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The Journal of Neuroscience, November 15, 2000, 20(22):8365-8376
Inositol 1,4,5-Trisphosphate (IP3)-Mediated
Ca2+ Release Evoked by Metabotropic Agonists and
Backpropagating Action Potentials in Hippocampal CA1 Pyramidal
Neurons
Takeshi
Nakamura,
Kyoko
Nakamura,
Nechama
Lasser-Ross,
Jean-Gaël
Barbara,
Vladislav M.
Sandler, and
William N.
Ross
Department of Physiology, New York Medical College, Valhalla, New
York 10595
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ABSTRACT |
We examined the properties of
[Ca2+]i changes that were evoked by
backpropagating action potentials in pyramidal neurons in hippocampal
slices from the rat. In the presence of the metabotropic glutamate
receptor (mGluR) agonists t-ACPD, DHPG, or CHPG, spikes caused Ca2+ waves that initiated in the proximal
apical dendrites and spread over this region and in the soma.
Consistent with previously described synaptic responses (Nakamura et
al., 1999a
), pharmacological experiments established that the waves
were attributable to Ca2+ release from internal
stores mediated by the synergistic effect of receptor-mobilized
inositol 1,4,5-trisphosphate (IP3) and spike-evoked Ca2+. The amplitude of the changes reached several
micromoles per liter when detected with the low-affinity indicators
fura-6F, fura-2-FF, or furaptra. Repetitive brief spike trains at
30-60 sec intervals generated increases of constant amplitude.
However, trains at intervals of 10-20 sec evoked smaller increases,
suggesting that the stores take 20-30 sec to refill. Release evoked by
mGluR agonists was blocked by MCPG, AIDA, 4-CPG, MPEP, and LY367385, a
profile consistent with the primacy of group I receptors. At threshold
agonist concentrations the release was evoked only in the dendrites;
threshold antagonist concentrations were effective only in the soma.
Carbachol and 5-HT evoked release with the same spatial distribution as
t-ACPD, suggesting that the distribution of
neurotransmitter receptors was not responsible for the restricted range
of regenerative release. Intracellular BAPTA and EGTA were approximately equally effective in blocking release. Extracellular Cd2+ blocked release, but no single selective
Ca2+ channel blocker prevented release. These
results suggest that IP3 receptors are not associated
closely with specific Ca2+ channels and are
not close to each other.
Key words:
pyramidal neuron; dendrite; IP3 receptor; metabotropic receptor; BAPTA; EGTA; carbachol; bis-fura-2; furaptra; endoplasmic reticulum
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INTRODUCTION |
Synaptically activated action
potentials backpropagate over the dendrites of hippocampal pyramidal
neurons (Turner et al., 1991; Jaffe et al., 1992; Spruston et al.,
1995). There is evidence that the action potentials and the
[Ca2+]i increases
that are associated with them may be important in the induction of
long-term potentiation (LTP) and long-term depression (LTD; Magee and
Johnston, 1997; Markram et al., 1997).
When the spikes are activated intrasomatically or antidromically, the
associated [Ca2+]i
increase is primarily attributable to Ca2+
entry through voltage-sensitive Ca2+
channels. The magnitude and spatial distribution of the
[Ca2+]i increase
depends on the distribution of different
Ca2+ channels in the dendrites (Magee and
Johnston, 1995) and the amplitude and propagation pattern of the
backpropagating spikes (Callaway and Ross, 1995; Spruston et al.,
1995). Synaptic input, targeted to the dendrites, can enhance (Magee
and Johnston, 1997) or inhibit (Buzsáki et al., 1996; Tsubokawa
and Ross, 1996) spike backpropagation by shifting the membrane
potential or causing a conductance shunt in the dendrites. Some
modulators like carbachol (CCh, a muscarinic receptor agonist;
Tsubokawa and Ross, 1997), isoproterenol (a 
-adrenergic agonist;
Hoffman and Johnston, 1999), and serotonin (5-HT; Ross and Sandler,
1998) affect spike propagation by modulating dendritic conductances.
Serotonin (Sandler and Ross, 1999) and possibly other agonists can
affect spike-associated [Ca2+]i increases
by modulating directly the Ca2+ channels
that are opened by the action potentials.
In addition to these mechanisms, we recently showed that action
potential-associated
[Ca2+]i changes
could be enhanced by pairing backpropagating spikes with repetitive
synaptic activation (Nakamura et al., 1999a). The enhanced
[Ca2+]i increase
results from the synergistic action of
Ca2+ entering through spike-opened
Ca2+ channels and metabotropic glutamate
receptor (mGluR)-mobilized inositol 1,4,5-trisphosphate
(IP3) acting on IP3
receptors (IP3Rs) to release
Ca2+ from the endoplasmic reticulum (ER).
The magnitude of this increase is much larger than the increase
attributable to spikes alone, even when the amplitude of the spikes is
increased by neuromodulators.
During the course of these experiments we found that bath application
of the mGluR agonist 1-amino-cyclopentyl-1,3-dicarboxylate (t-ACPD) appeared to have the same effect as synaptic
activation in generating the synergistic release of
Ca2+ by action potentials in pyramidal
neurons (Nakamura et al., 1999a). Because other metabotropic agonists
coupled to IP3 mobilization are likely to have
the same effect, it was of interest to discover which agonists or
neurotransmitters could couple with action potentials to evoke
Ca2+ release. In addition, analytical
experiments with bath-applied agonists allow for more precise targeting
of different receptors, eliminate the spatial inhomogeneity of synaptic
stimulation, and bypass effects on presynaptic terminals or other
neurons in the slice. Several questions were of interest. Which kinds
of mGluRs participate in Ca2+ release?
Which Ca2+ channels opened by action
potentials promote release? What kinds of mechanisms are responsible
for the preferred release of Ca2+ in the
proximal apical dendrites? How close in space are the Ca2+ channels to the
IP3R channels and how close together are
different IP3Rs to each other? What levels of
[Ca2+]i and
IP3 are required to cause regenerative release?
These new experiments explore some of these questions.
Parts of this work have been published previously in abstract form
(Ross and Sandler, 1998; Barbara et al., 1999; Nakamura et al., 1999b,
2000).
