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Volume 16, Number 13,
Issue of July 1, 1996
pp. 4113-4128
Copyright ©1996 Society for Neuroscience
Cholinergic-Dependent Plateau Potential in Hippocampal CA1
Pyramidal Neurons
Douglas D. Fraser and
Brian A. MacVicar
Neuroscience Research Group, Faculty of Medicine, University of
Calgary, Calgary, Alberta, Canada T2N 4N1
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Cholinergic stimulation of the hippocampal formation results in
excitation and/or seizure. We report here, using whole-cell patch-clamp
techniques in the hippocampal slice (34-35°C), a
cholinergic-dependent slow afterdepolarization (sADP) and long-lasting
plateau potential (PP). In the presence of 20 µM carbachol, action potential firing evoked by
weak intracellular current injection elicited an sADP that lasted
several seconds. Increased spike firing evoked by stronger depolarizing
stimuli resulted in long-duration PPs maintained close to 
20 mV.
Removal of either Na+ or
Ca2+ from the external media, intracellular
Ca2+
([Ca2+]i) chelation with
10 mM
bis(2-aminophenoxy)ethane-N,N,N
,N-tetra-acetic acid,
or the addition of 100 µM
Cd2+ to the perfusate abolished both the sADP and
PP. The sADP was depressed and the PP was abolished by either 10 µM nimodipine or 1 µM
-conotoxin, whereas 1.2 µM tetrodotoxin was
ineffective. The involvement of a
Na+/Ca2+ exchanger was
minimal because both the sADP and PP persisted after equimolar
substitution of 50 mM Li+
for Na+ in the external media or reduction of the
bath temperature to 25°C. Finally, in the absence of carbachol the
sADP and PP could not be evoked when K+ channels
were suppressed, suggesting that depression of K+
conductances alone was not sufficient to unmask the conductance. Based
on these data, we propose that a Ca2+-activated
nonselective cation conductance was directly enhanced by muscarinic
stimulation. The sADP, therefore, represents activation of this
conductance by residual
[Ca2+]i, whereas the PP
represents a novel regenerative event involving the interplay between
high-voltage-activated Ca2+ channels and the
Ca2+-activated nonselective cation conductance.
This latter mechanism may contribute significantly to ictal
depolarizations observed during cholinergic-induced seizures.
Key words:
muscarinic stimulation;
Ca2+-activated
nonselective cation conductance;
HVA Ca2+ currents;
ictal
depolarization;
epilepsy;
slice-patch technique
INTRODUCTION
The hippocampal formation receives cholinergic
afferents from the medial septum/diagonal band that terminate on
dendritic segments located in both the cornu Ammonis and dentate gyrus
(Wainer et al., 1993
). This septohippocampal pathway is critical in the
generation of rhythmical slow activity (Bland, 1986; Vanderwolf, 1988)
and is implicated in some forms of learning and memory (Shen et al.,
1994). Cholinergic stimulation is also effective in generating limbic
seizures and, thus, injection of cholinergic agents is used as a model
for temporal lobe epilepsy (Lothman et al., 1991). Indeed, hippocampal
sclerosis after infusion of cholinergic agonists accurately mimics
numerous pathological indices of human status epilepticus (Wasterlain
et al., 1993).
The electrophysiological actions of cholinergic agonists on neurons
have been studied extensively in vitro and, for the most
part, are considered excitatory (Halliwell, 1990; Krnjevi
, 1993;
McCormick, 1993). For example, cholinergic stimulation causes a
sustained depolarization associated with an increased input resistance
(RIN) and depression of spike frequency
adaptation. These effects are attributed to a cholinergic-induced
depression of four potassium conductances, including the M-current
(IM) (Halliwell and Adams, 1982; Madison et
al., 1987), a fast inactivating current
(IA) (Nakajima et al., 1986), a slow
Ca2+-activated current
(IAHP) (Madison et al., 1987; Bernardo and
Prince, 1982), and a background leak current
(ILEAK) (Madison et al., 1987).
Voltage-dependent Ca2+ channels are also
modulated by cholinergic stimulation; high-voltage-activated (HVA)
channels are depressed (IHVA) (Misgeld et
al., 1986; Toselli and Lux, 1989), whereas low voltage-activated
channels are enhanced (ILVA) (Toselli and
Lux, 1989; Fraser and MacVicar, 1991). More recently, a nonselective
cation conductance has been demonstrated to contribute to the initial
cholinergic-induced depolarization (Benson et al., 1988; Shen and
North, 1992; Colino and Halliwell, 1993). A slow afterdepolarization
(sADP) has also been observed in the presence of cholinergic agents
(Bernardo and Prince, 1982; Gähwiler, 1984; McCormick and Prince,
1986; Müller et al., 1988; Hasuo and Gallagher, 1990; Andrade,
1991; Müller and Connor, 1991), however, multiple ionic
mechanisms may underlie this phenomenon. For instance, a
Na+/Ca2+ exchanger
(Friedman et al., 1992), a prolonged Ca2+
conductance (Blitzer et al., 1991), deactivation of a
K+ conductance by maintained
Ca2+ influx (Constanti and Bagetta, 1991;
Constanti et al., 1993), and a Ca2+-activated
nonselective cation conductance (ICAN)
(Schwindt et al., 1988; Hasuo et al., 1990; Caeser et al., 1993) are
all plausible mechanisms.
Although there are multiple reports of sADPs after muscarinic
stimulation, cholinergic-dependent plateau potentials (PPs) have not
been reported. This PP may represent a novel regenerative event
involving interactions between IHVA and
ICAN. It is conceivable that interplay
between these channels could contribute significantly to ictal
depolarizations observed during cholinergic-induced seizures (Lothman
et al., 1991). The aim of the present study, therefore, was to identify
the ionic mechanisms underlying the cholinergic-induced PP recorded in
hippocampal CA1 pyramidal neurons with whole-cell patch-clamp
techniques.
These results have been presented in abstract form (Fraser and
MacVicar, 1995).
MATERIALS AND METHODS
Hippocampal slice preparation. Sprague-Dawley rats,
postnatal 15-23 d, were decapitated and the brains were immersed in
chilled artificial CSF (aCSF) containing (in mM):
126 NaCl, 2.5 KCl, 2 MgCl2, 2 CaCl2, 1.25 NaH2PO4, 26 NaHCO
3, and 10 D-glucose,
pH 7.3. The hippocampi were isolated and attached to the stage of
a vibrating micro-slicer with cyanoacrylate glue. Tissue orientation
and stability were ensured by propping the hippocampi against an
immobilized block of 2% agar dissolved in physiological saline
followed by a light application of low-melt gelatin around the tissue
base. Transverse slices (150-400 µm) were prepared by sectioning the
hippocampus perpendicular to its septotemporal axis. The slices were
then transferred to a glass vial filled with oxygenated aCSF and
incubated at room temperature.
