Download Ionic Mechanism of the Slow Afterdepolarization Induced by

Ionic Mechanism of the Slow Afterdepolarization Induced by
Muscarinic Receptor Activation in Rat Prefrontal Cortex
SAMIR HAJ-DAHMANE AND RODRIGO ANDRADE
Department of Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine, Detroit,
Michigan 48201
INTRODUCTION
The prefrontal cortex receives a dense cholinergic innervation originating in the basal forebrain and to a lesser
extent brain stem cholinergic nuclei (Woolf et al. 1984).
Cholinergic stimulation of pyramidal cells in this region
produces an increase in cellular excitability that is the result
of several specific effects, including a membrane depolarization, a reduction in the afterhyperpolarization (AHP) that
follows a burst of spikes, and its replacement by a slow
afterdepolarization (sADP) (Andrade 1991). Behavioral
studies have shown that the cholinergic input to cortex plays
an important role in maintaining normal cortical function
(Aigner 1995; McKinney and Coyle 1991; Winkler et al.
1995) including memory (Broersen et al. 1995; GoldmanRakic 1990; Granon et al. 1995). Therefore these cellular
effects might correspond to some of the elementary events
through which acetylcholine (ACh) regulates cortical networks to allow for normal cognitive functioning.
We recently have shown that the muscarinic induced depolarization in this region is mediated by the activation of a
voltage-sensitive nonselective cation current (Haj-Dahmane
and Andrade 1996). However the mechanism responsible
for the sADP in this region was not elucidated. sADPs,
similar to those observed in prefrontal cortex, were also
observed after muscarinic (Constanti and Bagetta 1991;
Schwindt et al. 1988), a1-adrenergic (Araneda and Andrade
1991), 5-HT 2 (Araneda and Andrade 1991; Spain 1994),
and glutamate metabotropic receptor activation (Constanti
and Libri 1992; Greene et al. 1992) in several different
cortical regions. This suggested that the current underlying
the sADP might be an important target for neurotransmitter
regulation in cortex. In this study we examined the ionic
mechanisms responsible for the generation of the sADP in
prefrontal cortex.
METHODS
Preparation of brain slices
Cortical brain slices that included the medial prefrontal cortex
( Krettek and Price 1977 ) were prepared as previously described
( Andrade 1991 ) . Male albino rats ( 200 – 250 g ) were anesthetized with halothane and killed by decapitation. The brain was
removed and cooled in ice-cold, oxygenated Ringer solution
( composition in mM: 119 NaCl, 2.5 KCl, 1.3 MgSO4 , 2.5 CaCl2 ,
1 NaH2PO4 , 26.2 NaHCO3 , and 11 glucose ) , and the anterior
forebrain was isolated. The anterior pole of the brain was then
affixed to a stage with cyanoacrylate glue and sectioned into
400-mm-thick coronal slices with the use of a vibratome ( Lancer
series 1000, Ted Pella, Irvine, CA ) . The brain slices were then
placed on filter paper saturated with oxygenated Ringer solution
inside a chamber filled with a moist atmosphere of 95% O2-5%
CO2 at room temperature. After ¢1 h of recovery, slices were
transferred as needed to a recording chamber of standard design
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Haj-Dahmane, Samir and Rodrigo Andrade. Ionic mechanism
of the slow afterdepolarization induced by muscarinic receptor
activation in rat prefrontal cortex. J. Neurophysiol. 80: 1197–1210,
1998. The mammalian prefrontal cortex receives a dense cholinergic innervation from subcortical regions. We previously have
shown that cholinergic stimulation of layer V pyramidal neurons
of the rat prefrontal cortex results in a depolarization and the appearance of a slow afterdepolarization (sADP). In the current report we examine the mechanism underlying the sADP with the use
of sharp microelectrode and whole cell recording techniques in in
vitro brain slices. The ability of acetylcholine (ACh) and carbachol
to induce the appearance of an sADP in pyramidal cells of layer
V of prefrontal cortex is antagonized in a surmountable manner
by atropine and is mimicked by application of muscarine or oxotremorine. These results indicate that ACh acts on muscarinic receptors to induce the sADP. In many cell types afterpotentials are
triggered by calcium influx into the cell. Therefore we examined
the possibility that calcium influx might be the trigger for the
generation of the sADP. Consistent with this possibility, buffering
intracellular calcium reduced or abolished the sADP but had little
effect on the direct muscarinic receptor-induced depolarization also
seen in these cells. These results, coupled to the previous observation that calcium channel blockers inhibit the sADP, indicated that
the sADP results from a rise in intracellular calcium secondary to
calcium influx into the cell. The ionic basis for the current underlying the sADP (IsADP ) was examined with the use of ion substitution
experiments. The amplitude of IsADP was found to be reduced in a
graded fashion by replacement of extracellular sodium with Nmethyl-D-glucamine (NMDG). In contrast no clear evidence for
the involvement of potassium or chloride channels in the generation
of the sADP or IsADP could be found. This result indicated that
IsADP is carried by sodium ions flowing into the cell. However, the
dependence of IsADP on extracellular sodium was less pronounced
than expected for a pure sodium current. We interpret these results
to indicate that the sADP is most likely mediated by nonselective
cation channels. Examination of the current underlying the sADP
at different voltages indicated that this current was also voltage
dependent, turning off with hyperpolarization. We conclude that
the sADP elicited by muscarinic receptor activation in rat cortex
is mediated predominantly by a calcium- and voltage-sensitive
nonselective cation current. This current could represent an important mechanism through which ACh can regulate neuronal excitability in prefrontal cortex.
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S. HAJ-DAHMANE AND R. ANDRADE
( Nicoll and Alger 1981 ) . In this chamber the slices were held
submerged between two nylon nets and continuously perfused
( 2 – 4 ml /min ) with normal Ringer solution bubbled to saturation
with 95%O2-5% CO2 at 30 { 17C.
Electrophysiological recordings
Intracellular recording were obtained from pyramidal neurons
of layer V of the prelimbic and anterior cingulate subdivisions
of the medial prefrontal cortex with the use of patch clamp or
conventional sharp microelectrode recording techniques. For standard intracellular recording sharp microelectrodes were pulled from
1.2-mm OD omega-dot glass (Friedrich and Dimmock, Millville,
NJ) with a Flaming-Brown horizontal puller (Model PC80/PC,
Sutter Instrumentrs, Novato, CA) to give resistance ranging from
90 to 150 MV when filled with 2 M potassium methylsulfate.
