Department of Biology, Sezione di Fisiologia Generale, University of Ferrara, 44100 Ferrara, Italy
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ABSTRACT |
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Sacchi, Oscar,
Maria Lisa Rossi, and
Rita Canella.
Nicotinic EPSCs in Intact Rat Ganglia Feature Depression Except
If Evoked During Intermittent Postsynaptic Depolarization.
J. Neurophysiol. 83: 3254-3263, 2000.
The involvement of the postsynaptic membrane potential level in
controlling synaptic strength at the ganglionic synapse was studied by
recording nicotinic fast synaptic currents (EPSCs) from neurons in the
intact, mature rat superior cervical ganglion, using the two-electrode
voltage-clamp technique. EPSCs were evoked by 0.05-Hz supramaximal
stimulation of the preganglionic sympathetic trunk over long periods;
their peak amplitude (or synaptic charge transfer) over time appeared
to depend on the potential level of the neuronal membrane where the
nicotinic receptors are embedded. EPSC amplitude remained constant
(n = 6) only if ACh was released within repeated
depolarizing steps of the postganglionic neuron, which constantly
varied between 50 and
20 mV in consecutive 10-mV steps, whereas it
decreased progressively by 45% (n = 9) within 14 min when the sympathetic neuron was held at constant membrane
potential. Synaptic channel activation, channel ionic permeation and
depolarization of the membrane in which the nicotinic receptor is
localized must occur simultaneously to maintain constant synaptic
strength at the ganglionic synapse during low-rate stimulation (0.03-1
Hz). Different posttetanic (20 Hz for 10 s) behaviors were
observed depending on the mode of previous stimulation. In the neuron
maintained at constant holding potential during low-rate stimulation,
the depressed EPSC showed posttetanic potentiation, recovering ~23%
of the mean pretetanic values (n = 10). The maximum effect was immediate in 40% of the neurons tested and developed over a
3- to 6-min period in the others; thereafter potentiation vanished
within 40 min of 0.05-Hz stimulation. In contrast, no statistically
significant synaptic potentiation was observed when EPSC amplitudes
were kept constant by repeated
50/
20-mV command cycles
(n = 12). It is suggested that, under these
conditions, posttetanic potentiation could represent an attempt at
recovering the synaptic strength lost during inappropriate functioning
of the ganglionic synapse.
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INTRODUCTION |
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Sympathetic ganglia have frequently been used as
models for the study of neuronal synaptic transmission and of the
processes modulating synaptic function. Mammalian ganglia possess a
multitude of neuroregulatory mechanisms; some of them affect or are
affected by preganglionic cholinergic activity. Ganglionic posttetanic potentiation is one of the first use-dependent synaptic potentiations that have been observed, and neurogenic long-term potentiation represents a well-known posttetany effect even in the cholinergic sympathetic pathway (for a review see Briggs 1995).
Apart from problems in terminology, a relatively brief conditioning
preganglionic stimulation results in modifications of synaptic function
over periods ranging from milliseconds to hours. These effects have been studied electrophysiologically by using extracellular mass recordings from postganglionic trunks, or intracellular recordings under current-clamp conditions. The parameters conventionally utilized
to measure the relative efficacy of synaptic transmission were
therefore the amplitude of either the postganglionic compound action
potential or the excitatory postsynaptic potentials (EPSPs) recorded
intracellularly in a single neuron. Analysis of miniature EPSP (mEPSP)
amplitude distribution represented a complementary tool applied in an
attempt to separate the pre- and postsynaptic components (Koyano
et al. 1985
). Neurogenic nicotinic long-term potentiation (LTP)
has been observed to last for several hours in vitro in rat superior
cervical ganglia (SCG) (Briggs et al. 1985
; Brown
and McAfee 1982
) and frog sympathetic ganglia (Koyano et
al. 1985
; Kuba and Kumamoto 1990
); in rat
ganglia a brief tetanic preganglionic stimulation resulted in a two- to
threefold potentiation of the fast EPSP lasting up to 3 h
(Briggs and McAfee 1988
), and a similar strong
potentiation was observed in frog ganglia.