Whole-cell patch-clamp recording. Whole-cell current-clamp
recordings from neurons within hippocampal slices were obtained using
either the ``blind-patch'' technique (Blanton et al., 1989) or visual
guidance (Edwards et al., 1989). Slices were individually transferred
to a recording chamber located on an upright microscope (Standard 14;
Zeiss, Thornwood, NY) and submerged in rapidly flowing (1 ml/min)
oxygenated aCSF. Bath temperature was maintained at 34-35°C with a
Peltier unit and Cambion bipolar controller. Individual cells were
visualized through a 40×, 0.75 numerical aperture, water-immersion
objective (Zeiss). The objective was first modified by filing away the
metal shoulder around the lens (Brown and Flaming, 1992). This improved
the working space beside the lens, thereby increasing the angle of
electrode approach. The objective was then coated with SYLGARD for
temperature and electrical isolation. Patch electrodes (5-7 M
) were
pulled from 1.5 outer diameter thin-walled glass (150F-4, World
Precision Instruments) in two stages on a Narishige puller (PP-83;
Tokyo, Japan) and filled with intracellular solution (in
mM): 140 K-gluconate, 1.1 EGTA, 0.1 CaCl2, 10 HEPES, 2 Mg-ATP, and 0.3 Na-GTP,
pH 7.2. Intracellular Ca2+ concentration was
calculated to be 16 nM. In some experiments,
Cs-gluconate or KCl was substituted for equimolar K-gluconate (see
Table 1 for description of
intracellular solutions). Voltage recordings were obtained in bridge
mode (Axoclamp-2A, Axon Instruments, Foster City, CA) and were low-pass
filtered (4-pole Bessel) at 10 kHz (3 dB). Capacitance neutralization
was fully maximized, and the voltage drop across the electrode patch
was subtracted by bridge circuit potentiometer. Data were digitized and
analyzed using computer software (pClamp or Axotape) via a Tl-1 A/D
interface (Axon Instruments). All data are presented as mean ± SE.
Measurement of electrophysiological properties. Membrane
characteristics were determined after patch rupture and sampled
throughout the experiment. Series resistance was determined via a
bridge potentiometer by balancing the voltage drop across the patch in
response to a negative current pulse (30 pA; 10 msec).
RIN was calculated from a steady-state
potential response, free of membrane rectification, to a
hyperpolarizing current step. The membrane time constant (
) was
calculated as the time necessary to reach 1 e1 (63%) of the maximum voltage
deflection. Action potential threshold
(APTH) was measured as the membrane
potential at the base of the action potential, whereas, action
potential amplitude (APAMP) was measured as
the voltage difference between the threshold and peak amplitude. Action
potential duration (APDUR) was measured at
the threshold potential.
RESULTS
The results in this paper were obtained from 211 CA1 pyramidal
neurons in the hippocampal slice preparation, recorded with whole-cell
patch-clamp techniques. The average series resistance was 14.0 ± 0.2 M (range 8-20; n = 211); recordings with series
resistance >20 M were discarded. The resting membrane potential,
input resistance, membrane time constant, and action potential
characteristics were similar to those reported previously (see Table 1)
(Stabel et al., 1992; Williams et al., 1994).
Cholinergic stimulation of CA1 pyramidal neurons
Hippocampal CA1 pyramidal neurons were stimulated by bath
application of carbachol, a nonhydrolyzable cholinergic agonist.
Consistent with previous reports, 20 µM
carbachol depolarized the membrane potential by 3-11 mV
(n = 188) and increased RIN
by 16 ± 1% (range 5-30; 50 neurons sampled). Spike frequency
adaptation was reduced, and the action potential afterhyperpolarization
was abolished (Fig. 1A). In thicker slices
(300-400 µm), an increase in synaptic noise was commonly observed
with carbachol application that could be differentiated into discrete
excitatory and inhibitory postsynaptic potentials (data not shown)
(MacVicar and Tse, 1989; Pitler and Alger, 1992; Behrends and
Bruggencate, 1993). These effects were antagonized by coapplication of
atropine (n = 7/7), indicating that carbachol acted
specifically at muscarinic receptors, as shown previously (Halliwell,
1990). At a concentration of 20 µM, however,
carbachol failed to induce rhythmic membrane potential oscillations or
theta activity, reported previously for higher concentrations (>50
µM) (Konopacki et al., 1987; MacVicar and Tse,
1989).
Fig. 1.
In the presence of carbachol, a nonhydrolyzable
cholinergic agonist, depolarizing current injection resulted in either
an sADP or a PP. A, Typical responses of a
hippocampal CA1 pyramidal neuron to hyperpolarizing and depolarizing
current injection. Neither an sADP nor a PP was observed in the absence
of cholinergic agonists. The resting membrane potential of this neuron
was 66 mV. After application of 20 µM
carbachol, the membrane potential depolarized to 60 mV, spike
frequency adaptation was reduced, and the action potential
afterhyperpolarization was abolished. In addition, an sADP
(arrow) was now evident after cessation of the current
stimulus. After a 20 min wash of carbachol, the membrane potential
repolarized to 64 mV, spike frequency adaptation was again observed,
and the sADP was absent. At least one component of action potential
afterhyperpolarization, however, failed to return after wash of
carbachol. B, In a different CA1 pyramidal neuron, 0.8 sec
depolarizing current injection resulted in a typical pattern of
repetitive action potentials. The membrane potential returned to the
baseline level after cessation of the stimuli. In the presence of 20 µM carbachol, however, spike firing evoked by
identical stimuli resulted in not only a slow afterdepolarization
(sADP; arrow), but also a long-lasting plateau
potential (PP; arrow). Notice the low-amplitude
oscillations superimposed on the latter portion of the PP. Both the
sADP and PP were reversed after carbachol had been washed from the
slice for 20 min. Action potentials were truncated by the digitization
rate.
[View Larger Version of this Image (31K GIF file)]
Cholinergic-dependent sADP and PP
In the absence of a cholinergic agonist, positive current
injection (
0.1 nA) resulted in repetitive action potential firing in
all CA1 pyramidal neurons tested (Fig. 1A,B;
n = 196). After cessation of current injection, the
membrane potential rapidly returned to resting levels. In contrast,
after a 5 min application of 20 µM carbachol,
weak current injection (~0.1 nA; 0.8 sec) resulted in repetitive
firing at higher frequency, which was commonly superimposed on a
depolarizing ramp. After cessation of evoked action potentials, a
depolarizing afterpotential was observed that declined slowly over
several seconds (Fig. 1A,B; n = 188/188).
This potential was termed an sADP. The average duration and maximal
amplitude of the sADP was 6.1 ± 0.2 sec (range 2.8-8.5;
n = 50) and 9 ± 1 mV (range 3-20; n = 50), respectively. Injection of stronger current (0.2 nA) for the
same duration resulted in higher frequency action potential firing
superimposed on a steep depolarization that developed into a
regenerative PP (Fig. 1B; n = 179/188). The
average duration of the PP evoked under the above conditions was 9.9 ± 0.6 sec (range 2.2-21.5; n = 50), whereas the average
membrane potential of the PP was 19 ± 1 mV (range 25 to 11;
n = 50; measured at 0.2 sec after cessation of the
stimuli). Oscillations in the membrane potential were commonly observed
superimposed on the PP (Fig. 1B), probably consisting of
Na+ and/or Ca2+
oscillations well described by others (MacVicar, 1985; Alonso and
Llinas, 1989; Leung and Yim, 1991; García-Muñoz et al.,
1993). Both the sADP and PP were antagonized by coapplication of 1 µM atropine, again suggesting the specific
involvement of muscarinic receptors (Fig. 2;
n = 7/7).
