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The Journal of Neuroscience, April 1, 2003, 23(7):2686
Dopamine D1-Class Receptors Selectively Modulate a Slowly
Inactivating Potassium Current in Rat Medial Prefrontal Cortex
Pyramidal Neurons
Yan
Dong1 and
Francis
J.
White2
Departments of 1 Neuroscience and
2 Cellular and Molecular Pharmacology, Finch University of
Health Sciences/The Chicago Medical School, North Chicago, Illinois
60064
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ABSTRACT |
The dopamine (DA) innervation of medial prefrontal cortex (mPFC)
regulates cognitive activity in a complex manner. Alterations of DA
function, particularly via the DA D1 receptor class (D1R), are
implicated in both schizophrenia and drug addiction, yet the precise
roles of DA in modulating mPFC excitability remain unclear. We focused
on DA modulation of voltage-gated K+ current (VGKC)
in acutely dissociated rat mPFC pyramidal neurons. We defined three
components of the whole-cell VGKC according to biophysical and
pharmacological properties. The A-type current (IA), with rapid activation and
inactivation kinetics, was completely inactivated by prolonged holding
of the membrane potential at 
40 mV and was sensitive to the
K+ channel blocker 4-aminopyridine (4-AP) but not
tetraethylammonium (TEA) or dendrotoxin (DTX). The slowly inactivating
K+ current (ID),
with rapid activation but relatively slow inactivation, was the major
contributor to VGKC and was completely inactivated at 40 mV and
sensitive to TEA and DTX but less so to 4-AP. The very slowly
inactivating K+ current
(IK) was elicited by command steps to
more depolarized potentials from a prolonged holding potential of 40
mV and was sensitive to all three blockers. Stimulation of DA D2
receptors failed to alter any component of whole-cell VGKC. Stimulation
of DA D1Rs selectively suppressed
ID, an effect mimicked by the adenylyl
cyclase activator forskolin, the active cAMP analog Sp-cAMP, and the
protein phosphatase inhibitor okadaic acid. Inhibition of protein
kinase A (PKA) with either PKI or Rp-cAMP abolished D1R
modulation. Thus, the DA D1R/cAMP/PKA signaling pathway mediates
modulation of ID by DA in rat mPFC
pyramidal neurons.
Key words:
dopamine; prefrontal cortex; potassium current; dopamine D1 receptors; drug addiction; schizophrenia
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Introduction |
The prefrontal cortex (PFC) is a
major association area connected to all areas of neocortex and to
various allocortical, limbic, and other regions. This provides the PFC
the ability to prioritize and reference stimuli to internal
representations, direct attention, and monitor temporal event
sequencing (Fuster, 2001
; Miller and Cohen, 2001). Dopamine (DA) is
essential for proper information processing in the PFC (Goldman-Rakic
et al., 2000). In particular, an optimal level of DA D1-class receptor
(D1R) stimulation is required. When PFC D1R stimulation is either
significantly reduced or augmented, disruption of working memory
results (Sawaguchi and Goldman-Rakic, 1991; Williams and Goldman-Rakic,
1995; Zahrt et al., 1997; Seamans et al., 1998).
The mesocortical DA projection arises primarily from the ventral
tegmental area (VTA) and terminates mainly on pyramidal neurons in deep
layers V and VI of medial PFC (mPFC) (Bjorklund et al., 1978; Emson and
Koob, 1978; Berger et al., 1991; Carr et al., 1999). Many of these
neurons project to the VTA and nucleus accumbens, providing the means
by which the PFC can modulate the mesoaccumbens DA system (Sesack and
Pickel, 1992; Carr and Sesack, 2000). Alterations of DA function within
these systems have been implicated in various neuronal disorders,
particularly schizophrenia (for review, see Lewis, 1995; Knable and
Weinberger, 1997) and drug addiction (for review, see Vanderschuren and
Kalivas, 2000; Tzschentke, 2001).
Despite clear evidence that DA modulates behavioral output from the
PFC, specific knowledge of the mechanisms by which DA alters the
activity of PFC pyramidal neurons remains limited. Indeed, the
electrophysiological actions of DA within the PFC have long been an
enigma (for review, see Yang et al., 1999). Recent current- and
voltage-clamp recordings have begun to provide clues about the targets
of DA modulation. DA was reported to decrease persistent
Na+ current in mPFC pyramidal neurons
(Geijo-Barrientos and Pastore, 1995), although others observed the
opposite effect (Yang and Seamans, 1996; Gorelova and Yang, 1997) or no
modulation (Maurice et al., 2001). DA D1R stimulation was recently
shown to suppress the transient Na+
current in acutely dissociated rat mPFC neurons (Maurice et al., 2001)
and to remove outward rectification in PFC neurons recorded in brain
slices (Yang and Seamans, 1996; Gorelova and Yang, 1997). A decrease in
excitability of PFC neurons has recently been attributed to DA D2-class
receptor (D2R) stimulation (Gulledge and Jaffe, 1998).
Here we focused on DA receptor modulation of voltage-gated
K+ currents (VGKCs) in acutely dissociated
rat mPFC pyramidal neurons. VGKCs are responsible for setting the
resting potential, repolarizing and hyperpolarizing the cell, and
shaping voltage trajectories in the subthreshold voltage range for
action potentials (Hille, 2001). As such, they may provide a specific
target by which DA may regulate cognitive function.
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Materials and Methods |
Acute dissociation. Pyramidal neurons from mPFC
layers V and VI were acutely dissociated from brain slices obtained
from 4- to 5-week-old rats. Rats were anesthetized with methoxyflurane (Mallinckrodt Veterinary Incorporated, Mundelein, IL) and
decapitated. Brains were quickly removed, blocked, and sliced on a DSK
microslicer (Campden Instrument) in a 1-2°C sucrose
solution containing (in mM): 234 sucrose, 2.5 KCl, 1 Na2HPO4, 11 glucose,
4 MgSO4, 0.1 CaCl2, and 15 HEPES, pH 7.3, 300 mOsm/l. Coronal slices (400 µm) were incubated for
1-4 hr at room temperature in a sodium bicarbonate-buffered Earle's
balanced salt solution bubbled with 95%O2/5%
CO2 and containing (in mM):
1 kynurenic acid, 1 pyruvic acid, 0.1 N-nitroarginine, and
0.005 glutathione, pH 7.4, 300 mOsm/l. Individual slices were placed in
a Ca2+-free buffer containing (in
mM): 140 Na-isethionate, 2 KCl, 4 MgCl2, 23 glucose, and 15 HEPES, pH 7.4, 300 mOsm/l, and the mPFC was isolated under a dissecting microscope. The
mPFC tissue was then placed into an oxygenated, HEPES-buffered HBSS
containing 1.5 mg/ml protease (type XIV) at 35°C for 30 min. The
enzyme chamber also contained (in mM): 1 kynurenic acid, 1 pyruvic acid, 0.1 N-nitroarginine, and
0.005 glutathione, pH 7.4, 300 mOsm/l.
