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The Journal of Neuroscience, July 15, 1999, 19(14):6079-6089
Shunting versus Inactivation: Analysis of Presynaptic Inhibitory
Mechanisms in Primary Afferents of the Crayfish
Daniel
Cattaert1 and
Abdeljabbar
El Manira2
1 Laboratoire Neurobiologie et Mouvements, Centre
National de la Recherche Scientifique, 13402 Marseille Cedex 20, France, and 2 The Nobel Institute for Neurophysiology,
Department of Neuroscience, Karolinska Institutet, S-171 77 Stockholm,
Sweden
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ABSTRACT |
Primary afferent depolarizations (PADs) are associated with
presynaptic inhibition in both vertebrates and invertebrates. In the
present study, we have used both anatomical and electrophysiological techniques to analyze the relative importance of shunting mechanisms versus sodium channel inactivation in mediating the decrease of action
potential amplitude, and thereby presynaptic inhibition. Experiments
were performed in sensory afferents of a stretch receptor in an
in vitro preparation of the crayfish. Lucifer yellow
intracellular labeling of sensory axons combined with GABA
immunohistochemistry revealed close appositions between
GABA-immunoreactive (ir) fibers and sensory axons. Most contacts were
located on the main axon at the entry zone of the ganglion, close to
the first branching point within the ganglion. By comparison, the
output synapses of sensory afferents to target neurons were located on
distal branches. The location of synaptic inputs mediating spontaneous PADs was also determined electrophysiologically by making dual intracellular recordings from single sensory axons. Inputs generating PADs appear to occur around the first axonal branching point, in
agreement with the anatomical data. In this region, small PADs (3-15
mV) produced a marked reduction of action potential amplitude, whereas
depolarization of the membrane potential by current injection up to 15 mV had no effect. These results suggest that the decrease of the
amplitude of action potentials by single PADs results from a shunting
mechanism but does not seem to involve inactivation of sodium channels.
Our results also suggest that GABAergic presynaptic inhibition may act
as a global control mechanism to block transmission through certain
reflex pathways.
Key words:
presynaptic inhibition; primary afferent depolarization; crayfish; synaptic transmission; chloride conductance; sodium channels
inactivation
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INTRODUCTION |
Presynaptic inhibition of sensory
transmission is associated with primary afferent depolarizations (PADs)
(Clarac et al., 1992 ; Nusbaum et al., 1997; Rudomin et al., 1998).
During locomotion in vertebrates, sensory afferents terminals receive
phasic PADs that can trigger antidromic action potentials (Gossard et
al., 1989, 1990). These PADs are likely to involve the activation of GABAA receptors that increase the conductance to chloride,
which has an equilibrium potential around  30 mV (Allen and Burnstock, 1990). In the crayfish locomotor system, PADs decrease the amplitude of
the afferent action potentials, thereby depressing monosynaptic EPSPs
in target neurons (Cattaert et al., 1992). A similar mechanism occurs
in sensory afferents in the locust (Burrows and Matheson, 1994). In
crayfish, PADs elicit antidromic action potentials that do not elicit
any postsynaptic effects (El Manira et al., 1991; Cattaert et al.,
1992).
The study of presynaptic inhibitory mechanisms in the primary afferents
of mammals is difficult to achieve directly using intracellular
recordings because of the very small diameter of the afferent axons
(0.5-1.0 µm) and boutons (3-6 µm) where PADs are likely to be
produced. Most of the data are derived from anatomical observations
(Fyffe and Light, 1984; Maxwell et al., 1990), measurements of
excitability changes by using extracellular electrodes (Rudomin et al.,
1998; Lomeli et al., 1998), and intra-axonal recordings of PADs
at a long distance from the axoaxonic synapse (Jimenez et al., 1988).
Two mechanisms, based on simulation studies, have been proposed to
account for presynaptic inhibition associated with PADs: a shunting
mechanism (Segev, 1990) and inactivation of sodium channels (Graham and
Redman, 1994). In mammals, however, experimental evidence for either of
these mechanisms is still lacking.
The crayfish locomotor system provides a convenient experimental model
to analyze the relative importance of these two mechanisms in
presynaptic inhibition as a result of PADs. The three phenomena associated with PADs and with presynaptic inhibition in vertebrate (depolarization, increase of chloride conductance, and antidromic spikes) are also present in the primary afferents of a chordotonal organ in the crayfish (Cattaert et al., 1992). In these afferents, spontaneous PADs occur either randomly in quiescent preparations or at
a fixed phase of the locomotor cycle in preparations displaying locomotor activity (El Manira et al., 1991; Cattaert et al., 1992). The
possibility of recording intracellularly from the regions where the
GABA synapses that produce PADs are located offers a significant
advantage in the analysis of the mechanisms of presynaptic inhibition
in this invertebrate system. In this paper, using electrophysiological and immunohistochemical techniques, we have localized the region of the
sensory axon where these spontaneous PADs are produced and have
determined the effect of PADs on spike conduction. Our findings suggest
that PADs mediate their inhibitory effects mainly through shunting
mechanisms, likely occurring on the main axon of the sensory neuron
arborization, at some distance from output synapses. In such an
organization, GABAergic PADs would therefore inhibit synaptic
transmission in all output synapses and do not seem to mediate a
selective control at different branches of single sensory axons.