Fig. 2.
Both the sADP and PP were depressed by
coapplication of the muscarinic receptor antagonist atropine. In
control, the resting membrane potential of this neuron was 65 mV, and
neither the sADP nor the PP was observed after action potential firing.
The inset illustrates the typical firing pattern of this CA1
pyramidal neuron. The calibration bars in the inset
represent 40 msec and 20 mV. In the presence of 20 µM carbachol, the membrane potential
depolarized to 61 mV, and both the sADP and PP were observed after
the spike firing. Coapplication of 1 µM
atropine depressed both the sADP and PP, implicating the involvement of
muscarinic receptors.
[View Larger Version of this Image (20K GIF file)]
Each PP consisted of at least four distinct phases (Fig.
3A, arrows). The first phase
consisted of a steep depolarizing shift that accompanied action
potential firing during intracellular current injection. The second
phase consisted of the prolonged PP. The third phase, lasting up to
several seconds, was a repolarization that commonly hyperpolarized
beyond the resting membrane potential. The final phase consisted of
this hyperpolarization, which, over a prolonged period of time,
returned to the baseline. The data in Figure 3A also
demonstrate the reproducibility of the PP waveform in any one cell. In
this neuron, five PPs were evoked, separated by 10 min between
stimuli.
Fig. 3.
The cholinergic-dependent PP consisted of at least
four distinct phases, and the likelihood of generating a PP increased
with the number of action potentials elicited. A, In the
absence of cholinergic stimulation, the resting membrane potential of
the neuron illustrated was 67 mV. Application of 20 µM carbachol depolarized this neuron to 62
mV. In addition, action potential firing evoked by depolarizing current
injection (0.1 nA; 0.8 sec) elicited a long-lasting plateau potential
in the CA1 pyramidal neuron illustrated. Notice that the spikes are
superimposed on a depolarizing ramp that jumped suddenly to a
depolarized membrane potential of 20 mV (phase 1;
arrow). The PP remained at positive membrane potentials for
a sustained period, long after cessation of the current injection
(phase 2; arrow). After a prolonged
period, the PP repolarized, surpassing the resting potential
(phase 3; arrow). Finally, the membrane
potential remained at a hyperpolarized potential for a period of ~19
sec before a gradual return to the resting potential
(phase 4; arrow). The PP was evoked five
times and digitized on line at five different rates to illustrate not
only the distinct phases of the PP, but also the reproducibility of the
waveform. Action potentials were truncated by the digitization rate.
B, In the presence of 20 µM
carbachol, the likelihood of generating a PP increased with the
magnitude of depolarizing current injection. The responses to three
amplitudes of current injection (0.1, 0.2, 0.3 nA; 0.8 sec) are
illustrated. The resting membrane potential of the neuron before
cholinergic stimulation was 68 mV, whereas in carbachol the neuron
depolarized to 59 mV. C, The probability of generating a
PP in carbachol also increased with the duration of depolarizing
current injection. Depolarizing current injection for three durations
(0.2, 0.4, 0.6 sec; 0.3 nA) is illustrated. The resting membrane
potential of the neuron before cholinergic stimulation was 72 mV,
whereas in carbachol the neuron depolarized to 65 mV.
[View Larger Version of this Image (31K GIF file)]
Increasing the number of evoked action potentials enhanced the sADP
amplitude until a critical threshold for PP genesis was achieved. For
example, gradually increasing the intensity of the injected current
resulted in increased spike firing (Fig. 3B). As a result,
the amplitude of the sADP increased accordingly until the PP was
elicited. Conversely, for a current pulse of a given amplitude,
increasing the duration of the stimuli also increased the number of
action potentials, thereby enhancing the sADP amplitude (Fig.
3C). A critical threshold for PP genesis was also obtained.
Even though a clear transition from sADP to PP genesis was observed,
the two phenomena may be related because both required cholinergic
stimulation and followed a burst of action potentials.
The amplitude of the sADP and the duration of the PP were dependent on
the holding potential (Fig. 4). As before, in the
absence of a cholinergic agonist, the membrane potential returned to
resting levels after a burst of evoked action potentials (Fig.
4A). In the presence of carbachol, however, both the sADP
and PP were observed after identical stimuli (Fig. 4B). The
membrane potential in this case was maintained at 60 mV with positive
DC current injection. The sADP and PP were differentially affected when
evoked from more negative membrane potentials. For example, the sADP
was hardly detectable below 75 mV, and the duration of the PP was
reduced with more negative membrane potentials (Fig. 4C-E;
n = 6/6). The graph depicts the PP duration versus
membrane potential (Fig. 4F; n = 3). The
duration was measured from the end of the current pulse to the point at
which the PP crossed or reached the prestimuli membrane potential.
Notice the nonlinear DC current injection, also depicted in Figure
4F (n = 3), required to maintain the
membrane at negative potentials. This indicates the activation of a
steady-state conductance at these negative membrane potentials; the
most likely candidate in this cell type is the
hyperpolarizing-activated cation conductance (Halliwell and Adams,
1982; Stabel et al., 1992).
Fig. 4.
The amplitude of the sADP and the duration of the
PP decreased with negative membrane potentials. A, Typical
response of a hippocampal pyramidal neuron to hyperpolarizing and
depolarizing current injection. Neither an sADP nor a PP was observed
in the absence of cholinergic stimulation. The resting membrane
potential of this pyramidal neuron was 66 mV. B, In the
presence of 20 µM carbachol, the neuron
depolarized to 63 mV. After depolarizing current injection, both an
sADP and a PP were evoked from a membrane potential of 60 mV.
C-E, Decreasing the membrane potential with negative DC
current injection decreased the amplitude of the sADP. In contrast, the
PP was still generated, however, the duration decreased with lowered
membrane potentials. F, A plot illustrating the PP duration
and negative DC current injection versus membrane potential. The data
were tabulated from three neurons.
[View Larger Version of this Image (37K GIF file)]
Ionic dependency of the sADP and PP
The ion(s) mediating the sADP and PP were investigated either by
substitution experiments or by including specific channel blockers in
the perfusate. In the first experiment, a role for
Na+ was investigated by replacing extracellular
Na+ with equimolar choline. When extracellular
Na+ was reduced from 152 to 26 mM, both the sADP and PP were reversibly
depressed (Fig. 5A; n = 6/6).
