Download 1S,3R-ACPD Induces a Region of Negative Slope Conductance in

1S,3R-ACPD Induces a Region of Negative Slope Conductance in the
Steady-State Current-Voltage Relationship of Hippocampal
Pyramidal Cells
ANITA LÜTHI, BEAT H. GÄHWILER, AND URS GERBER
Brain Research Institute, August Forel-Strasse 1, CH-8029 Zurich, Switzerland
INTRODUCTION
The suppression of K/ conductances is a major effect
associated with the activation of postsynaptic metabotropic
glutamate receptors (mGluRs) and muscarinic receptors in
hippocampal pyramidal cells. At least four K/ currents,
after-hyperpolarizing (IAHP ), IM , Ileak , and Islow , are decreased
with the stimulation of these G protein-coupled receptors,
leading to an enhancement in cellular excitability (Brown
and Adams 1980; Charpak et al. 1990; Gerber and Gähwiler
1994; Lüthi et al. 1996). Under voltage-clamp conditions,
this effect is in part reflected by a slowly developing inward
current associated with a decrease in input conductance. In
CA3 cells, the response is observed both with exogenous
application of agonists as well as with synaptic stimulation
of the receptors (Charpak and Gähwiler 1991; Gähwiler and
Brown 1985; Gerber and Gähwiler 1994; Gerber et al. 1993).
Synaptic responses mediated by mGluRs and metabotropic cholinergic receptors are highly voltage dependent, disappearing at potentials below 070 mV while disproportionately increasing positive to resting potential (Charpak and
Gähwiler 1991; Gähwiler and Brown 1985; Wang and
McCormick 1993). For mGluRs, an increase in amplitude
of more than fourfold is observed when responses are evoked
at a potential of 054 mV compared with resting potential
(about 064 mV) (Charpak and Gähwiler 1991). The increase
in driving force for K/ ions and the partial activation of IM
(Brown 1988; Gähwiler and Brown 1985) cannot quantitatively account for this voltage dependence. Activation of
mGluRs at depolarized potentials may therefore suppress
additional K/ currents or induce depolarizing inward currents. Alternatively, intrinsic voltage-dependent nonlinearities in the mGluR-mediated response may contribute to the
increase of the slow inward current on depolarization. For
example, the gating of K/ channels by an inhibitory G protein or second messenger may be sensitive to changes in
membrane potential.
Activation of G protein-coupled receptors often results in
more pronounced effects on membrane currents with moderate than with strong depolarization, as demonstrated for the
inhibition of N-type Ca2/ channels by norepinephrine, glutamate (acting at mGluRs), g-amino-butyrate, luteinizing-hormone-releasing hormone, and kappa opioid receptor agonists
(Anwyl 1991; Bean 1989; Boland and Bean 1993; Dolphin
1996; Luebke and Dunlap 1994; Swartz and Bean 1992).
This is thought to result from a G protein-induced conversion
of the channels into a gating mode that is less ‘‘willing’’ to
open on depolarization (Bean 1989; Boland and Bean 1993;
Diversé-Pierluissi et al. 1995). For the gating of K/ conductance by mGluRs, the nature of the transduction process is
unclear and the characterization of a voltage dependence
should provide insights concerning the mechanisms coupling
receptors to channels.
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Lüthi, Anita, Beat H. Gähwiler, and Urs Gerber. 1S,3R-ACPD
induces a region of negative slope conductance in the steady-state
current-voltage relationship of hippocampal pyramidal cells. J.