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MATERIALS AND METHODS |
Experimental animals. Experiments were performed on
male and female crayfish (Procambarus clarkii;
n = 14) weighing 25-30 gm. The animals were purchased
from a commercial supplier (Chateau Garreau, Landes, France) and kept
in circulating fresh water at 18-20°C.
In vitro preparation. An in vitro
preparation of the thoracic locomotor nervous system was used as
described previously (Sillar and Skorupski, 1986; Chrachri and Clarac,
1989). This preparation consists of the last three thoracic ganglia
(3-5) along with the motor nerves from the fifth ganglion to the
promotor, remotor, levator, and depressor muscles (Fig.
1A). The coxo-basal
chordotonal organ (CBCO) stretch receptor was also dissected and kept
intact. The preparation was pinned with the dorsal side up in a
silicone elastomer (Sylgard)-lined Petri dish. The fourth and fifth
ganglia were desheathed to improve the superfusion of the central
neurons and allow intracellular recordings from CBCO axon terminals
(CBTs) (Fig. 1B). The nervous system was continuously
superfused with oxygenated control saline containing (in
mM): 195 NaCl, 5.5 KCl, 13.5 CaCl2, 2.5 MgCl2, and 10 Tris, at pH 7.6.
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Figure 1.
Experimental setup. A, The
in vitro preparation of the crayfish thoracic locomotor
system consisting of ganglia 3-5 (G3, G4, G5) dissected
together with motor nerves of the proximal muscles and the coxo-basal
chordotonal organ (CBCO). B, Enlargement
of the region indicated by the dashed-line box showing a
sensory axon terminal and postsynaptic motoneurons in the ganglion.
ME1, Proximal electrode; ME2, distal
electrode. The terms proximal and distal will always refer to this
relative disposition. Distal processes of a sensory terminal are closer
to the motoneurons (MN). C,
Staining of all CBTs in the left 5th ganglion of crayfish. The
localization of the CBTs within the ganglion is represented in
C1. Most of the CBTs have the same anatomy, and all of
them are more or less superimposed (2). A single
CBT on which GABA-ir boutons were found was reconstructed from slices
(3). The box represents the zone
in which close appositions between sensory axons and GABA-ir fibers
were found (Fig. 2A).
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Electrodes and recordings. Sensory nerve activity was
recorded with platinum "en-passant" electrodes connected
to homemade amplifiers (gain ×10,000-100,000). Intracellular
recordings from CBTs within the ganglion (Fig. 1B)
were performed with micropipettes filled with either K-acetate (2 M, 30-40 M ) or Lucifer yellow (5% in 3 M
LiCl, 60-100 M). An Axoclamp 2A amplifier (Axon Instruments, Foster
City, CA) was used in current-clamp mode, with K-acetate-filled electrodes, and the level of saline was as low as possible to reduce the micropipette capacitance. CBTs were identified on the basis
of two criteria. First, injection of depolarizing current pulses
elicited spikes in CBTs that were correlated in a one-to-one manner
with extracellular spikes recorded in the CBCO sensory nerve. Second,
orthodromic spikes produced by the CBCO sensory neurons were correlated
with intracellular spikes at fixed time in CBTs. The CBTs analyzed in
this study fired action potentials attributable to the existence
of spontaneous activity in CBCO sensory neurons. An eight-channel
stimulator (A.M.P.I., Jerusalem, Israel) was used to trigger
intracellular pulses in CBTs during the identification procedure. Data
were displayed and printed on a four-channel digital oscilloscope
(Yokogawa, Tokyo, Japan) and stored on tape (Biological DTR 1800, Claix, France). Results are based on recordings from 16 identified CBTs
(11 dual and 5 single intracellular recordings).
Immunohistochemistry. Populations of sensory axons were
labeled with Lucifer yellow (Fig. 1C) using the following
procedure. The CBCO sensory nerve was cut and placed in a concentrated
solution of Lucifer yellow (potassium salt) for 12-18 hr at 5°C. The
preparation was then fixed for 3 hr in a solution containing 2.5%
glutaraldehyde, 1% paraformaldehyde, and 0.2% picric acid in a 0.1 M phosphate buffer at pH 7.4, and for 3 hr in an acetic
"Bouin" solution containing 250 ml saturated picric acid, 750 ml
37% paraformaldehyde, and 50 ml glacial acetic acid. The preparation
was then rinsed for 15 min in a 0.9% NaCl solution containing 0.05 M Tris at pH 7.6. Slices (30 µm thick) were made,
dehydrated in a series of alcohols, and stored in 95% alcohol.