A small amplitude sADP was still observed in low extracellular
Na+ (6 ± 1 mV; range 3-10; n = 6), however, the amplitude of the depolarizing current injected was
twofold greater. In a similar experiment, both the sADP and PP were
reversibly abolished when extracellular Ca2+ was
reduced from 2 to 0.1 mM (Fig. 5B;
n = 4/4). In this experiment, extracellular
Mg2+ was simultaneously increased from 2 to 10 mM to maintain the divalent cation surface charge
(Hille, 1984). Finally, a role for Cl was also
investigated by equimolar substitution of 70 mM
KCl for K-gluconate in the pipette solution (Table 1; n = 8). Even though the estimated Cl reversal
potential was shifted to 17 mV, characteristic sADPs and PPs were
still elicited in carbachol after depolarizing current injection and
action potential firing (data not shown; n = 8/8).
Hence, Na+ and Ca2+ influx
were both critical for genesis of the sADP and PP, whereas the
involvement of Cl was negligible.
Fig. 5.
Both the sADP and PP were dependent on
Na+ influx, independent of TTX-sensitive channels
and Ca2+ influx via HVA channels. A,
In 20 µM carbachol, depolarizing current
injection evoked both an sADP and a PP. Lowering external
Na+ from 152 to 26 mM reversibly
depressed the sADP and abolished the PP, suggesting that
Na+ influx was required for these
afterpotentials. The resting potentials before and after cholinergic
stimulation were 68 and 59 mV, respectively. The calibration bars
in the inset represent 40 msec and 20 mV. B,
In a different pyramidal neuron, both the sADP and PP were evoked by
intracellular current injection. Lowering external
Ca2+ from 2 to 0.1 mM
reversibly abolished both the sADP and PP. The concentration of
Mg2+ was simultaneously increased from 2 to 10 mM to maintain divalent cation charge screening.
This finding suggests that Ca2+ influx was also
necessary for these potentials. The resting potentials of this neuron
before and after cholinergic stimulation were 67 and 60 mV,
respectively. The calibration bars in the inset represent 40 msec and 20 mV. C, In 20 µM
carbachol, an sADP and a PP were observed after evoked action
potentials. Coapplication of 1.2 µM TTX, a
concentration sufficient to block action potentials, failed to depress
either the sADP or the PP. Notice that the PP followed a slow
regenerative potential, presumably
Ca2+-dependent, whereas the sADP did not.
Activation of voltage-dependent Na+ channels,
therefore, was not required for either of these afterpotentials. The
resting membrane potentials before and after carbachol were 65 and
62mV, respectively. D, In carbachol, an sADP and a PP were
elicited by intracellular current injection. Coapplication of 100 µM Cd2+ abolished both
the sADP and PP. Activation of HVA Ca2+ channels,
therefore, was necessary to evoke these potentials. The resting
membrane potential in control was 69 mV, whereas 20 µM
carbachol depolarized the membrane potential to 62 mV.
[View Larger Version of this Image (39K GIF file)]
To determine whether voltage-activated Na+
channels were involved in sADP and PP genesis, 1.2 µM tetrodotoxin (TTX) was included in the
perfusate (Fig. 5C; n = 5). In all neurons,
both the sADP and PP were still observed after application of TTX. In
the presence of TTX, not only were fast action potentials blocked, but
the low-amplitude oscillations observed superimposed on the PP were
also abolished (MacVicar, 1985; Alonso and Llinas, 1989; Leung and Yim,
1991). In some neurons, a small amplitude sADP was observed after
current injection in the absence of regenerative potentials (Fig.
5C; n = 2/5). The average duration and
maximal amplitude were 4.8 sec and 4 mV, respectively. The PP, however,
was only observed after the depolarizing stimuli activated slow,
regenerative potentials that were presumably
Ca2+-dependent (Fig. 5C;
n = 5). These data clearly eliminate the involvement of
TTX-sensitive, voltage-activated Na+ channels in
genesis of the sADP and PP. To determine whether HVA
Ca2+ channels were required for genesis of
the sADP and PP, 100 µM
Cd2+ was included in the perfusate (Fig.
5D). In all neurons (n = 4), both the sADP
and PP were blocked by coapplication of Cd2+,
suggesting a critical role for HVA Ca2+ channels
in genesis of these afterpotentials. In fact, the sADP and PP could not
be evoked, even if the amplitude and duration of current injection was
increased.
To determine the HVA channel subtypes involved in sADP and PP
genesis, blockers of either L- or N-type channels were included in the
perfusate (Fig. 6). In the presence of 20 µM carbachol, both a sADP and PP were evoked
after intracellular current injection and spike firing. The addition of
10 µM nimodipine, an L-type channel blocker, to
the perfusate depressed the sADP and abolished the PP (Fig.
6A; n = 4/4). Although an sADP of 11 ± 4 mV
(range 6-22; n = 4) was still observed, the amplitude
of the depolarizing current had been increased twofold. Similarly,
coapplication of 1 µM -conotoxin-GVIA, an
N-type channel blocker, also depressed the sADP and abolished the PP
(Fig. 6B; n = 4/4). As above, an sADP of 10 ± 2 mV (range 6-17; n = 4) was still observed,
however, these stimuli were also increased twofold. The N-type channel
blocker -conotoxin-GVIA was coapplied with 0.1 mg/ml cytochrome C to
inhibit nonspecific binding. Cytochrome C, however, had no measurable
effect on either the sADP or PP (Fig. 6B; n = 4). Interestingly, neither nimodipine nor -conotoxin-GVIA
abolished the sADP, suggesting that both L- and N-type channels
participate in elevating intracellular Ca2+
([Ca2+]i) to a critical
level for PP genesis. These data, however, cannot rule out the minor
involvement of other HVA Ca2+ channels in sADP
and PP genesis.
Fig. 6.
The sADP and PP required
Ca2+ influx through L- and N-type
Ca2+ channels. A, In a CA1 pyramidal
neuron, intracellular current injection revealed an sADP and a PP in
the presence of 20 µM carbachol. The resting
membrane potentials before and after cholinergic stimulation were 64
and 60 mV, respectively. The sADP was reduced and the PP could not be
evoked after coapplication of the L-type channel blocker nimodipine.
B, In another pyramidal neuron, both the
cholinergic-dependent sADP and PP were also evoked by intracellular
current injection. The resting membrane potentials before and after
cholinergic stimulation were 66 and 61 mV, respectively. Both the
sADP and PP were reversibly depressed by coapplication of the N-type
channel blocker -conotoxin-GVIA.
[View Larger Version of this Image (20K GIF file)]
Taken together, these data demonstrated that both
Na+ and Ca2+ influx was
critical for sADP and PP genesis. Moreover, Ca2+
influx occurred via HVA Ca2+ channels,
predominantly of the L and N subtypes, whereas
Na+ influx occurred independently of
TTX-sensitive channels.
Ionic mechanisms underlying the sADP and PP
To elucidate whether Ca2+ influx functioned
primarily as a charge carrier or as an intracellular messenger,
[Ca2+]i was chelated by
inclusion of 10 mM
bis(2-aminophenoxy)ethane-N,N,N,N-tetra-acetic acid
(BAPTA) in the pipette solution (Table 1). Application of 20 µM carbachol not only failed to depolarize
these BAPTA-containing neurons, but neither the sADP nor the PP were
observed after action potential firing evoked by depolarizing current
injection (Fig. 7B; n = 4/4).