In all these experiments a detailed phenomenological description was
not accompanied by a parallel understanding of the basic aspects
underlying potentiation, especially as concerns the biophysical properties of the postsynaptic receptors; most observations were actually performed under current-clamp conditions, and the postsynaptic properties were tested by applying exogenous neurotransmitter. In the
present experiments the nicotinic synaptic transmission was studied in
intact rat ganglia, before and after preganglionic tetanization, by
using the two-microelectrode voltage-clamp technique. Large synaptic
currents are thus recorded while precisely controlling the membrane
potential of the postganglionic neuron under conditions in which ACh is
released from naturally developed nerve terminals (Sacchi et al.
1998). Control of the postsynaptic membrane potential appeared
to be an additional, and unexpected, factor of crucial importance in
maintaining synaptic transmission efficacy, which could only be kept
constant, even at very low rate of ACh release, by preserving precise
timing between ACh exposure and potential modification of the membrane
hosting the ACh receptor. The application of ACh onto a recurrently
depolarized neuron, and exclusively during the
depolarization, appeared to be a prerequisite for maintaining a
synaptic current of constant amplitude at a low preganglionic stimulation rate; if these processes were dissociated, a progressive deterioration of synaptic efficacy ensued. This depression is determinant in synapse posttetanic behavior.
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METHODS |
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Experiments were performed on superior cervical ganglia isolated
from rats (male or female; 130-150 g body wt) during urethan anesthesia (1.5 g kg1 ip) and maintained in
vitro at 37°C. In some experiments the ganglion was dissected from
the animal 2 h after the initial anesthesia and sectioning of the
sympathetic trunk; in others the isolated ganglia were maintained for
2 h unstimulated in vitro before starting with the
electrophysiological experiment; the latter conditions did not appear
to affect subsequent behavior. After surgery, the animals were killed
with an overdose of anesthetic. Ganglia, including the preganglionic
sympathetic trunk and postganglionic nerves, were mounted on the stage
of an upright microscope (Zeiss UEM); superficial neurons were
identified at a magnification of ×500 by using diffraction
interference optics and impaled with two independent glass
microelectrodes filled with neutralized 4 M K+
acetate and having resistances of 30-40 M
. In a few experiments 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(tetrapotassium salt, BAPTA; Sigma) was dissolved in the microelectrode
filling solution to a final concentration of 170 mM. Recordings were
obtained under two-electrode voltage-clamp conditions as previously
described (Belluzzi et al. 1985
). Ganglia were
continuously superfused with a medium pregassed with 95%
O2-5% CO2 to a final pH
7.3 and had the following ionic composition (in mM): 136 NaCl, 5.6 KCl,
5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 14.3 NaHCO3, and 5.5 glucose. Choline chloride
10
5 M and atropine sulfate 1-2 × 10
6 M were added to the saline, unless
otherwise stated. In some cases the normal solution was diluted 1:1
with a synthetic culture medium (Dulbecco's modified Eagle medium)
keeping the calcium concentration constant at 5 mM. The bath was
grounded through an agar-3 M KCl bridge.
To activate the preganglionic input, supramaximal single current pulses
of 0.3 ms duration were applied at 0.05 Hz to the cervical sympathetic
trunk through a fine suction electrode, either while the neuron was
kept at a constant holding potential (usually 50 mV) or during the
application of repetitive cycles where the postsynaptic membrane
potential was commanded to different voltages. Under the latter
conditions, the usual protocol was to jump every 20 s to a
different test potential in the
20/
50-mV range, in 10-mV steps, 40 ms before the preganglionic shock was delivered and returning to the
holding level 60 ms after the stimulus. Each cycle started with the
20-mV command pulse and was continuously repeated, so that the EPSC
was observed at the same membrane potential level only once every
80 s. Great attention was taken during the preliminary procedures
to avoid any stimulation of the presynaptic fibers; only direct
stimulation of the neuron through the current electrode was used to
recognize the cell and evaluate the quality of the recording, whereas
preganglionic stimulation started exclusively with the onset of the
experiment. Preganglionic tetanization was represented by an isolated
10-s train of 20-Hz supramaximal stimuli while the neuron was clamped
at
50 mV. The ACh equilibrium potential (EACh) was evaluated at the end of the
experiment by extrapolating to zero current the current-voltage
(I-V) excitatory postsynaptic current (EPSC) peak amplitude
relationship obtained over the
80/
30-mV voltage range
(Sacchi et al. 1998
), or by occasionally adding the
80/
60-mV sequence to the normal 0.05-Hz
50/
20-mV cycles during
the course of the experiment.