GABA immunostaining was performed after rehydration of the slices using
rabbit monoclonal GABA antibody (Sigma, St. Louis, MO) revealed with a
rhodamine tetramethyl-rhodamine-isothiocyanate fluorochrome
anti-rabbit antibody conjugate (Sigma). Sections were examined using a
confocal microscope.
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RESULTS |
Immunohistochemical localization of GABAergic inputs to
sensory axons
We have shown previously that spontaneous PADs occurring in CBTs
are blocked by the GABA receptor antagonist picrotoxin and that local
application of GABA on the CBTs induces a depolarization of membrane
potential associated with a decrease in input resistance that is also
blocked by picrotoxin (El Manira and Clarac, 1991; Cattaert et al.,
1992). PADs and GABA-mediated depolarizations have a similar reversal
potential (approximately 35 mV) (Cattaert et al., 1992), and their
amplitude increases when the chloride reversal potential is shifted
toward more depolarized values (El Manira and Clarac, 1991; Cattaert et
al., 1994). To determine the location of GABAergic contacts, labeling
of CBTs with Lucifer yellow (Fig. 2,
green) combined with GABA-immunohistochemistry (Fig. 2,
red) was performed. The preparations (n = 3)
were scanned using a confocal microscope, and the presence of close
appositions between GABA-ir and Lucifer yellow-filled axons was
analyzed. GABA-ir processes appeared to surround each CBT (Fig.
2A; asterisk in B), close to
the first branching point of the sensory axon in the ganglion (Fig.
1C, box). Sites of close apposition appear as
yellow spots in the confocal microscopy image. These spots result from
a merge of the green and red color within a very small volume in the
same focal plane. This indicates that these close appositions between
the sensory terminal (in green) and the GABA-ir fibers (in red)
presumably correspond to synaptic contacts, although an ultrastructural
analysis is required to confirm the presence of synaptic inputs. These
appositions were located primarily on the main axon and consisted of
five to seven contact points (Fig. 2C, yellow
spots, arrows), which are likely to represent the
superposition of CBTs and presynaptic terminals of GABAergic fibers.
However, some CBTs (20%) did not have any close appositions with
GABA-ir fibers, suggesting that not all CBT sensory axons receive
GABAergic inputs. No close appositions were found on the distal thin
processes of CBTs [i.e., sites closer to the motoneurons (MNs)].
These results thus indicate that GABAergic inputs occur close to the
first branching point of CBTs in the ganglion.
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Figure 2.
Localization of GABA-immunoreactive
boutons on Lucifer yellow-filled CBCO sensory axons, using confocal
microscope analysis of a 30-µm-thick slice. Only projection views are
given. A, Global view of the slice showing CBCO sensory
axons backfilled with Lucifer yellow (green) and
GABA-immunoreactive structures (red). Scale bar, 5 µm.
B, Details of GABA-immunoreactive boutons particularly
abundant in the vicinity of a CBCO axon (asterisk)
stained by Lucifer yellow. Note, however, that GABA-immunoreactive
boutons are aligned and follow the CBCO axon. Scale bar, 2 µm.
C, Detailed view of five GABA-immunoreactive boutons
(arrows) that are in close apposition with the best
stained CBCO axon. Scale bar, 2 µm.
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Zones of possible synaptic contacts between CBCO sensory terminals and
target motoneurons were analyzed by performing three-dimensional (3D)
reconstruction of pairs of sensory afferents and monosynaptically connected MNs. After physiological characterization of the existence of
monosynaptic inputs, the pairs of sensory and motor neurons were
intracellularly injected with Lucifer yellow (Fig.
3) (n = 3). The
preparations were fixed, dehydrated, cleared, and subsequently scanned
using a confocal microscope. Figure 3A shows a CBT (asterisk) in the
ganglion, after the first branching point (not shown in the
photograph), and branches from the neurite (thick processes) of a MN.
The CBT makes close contacts (i.e., the two processes are in the same
focal plane) on both the main (small arrows and large box) and the
small branches (open arrow) of the MN. These contacts may correspond to
zones of synaptic interaction (Fig. 3B,C). These results
suggest that output synapses are located more distally in relation to
GABAergic synapses that mediate PADs.
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Figure 3.
Localization of zones of synaptic contacts between
sensory axons and target motoneurons. A, A
monosynaptically connected CBT and an MN were recorded intracellularly
and injected with Lucifer yellow. The two neurons were reconstructed in
3D using a laser-scanning confocal microscope. Zone of close
appositions were found after the branching point, both on the main
neurite (small arrows) and on small branches of the MN
(open arrow). Scale bar, 20 µm. B, High
magnification of a possible zone of close apposition between a sensory
process and the main neurite of the MN. Scale bar, 4 µm.
