1Department of Psychology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada; and 2Neuroscience Research, Eli Lilly Co., Indianapolis, Indiana 46285-0510
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ABSTRACT |
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Gorelova, Natalia A. and
Charles R. Yang.
Dopamine D1/D5 Receptor Activation Modulates a Persistent Sodium
Current in Rat Prefrontal Cortical Neurons In Vitro.
J. Neurophysiol. 84: 75-87, 2000.
The
effects of dopamine (DA) on a persistent Na+
current (INaP) in layer V-VI
prefrontal cortical (PFC) pyramidal cells were studied using whole cell
voltage-clamp recordings in rat PFC slices. After blocking
K+ and Ca 2+ currents, a
tetrodotoxin-sensitive INaP was
activated by slow depolarizing voltage ramps or voltage steps. DA
modulated the INaP in a
voltage-dependent manner: increased amplitude of
INaP at potentials more negative than
40 mV, but decreased at more positive potentials. DA also slowed the
inactivation process of INaP. The
D1/D5 dopamine receptor agonists SKF 38393, SKF 81297, and
dihydrexidine (3-10 µM), but not the dopamine D2/D3 receptor agonist
qiunpirole (1-20 µM), mimicked the effects of DA on
INaP. Modulation of
INaP by D1/D5 agonists was blocked by
the D1/D5 antagonist SCH23390. Bath application of specific protein
kinase C inhibitor, chelerhythrine, or inclusion of the specific
protein kinase C inhibiting peptide[19-36] in
the recording pipette, but not protein kinase A inhibiting
peptide[5-24], blocked the effect of D1/D5
agonists on INaP. In current-clamp recordings, D1/D5 receptors activation enhanced the excitability of
cortical pyramidal cells. Application of the D1/D5 agonist SKF 81297 induced a long-lasting decrease in the first spike latency in response
to depolarizing current ramp. This was associated with a shift in the
start of nonlinearity in the slope resistance to more negative membrane
potentials. We proposed that this effect is due to a D1/D5
agonist-induced leftward shift in the activation of
INaP. This enables DA to facilitate
the firing of PFC neurons in response to depolarizing inputs.
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INTRODUCTION |
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Mammalian medial prefrontal cortex (mPFC) is
functionally involved in higher cognitive processes that underlie
planning and organization of forthcoming behavior (Fuster
1995; Goldman-Rakic 1995
). The mPFC in both
primates and rodents receives a mesocortical dopaminergic projection
that arises from the ventral tegmental area (VTA) of the midbrain
(Björklund and Lindvall 1984
). Dopamine (DA), via D1/D5 receptor activation in the mPFC, has been shown to
modulate behavior that requires working memory (Brozowski et al.
1979
; Murphy et al. 1996
;
Sawaguchi and Goldman-Rakic 1991
). During short-term
working memory processing, a DA-dependent increase in active firing of
PFC neurons has been shown. This sustained period of firing typically
occurred during a brief delayed period when previously acquired memory
has to be "held" temporarily and be used later to guide forthcoming
behaviors (Fuster 1995
; Goldman-Rakic 1995
). An optimal level of DA in the mPFC is critical for this type of cognitive functions (Kimmberg et al. 1997
;
Murphy et al. 1996
; Zahrt et al. 1997
).
Too high or too low level of DA present in the mPFC can significantly
disrupt cognitive processes that utilize working memory.
An understanding of the ionic bases of DA actions that
mediate an increase in firing of mPFC pyramidal neurons is still
incomplete. In vivo electrophysiological studies in mPFC pyramidal
neurons show that microiontophoretic application of DA, or stimulation of the VTA, primarily suppress an on-going spontaneous firing activity
(Bunney and Aghajanian 1976; Pirot et al.
1992
; Sawaguchi and Matsumura 1985
;
Sesack and Bunney 1989
; Yang and Mogenson 1990
). In contrast, a very low level of DA potentiates the
firing activity induced by iontophoretic application of glutamate and acetylcholine in cortical neurons (Cépeda et al.
1992
; Yang and Mogenson 1990
). Recent in vitro
intracellular recordings in rat mPFC slices have revealed that DA
modulates voltage-dependent Na+,
K+, and Ca2+ currents to
alter neuronal excitability and, ultimately, synaptic integration
(Geijo-Barrientas and Pastores 1995
; Gulledge and Jaffe 1998
; Penit-Soria et al. 1987
; Shi
et al. 1997
; Yang and Seamans 1996
; reviewed in
Yang et al. 1999
). Several of these currents are active
at the subthreshold voltage range and play an active role in
determining firing threshold and amplification of subthreshold synaptic inputs.
In mammalian neocortical neurons, one of the major ionic conductances
that are active at the subthreshold voltage range is the slowly
inactivating, or "persistent," Na+ current
(INaP) (Connors et al.
1982; Crill 1996
; Stafstrom et al.
1985
). Results from single Na+ channel
recordings in acutely isolated cortical pyramidal neurons suggest that
the slow INaP, as well as the
transient Na+ current (responsible for spike
firing), is conducted via a single population of
Na+ channels. The Na+
channels can conduct the two currents by undergoing two kinetically different gating modes (Alzheimer et al. 1993
;
Brown et al. 1994
; Moorman et al. 1990
).