Neurons were impaled by delivering a high-voltage pulse through
the electrode or by ‘‘ringing’’ induced by overcompensating the
capacity compensation circuit of the amplifier. Data were collected
only from neurons with a stable resting membrane potential more
negative than –60 mV and overshooting spikes. Whole cell recordings were performed with the use of the blind tight seal technique (Blanton et al. 1989). The recording pipettes were pulled
from 1.2-mm OD borosilicate glass with the use of a FlamingBrown horizontal puller to give resistances ranging from 4 to 6
MV when filled with an internal solution of the following composition (in mM): 125 KMeSO4 , 5 NaCl, 1 MgCl2 , 10 N-2-hydroxyethylpiperazine-N *-2-ethanesulfonic acid (HEPES), 0.02 ethylene
glycol - bis( b - aminoethyl ether) - N, N, N *, N * - tetraacetic acid
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( EGTA), 2 Na2ATP, and 0.5 Na3 guanosine 5 *-triphosphate
(Na3GTP). The pH was adjusted to 7.3–7.4 with KOH. In some
experiments, the calcium buffering capacity of the pipette solution
was increased with 10 mM EGTA or bis-(o-aminophenoxy)N,N,N *,N *-tetraacetic acid (BAPTA) and added calcium to bring
the free calcium concentration to Ç10 nM. In some experiments,
potassium methylsulfate was substituted by cesium gluconate or
cesium methanesulfonate in the internal solution to examine the
possible role of potassium current in the sADP. In these cases the
pH of the internal solution was adjusted to 7.3–7.4 with CsOH
and the osmolarity to Ç5 mosM l – 1 lower than the osmolarity of
standard Ringer ( Ç295 mosM – 1 ). With all these solutions access
resistance measured with the use of the bridge compensation circuit
of the amplifier ranged from 10 to 25 MV.
Voltages were measured and current was injected with the use
of an Axoclamp 2A amplifier (Axon Instruments, Foster City,
CA). Electrical signals were filtered at 5–10 kHz and recorded
on-line with the use of a paper chart recorder (Model 3200, Gould
Instruments, Valley View, OH). Fast events such as voltage steps
and actions potentials were digitized with the use of an Intel processor-based computer equipped with a 12-bit A/D converter under
the control of pClamp 5.5 software (Axon Instruments). Voltageclamp experiments were conducted with the use of either continuous or discontinuous voltage-clamp techniques. In the case of continuous voltage clamping, series resistance was corrected by
Ç70%. In the case of discontinuous voltage-clamp recordings, the
headstage output was monitored on an oscilloscope, and the sampling frequency was adjusted to obtain complete discharge of the
electrode between cycles. In some experiments a ‘‘hybrid’’ current/
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FIG . 1. Acetylcholine (ACh) and carbachol induce a slow afterdepolarization (sADP). A: response of a pyramidal neurons
recorded in current-clamp mode to a long depolarizing pulse under control conditions (left), after a brief pressure application
of ACh (middle), and after recovery (right). Note the blockade of the afterhyperpolarization (AHP) and its replacement by
an sADP after pressure application of ACh. Inset: spiking activity during the pulse with the use of an expanded timescale
(calibration 20 mV, 2 nA, 100 ms). Resting membrane potential, –69 mV. B: bath application of carbachol (30 mM) mimics
the effect of pressure-applied ACh in a different pyramidal neuron. Resting membrane potential, –75 mV. C: camera Lucida
drawing of a neurobiotin-filled cell exhibiting the typical electrophysiological profile of the regular spiking cells used in this
study. The response of this cell to muscarine is illustrated in Fig. 4.
NONSELECTIVE CATION CURRENT MEDIATES SADP IN CORTEX
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voltage-clamp protocol was used to record aftercurrents after a
stimulus, generally a calcium spike. In these experiments the amplifier was switched from voltage clamp to current clamp 200–500
ms before the constant current stimulus and back to voltage clamp
50–250 ms after the end of the stimulus under the control of the
Master 8 stimulator.
Drugs were applied by bath application, dissolved in the Ringer
at known concentration. In a few experiments, a patch pipette
containing 100 mM ACh was placed immediately above the slice
as close as possible to the cell of interest, and ACh was ejected
by a brief pressure pulse. The pressure and duration used for
ejecting ACh were adjusted to produce a small droplet at the tip
of each pipette under visual inspection under the dissecting microscope used to visualize the slice. These parameters were subsequently optimized to produce a reliable inward current or depolarization with the ejection pipette in place. The low-sodium Ringer
was prepared by substituting N-methyl-D-glucamine (NMDG) for
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sodium chloride, and the pH was adjusted to 7.4 with the use of
HCl. In those experiments where extracellular sodium was reduced
to 10 mM, the control Ringer was modified by omitting the
NaH2PO4 and replacing it with 10 mM HEPES. The pH of this
solution was adjusted to 7.4 with the use of HCl, and pure oxygen
was bubbled to equilibrium. Osmolarity was adjusted to match that
of the control solution with the use of sucrose. This solution became
our control solution in these experiments. To reduce extracellular
sodium to 10 mM this control solution was replaced by a lowsodium solution prepared following the same protocol except that
NMDG was substituted for all but 10 mM of the sodium. The lowchloride Ringer was prepared by substituting sodium isethionate
for sodium chloride. Most of the compounds used in this study
were obtained from Sigma (St. Louis, MO). Tetrodotoxin (TTX)
was from Calbiochem (La Jolla, CA). Data were analyzed and
plotted with the use of Origin (Microcal Software, Northampton,
MA). Numerical data are presented as means { SE. Statistical
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FIG . 2. sADP is mediated by a slow inward aftercurrent. A1a: burst of spikes followed by an AHP under control
conditions. The current underlying this AHP can be examined with a ‘‘hybrid’’ current/voltage-clamp protocol. A1b: after
bath administration of carbachol (30 mM) this AHP is replaced by sADP. This sADP can be seen to be mediated by a slow
inward aftercurrent (IsADP ) when recording with a hybrid current/voltage-clamp protocol. A1c: a similar IsADP can be observed
following 2 depolarizing constant current pulses in the presence of tetrodotoxin (TTX; 1 mM) in the same cell. Previous
studies have shown that the transient depolarizations ( A1c) elicited in these cells by the depolarizing pulses is mediated at
least in part by the activation of low-threshold calcium currents (Sutor and Zieglgänsberger 1987). A1d: in the same cell,
a depolarizing step to –20 mV under voltage clamp can also elicit IsADP . 1a and 1b, insets: spiking induced by the current
pulse illustrated with an expanded timescale. Inset 1c depicts a low-threshold calcium spike (calibration 100 ms, 20 mV, 2
nA). Resting membrane potential, –69 mV. B: carbachol induces IsADP in a concentration-dependent manner. Cells were
depolarized for 800 ms from –65 to –30 mV to allow calcium into the cell, and the peak amplitude of the resulting inward
aftercurrent was plotted. This plot reflects data obtained in 4 cells tested. The raw data were fitted with an occupational
model to an EC50 of 3.2 mM and the Hill coefficient of 1.6. Right traces: examples of the traces used to construct the doseresponse curve. Holding potential, –65 mV; holding current at rest, 210 pA.