After enzymatic treatment, the tissue was rinsed several times in
Ca2+-free buffer and triturated with a
graded series of fire-polished Pasteur pipettes. The cell suspension
was placed in a 35 mm Lux Petri dish (Nunc, Naperville,
IL), which was mounted on an inverted microscope. Cells were then given
several minutes to settle before recording.
Whole-cell recordings. Whole-cell recordings were performed
using standard techniques. Electrodes were pulled from
Corning (Corning, NY) 7052 glass (Flaming/Brown P-97
puller; Sutter Instrument Co., Novato, CA) and
fire-polished (MF-83 microforge; Narishige, Hempstead, NY)
just before use. The intracellular recording solution for recording
outward K+ currents contained (in
mM): 60 K2SO4, 60 N-methyl-glucamine, 10 HEPES, 5 BAPTA, 12 phosphocreatine, 3 MgATP, 0.2 Na3GTP, 2 MgCl2,
and 0.5 CaCl2, pH 7.2, 275 mOsm/l. The normal
extracellular recording solution contained (in
mM): 140 Na-isethionate, 10 HEPES, 12 glucose,
17.5 sucrose, 1-4 KCl, 4 MgCl2, 0.01 TTX, pH
7.35, 300 mOsm/l. All reagents were obtained from
Sigma (St. Louis, MO) except ATP and GTP
(Boehringer Mannheim, Indianapolis, IN); BAPTA,
Sp-8-Brom-cAMP, Rp-cAMP, H8, active and inactive forskolin, and
okadaic acid (OA) were from Calbiochem (La Jolla, CA).
Extracellular recording solutions were applied via one of a series of
four glass capillaries (~250 µm inner diameter) in which
gravity-fed flow was regulated by electronic valves (Bio-logic).
Recordings were obtained with an Axon Instruments (Foster
City, CA) 200A patch-clamp amplifier and controlled and monitored with
a Pentium PC running pCLAMP (version 8.0) with a 125 kHz interface
(Axon Instruments). Electrode resistances were ~1-4
M
in bath. After formation of the gigaohm seal and subsequent cell
rupture, series resistance was compensated (70-80%) and periodically
monitored. Recordings were restricted to neurons with pyramidal soma
and small remnants of the apical dendrites. The neuron was not included
in analysis unless its whole-cell capacitance was between 8 and 14 pF
and its series resistance was below 8 M and steady (<10%
oscillation). Recordings were performed at room temperature
(22-24°C).
Data analysis. Decay time constants were determined by
fitting current recording with single, double, or triple exponential functions of the form I = A0 + A1
exp(t/
1) + A2
exp(t/2) + A3
exp(t/3).
A0 to
A3 are amplitude coefficients.
1 to 3 refer to time
constants. Dose-response data were fit with a Langmuir isotherm of the
form C/(C + IC50), where
C is the concentration of blockers. A two-site model of
pharmacological blockade was fit with
I/Imax = A/[1 + EXP(LOG(C)/B)] + (1 A)/[1 + EXP(C)/D], where
C is the concentration of blockers, and B and
D are two IC50 values. Recovery of
VGKC was fit with I/Imax = A × [1 EXP(t/1)] + (1 A)/[1 EXP(t/2)], in which I
is the current, t is time, and 1
and 2 are the time constants of recovery from
inactivation. Statistica (Statsoft, Tulsa, OK) was used
for statistical analysis. Origin (Microcal Northampton,
MA) was used to plot the current traces and graphs. Box-whisker was
used to plot most of the graph presentation because of the small sample
size. The box plot presented the distribution as a box, with the median
as a central line and the hinges as the edge of the box, which divided
the top and bottom halves of the distribution in two. The inner fence,
started from the edge of the box, runs to the limits of the
distribution, excluding outliers, which are defined as points that are
two times the interquartile range beyond the inner fence. The
outliers are shown as open circles. The averaged data are typically
shown as mean ± SE.
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Results |
Three components of VGKC in mPFC pyramidal neurons
Whole-cell VGKC was elicited in mPFC neurons using multiple
protocols. In a protocol with short depolarization duration, the membrane potential was held at 70 mV. A hyperpolarization prepulse (2 sec) to 100 mV was used to deinactivate inactivated
K+ channels that could not be recovered by
holding at 70 mV. The following 100 msec depolarization steps (from
100 to + 30 mV with 10 mV increments) elicited the whole-cell VGKC
(Fig. 1A). This
protocol elicited similar traces in most of the recorded neurons
(n = 16 of 20) in which whole-cell current showed a
rapid activation and slow inactivation. A fast inactivating component was observed in 20% of the recorded neurons (n = 4 of
20) (Fig. 2A), as
discussed later. Depolarization steps inactivated whole-cell currents
as shown in Figure 1B (n = 6). The
membrane potential was held at 70 mV. The current that was elicited
by a test step to +30 mV showed a voltage-dependent inactivation after
the 600 msec prepulse (stepped from 100 to +30 mV with 10 mV
increments). We chose this short prepulse to generate a
pseudo-steady-state inactivation instead of true steady-state
inactivation for technical convenience. The peak current-voltage
(I-V) curve is summarized in Figure
1C. Whole-cell VGKC in this neuron began to activate at 60
mV, suggesting that VGKC may be activated at physiological resting
membrane potentials (70 to 50 mV). A different set of protocols
(Fig. 1D,E) was used to isolate
individual components of VGKC by taking advantage of their inactivating
kinetics. The membrane potential was held at 70 mV. The first
protocol started with a hyperpolarization prepulse (4 sec) to 100 mV,
which deinactivated most inactivated K+
channels. The following 4 sec test steps from 70 to +30 mV (at 10 mV
increments) elicited the whole-cell current (n = 5)
(Fig. 1D). The second protocol started with a
depolarization prepulse (10-20 sec, 40 mV), which inactivated
components of the current with relatively short inactivation kinetics
(several seconds). The following test steps (from 70 to +30 mV at 10 mV increments) elicited relatively small currents with little
inactivation (n = 5) (Fig. 1E). We
operationally defined these noninactivating traces as
IK. This current did not change when
extracellular Cl
was replaced by
gluconate (n = 4; data not shown), excluding involvement of Cl channels. The slowly
inactivating current was obtained (Fig. 1F) after
subtraction of IK from the whole-cell
currents shown in Figure 1D. This current activated
rapidly and inactivated slowly and was operationally termed
ID. The I-V relationship of
IK and ID are
plotted in Figure 1G. Both currents begin to activate at approximately 40 mV (n = 5 for each group).