In the same slices, however, both the cholinergic-dependent sADP and PP
were evoked in pyramidal neurons dialyzed with control intracellular
solution (Fig. 7A; n = 4/4). These data
demonstrate that elevations in
[Ca2+]i were critical for
genesis of the sADP and PP. Hence, the primary role of
Ca2+ influx was to serve as an intracellular
messenger, not as a charge carrier.
Fig. 7.
Both the sADP and PP were blocked by intracellular
BAPTA. A, Typical responses of a hippocampal pyramidal
neuron to hyperpolarizing and depolarizing current injection. Neither
an sADP nor a PP was observed in the absence of cholinergic stimulation
(A1). The calibration bars in the inset represent
40 msec and 20 mV. In the presence of carbachol, however, depolarizing
current injection evoked a long-lasting PP (A2). This
pyramidal neuron was loaded with pipette solution containing 1.1 mM EGTA and 0.1 mM
Ca2+ and, thus, intracellular
Ca2+ was bufferred to ~16
nM. B, A different pyramidal neuron,
recorded from the same slice as the neuron illustrated in A,
loaded with 10 mM BAPTA. In control aCSF, neither
an sADP nor a PP was observed (B1). The calibration bars in
the inset represent 40 msec and 20 mV. In the presence of
carbachol, neither an sADP nor a PP could be evoked, suggesting that
elevated [Ca2+]i was
necessary for these potentials (B2).
[View Larger Version of this Image (23K GIF file)]
The goal of the next set of experiments was to determine whether the
sADP and PP could be evoked by maximizing Ca2+
influx via K+ channel suppression. Two methods
were used independently to suppress K+ channels.
First, external K+ channel blockers [10
mM tetraethylammonium (TEA), 5 mM 4-aminopyridine (4-AP), 100 µM Ba2+; Fig.
8A; n = 9] were included in
the perfusate, and when applied to pyramidal neurons, depolarized the
membrane potential by 9 ± 1 mV (range 5-10; n = 9).
Second, 40 mM Cs+ was
included in the pipette solution
([Cs+]i; Fig.
8B; Table 1; n = 11). Both conditions would
independently increase Ca2+ influx, and yet
neither the sADP nor the PP could be elicited from a membrane potential
maintained at 60 mV with negative DC current injection. Even though
K+ channels were suppressed, application of 20 µM carbachol still depolarized the membrane
potential by an additional 4 ± 1 mV (range 2-7; n = 17) (Benson et al., 1988; Colino and Halliwell, 1993). In the presence
of carbachol, both the sADP and PP were now easily evoked from a
membrane potential of 60 mV. These afterpotentials followed
regenerative Ca2+ spikes and, as before, were
reversibly depressed by reduction of extracellular
Na+ from 152 to 26 mM (Fig.
8A,B; n = 17/17). A small sADP, however, was
still observed in low extracellular Na+ with a
maximal amplitude of 5 ± 1 mV (range 3-13; n = 17);
however, the amplitude of the depolarizing current injection was
twofold greater. Interestingly, the membrane potential of the PP
measured at 0.2 sec after cessation of the stimuli was 10 mV more
positive when K+ channels were suppressed (9 ± 2 mV; range 21 to 3; n = 17; vs control 19 ± 1 mV). These experiments demonstrate that Ca2+
influx alone was not sufficient for sADP and PP genesis. Moreover,
Na+ was the main charge carrier underlying the
sADP and PP, whereas deactivation of a K+
conductance by maintained Ca2+ influx was
excluded as a contributing mechanism (Constanti and Bagetta, 1991;
Constanti et al., 1993). Finally, the membrane potential of the PP in
the presence of K+ channel blockers (9 mV) was
consistent with activation of a Ca2+-activated
nonselective cation conductance with little contribution of concurrent
outward conductances.
Fig. 8.
Blockers of K+ channels
failed to unmask either the sADP or the PP in the absence of
cholinergic stimulation. A, The sADP and PP were not
observed after application of 1.2 µM TTX and
K+ channel blockers (10 mM
TEA, 5 mM 4-AP, 100 µM
Ba2+). Cholinergic stimulation, however, revealed
a PP that was reversibly depressed by lowering
Na+ from 152 to 26 mM in
the external media (A1). Notice that robust
Ca2+ spikes remained in low
Na+, whereas the PP was reversibly depressed
(A2). B, In addition, neither the sADP nor the PP
was observed in pyramidal neurons loaded with 40 mM intracellular Cs+ and
bathed in 1.2 µM TTX. As above, cholinergic
stimulation revealed a PP that was reversibly depressed by lowering
Na+ in the external media (B1). In
this example, robust Ca2+ spikes also remained in
low Na+, whereas the PP was reversibly depressed
(B2).
[View Larger Version of this Image (32K GIF file)]
In the following experiments, the involvement of a
Na+/Ca2+ exchanger was
tested in two ways (Crépel et al., 1994). First, 50 mM LiCl was substituted for equimolar NaCl in the
extracellular solution (Fig. 9A;
n = 4). Immediately after ion substitution, the
membrane potential depolarized by 9 ± 1 mV (range 6-11 mV;
n = 4) corresponding to a
Li+-induced depression of the
Na+/K+ ATPase (Glynn,
1993). Negative DC current was applied via the recording pipette (60 ± 9 pA; range 80 to 40 pA; n = 4), however, to
maintain the membrane potential at the same presubstitution level.
Depolarizing current was then injected and, as described previously,
both the sADP and PP were evoked after spike firing in all neurons
tested (n = 4/4). Because the
Na+/Ca2+ exchanger is also
depressed by Li+ (Reuter and Porzig, 1995), this
experiment implies that the
Na+/Ca2+ exchanger played
little, if any, role in genesis of the sADP and PP. An alternative
explanation favored by this experiment is the
Ca2+-activated nonselective cation channel that
is permeable to Li+ (Yellen, 1982). In the second
experiment, spike firing elicited by depolarizing current injection
evoked both the cholinergic-dependent sADP and PP. The bath temperature
was then lowered from 35 to 25°C, and action potential firing was
elicited as described previously (Fig. 9B; n = 3). In all cases, a decrease of 10°C did not depress either the
sADP or the PP. Because the
Na+/Ca2+ exchanger is
extremely temperature-sensitive (Kimura and Reeves, 1979; see
Crépel et al., 1994), this second set of experiments also implies
that the Na+/Ca2+ exchanger
played a negligible role in genesis of the sADP and PP. Interestingly,
if the bath temperature was lowered to 21-23°C, the PP could not be
elicited (data not shown; n = 3).
Fig. 9.
The sADP and PP were independent of a
Na+/Ca2+ exchanger.