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S. HAJ-DAHMANE AND R. ANDRADE
FIG . 3. Ability of carbachol to induce sADP is
blocked by atropine. Effect of carbachol under control conditions (top traces) and in the presence of
100 nM of the selective muscarinic antagonist atropine (bottom traces). Bath administration of atropine blocks the ability of carbachol to induce sADP
in a surmountable manner. Cell resting membrane
potential, –72 mV.
RESULTS
Recordings were obtained from ú300 cells from layer V
of the dorsal anterior cingulate and prelimbic subdivisions
of the rat medial prefrontal cortex (Krettek and Price 1977).
As previously described, the vast majority of neurons encountered in these recordings could be classified as regular
spiking neurons based on their electrophysiological characteristics (Haj-Dahmane and Andrade 1996). We previously
have shown that, as elsewhere in cortex (Connors and Gutnick 1990; Connors et al. 1982; Foehring et al. 1991; McCormick et al. 1985), these regular spiking neurons correspond
to morphologically identified pyramidal cells (Fig. 1C). All
experiments reported herein were conducted on this cell population.
Pyramidal neurons in medial prefrontal cortex respond to
a suprathreshold constant current-depolarizing pulse with a
burst of spikes, which is followed by an AHP. As illustrated
in Fig. 1A, a brief pressure administration of ACh elicits a
marked reduction in this AHP and its replacement by sADP
(n Å 10 cells, Fig. 1A). This effect can be elicited repeatedly
on the same cell with little evidence of desensitization or run
down for ¢30 min after breaking into the cell. Essentially
identical results are obtained after bath administration of the
poorly hydrolyzable ACh analogue carbachol (3–100 mM,
Fig. 1B) (n ú100 cells). Concurrent with the appearance
of this sADP, pressure application of ACh or bath administration of carbachol also elicits a membrane depolarization
that is mediated through a nonselective cation current (not
shown) (Haj-Dahmane and Andrade 1996). When examining the sADP in current clamp, this depolarization was neutralized by injection of hyperpolarizing current to maintain
a constant membrane potential.
FIG . 4. Selective muscarinic cholinergic agonists oxotremorine and muscarine also induce
sADP. A: depolarizing pulses capable of triggering a burst of action potential were delivered
under control condition (left trace), in the presence of oxotremorine (30 mM) (middle trace),
and after bath application of atropine (1 mM)
(right trace). Note that oxotremorine induces
sADP and that this effect is completely reversed
after administration of atropine. Resting membrane potential, –73 mV. B: in a different cell,
a depolarizing pulse delivered in the presence
of muscarine induces a strong sADP, which
reached threshold and generated sustained firing
activity (middle trace). This effect is completely
reversed by atropine (right trace). Resting
membrane potential, –68 mV.
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comparisons used Student’s paired or unpaired two-tailed t-test or
analysis of variance; P õ 0.05 was considered to be statistically
significant.
NONSELECTIVE CATION CURRENT MEDIATES SADP IN CORTEX
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With the use of a hybrid current/voltage-clamp protocol,
in which the cell is voltage clamped immediately after a
depolarizing stimulus, it is possible to examine directly the
current underlying the sADP. Fig. 2A illustrates one such
experiment. Under control conditions a burst of spikes is
followed by a slow outward aftercurrent that corresponds to
the current responsible for the AHP. Carbachol administration reduces this outward aftercurrent and induces the appearance of a slow inward aftercurrent. This inward aftercurrent is responsible for the sADP observed in current-clamp
conditions and will be referred to as IsADP . Action potentials
are not required for triggering this IsADP because this current
can also be triggered by depolarizing pulses capable of opening low-threshold calcium channels (Fig. 2A1c) or, when
recording entirely in voltage clamp, by depolarizing steps to
potentials positive to –40 to –50 mV (Fig. 2A1d).
kinetics of the sADP could be detected over the range of
stimulus strengths examined in this study.
As illustrated in Fig. 3, atropine at a concentration as low
as 100 nM completely blocks the ability of carbachol (10 mM)
to elicit an sADP (Fig. 3, n Å 5 cells tested), and this antagonism can be surmounted by increasing the carbachol concentration. These results indicate that carbachol induces sADP by
stimulating muscarinic receptors. Consistent with these findings, the selective muscarinic cholinergic agonists muscarine
(30 mM, n Å 6 cells tested, Fig. 4A) and oxotremorine (30
mM, n Å 4 cells tested, Fig. 4B) are also capable of eliciting
the appearance of sADP. Recovery from these agonists is
slower than with carbachol but can be easily obtained by the
addition of atropine to the bath (Fig. 4). Because carbachol
washes out faster than the other muscarinic agonists tested, it
was used to induce the sADP in the rest of the study.
Pharmacology of the receptors mediating the sADP
Properties of the muscarinic-induced sADP
The concentration-response relationship for the carbacholinduced IsADP was determined by examining the effect of
increasing concentrations of carbachol on the amplitude of
the sADP after a constant depolarizing step. As illustrated
in Fig. 2B, under control conditions a depolarizing step is
followed by an outward aftercurrent that decays completely
within 1–2 s from the end of the depolarizing step. Addition
of low micromolar carbachol to the bath reduces this outward
aftercurrent. Concomitant with this reduction carbachol induces the appearance of a slower inward aftercurrent (IsADP ).
This inward aftercurrent decays slowly and generally incompletely over several seconds and thus exhibits a slower time
course than the outward aftercurrent. Increasing the carbachol concentration results in a concentration-dependent increase in the amplitude of IsADP (Fig. 2B) that reaches a
maximum at concentrations ú30 mM. The EC50 for carbachol in these experiments is 3.2 mM (n Å 4 cells). For
a given carbachol concentration it is generally possible to
increase the amplitude of the sADP by increasing the intensity of the stimulus to allow more calcium to enter the cell,
but this effect shows saturation. No obvious changes in the
As indicated above, action potentials are not necessary
for generating the sADP. A depolarizing pulse capable of
triggering low-threshold calcium spikes or depolarizing voltage steps positive to –40 to –50 mV capable of opening
low-threshold calcium channels (Sutor and Zieglgänsberger
1987) is as effective as a burst of action potentials in inducing an IsADP (Fig. 2A). These results, combined with the
previous observation that the carbachol-induced sADP is
reduced by lowering extracellular calcium and inhibited by
calcium channel blockers (Andrade 1991), suggested that
this afterpotential depends on calcium influx into the cell.