Furthermore activation of both protein kinase A (PKA) and C (PKC) have
been shown to modulate the transient Na+ current
as well as the INaP (Astman et
al. 1998
; Li et al. 1992
; Numan et al.
1991
; Smith and Goldin 1997
; Taverna et
al. 1999
; West et al. 1991
).
In the rat PFC, at least two families of dopamine receptors are shown
to be present (see Yang et al. 1999 for a review). They belong to the D1/D5 and D2/D3/D4 receptor families. Using current-clamp recordings in our previous study, we have shown that in mPFC pyramidal cells, D1/D5 receptor stimulation (by SKF38393) augments the duration of a Na+ plateau potential and shifts the
activation threshold of this potential to a more negative voltage. This
finding suggests that D1/D5 receptor stimulation may lower the
activation threshold and slow the inactivation of the
INaP (Yang and Seamans
1996
). These results have led us to hypothesize that DA may
modulate the kinetics of the Na+ channel that
conducts INaP and gives rise to the
Na+ plateau potential. Given that D1/D5 receptor
is coupled to G proteins, it is also likely that D1/D5 receptor
stimulation led to activation of the G-protein-coupled PKA or PKC
intracellular pathway to modulate Na+ channels
that conduct INaP .
In the present study, we have used whole cell voltage-clamp techniques
to determine the mechanisms, the DA receptor subtypes, and the
second-messenger pathway that DA activate to modulate the
INaP in layer V-VI mPFC pyramidal
cells in rat brain slices. Our results show that stimulation of D1/D5
receptor induces a PKC-mediated shift in the activation of
INaP to more negative voltages and
slows the inactivation of this INaP.
Thus in response to subthreshold depolarizing inputs, these actions of
DA on INaP will ensure pyramidal mPFC
neuron to reach firing threshold and to trigger repetitive firing.
Preliminary data has been published in an abstract form
(Gorelova and Yang 1997).
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METHODS |
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Brain slice preparations
The experiments were performed in brain slices prepared from
young adult (5-7 wk old, 80-150 g) male Spraque-Dawley rats. Similar
in all our previous studies, acute decapitation by a guillotine with no
prior anesthesia was performed using Decapicones plastic restrainer
(Braintree, MA). The brains were quickly removed and placed in ice-cold
oxygenated (95% O2-5%
CO2) artificial cerebrospinal fluid (ACSF) for
1-2 min. The ACSF contained (in mM): 125 NaCl, 3 KCl, 2.4 CaCl2, 1.3 MgCl2, 26 NaHCO3, and 10 glucose. The lateral portions of
the cortex from both hemispheres were trimmed away, leaving the medial
portions of the frontal cortex of both hemispheres still joined
together by the corpus callosum. Four-hundred-µm-thick bilateral PFC
slices were then cut on a vibratome (Campden, World Precision
Instruments). The slices were placed in continuously oxygenated ACSF at
room temperature. After 1 h of incubation, a single slice was
transferred to a submersion recording chamber (Medical System Corps)
when recording commenced.
Recordings
The patch-clamp technique in whole cell configuration was
used to study the effects of DA and its agonists on the
INaP. Patch pipettes were fabricated from
borosilicate tubing (1.5 mm OD, 1.1 mm ID) on a horizontal
microelectrode puller (P-87, Sutter Instruments). They had a resistance
of 3-5 M when filled with the patch pipette solution.
In voltage-clamp experiments, to pharmacologically
isolate the INaP, the patch pipette solution
was slightly modified from that used by Fleidervish et al.
(1996). The internal pipette solution contained (in mM): 135 CsCl, 2 MgCl2, 1 EGTA, 10 HEPES, 2 Na2ATP, and
0.3 Na2GTP, adjusted to pH 7.3 by CsOH and had an
osmolality of 285-295 mOsm (Advanced Instruments). In addition, 200 µM Cd2+ was added to the ACSF to block Ca2+
currents. Under these recording conditions, two protocols were used to
activate the INaP:
DEPOLARIZING VOLTAGE RAMP.
The membrane potential was clamped at 70 or
80 mV, and slow (20-50
mV/s) voltage ramps from
70 to 10 mV, or
80 to 0 mV were applied.
DEPOLARIZING VOLTAGE STEPS.
Depolarizing voltage steps were applied (from various holding
potentials, 1-s duration) to examine the steady-state
INaP mainly in the voltage range from
70 to
50 mV. At membrane potentials more positive than
50 mV,
unclampable fast Na+ currents were elicited and
they obscured the measurement of the INaP.
Drug applications
All drugs were bath-applied. Stock solutions of tetrodotoxin
(TTX, Alomone Lab., Israel), SKF 38393, SKF 81297 (RBI), PKC inhibitor
chelerythrine (RBI), PKC inhibitor
peptide[19-36] (Calbiochem) and PKA inhibitor
peptide[5-24] (Calbiochem) were prepared in
de-ionized water and stored as frozen aliquots at 20°C. The full
agonist for dopamine D1/D5 receptors, dihydrexidine (Tocris), was
freshly dissolved in ethanol prior to application. Stock solution of DA
was also prepared fresh just before application for each experiment. To
reduce oxidation of DA, sodium metabisulfate (0.002% final
concentration in ACSF) was used. Vehicle controls showed no apparent
changes in INaP or neuronal
excitability. All drugs were diluted to desired concentrations in the
perfusate immediately before application.