C, High magnification of a zone of close apposition
between a branch of the sensory axon and a neurite of the MN. Scale
bar, 10 µm.
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Localization of the transition between active and passive spike
propagation in a CBT
We have demonstrated previously that action potentials are
passively conducted in the distal processes of CBCO sensory axons (Cattaert et al., 1992). To determine the site at which action potentials start to be passively propagated, and whether PADs are
produced in an area of active or passive conduction, we performed experiments (n = 13) with dual intracellular recordings
from single CBTs. The distance between the two intracellular recording
electrodes was measured through the microscope during the experiment.
The location of the recording site was revealed by short injection of
Lucifer yellow into the CBT from the most proximal electrode.
The CBTs have a simple morphology that allows a direct comparison of
the data obtained in different experiments. The CBTs enter the
ganglion, turn rostrally, and give rise to some small lateral branches
(Fig. 4A). Overshooting
action potentials were recorded intracellularly at axonal sites up to
100 µm before the first branching point (amplitude = 76 ± 3 mV at a resting potential of 71 ± 4 mV; n = 5). The half-spike width of the action potentials was 1.20 ± 0.05 msec. In this area of the CBT (80-100 µm after the first branching
point), the spikes appeared to be actively conveyed because no change
in their shape (time-to-peak and rising-phase slope, and repolarization
phase time) was observed over this distance (Fig.
4B). A slight reduction of the spike amplitude (~3
mV) and a small conduction delay (150 µsec), however, were generally
observed (n = 3 of 5) (Fig. 4B).
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Figure 4.
Active propagation of spikes in CBCO sensory axons
in the region of the first branching point. A, Schema of
the experimental procedure using dual intracellular recordings (ME1,
100 µm before the branching point; ME2, 100 µm after the branching
point) from a single sensory axon in the region of the first branching
point (arrow). B, Superimposed
intracellular recordings from ME1 and ME2 (CBT, bottom
trace) and extracellular recordings from the CBCO sensory nerve
(CBn, top trace).
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When action potentials were recorded 150 µm more distally than the
first branching point, their amplitude was smaller, their rising phase
was slower, and their half-spike width was wider (1.43 ± 0.05 msec, n = 8) than that of the same action potential recorded in the region of the first branching point (compare thick and
thin traces in Fig. 5B,C). All
of these changes were statistically significant
(p < 0.01; t test). Note that ME1
location in Figure 5 corresponds roughly to ME2 location in Figure 4.
The half-spike widths were similar in both recordings (1.23 msec in
Fig. 4, ME2, and 1.22 msec in Fig. 5, ME1),
although their amplitude was slightly different (70 mV in Fig. 4,
ME2, and 63 mV in Fig. 5, ME1).
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Figure 5.
Comparison of the PAD and spike propagation in
passive sites of CBCO axons. A, Diagram of the
experimental procedure used. The more proximal microelectrode
(ME1) was recorded from a site located just after the
first branching point. The more distal microelectrode
(ME2) was recorded 150 µm more distally in the main
branch. The drawing was reconstructed after Lucifer yellow staining.
B, Simultaneous intracellular recordings from ME1 and
ME2. C, Enlarged superimposed ME1 (gray
trace) and ME2 (black trace) traces during a
spike. The recording of the more distal microelectrode displays a
smaller spike with an increased time to peak, therefore indicating that
the spike was passively propagated between recordings sites of ME1 and
ME2. To make these differences more obvious, the mid-amplitude of both
recordings have been aligned (dashed line), and the
spike recorded with ME1 has been moved slightly to the right to make
both spikes cross the mid-amplitude line at the same point.
D, Same arrangement as in C but of a PAD
instead of a spike. The amplitude of the distally recorded PAD
(ME2, black trace) is slightly smaller than the more
proximal recording of the same PAD (ME1, gray
trace).
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Electrophysiological localization of GABAergic synaptic input
mediating PADs
GABAergic PADs occur either randomly in quiescent preparations or
at a fixed phase of the locomotor cycle in preparations displaying
locomotor activity (El Manira et al., 1991; Cattaert et al., 1992). The
electrophysiological localization of GABAergic synapses was determined
by comparing the amplitudes of spontaneous PADs and their effect on
spike amplitude at two intracellular recording sites along the CBT. In
the experiment reported in Figure 5, the proximal electrode (ME1) was
placed 50 µm after the first branching point (active propagation
zone), and the second electrode (ME2) was placed 150 µm more distal
(passive zone). The amplitude of PADs was slightly larger at ME1 than
ME2 (Fig. 5B-D). The reduction of spike amplitude was more
pronounced, however, than that of the PADs (Fig. 5C,D).
Figure 6 shows the time course of the
change of the amplitude of action potentials relative to PADs. The
decrease of the amplitude of afferent action potentials displayed a
mirror image of PADs, with the maximum reduction occurring at the peak amplitude of PADs (Fig. 6A,B).