Calcium-dependent ADPs similar to those studied here
were previously reported to be mediated by either calcium
(Hounsgaard and Kiehn 1989) or calcium-activated (or inactivated) currents (Kramer and Levitan 1988; Partridge and
Swandulla 1988; Yoshimura et al. 1987). To distinguish
between these possibilities recordings were obtained with
the use of intracellular solutions with differing calcium-buffering capacities. When recordings were conducted with the
use of an intracellular solution of low calcium-buffering capacity (20 mM EGTA), low-threshold calcium spikes reli-
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FIG . 5. Increase in intracellular concentration of
calcium is required for generation of sADP. A: current-clamp recording obtained from a pyramidal cell
with an intracellular solution containing 20 mM ethylene glycol-bis( b-aminoethyl ether)-N,N,N *,N *-tetraacetic acid (EGTA). Under this condition 2 lowthreshold calcium spikes in the presence of carbachol
(30 mM) reversibly induce sADP. B: recording of a
pyramidal neurons obtained with an internal solution
c o nt a i n in g 1 0 m M b is - ( o -a m i n op h e n ox y) N,N,N *,N *-tetraacetic acid (BAPTA; estimated intracellular free calcium concentration, 10 nM). Buffering of intracellular calcium with BAPTA completely
blocked the carbachol-induced sADP but failed to affect the membrane depolarization elicited by carbachol (bottom trace). The break in the voltage trace
corresponds to the point in time when we tested for
the sADP. C: summary plot depicting the amplitude of
the carbachol-induced sADP observed with differing
intracellular calcium buffering conditions, 20 mM
EGTA, n Å 11 cells; 10 mM EGTA, n Å 11 cells, 10
mM BAPTA, n Å 6 cells. * P õ 0.01 vs. 20 mM
EGTA.
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S. HAJ-DAHMANE AND R. ANDRADE
ably produced sADP in the presence of carbachol (30 mM)
(Fig. 5A). In contrast, when the calcium-buffering capacity
of the intracellular solution was increased by 10 mM EGTA
or 10 mM BAPTA, the sADP triggered with the use of the
same stimulation protocol was greatly reduced or completely
suppressed (Fig. 5B). In this experiment the amplitude of
the sADP was reduced from 16.3 { 4.0 mV (n Å 11 cells)
in 20 mM EGTA to 3.0 { 0.47 mV in 10 mM EGTA (n Å
11 cells, P Å 0.008) and 0.4 { 0.24 mV in 10 mM BAPTA
(n Å 6 cells, P Å 0.003). In contrast to these results buffering intracellular calcium had no effect on the carbacholinduced depolarization (Fig. 5B) (Haj-Dahmane and Andrade 1996), indicating that the inhibition of the sADP was
not simply secondary to a loss of muscarinic receptor functioning. These results indicated that the carbachol-induced
sADP most likely resulted from a calcium-activated (or
-inactivated) current.
Ionic mechanism underlying the sADP
Previous studies have shown that muscarinic agonists depress the AHP in cortex (McCormick and Prince 1986;
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Schwindt et al. 1988) and hippocampus (Benardo and Prince
1982; Nicoll et al. 1990). Thus muscarinic agonists could
induce sADP simply by unmasking a constitutively present
inward aftercurrent. Alternatively, muscarinic receptor stimulation could activate a previously silent current whose expression depends on an influx of calcium into the cell. To
distinguish between these possibilities recordings were obtained with the use of a cesium-based intracellular solution.
As illustrated in Fig. 6A1 , under control conditions depolarizing steps to potentials above –50 mV are followed by a
slow outward aftercurrent mediated by a calcium-activated
potassium current (Schwindt et al. 1988). Substitution of
potassium by cesium in the intracellular solution completely
blocks the outward aftercurrents (Fig. 6, A2 and A3 , n Å 5
cells tested) but fails to unmask an sADP (Fig. 6A2 ). This
failure of cesium to unmask sADP does not reflect merely
an inability of the cesium-loaded cell to express the sADP
because an sADP comparable with that seen under control
conditions is observed after administration of carbachol (30
mM, Fig. 6B, n ú 50 cells). Depolarizing steps to potentials
positive to –30 to –40 mV in contrast generally resulted in
the appearance of a faster ADP. However this early ADP
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FIG . 6. Blockade of the current underlying the AHP fails to unmask IsADP . A1 :
whole cell voltage-clamp recording obtained with potassium methylsulfate-based
intracellular solution. Depolarizing voltage
step from –70 to –40 mV (left trace),
–30 mV (middle trace), and –20 mV (right
trace) induce outward aftercurrents (IAHP )
that correspond to the potassium current(s)
responsible for the AHP. A2 : recordings
similar to those in A1 but obtained with a
cesium methanesulfonate-based intracellular solution. Intracellular injection of cesium completely blocks the outward
aftercurrent that follows the depolarizing
step but fails to induce a slow inward
aftercurrent (IsADP ). A3 : comparison between the outward aftercurrent elicited by
stepping to different depolarized potential
with potassium ( ●, n Å 5 cells) or cesium
( s, n Å 5 cells) as the predominant intracellular cation. Aftercurrents were measured 50–100 ms after the end of the depolarizing step. B: whole cell voltage-clamp
recording obtained with a cesium methanesulfonate-based intracellular solution. A
depolarizing step to –30 mV was applied
under control condition (left trace), in the
presence of carbachol (middle trace), and
after recovery (right trace). Bath application of carbachol induces IsADP . All records
were obtained with a discontinuous voltage
clamp.
NONSELECTIVE CATION CURRENT MEDIATES SADP IN CORTEX
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decayed within 100–200 ms and was clearly temporarily
distinct from the sADP reported (Haj-Dahmane and Andrade
1997).
Essentially identical results on the effects of cesium on
the sADP were obtained when the experiments were repeated
in current clamp. In the presence of TTX (1 mM), cesiumloaded cells respond to a suprathreshold-depolarizing pulse
with an all-or-none calcium action potential. This calcium
spike was followed by a fast ADP lasting 200–300 ms (Fig.
