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The Journal of Neuroscience, May 1, 2002, 22(9):3454-3462
Nuclear Calcium Signaling Evoked by Cholinergic Stimulation in
Hippocampal CA1 Pyramidal Neurons
John M.
Power and
Pankaj
Sah
Division of Neuroscience, John Curtin School for Medical Research,
Australian National University, Canberra, ACT 0200, Australia
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ABSTRACT |
The cholinergic system is thought to play an important role in
hippocampal-dependent learning and memory. However, the mechanism of
action of the cholinergic system in these actions in not well understood. Here we examined the effect of muscarinic receptor stimulation in hippocampal CA1 pyramidal neurons using whole-cell recordings in acute brain slices coupled with high-speed imaging of
intracellular calcium. Activation of muscarinic acetylcholine receptors
by synaptic stimulation of cholinergic afferents or application of
muscarinic agonist in CA1 pyramidal neurons evoked a focal rise in free
calcium in the apical dendrite that propagated as a wave into the soma
and invaded the nucleus. The calcium rise to a single action potential
was reduced during muscarinic stimulation. Conversely, the calcium rise
during trains of action potentials was enhanced during muscarinic
stimulation. The enhancement of free intracellular calcium was most
pronounced in the soma and nuclear regions. In many cases, the calcium
rise was distinguished by a clear inflection in the rising phase of the
calcium transient, indicative of a regenerative response. Both calcium
waves and the amplification of action potential-induced calcium
transients were blocked the emptying of intracellular calcium stores or
by antagonism of inositol 1,4,5-trisphosphate receptors with
heparin or caffeine. Ryanodine receptors were not essential for the
calcium waves or enhancement of calcium responses. Because rises in
nuclear calcium are known to initiate the transcription of novel genes, we suggest that these actions of cholinergic stimulation may underlie its effects on learning and memory.
Key words:
gene; learning; memory; nucleus; IP3; acetylcholine; ryanodine
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INTRODUCTION |
Acetylcholine is an important
neuromodulatory transmitter affecting hippocampal-dependent learning
and memory (Hagan and Morris, 1988 ). Loss of cholinergic function has
been hypothesized to underlie age-related learning impairments and
memory loss that accompanies Alzheimer's disease (Bartus et al., 1982;
Kasa et al., 1997), and activation of the cholinergic system has been
shown to modulate processing in sensory and visual cortex (Furey et
al., 2000; Shulz et al., 2000). However, the mechanisms by which the
cholinergic system affects neuronal processing are not understood. It
is well known that acetylcholine, acting via muscarinic receptors,
modulates a number of potassium conductances in hippocampal pyramidal
neurons, including those that mediate spike frequency adaptation
(Nicoll et al., 1989). These actions of acetylcholine have been
suggested to underlie the mechanism of action of acetylcholine on
learning and memory (Hasselmo and Bower, 1993; Disterhoft et al.,
1999).
The long-term changes that underlie the final mechanisms in learning
and memory involve changes in gene transcription (Kandel and Pittenger,
1999). These changes are in large part initiated by rises in cytosolic
calcium (Ghosh and Greenberg, 1995; Finkbeiner and Greenberg, 1998),
which activates a number of signaling pathways. Under resting
conditions, cytosolic free calcium
[Ca2+]i is
maintained at 50-100 nM and can rise either as a result of influx from the extracellular space or by release from
intracellular stores (Tsien and Tsien, 1990; Berridge, 1998). The
pathways by which calcium can enter neurons from the extracellular
space are well characterized and include voltage-gated calcium channels and receptor-operated calcium channels, such as NMDA receptors. Much is understood about the activation of these pathways, their modulation, and the downstream consequences of the calcium rises that
follow (Ghosh and Greenberg, 1995; Berridge, 1998). Thus, rises in
cytosolic calcium attributable to influx via either L-type voltage-dependent calcium channels or NMDA receptors has been shown to
cause translocation of calmodulin to the nucleus and initiation of gene
transcription by phosphorylation of cAMP response element-binding
protein (CREB) (Deisseroth et al., 1996, 1998). Calcium is also
sequestered within the smooth endoplasmic reticulum (Henzi and
MacDermott, 1991) and can be released by activation of either inositol
1,4,5-trisphosphate (InsP3) or ryanodine
receptors (RyRs) (Tsien and Tsien, 1990; Berridge, 1998), both of which are present in abundance in central neurons (Sharp et al., 1993). Release of calcium from intracellular stores and the consequent rise in
nuclear calcium levels has been shown to be effective in initiating
gene transcription (Dolmetsch et al., 1998; Li et al., 1998; Hardingham
et al., 2001). Notably, in hippocampal neurons, a rise in nuclear
calcium without translocation of CaM is sufficient to activate the CREB
transcription pathway (Hardingham et al., 2001).