Large synaptic currents were recorded with good control of the membrane potential at any tested voltage; single currents were filtered at 5 kHz with an 8-pole Bessel filter, digitized at 10 kHz with a 12-bit A/D interface (Digidata 1200A operating pCLAMP software, Axon Instruments) and stored on disk for further analysis. Long-lasting current tracings were filtered at 5 kHz and digitized continuously on tape (Biologic, DTR-1200; 0-10 kHz). Data were analyzed on Pentium personal computers (AST) with pCLAMP (Axon Instruments) software package.
Statistical analysis was performed on absolute or normalized values (with respect to the 1st EPSC of each series) by using unpaired t-tests when comparing isochronal data points under two different experimental conditions. The difference between the time course over time of EPSC peak values was assessed by comparing (t-test) the regression coefficients (b) across the two series of time points when two different voltage-clamp protocols were applied. The level of significance is indicated in the text.
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RESULTS |
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General EPSC properties at low stimulation rate and after tetanization
Figure 1 shows the results obtained
from a rat sympathetic neuron, in which the nicotinic EPSC was recorded
for an unusually long-lasting period, making it possible to test a mix
of different preganglionic stimulation rates and postganglionic
membrane potential modifications in the same cell. The supramaximal
preganglionic stimulation at 0.05 Hz started with the neuron constantly
held at 50 mV, in a bathing medium enriched with a tissue culture medium. The EPSC peak amplitude was not maintained, but decreased by
~43% of its maximum value within 15 min. A 20-Hz tetanus of 10 s duration (1st arrow) only partially and transiently compensated for
this initial decay: the EPSC amplitude tested systematically at
50 mV
every 20 s slowly increased but thereafter returned to the
pretetanus values within 45 min. A different protocol was then applied
to the neuron, so as to observe the EPSC at different membrane
potential levels over the
50/
20-mV range (and later over the
70/
20-mV range), whereas keeping the 0.05-Hz preganglionic stimulation rate constant. Cutting off perfusion with the enriched medium and returning to normal saline solution (*) did not apparently modify the synaptic efficacy. A second tetanus (2nd arrow) was applied
two h after onset of recording with minor effect on EPSC amplitude,
compared with that observed after the first tetanus. The experiment was
concluded with a final period at a constant
50-mV holding potential.
The 0.05-Hz stimulation rate was maintained throughout; during this
period neither the EPSC decay time constant at the different membrane
potentials tested nor the ACh equilibrium potential were significantly
modified. This general behavior was also confirmed when, instead of
EPSC peak amplitude, synaptic charge displacement was taken into
account. Three different factors are apparently involved in controlling
synaptic efficacy during constant rate preganglionic stimulation,
namely 1) the level of the neuron holding potential,
2) repeated voltage modifications of the postsynaptic
membrane potential, and 3) application of a preganglionic
tetanus. Each of these aspects is analyzed separately.
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EPSC peak amplitude during low rate preganglionic stimulation
The major effect observed in the previous experiment is possibly
the initial fade in EPSC amplitude during 0.05-Hz stimulation, whereas
the neuron membrane potential was kept constant. This observation was
confirmed in a group of nine neurons maintained at a stable holding
potential of 50 mV, in which the mean EPSC peak amplitude
progressively declined by 45% during a 14-min period of 0.05-Hz
preganglionic stimulation, with an apparent exponential time course
(
= 6-8 min; Fig. 2,
A and D,
). This
result cannot be readily explained by modifications in the ACh volley
output from the presynaptic terminals, because it remained virtually constant in neurochemical experiments on the isolated rat SCG, even
during a 60-min stimulation period at 10 Hz (Sacchi et al. 1978
).