Neurophysiol. 77: 221–228, 1997. Synaptic responses mediated
by metabotropic glutamate receptors (mGluRs) display a marked
voltage-dependent increase in amplitude when neurons are moderately depolarized beyond membrane potential. We have investigated the basis for this apparent nonlinear behavior by activating
mGluRs with 1S,3R-1-aminocyclopentane-1,3-dicarboxylate
(1S,3R-ACPD; 10 mM) in CA3 pyramidal cells from rat hippocampal slice cultures with the use of the single-electrode voltage-clamp
technique. Under control conditions, cells depolarized from resting
potential by 10–20 mV responded with delayed outwardly rectifying currents due to activation of voltage- and Ca2/-dependent K/
conductances. In contrast, in the continuous presence of 1S,3RACPD, small depolarizations (10–20 mV) induced a delayed inward current. The steady-state current-voltage relationship for this
response displayed a region of negative slope conductance at potentials between 055 and 040 mV. The reversal potential of the
corresponding 1S,3R-ACPD-sensitive tail currents (093.0 { 2.2
mV, mean { SE) was close to the potassium reversal potential,
consistent with an mGluR-mediated suppression of K/ current.
When external K/ concentration was increased to 8 mM, there was
a positive shift in reversal potential to 076.9 { 5.1 mV. The
depolarization-induced inward current in the presence of 1S,3RACPD was blocked by Ba2/ (1 mM). The response was not dependent on changes in intracellular Ca2/ concentration and was insensitive to bath-applied Cs/ (1 mM), ruling out a contribution of
Ca2/-dependent currents or the inward rectifier IQ . Furthermore,
the effect of 1S,3R-ACPD was not mimicked by inhibiting afterhyperpolarizing current and M current with low-Ca2/ saline (0.5 mM
Ca2/, 10 mM Mg2/) containing 10 mM tetraethylammonium chloride. A comparison of the responses induced by 1S,3R-ACPD and
N-methyl-D-aspartate showed that both induce an inward current
with small depolarizations from resting potential but with different
kinetics and Mg2/ sensitivity. These results indicate that the suppression of K/ currents in response to activation of mGluRs is
markedly voltage dependent, increasing at depolarized potentials
and decreasing at hyperpolarized potentials. The negative slope
conductance at membrane voltages positive to resting potential
may underlie the amplification of mGluR-mediated responses when
the membrane potential approaches action potential threshold.
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A. LÜTHI, B. H. GÄHWILER, AND U. GERBER
The aims of this study were to electrophysiologically characterize the voltage dependence of the metabotropic glutamatergic response in hippocampal pyramidal cells and to
evaluate the physiological implications. Preliminary results
have appeared in abstract form (Lüthi et al. 1995).
METHODS
Hippocampal slice cultures
Slices 400 mm thick were obtained from 6-day-old rat pups and
cultured by means of the roller tube technique for 3–6 wk as
described previously (Gähwiler 1981).
Electrophysiological recordings
Drugs and chemicals
Drugs were applied in the bathing fluid and were purchased
from the following sources: tetraethylammonium chloride (TEA)
and muscarine from Sigma (St. Louis, MO); 1S,3R-1-aminocyclopentone-1,3-dicarboxylate (1S,3R-ACPD) and N-methyl-D-aspartate (NMDA) from Tocris Neuramin (Bristol, UK); bis-(o-aminophenoxy)-N,N,N*,N*-tetraacetic acid (BAPTA) from Molecular
Probes (Eugene, OR); and tetrodotoxin from Sankyo (Tokyo,
Japan).
RESULTS
Negative slope conductance in the steady-state currentvoltage relationship in cells bathed in 1S,3R-ACPD
The effect of mGluR activation on the steady-state I-V
relationship of CA3 pyramidal neurons was determined by
bath applying 1S,3R-ACPD (10 mM), a specific agonist for
mGluRs. At this concentration, 1S,3R-ACPD effectively
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FIG. 1. 1S,3R-1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD)
induces a region of negative slope conductance in the current-voltage (IV) relationship of voltage-clamped CA3 pyramidal cells. A: in the presence
of 1S,3R-ACPD a depolarizing step command to 050 or 040 mV induces
an inward current. In addition, 1S,3R-ACPD decreases the input conductance of the cell, as reflected by the diminished current responses to hyperpolarizing voltage commands. Voltage protocols are depicted between the
current traces. B: pooled data (n Å 13) illustrating the steady-state I-V
relationship in the presence and absence of 1S,3R-ACPD.
