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The Journal of Neuroscience, February 1, 2001, 21(3):771-781
Synaptically Driven Calcium Transients via Nicotinic Receptors on
Somatic Spines
Richard D.
Shoop1,
Karen T.
Chang1,
Mark H.
Ellisman2, 3, and
Darwin K.
Berg1
Departments of 1 Biology and
2 Neurosciences and the 3 National Center for
Microscopy and Imaging Research, University of California, San Diego,
La Jolla, California 92093-0357
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ABSTRACT |
Dendritic spines commonly receive glutamatergic innervation
at postsynaptic densities and compartmentalize calcium influx arising
from synaptic signaling. Recently, it was shown that a class of
nicotinic acetylcholine receptors containing 
7 subunits is
concentrated on somatic spines emanating from chick ciliary ganglion
neurons. The receptors have a high relative calcium permeability and
contribute importantly to synaptic currents, although they appear to be
excluded from postsynaptic densities. Here we show that low-frequency
synaptic stimulation of the 7-containing receptors induces calcium
transients confined to the spines. High-frequency stimulation induces a
transient calcium elevation in the spines and a more sustained
cell-wide elevation. The high-frequency transient elevation again
depends on 7-containing receptors, whereas the sustained elevation
can be triggered by other nicotinic receptors and depends on calcium
release from internal stores and probably influx through voltage-gated
L-type calcium channels as well. Retrograde axonal stimulation of the
neurons at high frequency mimics synaptic stimulation in producing
sustained cell-wide calcium increases that depend on L-type channels
and release from internal stores, but it does not produce calcium
transients in the spines. Thus frequent action potentials are
sufficient to generate the cell-wide increases, but 7-containing
receptors are needed for spine-specific effects. Patch-clamp recording
indicates that 7-containing receptors preferentially desensitize at
high-frequency stimulation, accounting for the inability of the
stimulation to sustain high calcium levels in the spines. The spatial
and temporal differences in the patterns of calcium elevation could
enable the neurons to monitor their own firing histories for regulatory purposes.
Key words:
nicotinic; spines; calcium; acetylcholine receptors; ciliary ganglion; 7
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INTRODUCTION |
Dendritic spines have been the
subject of intense study because they first were recognized as
primary sites of synaptic excitation and likely sites of synaptic
plasticity in the CNS (for review, see Harris, 1999
). Their
raison d'être appears to be an ability to segregate
electrical and biochemical components of synaptic signaling such that
the synaptic current can spread to other compartments whereas chemical
effects are confined mainly to the spine of origin (Koch and Zador,
1993; Yuste and Denk, 1995). This has been demonstrated best for
synaptically driven calcium elevations in hippocampal spines (Muller
and Connor, 1991; Yuste and Denk, 1995; Svoboda et al., 1996), where
the calcium transients mediate synaptic plasticity (for review, see
Malenka and Nicoll, 1999). Constraining calcium elevations to
individual spines serves a dual purpose: it permits limited calcium
influx to achieve a high, local concentration for maximum regulatory
impact on adjacent molecules, and it spatially confines the regulatory
effects to one or a few nearby spines. The spatial constraint may be
essential for maintaining the input specificity of synaptic plasticity
(Koch and Zador, 1993; Yuste and Denk, 1995) and for reducing the risk
of excitotoxicity (Choi, 1992).
Most spines that have been analyzed to date receive glutamatergic input
and depend on NMDA receptors for much of the calcium influx that has
been observed. An interesting exception is that of somatic spines on
chick ciliary ganglion neurons. Recent studies show that ciliary spines
are heavily endowed with nicotinic acetylcholine receptors containing
7 subunits (Shoop et al., 1999). Such receptors (7-nAChRs) have a
high relative calcium permeability, equivalent to that of NMDA
receptors (Bertrand et al., 1993; Seguela et al., 1993), and generate
large synaptic currents when activated (Zhang et al., 1996; Ullian et
al., 1997). Surprisingly, 7-nAChRs are excluded from postsynaptic
densities (Jacob and Berg, 1983), which instead contain a less abundant
nAChR subtype having 3 and other subunits (3*-nAChRs; Jacob et
al., 1984; Vernallis et al., 1993). During development the 7-nAChRs
are required for reliable, synchronized nicotinic transmission through
the ganglion (Chang and Berg, 1999). The receptors remain in abundance
throughout adulthood but become less important for acute ganglionic
transmission at later times, when 3*-nAChRs suffice (Chiappinelli,
1983; Chang and Berg, 1999) in combination with electrical synapses
that form between preganglionic calyces and postganglionic ciliary
neurons (Martin and Pilar, 1964a). The calcium permeability of
7-nAChRs, their abundance and positioning on somatic spines, and
their apparent dispensability for acute transmission in adulthood
suggest that the receptors play other signaling roles. A prime
candidate would be regulation of calcium-dependent events in the
postsynaptic cell. By translating firing history into specific temporal
and spatial patterns of calcium elevation, the receptor could produce
distinctive regulatory effects. We have used calcium imaging and rapid
multi-photon laser-scanning fluorescence microscopy to examine the
contributions of 7-nAChRs to synaptically driven calcium transients
in the postsynaptic cell and to assess how the resulting patterns
reflect the frequency and duration of synaptic input to the neurons.
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MATERIALS AND METHODS |
Dye loading of ciliary ganglia. Ciliary ganglia were
dissected with preganglionic and postganglionic nerve roots intact from embryonic day 15 (E15) chick embryos and from 3-week-old chickens, and
each postganglionic nerve root was trimmed to a length of 2-4 mm.
Neurons in the ganglia were backloaded with the calcium fluorescent
indicator dye Oregon green-1 conjugated to 10,000 MW dextran (Molecular
Probes, Eugene, OR). The dye was chosen in part because of its
relatively low affinity for calcium
(KD = 398 nM[SCAP], as reported by Molecular Probes for
the batch that was used) so that bound calcium could be expected to
dissociate rapidly and not artificially prolong the calcium signal that
is observed. The methods for dye loading have been described previously (Brain and Bennett, 1995) and were followed here with minor
modifications. Briefly, a drawn out Pasteur pipette was used to apply a
saturated solution of Oregon green-1-dextran to the postganglionic
nerve root for 1.5-3 hr at 37°C. An oxygenated phosphate-free
buffer containing (in mM) 170 Na+, 5 K+, 5 Ca2+, 2 Mg2+,
169 Cl
, 20 HCO3, and 11 glucose,
pH 7.4, was used both to perfuse ganglia and to dissolve the dye. After
backfilling, the ganglia were perfused for an additional 10 min and
then mounted for stimulation and recording in the oxygenated buffer at
37°C.
