Synaptic Mechanisms Section, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20892
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ABSTRACT |
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Stanley, Elis F..
Presynaptic Calcium Channels and the Depletion of Synaptic Cleft
Calcium Ions.
J. Neurophysiol. 83: 477-482, 2000.
The entry of calcium ions (Ca2+)
through voltage-gated calcium channels is an essential step in the
release of neurotransmitter at the presynaptic nerve terminal. Because
the calcium channels are clustered at the release sites, the flux of
Ca2+ into the terminal inevitably removes the ion from the
adjacent extracellular space, the synaptic cleft. We have used the
large calyx-type synapse of the chick ciliary ganglion to test for
synaptic cleft Ca2+ depletion. The terminal was voltage
clamped at a holding potential (VH) of 80
mV and a depolarizing pulse was applied to a range of potentials (
60
to +60 mV). The voltage pulse activated a sustained inward calcium
current and was followed, on return of the membrane potential to
VH, by an inward calcium tail current. The
amplitude of the tail current reflects both the number of open calcium
channels at the end of the voltage pulse and the Ca2+
electrochemical gradient. External barium was substituted for calcium
as the charge-carrying ion because initial experiments demonstrated
calcium-dependent inactivation of the presynaptic calcium channels.
Tail current recruitment was compared in calyx nerve terminals that
remained attached to the postsynaptic neuron and therefore retained a
synaptic cleft, with terminals that had been fully isolated. In
isolated terminals, the tail currents exhibited recruitment curves that
could be fit by a Boltzmann distribution with a mean
V1/2 of 0.4 mV and a slope factor of 5.4. However, in attached calyces tail current recruitment was skewed
to depolarized potentials with a mean
V1/2 of 11.9 mV and a slope factor of
12.0. The degree of skew of the recruitment curve in the attached
calyces correlated with the amplitude of the inward current evoked by
the step depolarization. The simplest interpretation of these findings
is that during the depolarizing pulse Ba2+ is removed from
the synaptic cleft faster than it is replenished, thus reducing the
tail current by reducing the driving force for ion entry.
Ca2+ depletion during presynaptic calcium channel
activation is likely to be a general property of chemical transmission
at fast synapses that sets a functional limit to the duration of
sustained secretion. The synapse may have evolved to minimized cleft
depletion by developing a calcium-efficient mechanism to gate
transmitter release that requires the concurrent opening of only a few
low conductance calcium channels.
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INTRODUCTION |
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The influx of Ca2+
through presynaptic calcium channels is an essential step in the action
potential-dependent secretion of neurotransmitters (Katz
1969; Llinas et al. 1981
). The calcium channels
are located primarily, if not exclusively, at the transmitter release
sites or "active zones" (see Wu et al. 1999
;
Stanley 1997
) therefore these ions enter the terminal
from the narrow synaptic cleft. Thus nerve terminal activity
necessarily results not only in accumulation of Ca2+ in the
cytoplasm but also in its removal from the synaptic cleft. Little is
known, however, about the dynamic changes of Ca2+ in the
synaptic cleft. Depletion of Na+ and K+
(Attwell and Iles 1979
) and Ca2+
(Engelman and Montague 1999
; Vassilev et al.
1997
) in the cleft has been hypothesized to result from the
flux of these ions through the pre- or postsynaptic membrane but direct
evidence is very limited. This question is difficult to test
experimentally because of the small size and general inaccessibility of
most presynaptic nerve terminals to direct recording under voltage
clamp. Even in the few large terminals that can be voltage clamped, it
is difficult to distinguish effects due to ion concentration changes from those on the calcium channels themselves.
We tested for synaptic cleft Ca2+ (CaSC2+)
depletion by examining the amplitude of the nerve terminal calcium tail
current (ICa,t) in fully isolated and intact
calyx-type presynaptic nerve terminals at the chick ciliary ganglion.
This calyx nerve terminal has advantages of large size, numerous
distinct transmitter release sites (de Lorenzo 1960;
Stanley and Goping 1991
), and highly clustered N-type calcium channels (Haydon et al. 1994
; Stanley
1991
; Stanley and Goping 1991
; Yawo and
Chuhma 1994
; Yawo and Momiyama 1993
). In addition, a major advantage of the chick calyx presynaptic terminal preparation is that it is possible to compare ion currents in nerve
terminals that remain attached to the postsynaptic neuron and therefore
retain a synaptic cleft, with currents in terminals that have been
fully dissociated. This has allowed us to distinguish effects of
Ca2+ flux on the presynaptic terminal itself from those
that result from the attachment of the terminal to the postsynaptic
cell. Furthermore, the excellent visualization of dissociated synapses (Haydon et al. 1994
; Stanley 1991
;
Stanley and Goping 1991
), greatly facilitates the
selection of calyces that are free of neighboring neurons or glia and
structurally favorable for patch clamp recording. We present evidence
that supports the occurrence of ion depletion in the synaptic cleft.