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Figure 1.
Whole-cell VGKCs in acutely dissociated mPFC
pyramidal neurons. A, After holding the membrane
potential at 70 mV and administering a 2 sec prepulse to 100 mV,
test protocols from 100 to 30 mV with 10 mV increments elicited the
whole-cell currents (n = 16 of 20).
B, Whole-cell VGKCs could be partially inactivated by a
depolarizing prepulse. After a holding potential at 70 mV and a 600 msec prepulse from 100 to 30 mV (10 mV increments), 100 msec test
steps to 30 mV elicited currents showing a (pseudo) steady-state
inactivation (n = 6). C, Current was
measured at the time point indicated as an open or
filled circle in A and B.
The activation and inactivation observed in A and
B were plotted as I-V curves.
D, Whole-cell VGKC was elicited by a similar activation
protocol but with longer time course (4 sec) (n = 5). E, After the holding potential at 40 mV, the same
test steps as in D elicited currents showing little
inactivation. This current is operationally termed
IK (n = 5).
F, A slowly inactivating current was obtained by
subtraction of traces in E from D. This
slowly inactivating component is operationally termed
ID. G,
I-V curves of IK and
ID indicate that both currents begin
to activate at approximately 40 mV (n = 5).
Currents were measured at the time points indicated by
open or filled circles in
E and F.
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Figure 2.
A-type K+ component of
whole-cell VGKC. A, A rapid inactivating component could
be observed in a small population of neurons (n = 4 of 20) using the protocol described in Figure 1A.
The rapidly inactivating component became more obvious at more
depolarized steps. Because both activation and inactivation of this
component are rapid and consistent with A-type current, it is
operationally called IA.
B, Activation curves for the early and later components
are plotted with the values measured at the time points indicated by
filled and open circles, respectively.
C1 and C2 show the activation of the same
neuron before and after TEA perfusion, respectively. In most neurons,
whole-cell VGKC activation protocols alone failed to show obvious
IA. However, after perfusion with 10 mM TEA, IA was
revealed (n = 4). D, Recovery
protocol shows that IA deinactivates
more rapidly than ID. After the
holding potential at 70 mV, a prepulse to 40 mV inactivated most of
the VGKC. A second hyperpolarizing prepulse to 100 mV recovered the
K+ channels from the inactive state. The time course
for this hyperpolarization prepulse varied from 10 msec to 1 sec with a
nonlinear increment. IA could be
recovered with a brief hyperpolarization step but contributes <25% of
the whole-cell current (n = 4). E,
The recovery time and the current amplitudes (measured at the time
points indicated with open and filled
circles in D) were plotted. The relationship of
the early component (IA
+ ID) could be fit with
two exponentials (1 = 18 msec;
2 = 890 msec). The relationship of the late
component could be fit with one exponential ( = 900 msec)
(n = 5). From F1 to
F4, IA was isolated
biophysically (n = 4). F1, A neuron
with obvious IA component was
selected. By running a regular protocol, the whole-cell VGKC was
elicited. This current contained
IA,
ID, and IK.
F2, The same testing step as in F1 after
a 200 msec depolarization prepulse elicited a K+
trace. Because the prepulse had inactivated
IA, the following trace contained only
ID and
IK. F3, The
IA component was isolated after
subtraction of the trace in F2 from F2.
The trace was shown in F4 in a higher
magnification.
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In 20% of the recorded neurons (n = 4 of 20), the
protocol shown in Figure 1A elicited a whole-cell
current with a rapid (several milliseconds) inactivating component
(Fig. 2A). Because the kinetics of this rapidly
inactivating component are similar to that of A-type currents
identified previously in cortical pyramidal neurons (Foehring and
Surmeier, 1993), we operationally defined it as the A-current or
IA. When IA
is present, it contributes only to the initiation of whole-cell VGKC
because it inactivates completely within the first several 10 msec. The I-V curve of both early and late
components measured from the same neuron is plotted in Figure
2B. Here, both components of the current begin to
activate at 40 mV. In general, the whole-cell VGKC in mPFC neurons
begins to activate at 42 ± 3 mV (n = 15). A
lack of obvious IA in most recorded
neurons could be explained if IA is too
small and is totally obscured by ID. To
test this hypothesis, we applied 1 mM TEA, which
is an effective blocker of non-A-type K+
current, to neurons that showed no obvious
IA in whole-cell VGKC (Figs.
1A, 2C1). The hidden
IA was revealed (Fig. 2C2) in
this neuron after ID was inhibited by TEA
perfusion. A similar effect was observed in three other neurons.
Obviously the limits of pharmacological isolation of
K+ currents must be noted. Low
concentrations of TEA (1 mM) blocked only ~50%
of ID, whereas higher concentrations (50 mM) of TEA also blocked
IA (see Fig. 4A).
Biophysical isolation of IA and
ID
To characterize further IA in mPFC
neurons, biophysical methods were used to isolate
IA and ID.
Deinactivation by hyperpolarization was shown by a recovery protocol in
which the membrane potential was held at 70 mV. A 4 sec
depolarization step to 40 mV inactivated both
IA and ID.
The membrane potential was then stepped to the recovery voltage (100
mV) for varying durations (5, 10, 20, 50, 100, 200, 500, 1000 msec). A
test step to +30 mV elicited the whole-cell VGKC (Fig.
2D). IA was recovered
from inactivation with relatively short hyperpolarization steps and
contributed only slightly to the whole-cell VGKC. Recovery of
ID required relatively long
hyperpolarization steps. After full recovery, ID totally obscured
IA. The currents were measured from 10 msec after onset of the current, at which
IA reached its peak, as well as from 400 msec after onset current, at which IA was
completely inactivated. The relationships between these two currents
and their recovery time are plotted in Figure 2E. The
early current from this cell was well fit with two exponentials with
time constants of 18 and 890 msec, respectively. The late current was
well fit with one exponential with a time constant of 900 msec,
consistent with the 890 msec time constant during the early current.
The averaged time constants are 39 ± 10 msec (n = 5) and 925 ± 207 msec (n = 5). Clearly,
IA and ID
could be distinguished by different recovery kinetics.
Another protocol was also used to isolate
IA from whole-cell VGKC by taking
advantage of the rapid inactivation of IA
(Fig. 2E). In the first part of this protocol, the
test step to +30 mV after prolonged holding at 100 mV elicited
whole-cell VGKC, which includes IA,
ID, and IK
(Fig. 2F1). In the second part of this protocol, a
200 msec prepulse to 30 mV fully inactivated IA but was short enough to maintain most
of ID and IK
because of their slow inactivation. So the following test step from
70 to +30 mV elicited a current including only
ID and IK
(Fig. 2F2). Subtraction of whole-cell
K+ current composed of
ID and IK
(Fig. 2F2) from the whole-cell K+ current composed of
IA, ID, and
IK (Fig. 2F1) resulted
in pure IA (n = 4) (Figs.