Calcium imaging. Visualization of the calcium dye was
performed with a high-speed multi-photon laser-scanning microscope
designed and built at the National Center for Microscopy and Imaging
Resources Center at the University of California, San Diego (Fan et
al., 1999). This system uses a femtosecond pulsed laser system
(Spectra-Physics, Mountain View, CA) and is built around a highly
modified version of the Nikon RCM 8000 video-rate confocal instrument
(Tsien and Bacskai, 1995). The microscope system used in these studies
was the development prototype for the Bio-Rad RTS2000 (Bio-Rad
Laboratories, Hercules, CA). The instrument has the normal advantages
of a typical multi-photon confocal microscope (e.g., decreased photo
damage, greater depth of visualization) as well as the additional
advantage of being able to capture images faster than video-rate. Most
of the images that were recorded for this study were taken at 18.5 msec/frame. To examine rapid rise times, we achieved a greater sampling frequency (2-8 msec/frame) by reducing the frame size or by
using line scanning. Data were transferred directly from a real-time
image- processing subsystem (Datacube) to a hard disk array developed
specifically for this instrument by Nikon . These operations were
managed by a user interface running under Windows NT. Some analysis of
data was performed as data streamed from the preparation, allowing
calcium fluctuations to be tracked during an experiment. Subsequent
processing of the data was accomplished with the RCM software created
specifically for this instrument. Subsequent analysis was performed
with RCM software, NIH Image, and CricketGraph.
Ganglia were stimulated by using a suction electrode attached to either
the preganglionic or postganglionic nerve root and a Master-8 (AMPI,
Jerusalem, Israel) controller device to deliver trains of electrical
pulses. Stimulation was triggered by the RCM software, allowing the
visual data to be time-locked with the stimulus data. An individual
stimulus within a train was typically 1 msec in duration at 12-15 V
(the minimal voltage required for robust stimulation was determined
experimentally). Atropine at 100 nM (Calbiochem, La Jolla,
CA) was included routinely in the bath solution during recording to
prevent the activation of muscarinic receptors. When 7-nAChRs were
to be blocked selectively, -bungarotoxin (-Bgt; Molecular Probes)
was added to a final concentration of 100 nM at least 30 min before stimulation of the ganglion. When 7-nAChRs were to be
imaged as part of the experiment, fluorescently conjugated -Bgt
(rhodamine--Bgt; Molecular Probes) was applied at 100 nM
to the ganglion, and images were collected after 10-15 min at 37°C.
When calcium release from internal stores was to be blocked, ganglia
were incubated with either 10 µM ryanodine or 1 µM thapsigargin (Calbiochem) for > 30 min before
stimulation. Nifedipine at 10 µM (Calbiochem) was used to
block L-type voltage-gated calcium channels. When ganglia were
stimulated via the postganglionic nerve root, 20 µM
D-tubocurarine (Calbiochem) was included in the bath to
prevent any intraganglionic nicotinic signaling.
Ganglia could be imaged continuously for ~22 sec at 18.5 msec/frame (>1100 frames) before significant photobleaching occurred. Neurons were excluded from the data set if they underwent bleaching or
if their calcium levels did not return to baseline after stimulation. When calcium levels were to be compared quantitatively in different regions of the same cell, the mean fluorescence intensity was measured
for defined circles with diameters of 2.5 µm positioned on the neuron
perimeter. Circles were chosen either to include ("on-spine") or to
exclude ("off-spine") membrane regions later shown to be rich in
-Bgt binding sites, indicative of 7-nAChR clusters correlated
with spine mats (Shoop et al., 1999, 2000). Calcium levels were
compared for the same neuron over space, time, or incubation condition,
but only relative rather than absolute levels were compared among
neurons because of variations in extent of dye loading and fluorescence
yield. For each set of experiments the gain of the photomultiplier was
adjusted to ensure operation in the midrange. The time course of decay
for the calcium signal was fit to either a first- or second-order
exponential with the Origin software (Microcal Software, Northampton,
MA). The best fit was determined by performing an F test of significance.
Imaging on dissociated neurons. Voltage-gated calcium
channels and 7-nAChRs were colabeled on freshly dissociated E15
ciliary ganglion neurons by incubating Alexa-488 conjugated -Bgt
(Molecular Probes) for 45 min immediately after the neurons were
dissociated and plated in culture medium as previously described (Shoop
et al., 2000). After three 5 min rinses in culture medium, the neurons were fixed in 4% paraformaldehyde in phosphate buffer for 30 min at
room temperature, rinsed five times in PBS (0.15 M
NaCl and 0.01 M
Na2HPO4, pH 7.4), and
permeabilized in PBS containing 0.1% (v/v) Triton X-100; they were
incubated for 3 hr at room temperature in PBS with 5% normal donkey
serum containing a 1:200 dilution of a rabbit antibody directed against
the 1c subunit of L-type voltage-gated calcium
channels (Chemicon, Temecula, CA) or rabbit IgG as a negative control.
After five rinses in PBS the neurons were incubated for 1 hr in the
secondary antibody, Cy3-conjugated anti-rabbit (1:200 dilution; Jackson
ImmunoResearch Laboratories, West Grove, PA), in PBS with 5% normal
donkey serum and 0.1% Triton X-100.
Labeled cells were viewed with a Bio-Rad MC1024 confocal microscope
with a 63×, 1.4 numerical aperture objective lens. Optical sections
were taken at 0.75 µm intervals through the neuron, and the final
volume was assembled digitally with Lasersharp software (Bio-Rad). A
final projection was reconstructed from this volume with NIH Image
software (National Institutes of Health, Bethesda, MD).
Electrophysiology. Synaptic currents and postsynaptic action
potentials were recorded from neurons in dissected E15 whole ciliary
ganglia with patch-clamp techniques as previously described (Chang and
Berg, 1999). Briefly, E15 ciliary ganglia were excised with the
preganglionic nerve root attached and then trimmed and treated with
enzymes to loosen connective tissue. A suction electrode was used to
stimulate the preganglionic nerve root while whole-cell patch-clamp
recording was used from individual neurons in situ at 37°C
to record either the resulting synaptic current in voltage-clamp mode
or the resting membrane potential and elicited postganglionic action
potentials in current-clamp mode. Cells were discarded if the resting
potential at the end of the recording session was more positive than
50 mV or if the cell failed to follow presynaptic stimulation
consistently with postsynaptic action potentials during the first 5 sec
stimulation period at 1 Hz. The extracellular perfusion solution
(recording medium) contained (in mM): 120 NaCl, 4 KCl, 10 glucose, 2 CaCl2, 1 MgSO4, 30 NaHCO3, and 1 NaH2PO4, pH 7.4. Atropine
(100 nM) was added routinely, and the solution was warmed to 37°C and gassed with 95% O2/5%
CO2. The intracellular solution (in the patch
pipette) contained (in mM): 140 KCl, 5 glucose, 2 EGTA-KOH, 1 MgCl2, and 10 HEPES, pH 7.2. Other
aspects were as described previously (Chang and Berg, 1999).