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Quite a different picture arose when the "presynaptic" part of the
experiment was left unchanged but the EPSC was evoked at variable
membrane potential levels of the postsynaptic neuron; the holding
potential was kept at 50 mV while the EPSC was now evoked during
voltage commands of 100 ms duration given every 20 s, and varying
in 10-mV steps within the range
20/
50 mV, so as to measure the EPSC
at
50 mV once every 80 s. Under these conditions the EPSC
amplitudes at the different membrane potentials were virtually
unchanged over the 14-min low-frequency stimulation that produced a
clear-cut EPSC rundown in the absence of postsynaptic membrane
potential migrations. A typical experiment is partially illustrated in
Fig. 2, B and C, whereas in Fig. 2D
the open circles show the
50-mV mean values (n = 6).
Point-by-point comparisons (unpaired t-test) indicate a
significant difference (P < 0.05) after 14 min of
stimulation when absolute data are considered; the same level of
significance is obtained after 3-min stimulation (with P
becoming <0.001 after 4 min) if normalized data are used. The
regression coefficient (b) was not significantly different from zero when the protocol with recurrent
50/
20-mV cycles was applied, but became highly significant (P < 0.001)
when the holding potential was kept constant. Comparison between the
two b values indicates that the time course of EPSC
amplitude over time is significantly different (P < 0.001) during the application of the two voltage-clamp protocols used
in Fig. 2D.
During this period the EPSC basic properties, namely current decay time
constant and ACh equilibrium potential, were stable over time,
independent of any EPSC decay. The protective effect of the
50/
20-mV cycles was active as long as this sequence was continuously applied, but vanished when this procedure was
discontinued, returning to a constant holding potential. At this time
the expected synaptic rundown started to develop with the usual time
course (not shown).
Determinants controlling EPSC amplitude over time
The EPSC rundown observed while the neuron was held at constant
membrane potential occurred only if the preganglionic low rate
stimulation was maintained throughout. In the experiment illustrated in
Fig. 2E (mean values from 4 neurons), the initial EPSC
amplitude was estimated with a single cycle of the 50/
20-mV protocol, and thereafter the neuron was kept unstimulated for 13 min.
When synaptic stimulation was resumed, the first EPSCs were similar or
even larger than the starting ones, but their rundown was only delayed
if the holding potential remained unvaried during the following 0.05-Hz
preganglionic stimulation period (the regression test applied to the
data after the silent period is significant: P < 0.001). A similar behavior was observed in the isolated ganglion kept
unstimulated in vitro for 2 h before starting with the experiment
or in the preparation maintained denervated in the animal for 2 h
before dissection. This silent, resting period did not exert any
control over EPSC amplitude and subsequent synaptic stability, which
was exclusively dependent on the mode of preganglionic stimulation and
postsynaptic membrane pulsing, as in the freshly dissected preparation.
The origin of the synaptic decay was investigated in further
experiments testing ionic and voltage effects. The presence of nutrients in the bath, the absence of atropine, the external calcium concentration varying in the 2- to 5-mM range, a higher holding potential level (up to 80 mV) or reduction of preganglionic
stimulation to 0.03 Hz all proved ineffective in preventing development
of the EPSC rundown.