Data analysis
In voltage-clamp recordings, a "leak" current was estimated off-line by fitting the linear portion of the current during a depolarizing ramp to a straight line using pClamp6. It was then subtracted off the trace digitally using SigmaPlot software. Group data are presented as means ± SE.
In current-clamp recordings from regular spiking neurons or intrinsic
bursting neurons (neuronal response types were classified according to
Yang et al. 1996), the threshold of action
potential generation was examined by injecting current ramps (800 ms)
with an amplitude just sufficient to evoke single or doublet action potentials. In between these current ramps (applied every 30 s), we monitored the input resistance of the cell by its voltage response to a small hyperpolarizing current pulses (100 ms; 25 pA). The slope of
the voltage trace during the ramp protocol in the voltage range from
80 to
65 mV (where the INaP was
largely inactive) was also used to calculate the slope resistance.
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RESULTS |
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Voltage-dependent changes of the INaP in layer V-VI pyramidal neurons
Whole cell Na+ currents were recorded using
Cs+-filled electrodes (to block
K+ currents) in the presence of extracellular
Cd2+ (to block Ca2+
currents) in layer V-VI mPFC neurons. Only neurons that had a resting
membrane potential more negative than 65 mV and spike height
exceeding 80 mV immediately on achieving whole cell mode were taken
into consideration. After switching from current- to voltage-clamp
mode, employing a slow depolarizing voltage ramp (34 mV/s) resulted in
an initiation of a negative slope conductance at membrane potentials
more positive than
60 mV. After leak subtraction, a net inward
current with a mean peak current of 290 ± 75 pA (range: 180-500
pA, n = 20) between
35 and
30 mV was revealed (Fig. 1). The inward current had a tendency to
increase gradually (i.e., "run-up") during the first 3-5 min after
achieving whole cell recording. Then it stabilized and in most cases,
the inward current did not show any sign of a "run down" during the
next 40-60 min into the experiment.
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Addition of 1 µM TTX to the bath (n = 4) completely
abolished this inward current, thus strongly suggesting that it
represents a TTX-sensitive INaP (Fig.
1A). At membrane potentials more positive than 25 mV, an
outward current was revealed in all cells recorded. This outward
current has previously been characterized as a nonselective cationic
current (Alzheimer 1994
).
The amplitude of inward current evoked by the ramp depolarization was
dependent on the rate of voltage ramp: the slower the rate of the
voltage ramp, the smaller the peak amplitude of the ramp current (Fig.
1B). This is consistent with the data that have been
obtained in mouse layer V pyramidal neurons in neocortical slices
(Fleidervish and Gutnick 1996) and suggests that
INaP in rat layer V-VI mPFC pyramidal
neurons is also subjected to inactivation during slow voltage ramp.
Holding cells at more negative potentials than
70 mV (up to
90 mV)
and employing depolarizing voltage ramps from these potentials did not
change the peak amplitude or voltage dependence of inward ramp current
(not shown).
Effects of DA on INaP and a "leak" current
Using the voltage-ramp protocol, DA (1-30 µM) did not induce a
consistent change in the peak amplitude of the
INaP. From the 10 cells tested, DA
increased the peak amplitude in 2 cells, reduced it in 1 cell, and did
not induce any changes in 7 cells. However, in 9 of 10 cells tested
using the depolarizing voltage-ramp protocol, application of 1-30 µM
DA induced voltage-dependent changes in the
INaP: there was an increase
INaP amplitude at potentials more negative than 40 mV and a decrease
INaP amplitude at potentials more
positive than
20 mV (Fig. 2). For
example, at a membrane potential of
50 mV, DA increased the
INaP by 30 to 50% (mean = 34 ± 9%, n = 10; Fig. 2, A-C).
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In 6 of 10 cells tested, DA also induced an increase of a
voltage-independent leak current (Fig. 2D). Following
subtraction of this leak current, a leftward shift in the voltage
dependence of ramp INaP was also
observed (Fig. 2E). This DA-sensitive leak current is
expressed as an increase in the slope of the linear portion of the
current trace in the voltage range between 80 and
65 mV (where the
INaP was inactive; Fig.
2D). To assess the reversal potential of the "leak"
current affected by DA in the six cells, DA (30 µM) was applied when
all Na+ currents were blocked by adding 1 µM
TTX in the perfusate. Slow depolarizing voltage ramp from
100 to
10
mV was then injected. In five of these cells, DA induced an increase in
the leak current that has a reverse potential of
30 mV (Fig.
3). In most cases, changes in the leak
current were completely reversible on 15-20 min of wash, while the
DA-induced shift in activation of the
INaP persisted for the duration of the
recording in the experiment (
1 h).
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Effects of DA on the inactivation kinetics of the INaP
Since INaP is subjected to a
time-dependent inactivation during the slow depolarizing ramp
(Fleidervish and Gutnick 1997), the ramp
INaP current is a result of an
interplay of activation and inactivation processes. Taking this into
consideration, a leftward "shift" in the voltage dependence of ramp
Na+ current may be due to a combination of a
leftward shift in activation, a slowing (time dependence) of
INaP inactivation, and a rightward shift in steady-state inactivation.