The relative declines in 7-nAChR and 3*-nAChR responses during a
stimulus train were calculated on the basis of the following. Normally
90% of the peak synaptic current is generated by 7-nAChRs, in the
absence of previous desensitization (Zhang et al., 1996; Liu and Berg,
1999b). At later times the 3*-nAChR response dominates. Accordingly,
measurement of synaptic current 7.5 msec after the peak response was
used to determine the decline in the 3*-nAChR response as a function
of stimulus number, because the 7-nAChR response had declined
to < 3% of its original value by then and the 3*-nAChR
response was still large enough to measure accurately. Curve fitting
for each individual trial to distinguish 3*- and 7-nAChR
components as done previously (Zhang et al., 1996) was impractical here because of the small amplitude of the signals toward
the end of the stimulus train. Once the relative decline in 3*-nAChR
response as a function of stimulus number was known, the information
was used to calculate how the 3*-nAChR portion of the peak current
would have changed with stimulus number; this, in turn, was used to
determine (by subtraction) the amplitude of the 7-nAChR component of
the peak as a function of stimulus number. In this way both the
3*-nAChR and 7-nAChR components of the synaptic response were
calculated for each stimulus and then expressed as a fraction of their
respective values seen at the outset (i.e., generated by the first
stimulus in the train) for normalization.
In some cases whole-cell patch-clamp recording was used to
measure the nicotinic responses of freshly dissociated E14-E15 ciliary
ganglion neurons before and after exposure to individual drugs. In
these cases the techniques and procedures were as previously described
(Liu and Berg, 1999a,b).
Materials. White leghorn chick embryos were obtained from
McIntyre Poultry (Lakeside, CA) and maintained at 37°C in a
humidified incubator. Three-week-old chickens were purchased from the
same source and killed on the day of arrival for experiments.
All animal care and handling was in strict accordance with the policies
of the University of California, San Diego, Committee on Animal
Subjects and the American Association for Accreditation of Laboratory
Animal Care. The University holds a currently approved National
Institutes of Health Assurance and a United States Department of
Agriculture License. Reagents were purchased from Sigma (St. Louis, MO)
unless otherwise indicated.
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RESULTS |
Low-frequency synaptic stimulation induces calcium transients
confined to spines
Rapid calcium imaging was used to examine the effects of
synaptic stimulation in situ on intracellular calcium levels
of chick ciliary ganglion neurons. Ganglia were dissected from E15
embryos and backfilled via the postganglionic nerve root with the
calcium indicator fluor Oregon green-1 conjugated to dextran. After
rinsing, the ganglia were stimulated synaptically with a suction
electrode positioned on the preganglionic nerve root. Individual
fluorescence images were collected at a rapid rate (18.5 msec/frame)
with a modified laser-scanning microscope, allowing for the
visualization of calcium entry into a single optical cross section of
an entire neuron. Relatively large neurons were chosen for the imaging
and, therefore, were likely to be ciliary instead of choroid neurons (McNerney et al., 2000). Background fluorescence, defined as the signal
20 msec before stimulation, was low in the neurons (Fig. 1A). A single stimulus
caused a detectable rise in the calcium levels within the first 18.5 msec after stimulation, and it was confined to regions immediately
underlying the cell membrane at specific sites (Fig.
1B). Within 1 sec after stimulation the calcium levels had returned to baseline (Fig. 1C). When stimulated
with a train of stimuli at 1 Hz, the pattern reproduced. Eight
successive trials were averaged to show more clearly the baseline
levels before stimulation (Fig. 1D), during the first
18.5 msec after stimulation (Fig. 1E), and after the
calcium level returned to baseline (Fig. 1F). The
images indicate that the 1 Hz train of stimuli caused localized,
transient increases in the concentration of free calcium immediately
beneath certain regions of the plasma membrane.
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Figure 1.
Fluorescence imaging of calcium transients induced
in spine regions by low-frequency synaptic stimulation. Neurons in
dissected E15 ciliary ganglia were filled with Oregon green-1-dextran
via the postganglionic nerve root and then imaged with a multi-photon
laser-scanning microscope at 18.5 msec/frame before, during, and after
synaptic stimulation through the preganglionic nerve root.
A-C, Single frames of a neuron taken 20 msec before,
immediately after, and 1 sec after delivering a single synaptic
stimulus. D-F, Averaged images of eight successive
responses to 1 Hz stimulation taken 20 msec before, immediately after,
and 800 msec after each stimulus. G, Labeling with
rhodamine-conjugated -Bgt for 10 min at the end of the experiment to
identify 7-nAChR clusters defining spine mats. The same neuron is
shown throughout. A single stimulus produces calcium transients in
discrete regions along the perimeter. Averaging the responses
intensifies the signal and shows that the calcium transients
consistently correlate in location with spine mats defined by
7-nAChR clusters. A total of 20 neurons was examined in this manner
and gave similar results. Arrow, A spine mat (on-spine);
arrowhead, a region lacking detectable spines
(off-spine). Scale bar, 10 µm.
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The nature of the regions displaying the transient calcium increases
was examined by labeling the neurons with rhodamine--Bgt after the
calcium imaging had been completed. The rhodamine--Bgt binds
to 7-nAChRs, which are concentrated on somatic spines folded into
distinct mats on ciliary neurons (Shoop et al., 1999). As a result, the
large clusters of fluorescent -Bgt labeling on the cell surface
indicate the location of spine mats. Because the receptors are
concentrated and bind -Bgt with high affinity (Couturier et al.,
1990; Schoepfer et al., 1990), they can be detected in the ganglion
with confocal microscopy under these conditions without having to rinse
away unbound toxin and risk moving the preparation. It was clear that
the somatic spine mats, defined by rhodamine--Bgt binding to
7-nAChRs, exactly coincided with the regions displaying the calcium
transients (Fig. 1G).
The time course of the calcium transients in discrete regions was
examined in more detail by quantifying the fluorescence in a
2.5-µm-diameter circle either overlying a spine mat ("on-spine") or overlying an equivalent portion of the surface membrane lacking detectable 7-nAChR labeling ("off-spine"). In such experiments, continuous imaging of the calcium signal was performed during the 1 Hz
train of synaptic stimulation as above, and then the cells were labeled
with rhodamine--Bgt to define the spine mats and permit selection of
appropriate on-spine (e.g., Fig. 1G, arrow) and
off-spine (e.g., Fig. 1G, arrowhead) regions of
the images for analysis. Examined in this manner, on-spine regions
showed rapid increases in calcium after each stimulus of the 1 Hz train (Fig. 2A). The
increases reached a maximum within the first 18.5 msec (first frame)
and declined over the next several frames to reach baseline in < 1 sec. Very little increase in calcium could be detected in the
off-spine region (Fig. 2B) or in the cytoplasm away
from the plasma membrane (data not shown). Thus the calcium transients
consistently occurred on-spine, and, although the amplitude of the
transients at a given on-spine location varied from stimulus to
stimulus, there seemed to be no failures. If an on-spine region responded to one stimulus in the train (as most did), it responded to
all stimuli in the train.
