Division of Life Sciences, University of Texas at San Antonio, San Antonio, Texas 78249
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ABSTRACT |
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Gulledge, Allan T. and David B. Jaffe. Multiple Effects of Dopamine on Layer V Pyramidal Cell Excitability in Rat Prefrontal Cortex. J. Neurophysiol. 86: 586-595, 2001. The mechanisms underlying the inhibitory effects of dopamine (DA) on layer V pyramidal neuron excitability in the prelimbic region of the rat medial prefrontal cortex were investigated. Under control conditions, DA depressed both action potential generation (driven by somatic current injection) and input resistance (RN). The presence of GABAA receptor antagonists blocked DA-induced depression of action potential generation and revealed a delayed increase in excitability that persisted for the duration of experimental recording, up to 20 min following the washout of DA. In contrast to spike generation, disinhibition did not affect the transient depression of RN produced by DA, suggesting independent actions of DA on spike generation and RN. Consistent with the hypothesis that DA acts to decrease pyramidal cell output via a GABAergic mechanism, DA increased the frequency of spontaneous inhibitory postsynaptic currents in both the absence and presence of TTX. Furthermore focal application of GABA to a perisomatic region mimicked the inhibitory effect of DA on spike production without affecting RN. Focal application of bicuculline to the same location reversed the inhibitory effect of bath-applied DA on spike generation, while again having no effect on RN. The depression of RN by DA was both occluded and mimicked by the Na+ channel blocker TTX, suggesting the involvement of a Na+ conductance in reducing pyramidal cell RN during the acute presence of DA. Together these data demonstrate that the acute presence of DA decreases pyramidal neuron excitability by two independent mechanisms. At the same time DA triggers a delayed and longer-lasting increase in excitability that is partially masked by synaptic inhibition.
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INTRODUCTION |
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The mammalian prefrontal
cortex (PFC) is important for a form of short-term memory often
described as "working memory" (Bauer and Fuster
1976; Goldman et al. 1971
; Granon
et al. 1994
), and the ability of the PFC to facilitate working
memory tasks is dependent on dopaminergic input from the ventral
tegmental area (VTA) (Brozoski et al. 1979
;
Bubser and Schmidt 1990
). In the rat, the cortical area
most associated with both working memory behaviors and mesocortical dopamine (DA) input is the prelimbic region of the medial PFC (mPFC)
(Fritts et al. 1998
; Seamans et al. 1995
,
1998
). This area receives dense dopaminergic afferents
(Berger et al. 1976
; Descarries et al.
1987
; Emson and Koob 1978
; Van Eden et
al. 1987
), and pyramidal and nonpyramidal neurons in this
region can express members of both the D1 and D2 DA receptor families
(Gaspar et al. 1995
; Vincent et al. 1993
,
1995
).
In primates and rats, local depletion of DA in the PFC produces
deficits in working memory task completion (Brozoski et al. 1979; Bubser and Schmidt 1990
). Moreover
overstimulation of prefrontal DA receptors impairs working-memory
performance (Arnsten et al. 1994
, 1995
; Druzin et
al. 2000
; Murphy et al. 1996
; Romanides et al. 1999
; Zahrt et al. 1997
). Because these
data suggest that proper working memory function depends on the
maintenance of an optimal level of prefrontal DA, it is important to
understand the effects of DA on the physiology of individual prefrontal neurons.
At the cellular level there appear to be two seemingly conflicting
effects of DA. Unit and intracellular recordings in vivo have shown
that DA, whether released by VTA stimulation or exogenously applied,
inhibits spontaneous and evoked firing of PFC neurons (Bernardi
et al. 1982; Ferron et al. 1984
; Godbout
et al. 1991
; Mantz et al. 1988
; Pirot et
al. 1992
, 1996
; Sesack and Bunney 1989
). In
agreement with these data, several in vitro studies have found DA
inhibits the excitability of rat prefrontal pyramidal neurons
(Geijo-Barrientos and Pastore 1995
; Gulledge and
Jaffe 1998
; Zhou and Hablitz 1999
). In contrast,
a number of other in vitro studies have reported dramatic enhancement
in the excitability of prefrontal pyramidal cells by DA (Ceci et
al. 1999
; Gorelova and Yang 2000
; Henze
et al. 2000
; Penit-Soria et al. 1987
;
Yang and Seamans 1996
).