To determine if DA affects the time dependence of inactivation of the
INaP, depolarizing voltage steps from
80 to
30 mV with increasing duration were injected prior to
application of a slow depolarizing voltage ramp from
80 to 0 mV
(Fleidervish et al. 1996
). We have used prepulse voltage
step only to
30 mV to avoid activation of the cationic outward
current. Prepulses with duration 1 s induced a duration-dependent
reduction of the peak amplitude of the
INaP (Fig.
4A).
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Figure 4, B and C, shows that DA significantly reduced this suppression of the INaP by the depolarizing prepulse steps. Before DA application, a 3-s depolarizing prepulse reduced the peak amplitude of INaP by 33 ± 3% (n = 3). After bath application of DA (10 µM), the INaP was reduced by only 24 ± 4% (n = 3; P < 0.05). With a 12-s prepulse, INaP was reduced by 54 ± 3% in the control but only by 46 ± 3% after DA. Thus DA slow down further the slow inactivation of the INaP.
We also used a depolarizing step protocol to test whether a DA-induced
leftward shift in voltage dependence of ramp Na+
current is primarily due to a possible DA modulation of the
steady-state inactivation of the INaP
or slowing of its inactivation by DA. Cells were voltage-clamped
continuously at either 70 or
90 mV, and then depolarizing voltage
steps (1 s) were applied to bring the potentials to
55 mV (Fig.
4D). At
55 mV, the recorded inward current was not
obscured by the appearance of any unclamped fast Na+ current. The inward Na+
currents obtained during depolarizing steps from
70 to
55 mV and
from
90 to
55 mV were increased by DA by 34 ± 6%
(n = 3) and by 32 ± 7% (n = 3),
respectively (Fig. 4D). The fact that there was no
significant difference in the DA-induced changes in the current when
the cell was held at
90 or
70 mV suggests that the DA-induced
increase in INaP at subthreshold
membrane potentials is not due to a change in the
steady-state inactivation. Hence the DA-induced shift in
voltage-dependence of the INaP is not due to a possible DA-induced modulation of the
steady-state inactivation.
The DA-induced increase in inward Na+ current can be seen not only at the end but also at the beginning of the voltage step (Fig. 4D) where the inactivation process (in response to depolarization) has not yet played a significant role. It suggests that although DA-induced changes in time dependence of inactivation can contribute to the shift in voltage dependence of the ramp INaP, the DA-induced increase of ramp INaP at subthreshold membrane potentials are mainly not due to slowing of the INaP inactivation by DA. Taking together, these data suggest that DA-induced leftward shift in the voltage dependence of the INaP is primarily due to a leftward shift in the activation of the INaP by DA.
Effects of DA receptor agonists on INaP
Bath application of the D1/D5 agonists SKF 39393, SKF 81297, or dihydrexidine (1-10 µM) did not induce consistent changes in the peak amplitude of the INaP activated by a depolarizing voltage ramp. Of 34 cells tested, the peak amplitude of the INaP (evoked by a depolarizing voltage ramp) was reduced by the D1/D5 agonists in 12 cells, increased in 5 cells (e.g., Fig. 5C), and not changed in 17 cells.
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In 30 of 34 cells tested, the D1/D5 receptor agonist applications shifted the voltage dependence of ramp INaP to more negative potentials despite having different effects on the peak amplitude of INaP in some of these cells (Fig. 5, A and C). Since SKF38393, SKF81297 and dihydrexidine are equally effective in inducing a leftward shift in the activation of the ramp INaP, we have pooled the D1/D5 agonists data together for analysis.
At 50 mV, INaP activated by slow
depolarizing ramp was increased by 27 ± 12% (n = 34 cells). As in the case with DA, the D1/D5 agonist-induced leftward
shift in the activation of ramp INaP
in most cases lasted for 30 min (Fig. 5, B and
D). However, when the brain slices were preincubated for 10 min with D1/D5 receptor antagonist SCH23390 (9 µM), application of
the D1/D5 agonist dihydrexidine (3 µM) no longer induced any
significant shift in the voltage dependence of ramp
INaP (not shown). Ramp INaP measured at
50 mV was not
changed (
1.3 ± 4.2%, n = 4). The D2/D3 agonist
quinpirole at a concentration of
20 µM failed to affect the peak
amplitude of INaP or to induce any
shift in the voltage dependence of
INaP evoked by a voltage ramp
(n = 11). Taken together, these results strongly
suggest that DA affects the activation of the
INaP through D1/D5 receptor stimulation.
Similar to the effects induced by DA, D1/D5 agonists also slowed the inactivation of the INaP. Figure 6 illustrates an experiment when the INaP activated by a long (7.5 s) depolarizing pulse was used to record the full extent of the inactivation of INaP. The INaP evoked following D1/D5 agonist application was normalized against baseline current in the control (Fig. 6A). Following exposure to SKF81297 (5 µM), the time constant (tau) of full inactivation of the INaP was enhanced by 43 ± 16% (n = 6).
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Effects of D1/D5 receptor stimulation on the activation of the INaP required PKC but not PKA
Activation of PKC has been shown to induce a leftward shift in the
voltage dependence of ramp-activated
INaP and slows the inactivation of the
transient Na+ current (Astman et al.