In addition to its actions on potassium currents, acetylcholine, acting
at muscarinic receptors, also stimulates the formation of
InsP3 and diacylglycerol (Fowler and Tiger, 1991;
Fisher et al., 1992). In non-neuronal cells, receptor-mediated
generation of InsP3 results in release of calcium
from intracellular stores, often in an oscillatory manner (Henzi and
MacDermott, 1991; Berridge, 1998). Here we show that activation of
muscarinic acetylcholine receptors on hippocampal pyramidal neurons
leads to rises of cytosolic calcium that are initiated in the apical
dendrite and propagate as a wave to the soma in which they invade the
nucleus. These calcium rises are attributable to release of calcium
from InsP3-sensitive intracellular calcium
stores. Because of the calcium dependence of the
InsP3 receptor, this mechanism also acts to
amplify nuclear calcium rises in response to trains of action
potentials. Thus, this mechanism functions as a nuclear detector of
cholinergic activation and may explain how memory formation is enhanced
during cholinergic activation.
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MATERIALS AND METHODS |
All experiments were done on transverse hippocampal slices
prepared from 17- to 28-d-old rats using standard methods (Sah and
Isaacson, 1995). For recording, slices were transferred to the stage of
a BX50 microscope (Olympus Optical, Tokyo, Japan) and superfused
with artificial CSF (aCSF) containing (in mM): 119 NaCl, 2.5 KCl, 1.3 MgCl2, 2.5 CaCl2, 1.0 Na2PO4, 26.2 NaHCO3, and 11 glucose (equilibrated with 95%
CO2-5% O2 and heated to 33°C). Whole-cell recordings were made from the soma of pyramidal neurons in area CA1 using infrared differential interference
videomicroscopy. Patch pipettes (2-5 M ) were fabricated from
borosilicate glass and filled with an internal solution containing 135 mM KMeSO4, 8 mM NaCl, 10 mM HEPES, 2 mM Mg2-ATP,
0.3 mM Na3-GTP, pH 7.3 with KOH
(osmolarity 280-290 mOsm), and 50 µM Oregon green
BAPTA-1 (Molecular Probes, Eugene, OR). Cells were selected close to
the surface of the slice. We estimate that the depth of the cell body and proximal dendrite were within 50 µm from the surface.
Electrophysiological signals were recorded with an Axopatch 1D
amplifier (Axon Instruments, Foster City, CA), digitized at 20 kHz with
an ITC-16 board (InstruTech, Port Washington, NY), and controlled using
custom software running under Igor Pro (WaveMetrics, Lake Oswego, OR)
or Axograph (Axon Instruments). Electrophysiological data were analyzed
using Axograph.
Whole-field fluorescence measurements were made using a
monochromator-based imaging system (Polychrome II; T.I.L.L. Photonics, Martinsried, Germany). Neurons were visualized using a 60× water immersion objective (0.9 numerical aperture; Olympus Optical) and
illuminated with 488 nm light. Images were acquired with an in-line
transfer, cooled CCD camera (T.I.L.L. Photonics) in which the scan
lines were binned by two in both horizontal and vertical directions,
giving a spatial resolution of 0.33 µm per pixel. During collection
of image sequences, the exposure time was 10 msec per frame to improve
temporal resolution and minimize photobleaching of the dye. When
examining calcium transients in response to action potentials, frames
were collected at either 16 or 33 Hz. During bath application of
agonists, frames were collected at 1 or 2 Hz. Single action potentials
were evoked by a 10 msec current pulse (200-700 pA), and trains of
action potentials were evoked by either a 300 msec current pulse or a
train of 10 msec current pulses. Images were analyzed offline using
Vision (T.I.L.L Photonics). Small rectangular regions (~10 × 10 pixels) were selected over the nucleus, extranuclear soma, and the
proximal dendrite, and the fluorescence over this region was averaged.
Confocal fluorescence images were obtained using a Zeiss (Oberkochen,
Germany) Axioskop 2FS, with a 510 laser scanning head and equipped with
an argon laser. Confocal fluorescence images were acquired in line scan mode in which the selected line encompassed nuclear, somatic, and
proximal apical dendritic regions at a resolution of 10-20 pixels/µm. The laser power was reduced to 1% to prevent bleaching. The detector pinhole aperture was set to give a vertical resolution of
<2 µm. Lines were acquired at 20 Hz. Small segments (~3 µm) were
selected over the nucleus, extranuclear soma, and the proximal dendrite, and the fluorescence over this region was averaged.
Kinetic sequences were then constructed over time for each of the
selected regions. Calcium signals were measured as the relative change
in fluorescence over baseline fluorescence
( F/F). Additional experiments were
performed using fura-2 and Fluo4 (Molecular Probes). Fura-2 calcium
signals were quantified as background subtracted ratio of fluorescence
signals using excitation wavelengths of 340 and 380 nm
(F340/F380).
Most recordings concentrated on the soma and proximal 25-50 µm of
the apical dendrite.
Muscarine or carbachol (Sigma, St. Louis, MO) were either bath applied
(10-20 µM) or applied by focal pressure application (2-100 µM in aCSF) through a patch pipette. Focal
pressure was applied either by manually applying pressure through via a
5 ml syringe or via a Picospritzer (30 psi, 50-350 msec; Parker
Hannifin, Fairfield, NJ). In some experiments, 500 µg/ml low
molecular weight heparin (Sigma), 20 µM ruthenium red
(Sigma), and/or 50 µM caged myo-inositol
1,4,5-trisphosphate (Molecular Probes) were added to the internal
solution. The lamp used for the photolysis of caged
InsP3 was a pulsed xenon arc lamp (T.I.L.L.