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MATERIALS AND METHODS |
Transverse hippocampal slices (300 µm thick) were prepared
from 2- to 3-week-old Sprague Dawley rats as previously described (Tsubokawa and Ross, 1997; Nakamura et al., 1999a). Animals were anesthetized with methoxyflurane before decapitation. Slices were incubated for at least 1 hr in normal artificial CSF (ACSF) composed of
(in mM): 124 NaCl, 2.5 KCl, 2 CaCl2,
2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10-20 glucose, bubbled with a
mixture of 95% O2/5% CO2,
making a final pH of 7.4. During experiments the ACSF was superfused
over the slice at a rate of 1 ml/min. Tight seals on CA1 pyramidal cell
somata and electrophysiological recordings were made as previously
described (Sakmann and Stuart, 1995; Nakamura et al., 1999a). The
standard patch pipette solution contained (in mM): 140 K-gluconate, 4 NaCl, 4 Mg-ATP, 0.3 Na-GTP, and 10 HEPES, pH-adjusted to
7.2-7.4 with KOH. For some experiments we added 14 mM
Na2-phosphocreatine. This solution was
supplemented with 0.1-1.0 mM of a selected calcium
indicator (fura-2, bis-fura, fura-6F, fura-2-FF, furaptra; all from
Molecular Probes, Eugene, OR). Typical osmolarity was 300 mOsm. AP-5
[(±)-2-amino-5-phosphonopentanoic acid], CNQX
(6-cyano-7-nitroquinoxaline-2,3-dione), MCPG
[(R,S)-
-methyl-4-carboxyphenylglycine], t-ACPD, ryanodine, serotonin, -methyl-serotonin,
phenylephrine, dopamine, nimodipine, nifedipine, nitrendipine,
-conotoxin GVIA, BAPTA
[1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic
acid], EGTA, IP3
(D-myo-inositol 1,4,5-trisphosphate),
CPA (cyclopiazonic acid), TTX (tetrodotoxin), ruthenium red, and
low-molecular-weight heparin were obtained from Sigma/RBI (St. Louis,
MO). DHPG [(S)-3,5-dihydroxyphenylglycine], CHPG
(2-chloro-5-hydroxyphenyglycine), AIDA (1-aminoindan-1,5-dicarboxylic acid), 4-CPG (4-carboxyphenylglycine), MPEP
[2-methyl-6-(phenylethynyl)-pyridine], and LY367385
[(+)-2-methyl-4-carboxyphenylglycine] were obtained from Tocris
(Ballwin, MO). -Agatoxin-IVA was obtained from Peptides International (Louisville, KY). Thapsigargin and
3-F-IP3
(3-deoxy-3-fluoro-D-myo-inositol 1,4,5-trisphosphate) were obtained from Calbiochem (San Diego, CA).
Caffeine was obtained from Wako Chemicals (Tokyo, Japan). Adenophostin
A was a generous gift from Sankyo (Tokyo, Japan).
Nimodipine, nitrendipine, and nifedipine were prepared as stock
solutions in ethanol or DMSO at 1000× final concentration and diluted
into ACSF. In separate experiments we found that this concentration of
ethanol or DMSO had no measurable effect on the electrical or optical
parameters that were measured in these experiments. Dopamine was
prepared daily as a 30 mM stock solution containing 0.2%
ascorbic acid. For experiments this solution was diluted 100× in ACSF.
Control experiments demonstrated that 0.002% ascorbic acid in ACSF did
not change the electrical or optical properties of pyramidal neurons.
For ACSF containing 10-20 mM caffeine, NaCl was reduced by
a comparable amount to preserve osmolarity. The other compounds were
added directly to the ACSF or pipette solution at the indicated concentrations.
In a typical experiment a train of backpropagating action potentials
was evoked with a train of 1 msec depolarizing pulses from the patch
electrode. Time-dependent
[Ca2+]i
measurements from different regions of the pyramidal neuron were made
as previously described (Lasser-Ross et al., 1991; Nakamura et al.,
1999a). Electrical traces were recorded simultaneously and matched to
the optical recordings. Data were taken and analyzed with Windows-based
software written in our laboratory. Images were taken at 33 msec
intervals. Electrical records were sampled at 200 µsec intervals.
During the experiments the solution changes were made by switching
among different sources without changing the flow rate. In our system
the chamber composition was changed by 90% in ~4 min.
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RESULTS |
Spike-evoked Ca2+ release in the presence of
t-ACPD
Backpropagating action potentials induce
[Ca2+]i increases
at all pyramidal cell locations by opening voltage-sensitive
Ca2+ channels in the dendrites (Jaffe et
al., 1992). This is illustrated in the first column of Figure
1, which shows the
[Ca2+]i change
that is associated with a pair of action potentials evoked by brief
intrasomatic pulses. The rapid rise in
[Ca2+]i occurs at
essentially the same time at all dendritic locations, reflecting the
high velocity of spike backpropagation (Spruston et al., 1995).
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Figure 1.
Backpropagating action potentials evoke
Ca2+ waves in the presence of t-ACPD.
In control ACSF (first column) a pair of spikes
initiated with brief intrasomatic pulses caused rapid, almost
simultaneous, increases in [Ca2+]i
( F/F) at all locations in the
cell. This increase is shown in two ways. In the top
panel the amplitude changes are indicated by a change in color.
Positions along the ordinate correspond to the pixels
overlaid on the cell image in the fourth column.
Positions along the abscissa correspond to the same time
scale as in the bottom panel. Below the
pseudocolor image the same data are plotted as time-dependent changes
for the two regions of interest (red and
green boxes) indicated on the image of
the cell. In ACSF containing 30 µM t-ACPD,
the same pair of spikes caused the same synchronous increases in
[Ca2+]i, followed by larger
increases at different times in different locations. The pseudocolor
image shows that this secondary increase propagated as a wave that
initiated at a location ~50 µm from the soma. This wave did not
propagate into the distal apical dendrites nor into the basal
dendrites. Note that the pseudocolor scale has been changed to include
the larger-amplitude secondary response. Five spikes (third
column) caused a similar secondary response that initiated
earlier and more synchronously at different dendritic locations.
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To isolate the effects of metabotropic agonists, we added 10 µM CNQX and 100 µM AP-5 to the ACSF to
block ionotropic glutamate receptors in all of the experiments
described in this paper. When 30 µM t-ACPD was
added to this solution and superfused into the bath, the cell
depolarized 5.5 ± 0.3 mV (n = 21). The resting fluorescence of the bis-fura-2-filled cell, at dendritic locations within 30 µm from the soma, decreased by 3.8 ± 0.4%
(n = 13) in the first few minutes after the solution
change. At later times it was difficult to assess the change because
the indicator concentration was increasing slowly and these
measurements were made at a single excitation wavelength. Assuming that
the resting
[Ca2+]i in the
cell was ~100 nM and that the
KD for bis-fura-2 binding to
Ca2+ is 370 nM
(Molecular Probes catalogue), then this fluorescence decrease
corresponds to an increase in
[Ca2+]i of <20
nM (Lev-Ram et al., 1992). This small increase
could result from entry through voltage-sensitive
Ca2+ channels opened by the potential
change near resting potential (Magee et al., 1996) or through a
nonselective cation conductance activated by group I mGluRs (Congar et
al., 1997). We did not investigate the mechanism of this increase in
the present experiments. When t-ACPD is applied rapidly to
the bath (Bianchi et al., 1999) or puffed onto the cell (Jaffe and
Brown, 1994), larger
[Ca2+]i increases
are recorded. However, these increases were not observed in our
experiments in which the bath composition was changed slowly by being
superfused at ~1 ml/min.