The purpose of the present study was to determine the mechanisms
underlying the inhibitory effects of DA on action potential generation
and input resistance (RN) of pyramidal
neurons in the prelimbic region of the rat mPFC. Several factors
suggest that DA may inhibit prefrontal pyramidal cell excitability
indirectly by modulating GABAergic inhibition. First, anatomical
studies have established direct DA innervation of GABAergic neurons in the PFC of both rats and monkeys (Benes et al. 1993,
1996
; Sesack et al. 1995a
,b
, 1998
; Smiley
and Goldman-Rakic 1993
; Verney et al. 1990
).
Second, DA can directly modulate the physiology of nonpyramidal,
presumably GABAergic neurons in the rat PFC (Zhou and Hablitz
1999
). Third, results from in vitro and in vivo studies demonstrate dopaminergic enhancement of extracellular GABA levels in
the rat mPFC (Bourdelais and Deutch 1994
; Del
Arco and Mora 2000
; Grobin and Deutch 1998
;
Retaux et al. 1991
). DA-mediated increases in GABA
release could be expected to both inhibit action potential generation
and reduce RN in layer V pyramidal neurons.
We tested the hypothesis that DA indirectly inhibits pyramidal
cells by increasing local GABAergic tone within the rat prelimbic PFC.
Evidence is presented suggesting that DA's inhibitory actions on layer
V pyramidal neurons are mediated through at least two distinct
mechanisms and that this inhibition can mask a delayed increase in
excitability. In addition, we show that dopaminergic reduction of
RN is TTX sensitive, suggesting
dopaminergic modulation of a Na+ conductance. A
preliminary report has been presented in abstract form (Gulledge
and Jaffe 1999).
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METHODS |
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Brain slices
Animals were housed and handled in accord with the guidelines
set by The American Physiological Society. Following decapitation of
unanesthetized, 17- to 30-day-old Sprague-Dawley rats (Harlan, Indianapolis, IN), brains were removed and immersed in cold (0°C) artificial cerebral spinal fluid (ACSF, described in the following text) where NaCl was replaced with choline chloride. Coronal slices (300 µm) containing the prelimbic region of the mPFC were prepared with a vibratome. Brain slices were immediately (<5 min following decapitation) placed in a holding chamber of ACSF containing (in mM)
124 NaCl, 26 NaHCO3, 2.5 KCl, 1.25 Na2HPO4, 2 MgCl2, 2 CaCl2, and 10 dextrose. Slices were allowed to incubate for 1 h at room temperature
(~23°C) before being transferred, as needed, into a submerged
recording chamber perfused with oxygenated ACSF (~2 ml/min). In some
experiments, CaCl2 was removed and the amount of
MgCl2 was increased to 6 mM. In other
experiments, where spontaneous inhibitory postsynaptic currents (IPSCs)
were monitored, the level of KCl was raised to 5.5 mM. All ACSF
solutions were oxygenated continuously with a mixture of 95%
O2-5% CO2. Experiments
were performed at ~31°C.
Electrophysiology
Whole cell recordings were made from visually identified layer V
pyramidal cells from the prelimbic region of the rat mPFC using
infrared video/differential interference contrast microscopy (Stuart et al. 1993) with a ×40 water-immersion
objective. To ensure that pyramidal cells did not have cut apical
dendrites, slices were chosen so that the apical dendrites were either
in the same plane as the soma or descended into the slice. Patch pipettes (3-5 M
) were filled with (in mM) 120 K-gluconate, 10 HEPES, 0.1 EGTA, 20 KCl, 2 MgCl2, 3 Na2ATP, and 0.3 NaGTP (pH = 7.3). In some
experiments, as noted, K-gluconate was replaced with KCl.
Current- or voltage-clamp recordings were made using an Axoclamp
2B amplifier (Axon Instruments) and were digitized at 1, 3, 5, or 25 kHz using an ITC-16 interface (Instrutech) connected to a Power
Macintosh computer running AxoData (Axon Instruments) acquisition
software. Whole cell pipette series resistance was <20 M and was
bridge-compensated. No corrections were made for junction potentials,
which should be ~10 mV with K-gluconate. Analyses of electrical
responses were performed off-line using custom software written with
Igor Pro (Wavemetrics). Only one neuron, experiencing a single drug
treatment, was used from each brain slice. In all cases, only cells
with a resting potential of at least
60 mV, and stable baseline
responses were given drug treatments.
Experimental paradigm
Trains of action potentials were evoked by depolarizing current
steps (1 s duration, 100-300 pA) at 0.1 Hz. Current intensities were
adjusted so that they evoked about six action potentials before any
drug treatment (Gulledge and Jaffe 1998).