Photonics), which illuminated the entire field of view and discharged
~80 J in 2 msec. Light from both the monochromator and the flash were delivered to the BX50 microscope via a quartz light guide and a custom
epifluorescence attachment provided by T.I.L.L. Photonics and then to
the cells through the objective. Atropine, ryanodine,
dantrolene, and cyclopiazonic acid (CPA) were purchased from Sigma.
Thapsigargin,
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 2-amino-5-phosphono- valeric acid
(APV), and  -methyl-4-carboxyphenylglycine [(+)-MCPG] were
purchased from Research Biochemicals (Natick, MA). Tetrodotoxin (TTX)
was purchased from Alomone Labs (Jerusalem, Israel).
Statistical comparisons were made using a paired Student's
t test or as otherwise indicated. All data are presented as
mean ± SEM.
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RESULTS |
Whole-cell recordings were obtained from CA1 hippocampal pyramidal
neurons. All cells had resting potentials more negative than  55 mV.
The nucleus could be seen as an area of higher fluorescence within the
soma (Fig. 1, 3). The higher
fluorescence is attributable to the lack of organelles within the
nucleus, resulting in a greater ratio of free intracellular space to
total volume compared with the soma (O'Malley, 1994) and not
attributable to differences in resting calcium. In confirmation of
this, ratiometric imaging with fura-2 AM did not show a difference in
the 340/380 ratio between the nucleus and the soma (Fig.
1e). Activation of muscarinic acetylcholine receptors by
bath application of muscarine (10-20 µM)
caused a transient rise in cytosolic calcium, as indicated by an
increase in Oregon green fluorescence. The rise in calcium began as a
focal increase in the proximal apical dendrite (30-50 µm from the
soma) and propagated as a wave toward the soma and into the nucleus
(Fig. 1). Backpropagation of the calcium wave was observed on occasion;
however, the extent of the backpropagation was usually limited to a few
micrometers. On some occasions, calcium waves were also
propagated from basal dendrites (n = 6). The average speed of wave propagation was 26 ± 2 µm/sec (n = 35). Entry of calcium into the nucleus was associated with a slight
delay (Fig. 1d,e). The movement of free calcium
from the dendrite to the soma cannot be attributable to simple
diffusion because the amplitude of the calcium transient was not
decremental (Fig. 1c). The time course of the calcium rise
was fastest in the dendrite with a half-width of 1.18 ± 0.14 sec
and became broader as it propagated to the soma; the half-width of the
calcium transient at the soma was 2.67 ± 0.40 and 3.48 ± 0.57 sec at the nucleus (n = 10). In many cases (18 of
42), repetitive rises in calcium were observed in the continued
presence of agonist, occurring at a frequency of ~0.03 Hz (data not
shown). Each cycle of the oscillation generally took the form of a wave
that originated in the dendrite and propagated to the soma and nucleus.
Membrane depolarization was not required for the rise in cytosolic
calcium because muscarine was equally effective in raising calcium when
cells were voltage clamped between 60 and 70 mV (28 of 34) as when
cells were held in current clamp mode (19 of 22). The calcium rises
were rarely associated changes in whole-cell current, suggesting that
the calcium was released from intracellular calcium stores (Fig.
1e). Bath application of cholinergic agonists has been
reported to produce oscillatory network activity in hippocampal slices
(Fellous and Sejnowski, 2000). Calcium waves were not initiated by
network activity because calcium waves were evoked when recurrent
synaptic activity was blocked with tetrodotoxin (500 nM; two of three) (Fig.
2a). Furthermore, brief focal
pressure application of muscarine onto the soma and proximal apical
dendrite of neurons reliably and repeatedly evoked calcium waves (16 of
18), even when ionotropic and metabotropic glutamate and GABA receptors
were blocked (two of two) (Fig. 2b) or when voltage-gated
calcium channels were blocked with 5 mM NiSO4 (two of two) (Fig. 2c). The
onset of the calcium rise occurred 828 ± 134 msec after
Picospritzer application of agonist muscarine. The half-width of the
calcium transients evoked by focal application was similar to that of
bath-applied agonist (dendrite, 0.37 ± 0.02 sec; soma, 1.67 ± 0.15 sec; nucleus, 2.33 ± 0.16 sec). Focal application of
muscarine to the distal dendrites (>150 µm from the soma) did not
evoke a calcium rise (n = 2) in either the distal dendrites or the proximal dendrites and soma.
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Figure 1.
Muscarinic stimulation evokes calcium
waves in hippocampal pyramidal neurons. A fluorescent image of a cell
loaded with 50 µM Oregon Green BAPTA-1 is shown in
a. The boxes indicate the regions in
which fluorescence measurements were taken. The nucleus can be seen as
a bright area in the soma (area 1). b, Selected
pseudocolor frames are baseline-subtracted images
(F) of the cell shown in a
during bath application of 20 µM muscarine. The first
detectable increase in fluorescence is labeled as time 0, and
subsequent frames at the indicated times are shown below.
c, Rises in calcium, plotted as
F/F, measured over the regions
indicated in a are shown over time. The calcium
transient had the fastest time course in the dendrite and became slower
as it propagated to the soma and nucleus. d, Different
cell loaded with the ratiometric indicator fura-2 AM (100 µM). The panel above shows image recorded
using the isobestic 360 nm excitation wavelength. The
boxes indicate the regions in which fluorescence
measurements were made. The selected pseudocolor frame below shows the
calcium wave propagating into the soma before entry into the nucleus.
e, Rises in calcium plotted as the ratio
F340/F360
over time for the three regions (dendrite, soma, and nucleus) indicated
in the frames above. Note that [Ca2+]i
is similar in the soma and nuclease, and there is a clear delay between
the calcium rises in the extranuclear soma surrounding the nuclear
region and the calcium rise in the nucleus itself.