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The single-electrode voltage clamp was used to record from
CA3 pyramidal cells (Axoclamp2 amplifier, Axon Instruments,
Foster City, CA). For this purpose, cultures were transferred to a
temperature-controlled recording chamber that was mounted on
the stage of an inverted microscope (Axiovert 35M, Zeiss, Jena,
Germany) and superfused with a saline solution (337C) containing
(in mM) 149.2 Na/, 2.7 K/, 147.2 Cl0, 2.8 Ca2/, 0.5 Mg2/, 11.6
HCO03 , 0.4 H2PO04 , 5.6 D-glucose, and 0.0005 tetrodotoxin. For
experiments with high K/ (8 mM), NaCl content was reduced
correspondingly. When Ba2/-containing solutions (1 mM) were
used, H2PO04 was omitted from the saline to avoid precipitation of
phosphate salts and pH was readjusted. Low-Ca2/ saline contained
0.5 mM Ca2/ and 10 mM Mg2/ instead of the concentrations given
above. Solutions were brought to a pH of 7.4 by bubbling with
95% O2-5% CO2 and monitored with Phenol Red (10 mg/l).
For microelectrode recordings, thin-walled electrodes were filled
with a solution containing (in M) 2 KCl, 2 CsCl, or 1 KMeSO4 ,
resulting in a tip resistance of 30–50 MV for KCl and CsCl and
60–80 MV for KMeSO4 , respectively. During voltage-clamp recordings, the switching rate ranged between 1.7 and 2.0 kHz and
headstage output was continuously monitored to ensure adequate
settling time between samples. After voltage steps, the clamp stabilized within 5–10 ms. Input resistance was assessed by applying
0.5-s hyperpolarizing voltage commands of 10 mV.
Microelectrode signals were digitized at 1–2 kHz, stored, and
analyzed off-line on a PC with the use of pClamp version 6.0.1
(Axon Instruments, Foster City, CA) and Fig.P (Biosoft, Cambridge, UK) software. Quantitative data are given as means { SE.
Statistical analysis was performed with the use of Student’s t-test.
suppresses K/ currents in pyramidal neurons of organotypic
slice cultures (Lüthi et al. 1994). Under control conditions,
increasing the amplitude of hyperpolarizing steps in 10-mV
increments to 090 mV evoked passive current responses
(Fig. 1). In contrast, depolarizing pulses resulted in rapid
activation of transient currents followed by slowly developing outward currents that reached a maximal amplitude
after 200–600 ms, as described previously (Brown and Griffith 1983; Brown et al. 1990; Segal and Barker 1984).
In this study, we have focused on the characterization of
metabotropic actions on the steady-state components of the
depolarization-induced responses. Bath application of 1S,3RACPD (10 mM) for 1–2 min at 060 mV induced an inward
current of 0181 { 35 pA (n Å 19), as demonstrated earlier
(Charpak and Gähwiler 1991; Charpak et al. 1990; Gerber et
al. 1993). In marked contrast to the control outward currents
evoked by positive voltage steps, identical depolarizing voltage commands from 060 mV in the presence of 1S,3RACPD induced additional inward current despite the already
fully developed mGluR response. Hyperpolarizing steps in
the presence of 1S,3R-ACPD evoked passive current responses of decreased amplitude compared with control
(44.8 { 7.1% of control for 10-mV hyperpolarizing steps,
n Å 12; 72.3 { 6.9% of control for 30-mV hyperpolarizing
ACPD INDUCES NEGATIVE SLOPE CONDUCTANCE IN I-V RELATION
Kinetics of the 1S,3R-ACPD-dependent current induced on
depolarization
The development of the depolarization-induced inward
current in the continuous presence of 1S,3R-ACPD was kinetically best described by a single-exponential fit giving a
time constant of 800 { 262 ms (at 050 mV, n Å 10). At
040 mV, the current peaked earlier, with a time constant of
281 { 49 ms (n Å 5). The 1S,3R-ACPD-sensitive inward
current at 050 or 040 mV tended to decrease when the
depolarization was maintained for ú5 s, suggesting desensitization of the metabotropic response.
