AG Molekulare Zellphysiologie, Institut für Physiologie der Charité, Humboldt Universität zu Berlin, D-10117 Berlin, Germany
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ABSTRACT |
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Egorov, Alexei V., Tengis Gloveli, and Wolfgang Müller. Muscarinic Control of Dendritic Excitability and Ca2+ Signaling in CA1 Pyramidal Neurons in Rat Hippocampal Slice. J. Neurophysiol. 82: 1909-1915, 1999. The cholinergic system is critically involved in synaptic models of learning and memory by enhancing dendritic [Ca2+]i signals. Diffuse cholinergic innervation suggests subcellular modulation of membrane currents and Ca2+ signals. Here we use ion-selective microelectrodes to study spread of carbachol (CCh) after focal application into brain slice and subcellular muscarinic modulation of synaptic responses in CA1 pyramidal neurons. Proximal application of CCh rapidly blocked the somatic slow afterhyperpolarization (sAHP) following repetitive stimulation. In contrast, the time course of potentiation of the slow tetanic depolarization (STD) during synaptic input was slower and followed the time course of spread of CCh to the dendritic tree. With distal application, augmentation of the somatic STD and of dendritic Ca2+ responses followed spread of CCh to the entire apical dendritic tree, whereas the sAHP was blocked only after spread of CCh to the proximal dendritic segment. In dendritic recordings, CCh blocked a small sAHP, augmented the STD, and rather reduced dendritic action potentials. Augmentation of dendritic Ca2+ signals was highly correlated to augmentation of the STD. The NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV) blocked ~55% of the STD in control and during CCh application. In conclusion, muscarinic suppression of the proximal sAHP can augment firing and thereby Ca2+ responses. Dendritic augmentation of the STD by blockade of the sAHP and direct enhancement of N-methyl-D-aspartate (NMDA) receptor-mediated currents potentiates Ca2+ signals even when firing is not affected due to suprathreshold input. In this way, subcellular muscarinic modulation may contribute to parallel information processing and storage by dendritic synapses of CA1 pyramidal neurons.
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INTRODUCTION |
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Synaptic plasticity like long-term potentiation is
an attractive model for learning and memory. The power of memory
storage is enforced not only by parallel processing by a huge number of neurons but also by parallel processing of 30.000-100.000 individual synaptic contacts onto a single pyramidal cell (Müller and
Connor 1991b). For most models, induction of synaptic
plasticity requires free Ca2+ to reach certain thresholds
at particular postsynaptic sites functionally linked to
N-methyl-D-aspartate (NMDA) receptors. Muscarinic transmission facilitates intradendritic accumulation of free
Ca2+ during excitatory input by uncoupling intracellular
Ca2+ from activation of small-conductance inhibitory
potassium channels mediating accommodation of firing and a slow
afterhyperpolarization (sAHP) following repetitive discharge
(Kohler et al. 1996
; Lancaster et al.
1991
; Müller and Connor 1991a
). This
mechanism involves M2-receptors with high affinity for muscarinic
agonists and requires Ca2+/calmodulin-dependent protein
kinase II (CaMKII) (Müller and Misgeld 1986
;
Müller et al. 1992
). Stronger muscarinic
activation augments NMDA receptor-mediated current responses
(Markram and Segal 1992
). This is likely to contribute
to enhancement of Ca2+ signals. In this way, muscarinic
transmission may switch between stable synaptic transmission and
inducibility of synaptic changes. The above-mentioned mechanisms work
not only on entire neurons but also with focal application of glutamate
and acetylcholine onto dendritic segments (Egorov and
Müller 1999
). This suggests that the diffuse cholinergic
innervation across the entire dendritic tree of pyramidal cells may be
able to modulate dendritic subcompartments independently, thereby
increasing the degrees of freedom and therefore the power of synaptic
information processing and storage. To address this issue we used
Ca2+ imaging in combination with somatic and dendritic
intracellular recording to study the modulation of synaptically evoked
responses of free intradendritic Ca2+ and of the membrane
potential by proximal and distal muscarinic activation after focal
application of the cholinergic agonist carbachol (CCh).