RN was determined within the linear
range of the steady-state voltage-current relationship, generated by a
series of hyperpolarizing and depolarizing current injections (usually
50 to +50 pA). When a full range of steps could not be obtained, for
example during brief pressure application of drugs (described below),
RN was calculated from the response to
single hyperpolarizing current steps (
50 or
100 pA). DA-induced changes in resting potential were compensated with a bias current to
maintain baseline levels during these measurements.
After baseline measurements of action potential number and
RN, DA (10 µM) was bath-applied for
periods of ~5 min. Two gravity-fed perfusion lines were used to
change solutions. New solutions reached the recording chamber in ~2
min and were removed in ~5 min (Gulledge and Jaffe
1998). In some experiments, as noted, drug solutions loaded
into patch pipettes were pressure-ejected into the slice for periods of
10-30 s (see Fig. 5). Both control and drug ACSF solutions were
continuously oxygenated throughout experiments.
In some control experiments, excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) were evoked with a monopolar stimulating electrode placed into layer V near the soma of the postsynaptic pyramidal cell. EPSPs were isolated using 10 µM bicuculline while IPSPs were recorded in the presence of 2 mM kynurenate. Evoked potentials were generated with a 50 µs pulse from a stimulus isolation unit controlled by a Master 8 pulse generator (AMPI, Israel).
Stock solutions of DA and bicuculline methiodide were made fresh daily
and mixed into oxygenated ACSF as needed. Stock solutions of TTX were
kept as frozen aliquots until just prior to use. Ascorbate (10 µM)
was added to all DA solutions to reduce oxidation. We previously found
that this concentration of ascorbate has no effect on pyramidal neuron
physiology (Gulledge and Jaffe 1998). All drugs were
purchased from Sigma (St. Louis, MO).
Analysis and statistics
The effects of DA on excitability were determined by analyzing the number of spikes generated from the average of five traces obtained immediately before, at the end of, and five minutes after DA application. IPSCs were identified and selected as inward currents with fast rise times and an amplitude threshold of at least three standard deviations from the mean current noise. Fast rise times were selected from events >1.5 to 3 times the standard deviation of a sweep's second derivative. These parameters were adjusted on a cell-to-cell basis to maximize the accuracy of IPSC detection (to limit false hits) but were kept constant for all phases of an individual cell's analysis. Statistical significance was determined using a two-tailed Student's t-test for paired or unpaired samples, as appropriate. Numerical values are expressed as means ± SE.
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RESULTS |
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As previously reported (Gulledge and Jaffe 1998),
bath application of DA (10 µM) decreased both the number of action
potentials generated by somatic current injection and the
RN of layer V pyramidal cells in
slices of rat mPFC (Fig. 1). The
inhibitory effects of DA on spike generation and
RN were evident within 3 min of
application and reversed ~5 min following drug removal (Fig.
1C). In eight cells, DA significantly decreased spike
generation by 81 ± 8% (df = 7, t = 7.1, P < 0.05; Fig. 1, A, C, and
E), while the latency to the first action potential (for 6 cells that still fired
1 spike in the presence of DA) increased from
52 ± 6 to 74 ± 11 ms (mean increase = 41 ± 9%;
df = 5, t = 3.3, P < 0.05; data
not shown). DA reduced RN from a
baseline mean of 63 ± 9 M
by 15 ± 3% (df = 7 , t = 4.3, P < 0.05; Fig. 1,
B, D, and F). Dopaminergic reduction
of RN was symmetrical with respect to
both depolarizing and hyperpolarizing directions. There was no
significant difference, during DA application or following drug
removal, between mean steady-state voltage responses pooled from
depolarizing versus hyperpolarizing current steps (Fig. 1, D
and G).
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As previously reported (Bernardi et al. 1982;
Geijo-Barrientos and Pastore 1995
; Gulledge and
Jaffe 1998
; Law-Tho et al. 1994
; Penit-Soria et al. 1987
; Shi et al. 1997
)
DA application depolarized layer V pyramidal cells by 2.0 ± 0.5 mV (resting Vm =
70 ± 1 mV).
This was not correlated with DA-mediated effects on
RN (Fig. 1H) and did not
significantly influence the inhibitory effects of DA on spike
generation (Gulledge and Jaffe 1998
).
GABAA antagonists occlude the inhibitory effect of DA on spike generation
To determine if DA inhibits pyramidal cell excitability by
modulating GABAergic inhibition, DA was applied in the presence of
GABAA blockers (Fig.