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Figure 2.
Calcium waves are not the result of enhanced
network activity. a, Rises in calcium, plotted as
F/F, to bath application of muscarine
in the presence of TTX (500 nM) are shown in
a. b, Focal application of muscarine onto
the soma and proximal dendrite readily evoked calcium waves, even when
glutamatergic and GABAergic synaptic activity was blocked by bath
application of APV, CNQX, MCPG, picrotoxin, and CGP 588458. Rises in
calcium are plotted as F/F and are
shown at the top. This neuron was recorded in
current-clamp mode. The simultaneously recorded voltage is shown below.
The timing of the Picospritzer activation is indicated by the
arrowhead and the vertical line. Note the
delay between application of muscarine and the onset of the calcium
wave. c, Calcium waves do not depend on extracellular
calcium because a calcium wave could be evoked in 5 mM
NiSO4. Rises in calcium are plotted as
F/F and are shown for the dendrite,
soma, and nucleus, along with the simultaneously recorded whole-cell
current.
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Although the kinetics of nuclear calcium rises measured with
whole-field fluorescence imaging are consistent with calcium rises
occurring in the nucleus itself, it is possible that the nuclear
fluorescence signal is contaminated by somatic signals above and below
the nucleus. To address this possibility, muscarinic calcium waves were
measured with a confocal imaging system (Fig. 3). The confocal line scan data clearly
show the delayed invasion of the calcium wave into the nucleus. In
addition, the slow kinetics of the nuclear calcium rise observed with
confocal imaging are in complete agreement with the whole-field
fluorescence data. The half-width of the calcium rise was 0.66 ± 0.03 sec at the apical dendrite, 2.19 ± 0.20 sec at the soma, and
3.02 ± 0.05 sec at the nucleus (n = 3).
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Figure 3.
Calcium waves invade the nucleus. A confocal
fluorescent image of a cell loaded with 50 µM Oregon
Green BAPTA-1 is shown on the left of a.
The nucleus can be seen as a bright area in the soma. The
vertical red line through the cell indicates the line of
interest used in subsequent line scan time series. The
baseline-subtracted time series (F) displayed
in pseudocolor after focal application of carbachol (20 µM) is shown on the right. The
vertical white line indicates the time of the first
detectable rise in calcium. Dashed white lines mark the
nucleus. b, Rises in calcium, plotted as
F/F, measured over the dendritic
(D), somatic (S), and
nuclear (N) regions indicated in a
are shown over time. Note the latency between the initiation of the
calcium wave and its subsequent nuclear invasion.
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Action potentials in hippocampal pyramidal neurons initiated rises in
both somatic and nuclear calcium. As shown previously (Jaffe et al.,
1992; Markram et al., 1995; Sah and Clements, 1999), [Ca2+]i peaked at
a higher level and decayed more rapidly in the dendrites than in the
soma. Compared with extranuclear somatic calcium, however, the rise in
nuclear calcium was smaller in amplitude (Schiller et al., 1995) and
had a slower time course than that of the extranuclear soma (Fig.
4). This is likely to be attributable to
the diffusional barrier presented by the nuclear pores in the nuclear
membrane (Rogue and Malviya, 1999). Because voltage-activated calcium
currents are modulated by muscarinic receptors (Gahwiler and Brown,
1987), we next tested whether action potential-induced calcium rises
are affected during muscarinic stimulation. Calcium transients in one
cell recorded from the soma and the apical dendrite in response to a
single action potential (Fig.
5a) and an action potential
train (Fig. 5b) are shown in Figure 5. After muscarinic stimulation, the peak
[Ca2+]i in
response to single action potentials was typically smaller (Fig.
5a) than the control response. The median peak
F/F in the presence of agonist was reduced by
26% in the nucleus (p < 0.001; n = 38), 27% in the soma (p < 0.002; n = 38), and 19% in the proximal dendrite
(p < 0.001; n = 36; Wilcoxon
signed rank test). In contrast, during action potential trains, there
was a large amplification of the calcium response; both the amplitude
and duration of the calcium response were larger (Fig. 5b).
Average peak F/F increased by 145 ± 39%
in the nucleus, 78 ± 25% in the soma, and 39 ± 17% proximal dendrite (n = 10). To test whether the
enhancement of the action potential-induced calcium rise in the
presence of muscarine was simply attributable to the reduction of spike
frequency adaptation and corresponding increase in the number of action
potentials (Müller et al., 1988; Tsubokawa and Ross, 1997), we
examined the effect of trains of action potentials evoked by four 10 msec current pulses given at 20 Hz. Both the amplitude and duration of
the calcium response evoked by four spike trains of action potentials
were larger during muscarinic stimulation (Fig. 5c). Average
peak F/F increased by 56 ± 21% in the
nucleus (p < 0.05; n = 28),
61 ± 23% at the soma (p < 0.01;
n = 28), and 10 ± 7% in the proximal dendrite.