Depolarization-induced 1S,3R-ACPD current is due to a
suppression of K/ current
The negative slope region in the I-V relationship in the
presence of 1S,3R-ACPD could be explained by 1) a de-
crease in 1S,3R-ACPD-sensitive K/ currents or 2) an induction of an inward current.
The reversal potential of the depolarization-evoked current in the presence of 1S,3R-ACPD was determined by
measuring the amplitude of the 1S,3R-ACPD-sensitive instantaneous currents as a function of the holding potential.
Tail currents were evoked with the use of a two-pulse procedure (Hodgkin and Huxley 1952). Cells were voltage
clamped at 040 mV for 1 s to activate the 1S,3R-ACPDsensitive current, and the membrane potential was then
stepped to between 090 and 050 mV for 1 s. The family
of currents generated in this way is shown in Fig. 3. The
1S,3R-ACPD-sensitive tail currents (Fig. 3C) were isolated
by digitally subtracting the control tail currents (Fig. 3A)
from the tail currents in the presence of 1S,3R-ACPD (Fig.
3B). The amplitude of these responses, measured 20 ms after
the voltage step, was plotted against the clamp potential,
resulting in a linear relationship (Fig. 3E, ●). The fitted
curve intersects the zero-current line at 093.0 { 2.2 mV
(n Å 6), close to the reversal potential for K/ ions in CA3
cells, as previously determined (about 0100 mV, Lüthi et
al. 1996). When extracellular K/ was raised from 2.7 to 8
mM, the reversal potential for the current was shifted positively to 076.9 { 5.1 mV (n Å 3; Fig. 3, D and E). This is
in reasonable agreement with the predicted potential of 072
mV according to the Nernst equation.
Three additional series of experiments supported the
conclusion that the 1S,3R-ACPD-sensitive current is carried by K / ions. First, in cells internally perfused with
2 M CsCl, 1S,3R-ACPD-dependent current was not
detected at depolarized potentials (n Å 6, not shown).
Second, in the presence of 1 mM extracellular Ba2/ (in
low-Ca2/ saline), the effect of 1S,3R-ACPD on the I-V
relationship was markedly reduced (Fig. 4). Third, when
electrodes were filled with 2 M KMeSO4 instead of 2
M KCl to change the reversal potential for Cl0 in the
hyperpolarizing direction (Streit et al. 1989), the action
of 1S,3R-ACPD remained unaltered, indicating that a Cl0
conductance was not involved (n Å 5, not shown).
FIG. 2. Depolarization induces an inward current in the continuous presence of muscarine. Top 2 traces: membrane
currents. Bottom: membrane potential. Current responses to a step depolarization from 060 to 040 mV and to a step
hyperpolarization from 060 to 080 mV are shown. A: under control conditions, depolarization induces a delayed outward
current and hyperpolarization induces a passive current response. B: in the presence of muscarine, depolarization evokes an
inward current. Hyperpolarizing responses are largely passive but occasionally show a small outward current. C: pooled data
showing the steady-state I-V relationship in the presence and absence of muscarine (n Å 5), and the occlusion of the 1S,3RACPD response by muscarine (n Å 3).