2). In 17 cells treated with 2 mM
kynurenate (to block fast excitatory synaptic transmission and the
subsequent activation of inhibitory neurons) and either 100 µM
picrotoxin and 10 µM bicuculline or 20 µM bicuculline alone, DA
failed to decrease the number of spikes generated by depolarizing
current steps (mean change in DA = +20 ± 13% of baseline
value; P > 0.05). The enhanced excitability became
significant ~10 min after the washout of DA (mean change in
"wash" = +59 ± 13%; df = 16, t = 4.3, P < 0.05; Fig. 2, B and C) and
lasted for the duration of the recording (20 min following DA
removal). The inhibitory effect of DA was not affected by the presence
of kynurenate alone (Gulledge and Jaffe 1998
).
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In contrast, GABAA antagonists did not interfere
with DA's effects on RN or
Vm. Under conditions where fast
synaptic transmission was blocked, pyramidal cell
RN was significantly higher than in control conditions (with GABAA and glutamate
receptors blocked, mean RN = 105 ± 11 M; df = 23, t = 2.3, P < 0.05). DA application reversibly decreased
RN of these neurons by 10 ± 3%
(df = 16, t = 3.1, P < 0.05; Fig.
2D). This effect was not statistically different from that
of DA in control conditions. Likewise disinhibition did not affect the
depolarization produced by exposure to DA (mean baseline
Vm =
71 ± 1 mV; mean
change = +1.6 ± 0.4 mV; df = 15, t = 3.3, P < 0.05). These data suggest that DA's effect
on action potential generation is via a mechanism independent of its
effects on RN and
Vm.
Dopamine and spontaneous IPSCs
We next tested the hypothesis that DA affects the frequency or
amplitude of GABAergic IPSCs. In 17 neurons, spontaneous IPSCs (sIPSCs)
were recorded in the presence of 2 mM kynurenate (Fig. 3, A-C). Because the reversal
potential for Cl in cells filled with
K-gluconate-based pipette solutions is close to the resting potential,
intracellular Cl
concentration was increased
with a KCl-based pipette solution (see METHODS).
Additionally, in some experiments, extracellular KCl was increased to
5.5 mM. Mean sIPSC frequency was 7 ± 1 Hz. Bath-applied DA (10 µM) reversibly increased the frequency of sIPSCs by 22 ± 10%
(df = 16, t = 2.3, P < 0.05; Fig.
3C) but did not significantly change sIPSC amplitude (mean
change = +13 ± 8%).
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Previous in vitro studies indicate that DA modulates either
spike-mediated (Retaux et al. 1991; Zhou and
Hablitz 1999
) or spike-independent (Penit-Soria et al.
1987
) GABA release onto prefrontal neurons. To confirm if DA
enhances spike-independent GABA release, we recorded miniature IPSCs
(mIPSCs) in 10 cells exposed to 1 µM TTX and 2 mM kynurenate (Fig. 3,
D-F). During these experiments, extracellular KCl was
maintained at 2.5 mM. Baseline mIPSC frequencies in TTX (5 ± 1 Hz) were not significantly different from sIPSC frequencies, described
in the preceding text. Bath application of 10 µM DA significantly
enhanced mIPSC frequency by 29 ± 11% (df = 9, t = 2.4, P < 0.05; Fig. 3F)
while not significantly affecting mIPSC amplitude (mean change = +19 ± 12%). In contrast to sIPSCs, DA-mediated increases in
mIPSC frequency persisted following washout of DA [mean change in
"wash" = +29 ± 9% (df = 9, t = 5.7, P < 0.05)].
Calcium independence
If DA can enhance TTX-insensitive release of GABA, DA might still depress spike generation under conditions in which spike-mediated transmitter release is reduced. We tested this possibility by conducting experiments where evoked synaptic transmission was blocked by removing calcium from the ACSF while at the same time increasing external magnesium to 6 mM. To test whether transmission was indeed blocked, several experiments were performed to demonstrate that Ca2+ removal blocked evoked IPSPs and EPSPs. Synaptic responses were produced by 50 µs stimulation of a monopolar stimulating electrode placed in layer V near the soma of the recorded pyramidal cell in the presence of either 2 mM kynurenate (IPSPs, n = 3) or 10 µM bicuculline (EPSPs, n = 3). The removal of Ca2+ effectively, and reversibly, eliminated evoked responses in all six cells (data not shown).
Under Ca2+-free conditions, DA had the same
effect on pyramidal cell excitability as under control conditions (Fig.