In addition to the increase in peak amplitude, there was a dramatic
prolongation of the calcium transient. Under control conditions,
[Ca2+]i began to
decline immediately after the last action potential. However, during
muscarinic stimulation, the calcium transient showed a pronounced
plateau phase and greatly outlasted the action potential train (Fig.
5b,c). During muscarinic stimulation, the latency
from the last action potential to the peak of the calcium transient
increased from 18 ± 8 to 304 ± 58 msec in the nucleus (p < 0.001; n = 28) and from
4 ± 7 to 122 ± 29 msec over the soma (p < 0.001; n = 28). The
integrated area of the calcium transient evoked by four action
potentials increased by 147 ± 47% at the nucleus
(p < 0.005), 190 ± 59% at the soma
(p < 0.004), and 42 ± 18% in the apical
dendrite (p < 0.02; n = 26)
during muscarinic stimulation. In many cases, a clear inflection could
be seen in the rising phase of the calcium transient, which is
reminiscent of a regenerative response (Fig. 5c). This
inflection was never observed under control conditions, even with
longer trains of action potentials. The muscarinic amplification of the
calcium response during action potential trains was unlikely to result from blockade of the afterhyperpolarization (AHP) because
isoprenaline (10 µM), which also blocked the
AHP, did not cause any regenerative rises in calcium in response to
spike trains (n = 4; data not shown) (Sah and
Clements, 1999). All effects of muscarinic agonists were blocked by
atropine and fully reversible upon washout (n = 5).
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Figure 4.
Action potential-evoked calcium rises in the
nucleus are smaller and have a slower time course than transients
recorded in extranuclear soma. a, Cell loaded with
fura-2 AM. The squares show the regions selected for
measurement of calcium transients. b, Calcium transients
evoked by a single action potential are shown recorded from the
proximal dendrite, extranuclear soma, and nuclear soma. The
traces have been normalized and superimposed on the
right. c, Calcium rises in the nucleus
are smaller in amplitude, have a slower time-to-peak, and have a slower
decay to baseline. Average data are shown in the histograms below.
*p < 0.05.
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Figure 5.
Action potential-evoked
regenerative rises in free calcium in response to muscarinic
stimulation. Action potentials were evoked by a single or train of
current injections as shown in the traces at the
left. Calcium transients, plotted as
F/F, recorded over the nucleus,
extranuclear soma, and dendrite are shown to the right.
a, the calcium transient in response to a single action
potential (thin traces) was reduced in the presence of
muscarine (thick traces). b, In response
to a 200 msec current injection, which evokes a train of action
potentials, the calcium response showed a regenerative amplification in
the nucleus, extranuclear soma, and the proximal dendrite during
muscarinic stimulation. c, The amplification is not
attributable to increased number of action potentials evoked in
muscarine because a similar amplification is seen when only four action
potentials are evoked at a frequency of 20 Hz. The responses in control
Ringer's solution are shown as thin traces, and the
response in the presence of muscarine is shown as a thick
trace. Note that, in the presence of muscarine, the rising
phase of the calcium transients greatly outlast the action potential
train, indicated by the solid bar below the
traces.
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Synaptic activation of muscarinic receptors
The hippocampal formation receives a dense innervation by
cholinergic fibers from the septal nucleus (Lewis and Shute, 1967). Activation of cholinergic fibers with a brief tetanus (33 Hz; 300-600
msec) in stratum oriens evokes a slow depolarizing synaptic potential
and blockade of the AHP by activation of muscarinic receptors (Cole and
Nicoll, 1984). In neurons in which synaptic stimulation produced a
reduction of the current underlying the slow AHP, calcium waves were
also observed (n = 14) (Fig.
6a). Similar to bath-applied
carbachol and muscarine, the calcium rise from synaptic stimulation
produced a focal rise in calcium in the proximal segment of the apical
dendrite, which began 803 ± 207 msec after the first stimulus and
propagated toward the soma at 32 ± 4 µm/sec (n = 8). Similar to results obtained with agonist application,
backpropagation of the calcium wave was spatially limited (Fig.
6b,c). After reaching the soma, as with bath
application of agonists, the calcium wave invaded the nucleus. The
half-width of the calcium transient was 0.69 ± 0.21 sec in the
proximal dendrite, 1.11 ± 0.17 sec in the soma, and 1.68 ± 0.18 sec in the nucleus (n = 4).
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Figure 6.
Synaptic stimulation of
cholinergic afferents evokes calcium waves that invade the nucleus.
a, A fluorescent image of a cell loaded with 50 µM Oregon Green BAPTA-1. b, The selected
pseudocolor frames are baseline-subtracted images
(F) taken at 300 msec intervals after synaptic
stimulation (10 pulses at 33 Hz). c, Rises in calcium,
plotted as F/F, measured at the soma
(s), nucleus (n), and
different distances from the soma (in micrometers), are shown in the
top traces. The bottom trace shows the
whole-cell current over the same time period. A 30 mV hyperpolarizing
step was applied during the tetanus to ensure that voltage-gated
channels were not activated during the tetanus. d, In
neurons in which calcium waves evoked by brief (10-20 pulses at 33 Hz)
synaptic stimulation did not propagate to the nucleus, the propagation
distance could be increased by increasing the number of stimuli (data
not shown) or application of eserine. e, Synaptic
stimulation of cholinergic afferents amplifies of action
potential-induced calcium transients. The nuclear calcium rise to a
train of four action potentials, plotted as
F/F, was greater when action
potentials immediately followed synaptic stimulation (33 Hz, 1 sec;
thick traces) than without previous synaptic stimulation
(thin traces). Sequential application of eserine, MCPG,
and atropine in the presence of CNQX and APV demonstrate that the
amplification was attributable to activation of cholinergic
synapses.