In the presence of 30 µM t-ACPD the
backpropagating spikes evoked a secondary increase in
[Ca2+]i that was
much larger than in control ACSF (Nakamura et al., 1999a). With one or
two action potentials the secondary increase propagated as a wave in
the apical dendrites (Fig. 1, second column). The wave did
not propagate into the distal dendrites although the sharp spike-evoked
[Ca2+]i increase
could be observed in this region. In this figure the wave had a clear
initiation point ~50 µm from the soma. The rapid rise in
[Ca2+]i at the
initiation point began ~0.5 sec after the spikes. In five cells in
which release was activated by a single spike, the delays were 0.15, 0.35, 0.5, 0.8, and 2.1 sec. When there was a single initiation site,
this location was always in the proximal apical dendrites. In several
cells two initiation points could be observed. In two of these cells
the second initiation site was in the basal dendrites, within 10 µm
of the soma. However, these waves did not propagate basally >20 µm
from the soma. When five action potentials were stimulated (Fig. 1,
third column), the initial sharp
[Ca2+]i increase
was larger and the secondary response appeared more synchronously at
all locations at which it was observed and with a much shorter delay.
This was the consistent pattern in >100 pyramidal neurons.
To test whether the action potentials or just the brief stimulating
pulses were responsible for both the rapid and secondary [Ca2+]i increases,
we added TTX (1.0 µM) to the superfusate (Fig.
2). In this solution the action
potentials were blocked, the rapid spike-evoked
[Ca2+]i increase
was eliminated, and there was no secondary increase (n = 2). However, when a strong 0.2 sec depolarizing pulse was given, both
a rapid increase, time-locked to the pulse, and a secondary increase
were recorded. Washing out the t-ACPD left only the rapid
[Ca2+]i increase.
These results suggest that it was not action potentials, specifically,
that caused release but, rather, the membrane depolarization.
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Figure 2.
Membrane depolarization sufficient to cause a
significant increase in [Ca2+]i is
required to evoke Ca2+ release. In control ACSF a
train of 10 backpropagating spikes initiated by 1 msec depolarizing
pulses caused a rapid increase in
[Ca2+]i at a dendritic location ~30
µm from the soma. When 30 µM t-ACPD was
added to the ACSF, the same 10 spikes caused a secondary
[Ca2+]i increase. The addition of 1.0 µM TTX blocked the spikes and eliminated both the primary
and secondary [Ca2+]i increases.
Substituting a single 200 msec pulse for the train of brief pulses
restored both components of the
[Ca2+]i transient. Finally, washing
out t-ACPD left a rapid
[Ca2+]i increase similar to that
caused by spikes in the same solution.
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Responses with different indicators and different
stimulus intervals
The large magnitude of the secondary
[Ca2+]i increases
(often >50% F/F, using 100-300
µM bis-fura-2 in the pipette) suggested that
the released Ca2+ might be saturating the
indicator. To examine this possibility, we tested different indicators
resembling bis-fura-2. These indicators have similar spectral
characteristics (Molecular Probes catalogue), and their properties
could be assayed with the same fluorescence filter set. Figure
3 shows results with five indicators
having different KD values. The
KD values were obtained from the
Molecular Probes catalogue except for fura-2-FF (Schneggenburger et
al., 1999) and furaptra (Naraghi, 1997). For each indicator the figure shows representative fluorescence changes recorded from a small region
~30 µm from the soma (fura-2, n = 9; bis-fura-2,
n > 100; fura-6F, n = 68; fura-2-FF,
n = 2; furaptra, n = 2). The smaller gray traces show the change that follows a train of 20 action potentials in normal ACSF. These traces all have been scaled to the
same amplitude on the assumption that they all represent the same
magnitude of
[Ca2+]i change.
This assumption is not exactly true, because the indicators differ in
their buffering power and nonlinearity in response to Ca2+. However, the similar time courses
for all of the traces except that recorded with fura-2 suggest that the
error is not large. Note that the percentage of change in fluorescence
is very different between the high-affinity fura-2 and the low-affinity
furaptra. In the presence of 30 µM
t-ACPD the same train of action potentials evoked much
larger fluorescence increases (Fig. 3, darker traces). The
rounded tops of the traces recorded with fura-2 and bis-fura-2 (Fig. 3,
arrows) suggests that these responses saturated the
indicators. Much larger relative changes were recorded with the
low-affinity indicators fura-6F, fura-2-FF, and furaptra. None of these
responses appeared to saturate the indicators. The larger relative
response with furaptra (n = 2) may reflect the fact
that this indicator interfered less with the regenerative release
process as well as the fact that it was linear throughout the range of
physiological fluorescence changes. The 7% fluorescence change
detected with furaptra corresponds to a
[Ca2+]i increase
of ~3 µM (Lev-Ram et al., 1992; Nakamura et
al., 1999a). The 1% increase in normal ACSF corresponds to a change of
~0.4 µM. This is close to the magnitude
reported by Helmchen et al. (1996) for a train of action potentials
that used very low concentrations of fura-2.
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Figure 3.
Fluorescence changes evoked by backpropagating
action potentials, using indicators of different
Ca2+ affinities. The different indicators and their
approximate dissociation constants are shown at the top.
The gray traces show the increases in normal ACSF that
were observed at a location ~30 µm from the soma. All of these
increases have been scaled to the same size, although the amplitudes
(F/F) varied from 45 to 1%.
The black traces show the fluorescence increases that
were observed in ACSF containing 30 µM
t-ACPD. The increases that were recorded with fura-2 and
bis-fura-2 have flat tops (arrows) suggesting indicator
saturation. The ratio of increases in t-ACPD to those in
normal ACSF that were recorded with the lower-affinity indicators is
much larger.
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For pharmacological experiments in the rest of the paper, in which
measuring the relative magnitude of the fluorescence change under
different conditions was important, we used fura-6F. This indicator is
relatively linear in the physiological range, does not buffer the
responses significantly, and gives signals with a satisfactory
signal-to-noise ratio. For experiments designed to determine
qualitatively whether release occurred, we used low concentrations of
bis-fura-2. This indicator gives signals that can be recorded with a
good signal-to-noise ratio over most of the cell, is not toxic to the
cells, and bleaches <1% in a typical trial.
To assess the effect of different pharmacological agents on the release
process, we believed that it was important to have stable conditions in
which experiments produced repeatable results. Therefore, we tested
whether trains of spikes at different trial intervals consistently
could evoke
[Ca2+]i changes of
similar amplitude. Figure 4 shows
representative experiments that used 300 µM fura-6F as
the indicator. In each case a train of 5-10 action potentials at 30 msec intervals was evoked, first in normal ACSF and then with 30 µM t-ACPD added to the bath. In normal ACSF,
10 action potentials evoked a peak increase of 4%
F/F in a region in the proximal apical
dendrites. In t-ACPD the spikes evoked
Ca2+ release, and the peak fluorescence
increase was 18-20%. After the baseline response was established, in
each of the panels (Fig. 4A-D) the interval between
trials was varied. When the interval was 10 sec (Fig.