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METHODS |
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Preparation
Transverse hippocampal slices (300 µm thick) were prepared
from adult ether-anesthetized Wistar rats (180-200 g) using a Campden manual vibratome slicer (Loughborough, England) and standard techniques (Misgeld et al. 1989; Müller and Misgeld
1986
). The slices were continuously perfused with oxygenated
(95% O2-5% CO2)
artificial cerebrospinal fluid (ACSF) containing (in mM) 129 NaCl, 3 KCl, 1.6 CaCl2, 1.8 MgCl2,
21 NaHCO3, 1.25 NaH2PO4, and 10 glucose (37°C, pH 7.4).
Measurement of [CCh]o
For measurement of spread of the cholinergic agonist CCh, we
used nominally K-selective microelectrodes (Corning Nr. 477317) backfilled with saline containing 100 mM CCh and 5 mM KCl. The resin
was drawn into the presilanized ion-sensitive barrel of theta glass.
With these high-impedance electrodes (2-50 G), the sum of a
Nernst-diffusion potential of ion activity and the extracellular dc-potential was monitored. Signals were fed into a differential amplifier (3 channels, 2 with input impedance
>1014
), which subtracts the dc-potential of
the reference channel, filled with 154 mM NaCl, from the ion-sensitive
barrel signal. To suppress interference of disturbing ions in slices
(choline), we performed all measurements and calibrations in the
presence of 0.5 mM choline (Müller et al. 1988
)
and 10 µM atropine. CCh-sensitive electrodes were accepted as long as
they responded with >14 mV for a CCh concentration change from 100 to
1,000 µM in the presence of 0.5 mM choline. Typical calibration
responses are given in Fig.
1A. Using the water immersion
objective as for the other experiments, the application micropipette
and the CCh-sensitive electrode were both positioned in brain slice in
the same depth below the upper surface, i.e., where cells were recorded
to obtain good optical resolution (50-120 µm). CCh-induced rises in
[K+]o were blocked by
atropine (10 µM).
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Intracellular recording techniques
Intracellular recordings were obtained using sharp
microelectrodes pulled on a Brown-Flaming puller (Sutter Instruments,
Novato, CA) from 1.2-mm borosilicate glass. Tips were filled with 5 mM Fura-2 (K+ salt, Molecular Probes, Eugene, OR)
dissolved in 100 mM KCl, and electrodes were backfilled with 2 M
K-acetate. Electrode resistance was initially 150-200 M that
dropped within 20 min to 70-120 M
when electrode solutions
equilibrated. Intracellular recordings were made with a Neuro Data
IR-283 current-clamp amplifier. After impalement of a neuron, Fura-2
was injected iontophoretically (0.5-1 nA, 5-15 min) until a signal
over background of at least 3 was obtained. Neurons with resting
potentials positive to
60 mV were rejected. Schaffer collaterals were
stimulated over a width of up to 400 µm by bipolar glass-insulated
silver electrodes. Eletrophysiological data were recorded with an
IBM-compatible PC, an ITC-16 AD interface (Instrutech, Elmont, NY), and
WinTIDA data acquisition software (HEKA, Lambrecht/Pfalz, Germany).
Eventually, the microelectrode was withdrawn to avoid mechanical
problems and only Ca2+ responses were recorded.
Ca2+ imaging
Ratio imaging of Ca2+ was performed as
described previously (Müller and Connor 1991a,b
).
In brief, cells were imaged with an upright microscope (Axioskop,
Zeiss, Jena, Germany) and a long-distance water immersion objective
(Achroplan 63×/0.9). Digitized images were taken with a cooled
charge-coupled device camera system (Photometrics, Tucson, AZ) with a
Macintosh IIfx computer controlling image acquisition and display.
Cytosolic-free Ca2+ concentrations were
determined from background-corrected image pairs taken at 350- and
380-nm excitation using the ratio method (Grynkiewicz et al.
1985
).
Drugs
CCh (1-50 µM), atropine (1-10 µM, both from Sigma,
Deisenhofen, Germany), and DL-2-amino-5-phosphonovaleric
acid (APV, 30 µM, Tocris Cookson, Bristol, UK) were bath applied by
continuous perfusion or by bolus application (Müller et
al. 1988). Focal pressure pulse application of CCh (1-10 mM in
ACSF, 1 bar, 10-50 ms) was performed through micropipettes with 2-3
µm tip diameter.