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METHODS |
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Isolation of calyx nerve terminals
Ciliary ganglia were removed from 15-day chick embryos,
enzymatically dissociated, and plated on plain coverslips as previously described (Haydon et al. 1994; Stanley and Goping
1991
; Stanley and Mirotznik 1997
). The
coverslips were incubated for 30 min at 37°C to allow the cells and
cell fragments to attach and were rinsed and maintained at 20°C until used.
Patch clamp recording
The dissociated ganglia were placed in a Leiden chamber filled
with 0.6 ml extracellular medium containing (in mM) 165 NaCl, 5 CaCl2 (or BaCl2), 0.8 MgCl2, 10 HEPES, 2 4-aminopyridine (RBI), 3 × 105 tetrodotoxin (7.35 pH, adjusted with
NaOH; osmolarity ~295 mOsm). The cells were imaged on an inverted
microscope (Nikon Diaphot) under high power (×40 oil, 1.4 NA lens).
Only compact calyx nerve terminals, as confirmed by direct visual
inspection, were used in this study. Data presented in this report were
from calyx recordings selected for good space-clamp characteristics
from 25 individual recordings. Patch electrodes of 3-5 M
resistance
were filled with intracellular solution containing (in mM) 145 Cs
gluconate, 20 CsCl, 1.0 MgCl2, 10 HEPES, 10 EGTA,
2 ATP-Mg (7.35 pH, adjusted with CsOH; osmolarity ~305 mOsm). Whole
cell currents were recorded using an Axopatch 200A amplifier under the
control of pClamp 6 software (Axon Instruments) with a minimum of a 5-s
intertrial interval. Data were filtered at 5 kHz online and sampled at
100 µs intervals. Electrode and cell capacitance were electronically compensated. Leak currents were subtracted online by a P/6 protocol. All experiments were carried out at room temperature (22-24°C). Small variations in residual leak current were subtracted during analysis using a portion of the traces free from pulse-evoked currents.
Before presentation the traces were digitally filtered at 2 kHz and
residual transient artifacts were blanked.
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RESULTS |
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Experimental strategy
A strategy was developed to test for depletion of synaptic cleft
divalent ion (XSC2+; where
X2+ is the ion carrying the charge through
the calcium channel) in the calyx synapse. A long step depolarization
from VH was given to open presynaptic
calcium channels and evoke a continuous flux of ions from the external
medium into the nerve terminal via the synaptic cleft. When the calcium
current reaches steady state during the long step depolarization, the
concentration distribution of X2+ will
depend on the number of calcium channels activated and their unitary
conductance, the overall (bath to cytoplasm) electrochemical driving
force (EX), and on the resistances to ion
flux along the total pathway. As a simple model,
EX can be divided into two components:
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Each trial consisted of two depolarizing pulses: a long pulse of 100 ms
followed 10 ms later by a short test pulse of 4 ms. The long pulse was
applied at a range of membrane potentials to evoke sustained calcium
currents of different amplitudes and to evoke a tail current
(IX,t1) on return of the membrane potential to
VH. The subsequent short test pulse was always
to +80 mV and evoked a second tail current,
IX,t2. Cleft ion depletion is minimal during
this second pulse because the membrane potential is ~15 mV more
depolarized than the apparent current reversal potential
(Stanley 1991; Yawo and Momiyama 1993
). The amplitude of IX,t2 was used to monitor any
sustained effects of the long pulse on the open probability of the
calcium channels themselves.
Tail currents in isolated nerve terminals in external calcium
It was first necessary to determine if the long depolarizing pulse
had any direct effect on the presynaptic calcium channels. This was
tested in fully isolated nerve terminals that lack a synaptic cleft. In
such terminals, the inward calcium current during the long pulse
exhibited a decay (Fig. 1A)
and ICa,t1 recruitment extended over a
fairly broad range of potentials (Fig. 1C). In addition,
ICa,t2 was inhibited by an amount that was
proportional to the amplitude of the current at the end of the long
pulse (ISS; Fig. 1, B and
C). A current-dependent inhibition of
ICa,t2 was observed in 5/5 calyces examined.