A, Intracellular current injection revealed an sADP and a PP
in the presence of 20 µM carbachol. The resting
membrane potentials before and after cholinergic stimulation were 70
and 62 mV, respectively. Both the sADP and PP were still observed
after substitution of 50 mM
Li+ for equimolar Na+ in
the external media. After Li+ substitution, the
membrane depolarized an additional 9 mV; however, negative DC current
was injected to maintain the membrane potential at the presubstitution
value. B, In a different neuron, intracellular current
injection also revealed an sADP and a PP in the presence of 20 µM carbachol. The resting membrane potentials
before and after cholinergic stimulation were 66 and 61 mV,
respectively. Both the sADP and PP were still observed after a
reduction in bath temperature from 35 to 25°C. The neuron depolarized
after temperature reduction, however, negative DC current was injected
to maintain the membrane potential at the same value as at
35°C.
[View Larger Version of this Image (35K GIF file)]
Taken together, these experiments indicate that both the sADP and PP
require three events: cholinergic stimulation, an elevation in
[Ca2+]i via HVA
Ca2+ channels (L- and N-type), and activation of
the Ca2+-activated nonselective cation
conductance.
Effects of individual K+ channel blockers on the sADP
and PP
Although the sADP and PP could not be unmasked by
K+ channel suppression, once elicited, these
waveforms are likely to be modulated by concurrent outward
K+ conductances. The effects of individual
K+ channel blockers, therefore, were tested on
the sADP and PP to determine whether individual
K+ conductances influenced the shape and/or
duration of the afterpotential waveforms. In the absence of carbachol,
the addition of 10 mM TEA to the perfusate
resulted in an initial 5 ± 2 mV (range 2-7 mV; n = 3)
depolarization of the membrane potential. When the membrane potential
was stable, spike firing elicited by intracellular current injection
revealed a brief PP, probably mediated by HVA
Ca2+ channels activated by action potential
firing (Fig. 10A; n = 3/3)
(Bourque et al., 1986). After cessation of the stimuli, neither an sADP
nor a PP was observed. In a different pyramidal neuron, both the
cholinergic-dependent sADP and PP were easily evoked after action
potential firing. Coapplication of 10 mM TEA,
however, reduced the amplitude of the sADP and the duration of the PP
(Fig. 10A; n = 4/4). The duration of the PP
was reduced from 11.6 ± 4.1 sec (range 10-21.4; n = 4) in carbachol to 6.9 ± 2 sec (range 1.3-10.4; n = 4) in the presence of TEA. Furthermore, the membrane potential of the
PP measured at 0.2 sec after cessation of the stimuli was increased
from 15 ± 3 mV (range 20 to 9; n = 4) in
carbachol to 8 ± 3 mV (range 4 to 0) in the presence of TEA.
Because TEA effectively increased Ca2+ influx via
K+ channel depression, it seems reasonable to
conclude that TEA inhibited the Ca2+-activated
nonselective cation channel directly (see Crépel et al.,
1994).
Fig. 10.
Individual K+ channel
blockers modulate the PP waveform. A, Action potential
firing in the presence of 10 mM TEA resulted in a
brief plateau potential that was presumably
Ca2+-dependent (A1). Neither an sADP
nor a PP was observed after cessation of the depolarizing stimulus. The
calibration bars in the inset represent 40 msec and 20 mV.
In a different neuron, both the sADP and PP were observed in the
presence of 20 µM carbachol (A2).
Coapplication of 10 mM TEA abolished the sADP and
decreased the duration of the PP. B, In the presence of 5 mM 4-AP, individual action potentials activated
brief afterdepolarizations that presumably were
Ca2+-dependent (B1). After cessation
of the depolarizing current stimuli, a brief afterpotential was
commonly observed. Neither the sADP nor the PP was observed. The
calibration bars in the inset represent 40 msec and 20 mV.
In another neuron, a PP was evoked in the presence of 20 µM carbachol (B2). Coapplication of
5 mM 4-AP slightly prolonged the PP.
C, Application of 100 µM
Ba2+ to the perfusate resulted in a brief
afterpotential and subsequent spike broadening (C1). Brief
afterpotentials were also observed after depolarizing current stimuli,
however, neither the sADP nor the PP was evoked. In the presence of 20 µM carbachol, a different neuron exhibited an
sADP and a PP after cessation of stimuli (C2). Coapplication
of 100 µM Ba2+ to the
perfusate increased the PP duration.
[View Larger Version of this Image (30K GIF file)]
In separate experiments, the addition of 5 mM
4-AP to the perfusate, in the absence of carbachol, resulted in a 3 ± 1 mV (range 1-4 mV; n = 3) depolarization of the
membrane potential. This K+ channel blocker also
increased Ca2+ influx, but instead of a single
PP, which was observed after TEA perfusion, a transient potential
followed each action potential (Fig. 10B; n = 3). After a burst of evoked action potentials, only a brief
afterpotential was observed in 4-AP. In a different pyramidal neuron, a
PP was evoked in the presence of 20 µM
carbachol that was slightly prolonged by coapplication of 4-AP (Fig.
10B; n = 3/3). The duration of the PP was
11.3 ± 0.9 sec (range 9.7-12.8; n = 3) in carbachol
versus 14.3 ± 1.1 sec (range 11.4-15; n = 3) in 4-AP.
In addition, the membrane potential of the PP measured at 0.2 sec after
cessation of the stimuli was increased from 20 ± 2 mV (range 22 to
17; n = 3) in carbachol to 14 ± 1 mV (range 17
to 13) in the presence of 4-AP. The small increase in duration may
reflect increased Ca2+ influx stimulated by
K+ channel suppression.
In the absence of carbachol, the addition of 100 µM Ba2+ to the perfusate
resulted in a 4 ± 1 mV (range 2-5; n = 3)
depolarization of the membrane potential. This K+
channel blocker increased Ca2+ influx as
evidenced by increased spike broadening (Fig. 10C;
n = 3) (Bourque et al., 1986). At this concentration,
however, it is unlikely that Ba2+ permeated the
HVA Ca2+ channels, because
Ba2+ has a much lower binding affinity within the
pore and therefore is repelled out of the channel by
Ca2+ (Hess and Tsien, 1984). Even with increased
Ca2+ influx, neither an sADP nor a PP was evoked
after current injection. In another pyramidal neuron, an sADP and a PP
were observed after current injection in the presence of 20 µM carbachol. Coapplication of 100 µM Ba2+ greatly prolonged
the PP duration (Fig. 10C; n = 3/3). In
carbachol, the average duration of the PP was 8.8 ± 2.1 sec (range
5.9-13.7; n = 3), whereas in
Ba2+ the duration increased to 17.2 ± 3.1 sec
(range 13.6-22.9; n = 3). In addition, the membrane
potential of the PP measured at 0.2 sec after cessation of the stimuli
was increased from 17 ± 1 mV (range 20 to 16; n = 3) in carbachol to 12 ± 1 mV (range 14 to 10) in the presence
of Ba2+. These data imply that a
Ba2+-sensitive conductance contributed to the
shape of the PP waveform. A likely candidate expressed in hippocampal
pyramidal neurons that opposes prolonged depolarizations and is
Ba2+-sensitive is IM
(Halliwell and Adams, 1982). Additional K+
channels are, however, also suppressed by this divalent cation.