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RESULTS |
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Spread of CCh after focal application
We have shown previously that focal application of glutamate to
apical dendrites of CA1 pyramidal neurons evokes spatially restricted
increases of
[Ca2+]i that are
rapidly augmented by focal application of muscarinic agonists
(Egorov and Müller 1999). Here we address
subcellular muscarinic modulation of synaptic responses. Our hypothesis
is that muscarinic modulation of the
Ca2+-dependent sAHP and of other currents locally
augment excitability and Ca2+ responses while
suppression of a strong proximal sAHP will globally affect the neuronal
behavior. To correlate effects with the spatiotemporal pattern of
activation of muscarinic receptors by CCh, we determined the
concentration of CCh in the extracellular space and its dependence on
the distance from the application site and on the time after application with CCh-sensitive microelectrodes. Figure 1 demonstrates calibration responses of the ion-selective electrode to CCh
(A), the recording situation (B), and
representative recordings of CCh concentration time courses at various
distances from the application pipette (duration of pressure
application 15-45 ms, C). During the initial 2-3 s a high
CCh concentration of 10-50% of the pipette concentration is achieved
in an area of ~120 µm diameter, depending on the duration of the
pressure application of 15-45 ms. Due to time needed for diffusional
spread, there is a delay of onset of the CCh signals that increases
with distance up to several seconds. Spread into an increasing volume
can explain the strong radial gradient of peak concentration of >100:1
at 10 µm versus 300 µm distance from the application site.
Effects of proximal CCh application
Focal application of CCh to the soma completely blocked the sAHP
in the soma within 1 s (n = 4) and a depolarizing
afterpotential became frequently evident within ~10 s (Fig.
2, filled arrow). Enhancement of the slow
tetanic depolarization (STD) during repetitive synaptic input was
observed to start within 1 s and to grow continuously for the
following 60 s (open arrow), i.e., when CCh has diffused from the
proximal area to all sites of the dendritic tree. Depending on
depolarization, decrease of action potential amplitude during the train
was enhanced (cf. Fig. 5) and action potential half-width was increased
by CCh (Nakajima et al. 1986).
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Effects of distal CCh application
Figure 3A shows representative somatic membrane potential responses to repetitive synaptic input (Schaffer collaterals 50 Hz, 1 s) in control and after focal application of CCh to the apical dendrite at ~280 µm from the soma. One-three seconds after CCh application, i.e., when CCh was able to activate muscarinic receptors only at a distal dendritic region of 60-120 µm in radius (cf. Fig. 1), the synaptic membrane potential response was not changed in the soma (B). Forty seconds after CCh application (C), i.e., when CCh has diffused to the soma (cf. inset), the sAHP following repetitive discharge is strongly reduced, and the slow depolarization during repetitive synaptic input is enhanced, whereas the decrease of amplitude of action potentials during the train is not affected. With weak train stimulation eliciting action potentials at ~50-70% of excitatory postsynaptic potentials (EPSPs) in control, CCh increased the number of action potentials evoked by this stimulus by 20-50%, depending on enhancement of the slow depolarization during the train and on the up to several minutes lasting depolarizing shift of the resting membrane potential.
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Figure 3B demonstrates the Ca2+ profile along the main apical dendrite before and during synaptic stimulation via Schaffer collaterals in another CA1 pyramidal cell. Peak Ca2+ concentration is observed in the proximal part of the dendrite with moderate and steep gradients toward the distal dendrite and soma, respectively. Focal application of CCh to the apical dendrite at 350 µm from the soma augmented the intradendritic Ca2+ response to synaptic input only after a delay of >10 s with a maximal effect at ~35 s (n = 16). This effect was most prominent at the proximal site of higher Ca2+, and its time course was in agreement with the spread of CCh to this proximal site. This result might be due to a Ca2+ dependence of the muscarinic modulation or due to a special importance of the proximal membrane. With distal synaptic Ca2+ responses, we observed an effective augmentation again only after diffusion of CCh to the proximal membrane (including the entire apical dendritic tree, n = 5). Figure 4 demonstrates augmentation of a dendritic Ca2+ signal at ~300 µm from the soma at 18 and 35 s after distal application of CCh (45 and 15 ms), exhibiting a strong correlation with the somatic/apical-proximal CCh concentration time course.
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Dendritic electrophysiology
To directly address muscarinic modulation of dendritic membrane
potential responses, we performed intradendritic recordings with sharp
microelectrodes. Dendritic recordings showed that decay of action
potential amplitudes during synaptic train input became more rapid and
stronger with distance from the soma in addition to a decrease of
action potential amplitude (Fig. 4Aa) (cf. Spruston et al. 1995). In our dendritic recordings, synaptic stimulation hardly evoked compound spiking or burst discharge (cf. Andreasen and Lambert 1995a
,b
). The mean of the sAHP following
synaptically evoked repetitive discharge was significantly smaller in
dendritic (200-300 µm) as compared with proximal recordings. Bath
application of CCh (1-50 µM) suppressed the dendritic sAHP elicited
either by synaptic (n = 18) or by direct stimulation
(n = 5, Fig.