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Synaptically evoked calcium waves were not blocked by the addition of
CNQX and APV (eight of nine cells) or the metabotropic glutamate
receptor antagonist MCPG (1 mM; n = 2) but
were blocked by the muscarinic antagonist atropine (1 µM; five of six). Consistent with the waves
being evoked by acetylcholine, rises in response to synaptic
stimulation were enhanced by increasing the number of stimuli or by
addition of the acetylcholinesterase blocker eserine (1-2 µm;
n = 6) (Fig. 6d).
In cells in which cholinergic stimulation reduced the AHP, an
amplification of action potential-induced calcium rise was also observed. The amplitude of the calcium transient to a train of action
potentials (4-10 spikes 20 Hz) after synaptic activation was 22 ± 3% greater in the nucleus, 30 ± 14% greater in the soma, and
17 ± 14% greater in the proximal dendrite than the additive response of the unpaired tetanus and action potential train
(n = 3) (Fig. 6e). These effects of
cholinergic stimulation were unaffected by glutamate receptor
antagonists, potentiated by eserine, but fully blocked by atropine
(n = 3) (Fig. 6e), showing that they are
attributable to activation of muscarinic receptors.
Involvement of intracellular calcium stores
All five types of muscarinic receptor (m1 to m5) are expressed in
hippocampal pyramidal neurons (Levey et al., 1991, 1995). Because m1
and m3 receptors are coupled to phospholipase C and lead to generation
of InsP3 (Caulfield, 1993), we tested whether the
changes in calcium dynamics resulting from muscarinic stimulation are
attributable to activation of InsP3-sensitive
calcium stores. The uptake of calcium into
InsP3-sensitive and ryanodine-sensitive stores is
mediated by a Ca2+-ATPase (Henzi and
MacDermott, 1991), which can be blocked by CPA and thapsigargin, thus
emptying these stores. Application of muscarine in the presence
thapsigargin (100 nM; n = 3) (Fig. 7a,b) or CPA (30 µM; n = 5) (Fig.
7c,d) led to a depolarization and blockade of the
AHP and reduction in spike frequency adaptation. However, calcium waves
(Fig. 7a,c) and amplification of action potential-evoked calcium rises (Fig. 7b,d) were
blocked after application of thapsigargin or CPA. To confirm that CPA
had indeed emptied the InsP3-sensitive stores,
neurons were loaded with 20-50 µM caged
InsP3. Photolytic uncaging of
InsP3 readily evoked a large calcium rise in all
cells. This response to InsP3 was blocked by CPA
(Fig. 7e), confirming that
InsP3-sensitive stores had been depleted. It
should be noted that intracellular calcium stores were not depleted
after muscarinic stimulation because the calcium response in response
to uncaging of InsP3 after muscarinic stimulation was not blocked (Fig. 7e).
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Figure 7.
Intracellular calcium stores are required for
generation of calcium waves and the amplification of action
potential-evoked calcium rises. a, In the presence of
thapsigargin (100 nM), application of muscarine reduced
spike frequency adaptation and blocked the slow afterhyperpolarization
(inset). Action potentials were evoked using a 200 msec,
300 pA depolarizing current injection. b, Oregon Green
fluorescence recorded in response to a train of action potentials
(shown in a) from the soma and proximal dendrite before
(thin traces) and after (thick traces)
application of muscarine. No regenerative rises in calcium were seen
after the application of muscarine. c, Calcium rise in
response to muscarinic stimulation is completely blocked in the
presence of the calcium ATPase blocker cyclopiazonic acid.
d, the amplification of action potential-induced calcium
rises in the presence of muscarine are also blocked by cyclopiazonic
acid. In each case, the control response is shown as the thin
line, and the response in muscarine is shown as the
thick trace. e, Photolysis of caged
InsP3 causes a rapid rise in cytosolic calcium that is only
slightly affected after muscarinic stimulation (thick
trace) but blocked by application of CPA, confirming that
InsP3-sensitive stores have been emptied in CPA.
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Caffeine at high concentrations reversibly antagonizes the actions of
InsP3 (Parker and Ivorra, 1991). Application of
muscarine in the presence of caffeine (10 mM) blocked the
AHP. However, no calcium waves or regenerative calcium rises in
response to action potential trains were seen (Fig.