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Figure 6.
Time course of the decrease of the amplitude of
action potential in relation to PADs. A, The amplitude
of afferent action potentials decreases during the occurrence of PADs.
B, The maximum decrease in the amplitude of action
potentials coincides with the peak of PADs.
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The analysis of PAD and spike amplitude is shown in Figure
7. In this experiment, the spike
amplitudes ranged from 50 to 63 mV in ME1 and from 40 to 56 mV in ME2.
The variation of the spike amplitude was attributable to a continuous
discharge of PADs that shunted the spikes (Fig. 5B). The
amplitude of the spikes recorded more distally in the branch was
linearly related to the amplitude of spikes recorded proximally (Fig.
7A), except for very small spikes (i.e., those that occurred
during the largest PADs). The distribution histogram of spike amplitude
in both intracellular recording sites had similar shapes, but the
distal spike histogram was shifted by 10 mV toward smaller amplitudes
(Fig. 7B). The PADs also displayed a large variation in
their amplitude (0.8 to 4 mV in ME1 and 0.5 to 3.5 mV in ME2). The
amplitude of the PADs recorded with ME1 was linearly related to the
amplitude of the PADs recorded with ME2 (Fig. 7C),
indicating that both electrodes recorded the same events. Most of the
points in the diagram are below the (y = x) curve plot (Fig. 7C), showing that the PADs recorded with ME2 had a smaller amplitude than those recorded with ME1
(Fig. 7C,D). To compare the propagation of spikes with that
of PADs, we calculated the ratio of the amplitude for PADs and spikes
recorded by ME2 with that recorded by ME1. This ratio was constant
(86 ± 0.049% SEM, n = 1000) (Fig. 7E)
for spikes, whereas that of PADs was larger and more variable (92 ± 0.84% SEM, n = 250) (Fig. 7E),
indicating that PADs show less attenuation than spikes (see below).
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Figure 7.
Comparison of PAD and spike propagation in passive
sites of CBCO axons: statistical analysis. The results are analyzed
from the experiment shown in Figure 5. A,
B, The spike amplitude at the distal recording site is
always smaller than the proximal one. A, Graph plotting
the amplitude of distal against proximal spike. The line
is the linear regression curve (r = 0.93).
B, Histograms of spike amplitudes recorded proximally
(from ME1 in Fig. 5; open bars) and distally (from ME2
in Fig. 5; filled bars). C,
D, The same analysis as in A and
B but for the amplitude of PADs. C, Graph
plotting distal against proximal PAD amplitude. The relationship is
linear (dashed line represents the regression line;
r = 0.84). The continuous line
represents the equation y = x. The
fact that most of the points are below this line indicates that PADs
display a smaller amplitude at the distal than at the proximal
recording site. D, The distal and proximal PAD amplitude
histograms largely overlap, the distal one being slightly displaced to
the left. E, Graph of the amplitude ratio of PADs and
spikes recorded at two sites in the same sensory axon.
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To determine the location of synaptic inputs mediating PADs in CB
sensory axons, the reduction of the action potential amplitude as they
propagated from ME1 to ME2 was compared in the absence and presence of
PADs (n = 8) (Fig. 8).
The two electrodes were positioned after the branching point (same
experiments as Fig. 7). In both the absence and presence of PADs, the
amplitude of action potentials was reduced to 86% of the initial value
because of the passive propagation between ME1 and ME2 recording sites (Fig. 8A,B). This indicates that no GABAergic
synaptic input occurs at regions between ME1 and ME2 (50-100 µm more
distal to the branching point), because action potentials were not
shunted as they propagated from the ME1 to ME2 recording site (Fig.
8C1,2). Subsequently, the synaptic inputs mediating PADs
were likely to occur at regions before the ME1 recording site (Fig.
8C1, around the branching point), and the shunted action
potentials would then propagate passively toward the distal processes
without being shunted farther. These results, together with
those of the GABA immunohistochemistry, show that the synaptic inputs
mediating PADs in CBTs are located close to the branching point of the
sensory terminals.
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Figure 8.
PADs reduce spike peaks at sites more proximal
than ME1. A, Simultaneous intracellular recordings from
ME1 and ME2 (same electrode positions as in Fig. 5). The first spike
occurs in the absence of a PAD (1), whereas the
second spike occurs in the presence of a PAD (2)
and shows a reduced peak. B, Superimposed traces of
proximal (ME1) and more distal (ME2)
recordings from spike 1 (in the absence of PADs) and spike 2 (in the
presence of PADs). In both the absence and presence of PADs, the
amplitude of action potentials was reduced to 86% of the initial value
because of the passive propagation between ME1 and ME2 recording sites,
indicating that PADs did not produce any shunting of action potentials
in the sensory axon between ME1 and ME2 recording sites.