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Figure 2.
Time courses of synaptically driven calcium
transients at identified sites on the postsynaptic neuron. The amount
of calcium fluorescence in a 2.5-µm-diameter circle either overlying
a spine mat (on-spine) or overlying part of the cell surface devoid of
spines (off-spine) was quantified from a series of images collected
continuously during synaptic stimulation at 1 Hz (18.5 msec/image). The
cell that was analyzed was the same as that in Figure 1, and the
on-spine and off-spine regions that were measured were those indicated
by the arrow and arrowhead, respectively,
in Figure 1G. A, Continuous imaging
on-spine. B, Continuous imaging off-spine.
C, Eight averaged responses from A. Error
bars are shown for each individual time point. The
arrowheads indicate the time of stimulation.
D, Eight averaged responses from B with
the corresponding error bars. Synaptic stimulation at 1 Hz elicits
calcium transients that reach maximum during the first 18.5 msec
poststimulation and quickly return to baseline; they are confined
mainly to the spine mat area because very little change is seen in the
off-spine region. Similar results were obtained with 18 neurons that
were tested. Calibration: A, B, 1 sec; C,
D, 200 msec.
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For greater clarity, all eight responses elicited by an 8-sec-long 1 Hz
train of stimuli were averaged both for on-spine (Fig. 2C)
and off-spine (Fig. 2D) regions. A significant,
rapidly rising and rapidly decaying calcium elevation is apparent in
the on-spine region, whereas only a small, slowly decaying elevation
can be distinguished in the off-spine region. As indicated by the
figure, all of the rise on-spine occurred during the first 18.5 msec, represented by the time between the stimulus and the peak response. In
fact, reducing the area of the circle that was examined so that the
image could be collected as rapidly as 8 msec after the stimulus still
yielded a peak on-spine response, indicating that it occurred very
quickly. The mean time constant of decay for the calcium transient
on-spine was 163 ± 25 msec (n = 18 cells).
High-frequency synaptic stimulation induces calcium elevations
throughout the soma
High-frequency stimulation produces a qualitatively different
pattern from that seen with 1 Hz. A 5 sec stimulation at 50 Hz produces
a sustained calcium elevation extending across the entire cell; the
elevation quickly collapses to baseline after stimulation ceases (Fig.
3A-C). The pattern depended
on the frequency of stimulation rather than on the number of stimuli
delivered because even after three stimuli at 50 Hz the cell-wide
increase was seen, although eight stimuli at 1 Hz failed to produce it. Comparing the time courses of calcium changes in on-spine and off-spine
regions shows a complex pattern. At 50 Hz on-spine the calcium level
quickly rises to a maximum and decays slowly in a biphasic manner
throughout the 5 sec stimulation period (Fig. 3D). Off-spine
the calcium level rises to a smaller maximum and decays slowly in a
monophasic manner (Fig. 3E). Analyzing the responses from a
number of cells yielded mean time constants for decay on-spine of
3.3 ± 1.4 sec (mean ± SEM; n = 16) and
15 ± 4 min (n = 16). The latter should be
considered only an estimate because it extends so far beyond the 5 sec
stimulation period that was examined. The faster decaying component
represented 37 ± 6% (n = 16) of the maximum
response. Analysis of off-spine regions yielded an estimated mean
single time constant for decay of 18 ± 3 min (n = 16), a value similar to that found for the slowly decaying component
on-spine. At the end of the 50 Hz stimulation the calcium elevation
collapsed to baseline, with time constants of 380 ± 200 and
560 ± 260 msec (n = 16) for on- and off-spine locations, respectively.
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Figure 3.
Cell-wide spread of calcium elevations at
high-frequency synaptic stimulation. The same cell shown in Figure 1
was stimulated synaptically at 50 Hz and analyzed as described in
Figure 2. A-C, Images collected 20 msec before, midway
through a 5 sec stimulation at 50 Hz, and 1 sec after the stimulation
was stopped. D, E, Continuous quantification of calcium
fluorescence in the on-spine and off-spine regions identified in Figure
1G while the neuron was stimulated synaptically at 50 Hz. High-frequency stimulation causes a rapid increase in calcium
fluorescence both on- and off-spine; the decay is biphasic with both a
fast and slow component on-spine, whereas it is monophasic with only a
slow component off-spine. Similar results were obtained with 16 neurons
that were tested. F, Stimulation of a neuron
continuously for 30 min at 50 Hz but imaged only briefly on-spine at 10 min intervals, as indicated. Arrowhead, Termination of
the stimulation. A smooth, slow decline in the calcium elevation occurs
over the 30 min stimulation period. G, Same neuron as in
F, allowed a brief recovery before being stimulated
again at 50 Hz to show that the cell remained competent. Scale bar for
A-C, 10 µm; calibration for D-G, 1 sec.
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A better estimate for the slowly decaying component during 50 Hz
stimulation was obtained by extending the stimulation period to 30 min.
Imaging the neurons briefly at widely spaced intervals to avoid
bleaching allowed a mean time constant of decay to be calculated for
the entire period (Fig. 3F). The value obtained, 17 ± 5 min (n = 4) for the slow on-spine
component, is in good agreement with that extrapolated from the 5 sec
stimulation period above. After a brief rest period 50 Hz stimulation
of the same neuron again produced the original pattern of calcium
elevation, indicating it had not been damaged by the procedure (Fig.
3G). Stimulation at 20 Hz produced on- and off-spine calcium
elevations qualitatively similar in time courses to those seen at 50 Hz
(data not shown). Taken together, the results indicate that both the spatial and temporal patterns of calcium elevation caused by
high-frequency synaptic stimulation are quite different from those
caused by low-frequency stimulation in the same neurons.
Calcium transients in spines depend on 7-nAChRs
Because 7-nAChRs are the major nicotinic receptor on ciliary
ganglion neurons and because the receptors are concentrated on the
spines and have a high relative permeability to calcium, it seemed
likely that they would play a major role in the calcium transients
elicited by synaptic stimulation. This was tested by comparing the
calcium transients induced by stimulation in the same neurons before
and after treatment with 100 nM -Bgt for 30 min to block
7-nAChRs. Under these conditions the synaptic currents generated by
3*-nAChRs are sufficient to elicit postsynaptic action potentials
approximately one-half of the time, averaged over the ciliary
population (Chang and Berg, 1999). At 1 Hz the toxin treatment almost
completely blocked calcium transients in the on-spine regions seen by
signal averaging (Fig.