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steps, n Å 12) due to the suppression of resting K/ conductances (Gerber and Gähwiler 1994). As a result, the outwardly rectifying steady-state I-V relationship of CA3 neurons under control conditions was converted to an S-shaped
curve exhibiting a region of negative slope conductance between 060 and 040 mV in the presence of 1S,3R-ACPD
(Fig. 1B). Similar findings were obtained when muscarinic
cholinergic receptors were activated. In the continuous presence of muscarine (10 mM), a 20-mV depolarization from
resting potential evoked an inward current with an amplitude
peaking at 040 mV (Fig. 2, A–C, n Å 5). Under these
conditions of muscarinic receptor activation, application of
1S,3R-ACPD (10 mM) induced no significant additional response (Fig. 2C, n Å 3).
The nonlinearity of the 1S,3R-ACPD-response was not
dependent on whether the step depolarization preceded drug
application, or vice versa. Thus the absolute holding current
level reached with a depolarization to 050 mV in the continuous presence of 1S,3R-ACPD was not significantly different
from that attained when 1S,3R-ACPD was applied to a cell
already held at 050 mV (0283 { 30 pA vs. 0275 { 29
pA, n Å 3, P ú 0.05, not shown).
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A. LÜTHI, B. H. GÄHWILER, AND U. GERBER
steady-state I-V relationship did not show a region of negative slope conductance and outward rectification was largely
maintained. A similar result was obtained when increases in
intracellular Ca2/ concentration were prevented with the
Ca2/ chelator BAPTA (100 mM in the pipette, n Å 6, not
shown). Moreover, in the presence of TEA (10 mM), an
effective blocker of IM in hippocampal pyramidal cells
(Storm 1989), depolarization did not induce inward currents,
even though TEA was effective in reducing delayed outward
currents. Furthermore, TEA did not occlude the depolarizaFIG. 3. Measurement of the reversal potential of the depolarization-induced 1S,3R-ACPD-dependent current. Tail currents were evoked with the
use of the voltage protocol shown in the inset. All current families were
leak subtracted with the use of the voltage protocol (dashed line) without
the conditioning depolarizing voltage step. A: current responses in the absence of 1S,3R-ACPD. B: current responses in the presence of 1S,3R-ACPD
(10 mM). C: 1S,3R-ACPD-sensitive tail currents obtained by subtraction of
the traces in B from the traces in A. D: tail currents as in C, but in the
presence of 8 mM external K/. E: plot of the 1S,3R-ACPD-sensitive tail
current amplitudes as a function of membrane potential yielded an extrapolated reversal potential of 093.0 { 2.2 mV (n Å 6). When external K/
concentration was raised to 8 mM, the response reversed at 076.9 { 5.1
mV (n Å 3). Correlation coefficients of the linear regressions were 0.98
and 0.99, respectively.
Inhibition of IAHP and IM does not induce a region of
negative slope conductance
Experiments were performed to check whether the nonlinearity of the 1S,3R-ACPD response apparent at depolarized
potentials was due to suppression of the K/ currents IAHP
and IM . The effects of 1S,3R-ACPD were therefore compared
with those of K/ channel blockers (Fig. 5). First, cells were
bathed in a solution containing low-Ca2/ (0.5 mM) and highMg2/ saline (10 mM) to prevent activation of Ca2/-dependent K/ currents. Although this solution was effective in
blocking the IAHP evoked by a 50- to 2,000-ms depolarizing
voltage command of 30–40 mV (not shown), the resulting
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FIG. 5. Effect of 1S,3R-ACPD (●) is not mimicked by K channel
blocking solutions. A: low-Ca2/ solution (0.5 mM Ca2/, 10 mM Mg2/) (n)
and tetraethylammonium chloride (TEA) (10 mM) in low-Ca2/ solution (h)
were tested. B: application of these solutions reduced delayed outward
currents, but did not result in a negative slope conductance in the I-V
relationships. The curve for 1S,3R-ACPD is shown for comparison.