4). In the absence of
Ca2+, DA significantly depressed action potential
generation by 68 ± 12% (Fig. 4, A and B;
df = 5, t = 5.6, P < 0.05) and
reduced RN by 11 ± 3% (Fig.
4C; baseline RN = 72 ± 14 M; df = 5, t = 4.7, P < 0.05). Additionally, DA depolarized these cells 1.5 ± 0.6 mV
(baseline Vm =
68 ± 1 mV;
df = 5, t = 2.5, P < 0.05). This
change in Vm, like the effects on
spiking and RN, was not significantly
different from changes seen in control cells treated with DA in normal
ACSF. These results support the hypothesis that DA inhibits spike
generation in pyramidal cells by enhancing TTX-insensitive GABAergic
synaptic inhibition while at the same time ruling out dopaminergic
modulation of voltage-gated Ca2+ channels.
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Exogenous GABA mimics the effect of dopamine on spike inhibition
The data presented in the preceding text point to a GABAergic
mechanism for DA-dependent depression of spike generation that is, at
least in part, independent of its modulation of
RN. Anatomical studies in primate PFC
(Sesack et al. 1998) suggest DA could preferentially increase GABA release near spike-generating sites in the axon. If this
is the case, exogenous GABA introduced near the axon should mimic the
inhibitory effects of DA on action potential generation while having
little or no effect on somatic RN. To
test this hypothesis, we performed experiments in which GABA (2 µM)
was pressure-ejected (10 to 30 s duration) to a location where we would
expect to affect the pyramidal neuron's axon initial segment (Fig.
5A) (Williams and
Stuart 2000
). In five cells, focal application of GABA
reversibly and repeatedly reduced the number of spikes produced by
depolarizing current injection. The mean change in spike number was
59 ± 5% (df = 4, t = 5.0, P < 0.05), an amount not significantly different from
the inhibition produced by bath-applied DA. More importantly, the
decrease in spike generation produced by focal GABA application was not
accompanied by a decrease in pyramidal cell
RN (Fig. 5, B-E),
suggesting that the effect of GABA was indeed localized and distal from
the soma or dendrites. The mean change in
RN measured from the voltage responses
to single hyperpolarizing current steps, was only
5 ± 2% (mean
baseline RN = 98 ± 8 M
;
df = 4, t = 2.1, P > 0.05).
Additionally, GABA application did not significantly impact pyramidal
cell membrane potentials (mean baseline
Vm =
71 ± 1 mV; mean
change = +0.7 ± 0.4 mV).
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If DA acts to reduce action potential generation by enhancing GABA
release at perisomatic locations, localized disinhibition should
prevent dopaminergic effects on spike generation. We first examined the
effect of focal bicuculline application (20 µM) into the same basal
region as described in the preceding text, under control conditions
(Fig. 6A). In four cells,
focally applied bicuculline produced modest facilitation of spike
generation (mean increase was only 1.1 ± 0.8 spikes). Next, as
observed previously, for seven pyramidal neurons exposed to 10 µM DA,
there was a 69 ± 8% decrease in spikes, a 15 ± 4%
decrease in RN (baseline
RN = 71 ± 13 M), and a
2.3 ± 0.3 mV depolarization of
Vm (baseline Vm =
72 ± 1 mV). With DA in
the bath, focally applied bicuculline (10-30 s) partially reversed the
effects of DA on spike generation, but not
RN (Fig. 6, B-D). The
number of spikes generated with bicuculline present was significantly
increased by 3.9 ± 0.7 spikes (Fig. 6C; df = 6, t = 5.5, P < 0.05), returning the
number of action potentials generated to within 10 ± 21% of
baseline values. Bicuculline's effect on spike number in the presence
of DA was significantly greater than under control conditions (df = 9, t = 2.6, P < 0.05).
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In contrast, in the presence of DA, focal application of bicuculline
did not significantly affect RN.
During bicuculline application, RN
remained depressed by 10 ± 4% from control values, an amount not
significantly different from found under control or disinhibited conditions (see Figs. 1F and 2D). Additionally,
focal bicuculline application did not significantly affect pyramidal
cell Vm (mean change in
Vm during bicuculline puff = 0.2 ± 0.2 mV). These data are consistent with the hypothesis
that DA inhibits pyramidal neuron excitability via modulation of
GABAergic synaptic transmission, that this modulation is localized to
the spike generating zone, and that DA's inhibitory effect on action
potential generation is, in part, independent of its effects on
RN and
Vm.