4A), the magnitude of the fluorescence increase was
reduced by 74.6 ± 5.5% from the peak value (n = 7). The percentage of reduction was not sensitive to the position of
the selected region over the first 50 µm of the apical dendrites. The
selected examples to the right show that the release transient was
delayed as well as reduced in size after repetitive stimulation at 10 sec intervals. After a 2 min wait the amplitude and delay recovered almost to their original values. Experiments at longer intertrial intervals showed less of a reduction. At 20 sec intervals the peak was
reduced 53.5 ± 4.0% (n = 4); at 30 sec intervals
the peak was reduced 23.5 ± 7.8% (n = 4); at 60 sec intervals the peak was reduced by 15.3 ± 3.5%
(n = 6). The reductions measured at 30 and 60 sec
intervals were not significantly different. Therefore, most experiments
were done at 30 or 60 sec intervals.
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Figure 4.
[Ca2+]i increases
observed after trains were stimulated at different intervals.
A, Trains evoked at 10 sec intervals. A train of 10 spikes at 30 msec intervals was evoked in normal ACSF. Trace
1 in the right panel shows the
response at the location on the cell image (left
panel). This pattern was repeated four times at an
intertrain interval of 30 sec. The peak response amplitudes for each of
these trials are plotted as the first four points in the
middle graph. When 30 µM
t-ACPD was added to the bath (horizontal
bar), the same spike train evoked a secondary
[Ca2+]i increase of larger amplitude.
This pattern was repeated several times. A representative response
(2) is shown in the right panel.
When the interval between trains was reduced to 10 sec, the amplitude
decreased. At the end of several minutes (4) the
amplitude was reduced ~60%, and the response was delayed. When the
intertrain interval was increased, the amplitude increased and the
delay was shortened (5). Washing out the
t-ACPD restored the initial response. B,
A similar experiment that used 20 sec intertrain intervals. The
amplitude reduction was less than that observed with 10 sec intervals.
C, A similar experiment that used 30 sec intertrain
intervals. There was almost no reduction in amplitude.
D, A similar experiment that used 60 sec intervals. For
these experiments there were only five spikes in the train. There was
no observed amplitude reduction from trial to trial.
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The strong peak reduction at 10 and 20 sec intervals suggests that the
stores did not replenish their Ca2+ in
this interval or that the receptors on ER did not recover from
inactivation (Hajnoczky and Thomas, 1994). Whichever the explanation,
these experiments show that the recovery time constant after
spike-evoked regenerative release is ~30 sec.
These experiments also suggest that the
Ca2+ entering the cytoplasm with the
spikes did not make a significant contribution to replenishing the
stores. The magnitude of the
[Ca2+]i increase
after five spikes in control conditions was five times less than the
magnitude in 30 µM t-ACPD when the trials were
conducted at 1 min intervals (Fig. 4D). Nevertheless,
this large-amplitude [Ca2+]i increase
could be evoked repeatedly over a 15 min interval. It seems unlikely
that the small
[Ca2+]i increase
attributable to the spikes could supply enough
Ca2+ to refill the stores. Rather, the
stores probably were refilled from the
Ca2+ that was released in the previous
trial. However, we cannot rule out the possibility that some of the
spike-evoked Ca2+ entry replenished
Ca2+ that leaked out or was pumped from
the cell. We also found that release could be evoked with only a few
spikes in the first trial in experiments in which the slice was
preincubated with 30 µM t-ACPD (data
not shown). In these experiments no action potentials were given to
load the stores. This result supports the idea that spikes are not
needed to fill the stores. This conclusion differs from that of some
previous investigators (Jaffe and Brown, 1994) who emphasized the
importance of filling stores before evoking release.
Mechanism of release
In experiments that used synaptic stimulation, we showed that
regenerative Ca2+ release was attributable
to mGluR-activated IP3 synergistically acting
with Ca2+ entering through spike-opened
Ca2+ channels to open
IP3Rs (Nakamura et al., 1999a). The similar shape
of the release transients evoked in t-ACPD suggests that the
same mechanism was at work in these new experiments. To test this
hypothesis, we repeated the pharmacological experiments of Nakamura et
al. (1999a) by using bath-applied t-ACPD. Figure
5A shows that spike-evoked
release was blocked by 20 µM CPA
(n = 6), which blocks the ER
Ca2+-ATPase (Seidler et al., 1989).
Release also was blocked by 10 µM ryanodine
(Fig. 5B; n = 5), which depletes the ER by
keeping the ryanodine receptor channel open (Rousseau et al., 1987).
Including ruthenium red, which blocks the ryanodine receptor in the
closed state (Smith et al., 1988), in the pipette did not block release but did prevent the action of 10 µM ryanodine
(Fig. 5C; n = 5). In addition, release was
prevented by including low-molecular-weight heparin (1 mg/ml,
n = 5; data not shown) in the pipette. Heparin blocks
the IP3Rs nonspecifically in a variety of cell
types (Ghosh et al., 1988; Kobayashi et al., 1988). Heparin did not
block the slow depolarization induced by t-ACPD, suggesting
that a non-IP3-dependent pathway requiring mGluR
activation mediated this voltage change.
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Figure 5.
Effect of different pharmacological agents on
spike-evoked release in the presence of 30 µM
t-ACPD. A, CPA blocks release. Cell
image, peak F/F graph, and insets are
similar to those shown in Figure 4. In normal ACSF a train of 20 backpropagating spikes evoked at 30 msec intervals caused a small
[Ca2+]i increase linked in time to the
spikes (1). When 30 µM
t-ACPD was added to the bath, a larger increase was
observed (2). Adding 20 µM CPA to
the solution reduced the [Ca2+]i
increase to the level that was observed in control conditions
(3). Each point on the graph
represents a separate trial evoked at 30 sec intervals. Some points
have been deleted because other protocols were tested at those times.
B, A similar experiment showing that 10 µM
ryanodine added to the ACSF blocked the secondary
[Ca2+]i increase. C, A
similar experiment showing that 10 µM ryanodine did not
block the secondary [Ca2+]i increase
when 120 µM ruthenium red was included in the patch
pipette. D, Summary histogram showing the effects of the
different agents. In addition to showing the results of parts
A-C of this figure, the histogram shows that 1 mg/ml of
heparin in the pipette blocked release, 20 mM caffeine that
was added to the ACSF blocked release, and preincubation with 3 µM thapsigargin blocked release.
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These results parallel those we found when mGluRs were activated by
synaptic transmission (Nakamura et al., 1999a). We also tested several
additional compounds that are known to affect intracellular stores.
Thapsigargin, like CPA, blocks the ER
Ca2+-ATPase (Thastrup et al., 1990).
Preincubation of the slice with 3 µM thapsigargin
(n = 3 of 4) prevented spike-evoked release (data not
shown). Caffeine sensitizes the ryanodine receptor and, at high
concentrations, opens the receptor at rest, depleting the stores
(Zucchi and Ronca-Testoni, 1997). Caffeine also blocks IP3Rs in some preparations (Parker and Ivorra,
1991). Caffeine (2-10 mM, n = 7;
data not shown) blocked t-ACPD-mediated release in all
experiments, an effect compatible with either mechanism. Caffeine by
itself caused only a 10-20% increase in the spike-evoked [Ca2+]i change in
normal ACSF without altering the time course of the [Ca2+]i transient
(Sandler and Barbara, 1999). This small increase may be attributable to
the effect of caffeine on the indicator (Muschol et al., 1999).