C, Schematic diagram representing the effect of the
location of PADs on the amplitude of action potentials. If PADs occurs
close to the first branching point (C1), the amplitude
of action potential does not show any further decrease as they
propagate from ME1 to ME2. If PADs occur more distal than the branching
point, the amplitude of action potentials is decreased further
(C2).
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Effect of PADs on sodium channel inactivation
We have shown previously that the GABAergic PADs are caused by an
increase in chloride conductance (El Manira and Clarac, 1991; Cattaert
et al., 1994), which reduces the peak of action potentials, thereby
depressing the amplitude of monosynaptic EPSPs in target neurons
(Cattaert et al., 1992). PADs have a reversal potential of between 35
mV and 30 mV (Cattaert et al., 1992). During bursts of PADs, it is
likely that some Na+ channels could be inactivated,
thus reducing the peak of the action potentials. To determine the
relative importance of Na+ channels in activation
versus shunting mechanisms in causing the decrease of the peak of
action potentials during occurrence of PADs, we performed experiments
in which intra-axonal recordings were made from sensory afferents
either before they reach the ganglion or in a region distal to the
first branching point, and we compared the effects of PADs on the
membrane potential and action potential properties (Fig.
9). In experiments (n = 4) in which recordings were made from sensory axons before they reach the ganglion, PADs induced depolarization of membrane potential but did
not cause any shunting of action potentials. In intracellular experiments in which recordings were made in a region distal to the
first branching point (where PADs are produced), PADs produced a
depolarization of the membrane potential and shunting of spikes. The
results of these experiments are shown in Figure 9. PADs recorded at an
axonal site before the sensory nerve reaches the ganglion (Fig.
9A, inset) had no effect on the spike peak (Fig.
9A,B). By contrast, when the recording electrode was
situated after (>100 µm) the first branching point (Fig.
8C, inset), the recorded PADs were able to
linearly reduce the spike peak (Fig. 9C,D). The failure of
membrane potential depolarization to affect the spike peak suggests
that small amplitude (<10 mV) PADs do not cause any inactivation of
Na+ channels. However, larger PADs could involve
Na+ channel inactivation. This question was examined
in the following experiments (Fig.
10).
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Figure 9.
Effect of PADs on spike peaks at two locations of
a CBCO sensory axon. A, Superimposed recordings from a
site 100 µm before the first branching point. Positioning of the
microelectrode and anatomy of the sensory terminal (reconstructed after
Lucifer yellow staining) are shown in the inset. The
thin and thick traces were obtained in
the absence and presence of a PAD, respectively. Note that peak values
of both recordings are identical. Vm = resting membrane potential. B, Diagram of spike peak
value against PAD amplitude. At this recording site, PADs have a very
small but positive effect on the spike peak: the larger the PAD, the
larger the spike. C, Superimposed recordings from a site
100 µm after the first branching point. Positioning of the
microelectrode and anatomy of the sensory terminal (reconstructed after
Lucifer yellow staining) are shown in the inset. The
thin and thick traces were again obtained
in the absence and presence of a PAD, respectively. Note that the peak
value in the presence of the PAD is reduced. D, Diagram
of spike peak value against PAD amplitude. At this more distal
recording site, PADs reduce spike peak linearly with respect to their
amplitude. Recordings in A and C are from
different experiments.
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Figure 10.
Shunting and inactivating mechanisms of PADs.
A, Intracellular recording from a CBCO sensory axon. Two
microelectrodes were placed in the same CBCO sensory axon. One was used
to inject current steps (recording not shown), and the other was used
to measure the voltage response. B, During the same
experiment as in A, spontaneously occurring PADs reduced
spike amplitudes. C, Comparison of the effects of
spontaneous PADs and current injection on spike amplitude. The peak
value is plotted against the membrane potential at the base of the
spike. Although 1- to 17-mV-amplitude PADs (responsible for membrane
potential at the base of spikes being in the range 77 to 60 mV)
induce a noticeable reduction of spike peak, the injection of
depolarizing current does not induce any visible reduction of the
peak until the membrane potential is increased to 55 mV and above
(open symbols). D, Relationship between
input resistance (Rin) and the imposed membrane
potential during the experiment.
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Dual intracellular recordings were performed from single CBTs
(n = 5) in the region of the first branching point
where synaptic inputs mediating PADs are located, and changes in spike
peak induced by depolarizing current were compared with those induced
by PADs. In the experiment presented in Figure 10, depolarization of
the membrane potential of CBTs up to 15 mV above the resting potential did not produce any decrease of the peak of the action potentials. Depolarization of CBTs does not produce any outward rectification. A
decrease of the peak of action potentials started to be seen when the
CBTs were depolarized by 22 mV (Fig. 9A). This effect could
be caused by inactivation of Na+ channels or an
increased conductance that produced shunting of action potentials, or
both. The input resistance decreased when the membrane potential
was depolarized (Fig. 10D). The duration of this
effect is ~1-2 sec, which is three to five times larger than the
duration of PADs. The membrane potential reached by the peak of action
potentials remained unchanged when the membrane potential of CBTs was
held between 90 and 55 mV. On the other hand, at the resting
potential, small PADs, which depolarized the membrane potential by
3-15 mV, produced a marked decrease of the spike peak (Fig.