The ionic currents generated during the depolarizing commands (and the
resulting intracellular messages) were possibly involved in this
unexpected behavior; in fact, activation of all the voltage-dependent currents described in the rat sympathetic neuron starts at 30 mV. In
Fig. 3A the sodium and calcium
currents associated with the
50/
20-mV command in Fig. 2D
were evoked 10 ms before or immediately after the EPSC by applying a
single 8-ms voltage step to
20 mV to a neuron held at
50 mV. The
timing of Na+ and Ca2+
injection was only slightly modified with respect to the original protective protocol, but this was sufficient to allow the development of a progressive decrease in EPSC amplitude. The importance of depolarization occurring while the synaptic channels were open (and the
absence of any immediate link between activation of ionic voltage-dependent currents and EPSC rundown) was confirmed in the
experiment illustrated in Fig. 3B; the
50/
20-mV command cycles preventing the EPSC change in Fig. 2D were precisely
reproduced, but preganglionic stimulation was no longer applied
within the voltage step but, rather, 10 s before. This
canceled the otherwise preventive action of the protocol on EPSC
amplitude. The protective effect on synaptic efficacy associated with
membrane depolarization, on the other hand, presented an evident
voltage threshold for onset of synaptic depression, because recurrent
50/
30-mV cycles proved inadequate to prevent EPSC rundown (Fig.
3C). Each distinct behavior, illustrated in Fig. 3,
A-C, for single neurons, was confirmed in at least three
other cells.
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The reversibility of EPSC decay was tested, at different times during
its development, by applying the 50/
20-mV protocol, which would
have prevented synaptic rundown if used early on. The most frequently
observed result was the blocking of any further synaptic change, but no
reversal of the effect of depression was seen during subsequent
stimulation (an example is given in Fig. 3B). Only
occasionally was this procedure alone able to generate some recovery in
the EPSC amplitude (Fig. 3D), and only in 1 neuron of 11 did
the recovery prove complete.
These data suggest that fast calcium movements in the postsynaptic
membrane and external calcium concentrations larger than 2 mM are
apparently irrelevant in controlling EPSC amplitude stability over
time. The opposite held true when the actual intracellular calcium
concentration was modified. Figure
4Aa shows an experiment during
which 170 mM BAPTA diffused from the microelectrodes positioned inside
the cell. The effects of this treatment are illustrated in Fig.
4Ab in a different neuron held at 50 mV under
current-clamp conditions. The fast calcium buffer actually reached an
internal concentration sufficient to cancel the effects of both the
potassium calcium-dependent currents operating in the sympathetic
neuron during the action potential:
IKCa (which mainly sustains the fast spike repolarization when the cell is maintained at
50 mV)
(Belluzzi and Sacchi 1991
) and
IAHP (which sustains the long-lasting
spike afterhyperpolarization) (Sacchi et al. 1995
).
Under BAPTA treatment, presynaptic stimulation resulted in a
progressive decrease in EPSC amplitude, despite the
50/
20-mV
voltage cycles (the behavior shown in Fig. 4Aa was confirmed
in 4 different neurons). Moreover, under these conditions there was no
indication that the basic properties of the synaptic current were
consistently affected during the accumulation of the buffer within the
postsynaptic neuron.
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The 0.05-Hz preganglionic stimulation frequency, used to test synaptic
efficacy itself, seems unlikely to contribute significantly to the
genesis of any synaptic potentiation. The effects on synaptic strength
were, however, so unexpected as to suggest the use of the protective
protocols even at higher frequencies. The experiment illustrated in
Fig. 2D was thus repeated simply by increasing preganglionic
stimulation at 1 Hz in the case of constant holding potential, whereas
the protective 0.05 Hz 50/
20-mV cycles were completed with the 1-Hz
rate within the interpulse intervals at
50 mV holding potential (thus
maintaining the overall 1-Hz rate throughout). The
50/
20-mV cycles
systematically started with the
20-mV pulse, so that the
50-mV/EPSC
was the fourth of the sequence; accordingly, the fourth EPSC of the
pure 1-Hz series (constant holding potential) was taken as the initial
reference value. The results of this experiment are illustrated in Fig. 4B. As in the case of the 0.05-Hz stimulation rate, the EPSC
peak amplitude (and synaptic charge transfer) rapidly decreased to 43%
of the initial values after 5 min stimulation when the holding potential was kept constant, whereas the
50/
20-mV cycles proved to
exert the protective effects on synaptic strength under these conditions as well. Point-by-point comparisons between the two experimental groups indicated a significant difference
(P < 0.05) after 80 s of 1-Hz stimulation
(P < 0.001 after 2 min). Despite the use of the
protective voltage-clamp protocol, the EPSC peak amplitude actually
decreased by ~10% during the first minute of stimulation, presumably
reflecting the adjustment of ACh release at the new rate; this level,
however, was thereafter maintained constant. Notably, the net effect on
EPSC depression at 1 Hz was of the same size as that observed at 0.05 Hz.