However, we have no direct evidence supporting this explanation. The
absence of regenerative spike-evoked release in the presence of 2-5
mM caffeine is interesting because this protocol has been reported to cause all-or-none
Ca2+-induced
Ca2+ release (CICR) transients in rat
sensory neurons (Usachev and Thayer, 1997). That mechanism,
mediated by ryanodine receptors, does not appear to be significant in
pyramidal neurons. Figure 5D summarizes the results by using
agents that modulate intracellular stores.
To reinforce the conclusion that release was mediated by the activation
of IP3Rs, we tested several compounds that are
relatively specific activators of this receptor. Adenophostin A at low
concentrations is a potent IP3R agonist
(Takahashi et al., 1994). When 0.3 µM adenophostin A was
included in the pipette, a train of action potentials evoked an
increase in dendritic
[Ca2+]i that
outlasted the train of spikes (n = 5; Fig.
6A, trace
1). The time course of the
[Ca2+]i change resembled that evoked
by t-ACPD and spikes (see, for example, Fig. 1). To confirm
that this increase was attributable to release of
Ca2+ and not some nonspecific effect, we
added 10 mM caffeine. Caffeine, as indicated
above, empties the stores and blocks IP3Rs. In
this condition the same train of spikes evoked a smaller
[Ca2+]i increase
that was linked in time to the action potentials (Fig. 6A, trace 2). In control experiments (data
not shown) we found that caffeine did not reduce the spike-evoked
[Ca2+]i increase.
Washing out the caffeine allowed the stores to refill. Because
adenophostin A was still in the cell, spikes again evoked release (Fig.
6A, trace 3). Figure 6B
shows similar experiments that used 100 µM
3-F-IP3 in the pipette instead of adenophostin A
(n = 3). This analog of IP3,
resistant to 3-kinase, is equipotent to IP3 in
releasing Ca2+ from stores in other
preparations (Kozikowski and Fauq, 1990). Finally,
IP3 itself, at a concentration of 500 µM in the pipette, also caused
Ca2+ release when it was paired with a
train of action potentials (Fig. 6C; n = 6).
These concentrations of agonists were close to the threshold
concentrations for this protocol because 0.1 µM
adenophostin A (n = 3), 30 µM
3-F-IP3 (n = 5), and 300 µM IP3 (n = 5) did not cause spike-associated Ca2+
release. Together, these results strongly support the conclusion that
release is mediated via the action of IP3 on the
IP3R.
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Figure 6.
Effect of IP3 and analogs in evoking
spike-associated Ca2+ release. A,
Adenophostin A (0.3 µM) was included in the patch
pipette. A train of 10 backpropagating spikes evoked at 30 msec
intervals caused a [Ca2+]i increase
that had an inflection (arrow) on the rising phase
(1). The addition of 10 mM caffeine
to the ACSF reduced the amplitude of the
[Ca2+]i increase and removed the
inflection (2). Washing out the caffeine restored
the larger amplitude and rounded time course of the
[Ca2+]i increase
(3). B, A similar experiment in
which 100 µM 3-F-IP3 was included in the
pipette. This analog also caused a spike-associated secondary
[Ca2+]i increase that was blocked by
10 mM caffeine. C, A similar experiment in
which 500 µM IP3 was included in the pipette.
IP3 also caused a secondary
[Ca2+]i increase that was blocked by
10 mM caffeine. Scale in cell image, 50 µm.
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One difference between release mediated by tonic activation of mGluRs
and release mediated by intracellular injection of
IP3R agonists is that the amplitude of
spike-associated Ca2+ release in the
presence of intracellular IP3R agonists was
smaller than the amplitude in the presence of t-ACPD.
Increasing the concentration of IP3R agonists did
not increase the amplitude of the
[Ca2+]i change.
This result might suggest that the IP3 agonists
are not activating the same release process as t-ACPD.
However, injection of IP3R activators at
concentrations high enough to cause release occluded the effect of
t-ACPD; i.e., when t-ACPD was added to the bath,
large amplitude spike-evoked release did not occur in the presence of
intracellular agonists (1.0 µM adenophostin A, n = 5; 100 µM
3-F-IP3, n = 3; 500 µM IP3, n = 2; data not shown). Lower concentrations of
IP3R agonists (0.1 µM
adenophostin A, n = 2; 30 µM
3-F-IP3, n = 3; 300 µM IP3, n = 3), which did not cause even a low level of release, did not occlude
the effect of t-ACPD. The reason for the small amplitude of
release with injected IP3R agonists is not clear.
The IP3R agonists did not occlude the resting
membrane depolarization caused by t-ACPD, an observation
that parallels the results with injected heparin.
Effects of calcium channel blockers
Two kinds of experiments indicate that the critical role of spikes
in the release process is to increase transiently the
[Ca2+]i in the
cells to act cooperatively with IP3 to open the
IP3R channels. First, blocking
Ca2+ entry with 200 µM
Cd2+ strongly reduced the immediate
[Ca2+]i increase
and prevented the secondary
[Ca2+]i increase
attributable to release (Fig. 7). Because
the immediate increase was time-locked to the spikes and its amplitude
was insensitive to CPA and ryanodine, it reflects
Ca2+ entry through voltage-dependent
Ca2+ channels. This figure also shows that
Cd2+ had no effect on the resting
bis-fura-2 fluorescence intensity, indicating that
Cd2+ did not enter the cell to quench the
indicator fluorescence. Second, in most experiments that used
bis-fura-2 as the indicator, one or two spikes did not cause release,
but a larger number of spikes were effective. More spikes increase the
peak [Ca2+]i level
reached in a train. In contrast, when furaptra was used, only one or
two spikes were necessary. This low-affinity indicator does not buffer
Ca2+ significantly and allows the peak
free [Ca2+]i to
reach higher levels. These experiments suggest that spikes produce a
threshold level of
[Ca2+]i that
cooperatively acts with IP3 to trigger
release.
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Figure 7.
Cd2+ (200 µM)
blocks both the primary and secondary spike-associated
[Ca2+]i increase in the presence of 30 µM t-ACPD. The middle panel
shows that Cd2+ did not affect the resting
fluorescence level in the cell, indicating that Cd2+
did not enter the cell to quench the indicator fluorescence. Scale is
in arbitrary units. The bottom panels show
representative responses. A small spike-associated response,
insufficient to evoke release, remained in
Cd2+-containing ACSF.
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Because the almost complete block of Ca2+
entry by Cd2+ was effective in preventing
release, it was of interest to see whether entry through any of the
major kinds of Ca2+ channels was
particularly effective in inducing release. Figure 8 shows experiments in which we tested
several antagonists that are relatively specific for different
Ca2+ channel types. At 100 µM, Ni2+ preferentially
blocks low-threshold T-type channels (Ozawa et al., 1989; Mogul and
Fox, 1991). When superfused over the preparation, Ni2+-containing ACSF reduced the
spike-evoked
[Ca2+]i increase
by 25.3 ± 1.1% (n = 3) as previously reported
(Christie et al., 1995). However, with the addition of 30 µM t-ACPD the spikes still were able
to evoke a large secondary
[Ca2+]i increase
(Fig. 8A; n = 4 of 5). Similar
results were obtained with 1-5 µM
-conotoxin-GVIA, a blocker of high-threshold N-type Ca2+ channels (McCleskey et al., 1987).