8a,b). After
washout of caffeine, a second application of muscarine led to
regenerative calcium rises (Fig. 8). Finally, cells were loaded with
the InsP3-receptor antagonist heparin (Ghosh et
al., 1988) by passive diffusion from the patch pipette. The response to
uncaging InsP3 was completely blocked by heparin
(Fig. 8d), showing that InsP3
receptors had been inhibited. Calcium waves were never observed when
muscarine was applied in the presence of heparin (n = 7) (Fig. 8c). The electrophysiological properties of cells
loaded with heparin were indistinguishable from control neurons;
application of muscarine to these cells caused a membrane
depolarization, blockade of the AHP (Fig. 8e), and reduced
spike frequency adaptation, showing that heparin had not disrupted
coupling of muscarinic receptors to G-protein-mediated second-messenger
systems. However, the regenerative changes in intracellular calcium
evoked by trains of action potentials were fully blocked in
heparin-loaded cells (Fig. 8c). In heparin-loaded cells,
muscarine reduced the average peak calcium rise to a train of action
potentials by 27 ± 5% in the nucleus and 29 ± 10% in the
soma. The integrated area of the calcium rise to a train of action
potentials was reduced by 16 ± 17% in the nucleus and 12 ± 18% in the soma. Together, these results indicate that calcium waves
and amplification of action potential-induced calcium rises is
attributable to regenerative calcium release from
InsP3-sensitive stores.
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Figure 8.
Inositol trisphosphate receptor activation is
required for generation of calcium waves and the amplification of
action potential-evoked calcium rises. a,
Muscarine-evoked calcium waves were reversibly blocked by 10 mM caffeine. Rises in calcium are plotted as
F/F in arbitrary units
(A.U.). Nuc, Nucleus;
Dend, dendrite. b, Superimposed
somatic calcium transients, plotted as
F/F, in response to a train of action
potentials are shown in the presence of caffeine (thin
trace) and after addition of muscarine (thick
trace). After washout of caffeine (traces on
right), reapplication of muscarine to the same cell now
evokes a large regenerative calcium rise. c, When
heparin (500 µg/ml) was included in the internal solution, calcium
waves (top traces) and action potential-induced
amplification (bottom traces) were not observed after
application of muscarine. d, In heparin-loaded cells,
InsP3 receptors are blocked, as shown by the lack of
response to uncaging of InsP3. e, Muscarine
application still blocked the AHP evoked by a train of action
potentials in heparin-loaded cells, showing that heparin did not
disrupt the activation of second-messenger systems.
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|
Because RyRs are also present in hippocampal pyramidal neurons (Sharp
et al., 1993), we tested whether the RyR-sensitive calcium stores were
also involved in the regenerative calcium response. Application of
muscarine in the presence of ryanodine (10 µM) failed to
produce a calcium wave or amplification of the action potential-induced
calcium rises (n = 5) (Fig.
9a,b). In contrast, waves were readily observable when muscarine was applied in the presence of the RyR antagonists ruthenium red (three of four) or
dantrolene (two of four). The calcium amplification of the action
potential train-induced calcium rise was also not occluded by ruthenium
red or dantrolene (Fig. 9a,b). Ryanodine at low
concentrations depletes RyR-sensitive calcium stores by locking the
RyRs in the open state (Rousseau et al., 1987). To test whether the
ryanodine blockade of the muscarinic calcium amplification may be the
result of depletion of the InsP3 store, we tested
the status of the InsP3 stores by uncaging
InsP3. The response to uncaged
InsP3 was blocked by ryanodine but not by
ruthenium red or dantrolene (Fig. 9c), indicating that
ryanodine and InsP3 receptors in CA1 neurons
share a common calcium pool. These results indicate that blockade of muscarinic calcium response by ryanodine is not attributable to the
block of ryanodine receptors.
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Figure 9.
Ryanodine receptors share the same receptor store
as InsP3 receptors but are not required for calcium waves
or amplification of action potential-induced calcium transients.
Application of ryanodine (10 µM) blocks muscarine-induced
calcium waves (a) and amplification of action
potential-induced calcium transients (b). In
contrast, neither calcium waves (top) nor amplification
(bottom traces) are blocked by dantrolene (100 µM). The control response is shown by the thin
lines, and the response in the presence of ryanodine and
dantrolene are shown as the thick traces.
c, Photolytic uncaging of InsP3 leads to
release of calcium from an intracellular store (baseline response) that
is abolished by application of ryanodine but is unaffected in the
presence of the ryanodine receptor antagonist dantrolene
(trace on right). A.U.,
Arbitrary units.
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DISCUSSION |
These results show that activation of muscarinic receptors on
hippocampal pyramidal neurons causes a rise of cytosolic calcium that
begins in the apical dendrite, propagates toward the soma as a wave,
and invades the nucleus. Pairing trains of action potentials with
muscarinic stimulation leads to an amplification of cytosolic and
nuclear calcium by its synergistic release from intracellular stores.
Potentiation of the calcium response to trains of action potentials in
the presence of muscarinic agonists has been reported previously in CA1
pyramidal neurons (Müller and Connor, 1991; Tsubokawa and Ross,
1997; Beier and Barish, 2000). However, this effect was suggested to be
attributable to a combination of reduced spike frequency adaptation and
enhancement of backpropagation of action potentials into the dendritic
tree (Müller and Connor, 1991; Tsubokawa and Ross, 1997) or
attributable to alterations in Ca2+
clearance from the cytosol (Beier and Barish, 2000). We showed that
both calcium waves and action potential-evoked release are inhibited by
antagonists of InsP3 receptors and by emptying
intracellular calcium stores with CPA or thapsigargin, indicating that
they involve the release of calcium from
InsP3-sensitive intracellular stores.