1998; Numann et al. 1991
; Sancini et al. 1999
; Taverna et al. 1999
). In the present
study, we have examined the possibility that D1/D5 receptor stimulation
activated an intracellular PKC pathway to induce a shift in the voltage
dependence of ramp INaP in two series
of experiments.
In the first series of experiments (n = 8), the PKC
inhibitor peptide[19-36] (20 µM) was added
to the patch pipette solution immediately before recording. In six of
eight cells tested after 13-30 min of diffusion of the inhibitory
peptide into the recorded cell, bath application of D1/D5 agonists no longer induced an increase in the ramp
INaP at membrane potentials more
negative than 40 mV (Fig. 7). When PKC
activity was blocked, amplitude of ramp
INaP measured at
50 mV at 10 min
after D1/D5 agonist application was even slightly reduced in most cases
(mean changes
4 ± 5%, n = 6). In the remaining
two cells, we observed a delayed shift in the activation of the
INaP, starting at 20 min after the
D1/D5 agonist application. The peak amplitude of the ramp
INaP was reduced slightly by the D1/D5
agonists following PKC inhibitor
peptide[19-36] treatment (Fig. 7, A
and B). Control experiments revealed that the PKC inhibitor peptide[19-36] alone could reduce the peak
amplitude of the ramp INaP in two of
four tested neurons, but the peptide alone failed to induce a shift in
the INaP activation.
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In the second series of experiments, another PKC inhibitor, chelerythrine (15 µM), was bath applied for 30-50 min. Subsequent application of D1/D5 agonists failed to induce a shift in the activation of ramp INaP to more negative potentials (n = 3, not shown). These data suggest that PKC may be involved in mediating the effects of D1/D5 receptor activation on the INaP evoked by a voltage ramp in rat mPFC pyramidal cells.
Since it was shown that DA also modulates the transient
Na+ current in pyramidal neurons of hippocampus
through the cAMP-dependent activation of PKA (Cantrell et al.
1997), we have tested the possible role of this intracellular
pathway in modulating ramp INaP by D1/D5 receptor stimulation. Inclusion of the PKA inhibitor
peptide[5-24] (20 µM) in the internal patch
pipette solution did not block the leftward shift in the voltage
dependence of ramp INaP in response to
application of D1/D5 agonists (Fig. 7, C and D,
n = 4). It resulted in an increase in the amplitude of
ramp INaP measured at
50 mV. Average
value of current increase was 21 ± 4%, which is not
significantly different from the value of current increase by D1/D5
agonist without the PKA peptide inhibitor in the pipette solution
(27 ± 12%). However, the D1/D5 agonist did reduce the peak
amplitude of the INaP (Fig. 7,
C and D). Collectively, these data suggest that
the PKC, but not PKA, pathway is likely to mediate the effects of D1/D5
receptor activation on the shift of voltage dependence of
INaP evoked by voltage ramp in rat
layer V-VI pyramidal cells from the mPFC.
Effect of D1/D5 receptor stimulation on excitability of cortical pyramidal neurons
INaP is known to play an
important role in setting the threshold for firing action potentials
(Crill 1996). Therefore we have investigated how
modulation of INaP by D1/D5 agonists
might affect the excitability of mPFC neurons in current-clamp
experiments. To avoid indirect effect of the D1/D5 agonists on the cell
excitability through modulating glutamate and/or GABA release
(Gulledge and Jaffe 1998
; Zhou and Hablitz
1999
), APV (50 µM), 6,7-dinitroquinoxaline-2,3-dione (DNQX;
10 µM), and bicuculline (10 µM) were added to the perfusate to
block the N-methyl-D-aspartate (NMDA), AMPA and
GABAA receptor channels, respectively.
Bath application of D1/D5 agonist SKF81297 (3-10 µM) induced a small
membrane depolarization (2-3 mV) accompanied by a transient reduction
in the input resistance (n = 4 of 8 cell tested) (see also Shi et al. 1997; Yang and Seamans
1996
). Under this condition, the number of spikes evoked by
injection of depolarizing current steps increased (Fig.
8A1). However, if the membrane
potential was current-clamped continuously (via DC injection) to
predrug value, injection of the same depolarizing current steps evoked less number of spikes (Fig. 8A1). With this bias current
continuous holding the cell, injection of a depolarizing current ramp
resulted in a biphasic change in the latency of the first spike
initiation (Fig. 8A, 2 and 3). An initial brief
increase was followed by a long-lasting pronounced decrease in first
spike latency. The late, long-lasting effect on spike firing was
associated with the starting of nonlinearity in the voltage slope at
more negative membrane potential and a decrease in spike firing
threshold, thus giving rise to an overall increase in neuronal
excitability (Fig. 8A, 2 and 3). The graph in
Fig. 8A3 shows the time course of the D1/D5 agonist-induced
responses to current ramp injection.
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In mPFC neurons that were not depolarized by SKF81297, they only showed a decrease in first spike latency evoked by a depolarizing current step and ramp (Fig. 8B, 1 and 2) and an increase in the number of spikes evoked by a depolarizing step (n = 4 of 8 cells tested; Fig. 8B1). These long-lasting effects were accompanied by no significant changes in input resistance (Fig. 8B3).