This toxin reduced the sharp spike-evoked
[Ca2+]i increase
by 26.7 ± 1.4% (n = 7) but did not prevent
release (Fig. 8B).
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Figure 8.
Selective Ca2+ channel blockers
do not prevent spike-associated Ca2+ release in the
presence of 30 µM t-ACPD.
A, A train of 10 backpropagating spikes evoked at 30 msec intervals caused a rapid [Ca2+]i
increase at a location close to the soma in the apical dendrites. The
amplitude of this change was approximately constant when these trains
were evoked at 30 sec intervals (1). The addition
of 100 µM NiCl2 to the ACSF reduced the
amplitude of this increase without changing the shape of the transient
(2). When t-ACPD was added to this
solution, a secondary [Ca2+]i increase
was observed (3). B-D, Similar
experiments are shown with 1 µM -CTX-GVIA, 10 µM nimodipine, and 400 nM -Aga-IVA. None
of these agents prevented release by t-ACPD
(B, D) or CCh (C).
-CTX-GVIA reduced the spike-associated
[Ca2+]i in normal ACSF, whereas
nimodipine and -Aga-IVA did not. For the experiment with
-Aga-IVA, fura-6F was the Ca2+ indicator,
resulting in faster transients.
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Almost similar results were obtained with dihydropyridine antagonists
of L-type Ca2+ channels. Nimodipine (Fig.
8C; 10-30 µM; n = 15), nifedipine (10 µM; n = 6),
and nitrendipine (10 µM; n = 1)
did not prevent spike-evoked Ca2+ release.
Interestingly, none of these antagonists had a significant effect on
the sharp spike-evoked
[Ca2+]i increases
in these slice experiments. This result differs from previous sharp
electrode experiments that found an L-type channel component in
spike-associated
[Ca2+]i increases
(Christie et al., 1995). Two control experiments confirmed that
dihydropyridine-containing ACSF was capable of blocking L-type
Ca2+ channels. In one, 10 µM nimodipine reduced the
[Ca2+]i increase
caused by a 1 sec depolarizing pulse (n = 3; data not
shown). This rules out the possibility that many of the L-type Ca2+ channels were washed out (Tombaugh
and Somjen, 1997). In the second, the same solution dilated isolated
rat blood vessels (Ungvari et al., 2000), confirming the effectiveness
of the nimodipine we purchased. Thus, it is likely that few L-type
channels are opened by backpropagating action potentials, although
these channels are found in CA1 pyramidal neurons (Westenbroek et al.,
1990; Magee and Johnston, 1995). One possible explanation for these results is that the activation kinetics of the L-type channels are slow
and the channels are not opened by the action potential (Mermelstein et
al., 2000). The important result, however, is that L-type channels are
not connected specifically to the IP3R-mediated release mechanism in pyramidal neurons. This conclusion applies even if
L-type Ca2+ channels are washed out,
because in that case there can be no connection between these
Ca2+ channels and any intracellular
receptor. A strong connection has been implicated between L-type
channels and ryanodine receptor-mediated release in other neurons
(Chavis et al., 1996).
Similar results were found with the P-type
Ca2+ channel blocker, -agatoxin-IVA
(Mintz et al., 1992). In five experiments 200-400 nM of
this toxin did not prevent release and had no significant effect on the
sharp spike-evoked
[Ca2+]i increase.
To test the potency of our sample, we confirmed that -agatoxin-IVA
could block the voltage-dependent
[Ca2+]i increase
in rat cerebellar Purkinje cells (n = 2; data not shown). We did not test the possibility that R-type
Ca2+ channels could be linked specifically
to the release process.
Effect of calcium buffers
The observation that several action potentials were effective in
causing Ca2+ release when one or two were
not suggests that a threshold
[Ca2+]i level
might be needed to trigger release. If this hypothesis is correct, then
buffering the peak
[Ca2+]i level with
high concentrations of indicator (Helmchen et al., 1996) will make it
more difficult to evoke release. Indeed, we found that 1.0 mM bis-fura-2 prevented synaptically evoked release, whereas 0.3 mM did not (Nakamura et al., 1999a). In new
experiments we found that the same conclusion applied to spike-evoked
release in the presence of t-ACPD (Fig.
9).
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Figure 9.
Effect of different Ca2+
buffers on the ability of spikes to evoke Ca2+
release in the presence of 30 µM t-ACPD.
When the pipette contained 0.5 mM bis-fura-2, release was
always observed. Increasing the concentration to 1.0 mM
bis-fura-2 prevented release. Different concentrations of EGTA and
BAPTA in the pipette were tested with electrodes also containing
100-200 µM of the low-affinity indicator fura-6F. BAPTA
(1.0 mM) and EGTA (2.0 mM) prevented release.
Numbers over the bars
indicate the cells that were tested.
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Release measured in the presence of different buffers also can reveal
information about whether Ca2+ ions acting
on particular IP3Rs diffuse far from their source (either Ca2+ channels or other
IP3Rs) or whether the receptors and channels are
close to each other. Because the rate constants for
Ca2+ binding to EGTA are much slower than
the rate constants for binding to BAPTA, a large difference in the
effectiveness of these two buffers has been taken to indicate that
Ca2+ does not go far before activating its
target (Adler et al., 1991). To test this possibility, we included EGTA
and BAPTA in the recording pipette at different concentrations. No
Ca2+ was added except for trace amounts.
We also included a low concentration of fura-6F to monitor the
[Ca2+]i change.
This low-affinity indicator (KD~6
µM) has only a weak buffering effect on the
spike-evoked
[Ca2+]i increase.
Figure 9 shows that the effective concentration of BAPTA and EGTA
differed by only a factor of two. This result suggests that the source
and target of Ca2+ in the release process
are not very close to each other.
Pharmacology of mGluRs
In previous experiments (Nakamura et al., 1999a) we showed that
synaptically activated Ca2+ release could
be blocked by 1 mM MCPG, a group I and II mGluR antagonist
(Schoepp et al., 1999). To determine more precisely the receptor types
participating in the release process, we tested a series of known mGluR
agonists and antagonists. For the agonists we determined the minimum
concentration that reliably evoked release after a train of 10-20
action potentials. We found that 20 µM t-ACPD
(n = 28), 5 µM DHPG
(n = 6), and 500 µM CHPG
(n = 9) were effective (Table
1). Of these, CHPG is reportedly the most
selective agonist, acting preferentially on
mGluR5 receptors (Doherty et al., 1997).
We found that the threshold agonist concentration capable of evoking
spike-associated release was not uniform in all regions of the cell.
When we used 0.5 mM CHPG, release was detected only in the
dendrites (n = 6 of 7). However, 1.0 mM CHPG evoked release in both the dendrites and
soma (n = 7 of 8; data not shown). Similarly, 20 µM t-ACPD preferentially evoked
release only in the dendrites (n = 16 of 29), whereas
30 µM t-ACPD consistently evoked
release in both locations (n = 29 of 30).