An InsP3-mediated dendritic calcium rise has been
shown recently in response to metabotropic glutamate receptors in CA1
pyramidal (Nakamura et al., 1999, 2000) and cerebellar Purkinje (Finch
and Augustine, 1998; Takechi et al., 1998) neurons. However, calcium waves evoked by metabotropic glutamate stimulation are confined to the
dendritic tree. In CA1 pyramidal neurons, calcium waves evoked by
metabotropic glutamate stimulation are also initiated in the proximal
apical dendrite, ~30-50 µm from the soma. Cholinergic stimulation
of distal dendrites did not evoke a calcium rise. Interestingly,
cholinergic stimulation of the proximal apical dendrite is necessary
for cholinergic blockade of the AHP (Egorov et al., 1999). This
initiation site may reflect a differential distribution of metabotropic
receptors and/or InsP3 receptors. Type I
metabotropic glutamate receptors, which generate
InsP3, are located on the dendrites (Lujan et
al., 1996). However, neither muscarinic receptors (Levey et al., 1995)
nor InsP3 receptors (Sharp et al., 1993) show any
obvious somatodendritic distribution gradient. The dendritic origin of
the calcium wave may instead be attributable to a faster rise in the
concentration of InsP3 attributable to the
smaller volume of the dendritic compartment. Interestingly, dendritic
spines, which contain IP3 receptors (Sala et al., 2001) and are thought
to compartmentalize calcium (Sabatini et al., 2001), first appear
30-50 µm from the soma.
Because InsP3 and calcium act as coagonists at
InsP3 receptors (Bezprozvanny et al., 1991; Finch
et al., 1991; Mak et al., 1999), propagation of the calcium wave into
the soma and nucleus may require both calcium and generation of
InsP3 in the soma by somatic cholinergic
receptors. Ryanodine receptors are not required for calcium waves or
the amplification of action potential-evoked calcium transients,
although they share a common intracellular calcium pool. Similarly, in
cerebellar Purkinje neurons, ryanodine and
InsP3-sensitive calcium stores share a common
calcium pool (Khodakhah and Armstrong, 1997). Although not necessary
for the regenerative calcium response, it is possible that
calcium-induced calcium release from ryanodine receptor activation also
occurs in concert with calcium release from InsP3 receptors.
Action potentials lead to a rise in free calcium by opening
voltage-dependent calcium channels (Markram et al., 1995). In the
presence of muscarinic agonists, there was a reduction in the calcium
transient in response to single action potentials. Activation of
muscarinic receptors has been shown previously to reduce the amplitude
of voltage-activated calcium currents in CA3 pyramidal neurons
(Gahwiler and Brown, 1987). The smaller peak amplitude of the calcium
transient in the presence of muscarinic agonists likely reflects this
reduction of the calcium current. In contrast to single spikes, trains
of action potentials evoked regenerative, prolonged rises in calcium in
the presence of muscarinic agonists. Type I and type II
InsP3 receptors show a bell-shaped dose-response
curve to free calcium (Bezprozvanny et al., 1991; Finch et al., 1991)
so that, in the presence of InsP3, increases in
free calcium cause a positive feedback and amplification of calcium
release (Mak et al., 1999). Thus, the regenerative component of the
calcium response is most likely attributable to calcium-induced calcium
release via InsP3 receptors (Taylor and Marshall,
1992; Nakamura et al., 1999). The observation that the regenerative increases is seen after action potential trains but not after single
action potentials (Yamamoto et al., 2000) is consistent with this idea.
The cholinergic system has been implicated in a variety of cognitive
tasks involving learning and memory. Its role in the pathophysiology of
memory loss such as in Alzheimer's disease is well recognized (Bartus
et al., 1982). Extracellular ACh concentrations rise in the hippocampus
by as much as fourfold during a variety of hippocampal-dependent
learning tasks (Stancampiano et al., 1999; Nail-Boucherie et al.,
2000). It is now clear that long-term synaptic changes associated with
memory and learning require new gene expression (Ghosh and Greenberg,
1995; Berridge, 1998), and rises in nuclear calcium levels form an
integral part of the mechanisms responsible for activation of cascades,
which lead to new gene transcription (Dolmetsch et al., 1998; Li et
al., 1998; Hardingham et al., 2001). Our finding that cholinergic
stimulation leads to rises in nuclear calcium and amplification of
action potential-induced calcium transients in the nucleus may explain
the role of cholinergic system in regulating memory. The nuclear
envelope is well known to be continuous with the smooth endoplasmic
reticulum and has been shown to have both calcium stores and
InsP3 receptors (Malviya et al., 1990; Nicotera
et al., 1990). Thus, it is possible that the rise in nuclear calcium
reported here may be attributable to calcium release from these stores
in the nuclear envelope. These results suggest that the cholinergic
stimulation amplifies and may improve signal-to-noise ratio for
stimulus transcription coupling dependent on nuclear calcium signals.
| |
FOOTNOTES |
Received Sept. 25, 2001; revised Jan. 7, 2002; accepted Jan. 30, 2002.
Correspondence should be addressed to Pankaj Sah, Division of
Neuroscience, John Curtin School of Medical Research, GPO Box 334, Canberra, ACT 2601, Australia. E-mail: pankaj.sah{at}anu.edu.au.
| |
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ABSTRACT
INTRODUCTION