TTX blocks the inhibitory effect of dopamine on RN
Our observation that DA still depressed
RN under disinhibited conditions
suggests that its effect on membrane conductance is independent of a
GABAergic mechanism. A likely candidate mechanism for DA's inhibitory
actions is through modulation of voltage-gated Na+ channels. When we examined spike threshold,
the second derivative of the spike rising phase, action potential
amplitude, and spike half-width, no significant effects of DA (or DA
washout) were observed in any of the preceding experimental conditions
(data not shown). However, it is possible that DA might modulate
subthreshold states of Na+ channels and/or a
persistent Na+ conductance, while not affecting
fast Na+ currents underlying spike kinetics
(Ahern et al. 2000; Cepeda et al. 1995
;
Hammarström and Gage 1999
).
In cortical pyramidal cells, voltage responses can be amplified in both
the depolarizing and hyperpolarizing directions, respectively, by the
activation and deactivation of inward Na+
currents active near rest (Cepeda et al. 1995;
French et al. 1990
; Schwindt and Crill
1995
; Stuart 1999
). Depression of this current
by DA would decrease steady-state voltage responses, resulting in
reduction of the measured RN. To test
this hypothesis, 10 µM DA was bath-applied to 15 cells in the
presence of 1 µM TTX (Fig. 7). Under
these conditions, DA failed to reduce pyramidal cell RN (baseline
RN = 83 ± 9 M
; mean
change = +1 ± 3%). This lack of effect in the presence of
TTX was significantly different from changes in
RN seen in control cells treated with
DA alone (Fig. 7D; df = 21, t = 4.6, P < 0.05). TTX did not occlude the DA-induced depolarization of Vm (baseline
Vm =
71 ± 1 mV; mean
change = +1.1 ± 0.5 mV; df = 14, t = 2.3, P < 0.05). Changes in
Vm with DA application in the presence
of TTX were not significantly different from the changes in
Vm observed with DA alone.
|
If DA decreases RN by inhibiting a
resting Na+ conductance, it follows that the
application of TTX should mimic the effect of DA on
RN and that TTX application itself
should hyperpolarize pyramidal cells. To test these predictions, in
eight cells RN was measured before and
after bath application of 1 µM TTX under conditions in which fast
synaptic transmission was blocked with bicuculline (20 µM) and
kynurenate (2 mM; Fig. 8). As was found for cells treated with DA alone, TTX application produced a decrease in
the voltage-current relationship in both the depolarizing and hyperpolarizing directions (Fig. 8B), resulting in a 17 ± 3% decrease in RN (mean baseline
RN = 125 ± 26 M; df = 7, t = 3.8, P < 0.05). The decrease
in RN seen with TTX application was
similar in magnitude to the effect seen in control cells treated with
DA alone (Fig. 8D). Bath application of TTX had no effect on
pyramidal cell Vm (baseline
Vm =
73 ± 1 mV; mean
change = +0.1 ± 0.6 mV). Therefore the effect of TTX on
RN, but not
Vm (see DISCUSSION), is
consistent with the reduction of a Na+
conductance.
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DISCUSSION |
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The major conclusions of the present study are that the acute presence of DA depresses action potential generation primarily via an increase in GABAergic inhibition, DA reduces RN by modulating a TTX-sensitive conductance independent of GABA receptor activation, and DA triggers a delayed increase in excitability that is, in part, masked by synaptic inhibition. Taken together, these data indicate that the overall effect of DA on pyramidal cell excitability may depend on phasic changes in DA levels and the tone of GABAergic inhibition.
Dopamine and action potential generation
Three lines of evidence presented here indicate that the inhibitory effect of DA on spike generation in layer V pyramidal neurons is mediated, in part, by an increase in GABAergic synaptic transmission. First, blockade of GABAA receptors occludes the inhibitory effect of DA on spike generation. Second, the inhibitory effect of DA on spike generation is mimicked by focal GABA application to sites near the spike-generating zone. Third, DA increases the frequencies of spontaneous and miniature IPSCs in layer V pyramidal cells. The GABAA-independent effect of DA on RN suggests a second mechanism for modulating membrane excitability (discussed in the following text).