2F3, 4).
We have described three components of the whole-cell VGKC,
IA, ID, and
IK, in mPFC neurons, similar to that
described in sensorimotor cortex pyramidal neurons (Foehring and
Surmeier, 1993). Another way to distinguish different components in a
combined current is to analyze current decay (Hoshi et al., 1990). An
outward current trace was elicited by a 200 msec step to +30 mV from a
holding potential of 100 mV (Fig.
3A, inset). The
point at which VGKC starts to decay is defined at 10 msec after the
onset of peak current. The digitized current was taken from the 10 msec
point to total decay. Using this protocol, the decay could be fit by two exponentials with time constants () of 15 and 250 msec. The two
components became more obvious when the current was converted to
logarithmic values (Fig. 3A). The late current was
extrapolated and subtracted from the early current. The VGKC was peeled
into two linear exponentials in the logarithmic coordinates (Fig.
3A). Another protocol with similar activation steps but
longer duration (4 sec) was used to generate the decay current (Fig.
3B, inset). The decay was analyzed with a similar
peeling process, and two decay time constants were obtained (200 msec
and 2.9 sec). No faster could be obtained because the step duration
is too long to catch short inactivating ( = 15 msec)
components. The fast (200 msec) in Figure 3B is
consistent with the slow (250 msec) in Figure 3A, which
suggests that they are the same component. The less than perfect
consistency may be attributable to the heterogeneous expression of VGKC
but is more likely the result of different time courses used to
truncate the current. In Figure 3B, the decay of the
250 msec component occupies the entire time course of the analysis and
should be more accurate.
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Figure 3.
Multiple components of whole-cell VGKC revealed by
inactivation kinetics. A, A cell with obvious
IA was selected for this example.
Whole-cell VGKC was elicited during a 200 msec step to 30 mV from 100
mV, as shown in the inset. The current amplitude was
digitized and put in a LOG coordinate. The late part, but not the early
part, of the current displayed a linear relationship with a time
constant of 250 msec. This linear relationship was then extrapolated
forward and subtracted from the early part of the digitized current,
which resulted in another linear relationship with a time constant of
15 msec. This exponential peeling of decay within the time course
demonstrated two components (n = 7).
B, Whole-cell VGKC was elicited during a longer (4 sec)
step to 30 mV from 100 mV, as shown in the inset.
Exponential peeling of the components of decay of this trace revealed
two time constants of 200 msec and 2.9 sec (n = 6).
C, Whole-cell VGKC was elicited during a 40 sec step to
30 mV from 100 mV, as shown in the inset. Exponential
peeling of the components of decay of this trace revealed two time
constants of 3.4 and 25 sec (n = 3).
D, A protocol as shown in the inset was
used to elicit the K+ tail current. Exponential
fitting revealed two components in the tail current (using the 40 mV
step as the example) with time constants of 8 and 43 msec
(n = 4). Taken together, these data support four
distinct inactivation time constants, one consistent with
IA, one consistent with
IK, and two as distinct components of
ID.
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Another similar protocol but with a much longer step (40 sec) generated
a VGKC trace that was well fit with two exponentials ( = 3.4 and 25 sec, respectively). Again the 3.4 sec in Figure 3C is consistent with the 2.9 sec in Figure
3B. Taken together, there are four components according to
the time constants of current decay: 12 ± 3 msec
(n = 7), 220 ± 26 msec (n = 6),
3.4 ± 0.5 sec (n = 6), and 33 ± 7 sec
(n = 3). The strategy to isolate these four time
constants is to progressively increase the voltage step duration. When
the step was much longer than a time constant, the component to this
decayed too quickly to be taken into account in such a long step.
On the contrary, the decay of the component with matched that
predominated in this analysis can be calculated accurately. The rapidly
inactivating (12 ± 3 msec) component is consistent with
IA. The very slowly inactivating (33 ± 7 sec) component is consistent with IK.
We propose that there are two components in
ID, with decay of 220 ± 26 msec
and 3.4 ± 0.5 sec, respectively. The pseudo-steady-state
inactivation data (see Fig. 5D) indicate the likelihood that
there are at least two components in ID,
consistent with our hypothesis.
We next examined current decay with a tail current protocol (Fig.
3D). This experiment was performed in 40 mM external K+ and
Cl was replaced with methanesulfonate.
The membrane potential was held at 100 mV. The tail currents were
elicited by stepping to 70 mV after 10 msec voltage commands to steps
from 60 to +30 mV with 10 mV increments. In cells with
IA, the tail current was well fit by two
exponentials with time constants of 8 and 43 msec (Fig. 3D).
Similar 2- deactivation was also observed in three other neurons (4 and 19 msec; 9 and 28 msec; 7 and 33 msec), suggesting that there are
at least two components involved in the tail current. Note that it is
difficult to correlate the deactivation values with inactivation
values.
Pharmacological isolation of K+ currents
The biophysical studies above suggest that mPFC pyramidal
neurons express at least four components of the whole-cell VGKC. Are
they attributable to different channels or to similar channels with
different gating properties? The following pharmacological studies
attempted to address this question.
Three pharmacological agents, TEA, 4-AP, and DTX, were applied to
ID and IK
currents. IK was elicited by a 100 msec
step to +10 mV from a holding potential of 40 mV (Fig.
4A2,B2,C2).
A whole-cell VGKC was elicited by a similar protocol except that the
prepulse was 100 mV. ID was generated by
subtraction of IK from this whole-cell
current (Fig. 4A1,B1,C1).
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Figure 4.
Pharmacological analysis also revealed multiple
components of the whole-cell VGKC. A modified step to 10 mV elicited
whole-cell VGKC when the holding potential was 100 mV but elicited
IK when the holding potential was 40
mV. ID was obtained with subtraction
of IK from whole-cell VGKC.
A1 and A2 show that
ID and
IK were blocked by different
concentrations of TEA. A3, A4, The
dose-response curves for TEA blockade of both
IK (A3) and
ID (A4) are best
fit with two Langmuir isotherms, suggesting two TEA-sensitive
components of these currents. B1 and B2
show that ID and
IK are blocked by different
concentrations of 4-AP. B3, B4, The
dose-response curve for 4-AP block of
IK (B3) is best fit by
two Langmuir isotherms, suggesting at least two 4-AP-sensitive
components of this current, whereas ID
(B4) appears to possess only one 4-AP-sensitive
component. C1 and C2 show that
ID and
IK are blocked by different
concentrations of DTX. C3, C4, The
dose-response curves for DTX blockade of
IK (C3) and
ID are best fit by two Langmuir
isotherms, suggesting at least two DTX-sensitive components of these
currents. For all graphs, three to six cells are represented at each
data point.