Because the second test pulse ended 14 ms after the long pulse and the
calyces were fully isolated, the reduction in
ICa,t2 is very unlikely to reflect
current-dependent changes in the driving force for Ca2+
entry, but must instead result from direct inhibition of calcium channel opening. This behavior is characteristic of calcium-dependent inactivation, as previously described for chick somatic (Cox and Dunlap 1994) and calyx (Yawo and Momiyama 1993
)
calcium currents. Thus the broad recruitment curve of
ICa,t1 and the relaxation in the long pulse
current can be at least partially accounted for by calcium-dependent
inactivation of the presynaptic calcium channels. Because both
calcium-dependent inactivation and cleft depletion are
current-dependent phenomena, the presence of the former greatly
complicates any test for the latter.
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Tail currents in isolated nerve terminals in external barium
To test for XSC2+ depletion without the
complication of the current-dependent inactivation, we substituted
Ba2+ as the charge carrying ion. This ion readily
permeates calcium channels but induces little, if any, current
dependent inhibition. In the fully isolated nerve terminals, the decay
in current during the conditioning pulse, which was seen with external
Ca2+, was much less evident with external
Ba2+ (Fig.
2A). Furthermore, there was no
current-dependent inhibition of the test pulse tail current,
IBa,t2 (Figs. 2, B and C
and 4B), consistent with insignificant inactivation of the
presynaptic calcium channels in external Ba2+
(Yawo and Momiyama 1993). Thus in isolated calyces, the
relationship of IBa,t1 to the amplitude of the
voltage pulse should reflect only the voltage-dependence of calcium
channel recruitment. IBa,t1 was described by a
Boltzmann curve (Fig. 2C, left panel) with a steep slope
factor of 5.4 mV.
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Tail currents in attached nerve terminals in external barium
We next examined IBa,t1 recruitment in calyces that remained attached to the postsynaptic ciliary neurons and therefore retained a synaptic cleft. We selected synapses with compact presynaptic calyx terminals by visual inspection. As in the isolated calyces, the barium current exhibited little decay during the long pulse (Fig. 3A). In addition, there was no current-dependent inhibition of IBa,t2 (Figs. 3, B and C and 4A). The recruitment of current as a function of voltage during the conditioning pulse in the attached calyces (Figs. 3C and 4B) was very similar to that in the fully isolated calyces; the steady-state current at the end of the long pulse, ISS, had a similar voltage threshold and reversal potential. However, the recruitment of IBa,t1 was significantly shifted to more depolarized potentials (Fig. 3C). Similar results were observed in five different attached calyces (Fig. 4C) and a Boltzmann distribution fitted to the pooled data (not shown) had a V1/2 of 11.9 mV and a slope factor of 12.0, twice that of the controls.
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DISCUSSION |
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These results demonstrate that in attached calyx nerve terminals the tail current recruitment curve is skewed toward depolarized potentials. This skew could have several different origins, the most likely of which are 1) a poor spatial control of the membrane potential, 2) a current-dependent inactivation of the calcium channels, 3) a shift in the activation properties of the presynaptic calcium channel, and 4) depletion of ions from the synaptic cleft. These possibilites are discussed below.
1) Difficulties achieving an effective space clamp would not
be surprising in a sheet-like structure such as a calyx. In fact, in
the first whole cell calcium currents recorded at this terminal we
selected calyces that exhibited extensive sheets completely enveloping
the postsynaptic neuron and these typically exhibited currents that
were poorly clamped across the entire calyx area (Stanley
1989; Stanley and Goping 1991
). However,
such recordings were readily identified by a shift toward more
depolarized potentials of both the threshold of calcium channel
recruitment and, even more markedly, in the extrapolation toward the
reversal potential of the steady-state current (Stanley and
Goping 1991
). This finding was attributed to the gradual
recruitment of the more distant fringes of the calyx (E. F. Stanley and A. Sherman, unpublished calculations). In the present study
we selected compact calyces and, as shown in Fig. 4B,
the threshold and reversal potentials of the current versus voltage
relations were indistinguishable from the fully isolated and rounded
calyces. Thus it is highly unlikely that the skew in
IBa,t1 in attached calyces can be attributed to poor voltage control.