In the absence of carbachol, the simultaneous addition of
Ca2+-activated K+ channel
blockers [100 nM iberiotoxin (ITX), 100 nM apamin] to the perfusate depolarized the
neurons by 3 ± 1 (range 0-4; n = 4) and completely
blocked the afterhyperpolarization after each action potential. In the
presence of these blockers, however, neither the sADP nor the PP could
be evoked (data not shown; n = 4/4). In different
pyramidal neurons, both ITX and apamin failed to affect either the sADP
or PP observed after carbachol application (n = 3/3).
These experiments indicate that Ca2+-activated
K+ conductances did not shape either the sADP or
PP waveforms.
Taken together, these experiments again illustrate that although
Ca2+ influx was increased via
K+ channel depression, the sADP and PP required
cholinergic stimulation. In addition, TEA may have an inhibitory affect
on the Ca2+-activated nonselective cation
channel, whereas a Ba2+-sensitive current (e.g.,
IM) was important for determining the
duration of the PP waveform.
Conductance changes accompanying the PP
In the final experiments, changes in
RIN were examined during the PP by
injection of low-amplitude current pulses (50 pA; 0.2 sec; 5 Hz; Fig.
11). In all neurons examined, the
RIN decreased during the PP by 81 ± 2%
(range 71-87; n = 16), measured within 2 sec after
cessation of the depolarizing current stimuli (Fig. 11A). In
the presence of TTX and K+ channel blockers, the
RIN measured during the PP also decreased
by 80 ± 3% (range 75-84; n = 3; Fig.
11B). Because similar changes in
RIN were obtained in the absence and
presence of both TTX and K+ channel blockers, the
contributions of slow Na+ oscillations and
K+ channel activation were minimal in analysis of
conductance changes immediately after cessation of stimuli. In all
cases, a reproducible pattern was observed, in which the
RIN observed immediately after cessation of
the depolarizing stimuli gradually diminished during the PP. Even
though the RIN increased significantly
during the PP, only a minor change in membrane potential was observed
(Fig. 11C). Finally, injection of large-amplitude current
pulses of long duration (0.4 nA; 1 sec) failed to terminate the PP
(Fig. 11D; n = 4/4).
Fig. 11.
The PP was associated with a large
RIN decrease and could not be terminated by
hyperpolarizing pulses. A, Cholinergic-dependent PPs
illustrated on two time scales. In the latter, brief current injection
(50 pA; 0.2 sec; 0.5 Hz) demonstrated a large decreased
RIN. B, PPs in the presence of
carbachol, TTX, and K+ channel blockers (10 mM TEA, 5 mM 4-AP, 100 µM Ba2+). Under these
conditions, brief current injection confirmed an
RIN decrease in the absence of slow
Na+ oscillations and suppression of
K+ channels. C, A graph depicting the
decreased RIN relative to control obtained
at the resting potential, versus duration of the PP. In the same graph,
membrane potential was also plotted versus PP duration. Both
RIN and membrane potential values were
obtained from the neuron illustrated in B (*). Notice that
the change in membrane potential was minimal, whereas the increase in
RIN was significant. D, Two
superimposed PPs evoked from a CA1 pyramidal neuron. In the broken
trace, hyperpolarizing current injection (0.4 nA; 0.8 sec) failed to
terminate the PP.
[View Larger Version of this Image (29K GIF file)]
DISCUSSION
In this study, we report a cholinergic-induced sADP and PP
recorded from hippocampal CA1 pyramidal neurons with the whole-cell
patch-clamp technique. We propose that the PP represents a novel
regenerative event involving the interplay between HVA
Ca2+ channels (L- and N-type) and the
Ca2+-activated nonselective cation conductance.
These ionic mechanisms may contribute significantly to ictal
depolarizations observed during temporal-lobe seizures of the
partial-complex subtype (Lothman et al., 1991).
Both the sADP and PP were depressed by removal of either
Na+ or Ca2+ from the
external media, and blocked by either extracellular
Cd2+ or chelation of
[Ca2+]i by inclusion of
BAPTA in the intracellular solution. Moreover, the sADP was depressed
and the PP was abolished by either the L-type channel blocker,
nimodipine, or the N-type channel blocker -conotoxin-GVIA.
Superfusion of TTX at a concentration sufficient to block fast action
potentials, however, did not suppress either the sADP or PP. These data
imply that both the sADP and PP rely on elevations in
[Ca2+]i via HVA
Ca2+ channels (L- and N-type) and
Na+ influx independent of TTX-sensitive
channels.
One possible mechanism contributing to the sADP and PP described here
is the Na+/Ca2+ exchanger
(Friedman et al., 1992). Our experiments, however, do not support a
role for this mechanism. First, equimolar substitution of extracellular
Na+ with 50 mM
Li+ did not affect either the sADP or the PP. The
Na+/Ca2+ exchanger does not
use Li+ as a charge carrier (Kimura et al., 1987;
Reuter and Porzig, 1995); however, the
Ca2+-activated nonselective cation channel is
permeable to Li+ (Yellen, 1982). Second, lowering
the temperature from 35 to 25°C also did not affect either the sADP
or the PP (see Crépel et al., 1994). Because the
Na+/Ca2+ exchanger is
highly temperature-sensitive (Kimura and Reeves, 1979), this latter
experiment also suggests that this mechanism plays a negligible role in
sADP and PP genesis. Another possible mechanism that may have
contributed to the sADP and PP is deactivation of a
K+ conductance by maintained
Ca2+ influx (Constanti and Bagetta, 1991;
Constanti et al., 1993). This mechanism was also ruled out by our
experiments because both the sADP and the PP were observed in the
presence of either extracellular K+ channel
blockers or [Cs+]i.
Furthermore, an increased conductance was observed during both the sADP
and PP, which is not consistent with this mechanism (Constanti and
Bagetta, 1991). Based on our experiments, a
Ca2+-activated nonselective cation conductance is
the prime candidate for sADP genesis (Schwindt et al., 1988; Hasuo et
al., 1990; Caeser et al., 1993). The PP, however, relied not only on
the Ca2+-activated nonselective cation
conductance, but also on prolonged activation of HVA
Ca2+ channels (Blitzer et al., 1991). After
Ca2+ was elevated to a critical level by spike
firing, the Ca2+-activated nonselective cation
channel was sufficiently activated to depolarize the membrane potential
positive enough to sustain Ca2+ influx, and the
regenerative potential was initiated. Hence, the sADP represents a weak
depolarization mediated by residual
[Ca2+]i, whereas the PP
represents a novel regenerative event relying on continual activation
of both HVA Ca2+ channels and the
Ca2+-activated nonselective cation
conductance.
A major question is whether the Ca2+-activated
nonselective cation conductance was directly enhanced by carbachol or
simply unmasked by cholinergic-induced suppression of
K+ conductances (Caesar et al., 1993). Our data
indicate that the Ca2+-activated nonselective
cation conductance was directly enhanced by cholinergic stimulation. In
the presence of either K+ channel blockers or
[Cs+]i, neither the sADP
nor the PP was observed. The sADP and PP were only elicited after
carbachol was added to the perfusate. Because HVA
Ca2+ channels are depressed by cholinergic
stimulation (Toselli and Lux, 1989), the
Ca2+-activated nonselective cation channel must
have been augmented by carbachol. Cholinergic-induced depression of
K+ channels would, however, facilitate genesis of
the sADP and PP by increasing the net inward current. Moreover, because
neither the sADP nor the PP was observed with K+
channel depression, the Ca2+-activated
nonselective cation channel must have a low open-channel probability in
the absence of cholinergic stimulation.