We next tested the ability of various mGluR antagonists to block
spike-evoked release in the presence of 30 µM
t-ACPD. As with the agonists, we found that the sensitivity
to the antagonists was not spatially uniform. Figure
10A shows that 1 µM MPEP blocked release in the soma but did not
block release in the dendrites (n = 3 of 3). However,
3-10 µM MPEP blocked release at both locations (Fig. 10B; n = 6 of 6). This
differential sensitivity to antagonists in the soma and dendrites,
measured at threshold concentrations, also was observed with AIDA
(n = 5 of 7 blocked only in the soma at 1 mM; n = 4 of 4 blocked at both
locations at 2 mM) and 4-CPG (n = 1 of 1 blocked only in the soma at 500 µM;
n = 2 of 2 blocked at both locations at 1 mM). Table 1 summarizes the results for MCPG,
AIDA, 4-CPG, MPEP, and LY367385. Because MPEP is reportedly an
mGluR5 antagonist (Gasparini et al., 1999) and
LY367385 is reportedly an mGluR1 antagonist
(Clark et al., 1997), these results suggest that both
mGluR1 and mGluR5 receptors
are coupled to IP3 mobilization on CA1 pyramidal
neurons.
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Figure 10.
MPEP has a differential effect in blocking
release in the soma and dendrites when it is applied at low
concentration. A,
[Ca2+]i increases in response to a
train of backpropagating spikes are shown at two locations, one at the
soma and one in the dendrites. When 30 µM
t-ACPD was added to the ACSF, a secondary increase of
larger amplitude was observed at both locations. When 1 µM MPEP was added to this solution, the secondary
increase in the soma was blocked, but the increase in the dendrites
remained. B, A similar experiment in which 10 µM MPEP was applied. In this case the secondary increase
was blocked at both locations.
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Some agonists and antagonists, like MPEP (Gasparini et al., 1999), are
reported to be very selective in distinguishing between mGluR1 and mGluR5
receptors. Others (Schoepp et al., 1999) are less selective. To examine
this selectivity, we tested several antagonists for their ability to
block the release induced by the putative mGluR5
agonist CHPG. Table 1 shows that AIDA, MPEP, and LY367385 were as
effective against 0.5 µM CHPG-induced release as they
were against 30 µM t-ACPD-induced release.
Because LY367385 is considered a mGluR1
antagonist, this result implies that either or both LY367385 and CHPG
cannot be completely selective for their putative receptor subtypes.
Consequently, these pharmacological experiments, by themselves, cannot
establish which of the two group I receptors are most prominent on CA1
pyramidal neurons.
Other metabotropic agonists
The experiments with t-ACPD established that
spike-evoked release of Ca2+ from internal
stores is mediated by IP3 produced by activation of mGluR. Because other neurotransmitters are known to activate receptors that mobilize IP3, we examined whether
these transmitters could participate in spike-evoked
Ca2+ release in pyramidal neurons. Figure
11 shows the effect of carbachol (CCh),
a nonhydrolyzable analog of acetylcholine. In this cell 3 µM CCh evoked release with approximately the
same amplitude and spatial distribution as 20 µM t-ACPD (n = 3).
The effect of 3 µM (n = 10) or
10 µM (n = 11) CCh was very
reliable, producing spike-evoked release in 20 of 21 cells. However, 3 µM was close to threshold, because 1 µM did not cause release (n = 6). CCh probably acts via M1 receptors (Hammer
and Giachetti, 1984) because release was blocked by atropine (1 µM, n = 3; 3 µM, n = 4; 10 µM, n = 2) and pirenzepine (0.5 µM, n = 3; data not shown).
M1 receptors couple to
IP3Rs (Hulme et al., 1990). Consistent with this
conclusion and with the similarity to t-ACPD-induced
release, we found that including low-molecular-weight heparin in the
pipette (1 mg/ml) blocked release in the presence of 10 µM CCh (n = 3). In addition,
release mediated by including 3-F-IP3 in the
pipette occluded the release mediated by 3 µM
CCh (n = 2), similar to the results with
t-ACPD. Therefore, it is likely that both t-ACPD and CCh release Ca2+ from the same
compartment in the same IP3-dependent manner.
Some evidence for the coupling of voltage-dependent
Ca2+ entry and muscarinic activation in
pyramidal neurons has been reported by other investigators (Irving and
Collingridge, 1998; Sah, 1999; Yamamoto et al., 2000).
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Figure 11.
CCh and t-ACPD evoke
spike-associated Ca2+ release of similar amplitude
and spatial distribution in pyramidal cells. In the presence of 30 µM t-ACPD a train of 20 spikes at 30 msec
intervals evoked a secondary increase in the soma and proximal apical
dendrites (panel 2). The colored
traces show the time-dependent increases at the indicated
locations on the cell image (panel 1). The
pseudocolor image shows the spatial distribution of the
[Ca2+]i increase at the end of the
spike train. Panel 3 shows the traces and spatial
distribution of the [Ca2+]i increase
after t-ACPD was washed from the bath. Panel
4 shows the traces and distribution after 3 µM
CCh was added. The traces and spatial distribution are similar to those
observed in t-ACPD. The graph on the
left shows the peak amplitude of the
[Ca2+]i change recorded at the
location of the white arrow during the entire
course of the experiment. The events at times 1,
2, and 3 are shown in the three
panels.
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We tested several other neurotransmitters that are known to couple to
IP3 mobilization in other preparations. Figure
12 shows typical experiments in which
we tested 30 or 100 µM phenylephrine (Phe, an
-adrenergic agonist; n = 5), 100 or 300 µM dopamine (DA; n = 7), and 10 µM 5-HT. In each case the agonists did not cause Ca2+ release from intracellular
stores. Subsequent application of either 10 µM
CCh or 30 µM t-ACPD did evoke
release, demonstrating that the ER was intact and capable of
release.
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Figure 12.
The metabotropic agonists Phe, DA, and 5-HT do
not evoke spike-associated Ca2+ release in pyramidal
neurons. A, In the presence of 100 µM Phe
a train of 10 backpropagating action potentials at 30 msec intervals
evoked only a small spike-linked
[Ca2+]i increase. Trials were repeated
at 30 sec intervals. One example (1) is shown at
the right. When Phe was washed out and replaced by 10 µM CCh, the same train evoked Ca2+
release (2). Scale under the
traces: Ordinate, 10%
F/F, 50 mV; abscissa, 1 sec. B, A similar experiment showing that 300 µM DA did not cause Ca2+ release.
C, A similar experiment showing that in most cases 10 µM 5-HT did not cause release.