10B). The effects of PADs on action potentials were
compared with those of current injection (Fig. 10C). These results were consistent in the five experiments performed and suggest
that the decrease of the peak of action potentials mediated by single
PADs does not involve Na+ channel inactivation but
results from a shunting mechanism through increased chloride conductance.
| |
DISCUSSION |
PADs occur locally and shunt afferent action potentials
Close apposition sites between GABA-ir and Lucifer yellow-filled
axons (Fig. 2) were found in the area predicted by the
electrophysiological analysis. With use of ultrastructural methods,
axoaxonal synapses, which represent the morphological substrate of
presynaptic inhibition, have been found in different species (Atwood
and Morin, 1970; Nakajima et al., 1973; Hirosawa et al., 1981; Fyffe
and Light, 1984; Wang-Bennett and Glantz, 1985; Watson et al., 1991;
Lamotte d'Incamps et al., 1998a). Most inhibitory axoaxonal synapses, however, have been found close to presynaptic release sites (Atwood et
al., 1984; Fyffe and Light, 1984; Lamotte d'Incamps et al., 1998a).
Inhibitory axoaxonal synapses distant from output synapses have been
described in the brain of crustacea (Wang-Bennett and Glantz, 1985). In
this case, inhibitory axoaxonal GABAergic synapses are located on the
terminal arbor between the axon and the output synapses. This situation
is more comparable to the CBTs. Confocal microscopy analysis
suggests that such output synapses from CBT onto MNs are located after
the branching point on the small branches (Fig. 3).
The fact that the inhibitory endings do not occur at or immediately
adjacent to the release sites suggests that the inhibitory action does
not directly act on the transmitter release machinery per se, but acts
by decreasing the amplitude of action potential and thereby reducing
transmitter release at the presynaptic terminals. This is supported by
the electrophysiological findings reported in this study (Fig. 7).
Because PADs did not produce any further reduction of action potentials
in sites more distal to the first branching point, the GABA synapse
responsible for PADs presumably acts before this first branching point,
i.e., where the CBT enters the ganglion. We have previously reported
the existence of a clear relationship between the amplitude of
presynaptic spikes and that of postsynaptic EPSPs (Cattaert et al.,
1992). The results of the present work indicate that the reduction of
spike amplitude by PADs is likely to be directly responsible for the
reduction of transmitter release. A direct effect onto the output
synapse is unlikely to occur because GABA synapses are located far from the output synapses.
The electrophysiological and immunohistochemical analyses suggest that
action potentials propagate passively after the first branching point
and that GABAergic synapses mediating PADs are located close to the
branching point. The increased chloride conductance during PADs will
shunt afferent action potentials as they propagate toward the
presynaptic terminals and thus reduce their efficiency in evoking
transmitter release. The fact that the GABAergic inputs appear to be
located primarily around the first branching point suggests that
synaptic transmission from sensory neurons is globally controlled.
GABAergic presynaptic inhibition does not seem to mediate selective
control of transmitter release from different branches of single
sensory axons. A local control of sensory information flow in different
branches of single sensory axons has recently been shown in the cat
spinal cord (Lomeli et al., 1998). In the crayfish, such a
mechanism may involve other receptors or may be limited to some sensory
axons. No presynaptic inhibition was observed in some CBTs (20%) in
which a specific filtering of afferent messages with respect to their
target is therefore possible.
Active and passive conduction in CBTs
The results presented in this study suggest that only passive
conduction occurs in the distal processes of CBTs; however, the precise
location of this transition is difficult to determine on the basis of
the electrophysiological analysis. Moreover, it is not known whether
the transition between the zone of active and passive conduction occurs
gradually or abruptly. Results from simulation studies (our unpublished
observations) have shown that the reduction of the orthodromic spike
peak in the distal part of a CBT can be observed even if the recording
is made from an active site. Therefore, the reduction of the spike peak
per se is not an indication that we are recording from a passive site, but rather that the recording is made close to a passive zone. Such a
reduction of spike peak is presented in Figure 4B, in
which the more distal electrode is likely to be still in an active
propagation zone. By comparison, purely passive propagation fulfills
three criteria: spatial attenuation, low-pass filtering, and absence of
any conduction delay. The latter criterion is often masked by the
membrane potential noise. However, because this is the case when
recording from sites 150 µm more distal to the first branching point
(Fig. 5), the passive propagation zone is likely to start ~150 µm
after the first branching point.