Previous studies have shown that PPs can be generated by either
voltage-activated Ca2+ or
Na+ channels. PPs generated by
Ca2+ channels alone, however, are observed only
when K+ channel blockers are present,
intracellular Ca2+ is chelated, and/or
Ba2+ ions are used as the charge carriers (Llinas
and Yarom, 1981; Bourque et al., 1986). PPs generated solely by
voltage-activated Ca2+ channels are depressed by
muscarinic agonists (Misgeld et al., 1986). This effect is consistent
with voltage-clamp studies demonstrating cholinergic-induced depression
of HVA Ca2+ channels (Toselli and Lux, 1989).
Alternatively, fast-inactivating Na+ channels may
gate in a bimodal manner, whereby bursts of channel openings are
prolonged (Taylor, 1993). In hippocampal pyramidal neurons, the role of
persistent Na+ channel openings in generating PPs
is unclear, because these events have been observed only in 0 Ca2+ external media (Leung and Yim, 1991;
García-Muñoz et al., 1993; Segal, 1994). PPs generated by
these Na+ channels are identified by their
sensitivity to the specific channel blocker TTX. In contrast to the
above mechanisms, the sADP and PP described here were evoked in
physiological solutions, requiring only cholinergic stimulation. The
expression of PPs in intact tissue, therefore, could occur as a result
of burst firing and activation of the cholinergic system.
The Ca2+-activated nonselective cation
conductance implicated in this study is present in a broad range of
cell types (Partridge and Swandulla, 1988) and is distinct from
voltage-gated cation channels. For example, voltage-gated cation
channels have been described in a variety of neuronal cell types (Hoehn
et al., 1993; Alzheimer, 1994) and participate in afterpotential
genesis after spike repolarization (Penington and Kelly, 1993). These
depolarizing afterpotentials are not sensitive to
[Ca2+]i, are only
milliseconds in duration, and mediate burst firing at rapid frequencies
(Deisz, 1996). Hence, the depolarizing afterpotentials mediated by
voltage-activated cation channels are clearly distinguishable from the
sADP and PP described in this paper.
Based on our findings, the sADP represents an afterpotential due
to the Ca2+-activated nonselective cation
conductance activated by Ca2+ influx via HVA
channels. The PP, however, represents a novel regenerative event
involving the interplay between HVA Ca2+ channels
and the Ca2+-activated nonselective cation
conductance. This interplay between channels may occur at a distal
dendritic site because both a Ca2+-sensitive
inward current (Benson et al., 1988; Colino and Halliwell, 1993) and
HVA Ca2+ channels (L-type, Westenbroek et al.,
1990; Magee and Johnston, 1995; N-type, Jones et al., 1989; Mills et
al., 1994) have been localized to dendritic processes. Dendritically
located currents may be one reason why the PP has not been reported
previously; for example, the PP may have been masked by a large somatic
current shunt produced by microelectrode impalement (Spruston and
Johnston, 1992; Staley et al., 1992). In addition, many studies using
whole-cell patch-clamp techniques have either chelated
[Ca2+]i or worked at room
temperature, where the PP was not observed. Under our experimental
conditions, the ability to evoke the sADP and PP was both reliable and
reproducible.
The sADP and PP described here involved intrinsic mechanisms directly
modulated by cholinergic stimulation. These included enhancement of the
Ca2+-activated nonselective cation conductance
and depression of several K+ conductances. It is
unlikely that other neurotransmitters (e.g., glutamate) or synaptic
interactions played a significant role in this study because both the
sADP and PP were observed in TTX. Moreover, thin slices (150 µm) were
commonly used where synaptic connections and recurrent collaterals were
minimal. Although other neurotransmitters and synaptic interactions
were presumably not involved in sADP and PP genesis in this study, they
likely play a significant physiological role. For example, previous
work in cortical neurons has demonstrated a cholinergic-sensitive slow
inward conductance potentiated by low-frequency synaptic stimulation of
white matter (Andrade, 1991). It is therefore conceivable that a PP
could also be evoked during synaptic stimulation. For instance,
cholinergic stimulation elevates intradendritic
Ca2+ accumulation in hippocampal pyramidal
neurons during repetitive firing (Müller and Connor, 1991) and
enhances Ca2+ influx via postsynaptic NMDA
receptors (Markram and Segal, 1992; Segal, 1992). This elevation in
dendritic [Ca2+]i could
activate the Ca2+-activated nonselective cation
conductance. Depolarization of the membrane would then activate HVA
Ca2+ channels, and a regenerative PP could be
initiated. Similar mechanisms may underlie long-duration rhythmic
bursts observed after either cholinergic or metabotropic stimulation of
hippocampal slices (Bianchi and Wong, 1994, 1995). In our hands,
stimulation of metabotropic glutamate receptors with
trans-(I)-1-amino-1,3-cyclopentanedicarboxylic
acid also resulted in sADP and PP generation (our unpublished
observations). Interestingly, both cholinergic and metabotropic
glutamate receptors are linked to phospholipase C via G-proteins,
suggesting a common final pathway.
Functional significance
Cholinergic agents are effective in generating hippocampal
seizures both in vivo and in vitro (Lothman et
al., 1991; Wasterlain et al., 1993). In the hippocampal slice
preparation, these same analogs generate prolonged depolarizations
(Bianchi and Wong, 1994) and exacerbate ictal depolarizations observed
during high K+-induced seizures (Yaari and
Jensen, 1989). In this paper, we have identified a novel regenerative
PP involving the interplay between HVA Ca2+
channels and the Ca2+-activated nonselective
cation conductance, and have also presented evidence that this latter
conductance was directly enhanced by cholinergic stimulation. Because
this PP has properties reminiscent of ictal depolarizations observed
during cholinergic-induced seizures, it seems reasonable to conclude
that these mechanisms could play a central role in hippocampal
epileptogenesis.
FOOTNOTES
Received Feb. 27, 1996; revised April 2, 1996; accepted April 8, 1996.
This work was supported by a grant from the Medical Research Council of
Canada (MRC). D.D.F. is a recipient of studentships from the MRC,
Alberta Heritage Foundation for Medical Research (AHFMR), and Savoy
Foundation for Epilepsy Research. B.A.M. is an AHFMR scientist and the
Ciba Geigy chair for schizophrenia research. We are grateful to Drs. S. Barnes and G. Spencer for critical comments on this manuscript and Drs.
A. R. McQuiston and W. F. Colmers for slice-patch advice.
Correspondence should be addressed to Douglas D. Fraser, Department of
Neuroscience, 3330 Hospital Drive N.W., Health Sciences Building,
University of Calgary, Alberta, Canada, T2N 4N1.
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