7, inset), but there was no evidence for sADP (or IsADP , n Å
40 cells, Fig. 7). As previously seen in voltage clamp, the
subsequent administration of carbachol (10–30 mM) elicited
the appearance of IsADP in most of the cells tested (29/40
cells, Fig. 7).
The results above suggest that muscarinic receptor activation induces the appearance of a calcium-dependent current,
which in turn is responsible for the sADP. The subsequent
experiments were designed to characterize its underlying
ionic mechanisms.
Several distinct ionic mechanisms could underlie sADP.
Previous studies in olfactory cortex identified a muscarinic
induced ADP similar to that seen in prefrontal cortex (Constanti and Bagetta 1991; Constanti et al. 1993) and concluded that it is mediated by the closure of potassium channels. Therefore we examined the possibility that a similar
mechanism could account for the muscarinic induced sADP
in prefrontal cortex. If the sADP were mediated through a
calcium-inactivated potassium conductance, the amplitude
of this afterpotential should diminish with hyperpolarization
and should reverse polarity near EK (approximately –104
mV under our experimental conditions). To test this prediction, we examined the voltage dependence of IsADP from
–50 to –120 mV. Voltages positive to –50 mV could not
be examined because the resulting calcium influx by itself
activates IsADP . As illustrated in Fig. 8, IsADP diminished with
hyperpolarization but remained inward even at –120 mV
(n Å 5 cells). In none of the cells tested did the peak IsADP
reverse below EK . In three of the five cells tested, the very
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late portion of IsADP , but not the peak, appeared to reverse
5–10 mV below EK .
There are multiple reasons that could account for our failure to observe reversal of IsADP at EK in control extracellular
potassium. Therefore we reexamined this issue with the use
of elevated extracellular potassium, a condition that should
favor observing the reversal of a current mediated by a decrease in potassium conductance. In this experiment we
raised extracellular potassium from 2.5 to 10 mM to shift
EK to near –65 mV. As expected, this resulted in the suppression of the outward potassium current responsible for the
AHP in these cells (Fig. 9A). However, even under these
conditions, bath application of carbachol still induces the
appearance of IsADP (Fig. 9B, n Å 6 cells tested). In three
of these cells it was possible to examine IsADP in the –60 to
–90 mV range (i.e., below EK under these conditions). In
all of these cells IsADP remained inward over this voltage
range. Moreover, no evidence of a reversing late component
of IsADP could be detected. From these observations and the
persistence of IsADP after intracellular cesium loading we
conclude that this current must be mediated largely by a
mechanism other than a decrease in potassium conductance.
sADP such as that studied here could in principle be mediated by changes in chloride conductance. Indeed, previous
studies in other neuronal cell types identified ADPs mediated
by calcium-activated chloride currents (Mayer 1985; Owen
et al. 1984). In our study the muscarinic-induced sADP can
be recorded at potentials very close to ECl estimated from
the chloride concentration in the intracellular solution. Thus
the carbachol-induced sADP is unlikely to be mediated by
a calcium-dependent chloride current. Nevertheless, because
a chloride gradient could conceivably be present at sites
distal from the recording electrode, we tested the effect of
reducing the extracellular chloride concentration on the amplitude of the carbachol-induced sADP. As illustrated in Fig.
10, lowering the extracellular concentration of chloride from
126.5 to 57.5 mM, which should shift ECl by Ç20 mV in
the depolarizing direction and effectively reverse a chloride-
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FIG . 7. Effect of carbachol on a cell recorded with a cesium gluconate-based intracellular solution in the presence of
TTX (1 mM). Under these conditions a brief depolarizing pulse delivered with a hybrid current/voltage-clamp protocol
results in an all-or-none calcium spike ( inset). Approximately 250 ms after the end of the depolarizing pulse the amplifier
was switched to voltage-clamp mode ( m ) to measure any slow aftercurrents after the calcium spike. Under control conditions
only a very small inward aftercurrent is detected at this time. However, after bath application of carbachol, a large, slow
inward aftercurrent (IsADP ) becomes evident. Discontinuous voltage clamp, holding potential, –71 mV; holding current at
rest, none.
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S. HAJ-DAHMANE AND R. ANDRADE
mediated potential at –65 to –70 mV, had no effect on the
amplitude of the carbachol-induced sADP (control Å 4 {
0.4 mV, low chloride Å 4.75 { 0.47 mV, n Å 4 cells tested).
These results indicate that the carbachol-induced sADP is
not mediated by a change in chloride permeability.
An alternative mechanism for the sADP could involve a
calcium-activated nonselective cation current. Such currents
were shown to mediate ADPs in a variety of preparations
(Caeser et al. 1993; Partridge and Swandulla 1988). To test
the possible involvement of nonselective cation currents we
examined the effect of lowering the extracellular concentration of sodium from 126 to 46 mM on the amplitude of
the carbachol-induced sADP. This reduction of extracellular
concentration should shift ENa in the negative direction by
Ç25 mV. As illustrated in Fig. 11, substitution of 60% of
extracellular sodium with NMDG reversibly reduced the amplitude of the carbachol-induced sADP. Overall, in a group
of five cells tested with the use of this protocol, 60% sodium
substitution with the use of NMDG reduced the sADP from
5.7 { 0.58 mV to 2.8 { 0.43 mV (n Å 5 cells, P õ 0.005).
The reduction in the amplitude of the sADP appeared to be
most prominent during the early part of the sADP. This is
most evident on superimposition of the traces obtained under
control condition and in low sodium. No change in the input
resistance was observed during the perfusion of low sodium.
The ion substitution employed above should have reduced
a pure sodium current in direct proportion to the reduction
in driving force, in this case Ç20%. However, we observed
a reduction of 50%. This could be explained if the sADP
were mediated by a nonselective cation current. However,
other explanations cannot be readily ruled out from this experiment. Therefore we reexamined the sodium dependence
of IsADP after cesium loading, a condition that would partially
isolate this current and minimize space-clamp artifacts. As
illustrated in Fig. 12, lowering extracellular sodium reduced
the amplitude of IsADP recorded in cesium-loaded cells, and
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this inhibition depended on the fraction of extracellular sodium replaced with NMDG (n Å 5 cells for 50% replacement
and n Å 4 cells for 93% replacement, Fig. 12B). These
effects were again somewhat larger than expected for sodium-selective channels and argued for a nonselective cation
current.
sADP mediated by a nonselective cation current should
be associated with an increase in chord conductance. Surprisingly, when we looked for changes in the apparent membrane
resistance during IsADP with the use of brief hyperpolarizing
steps, we observed the opposite, an apparent decrease in
membrane conductance (Fig. 13). This effect was even more
prominent when we repeated the experiment with the use of
depolarizing steps. This asymmetry suggested that the voltage steps were not simply measuring membrane conductance, but rather that the current was rapidly activating (and
deactivating) during the voltage steps. To test directly
whether IsADP was indeed voltage dependent, we reexamined
this current in –50 to –110 mV under conditions that would
isolate this current from other currents also expressed in the
cell. Under these conditions, IsADP was found to exhibit
marked voltage sensitivity (Fig. 14), deactivating quickly
with hyperpolarization in the subthreshold range (where our
conductance measurements were conducted). This voltage
dependence would predict the observed reduction in the amplitude of the conductance pulse observed during IsADP .