Shunting mechanism versus inactivation of
Na+ channels
We have shown previously that PADs result from the opening of a
GABA-activated receptor channel (Cattaert et al., 1992) and that local
application of GABA depolarizes the membrane potential of CBTs by ~20
mV and dramatically reduces the spike amplitude by 75%, because of a
decrease of input resistance by up to 67%. This decrease in input
resistance is probably responsible for most of the reduction of spike
amplitude. In this report, we have demonstrated that for small PADs
(<20 mV) only a shunting mechanism would be involved (Fig. 10). Larger
PADs, such as those occurring during locomotor-related bursts, could
possibly reduce the peak of orthodromic spikes by inactivating
Na+ channels. We were unable to obtain the
inactivation curve for Na+ in these axons because
the CBTs cannot be clamped because of their extensive branching.
However, an argument against such an inactivation process is that large
PADs often elicit antidromic spikes (El Manira et al., 1991; Cattaert
et al., 1992, 1994). This apparent contradiction is enhanced by the
fact that such antidromic spikes do not elicit any EPSP in postsynaptic
MNs (Cattaert et al., 1992). One would think that if inactivation
played the major role in presynaptic inhibition, such antidromic spikes
would produce EPSPs in the postsynaptic neurons, as do orthodromic
spikes; however, this is not the case. A simulation study has
demonstrated that the reduction of spike peak largely depends on where
inactivation of Na+ channels is produced relatively
to the passive zone (our unpublished observations). Spikes produced
near the transition between active and passive zones by large PADs will
not be propagated in both directions. However, in this situation, the
shunting mechanism is largely involved in preventing spikes being
conveyed toward the terminal endings (our unpublished observations).
These simulation studies suggest that in this case spikes are likely to
be triggered in sites situated more proximally to GABA synapses because
at the GABA synapse site, as local currents leak through the chloride channels, the spike is prevented from developing fully. Such mechanisms must also exist in mammals because antidromic discharges are produced during fictive locomotion in the cat (Dubuc et al., 1988; Gossard et
al., 1989). However, in this case, PADs are supposed to be produced
close to the output synapse (Fyffe and Light, 1984). In the latter
case, the question remains whether antidromic discharges produced by
PADs would produce an EPSP.
Our conclusion emphasizes the role of a shunting mechanism in
PAD-mediated presynaptic inhibition. This finding is different from
previous simulation studies in mammals (Graham and Redman, 1994;
Walmsley et al., 1995; Lamotte d'Incamps et al., 1998a,b). Simulation
studies, based on serial electronic microscopy reconstruction of Ia
sensory neurons (Walmsley et al., 1995), demonstrated that inactivation
could play the main role in presynaptic inhibition. This conclusion was
supported by several data. First, input synapses (likely GABAergic) are
located at some distance from the terminal but are too few to produce a
massive shunting. Second, PADs propagate easily in the branches and may
inactivate the incoming spike at some distance from the GABA synapse.
Recent studies in cat confirm this hypothesis (B. Lamotte d'Incamps,
unpublished observations).
Our results do not totally contradict these findings because, as we
proposed, both mechanisms (shunting and inactivation) are present,
depending on the PAD amplitude. Our electrophysiological data (Fig. 10)
demonstrate that small PADs (<10 mV) produce exclusively shunting,
whereas larger PADs such as those occurring during phasic bursts during
locomotion (up to 25 mV) will also induce inactivation of
Na+ channels. An important point concerns the
resting potential of the sensory terminal. In the crayfish it is
generally around 75 mV. Therefore small PADs cannot inactivate
Na+ channels. Another point concerns the number of
GABA synapses and the amount of shunting they produce. In the crayfish
CBCO terminals, we have demonstrated that a large shunt (67% decrease in input resistance) can be produced when most of the GABA synapses are
activated by local application of GABA via a pressure ejection micropipette (Cattaert et al., 1992). This finding was supported in the
present work by the demonstration that GABA-ir boutons were found
concentrated in the region of the first branching point. Although an
ultrastructural analysis is required before it can be concluded that
these boutons represent GABA synapses, the existence of so many
GABAergic synapses at that site is in accordance with the possibility
of a shunting mechanism.
| |
FOOTNOTES |
Received Nov. 30, 1998; revised April 19, 1999; accepted April 27, 1999.
This work was supported by Centre National de la Recherche Scientifique
(D.C.), by the Swedish Medical Research Council project 11562 (A.E.),
and by the Sweden/France exchange program. We thank Drs. F. Clarac, D. Parker, P. Wallén, and L. Vinay for their comments on this manuscript.
Correspondence should be addressed to D. Cattaert, Laboratoire
Neurobiologie des Réseaux, Centre National de la Recherche Scientifique, UMR 5816, Université de Bordeaux 1, Bat B2, Avenue des Facultés, 33401 Talence Cedex, France.
| |
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TOP
ABSTRACT
INTRODUCTION