In this study, we employed bicuculline methiodide to block
GABAA receptors. Because bicuculline blocks
channels mediating the slow afterhyperpolarization (sAHP)
(Khawaled et al. 1999), it is important to note that our
data are not consistent with an alternative hypothesis where DA
enhances the sAHP to decrease excitability. To our knowledge,
subcortical modulators such as DA typically depress the sAHP
(Malenka and Nicoll 1986
), leading to enhanced
excitability. Moreover involvement of a sAHP was ruled out by the fact
that DA increased the latency to the first spike produced by
depolarizing current injection. Additionally, we found that DA, in many
cases, depressed firing down to one or no spikes (see Fig. 1). A sAHP
strong enough to depress subsequent spikes is not likely to be
generated by a single action potential, and no indications of a sAHP
were evident following the limited number of spikes generated under
bicuculline-free conditions.
Our finding that bicuculline blocks DA-mediated spike inhibition in
vitro supports earlier in vivo results demonstrating bicuculline sensitivity of DA-induced inhibition of spontaneous firing of prefrontal neurons (Pirot et al. 1992). Additionally,
the dopaminergic enhancement of spontaneous IPSCs presented here is
similar to previous findings that DA can increase extracellular GABA
levels in PFC (Bourdelais and Deutch 1994
; Del
Arco and Mora 2000
; Grobin and Deutch 1998
;
Retaux et al. 1991
) and enhance IPSC frequency onto both
pyramidal and nonpyramidal cells (Zhou and Hablitz
1999
). We should note that in our experiments DA only had a
modest effect on IPSC frequency (~20% increase), while Zhou
and Hablitz (1999)
found DA to produce large increases in both
the frequency and amplitude of spontaneous IPSCs (~250 and ~155%,
respectively). Disparity between the results of these studies may
reflect differences in prefrontal region (anterior cingulate versus
prelimbic PFC) and cell type (layer I and III pyramidal and
nonpyramidal neurons versus layer V pyramidal cells). Finally, DA has
been shown to directly enhance the excitability of prefrontal
nonpyramidal neurons (Zhou and Hablitz 1999
), and
dopaminergic modulation of inhibitory neuron excitability is fully
consistent with a GABAergic mechanism for spike inhibition. However,
the observations that DA increases mIPSC frequency in the presence of
TTX and still depresses spike generation in
Ca2+-free ACSF suggest that increases in
nonpyramidal cell firing rates may not be required.
If DA modulates spike-independent GABA release, wouldn't a change in
RN be expected in the presence of TTX?
Two sets of experiments suggest that DA-induced modulation of GABA
release may have minimal impact on somatic
RN. First, mIPSC frequencies were only
modestly enhanced by DA (<30% increase) and IPSCs are only one of
many conductances contributing to the total membrane conductance.
Second, even direct application of exogenous GABA, at concentrations
sufficient to reduce spike generation, did not significantly depress
RN (see Fig. 5). This experiment
demonstrates that modulation of excitability at a site distant from the
soma, such as the initial node (Williams and Stuart
2000), can have little, if any, effect on total input conductance. This explanation is consistent with anatomical data demonstrating that some GABAergic neurons form axoaxonic synapses onto
pyramidal cells (Anderson et al. 1995
; Kawaguchi
and Kubota 1998
; Williams et al. 1992
) and that
GABAergic neurons are innervated by dopaminergic fibers (Benes
et al. 1993
; Sesack et al. 1995a
,b
, 1998
;
Smiley and Goldman-Rakic 1993
; Verney et al.
1990
).
Effects of dopamine on TTX-sensitive conductance
Dopaminergic reduction of RN was
not affected by GABAA antagonists but was
occluded after blockade of Na+ channels with TTX.
There are two possible explanations for the TTX sensitivity of
dopaminergic modulation of RN: DA
depresses the constitutive, TTX-sensitive release of another
neuromodulator that normally increases pyramidal cell
RN, or the acute presence of DA,
inhibits a resting Na+ current in layer V
pyramidal cells. How might the closing of a Na+
channel lead to an increase in conductance? Cortical pyramidal cells
have resting voltage-gated Na+ currents that
amplify both depolarizing and hyperpolarizing membrane responses
(French et al. 1990; Schwindt and Crill
1995
; Stuart 1999
). A reduction in this current
by DA would appear as a decrease in the steady-state voltage response
and be interpreted as a decrease in
RN.
If DA does reduce a resting Na+ conductance, why
doesn't DA, or the application of TTX itself, hyperpolarize layer V
pyramidal cells? Fig. 1H suggests that DA modulates
RN and
Vm independently. Additionally,
DA-induced depolarization of pyramidal neurons may mask any
hyperpolarizing effect of Na+ channel closure.