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IA was TEA insensitive (Fig.
2C2), but IK and
ID were reversibly blocked by TEA across a
broad concentration range (Fig.
4A3,A4). The relationship between
relative current (I/Imax) of
IK and LOG[TEA] was well fit with a
two-site model (Langmuir isotherm) with IC50 values of 8 µM and 4 mM
(Fig. 4A3). The dose-response curve for ID was also well fit with a two-site model
with IC50 values of 25 µM
and 10 mM (Fig. 4A4).
IA in mPFC pyramidal neurons was sensitive to 4-AP and blocked by 60% at low (1-5 mM)
concentrations (data not shown). Both IK
and ID currents were also sensitive to
4-AP in our study (Fig. 4B3,B4).
The relationship between relative current and LOG [4-AP] for
IK was well fit with a two-site model with
IC50 values of 60 µM and
6 mM (Fig. 4B3). The effects of 4-AP on ID currents are relatively
limited. The maximal blockade was ~35% when 10 mM 4-AP was applied. The dose-response curve was
well fit with a single-site model with an IC50 of
30 µM (Fig. 4B4).
IA was insensitive to low (1-10
nM) concentrations of DTX, but both
ID and IK
were blocked by DTX across a broad concentration range. The
dose-response curve for IK was best fit
with a two-site model with IC50 values of 800 pM and 52 nM (Fig.
4C3). The does-response curve for
ID was well fit with a two-site model with
IC50 values of 200 pM and
40 nM (Fig. 4C4). The two
IC50 values usually suggest two binding sites in
the channels or two kinds of channels. The similar pharmacology of VGKC
(multiple binding sites or multiple channels) was reported previously
in sensorimotor cortical neurons (Foehring and Surmeier, 1993).
DA modulation of VGKC
The whole-cell VGKC is composed of three components,
IA, ID, and
IK, each of which display different
biophysical and pharmacological properties. In this section of the
study, we examined DA receptor modulation of each VGKC component. In
this set of simplified protocols, IK was
elicited when the membrane potential was stepped to +30 mV (2 sec) from
prolonged (>10 sec) holding at 40 mV (Fig.
5A). The current amplitude was
measured at the time point 200 msec after current initiation. To elicit
ID, the membrane potential was held at
100 mV. The prepulse (100 msec) to +30 mV was used to inactivate
available IA. After a brief
hyperpolarization (1 msec) to 70 mV, the membrane potential was again
stepped to +30 mV, which elicited the combined current of
ID and IK
(Fig. 5B). Because IK is so
small compared with ID, and because
IK is insensitive to D1R stimulation (see
below), the combined current is regarded as
ID in the following functional studies.
The current amplitude was measured at the time point 100 msec after
onset. The detailed isolation of IA was
described in Figure 2F. Peak
IA was measured and was regarded as
IA amplitude. The relative current, or
IRelative, is defined as current
amplitude during perfusion of drug divided by current amplitude in
control
(IPerfusion/IControl).
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Figure 5.
Dopamine D1 receptor-mediated inhibition of VGKC.
A, Activation of D1 receptors by the selective agonist
SKF 81297 (0.1 and 1 µM) failed to alter
IK (n = 5). The
inset shows two traces of
IK in the presence and absence of SKF
81297 (0.1 µM), clearly indicating the lack of
effect of D1 receptor stimulation on this current. B,
Activation of D1 receptors with SKF 81297 produced a dose-dependent
suppression of ID
(n = 25), which was prevented by the D1 receptor
antagonist SCH 23390 (n = 5). Inset
traces clearly show the dose-dependent modulation.
C, Activation of D1 receptors by SKF 81297 (1 µM) failed to alter
IA (n = 5).
Inset traces show the overall effect of SKF 81297 and
the effect on the isolated IA.
D, The late component of VGKC (mostly
ID) was measured at the open
circle in inset; peak VGKC (mostly
IA) was measured at the beginning of
the current, as indicated by the filled circle in
inset. Perfusion of DA (20 µM) failed
to alter peak VGKC (mostly IA)
(n = 5; filled circles) but
significantly inhibited the late component of VGKC (mostly
ID) (n = 6;
open circles). E, The effects of DA and
SKF 81297 on the various components of VGKC are summarized in a
box-whisker plot. Concentrations of 0.1 µM SKF
81297 (n = 5), 1 µM SKF 81297 (n = 5), or 0.1 µM SKF 81297 with 1 µM SCH 23390 (n = 3)
failed to alter the amplitude of IK.
Perfusion of 0.1 µM SKF 81297 produces a 19 ± 3% inhibition of ID
(n = 15). Perfusion of 1 µM SKF
81297 produces a 34 ± 17% inhibition of
ID (n = 10).
Perfusion of 0.1 µM SKF 81297 together with 1 µM SCH 23390 does not alter the amplitude of
ID
(IRelative = 0.96 ± 0.03;
n = 5). Perfusion of 0.1 (n = 3) or 1 µM SKF 81297 (n = 5), or
SKF 81297 (0.1 µM) with SCH 23390 (1 µM) (n = 3), does not alter the
amplitude of IA. Perfusion of DA
does not alter IA
(IRelative = 0.93 ± 0.09;
n = 5) but inhibits
ID
(IRelative = 0.78 ± 0.06;
n = 6.).
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IK was insensitive to D1R stimulation.
Activation of D1Rs with the selective agonist SKF 81297 (0.1 and
1 µM) failed to induce any change in current
amplitude of IK (n = 5 for
each concentration) (Fig. 5A,E).
However, in the same neuron, SKF 81297 induced a significant inhibition
of ID amplitude (Fig. 5B). The
D1R-mediated inhibition of ID was dose
dependent. Perfusion of neurons with 0.1 µM SKF
81297 induced 19 ± 3% inhibition of
ID amplitude (n = 15;
p < 0.05; paired t test between
control and perfusion of SKF 81297,) whereas perfusion with 1 µM SKF 81297 induced 34 ± 5% inhibition
of ID amplitude (n = 10;
p < 0.05; paired t test between
control and perfusion of SKF 81297) (Fig.
5B,E). The D1R-mediated inhibition
(by SKF 81297, 0.1 µM) was reversible after
wash and was antagonized by the DA D1R-selective antagonist SCH
23390 (1 µM; n = 5;
IRelative = 0.98 ± 0.02) (Fig.
5B,E). In most neurons (6 of 7),
ID was also inhibited by the partial DA
D1R agonist SKF38393 (0.1 µM; data not shown).