DISCUSSION
One of the most striking effects of cholinergic stimulation
of pyramidal cells in the rat prefrontal cortex is the appearance of an sADP. In the current study this sADP could be
induced by brief applications of ACh or by bath administration of carbachol, muscarine, or oxotremorine. Because all
of these effects could be blocked by low concentrations of
atropine, these results indicate that the sADP is elicited by
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FIG . 8. Amplitude of the carbachol-induced IsADP decreases with hyperpolarization but fails to reverse at potentials negative to EK . A: carbachol-induced IsADP recorded at different membrane potentials.
The IsADP was triggered with 2 voltage steps
(200 ms) to –20 mV. Note that this
aftercurrent decreased in amplitude and decayed faster with hyperpolarization. In this
cell the late portion of IsADP was found to
reverse between –100 and –120 mV. B:
I-V relationship for the carbachol-induced
IsADP obtained from three different pyramidal cells chosen to illustrate the range of
IsADP amplitudes encountered in these experiments. Holding potential, –60 mV;
control holding current, 0.22 nA; holding
current in carbachol, 0.17 nA.
NONSELECTIVE CATION CURRENT MEDIATES SADP IN CORTEX
1205
FIG . 9. Carbachol induces a robust IsADP at EK in high extracellular potassium. A: superimposed traces illustrating the aftercurrents seen after 2-step
depolarizations (200 ms to –20 mV) from –65 mV in the presence of TTX
(1 mM). One of these records was obtained in 2.5 mM extracellular potassium
and shows the expected outward potassium aftercurrent. The second record
was obtained in 10 mM extracellular potassium. Under these conditions EK
shifts to approximately –68 mV, and consequently no outward aftercurrent is
detectable after the depolarizing stimuli. Holding potential, –65 mV; holding
current in 2.5 mM potassium, 320 pA; holding current in 10 mM potassium,
190 pA. B: bath administration of carbachol (30 mM), however, is still capable
of inducing a slow inward aftercurrent in high potassium. Holding potential,
–65 mV; holding current in 10 mM potassium, 180 pA; holding current for
carbachol in 10 mM potassium, 120 pA.
the activation of muscarinic receptors. The potency of carbachol for eliciting this response was comparable with that
previously reported for eliciting a membrane depolarization
in these same cells (Haj-Dahmane and Andrade 1996) and
for reducing Im in CA1 pyramidal neurons (Madison et al.
1987). However, it was considerably higher than that required to reduce the AHP in hippocampus (Madison et al.
1987).
Muscarinic receptor activation could elicit the appearance
of the sADP directly by inducing its expression or indirectly
by inhibiting the AHP and unmasking a constitutive sADP.
Because inhibition of the AHP with the use of intracellular
cesium failed to unmask sADP, we conclude that muscarinic
receptors induce the de novo expression of an sADP in these
cells. A similar conclusion was reached for the muscarinic
receptor-induced ADP in sensorimotor and olfactory cortises
based on the reversal potential of the AHP (Schwindt et al.
1988) and the inability of cAMP, which inhibits the AHP,
to induce an ADP (Constanti et al. 1993).
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FIG . 10. sADP does not depend on the extracellular concentration of
chloride. Superimposed records of sADPs elicited by depolarizing current
pulse (1.2 nA, 300 ms) before, during carbachol application, and after
recovery in normal (126.5 mM, top traces) and low-chloride Ringer solution (57.5 mM, bottom traces). Reduction of the extracellular concentration
of chloride had no effect on the amplitude of the sADP induced by bath
administration of carbachol (30 mM). Cell membrane potential, –69 mV.
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In this study we investigated the ionic basis for the sADP.
Previous studies have shown that the muscarinic receptorinduced sADP seen in prefrontal (Andrade 1991), sensorimotor (Schwindt et al. 1988), and olfactory (Constanti et
al. 1993) cortices as well as hippocampus (Caeser et al.
1993; Fraser and MacVicar 1996) depended on extracellular
calcium influx into the cell. This could indicate that the
sADP is triggered by a rise in intracellular calcium or that
calcium itself carries the sADP current (Hounsgaard and
Kiehn 1989). Buffering intracellular calcium with EGTA or
BAPTA greatly reduced or blocked the sADP. Because this
inhibition was observed in the absence of a reduction of the
muscarinic depolarization also present in these cells, this
inhibition was unlikely to reflect a nonspecific effect of the
calcium buffers on muscarinic receptor functioning. These
results, together with earlier results indicating a critical role
for calcium influx in the generation of the sADP (Andrade
1991), indicate that the sADP seen in prefrontal cortex is
triggered by a rise in intracellular calcium. The observation
that calcium chelation inhibits the sADP is consistent with
similar results in hippocampus (Fraser and MacVicar 1996)
but disagrees with the report that injections of EGTA or
BAPTA by sharp microelectrodes fail to reduce the ADP in
olfactory cortex (Constanti et al. 1993).
A rise in intracellular calcium could produce the sADP
through several distinct mechanisms, including changes in
sodium, potassium, or chloride permeabilities, the activation
of an electrogenic calcium pump, or a combination of these.
From these experiments we conclude that the mechanism
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S. HAJ-DAHMANE AND R. ANDRADE
responsible for the generation of the sADP is the activation
of a current carried by sodium, most likely a nonselective
cation current.