This depolarization, produced by DA, may not involve activation of DA
receptors but may result from nonspecific effects of DA on other
receptor types within the PFC (Shi et al. 1997).
DA-induced depolarization, however, does not explain why the
application of TTX alone does not hyperpolarize prefrontal neurons. It
is possible that TTX application modulates the relative endogenous
concentrations of DA and other neuromodulators that influence pyramidal
cell resting potentials, as well as
RN. Additionally, one must consider
that reducing a resting Na+ conductance would
likely decrease resting intracellular Na+
concentrations. In turn, a number of factors, including depolarization of the equilibrium potential for Na+, decreasing
the electrogenic contribution the
Na+/K+ ATPase, or reducing
outward current generated by Na+-activated
K+ currents (Schwindt et al.
1989
), could all compensate for small changes in
Vm produced by depressing the inward current.
Although the relationship between DA, TTX, and pyramidal cell
Vm is as yet unresolved, there is
substantial evidence linking DA with Na+ channel
regulation. Generally, DA has an inhibitory influence on
Na+ currents. In both striatal (Calabresi
et al. 1987; Cepeda et al. 1995
;
Schiffmann et al. 1998
), and hippocampal
(Cantrell et al. 1997
, 1999
) neurons, DA depresses
Na+ currents. While DA has purely inhibitory
effects on Na+ conductances in other cell types,
there is not yet consensus on the effect of DA on
Na+ currents in prefrontal cells. Our results are
most consistent with the observations of Geijo-Barrientos and
Pastore (1995)
and Geijo-Barrientos (1999)
, who
report DA-induced decreases of subthreshold Na+
conductances (see also Maurice et al. 2000
). In
contrast, the work of Yang and colleagues indicates that DA activates a
delayed increase in a persistent Na+ current
(Gorlova and Yang 2000
; Yang and Seamans
1996
). The most unifying hypothesis is that DA has a biphasic
effect on subthreshold Na+ conductances.
Differential experimental conditions, including whether excitability is
assessed in the acute presence of DA or after exposure to DA, or
variations in the level of synaptic inhibition, may dictate whether the
excitatory or inhibitory effects of DA are observed.
Dopamine and working memory
The data presented here point to at least three distinct
mechanisms for dopaminergic modulation of prefrontal pyramidal cells. First, DA inhibits action potential generation by enhancing spontaneous inhibitory synaptic input. Second, DA decreases
RN via reduction of a TTX-sensitive
conductance. Both of these inhibitory effects are expressed during
acute exposure to DA. Third, DA triggers a delayed and prolonged
enhancement of excitability, as has been previously reported
(Gorelova and Yang 2000; Henze et al.
2000
; Yang and Seamans 1996
). This increased
excitability may be related to the "rebound" excitation we
previously found when inhibition was intact (Gulledge and Jaffe
1998
). It is possible that prolonged increases in excitability,
observed only under disinhibited conditions, are normally masked by the
persistent increase in TTX-insensitive IPSCs reported here.
One hypothesis for working memory involves the formation of
reverberatory circuits that actively maintain information for a short
period of time (Amit 1995). Indeed, a well-established feature of prefrontal neurons is enhanced output during specific phases
of working memory tasks (Funahashi et al. 1989
;
Fuster and Alexander 1971
; Kubota and Niki
1971
). While computational models suggest that transient
changes in either membrane excitability or synaptic connectivity
(Camperi and Wang 1998
; Durstewitz et al.
1999
; Lisman et al. 1998
; Wang
1999
) may maintain sustained activity, a level of synaptic
inhibition is required to constrain firing to only those neurons
providing behaviorally significant output (Camperi and Wang
1999
). The acute presence of DA, through its inhibitory actions
on TTX-sensitive conductances and enhancement of GABAergic
transmission, may serve to gate appropriate firing by allowing output
only from those neurons receiving the strongest excitatory drive.
Delayed increases in excitability may regulate tonic levels of
excitability. In this way, DA release during working-memory tasks may
not only set the stage for reverberatory activity, but may also
constrain such heightened activity to behaviorally relevant neurons.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute of General Medical Sciences Grant GM-0P194-1751.
Present address of A. T. Gulledge: Div. of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200, Australia.
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FOOTNOTES |
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Address for reprint requests: D. B. Jaffe, Div. of Life Sciences, University of Texas at San Antonio, 6900 North Loop 1604 West, San Antonio, TX 78249 (E-mail: djaffe{at}utsa.edu).
Received 29 December 2000; accepted in final form 11 April 2001.
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REFERENCES |
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