2) The skew in IBa,t1
recruitment correlated with the amplitude of the steady-state inward
current at the end of the conditioning pulse
(ISS). A simple interpretation of this
finding is that the channels exhibit current-dependent inactivation, as
reported previously at calyx-type presynaptic nerve terminals
(Forsythe et al. 1998; Yawo and Momiyama
1993
, although ion depletion might have contributed to the
degree of current inhibition in these reports, see below). Whereas
calcium-dependent inactivation was noted as a reduction in
ICa,t2 in external Ca2+ (Fig.
1C), no similar effect was observed in Ba2+.
Thus the skew in IBa,t1 noted in attached
calyces cannot readily be attributed to current-dependent inactivation.
Herein we shall refer to the IBa,t1
recruitment curve obtained in the isolated calyces as the "control
recruitment curve."
3) A viable possibility is that attachment of the calyx
results in a depolarizing shift in the voltage-dependence of calcium channel steady-state open probability. Such a shift could conceivably result from an access resistance between the external medium and the
channels themselves because of the restricted space in the synaptic
cleft. Alternatively, in attached calyces calcium channel properties
might be modified by an interaction between the channels and a cleft or
postsynaptic protein. However, this shift in voltage-dependence hypothesis is weakened by the observed decline in voltage dependence in
the Boltzmann relation in the attached calyces, seen as a doubling of
the slope factor (Figs. 4C and
5A). This was analyzed as
follows. The fraction of the IBa,t1
inhibited at each test potential can be calculated by simply
subtracting the mean normalized IBa,t1 obtained in the attached calyces from the control
IBa,t1 recruitment curve (Fig. 5). We have
termed the resulting relation the "observed fractional inhibition
curve." The hypothesis that the skew in IBa,t1 is caused by a simple depolarizing
shift in steady-state activation can be tested graphically by comparing
the observed fractional inhibition curve with one generated by a
voltage shift of the control curve (Fig. 5A). That is,
the control IBa,t1 recruitment curve is
subtracted from the same curve displaced by +10 mV (the difference
between the V1/2 of the two Boltzmann
distributions; see Fig. 5A). The observed and
voltage-shifted fractional recruitment curves have different
characteristics (Fig. 5B). Whereas they are essentially
the same over the more negative potential range (20 to +5 mV), they
diverge markedly at more depolarized values. Thus a simple shift in
steady-state activation does not readily explain the form of the skewed
IBa,t1 recruitment curve. It is possible
that a skewed tail current recruitment curve might be generated by a
model with a resistance gradient in the synaptic cleft. However, there
is presently no basis for such a complex model which would, in any
case, be very difficult to test.
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4) The most interesting result from the analysis of the recruitment curves is obtained when the observed fractional inhibition curve is compared with the amplitude of the steady-state calcium current at the end of the long pulse, ISS (Fig. 4B). It is then readily apparent that the degree of inhibition of the tail current amplitude is proportional to ISS (Fig. 5B). This is strong evidence in support of a direct relation between the skew in IBa,t1 recruitment and the rate of calcium entry into the nerve terminal. The simplest interpretation of this finding is that with small inward currents the diffusion of ions from the bath to the synaptic cleft keeps pace with the influx of the ions into the terminal. However, with larger currents, Ca2+ is transferred from the synaptic cleft into the terminal (Fig. 6C) at a rate that is faster than its replacement from the external medium (Fig. 6, A and B). This results in a net CaSC2+ deficit and therefore a reduction in ESM and the tail current amplitude. Thus our results suggest that the rightward skew in IBa,t1 recruitment reflects a depletion of CaSC2+. If we assume that the inhibition of IBa,t1 at the peak of ISS is solely the result of ion depletion and therefore to a shift in EBa (and that other factors remain constant) we can estimate, using the Nearnst equation that cleft Ba2+ declines from 5 to ~3.4 mM.
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From these data alone one cannot determine where exactly in the
calyx-neuron junction depletion occurs. One possible site is the long
diffusion pathway from the edge of the calyx to the synapses, that is,
a deficit in the synaptic space rather than the synaptic cleft (Fig.
6A; but see Fujiwara and Nagaro 1989 for
evidence of pores in the calyx). If so, one might suppose that the
external ion depletion seen here is more a characteristic of extensive
calyx-type synaptic contacts than a general synapse phenomenon.
However, even if this is the case, these findings have biological
significance because restricted diffusion of Ca2+ in the
extracellular space may be a general feature of neuropil; a decline in
external Ca2+ has been detected during neural activity
using ion-sensitive electrodes (cf. Nicholson 1980
).