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DISCUSSION |
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In the present study in mPFC layer V-VI pyramidal neurons, slow depolarizing voltage ramp activated a TTX-sensitive INaP. DA induced a leftward shift in the voltage dependence of this ramp INaP. This has resulted in an increase of INaP in the subthreshold voltage range for spike generation but a decrease of this current at more depolarized membrane potentials. DA also slowed the development of a full inactivation of INaP during sustained depolarization. The DA effects on the INaP was mimicked by D1/D5 agonists SKF 81297, SKF38393, and dihydrexidine but not by the D2/D3 receptor agonist quinpirole. The effects of DA on the voltage-dependent activation of INaP were blocked by selective PKC, but not PKA, inhibitors. These data suggest that following D1/D5 receptor activation, DA exerted its effects on the voltage dependence of the INaP via intracellular mechanisms requiring the activation of PKC.
Several mechanisms have been proposed to account for the generation of
INaP in neurons.
INaP may be the result of
1) a "window" current arising from an overlap between
Na+ channel steady-state activation and
inactivation (Attwell et al. 1979); 2)
switching of an inactivating Na+ channel into a
noninactivating state
mode switching (e.g., transition of the
transient Na+ channel to noninactivating gating)
(Alzheimer et al. 1993
; Brown et al.
1994
; Keynes 1994
; Patlak and Ortiz
1986
); 3) slow closed-state inactivation if
Na+ channels, especially at intermediate membrane
potentials (Cummins et al. 1998
); and 4) the
presence of a distinct channel isoform with a different unitary
conductance (Crill 1996
; Magistretti et al.
1999
). According to the first three hypotheses,
INaP is conducted through the same
channels as the fast inactivating Na+ current.
Data from direct single channel recordings support the hypothesis that
the same Na+ channels from neocortical neurons
that give rise to fast transient Na+ current for
spike firing can also switch to a slowly inactivating/persistent mode
and give rise to INaP
(Alzeheimer et al. 1993
; Brown et al. 1994
). However, it is still unclear under what physiological
conditions the soma-dendritic Na+ channels of
mPFC neurons will preferentially switch to this slowly inactivating mode.
Our present results of the effects of DA on
INaP at first appeared to contradict
with reported findings of the effects of DA on the
INaP in striatal and mPFC pyramidal
neurons (Cépeda et al. 1995;
Geijo-Barrientos and Pastore 1995
). In striatal neurons in rat brain slices, whole cell patch-clamp recordings showed that DA
reduced the peak amplitude of a putative
INaP activated during a slow
depolarizing ramp protocol (Cépeda et al. 1995
). Since Ca2+ and K+ channels
were not blocked and the effects of DA on the activation and
inactivation relationships of the putative
INaP were not investigated in that
study, it is not possible to make a detail comparison with our present
results. This becomes more complicated especially when taking into
account that in some cells in our study, DA also reduced the peak
amplitude of the INaP in mPFC
pyramidal neurons.
In another study from mPFC pyramidal cells in rat brain slice
preparation, putative INaP was
recorded (also in the absence of any Ca2+ and
K+ channel blockers) using single sharp electrode
voltage clamp (Geijo-Barrientos and Pastore 1995). DA
(10 µM) was shown to suppress a persistent inward current during
depolarizing steps in the voltage range from
50 to
45 mV. It should
be noted that several ionic conductances contribute to the generation
of the inward current in this voltage range subthreshold to spike
generation. Activation of the low-threshold Ca2+
conductance (Sutor and Zieglgansberger 1987
) as
well as inactivation of the mixed K+ and
Na+ conductance (so called
IH, which was shown to be active at
resting membrane potential in cortical pyramidal neurons) (Spain
et al. 1987
) resulted in inward currents during depolarizing
steps to
50 mV. Furthermore leak currents, and outwardly rectifying
K+ currents (Yang et al. 1996
) can
affect the net amplitude of the inward current. Since neither
Ca2+ nor K+ current
blockers were used in the cited studies in the preceding text, it is
very difficult to assess which ionic current(s) was suppressed or
enhanced by DA.
The actions of DA are mediated via activation of multiple
second-messenger signal transduction pathways. D1-like receptor is
known to be coupled with the adenylate cyclase system (Kebabian et al. 1972). Subsequent increases in cAMP formation (via
stimulation of this cyclase) lead to activation of PKA and the
associated biochemical cascades downstream. An additional signal
transduction pathway coupled to D1-like receptors was shown by the
finding that rat striatal mRNA encodes the expression of D1-like
receptors coupled to phospholipase C (PLC) stimulation (Mahan et
al. 1990
). In rat brain and renal tissues, D1-like receptor
activation of PLC leads to increase in inositol phosphate (IP)
formation and diacyglycerol (DAG) formation (Felder et al.
1989
; Undie and Friedman 1990
; Undie et
al. 1994
). This resulted in a cAMP- independent increase in intracellular Ca2+ (by IP) and
activation of PKC (by DAG). In mice with D1a receptor subtype
"knock-out," there is a continual expression of D1
receptor-stimulated PI metabolism but not adenylate cyclase activation,
further suggesting that the D1-like receptor that couples to
stimulation of IP is distinct from the classic D1 receptor which is
coupled to stimulation of adenylate cyclase (Friedman et al.