Posttetanic effects are influenced by previous stimulation modality
Potentiation of the fast EPSC was induced by standard afferent
tetani at 20 Hz lasting 10 s. Posttetanic effects were
systematically observed in all neurons tested, but their development
was strongly affected by the modalities used during pretetanic
stimulation, as illustrated in Fig. 5 in
two typical recordings. Preganglionic tetanization was applied in two
groups of neurons after a 14-min period of 0.05 Hz supramaximal
stimulation, either while maintaining the postsynaptic membrane
potential at a constant 50-mV level, or during repetitive
50/
20-mV cycles. The results of these experiments are illustrated
in Fig. 6, with peak EPSC amplitudes
normalized to the initial values, for clarity. The expected synaptic
rundown developed during constant holding potential stimulation, as
previously shown in Fig. 2; the tetanus induced a potentiation of the
EPSC peak amplitude whose overall magnitude in 10 neurons was 22.8 ± 5.8% of the pretetanic values (Fig. 6,
; the difference between the final part of the pretetanic and the initial part of the
posttetanic curve is statistically significant, P < 0.01). The maximum effect was immediate in four cells and developed
over a period of 3-6 min duration in the others; thereafter, the EPSC
potentiation started to decline (Figs. 5A and 6). In a group
of six neurons, which could be recorded for a sufficiently long time,
posttetanic potentiation was virtually concluded after ~40 min of
0.05 Hz stimulation at
50 mV with the return of the EPSC amplitude to the levels that would have been reached in the absence of the tetanic
episode. No significant correlation was detected in these experiments
between the level of EPSC amplitude depression during the pretetanic
stimulation period versus posttetanic recovery. It is worth noting that
the original EPSC values observed at the beginning of low-frequency
stimulation were not resumed despite the preganglionic tetanization. If
the intracellular calcium concentration was controlled by BAPTA (Fig.
4A), the posttetanic potentiation was not evoked and EPSC
decayed independent of tetanus and of the protective
50/
20-mV
cycles. Quite a different description applies to the neuron sample in
which the EPSC amplitudes were kept constant by application of 0.05-Hz
preganglionic stimuli at different membrane potential levels within the
repeated
50/
20-mV command cycles. Under these conditions the
posttetanic effects were much less evident than in the previous case:
in five neurons a short-lived EPSC potentiation of ~4-5% at
50 mV
over the pretetanic (and initial) values was observed, after which EPSC
amplitude returned to the pretetanic levels and remained constant,
indefinitely, during further low-rate stimulation. Statistical analysis
on pooled data, however, was unable to detect any significant effect
related to the tetanic episode. The results of these experiments are
reported in Figs. 5B and 6 (in Fig. 6, open circles
represent the mean values of the
50-mV/EPSCs, n = 12;
small filled circles illustrate the mean EPSC amplitude time course in
4 neurons, in which the 0.05-Hz sequence at constant holding potential
was not intermingled with preganglionic tetanization).
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DISCUSSION |
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The new findings presented in this study are related to how
constant synaptic strength can be preserved at an intact and mature ganglionic synapse and the different effects of presynaptic
tetanization when synaptic efficacy is either depressed or maintained.
Depression is shown to occur during slow rate preganglionic stimulation
(0.05 Hz) with voltage constantly clamped near resting membrane
potential, but it does not occur if the neuron is held at 50 mV
without synaptic stimulation, nor in the preparation out of the animal or denervated in the animal for 2 h before experiment. Depression is also prevented during preganglionic stimulation at the same slow
rate, but including every 80-s stimulation while the postsynaptic cell
is held at
20 mV (as part of
20-,
30-,
40-,
50-mV cycles). Posttetanic potentiation apparently occurs if synaptic currents are
depressed and hardly at all if currents are not depressed.