In another neuron, whole-cell VGKC was elicited by a voltage step (1 sec) to +30 mV from a holding potential of 100 mV (Fig.
5C, inset). An obvious inhibition in the late
component (ID) of VGKC could be observed
when the neuron was perfused with SKF 81297 (0.1 and 1 µM)
(Fig. 5C, inset).
IA from this neuron was isolated from each
perfusion condition (Fig. 5C, inset),
displaying no response to D1R stimulation (Fig. 5C). In most
neurons, no alteration in IA amplitude was
observed when D1Rs were stimulated by SKF 81297 at concentrations of
0.1 µM (IRelative = 1.03 ± 0.04; n = 5,) or 1 µM (IRelative = 0.95 ± 0.05; n = 5,) (Fig.
5C,E).
No obvious alterations in whole-cell VGKC (or either component of VGKC)
were observed when D2Rs were stimulated by the selective D2R agonist
quinpirole (0.1 and 1 µM; n = 9; data not
shown). We next examined how DA, the endogenous agonist, affects VGKC. Whole-cell VGKC was elicited when the membrane potential was stepped to
+30 mV (4 sec) from a holding potential of 100 mV. In the neuron
shown in Figure 5D, inset, the very rapid
inactivating component (IA) was observed.
The amplitude of the peak current (mostly
IA) was measured at the time point 10 msec, as indicated by the filled circle. The amplitude of the late
component (mostly ID) was measured at the
time point 3 sec, as indicated by the open circle in Figure
5D, inset. Perfusion of neurons with DA (20 µM) did not alter peak VGKC (mostly
IA)
(IRelative = 0.93 ± 0.05;
n = 5), but induced a washable inhibition of the late component (mostly ID)
(IRelative = 0.78 ± 0.06;
n = 6) (Fig. 5D,E). A summary of DA-induced inhibition of VGKC (Fig. 5E)
indicates that (1) activation of D1R does not affect
IK and IA but
reliably inhibits ID, and (2) DA inhibits
VGKC via its action on D1Rs.
We next examined the effects of DA on the I-V curve of
whole-cell VGKC, which could be obtained by the protocols described in
Figure 1C. DA-induced (30 sec perfusion) inhibition of
current amplitude could be observed in both activation traces and
pseudo-inactivation traces (Fig.
6A). DA did not alter
activation but shifted inactivation in the hyperpolarizing direction
(Fig. 6B) [half-inactivation voltage
(Vh) of VGKC shifted 5.4 ± 1.2 mV in the hyperpolarizing direction by DA; n = 3]. We
argued above that DA modulates VGKC via its action on D1Rs, so it was
not surprising that stimulation of D1Rs by SKF 81297 induced a similar
left-shift of inactivation in VGKC, but activation remained intact
(Fig. 6C). It could also be observed that two
components (two Boltzmann equations) were involved in the
inactivation, which was interpreted as two components in the earlier
section of this study. It is worth mentioning that the variability of
Vh among neurons is high in our
preparations, which may be attributable to the diverse expression of
K+ channels/currents, the inadequate
voltage clamp, the incomplete compensation of junction potential, or
other factors. However, in all recorded neurons the
Vh consistently exhibited a left-shift (
Vh = 5.3 ± 1.1 mV;
n = 6) in responding to D1R activation, suggesting that
D1R suppression of VGKC might be mediated by the enhanced inactivation
of K+ channels.
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Figure 6.
The effects of dopamine (DA) and
SKF 81297 on the VGKC I-V relationship.
A1, A2, The activation protocol (as in
Fig. 1A) elicited VGKC before and during
perfusion of DA (20 µM). A3,
A4, The inactivation protocol (as in Fig.
1B) elicited VGKC in the same pyramidal mPFC
neuron before and during perfusion of 20 µM DA.
B, The I-V curve for whole-cell VGKC
from the neuron shown in A is plotted. DA clearly
produces a hyperpolarizing shift in the inactivation curve of VGKC in
mPFC neurons (n = 3 of 3). C, A
similar shift of the inactivation curve was observed during perfusion
of the D1 receptor agonist SKF 81297 (0.1 µM). The
inactivation of these neurons could be best fit with two Boltzmann
equations, suggesting that two components with distinct inactivation
kinetics are involved in the whole-cell VGKC.
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Mechanisms responsible for D1R modulation of
ID
D1Rs are positively coupled to the adenylyl cyclase/cAMP/protein
kinase A (PKA) signal transduction pathway (Stoof and Kebabian, 1984).
By activation of this pathway, D1Rs have been shown to suppress
whole-cell Na+ current in ventral and
dorsal striatal medium spiny neurons (Surmeier et al., 1992; Zhang et
al., 1998), PFC pyramidal neurons (Maurice et al., 2001), and
hippocampal pyramidal neurons (Cantrell et al., 1997, 1999). To
determine whether this signal transduction pathway is also responsible
for the D1R modulation of VGKC, we stimulated or inhibited each
component of this signal cascade and examined the role of this pathway
in D1R-mediated modulation of VGKC.
Perfusion of 20 µM 8-Br-Sp-cAMP, a membrane-permeable
cAMP analog with strong PKA activating effect, induced a significant inhibition of the late component of whole-cell VGKC (mostly
ID) but did not suppress the
fast-inactivating component (mostly IA) (Fig. 7A). Whole-cell VGKC
amplitude (measured at the end of the traces) was decreased by 26 ± 6% (n = 5; p < 0.05; paired
t test between control and perfusion of cAMP)
(Fig. 7A,F). Perfusion of 10 µM forskolin, a membrane-permeable activator of
AC, inhibited VGKC by 22 ± 4% (n = 6), whereas
incubation with H8 (10 µM), the nonspecific PKA
inhibitor, enhanced VGKC by 21 ± 5% (n = 5)
(Fig. 7B,F). Basal VGKC
could also be enhanced by PKI, a more specific PKA inhibitor
(n = 2; data not shown). OA was used to examine whether the K+ channel is the direct
substrate of PKA. OA is a strong inhibitor of multiple phosphatases,
inhibition of which blocks dephosphorylation of VGKC channels,
resulting in more phosphorylated channels. Perfusion of 100 nM OA suppressed VGKC (17 ± 4%;
n = 4) (Fig. 7C,F). The OA-induced inhibition of VGKC could not be washed out because of its
irreversible binding to phosphatases. The above experiments suggest
that (1) sequential activation of adenylyl cyclase, cAMP, and PKA is
sufficient for VGKC inhibition, (2) constitutive activity of PKA
tonically inhibits VGKC, and (3) K+
channels underlying VGKC exist in a dynamic balance between
phosphorylation and dephosphorylation states and can be modulated in
either direction.
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Figure 7.