1997
). In addition, while D1 receptors stimulation in the rat
amygdala induced a large increase in IP formation but no cAMP
formation, D1-like receptor activation in the frontal cortex and
striatum stimulated both transduction systems (Friedman et al.
1997
). These data suggest that there are also distinct
differences in the regional distribution of these two signal
transduction systems that are coupled to the D1 receptor in the brain
(Undie and Friedman 1990
).
Modulation of Na+ channel functions in mammalian
brain can be achieved by phosphorylation of the channels via activation
of the cyclic AMP-dependent protein kinase A (PKA), as well as by calcium-dependent PKC (Numann et al. 1991; Smith
and Goldin 1997
). Activation of PKA reduced the peak amplitude
of the fast Na+ current without affecting either
the kinetics or the voltage-dependent properties of the
Na+ channels (Gershon et al. 1992
;
Li et al. 1992
; Smith and Goldin 1997
).
Single-channel recordings in striatal and hippocampal neurons show that
DA, through activation of PKA but not PKC, reduced the open probability
of Na+ channels that conduct fast transient
Na+ current (Cantrell et al. 1997
;
Schiffmann et al. 1995
). Recently it was shown that
modulation of the fast Na+ current by D1/PKA
pathway is voltage dependent (Cantrell et al. 1999
) with
no detectable effect at
110 mV but a progressively increasing
suppressive effect at more depolarized membrane potentials. Since PKA
stimulation induced a small shift in the voltage-dependence of the
steady-state inactivation of this Na+ current,
the authors suggested that membrane voltage can directly alters the
extent or pattern of PKA phosphorylation.
In contrast, activation of PKC not only modulates the peak amplitude of
the transient fast Na+ current but also
substantially slows its inactivation. In MM14 muscle cells, slowing of
inactivation of Na+ current requires a lower
concentration of 1-oleoul-2-acetyl-sn-glycerol (OAG, a diacyglycerol
analog) than that needed for a reduction of the peak
Na+ current, suggesting that these events may be
caused by independent phosphorylation events (Numman et al.
1994). Activation of PKC was further shown to increase the
life-time of the single Na+ channel spent in the
open-state and the probability of its reopening during prolong
depolarization (Numann et al. 1991
). Moreover, recent
studies in rodent cortical neurons also show that a PKC-activating phorbol ester, and OAG, induced a shift in the activation of fast transient Na+ current and
INaP to more hyperpolarized membrane
potentials (Astman et al. 1998
; Taverna et
al. 1999
). A shift in activation has led to a significant
increase in the transient Na+ current and
INaP at membrane potentials more
negative than
40 mV and a decrease in their amplitudes at membrane
potentials more positive than
40 mV. The PKC-mediated increase in
Na+ currents in the subthreshold voltage range
for spike generation resulted in a subsequent increase in cortical
neuronal excitability (Astman et al. 1998
;
Sancini et al. 1999
; Taverna et al.
1999
). These PKC effects are remarkably similar to the effects
of DA and D1/D5 agonists on the INaP
in rat mPFC pyramidal cells as shown in the present study. Our present
finding shows that PKC inhibitors, but not a PKA inhibitor, blocked the
D1/D5 agonist-induced shift in the activation of
INaP. This finding further suggests that the shift in the activation of
INaP was mediated via D1/D5 receptor
activation of the PKC pathway.
INaP has been shown to play a
significant role in the modulation of neuronal excitability by setting
the threshold of firing and shaping of repetitive firing patterns as
well as in the nonlinear amplification of excitatory synaptic inputs
(Crill 1996; Schwindt and Crill 1995
;
Stuart 1999
; Stuart and Sakmann 1995
).
While a DA-induced leftward shift in the activation of
INaP results in a lowering of the
threshold of spike initiation, slowing of
INaP inactivation by DA may also be
responsible for increasing the neuronal ability to repetitive firing.
Since DA modulates not one but several ionic conductances at
subthreshold voltage range in mPFC pyramidal neurons, the net effect of
DA on spike initiation and neuronal excitability will depend on a
complex interplay of the INaP with
these conductances (Dilmore et al. 1999
; Yang and Seamans 1996
; Yang et al. 1999
).
An early transient effect of DA in reducing neuronal excitability due
to a reduction in input resistance was reported by Gulledge and
Jaffe (1998) and by Geijo-Barritos and Pastore
(1995)
. They showed that a significant DA-induced reduction of
slope resistance occurs when GABAa receptors were
not blocked adequately (Gulledge and Jaffe 1998
, 1999
),
and the suppression of evoked spikes by DA can be mimicked by D2/D3
agonist quinpirole (Gulledge and Jaffe 1998
). These data
suggest that DA-induced activation of GABAergic interneurons may also
take part in the control of pyramidal neuronal excitability
(Yang et al. 1999
; Zhou and Hablitz
1999
).
In some mPFC neurons in the present study, activation of D1/D5
receptors can also induce a transient decrease in input resistance with
no reduction in neuronal excitability. Our voltage-clamp data suggest
that this decrease in input resistance is likely to be due to an
increase in a leak current that has a reversal potential of
approximately equal to 35 mV. This reduction of input resistance by
DA in these cells was also accompanied by a small membrane
depolarization that can bring the membrane potential closer to the
point of activation of the INaP in
this group of mPFC neurons.