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2/
FIG. 4. Ba
(1 mM, 0.5 mM Ca2/, 10 mM Mg2/) reduces the effects
of 1S,3R-ACPD. A: representative current responses in a cell clamped at
060 mV to voltage commands from 090 to 040 mV for 2 s. B: plot of
current amplitudes in the absence (s) and presence (●) of 1S,3R-ACPD.
ACPD INDUCES NEGATIVE SLOPE CONDUCTANCE IN I-V RELATION
tion-induced actions of 1S,3R-ACPD (n Å 3, not shown).
These results suggest that the voltage-dependent action of
1S,3R-ACPD at depolarized membrane potentials cannot be
explained solely by inhibition of the voltage-dependent current IM and the Ca2/-dependent current IAHP .
Depolarization-evoked 1S,3R-ACPD-sensitive current is
Ca2/ independent
Comparison of the effects of 1S,3R-ACPD and NMDA
A region of negative slope conductance in the I-V relationship is a prominent feature associated with the activation of
NMDA receptors in the presence of Mg2/ ions (Mayer et
al. 1984; Nowak et al. 1984). The parallels observed in this
study with regards to the effects of 1S,3R-ACPD on the IV relationship prompted us to compare the actions of agonists for these two glutamate receptor types as a function of
the Mg2/ concentration (Fig. 6). When NMDA was applied
in the presence of Mg2/ (2 mM), a 10-mV depolarization
rapidly induced an inward current, as expected for immediate
depolarization-induced relief of the Mg2/ blockade in the
receptor pore (Fig. 6B). This rectification was, however, absent when extracellular Mg2/ was removed from the perfusate. In this case, NMDA-receptor-mediated currents were
observed as linear currents summating with the cellular response (Fig. 6C). In contrast, the 1S,3R-ACPD-sensitive current appeared not to be affected by Mg2/ in the concentration
range between 0 and 2 mM (Fig. 6, B and C). Repetitive
depolarizations from 060 to 050 mV evoked a delayed
inward current, showing that the 1S,3R-ACPD response recovered fully during the course of the decay of the outward
tail current.
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DISCUSSION
This study demonstrates that the reduction of K/ conductance in response to mGluR and muscarinic receptor activation exhibits pronounced voltage sensitivity. After full development of the mGluR-mediated suppression of K/ currents
at resting potential, moderate depolarization induced an additional inward current with a slow onset. This effect is apparent as a region of negative slope conductance in the steadystate I-V relationship of hippocampal pyramidal cells.
The negative slope conductance in the I-V relationship
with 1S,3R-ACPD is likely to correspond to the augmentation of evoked synaptic currents observed with depolarization from the resting potential (Charpak and Gähwiler 1991).
The holding current levels reached at the peak of the responses were independent of the sequence of depolarization
and drug application, indicating that the depolarization-induced enhancement of mGluR responses is independent of
the recent voltage history of the cell. Therefore both the
depolarization-evoked 1S,3R-ACPD-dependent inward current and the enhancement of synaptic responses on depolarization appear to reflect a slowly developing action on a
steady-state membrane current.
Identity of the underlying current
The 1S,3R-ACPD-sensitive tail currents reversed close to
the equilibrium potential for K/ ions. With low extracellular
[K/ ] the reversal potential was determined by extrapolation,
because it was not possible to experimentally reverse the
polarity of the current responses. This may reflect the weakened action of 1S,3R-ACPD at hyperpolarized membrane
potentials. When extracellular K/ was increased, however,
a reversal in the polarity of the tail currents was observed.
Furthermore, the 1S,3R-ACPD-induced current was significantly reduced in the presence of the K/-channel-specific
blockers, internal Cs/ or external Ba2/.
The negative slope conductance could also be due, in part,
to activation of 1S,3R-ACPD-gated inward currents. It has
previously been shown that 1S,3R-ACPD can activate cationic currents (Caeser et al. 1993; Crépel et al. 1994; Guérineau et al. 1995). In these studies, however, a cationic current response was only observed with strong depolarizing
steps positive to 030 mV, with high concentrations of 1S,3RACPD (100 mM) or under conditions of high external K/.