The adenylyl cyclase/cAMP/protein kinase A (PKA)
signaling system mediates DA D1 receptor inhibition of
ID. A, Sp-cAMP (20 µM), a membrane-permeable cAMP analog that activates
protein kinase A directly, inhibited
ID (n = 6) but not
IA. B, Forskolin (10 µM), a membrane-permeable stimulator of adenylyl
cyclase, also inhibited ID
(n = 6). Perfusion of the same neuron with H8 (10 µM), a membrane-permeable PKA inhibitor, increased
ID (n = 5).
C, The membrane-permeable protein phosphatase inhibitor
okadaic acid (100 nM) inhibited
ID (n = 4).
D, The PKI subunit (1 U/ml) was included in the pipette
solution and diffused into the cell when the whole-cell configuration
was formed. Once the cAMP/PKA pathway was fully blocked by PKI,
indicated as the VGKC reached steady state, the inhibitory effect of
SKF 81297 (1 µM) was also blocked
(n = 4). E, Perfusion of Rp-cAMP (50 µM), a membrane-permeable cAMP analog with strong
inhibitory effects on PKA (n = 5), also blocked the
inhibition by SKF 81297 (1 µM). F,
This plot summarizes the contribution of each component of the
AC/cAMP/PKA pathway to D1R-mediated modulation of VGKC. Perfusion of
Sp-Br-cAMP suppresses VGKC by 26 ± 6% (n = 5). Perfusion of forskolin suppresses VGKC by 22 ± 4%
(n = 6), whereas perfusion of H8 enhances VGKC by
21 ± 5%. Perfusion of okadaic acid suppresses VGKC by 17 ± 4% (n = 4). VGKCs become resistant to the
stimulation of D1R when the cell is treated with either PKI
(IRelative = 1.04 ± 0.05;
n = 4) or Rp-cAMP
(IRelative = 1.02 ± 0.05;
n = 5).
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Other signal transduction cascades are also reported to participate in
D1R signaling (Undie et al., 2000). Is the AC/cAMP/PKA pathway also
necessary in mediating D1R effects on VGKC? The next two experiments
addressed this question. The pipette solution contained 1 U/ml PKI
subunit, a PKA inhibitor. After cell rupture, PKI diffused into the
neuron, blocking PKA-mediated phosphorylation. After full blockade of
PKI was obtained (monitored as the amplitude of VGKC became stable),
perfusion with SKF 81297 no longer reduced VGKC
(IRelative = 1.04 ± 0.05;
n = 4) (Fig.
7D,F). Similar experiments were performed with another PKA inhibitor, Rp-cAMP, the nonfunctional cAMP analog that neutralizes intracellular cAMP activity so that PKA
activity is blocked. Perfusion of Rp-cAMP (50 µM) initially induced an enhancement of VGKC
(data not shown). After the PKA is fully inactivated, which was
indicated as VGKC became stable, SFK 81297 failed to suppress VGKC
(IRelative = 1.02 ± 0.05;
n = 5) (Fig.
7E,F). In sum, the above
results suggest that the AC/cAMP/PKA pathway is essential to D1R
modulation of VGKC.
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Discussion |
The effects of DA on mPFC pyramidal neurons have remained an
enigma for >25 years (for review, see Yang et al., 1999). Although recent studies have begun to elucidate the means by which DA modulates neuronal excitability in subcortical areas (for review, see Nicola et
al., 2000), such clarification has been slower to emerge in the mPFC,
perhaps because previous in vivo and in vitro
characterizations have been even more controversial than long-standing
disputes regarding striatal neurons. Our whole-cell voltage-clamp
studies provide the first direct demonstration of DA receptor
modulation of isolated VGKCs in pyramidal neurons of the mPFC. Although
this technical approach has the disadvantages of a lack of neuronal processes as a result of the dissociation process and a potential disruption of cytosolic messenger systems, it provides excellent voltage control and easy access to pharmacological agents, thereby allowing isolation of whole-cell VGKC and the ability to examine the
effects of DA on individual components of VGKC. Our results clearly
show that DA D1Rs, but not D2Rs, selectively decrease a slowly
inactivating K+ conductance via the
classical D1R/AC/cAMP/PKA signaling pathway.
Biophysical and pharmacological dissection of VGKCs
We identified at least three major contributors to whole-cell
K+ current in mPFC pyramidal neurons,
IA, ID, and
IK, as indicated by both biophysical and
pharmacological dissection. These findings are consistent with
previously described K+ currents in
sensorimotor cortical neurons (Foehring and Surmeier, 1993). Although
four time constants were obtained from studies of current decay, it is
difficult to know whether three or more types of
K+ channels are responsible for these
components as revealed by different biophysical properties. Multiple
K+ channels can express homogeneous
biophysics (Coetzee et al., 1999), and identical channels can express
heterogeneous biophysics, as a result of post-translational
modification and interactions with auxiliary subunits (Heinemann et
al., 1996; Holmqvist et al., 2001). Here we focused on current type as
opposed to distinct channel proteins.
The fast decaying component of the whole-cell VGKC ( = 12 ± 3 msec) is kinetically consistent with the fast inactivating component IA, whereas the very slow
decay component ( = 33 ± 7 sec) is kinetically consistent
with IK. The other two components ( = 220 ± 26 msec and 3.4 ± 0.5 sec) are consistent
with the slowly inactivating component
ID. Although the two components of
ID could not be isolated with
activation protocols (Fig. 1), they were dissociable during
inactivation in some neurons (Fig. 5D). This conclusion is
consistent with recent findings in pyramidal neurons from several
cortical regions, for example, in young neocortical neurons (Korngreen
and Sakmann, 2000; Bekkers and Delaney, 2001), visual cortical neurons
(Locke and Nerbonne, 1997), neocortex layer I neurons (Zhou and
Hablitz, 1996), and sensorimotor cortical neurons (Foehring and
Surmeier, 1993).
In dissociated mPFC neurons, IA was
often too small to be detected. Similar results were observed in
acutely dissociated sensorimotor cortical pyramidal neurons (Foehring
and Surmeier, 1993), cultured hippocampal pyramidal neurons (Murakoshi
and Trimmer, 1999), or dissociated striatal spiny neurons (Surmeier et
al., 1991), but in dissociated rat hippocampal neurons, A-current was
shown to prevail, and it contributed 61% of total
K+ currents (Martina et al., 1998). The
difference in expression of A-currents could be interpreted as cellular
diversity between hippocampal and mPFC pyramidal neurons. An
alternative interpretation is developmental regulation. Sensorimotor,
striatal, and mPFC neurons that exhibited small A-current were
dissociated from mature animals (4 weeks or older), whereas hippocampal
neurons that displayed predominant A-current were obtained from young
rats (11-16 d) (Martina et al., 1998). Both cultured and premature
neonatal striatal neurons exhibit large A-currents, whereas in mature
striatal neurons, the A-current is much less prominent (Surmeier et
al., 1991). Thus, different developmental ages could be responsible for
expression of different channel proteins.