As we have shown in a previous study (Yang and Seamans
1996), activation of D1/D5 receptors induced a long-lasting
increase in neuronal excitability which may be mediated by D1/D5
receptor actions on INaP (this study).
This delayed increase of excitability in mPFC neurons by DA has
recently been replicated independently by several investigators
(Ceci et al. 1999
; Gulledge and Jaffe 1999
; personal communication; Lavin and Grace
1999
). These findings suggest that for a given depolarizing
input, postsynaptic D1/D5 and D2/D3 dopamine receptor stimulation may
modulate time-dependent changes in ionic conductances that alter the
threshold for firing in mPFC neurons. In response to depolarizing
inputs, activation of D2/D3 receptors may induce an early transient
suppression of neuronal excitability by increasing a leak current,
while activation of D1/D5 receptors induces a late, prolonged
enhancement of neuronal excitability by shifting the activation of
INaP to a more negative potential,
plus slowing the rate of full inactivation of
INaP.
Functional significance of a DA modulation of INaP
Modulation of the activation and inactivation kinetics of
INaP by DA may result in a wide-range
of changes in neuronal signal integration. In mPFC pyramidal neurons,
subthreshold membrane depolarization by inward currents is known to be
opposed strongly by a slowly inactivating outward
K+ conductance (Yang et al. 1996).
Dopamine, via D1/D5 receptor activation, has been shown to attenuate
this slow K+ conductance, thus allowing a full
expression of the effects mediated by
INaP (Yang and Seamans
1996
; Yang et al. 1996
). Subthreshold EPSPs have
been shown to be amplified by TTX-sensitive
INaP, which are generated close to the
initial portion of the apical dendrites of deep layer somatosensory
cortical and hippocampal CA1 pyramidal neurons (Lipowsky et al.
1996
; Schwindt and Crill 1995
; Stafstrom et al. 1985
; Stuart and Sakmann 1995
). More
recently, Stuart (1999)
has also shown that
hyperpolarizing GABAergic inhibitory postsynaptic potentials (IPSPs)
can turn off the inward INaP, thus
resulting in a net enhancement of the outward GABAergic current to
ultimately produce an amplification of the GABAergic IPSP. The effects
of DA on INaP shown in the present
study suggest that DA may modulate both excitatory postsynaptic
potentials (EPSPs) and IPSPs via its interactions with the
INaP directly.
INaP also contributes to the
generation of a narrow bandwidth membrane voltage oscillation when
neocortical neurons are depolarized to the subthreshold membrane
potential range in a sustained manner (Alonso and Llinas
1989; Amitai 1994
; Gutfreund et al.
1995
; Llinas et al. 1991
; Stafstrom et
al. 1985
; Yang et al. 1996
). At the subthreshold
potentials, this TTX-sensitive oscillation is generated via
interactions of the INaP with a TEA-
or a dendrotoxin- sensitive slowly inactivation
K+ current (Gutfreund et al. 1995
;
Yang et al. 1996
) and other incoming synaptic inputs in
vivo (Cowan and Wilson 1994
; Nuñez et al. 1992
; Steriade et al. 1993
). This type of
oscillation has been suggested to be the cellular substrate that
underlie rhythmic synchronized activities recorded from cortical
networks during sensory information processing (Singer
1993
). Since the subthreshold oscillation itself cannot be
transmitted to neighboring neurons, in order for neuronal network
synchronization to occur, synaptic inputs could be used to generate
spikes that ride on top of the subthreshold oscillation. This may then
generate synchronized spike output of a specific frequency to the
neighboring interconnected neurons.
Finally, although not directly tested in the present study, it is
conceivable from our present findings that the dopamine-induced shift
of activation and inactivation kinetics of the
INaP will enhance the probability of
mPFC neurons to initiate spike firing at a designated time in response
to a subthreshold depolarizing input. In this way, specific spike
frequency can be transmitted to neighboring interconnected neurons
(Kritzer and Goldman-Rakic 1995; Levitt
et al. 1993
) that may be undergoing subthreshold oscillation at
the same frequency and phase. Outputs from these neurons may induce a
micro network of interconnected neurons to fire synchronously
(Lampl and Yarom 1993
; Yang et al. 1999
).
This may provide a cellular mechanism for "binding" the firing
activity between two or more interconnected mPFC neurons to a specific rhythm during working memory processing (Durstewitz et al.
1999
; Yang and Seamans 1996
; Yang et al.
1999
) when certain information has to be held for a short
period of time prior to their use to guide forthcoming behaviors.
Clearly experimental evidence is needed to support or refute such
theoretical framework of the functional roles of DA modulation of
INaP in synaptic integration and
neuronal network functions.
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ACKNOWLEDGMENTS |
---|
We thank M. Muhlhauser for editorial assistance.
This study is funded by the E.J.L.B. Foundation and the Medical Research Council of Canada.
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FOOTNOTES |
---|
Address for reprint requests: C. R. Yang, Neuroscience Research, Eli Lilly and Co., Lilly Corporate Ctr., Indianapolis, IN 46285-0510 (E-mail: cyang{at}lilly.com).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 30 November 1999; accepted in final form 15 March 2000.
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REFERENCES |
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