5A and B). CCh
dose-dependently enhanced the slow depolarization during synaptic train
input across the whole membrane, i.e., in the dendrites (+75% with 20 µM CCh, n = 8) as well as in the soma (+73%,
n = 5). Focal dendritic application of CCh (300 µm)
started to suppress the dendritic sAHP at 280 µm from the soma within 1 s (n = 5) with a maximal effect after 20-30 s,
whereas significant augmentation of the slow depolarization during
synaptic train was slightly delayed (~10 s) with respect to blockade
of the sAHP. The decay of dendritic action potential amplitudes during
repetitive discharge was eventually somewhat reduced
(Tsubokawa and Ross 1997
) but usually, concurrent to
the degree of slow membrane depolarization during stimulation, rather
enhanced by CCh (Fig. 5).
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NMDA receptors contribute to augmentation of Ca2+ signals
To study the importance of the enhancement of the STD during
synaptic train input for the augmentation of intradendritic
Ca2+ signals, we combined intradendritic
recording with Ca2+ imaging (n = 3). Figure 6 demonstrates a strong
correlation of the intradendritic Ca2+
accumulation with the STD in control (A), at a low and high
CCh concentration after bolus application into the bath perfusion (B and C) and recovery with wash out
(D). Particular enhancement was observed for the STD and the
Ca2+ concentration during the second half of the
train (peaks) versus the first half of the train (open symbol). Because
muscarinic enhancement of NMDA receptor-mediated responses as well as
muscarinic suppression of the Ca2+-dependent sAHP
can contribute to this muscarinic enhancement of the slow
depolarization, we blocked the NMDA-mediated component by the receptor
antagonist APV. Figure 7 shows muscarinic
enhancement of the STD and suppression of the sAHP in control
(A) and during superfusion with APV (30 µM, B).
Blockade of NMDA receptors by APV suppressed the STD (+11.3 ± 2.3 mV at end of train, mean ± SE, n = 12, to 5.2 ± 0.8 mV, n = 3)
(Collingridge et al. 1988; Herron et al.
1986
) while not affecting the sAHP. Muscarinic activation increased this depolarization in APV to +9.7 ± 2.5 mV, as
compared with +21 ± 6.4 mV in the absence of APV (20 µM CCh;
because we observed no difference between soma and apical dendrite,
data were pooled). The relative muscarinic augmentation of the STD was
not affected by APV (+86% without APV versus +87% in APV).
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DISCUSSION |
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Our results quantitatively characterize drug diffusion in the extracellular space after focal application of a drug that is not significantly degraded or taken up during the time span of interest. These results allow the determination of tissue regions with a drug concentration above certain levels of relevance. The correlation between augmentation of intradendritic Ca2+ signals and the proximal CCh concentration after distal as well as after proximal CCh application indicates an important role for the proximal membrane in addition to integrative contributions from local muscarinic modulation of the dendritic tree.
Immunocytochemistry demonstrated a clustering of L-type
Ca2+ channels at the base of major dendrites in
hippocampal pyramidal neurons (Westenbroek et al. 1990),
and Ca2+ influx through L-type
Ca2+ channels has been found to effectively
activate the sAHP (Marrion and Tavalin 1998
). Synaptic
stimulation evokes the strongest dendritic Ca2+
signals at this site, most likely due to dendritic action potentials. With synaptic stimulation missing threshold after initial firing due to
activation of the sAHP, suppression of the sAHP is sufficient to
increase firing and the intracellular Ca2+
accumulation, like with firing evoked by direct stimulation or glutamate application. With strong suprathreshold synaptic train stimulation, the number of action potentials elicited is not changed by
muscarinic activation. Then augmentation of the
Ca2+ signal is likely to depend on suppression of
the sAHP resulting in stronger slow depolarization, relief of the Mg
block of NMDA receptors, and stronger contribution of NMDA
receptor-mediated Ca2+ influx. The increase of
muscarinic effects during the initial 10 s after proximal
application reflects presumably diffusion of CCh to and contribution of
changes in dendrites up to ~150 µm from the soma. Because the
effects work within 1 s once CCh bound to the receptors, it is
unlikely that the time course is due to a slow cellular transduction process.