Thus in our experiments the contribution from cationic currents should be minor.
Possible mechanisms: voltage-dependent K/ current
versus intrinsic voltage sensitivity of 1S,3R-ACPD actions
The action of 1S,3R-ACPD could not be mimicked by
applying K/ channel blocking solutions that reduce the
1S,3R-ACPD-sensitive currents IM and IAHP . This suggests
that either the reduction of an unknown voltage-dependent
K/ current or an intrinsic voltage-dependent enhancement
of the action of 1S,3R-ACPD on K/ currents accounts for
the region of negative slope conductance.
On the basis of the activation kinetics and the sustained
action of the 1S,3R-ACPD effect, a putative current fulfilling
the properties required to produce the voltage-dependent response would have to be a member of the slowly or noninac-
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Experiments were performed in the presence of low-extracellular-Ca2/ saline (0.5 mM Ca2/, 10 mM Mg2/) to test for
a Ca2/ dependence of the 1S,3R-ACPD-dependent current.
Mg2/ itself was, however, found to reduce the amplitude of
the 1S,3R-ACPD-induced inward current observed at resting
potentials [80 { 18 pA (n Å 5) vs. 208 { 25 pA (n Å 8),
P õ 0.0025] and concomitantly diminished the effects of
1S,3R-ACPD on depolarization. The effects of Ca2/ at 2.8
and 0.5 mM were therefore compared (in the presence of
10 mM Mg2/). In both cases, the depolarization-induced
inward current was of comparable amplitude (n Å 8, P ú
0.05, not shown), indicating that the reduction of extracellular Ca2/ did not affect this action of 1S,3R- ACPD. In further
support for the Ca2/ independence of the 1S,3R-ACPD-sensitive current, intracellular perfusion of cells with high concentrations of the Ca2/ buffer BAPTA (100 mM) did not
prevent this effect of 1S,3R-ACPD (n Å 6, not shown). To
check for an involvement of an inward rectifier current (Halliwell and Adams 1982) 1S,3R-ACPD was applied in the
presence of 1 mM extracellular Cs/. The amplitudes of the
inward currents evoked with 10- to 20-mV depolarizing steps
did not significantly differ from the amplitudes of the
currents induced by 1S,3R-ACPD alone (026 { 41 pA vs.
035 { 24 pA at 050 mV; 0176 { 71 pA vs. 0247 { 53
pA at 040 mV, n Å 6, P ú 0.05). This suggests that Cs/sensitive inward rectifiers do not contribute to the current
induced on depolarization in the presence of 1S,3R-ACPD.
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tivating class of K/ currents, such as IM (Brown et al. 1990;
Storm 1993). The experimental results with high concentrations of TEA suggest that reduction of IM is not the primary
cause for the voltage-dependent action of 1S,3R-ACPD, although we cannot rule out a partial contribution by IM . Moreover, the involvement of a novel voltage-dependent tonic
current with an activation threshold near 055 mV current
appears unlikely. The current would have to attain a sufficient amplitude with moderate depolarizations for its suppression to fully occlude the sum of all the other depolarization-activated and tonic K/ currents.
The alternative possibility is an intrinsic voltage sensitivity in the transduction mechanism mediating the suppression
of a voltage-independent K/ conductance. Such a process
has been proposed to explain the voltage-dependent actions
of 1S,3R-ACPD and muscarinic agonists in cortical layer V
cells (Wang and McCormick 1993). Activation of mGluRs
and muscarinic receptors inhibits a voltage-independent K/
current contributing to the resting input conductance (Guérineau et al. 1994; Madison et al. 1987; McCormick and von
Krosigk 1992). The partial reduction of this conductance is
thought to account for the increase in input resistance measured with small hyperpolarizing voltage steps in the presence of agonists for these receptors (Guérineau et al. 1994).