Two lines of evidence support this conclusion. The first
is based on the exclusion of possible mechanisms. Previous
studies in olfactory cortex suggested that the sADP observed
in that region in response to muscarinic receptor activation
is mediated by a calcium-inactivated potassium conductance
(Constanti and Bagetta 1991; Constanti et al. 1993). Two
major lines of evidence lead us to reject the possibility that
this mechanism was responsible for the sADP seen in prefrontal cortex. First, a robust IsADP could be observed in the
presence of intracellular cesium, a manipulation that should
block most potassium channels. Although it is possible that
cesium might not block, or block very weakly, this particular
potassium channel, cesium is a fairly nonselective potassium
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FIG . 11. Amplitude of sADP depends on the extracellular concentration
of sodium. Intracellular recording of pyramidal neuron with sharp microelectrode containing potassium methylsulfate in the presence of TTX (1
mM). Two low-threshold calcium spikes were evoked by current injection
to allow calcium into the cell before (left trace), during (middle trace),
and after (right trace) removal of carbachol from the bath. The top set of
records was obtained in control Ringer solution, the middle set of records
was obtained in low-sodium Ringer, and the bottom set of records was
obtained after recovery to control extracellular sodium. Bottom right record
superimposes the sADP obtained in normal Ringer on that obtained in the
presence of low extracellular sodium. Note that a hyperpolarizing constant
current pulse was administered before the depolarizing stimuli to monitor
input resistance through the different manipulations. Decreasing extracellular sodium did not cause large changes in input resistance. This experiment
was conducted with sharp microelectrodes because this technique allows
for experiments lasting several hours, which was helpful because repeated
slow solution exchanges were required for this experiment.
channel blocker. Thus its failure to reduce IsADP suggests that
potassium channel closure is unlikely to be the predominant
mechanism underlying the sADP. Second, and most importantly, a robust IsADP persists as an inward current at and
below EK . Together these two lines of evidence indicate that
a calcium-inactivated potassium current is unlikely to be
primarily responsible for the generation of the sADP. However, they cannot completely exclude the possibility that a
calcium-inactivated potassium current could make a small
contribution to the generation of this afterpotential.
The sADP could also be mediated by a change in chloride
permeability or an electrogenic pump. A change in chloride
permeability could be ruled out as the fundamental mechanism responsible for the sADP because this afterpotential
was not affected by extracellular chloride substitution or, as
previously shown (Andrade 1991), by elevation of intracellular chloride. Similarly, a sodium/calcium electrogenic
pump also did not appear to be responsible for the sADP
because IsADP was voltage dependent and diminished, rather
than increased, on depolarization. Equally important, the amplitude of the sADP increased in amplitude until saturation
but did not increase in duration with increasing stimulus
strength. This is contrary to what was reported previously
for afterpotentials mediated by electrogenic pumps (Rang
and Ritchie 1968).
In contrast to the negative findings outlined above, the
sADP (or IsADP ) is reduced by replacing extracellular sodium
with NMDG, as expected for a current whose main charge
carrier is sodium. Moreover, the amplitude of this reduction
depends on the fraction of sodium replaced. This indicated
that IsADP is carried by sodium influx into the cell. It is
noteworthy that this dependence on extracellular sodium is
more pronounced than expected for a pure sodium current.
We interpret these observations to suggest that the sADP
is not mediated by a pure sodium current but rather by a
nonselective cation current. Technical limitations intrinsic
to slice preparations make it difficult to conduct a precise
determination of the contribution of different cations to this
current. However, a permeability to potassium and/or cesium could explain the observed deviations.
Although the results outlined above lead us to conclude
that IsADP corresponds to a nonselective cation current, we
were surprised to find that IsADP was associated with an apparent decrease in membrane conductance when assessed by
brief hyperpolarizing steps. Similar observations were made
by others for the muscarinic induced sADP in olfactory cortex (Constanti and Bagetta 1991; Constanti et al. 1993),
and the metabotropic glutamate receptor elicited sADP in
sensorimotor cortex (Greene et al. 1994). In the first of
these preparations, the apparent decrease in conductance was
attributed to a reduction in potassium conductance. However, as outlined above, this explanation could not account
for the sADP in prefrontal cortex.
Alternatively, the sADP in sensorimotor cortex was attributed to a mixed ionic mechanism involving both calciumactivated nonselective cation and calcium-inactivated potassium conductances. In this scenario the apparent decrease in
conductance could be explained by a predominantly somatic
localization of the potassium channels. Such a mechanism
would be similar to that proposed to mediate slow depolar-
NONSELECTIVE CATION CURRENT MEDIATES SADP IN CORTEX
1207
izations elicited by muscarinic and substance P receptors in
locus coeruleus (Shen and North 1992), muscarinic and
metabotropic glutamate receptors in hippocampus (Colino
and Halliwell 1993; Crepel et al. 1994, Guerineau et al.
1995), and neurotensin receptors in basal forebrain (Farkas
et al. 1994). One test for such a dual mechanism is to use
potassium channel blockers to isolate the nonselective cation
current. This strategy was used successfully in locus coeruleus to isolate a pure nonselective cation current that increased with hyperpolarization and was associated with a
conductance increase (Shen and North 1992). However, the
comparable experiment in our hands still resulted in a current
that decreased with hyperpolarization and was associated
with an apparent decrease in conductance. This finding argued against the idea that the closure of potassium channels
could account for the apparent increase in resistance observed during IsADP .
An alternative explanation for the apparent decrease in
conductance during the sADP could be that IsADP itself is
voltage dependent. Rapid voltage-dependent relaxations during the steps could give the appearance of decreases in conductance. In support of this possibility, the apparent decrease
in conductance during IsADP was found to be highly dependent on the polarity of the voltage step used. This asymetrical
behavior can be explained if depolarizing steps resulted in
an increase and hyperpolarizing steps resulted in a decrease
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in the amplitude of IsADP . To test this possibility we directly
examined the voltage dependence of IsADP . Consistent with
the idea that IsADP is voltage dependent, the amplitude of
IsADP recorded in isolation after cesium loading of the cells
was found to diminish steeply with hyperpolarization. Of
course, a variety of voltage-clamp artifacts, resulting from
the difficulty of clamping neurons with intact dendritic
arbors, could have conferred the appearance of voltage dependence to an otherwise voltage-independent cation current. However, in these same cells and with techniques identical to those used in the current study, it is possible to
demonstrate the presence of a well-behaved, voltage-insensitive, calcium-activated nonselective cation current (HajDahmane and Andrade 1997). Thus we conclude that IsADP
corresponds to a current carried most likely by voltage-dependent nonselective cation channels. We cannot exclude
the possibility that a small component of IsADP could involve
potassium or chloride channels. However, the involvement
of such channels is not required to explain the current observations.
sADPs similar to those studied here can be induced not
only by muscarinic stimulation elsewhere in cortex (Constanti and Bagetta 1991; Schwindt et al. 1988) but also by
serotonin (Spain 1994) acting on 5-HT 2 receptors (Araneda
and Andrade 1991), norepinephrine acting on a1 receptors
(Araneda and Andrade 1991), and metabotropic glutamate
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FIG . 12. Lowering extracellular sodium
reduces IsADP in proportion to the degree to
which sodium is replaced by N-methyl-Dglucamine (NMDG). A: hybrid current/
voltage-clamp protocol was used to record
IsADP . Recordings were obtained with a cesium-based intracellular solution, and calcium spikes were used to trigger IsADP under
control conditions and in low extracellular
sodium. Lowering extracellular sodium
concentration to 10 mM resulted in a large
reduction in IsADP . Discontinuous voltageclamp recording; holding potential, –70
mV; holding current at rest, –50 pA. B:
superimposition of traces depicting IsADP
under control conditions and in low extracellular sodium. C: summary plot illustrating the relationship between the amplitude
of IsADP elicited in the presence of 30 mM
carbachol and the percent substitution of
extracellular sodium by NMDG; n Å 5
cells; error bars correspond to SE.