4A,B). The residual
calcium transient seen in the presence of the toxin was comparable with
that seen for off-spine regions (Fig. 4C). The off-spine
signal was not reduced by -Bgt (Fig. 4D).
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Figure 4.
Effects of 7-nAChR blockade on synaptically
driven calcium transients. Neurons were tested for calcium elevations
during synaptic stimulation both before and after a 30 min exposure to
100 nM -Bgt to block 7-nAChRs; the results were
analyzed as in Figures 2 and 3. A-D, Averaged
sequential responses to eight stimuli at 1 Hz obtained from a single
neuron either on-spine or off-spine before and after -Bgt treatment,
as indicated. Note that the error bars (see Fig. 2) have been omitted
both here and in subsequent figures for clarity, but the time from
stimulus to peak response nonetheless represents the average of the
first 18.5 msec frame captured in each trial. E,
Complete blockade of the calcium transient by 20 µM
D-tubocurarine. Arrowheads indicate the time
of stimulation. F, G, Unaveraged responses elicited from
a single neuron on-spine before and after -Bgt treatment while it
was stimulated at 50 Hz. H, I, Control showing no change
in the amplitude or time course of the calcium transient elicited by 50 Hz stimulation when a cell was held for 30 min without -Bgt.
Blockade of 7-nAChRs almost completely eliminated both the rapid
on-spine calcium transient seen at 1 Hz and the rapidly decaying
on-spine transient seen at 50 Hz, reducing them to the kind of signal
seen at off-spine regions. Similar results were obtained with 14 neurons that were tested. J, Complete blockade of the
calcium elevation elicited by 50 Hz synaptic stimulation in the
presence of 20 µM D-tubocurarine.
Calibration: A-E, 200 msec; F-J, 1 sec.
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At 50 Hz stimulation the toxin blockade affected only the initial
component of the calcium elevation on-spine. The maximum response was
reduced and the decay phase was monophasic (Fig. 4F,G), resembling that seen above for off-spine
calcium elevations induced by 50 Hz stimulation. Control experiments
showed that no change of signal occurred in on-spine regions during the
test period when toxin was omitted from the incubation (Fig.
4H,I). Calculating the proportion of the peak
on-spine signal that remained after -Bgt treatment yielded a value
of 65 ± 8% (n = 14 cells), in good agreement
with that found above for the portion of the response that slowly
decays at 50 Hz stimulation. Including 20 µM
D-tubocurarine in the bath to block all nAChRs
completely eliminated the calcium transients induced by either 1 or 50 Hz (Fig. 4E,J). The toxin results demonstrate
that 7-nAChRs are essential for the transient calcium increases
elicited by 1 Hz synaptic stimulation in spines but contribute less
significantly to the extended increases seen at 50 Hz throughout the
cell. These latter cell-wide increases seen at high-frequency
stimulation apparently can be triggered by synaptic activation of
3*-nAChRs because they persist after toxin blockade. Similarly, the
small, transient increases seen in off-spine regions at low-frequency
stimulation also arise from activation of 3*-nAChRs.
Analysis of ganglia from older animals yielded the same kind of results
that were obtained with E15 ganglia. Thus ciliary neurons in ganglia
from 3-week-old chickens showed the rapid calcium transients confined
to on-spine regions at 1 Hz, and the transients were blocked almost
completely by -Bgt (Fig.
5A-C), as was the case for
neurons in E15 ganglia. Synaptic stimulation at 50 Hz produced
sustained calcium elevation throughout the cell; on-spine the elevation
decayed in a biphasic manner, and blockade with -Bgt removed the
more rapidly decaying component (Fig. 5D,E) as it did for
calcium elevations in E15 neurons synaptically driven at 50 Hz. The
calcium patterns that were observed, then, are not confined to early
developmental stages in the ganglion but rather can be seen at
posthatch times when the visual system and attendant reflexes are fully
functional.
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Figure 5.
Synaptically driven calcium transients in ciliary
neurons from posthatch chicks. Ciliary ganglia from 3-week-old chicks
were dissected, loaded with dye, stimulated, and imaged as described
for E15 ganglia in Figures 1-4. A-C, On-spine calcium
transients elicited by 1 Hz synaptic stimulation and shown either as a
continuous trace or as a signal averaged over eight responses either
before or after a 30 min exposure to -Bgt to block 7-nAChRs, as
indicated. D, E, On-spine calcium elevations
induced by 50 Hz synaptic stimulation before and after -Bgt
treatment. As in E15 ganglia, synaptic stimulation at 1 Hz induced
rapid calcium transients on-spine, and the transients were almost
completely dependent on 7-nAChRs; 50 Hz stimulation induced a
sustained elevation showing a biphasic decay, and 7-nAChR blockade
eliminated the rapidly decaying component. Similar results were
obtained with all six ganglia that were tested (one neuron per
ganglion). Calibration: A, D, E, 1 sec; B,
C, 200 msec. Arrowheads indicate the time of
stimulation.
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Frequency-dependent attenuation of synaptic current limits
7-nAChR contributions
The fact that 7-nAChRs rapidly desensitize in
situ suggests an explanation for the short-lived calcium
elevations that are -Bgt-sensitive on-spine: the 7-nAChRs quickly
may cease to contribute significantly at high-frequency stimulation.
This was examined by using patch-clamp recording from neurons in
situ to compare the stimulus-dependent attenuation of synaptic
currents in response to low- and high-frequency stimulation of the
presynaptic nerve root. At 1 Hz the peak whole-cell response showed no
significant decline during the 5 sec stimulation period (Fig.
6A). In contrast, at 50 Hz there was a rapid and large decrement in the whole-cell peak
current. When the portion of the peak response attributable to
7-nAChRs was calculated (normally 
90%; Liu and Berg, 1999b), it
was found to decline to approximately one-third within 1 sec (Fig.
6B). Although the 3*-nAChR response also declines,
possibly because of reduced transmitter release (Fig. 6C),
the decline was not as severe as that for 7-nAChRs.
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Figure 6.
Frequency-dependent changes in synaptic currents
and the reliability of synaptic transmission. E15 ganglia were
stimulated via the preganglionic nerve root at either 1 or 50 Hz for 5 sec while the postsynaptic responses were monitored with patch-clamp
recording. A, Individual synaptic responses recorded in
voltage-clamp mode at the beginning and end of the 5 sec stimulation
period either at 1 Hz (left pair) or at 50 Hz
(right pair). No change is seen in the amplitude or time
course of the synaptic current after the stimulation period at 1 Hz,
but a substantial decrement in amplitude and a change in shape are seen
for the synaptic current after 50 Hz. B, Relative
amplitude of the 7-nAChR portion of the synaptic current as a
function of stimulation time either at 1 Hz (open
circles) or 50 Hz (filled circles).