In these voltage-clamp experiments on the rat superior cervical
ganglion, however, it is hard to evaluate the true relevance of the
posttetanic effects, because there is no precise definition of either
the fast EPSC amplitude to be considered as a reference value or of the
procedures to determine any previous ganglion potentiation at the time
of the dissection and the modalities for its cancellation. Assuming
that the EPSC amplitude (or inward charge displacement), measured at
the first synchronous preganglionic stimulation of the isolated
ganglion, indicates the maximum strength of ganglionic transmission,
then the natural conclusion to be drawn from these experiments is that
synaptic potentiation following a tetanic episode is very limited or
absent. This is in contrast with previous observations in the same
preparation based on extracellular or current-clamp recordings
(Briggs and McAfee 1988; Briggs et al.
1985
). If the EPSC amplitude immediately preceding tetanization is considered as the reference value, then it should be argued that
tetanus results in a potentiation of the depressed ganglionic EPSC of
~20%. This is probably the result that most closely fits the general
description of LTP in other sympathetic ganglia, although we did not
observe long-term maintenance of this effect. EPSC amplitudes higher
than the initial ones were only occasionally observed in the present
experiments; this suggests that maximal potentiation of nicotinic
synaptic transmission was already present in the silent preparation at
the moment of its dissection from the animal, and that any subsequent
treatment in vitro was, under the most favorable conditions, only
adequate to preserve it from failing. The loss in synaptic efficacy was
not related to the possible deterioration of the ganglion in vitro, but
simply to the modalities of its stimulation or, more precisely, the
modalities by which endogenous ACh is applied to the postsynaptic
receptor, which is apparently sensitive to the actual transmembrane
potential present at the moment of its activation. If the synaptic
channels are opened at holding potentials kept constantly close to the presumed resting values, a failure in synaptic transmission
progressively ensues, which rapidly and permanently reduces the EPSC
amplitude by ~50%. This holds true over a wide range of stimulation
frequencies, from the very low ones used to prevent the occurrence of
any form of potentiation, to those high enough to mimic the
physiological discharge rate present in preganglionic sympathetic
fibers at the moment of dissection (reviewed by Jänig
1995
).
The mechanisms involved in the permeation of the ganglionic nicotinic
channel therefore appear to be of more general interest than the
effects on posttetanic potentiation, which are presumably limited or
absent at this synaptic station in which synaptic transmission physiologically occurs with a high safety factor (Sacchi and
Perri 1973).
Our results demonstrate that the block of synaptic depression is
apparently calcium dependent, but they do not demonstrate how or where
Ca2+ acts to be effective. The arguments are the
following: 1) BAPTA present in the postsynaptic neuron
prevents the protective effects on depression of the depolarizing
cycles; 2) depolarizing steps to 30 mV only, prevent
synaptic decay less effectively than do steps to
20 mV (most likely
due to less activation of calcium channels); 3) depression
is not dependent on extracellular [Ca2+] above
2 mM; and 4) timing of calcium movements is essential, i.e.,
synaptic channels must be open during depolarization. Depolarizing steps to
20 mV applied 10 ms before or after stimulation are not
effective. Full voltage cycles separated from ACh release by 10 s
are also ineffective. These results suggest that a postsynaptic increase in [Ca2+]i may
not be sufficient by itself to prevent depression at this synapse;
rather they indicate that there is an interaction of the effects that
occur at depolarized potential (in particular as concerns calcium
influx) and the channel characteristics. Hence maximum synaptic
conductance apparently occurs in the presence of a calcium-dependent
mechanism [e.g., the depolarization-dependent release-promoting factor
S coupling spatial facilitation in frog axon terminals (Dudel et
al. 1993
), or a calcium-dependent polyamine unblock
(Rozov et al. 1998
)], which requires depolarization
simultaneously with the opening of the synaptic channels. Such a
mechanism may well be dependent on very close colocalization of calcium
channels and synaptic channels and thus may not occur in nonsynaptic patches.