In addition to suppression of the sAHP, muscarinic activation can
enhance synaptic Ca2+ signals by direct
augmentation of NMDA receptor-mediated currents and of amplitude and
duration of action potentials. Our results with trains of
suprathreshold EPSPs demonstrate that the slow depolarization
during repetitive synaptic input is largely due to current flow through
NMDA receptor channels (Fig. 7). This slow depolarization effectively
contributes to dendritic Ca2+ signals and their
potentiation (Fig. 6), even in the absence of action potentials when
evoked by focal glutamate application (Egorov and Müller
1999). Augmentation of this slow depolarization requires higher
CCh concentrations and has been shown, in the case of glutamate
application, to be lithium sensitive. Both points support mediation by
M1 receptors as opposed to M2 receptors in suppression of the sAHP
(Markram and Segal 1992
; Müller and Misgeld 1986
).
We have shown previously that suppression of the sAHP is sufficient to
strongly augment Ca2+ signals evoked by direct
depolarization due to increased firing (Müller and Connor
1991a). Synaptically as well as antidromically evoked distal
dendritic action potentials have been demonstrated to be enhanced in
amplitude by CCh (Hoffman and Johnston 1999
; Tsubokawa and Ross 1997
). We have observed eventually
slight increases in spike amplitude, but often no change or a decrease.
While we elicited action potentials directly or orthodromically using a wide stimulation electrode and used sharp microelectrode versus perforated and whole cell patch-clamp recording, these differences are
not likely to explain the discrepancy. The decrease of action potential
amplitudes is presumably due to suppression of the sAHP and
augmentation of the synaptically evoked slow membrane depolarization, causing inactivation of Na+ channels. In
addition, Cantrell et al. and others have shown that high
concentrations of CCh (
5 µM) inhibit the sodium current through
activation of PKC with a Ki of ~8 µM
(Cantrell et al. 1996
; Mittmann and Alzheimer
1998
). With focal as well with bolus applications used in this
study, we obtained CCh concentrations in this range (10-50 µM) and
above, in contrast to CCh concentrations enhancing dendritic action
potentials (Hoffman and Johnston 1999
; Tsubokawa and Ross 1997
). In addition, there are
methodological differences that might stabilize the dendritic balance
between inward and outward currents and hence dendritic action
potentials; e.g., Tsubokawa and Ross as well as Hoffman and Johnston
reduced fast GABAergic inhibition using an antagonist, age of animals, strain, and temperature.
In summary, our results demonstrate that muscarinic input
modulates membrane excitability and synaptic Ca2+
signals subcellularly with an important proximal and a distributed dendritic component. The dendritic component clearly involves 1) suppression of the sAHP, allowing increased firing and
depolarization contributing to removal of the
Mg2+ block of NMDA channels; 2) direct
augmentation of NMDA receptor-mediated currents (Markram and
Segal 1992); and 3) inhibition of A-type K channels
(Nakajima et al. 1986
). Local augmentation of distal dendritic responses presumably has been below the threshold of detection due to widespread synaptic activation, electrotonic coupling,
and limited temporal and spatial resolution of the methods used. The
dendritic component became evident in somatic recordings only when
affecting a significant fraction (
150 µm in radius) of the
dendritic tree. The proximal membrane has a particular importance not
only for the somatic but also for the dendritic membrane potential and
Ca2+ signals, most likely due to clustering of Ca
channels linked to activation of a strong sAHP. The somatic membrane
potential is of particular importance for the input-output function of
the cell including backpropagation of action potentials into the
dendritic tree. Inhibition of Na+ and
Ca2+ channels at higher agonist concentration
(Cantrell et al. 1996
; Toselli and Lux
1989
) will limit intradendritic Ca2+
levels during strong excitation. In this way, muscarinic activation will facilitate induction of Ca2+-dependent
synaptic plasticity while avoiding deleterious
Ca2+ overload. Whether in vivo the diffuse
cholinergic input independently modulates proximal and distal membrane
regions remains to be investigated.
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ACKNOWLEDGMENTS |
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We thank A. Düerkop for excellent technical assistance.
This study was supported by Deutsche Forschungsgemeinschaft Grant Mu 809/6-2, a Graduiertenkolleg 238 grant, a Heisenberg stipend to W. Müller, and a Charité grant.
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FOOTNOTES |
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Address for reprint requests: A. V. Egorov, AG Molekulare Zellphysiologie, Institut für Physiologie der Charité, Humboldt Universität zu Berlin, Tucholskystr. 2, D-10117 Berlin, Germany.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 April 1999; accepted in final form 1 June 1999.
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REFERENCES |
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