Values (mean ± SEM) have been normalized to the initial response and
are shown at 100 msec intervals for the 1st sec and at 200 msec
intervals for the remaining 4 sec of stimulation. A large decline is
seen after the first few stimuli at 50 Hz. C, Relative
amplitude of 3*-nAChR responses as a function of stimulation time
either at 1 Hz (open circles) or 50 Hz
(filled circles). Values (mean ± SEM) have been
normalized to the initial response and plotted as in
C. The 3*-nAChR response declines with
stimulation frequency although proportionately not as much as does the
7-nAChR response. D, Action potentials elicited by 1 Hz stimulation for 5 sec and recorded in current-clamp mode.
E, Action potentials elicited by 50 Hz stimulation. No
failures were seen at 1 Hz, but a significant incidence of failures is
seen at 50 Hz.
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Despite the reduction in synaptic currents at high-frequency
stimulation, the synapses were still capable of triggering action potentials with some frequency. At 1 Hz, transmission was 100% reliable during the 5 sec test period as seen by the five action potentials (Fig. 6D), whereas at 50 Hz it was much
reduced but not eliminated (Fig. 6E). Some neurons
showed no failure in transmission for the first 10 stimuli at high
frequency (Chang and Berg, 1999), but all showed substantial failures
at later times. Examining six neurons yielded an average number of
75 ± 18 (mean ± SEM) for the number of action potentials
elicited by the 250 stimuli delivered at 50 Hz for 5 sec; 13 ± 6 action potentials occurred during the final 2 sec of stimulation. The
fact that at least some action potentials occurred in all cells
throughout the test period raises the possibility that voltage-gated
channels may contribute importantly to the cell-wide calcium signal
seen at high-frequency stimulation.
Sustained calcium increases depend on multiple calcium sources
Two potential sources of calcium, in addition to nAChRs, are
voltage-gated calcium channels and release from internal stores. Both
are known to contribute to calcium levels in the neurons (Rathouz et
al., 1996; Brain and Bennett, 1998). L-type calcium channels are
distributed all over the cell body, as revealed by immunostaining with
a monoclonal antibody directed against the 1c
subunit; no preferential localization is seen on spine mats defined by
staining for 7-nAChRs (Fig.
7A-C). Blockade of L-type channels with 10 µM nifedipine had no obvious
effect on calcium transients that were induced by 1 Hz synaptic
stimulation (Fig. 7D,E), but it did have a dramatic impact
on calcium elevations in response to 50 Hz. On-spine the elevations
were reduced to baseline shortly after the initial maximum seen at 50 Hz (Fig. 7F,G). The slowly decaying component was lost
completely, and the residual response had a mean time constant for
decay of 1.2 ± 0.1 sec (n = 9). The amplitude of
the residual response was 41 ± 5% (n = 9) of the
peak response before nifedipine treatment. Thus both the relative
amplitude and the time course of decay for the residual component
approximated those calculated above for the on-spine calcium transient
generated by 7-nAChRs at 50 Hz, although the small difference in
decay constants suggested some contribution of L-type channels to the
on-spine signal. The much more important role of L-type channels in the
sustained calcium signal can be seen readily in off-spine regions; in
these cases the 50 Hz signal was blocked almost completely by the
nifedipine treatment (Fig. 7H,I).
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Figure 7.
Contribution of voltage-gated L-type calcium
channels to synaptically driven calcium elevations. A,
Immunofluorescent detection of voltage-gated L-type calcium channels on
a dissociated E15 ciliary neuron, using an antibody to the
1c subunit and fluorescent secondary antibody.
B, Same neuron as in A costained with
rhodamine--Bgt to reveal the 7-nAChRs clusters representing
somatic spine mats. C, Control showing absence of
labeling for an E15 neuron incubated with rabbit IgG instead of the
1c antibody. The same gain and exposure times were used
for A-C. D-I, Continuous calcium
imaging of on-spine and off-spine regions of a neuron stimulated at 1 or 50 Hz, as indicated, before (control) and
after (nif) a 30 min incubation with 10 µM nifedipine to block voltage-gated L-type calcium
channels. Almost all of the slowly decaying cell-wide calcium elevation
was lost when L-type calcium channels were blocked; much of the
fast-decaying on-spine calcium transient remained. Similar results were
obtained for all nine neurons that were tested. Scale bar for
A-C, 10 µm; calibration for D-G, 1 sec.
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Calcium release from internal stores also was tested for a
contribution to the sustained cell-wide elevation induced by
high-frequency synaptic stimulation. A 30 min incubation with 10 µM ryanodine to block release from internal stores
reduced the slowly decaying component of the on-spine calcium elevation
caused by 50 Hz stimulation while having little, if any, effect on the
more rapidly decaying component (Fig.
8A,B). Off-spine the
ryanodine again decreased the amplitude of the slowly decaying calcium
component (Fig. 8C,D). No significant effect of ryanodine
treatment was seen for the calcium transients induced by 1 Hz
stimulation (data not shown). Treating ganglia with 1 µM thapsigargin to deplete internal calcium stores had the same effects as with ryanodine (data not shown). The
results suggest that calcium-induced calcium release selectively amplifies the cell-wide calcium elevations caused by high-frequency synaptic stimulation and has little effect on the transients attributed to 7-nAChR activation.
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Figure 8.
Contribution of release from internal stores to
synaptically driven calcium elevations. Shown is continuous calcium
imaging of on-spine (A, B) and off-spine (C,
D) regions of a neuron stimulated at 50 Hz before
(control) and after (ryan) a 30 min incubation with 10 µM ryanodine to block calcium
release from internal stores. Much of the slowly decaying cell-wide
calcium elevation was lost when the release from internal stores was
blocked; little effect was seen on the rapidly decaying on-spine
response. Similar results were obtained with all 10 neurons that were
tested. Calibration, 1 sec.
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The ryanodine experiments also provide information about the
distribution of receptors contributing to the calcium changes and about
the sequence of events that is involved. Ryanodine establishes blockade
early on with the protocols used, as demonstrated by the fact that it
is already maximally effective in reducing the off-spine calcium signal
generated by 50 Hz stimulation even at the earliest times (Fig.