It is more likely that it is at the synapse itself, the specialized
region where quantal transmitter release occurs (Fig. 6), that
Ca2+diffusion is the most impeded (see theoretical models
by Attwell and Iles 1979; Engelman and Montague
1999
; Vassilev et al. 1997
). Here the diffusion
is limited by the very close (~50 nm) apposition of the pre- and
postsynaptic membranes. It is highly likely therefore that any
extracellular CaSC2+ depletion can be attributed to the
diffusion path within the synapse region itself (Fig.
6B). Indeed, CaSC2+ depletion and that
of ions involved in postsynaptic conductances (Attwell and Illes
1979
; Vassilev et al. 1997
) is probably a
general feature of all chemical synapses and may have constrained the evolution of the synapse structure. Synaptic clefts may differ somewhat
in shape, usually a rod or a small patch, but the distance from the
edge to the center is usually ~250 nm.
We have no reason to suppose that CaSC2+ depletion
serves a useful function in synaptic transmission; but instead, that it
sets a physiological limit on chemical transmission. Of course,
transmitter release at fast synapses is typically induced by action
potentials and not by the sustained depolarizations used in this study.
Modeling studies suggest that depletion develops abruptly, within the
duration of an action potential, whereas recovery is equally rapid,
within 2 ms (Engelman and Montague 1999). Thus depletion
at an individual synapse would be expected to affect transmitter
release during a prolonged action potential, for example as a result of
the down-regulation of potassium channels, but would not affect
transmission during a fast train of impulses.
It is now generally accepted that transmitter release follows the
~4th power of the external calcium concentration (see Dunlap et al. 1995). Thus small changes in cleft calcium can result in large changes in transmitter secretion. There are three simple mechanisms that may have evolved to reduce extracellular ion depletion: first, minimizing the area of synaptic contact so that calcium ions can
readily diffuse into the cleft; second, providing a readily accessible
reserve of ions; and third, maximizing the efficiency of transmitter
release with respect to calcium influx. Minimizing the synaptic area is
clearly important, as mentioned above. A reserve of
CaSC2+ is, at this point, hypothetical. One possible
source could be Ca2+ bound to buffers in the cleft with a
low affinity (to allow rapid dissociation). Another source is the
secretory vesicle itself which is known to contain high concentrations
of calcium (~20 mM) which could be added to the cleft during
transmitter release. Finally, there may be transport systems on the
presynaptic membrane that restore the ion to the cleft. We have
recently used immunocytochemistry to show that calcium pumps are
preferentially located at the transmitter release sites (Juhaszova et
al. 2000
). Although strategically located to return Ca2+ to
the synaptic cleft, ATP-driven pumps are probably too slow to
significantly affect CaSC2+ depletion during an action
potential but might play a role in restoring the ion to the synaptic
space during the period between impulses.
Perhaps the best defense against depletion, however, is to design the
transmitter release mechanism so that it can be activated by a minimal
influx of Ca2+. With this adaptation, the nerve terminal
would only require calcium channels of low conductance and density,
thus minimizing CaSC2+ depletion. Recent studies
suggest that this is the case; calcium channels are located
strategically close to the release site and very few (~180) calcium
ions are required to trigger release (Stanley 1993,
1997
). The chick ciliary ganglion calyx terminal remains the
only synapse where properties of release site-associated single calcium
channels have been examined directly (Stanley 1991
,
1993
). Presynaptic calcium channel openings are exceedingly
transient and admit only a few hundred ions at a time. Thus calcium
channels in the nerve terminal may be adapted to minimize
CaSC2+ depletion by limiting ion influx at any single
point in the presynaptic membrane.
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ACKNOWLEDGMENTS |
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I am indebted to helpful suggestions from Drs. L. C. Schlichter, R. Mirotznik, X. Zheng, and all previous lab members (Synaptic Mechanisms Section) and to R. Harris, Synaptic Function and Formation Unit, Basic Neurosciences Division, Division of Intramural Research, National Institutes of Neurological Diseases and Stroke, National Institutes of Health.
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FOOTNOTES |
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Present address and address for reprint requests: Division of Cell and Molecular Biology, Toronto Western Research Institute, Main Pavilion 14-320, 399 Bathurst St., Toronto, ON M5T 2S8, Canada.
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 10 June 1999; accepted in final form 16 September 1999.
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REFERENCES |
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