The increased suppression of such a voltage-independent,
tonic K/ conductance at potentials positive to resting potential should counteract depolarization-induced passive currents and overcompensate outward rectification. The following experimental observations are consistent with a depolarization-enhanced suppression of a tonic K/ current following
the activation of mGluRs: 1) the K/ current sensitive to
1S,3R-ACPD was a steady-state membrane conductance, because the depolarization-induced enhancement of the mGluR
response was not dependent on the sequence of drug application or step depolarization; 2) the current was Ba2/ sensitive,
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similar to voltage-independent currents contributing to the
resting conductance (Storm and Helliesen 1989); and 3) the
suppression of passive membrane conductance, as assessed
with hyperpolarizing voltage steps, was larger with 10-mV
than with 30-mV negative commands (cf. Fig. 1).
In a previous investigation from our laboratory, it was
found that the 1S,3R-ACPD-sensitive leak current exhibited
a linear I-V relationship in the range between 0130 and 050
mV (Guérineau et al. 1994). In these experiments, however,
a fast (1 s) ramp protocol was used to assess instantaneous
changes in membrane conductance. Similarly, in the present
study we found that the early response to a step depolarization in the presence of agonist displayed linear characteristics
(not shown), whereas the voltage-dependent component had
markedly slower kinetics.
Involvement of second-messenger pathways
We have previously shown that the reduction in membrane
K/ conductance in response to the stimulation of mGluRs
is prevented when G protein function is disrupted (Gerber
and Gähwiler 1994; Guérineau et al. 1994). To date it has not
been possible, however, to identify other diffusible cytosolic
second messengers involved in the signal transduction process, raising the possibility that G proteins interact directly
with K/ channels in hippocampal pyramidal cells. Evidence
for direct gating of K/ channels by G protein components
has been found in the hippocampus (VanDongen et al. 1988).
Although the present study does not permit conclusions to
be drawn concerning the molecular mechanisms by which
K/ channels are modulated, our findings suggest that an
analogous process involving a voltage-dependent interaction
between G proteins and K/ channels may underlie the effect
of 1S,3R-ACPD on a tonic K/ current.
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FIG. 6. Comparison of the current responses to a
10-mV depolarizing step in the presence of 1S,3RACPD or N-methyl-D-aspartate (NMDA) at 2 different
Mg2/ concentrations. A: current responses in the presence of 2 mM Mg2/ before application of agonists.
B: in 2 mM Mg2/, depolarization evoked an instantaneous, desensitizing inward current in the presence
of NMDA, whereas 1S,3R-ACPD induced a slowly
developing inward current. C: in the absence of Mg2/,
depolarization evoked an outward current in the presence of NMDA and an inward current during 1S,3RACPD application. Reduced concentrations of NMDA
were chosen when Mg2/-free solutions were used to
maintain an amplitude of the inward current comparable with that obtained with 2 mM Mg2/.
ACPD INDUCES NEGATIVE SLOPE CONDUCTANCE IN I-V RELATION
Physiological significance
We thank Drs. D. A. Brown, D. A. McCormick, and S. M. Thompson
for helpful comments in the course of this study and for critical reading of
the manuscript. We are very grateful to L. Rietschin, L. Heeb, H. Kasper,
R. Schöb, and E. Hochreutener for excellent technical assistance.
This work was supported by Grant 31-45547.95 from the Swiss National
Science Foundation, by the Prof. Dr. Max Cloëtta Foundation (U. Gerber),
and by Sandoz Pharma AG, Basel (A. Lüthi).
Present address of A. Lüthi: Section of Neurobiology, Yale University
School of Medicine, 333 Cedar St., New Haven, CT 06510.
Address for reprint requests: U. Gerber, Brain Research Institute, August
Forel-Strasse 1, CH-8029 Zurich, Switzerland.
Received 15 May 1996; accepted in final form 29 August 1996.
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