8C,D). The fact that ryanodine is unable to reduce the
initial calcium elevations on-spine under these conditions although
less than one-half of it is generated by 7-nAChRs leads to several
conclusions. First, the compound seems to have no direct effect on
either 7- or 3*-nAChRs, because both receptor subtypes contribute
importantly and directly to the initial increase in on-spine calcium at
high-frequency stimulation; second, release from internal stores
apparently does not contribute to the initial signal on-spine; and
third, essentially all of the cell-wide off-spine signal generated at
50 Hz arises from L-type channels and internal release with little, if
any, coming directly from either 7- or 3*-nAChRs. This last
conclusion is consistent with the known distribution of the receptors
from imaging experiments (Shoop et al., 1999).
The strength of the nifedipine and ryanodine experiments depends on the
specificity of the compounds. Neither significantly impacted the
7-nAChR response, as seen from the calcium signals at 1 Hz. For
3*-nAChRs the specificity was tested directly by incubating
dissociated ciliary ganglion neurons in 100 nM -Bgt for
1 hr to block 7-nAChRs and then by using whole-cell patch-clamp recording to measure the 3*-nAChR response to 20 µM
nicotine before and during exposure to either 10 µM
nifedipine or ryanodine. Ryanodine had no significant effect on the
3*-nAChR response (
5% inhibition; n = 4 cells),
but nifedipine produced a substantial inhibition (58 ± 7%,
mean ± SEM; n = 5 cells). This raised questions about the mechanism by which nifedipine blocked the sustained calcium
increases seen at high-frequency synaptic stimulation.
We examined this further by testing the ability of retrograde
stimulation via the postganglionic nerve root to produce sustained cell-wide calcium elevations in the absence of synaptic input. The
ganglia were perfused with 20 µM
D-tubocurarine during such experiments to ensure that no
intraganglionic nicotinic signaling (if such exists) could influence
the outcome. Retrograde stimulation at 50 Hz produced a prominent
calcium elevation throughout the soma both at on- and off-spine
locations (Fig. 9A). The
elevation was sustained throughout the period of stimulation and showed no evident decay until stimulation was stopped. Ryanodine treatment significantly reduced the plateau response (Fig. 9B),
demonstrating that calcium release from internal stores contributed to
the signal. With 1 Hz stimulation only a small, transient calcium
increase was seen either on- or off-spine (Fig. 9C),
approximately equivalent to that seen on-spine after the blockade of
7-nAChRs. Ryanodine treatment almost completely blocked the calcium
transient induced by retrograde stimulation at 1 Hz (Fig.
9D). Blockade of the L-type voltage-gated calcium channels
with nifedipine completely blocked the calcium elevations induced
either by 1 or 50 Hz retrograde stimulation (Fig. 9E). The
fact that nifedipine was more effective than ryanodine at 50 Hz
suggests that L-type channels not only contribute directly to the
calcium signal but also may be needed to trigger ryanodine-sensitive
release from internal stores. In any case, the results indicate that
voltage-gated channels and release from internal stores define a
pathway for elevating intracellular calcium levels in the neurons and
that repetitive membrane depolarization is sufficient to activate the
pathway. High-frequency stimulation of 3*-nAChRs depolarizes the
membrane and may well use this same pathway to generate sustained
cell-wide calcium increases.
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Figure 9.
Calcium elevations elicited by retrograde
stimulation. E15 ciliary ganglia were backfilled with dye and
stimulated in a retrograde manner via the postganglionic nerve root at
either 1 or 50 Hz while individual cells were imaged for calcium
fluorescence on-spine. At 50 Hz the stimulation caused a sustained
calcium increase (A) that was blocked
substantially by ryanodine (B) and blocked
completely at the end of the experiment by nifedipine
(E). At 1 Hz the stimulation produced a small
averaged signal (eight responses) on-spine (C)
that also was decreased by ryanodine (D). The
arrowheads indicate the time of stimulus. Both L-type
channels (n = 5 neurons) and release from internal
stores (n = 10 neurons) contribute substantially to
the sustained cell-wide calcium elevations seen at high-frequency
stimulation. No difference was seen between on-spine and off-spine
regions with respect to calcium increases at either 1 or 50 Hz
retrograde stimulation (n = 21 neurons).
Calibration: A, B, E, 1 sec; C, D, 200 msec.
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DISCUSSION |
The principal findings reported here are that low-frequency
synaptic stimulation triggers calcium transients that are confined to
somatic spine mats on ciliary neurons and that the transients are
dependent on the activation of 7-nAChRs. High-frequency synaptic stimulation induces sustained cell-wide calcium elevations that can be
triggered by 3*-nAChRs and depend on release from internal stores
and very likely participation of voltage-gated L-type calcium channels
as well. The temporal and spatial differences of the two kinds of
patterns offer clear mechanisms by which the neuron could encode
information about transmission efficiency and firing frequency, and, as
such, enable the neuron to exploit calcium-dependent mechanisms for
self-regulation of future signaling capabilities.
The imaging analysis performed here was made possible by the rapid
-scanning feature of the recently developed multi-photon laser-scanning
microscope and by the distinct topography of the somatic spine mats on
ciliary neurons. Because the calcium transients elicited at
low-frequency synaptic stimulation rapidly decay, the signal would have
been much reduced or missed completely if captured at conventional
imaging rates. The fact that the spines are grouped in distinct mats or
clumps on the ciliary neuron soma also facilitated the on- versus
off-spine quantification. Previous studies have demonstrated that the
7-nAChRs are highly concentrated on the spines, that the spines are
arranged in discrete mats, and that fluorescent -Bgt labeling of
7-nAChRs is a good marker for spine location (Shoop et al., 1999,
2000). Key for success in correlating the location of spine mats and
calcium transients in the present experiments was the fact that the
preparation could be held rigidly for the duration with no detectable
spatial drift of the cell that was being imaged and the fact that the
7-nAChR density was sufficient to bind adequate fluorescent -Bgt
for detection even in the presence of unbound toxin.
A low-affinity calcium fluor was used for the present experiments so
that bound calcium would be released quickly and not artificially
extend the time courses measured for calcium elevations in the cells.
Because of large variations in the efficiency of dye loading, it was
not feasible to manipulate the dye level in a controlled way to test
directly whether the dye itself constrained the spatial or temporal
patterns being reported for the calcium transients. The fact that an
L-type channel blocker in the present experiments shortened the time
course of decay for the calcium transient, however, suggests that the
dye was not rate limiting in determining the duration. The dye
previously has been used successfully to monitor rapid calcium
transients in dendritic spines (Helmchen et al., 1999; Yuste et al.,
1999).
The finding that 7-nAChRs are responsible for synaptically driven
calcium transients in somatic spine mats is consistent with the high
relative calcium permeability of the receptors and their being
concentrated on the spines (Bertrand et al., 1993; Shoop et al., 1999).