We have no direct data on the basic properties of single channel
behavior, except as concerns its mean open time and the equilibrium potential of the permeating cationic current, both of which appear to
be unaffected by pre- and posttetanic events. Apparently, the synaptic
channels best operate in the presence of recurrent voltage shifts
moving the neuron membrane potential toward the ACh equilibrium potential. These are actually the physiological conditions encountered during normal synapse function: the synaptic current depolarizes the
cell until an action potential is fired, but the synaptic channels
remain open during the depolarizing voltage migrations because of their
slow closing kinetics (which, in this preparation, are scarcely
influenced by voltage) (Sacchi et al. 1998). If this peculiar coexistence between channel opening, its ionic permeation and
depolarization of the membrane hosting the receptor is not obeyed, the
synaptic current is expected to rapidly decrease over time in response
to constant amounts of neurotransmitter. These findings are reminiscent
of previous demonstrations, obtained under current-clamp conditions, in
which a precise timing between pre- and postsynaptic activity proved
crucial in controlling synaptic strength. When back-propagating
postsynaptic spikes were summed with subthreshold EPSPs, LTP of
subsequent single EPSPs was produced. Moreover, to strengthen the
synapse, EPSP must precede the spike by an interval of 10 ms, or less,
whereas synaptic depression is induced when this order was reversed
(Markram et al. 1997
). A similar conclusion arises from
the present experiments under voltage-clamp conditions, in which ACh is
applied in the presence or absence of membrane potential voltage jumps.
The previous observations were obtained in neocortical slices and
confirmed in different preparations including hippocampal CA3-CA3 cell
pairs in cultured slices (Debanne et al. 1998
),
Xenopus retinotectal synapses (Zhang et al.
1998
), and cultured hippocampal pyramidal neurons (Bi
and Poo 1998
). In these preparations
N-methyl-D-aspartate (NMDA) receptors are
involved in synaptic transmission; therefore the voltage-dependent unblock of NMDA receptors during spike depolarization, and the subsequent Ca2+ influx larger than that
contributed by the separate spike and EPSP in isolation (Koester
and Sakmann 1998
; Yuste and Denk 1995
), appear a
convincing explanation for LTP. These arguments, however, hardly apply
to the nicotinic receptor, in which voltage-dependent block or unblock
has never been described. They also provide some support to the
contention that the present findings are possibly related to the single
channel properties of the nicotinic receptor, rather than to
intracellular messages mediated by depolarization.
These properties are potentially of some interest in the organization and maintenance of neuronal functioning. It could be the case of a neuron in which the presynaptic input is normally developed and physiologically active at normal rates, but the neuron itself somehow becomes unable to discharge action potentials; or a neuronal network developed in culture, in the absence of stable pre- and postsynaptic spiking activity. Under these conditions, synaptic transmission would deteriorate through an unknown, merely use-dependent postsynaptic mechanism. The activity-dependent links between postsynaptic versus presynaptic events are well known; in the present example, however, the absence of spike discharge in the postsynaptic neuron would be the main trigger generating the synaptic fading. Similarly well understood is the relief of blocked NMDA-type receptors operated by depolarization; in the ganglion, this principle is apparently reversed, because it is no longer the depolarization that removes the block of the synapse but rather the absence of depolarizing episodes that induces its appearance. This new status is difficult to counteract, at least under the present experimental conditions in the isolated preparation. A possible instrument for recovering initial strength could actually be preganglionic tetanization, which would not represent a functionally relevant modification of the ganglionic synapse, but simply an attempt to recover the synaptic power lost during inappropriate functioning of the synaptic machinery.
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ACKNOWLEDGMENTS |
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We thank Dr. F. A. Edwards for many helpful comments on the manuscript.
This paper was supported by a grant from the Ministero della Università e della Ricerca Scientifica e Tecnologica within the national research project "Neurobiological Systems."
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FOOTNOTES |
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Address for reprint requests: O. Sacchi, Dipartimento di Biologia, Sezione di Fisiologia Generale, Via Borsari 46, I-44100 Ferrara, Italy.
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 17 September 1999; accepted in final form 11 January 2000.
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