition, perhaps from activation of
GABAB receptors or by creating a spatially extended inhibition
through activation of extrasynaptic GABAA receptors that cannot be
reproduced by electrical stimulation of the inhibitor axon. The fact that a
qualitatively differential inhibitory response profile was observed with bath
application of GABA versus standard presynaptic inhibition
demonstrates the spatial and temporal specificity of presynaptic inhibition at
the crayfish neuromuscular junction.
The crayfish excitor-inhibitor motor pair is an example of a system where a
single inhibitory axon delivers inhibition to virtually all of the output
synapses of its target. Local regulation of synaptic output has been
demonstrated to be possible in transmitter-releasing dendrites of olfactory
mitral cells that receive inhibitory GABA synaptic input from 20
interneurons at sites distributed along their length. Localized inhibition of
action potential-mediated Ca influx (and thus localized reduction of
transmitter release) in dendrites of mitral cells has been demonstrated with
localized release of caged GABA and by local stimulation of presynaptic
interneurons (Chen et al.,
2000
; Lowe, 2002
).
Thus, localized shunting inhibition can be used to selectively reduce output
from some but not all synapses by reducing Ca influx without causing complete
failure of action potentials. Presynaptic inhibition of transmitter release
has also been shown to contribute to gating transmission by a command neuron
to the appropriate phase of activity of a rhythmic pattern generator
(Hurwitz et al., 2004
). Thus,
the spatial and temporal specificity of action that can be achieved with fast
presynaptic inhibition at crayfish neuromuscular junction represents a
strategy utilized by other synapses to achieve task-specific control of
transmission in neuronal networks.
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Acknowledgments |
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References |
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