The on-spine calcium transients generated by 7-nAChR activation both
at 1 and 50 Hz appear to be mainly independent of contributions from
L-type channels and release from internal stores. At 1 Hz neither
nifedipine nor ryanodine had any effect on the transients although
-Bgt almost completely blocked them. At 50 Hz nifedipine had the
most effect, sharply reducing the sustained calcium elevation, but it
had little effect on the rapidly decaying -Bgt-sensitive component
generated by 7-nAChRs. The nifedipine-resistant component had the
same relative peak amplitude (proportion of peak current before
treatment) as did the proportion of the control response calculated to
be -Bgt-sensitive. A small difference in decay rate was observed for
the two, but this could arise either from a small contribution of
L-type channels to the rapidly decaying component or from the
limitations of calculating a decay constant for a small, rapidly
decaying component in the presence of a larger, more slowly decaying
one. If L-type channels and calcium stores contribute to local calcium
transients in spines, their contributions must be minor compared with
that of 7-nAChRs. In fact, the high local concentrations of calcium
produced in spines by 7-nAChRs transiently might inhibit nearby
voltage-gated calcium channels (Levitan, 1999). Endoplasmic reticulum
has been seen in ciliary spines (Shoop et al., 1999), but it may be
more important for calcium buffering.
The sustained cell-wide calcium elevations induced by
high-frequency synaptic stimulation depend on 3*-nAChRs, but not on 7-nAChRs, because the elevations can be blocked by
D-tubocurarine, but not by -Bgt. Although 3*-nAChRs
are much less abundant than are 7-nAChRs, they are distributed more
widely, being found both on spines and at postsynaptic densities on the
soma (Jacob et al., 1984; Wilson Horch and Sargent, 1995; Williams et
al., 1998; Shoop et al., 1999). The present measurements could not
resolve the site of origin for the sustained calcium elevations; the
speed of image collection was considerable, but not sufficient to
distinguish differences in the rates of rise for calcium increases at
different points along the cell surface.
The role of 3*-nAChRs in generating the sustained calcium elevations
may be primarily that of a catalyst, acting to depolarize the membrane.
Depolarizing the membrane would activate L-type calcium channels, and
these, in turn, apparently can induce calcium release from internal
stores, as the retrograde stimulation experiments demonstrate. The
immunofluorescence staining of L-type channels showed that they are
distributed all over the ciliary neuron soma, and membrane compartments
likely to serve as sites of calcium release can be found throughout the
soma as well (Pilar and Landmesser, 1976; Jacob and Berg, 1988). A
second mode of action for 3*-nAChRs that cannot be excluded yet
(given the partial inhibition of the receptors by nifedipine in the
present experiments) is that they may permit sufficient calcium influx
themselves to trigger some calcium-induced calcium release from
internal stores.
Calcium imaging in chick ciliary ganglion neurons in situ
has been used previously to examine the rate at which calcium levels in
the neurons return to normal after varying periods of high-frequency stimulation (Brain and Bennett, 1998). The instrumentation that was
used would not have detected the rapid on-spine calcium transients reported here, but the study was able to consider in some detail the
mechanisms influencing calcium release from internal stores and those
responsible for returning calcium levels to baseline in ciliary neurons
(Brain and Bennett, 1998). Recovery was not examined in the present
study, other than to determine mean time constants for return to
baseline after 50 Hz stimulation. The fact that very little decrement
occurred in the cell-wide calcium increase during the 5 sec stimulation
at 50 Hz demonstrates the stamina of the signaling system.
What might be the purpose of synaptically driven calcium
signaling in ciliary ganglion neurons? One possibility is that it helps
to guide developmental events. Spontaneous bursting activity has been
reported in a number of developing systems, including the visual system
and the somatic motor pathway, and it has been postulated to shape
neuronal development and circuit formation (Feller, 1999; Milner and
Landmesser, 1999). It is not known whether spontaneous bursting
activity occurs in the ciliary ganglion during embryogenesis, but the
fact that the ganglion represents a motor pathway makes this a
consideration. The mature calyx synapse also can support high-frequency
signaling (Dryer, 1994), but it is not known how often the synapse is
called on to do so in vivo. The present results indicate
that high-frequency signaling can produce sustained cell-wide calcium
elevations, raising the possibility of excitotoxicity. Chronic exposure
in ovo to nicotinic antagonists in general and -Bgt in
particular have pronounced and complex effects on neuronal survival and
development in the ciliary ganglion (Meriney et al., 1987). It has not
yet been possible, however, to dissect unambiguously the contributions
of ganglionic receptors and peripheral muscle receptors to these events.
The fact that the synaptically driven calcium transients persist into
posthatch chicks and retain many of the features seen earlier during
development suggests that the calcium signaling may play a regulatory
role in the mature system. An attractive possibility is that
synaptically driven calcium transients in the ciliary ganglion regulate
synaptic plasticity, as is the case in the CNS. Several forms of
synaptic plasticity have been reported for the calyx synapse on ciliary
neurons; these include facilitation, post-tetanic potentiation (PTP),
and long-term potentiation (LTP; Martin and Pilar, 1964b; Poage and
Zengel, 1993; Scott and Bennett, 1993a,b; Brain and Bennett, 1995). In
addition, 7-nAChRs on ciliary ganglion neurons undergo
activity-dependent rundown, as do NMDA receptors on hippocampal spines,
and the rundown depends on calcium influx and the ensuing balance of
activities between calcium/calmodulin-dependent protein kinase II and
calcineurin (Liu and Berg, 1999a). Thus calcium transients in somatic
spines may act locally to regulate signal detection in the spine over
the short term. Local calcium transients also may influence the shape
or stability of the spine itself, as demonstrated for dendritic spines
(for review, see Segal et al., 2000). Over the long range, synaptically
driven changes in calcium levels could regulate gene expression
(Mermelstein et al., 2000). Understanding these processes may provide
information about nicotinic signaling elsewhere in the nervous system
and offer new insight into the many functions of spines, whether
dendritic or somatic.
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FOOTNOTES |
Received July 12, 2000; revised Oct. 6, 2000; accepted Nov. 3, 2000.
This work was supported by National Institutes of Health Grants
NS12601, NS35469, and RR04050 and by Tobacco-Related Disease Research
Program Grant 9RT-0221. We thank Drs. Maryann Martone and Gary Fan
(University of California, San Diego) for advice on sample preparation
and image collection, and we thank Dr. Keith Brain (University of
Sydney, Sydney, Australia) for advice on retrograde dye filling of neurons.
Correspondence should be addressed to Dr. Darwin K. Berg, Department of
Biology 0357, University of California, San Diego, 9500 Gilman Drive,
La Jolla, CA 92093-0357. Email: dberg{at}ucsd.edu.
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TOP
ABSTRACT
INTRODUCTION