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The experiments with 5-HT were interesting. Although most cells did not
respond to 5-HT, in six experiments we found that spikes evoked
Ca2+ release in ACSF containing 10 µM 5-HT (Fig. 13;
n = 6 of 35). This
[Ca2+]i increase
initiated and propagated as a wave in the proximal apical dendrites,
similar to the release transients observed with t-ACPD and
CCh. Increasing the concentration of 5-HT to 30 µM did not increase the probability of
observing release (n = 0 of 3). Tests with 100 µM -methyl-5-HT, a selective
5-HT2 receptor agonist known to mobilize
IP3 (Richardson et al., 1985), also caused
release in only a fraction of the tested cells (n = 1 of 5). Because the effect of 5-HT was unreliable, we could not analyze its pharmacology nor the spatial distribution of release in more detail.
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Figure 13.
5-HT sometimes evokes spike-associated
Ca2+ release in pyramidal cell dendrites. In control
conditions a train of 10 spikes at 100 msec intervals evoked
spike-linked [Ca2+]i increases in the
soma and along the dendrite. When 10 µM 5-HT was added to
the ACSF, the spikes evoked a large secondary increase in the dendrites
that peaked at different times at the two marked locations. The
graph to the right of the cell image
shows the delay-to-peak of the secondary release along the cell axis.
Time 0 is the peak time at the initiation site. The
length scale (ordinate) also applies to the cell image.
The increase occurred earliest at a point ~90 µm from the soma and
then spread as a wave in both directions from that point. Washing out
the 5-HT restored the transients to the control response.
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DISCUSSION |
These experiments explore the effects of bath-applied and
pipette-injected pharmacological agents on the spike-associated release
of Ca2+ from intracellular stores.
Although the bath-applied agents affected the entire hippocampal slice,
several arguments suggest that the major effects were directly on the
tested pyramidal neurons and not mediated via other cells. First, all
of the measured properties of release evoked by metabotropic agonists
matched those of synaptically activated release (Nakamura et al.,
1999a). In those experiments the close correspondence of the site of
release to the site of synaptic activation indicated that the effect
was mediated directly by glutamate released from stimulated Schaffer
axon collaterals and not via intermediate cells. Second, astrocytes
primarily release glutamate when activated (Parpura et al., 1994). If
these cells or other glutamatergic neurons were stimulated in our
experiments, their effects on pyramidal neurons would have been
overwhelmed by the bath-applied glutamate agonists. Third, the
pharmacological profile of effective agonists (CHPG and CCh, but not
Phe and DA) matches the profile of known receptor types on CA1
pyramidal neurons (see below).
Mechanism of release
The properties of agonist-induced regenerative
[Ca2+]i increase
are consistent with our analysis of synaptically activated
Ca2+ waves (Nakamura et al., 1999a). These
results indicate that the activation of ryanodine-sensitive stores is
not necessary to evoke release, although ryanodine receptors are
present on the compartment that releases
Ca2+. In new experiments we showed that
the injection of adenophostin A, 3-F-IP3, and
IP3 could promote spike-associated release
without the need for metabotropic agonists. The effectiveness of these specific IP3R agonists is more direct evidence
for the involvement of the IP3R. In addition,
pharmacological experiments indicate that release was promoted by group
I mGluRs, M1 muscarinic receptors, and
5-HT2 receptors. All of these receptors are
coupled to the mobilization of IP3 (McKinney,
1993; Conn and Pin, 1997; Hoyer and Martin, 1997). One caution is that
high concentrations of IP3R agonists were
required to promote release (100 µM
3-F-IP3 and 500 µM
IP3). These levels are higher than are needed to
evoke release in other preparations (Hirota et al., 1995; Missiaen et al., 1996). This difference could mean that the
IP3Rs in pyramidal neurons are different from
receptors in other preparations or that intracellular kinases or other
degradative enzymes rapidly metabolize the injected
IP3R agonists. However, all of the injected IP3R agonists occluded the large-amplitude
spike-associated release mediated by bath-applied metabotropic
agonists. This result suggests that the exogenously applied
IP3R agonists also might be exerting some
inhibitory effect on the release mechanism. We have no additional information concerning this possibility.
Spike-evoked release reached levels of several micromoles per liter
when measured with low-affinity indicators. This suggests that resting
[Ca2+]i levels in
the stores are in the micromolar per liter range, consistent with
indicator measurements in other preparations (Miyawaki et al., 1997).
We found that large
[Ca2+]i increases
were observed consistently when short trains of spikes were evoked at
30-60 sec intervals, but lower amplitudes were recorded when they were
stimulated at 10-20 sec intervals. The simplest explanation of this
result is that the stores require ~20 sec to refill after
regenerative release. This time is comparable to the recovery time that
is observed in these cells after store depletion by caffeine puffs
(Garaschuk et al., 1997). However, we cannot rule out the possibility
that the IP3Rs are inactivated or desensitized
for brief periods after stimulation (Hajnoczky and Thomas, 1994).
Spike-evoked release in the presence of t-ACPD was most
prominent in the proximal apical dendrites (Nakamura et al., 1999a). Two new kinds of measurements reinforce the conclusion that this part
of the cell is particularly sensitive. First, we found that threshold
levels of mGluR agonists caused release only in the proximal apical
dendrites, whereas higher concentrations also caused release in the
soma. Mirroring this finding, we found that threshold levels of
antagonists selectively blocked release in the soma, sparing the
dendrites, whereas higher concentrations blocked release everywhere. In
addition, when a single initiation site was detected (see, for example,
Fig. 1), it was always in the proximal apical dendrites. Second, we
found that the same spatial distribution was determined with all of the
agonists that evoked release
group I mGluR, M1
muscarinic, and 5-HT2. This similarity suggests
that the distribution of metabotropic receptors on the surface of the
pyramidal neuron is not responsible for the spatial distribution of
regenerative release, because this would require the same nonuniform
distribution for all receptors. The distribution of
Ca2+ channels is also unlikely to be
responsible because (1) selective block of different
Ca2+ channel types did not affect the
pattern of release and (2) the same pattern was observed without spikes
when the release was evoked synaptically (Nakamura et al., 1999a). The
more likely explanations are either that IP3Rs
are distributed more densely on the ER in the proximal apical dendrites
or that the IP3Rs are more sensitive in this
region of the cell (Thomas et al., 2000).
Receptors contributing to spike-evoked
Ca2+ release
Using specific agonists and antagonists, we analyzed the receptors
contributing to the regenerative
[Ca2+]i increase.
Previously (Nakamura et al., 1999a), we found that t-ACPD, a
relatively nonselective mGluR agonist, reliably evoked release. In
these new experiments we found that DHPG, a group I agonist, and CHPG,
a mGluR5 agonist, also evoked release. These results are expected because pyramidal neurons express
mGluR5 receptors (Shigemoto et al., 1993; Romano
et al., 1995; Lujan et al., 1996). Consistent with these observations,
we found that the phenylgycine-related antagonists MCPG, AIDA, and
4-CPG blocked release evoked by t-ACPD. These compounds
block both group I receptors, although their relative affinity for
mGluR1 and mGluR5 varies (Schoepp et al., 1999). We were not able to determine whether only one
of these subtypes releases Ca2+ in
pyramidal neurons, because some of the pharmacological agents were not
as specific as previously reported. Nevertheless, the presence of
either receptor on pyramidal neurons is consistent with our other
results because both receptor subtypes are known to couple to
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ABSTRACT
INTRODUCTION