1208
S. HAJ-DAHMANE AND R. ANDRADE
receptors (Constanti and Libri 1992; Greene et al. 1992;
unpublished observations). This suggests that the appearance of sADP is a widespread response to activation of a
variety of metabotropic neurotransmitters receptors. Indeed,
we previously suggested that sADP might be one of the
common effects signaled by neurotransmitter receptors in
cerebral cortex coupled to G proteins of the Gq family (Araneda and Andrade 1991). If this were the case, it could be
expected that all of these sADPs should exhibit a common
underlying mechanism (Araneda and Andrade 1991). However, as outlined above, different groups studying the sADP
in cortex reached different conclusions regarding their origin. Thus, in olfactory cortex, the sADP was reported to be
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FIG . 13. The IsADP elicited by carbachol is accompanied with an apparent
increase in membrane resistance. A pyramidal neuron was voltage clamped
at –71 mV, and membrane conductance was assessed with hyperpolarizing
or depolarizing voltage steps (10 mV) before and after a jump to –20 mV
designed to allow calcium influx into the cell. A: under control conditions
the depolarizing jump is followed by an outward aftercurrent. As expected
for a current associated with an increase in potassium conductance, this
outward aftercurrent is associated with a conductance increase. B: after
carbachol administration, the outward aftercurrent is replaced by IsADP . B1 :
when membrane conductance was assessed with hyperpolarizing steps there
was an apparent decrease in membrane conductance during IsADP . B2 : this
apparent decrease in membrane conductance was even more pronounced
when assessed with depolarizing steps. Note that in both cases the largest
change in apparent membrane conductance occurred during the peak of the
current, not in a late portion of IsADP . Continuous voltage clamp; holding
current, 20 pA.
mediated by a decrease in potassium conductance (Constanti
and Bagetta 1991; Constanti et al. 1993), whereas in sensorimotor cortex (Greene et al. 1994; Schwindt et al. 1988), it
was attributed to a mixed ionic mechanism. In this study we
conclude that in prefrontal cortex the muscarinic-induced
sADP is mediated predominantly, if not exclusively, by an
increase in a voltage-dependent cation conductance. Further
studies will be required to determine whether different ionic
mechanisms underlie the ADP in different regions of cortex.
Interestingly, muscarinic or glutamate metabotropic receptor activation of CA3 neurons also results in the appearance of an ADP in hippocampal slice cultures (Caesar et al.
1993). Like the sADP in prefrontal cortex, this ADP was
reported to be mediated most likely by a calcium-activated
cation current. However, it differs from the sADP seen in
prefrontal cortex in being Ç10 times faster. In this regard,
the hippocampal ADP more closely resembles fast ADP
( fADP) also seen in prefrontal cortex (Haj-Dahmane and
Andrade 1997).
Muscarinic stimulation of pyramidal neurons of the prefrontal cortex results not only in the appearance of sADP
but also in the generation of a slow membrane depolarization
(Haj-Dahmane and Andrade 1996). We previously have
shown that this depolarization, like the sADP, is also mediated by a voltage-dependent nonselective cation current. Interestingly, in smooth muscle cells of the guinea pig ileum,
muscarinic activation also induces a calcium- and voltagedependent nonselective cation current (Inoue and Isenberg
1990a,b; Zholos and Bolton 1994, 1995) that mediates a
membrane depolarization and an inward aftercurrent not unlike those seen in prefrontal cortex. It is tempting to speculate
that in prefrontal cortex, like in guinea pig ileum, the muscarinic depolarization and sADP might also reflect different
manifestations of a unitary underlying ionic mechanism: the
depolarization corresponding to the activation of the current
at a basal intracellular calcium level and the sADP corresponding to the transient, calcium-induced enhancement of
this same current. Further studies will be needed to validate
or disprove this hypothesis. A similar current was also seen
in Aplysia bag cells, where it makes possible a long-lasting
afterdischarge (Wilson et al. 1996). The widespread distribution of this nonselective cation current(s) suggests that it
may play an ubiquitous role in regulating membrane excitability.
What could be the role for this sADP in prefrontal cortex?
Although ADPs are often associated with bursting activity,
in prefrontal cortex activation of IsADP fails to induce bursting. Instead, activation of IsADP leads to a long-lasting ADP
that, if large enough, produces self-sustained spiking activity
that greatly outlasts the original stimulus (Andrade 1991).
This behavior is reminiscent of that seen for some prefrontal
cortex cells during memory tests, where a cell continues to
fire long past presentation of a relevant stimulus (GoldmanRakic 1990). This sustained firing has been interpreted as
representing the neuronal substrate of working memory.
Given the well-established role of ACh on memory function
(Winkler et al. 1995), it is tempting to speculate on a possible relationship between these two phenomena. However,
careful experiments in vivo will be required to examine this
possibility. On a more general level, it is possible that the
NONSELECTIVE CATION CURRENT MEDIATES SADP IN CORTEX
1209
FIG . 14. Voltage dependence of IsADP
recorded with a cesium gluconate-based intracellular solution. A: representative traces
depicting IsADP at different holding potentials. IsADP was elicited by calcium spikes
in a hybrid current/discontinuous voltageclamp protocol. Holding current in carbachol at –50 mV, /25 pA. B: I-V relationship of the carbachol-induced IsADP obtained with a ‘‘hybrid’’ current/voltage
clamp protocol. The IsADP was triggered
with a calcium spike, and its amplitude was
measured at the peak at different membrane
potentials. Error bars represent the SE of 5
different determinations.
We thank Dr. Sheryl Beck for reading the manuscript.
This work was supported by National Institute of Mental Health Grant
MH-49355.
Address for reprint requests: R. Andrade, Dept. of Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine, 2309
Scott Hall, 540 East Canfield, Detroit, MI 48201.
Received 27 May 1997; accepted in final form 12 May 1998.
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