Pharmacological studies confirm our biophysical findings of multiple
currents involved in the whole-cell VGKC.
IK displayed two
IC50 values for all three blockers (4-AP, TEA,
and DTX) that we used. ID also displayed
two IC50 values for TEA and DTX. This suggests
that both ID and
IK contain at least two types of currents. ID displayed only one
IC50 for 4-AP, but the maximum blockade by 4-AP
is only ~50%, indicating that there is at least one component of
ID that is 4-AP insensitive. In principle,
the two types of currents, identified by the two
IC50 values, likely result from two distinct
types of channels, but this prediction only works well in homogenous
expression systems and may not hold in electrophysiological studies of
native neurons, in which two different
IC50 values are not sufficient to conclude that
there are two different channel types (Kirsch and Drewe, 1993). The
inactivation analysis shows that there are two components (two values) in ID, which is consistent with
the pharmacological results (two IC50 values).
There is no direct biophysical evidence indicating the composition (two
components) of IK, but the pharmacological
data show two components (two IC50 values). This
could indicate that IK contains two
components that share similar biophysical properties but differ in
binding to pharmacological agents. However, because the decay time
constant of the slow component in ID is
relatively large (3.4 sec), in the protocol that we used to isolate
IK, prolonged holding (10-20 sec) at 40
mV may not totally inactivate ID,
resulting in contamination of IK.
Therefore, the alternative interpretation is that the uninactivated slow component of ID may contribute to one
of the IC50 values of
IK. Both biophysical (two values) and
pharmacological (two IC50 values) analyses argue
that there are two components in ID, so in
theory the correlation of the values with the
IC50 values could be obtained by totally blocking
the component with small IC50 (with low
concentration) and calculating the of the rest of the current.
However, technical limitations prevented us from resolving this
question. In a small number of our experiments, low concentrations of
the blockers never "totally" blocked the one component, and when we
increased concentrations, the other component was also considerably affected.
Dopamine modulation of VGKC
The mesocortical DA input arises from the VTA and predominately
terminates on pyramidal neurons in deep layers of mPFC (Bjorklund et
al., 1978; Emson and Koob, 1978; Berger et al., 1991; Carr et al.,
1999). Both D1Rs and D2Rs are expressed in pyramidal neurons, although
the density of D2R appears to be considerably lower (Gaspar et al.,
1995; Vincent et al., 1995). It is widely accepted that D1Rs positively
couple to the AC/cAMP/PKA signal transduction pathway, whereas D2Rs
inhibit this transduction system (Stoof and Kebabian, 1984; Sibley and
Monsma, 1992). Thus, we expected VGKC to be modulated in opposite ways
by D1Rs and D2Rs. Surprisingly, most recorded neurons did not respond
to D2R agonists. Does this indicate a lack of D2R expression in mPFC
pyramidal neurons? Apparently not, because our parallel studies
demonstrate that when inwardly rectifying
K+ current (IRKC) and VGKC were
sequentially elicited in the same mPFC neuron, D2R agonists modulate
only IRKC but do not affect VGKC (Dong and White, 2001), suggesting
selective targeting of specific K+
channels for D2R activation.
D1R activation suppresses only ID in
mPFC neurons, suggesting that the channel(s) conducting
ID is selectively targeted by D1Rs as
opposed to channels conducting
IA and
IK. A similar inhibition of VGKC
induced by PKA activation was observed in the soma of striatal medium
spiny neurons (Surmeier and Kitai, 1993) as well as in dendrites of
hippocampal pyramidal neurons (Hoffman and Johnston, 1999). Delayed
rectifier K+ current
(IK), as well as the late
component of ID, is primarily responsible for action potential duration, whereas transient
K+ currents,
IA and the early component of
ID, preferentially control subthreshold responses to excitatory inputs and firing frequency (Debanne et al., 1997; Hille, 2001). Consistently, intracellular recordings from mPFC slices show that perfusion of either D1R-selective agonists or DA itself, but not D2R-selective agonists, reduces first
spike latency, lowers firing threshold, and increases firing frequency,
but does not alter the shape of the action potential (Yang and Seamans,
1996).
Mechanism of DA D1R modulation
Our studies indicate that D1Rs modulate
ID by activation of the AC/cAMP/PKA
signal transduction pathway. Low concentrations (0.1 µM) of D1R agonists induced similar inhibition,
which was abolished by the D1R-selective antagonist SCH 23390 (1 µM). Increasing intracellular cAMP levels with
either forskolin or Sp-cAMP mimicked D1R-mediated inhibition of
ID, suggesting that stimulation of the
AC/cAMP/PKA pathway is sufficient to modulate
ID. Neutralization of either cAMP or
PKA, with Rp-cAMP and PKI, respectively, abolished D1R-induced
inhibition of ID, suggesting that the
AC/cAMP/PKA pathway is essential in D1R-mediated modulation.
Phosphorylation is a common mechanism by which membrane channel
proteins are subject to modification. Indeed, phosphorylation-induced
inhibition of several types of K+ channels
has been reported in many other systems (for review, see Levitan,
1988).
The actions of DA on mPFC pyramidal neurons have long been
controversial. Why does DA increase excitability in some situations while decreasing it in others? Our study suggests that DA can influence
the activity of mPFC pyramidal neurons by modulation of VGKCs.
Activation of DA D1Rs can increase neuronal excitability by inhibition
of VGKC, whereas both D1R and D2R stimulation can suppress IRKC (Dong
and White, 2001), but DA may also decrease whole-cell sodium current to
decrease excitability (Maurice et al., 2001). Clearly, the actions of
DA on mPFC pyramidal neurons depend on the timing and strength of
synaptic inputs as well as on the membrane potential range at which
mPFC neurons are operating (Yang et al., 1999).
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FOOTNOTES |
Received Sept. 23, 2002; revised Jan. 9, 2002; accepted Jan. 10, 2002.
This work was supported by United States Public Health Service Grant DA
12618 and Senior Scientist Award DA 00456 from the National Institute
on Drug Abuse (F.J.W.). We thank Lori Baker and Kerstin Ford for
excellent technical assistance, Dr. Robert Foehring for his critical
comments on this manuscript, and Drs. Donald C. Cooper, D. James
Surmeier, and Xiu-Ti Hu for expert advice.
Correspondence should be addressed to Dr. Francis J. White, Department
of Cellular and Molecular Pharmacology, Finch University of Health
Sciences/The Chicago Medical School, 3333 Green Bay Road, North
Chicago, IL 60064. E-mail: francis.white{at}finchcms.edu.
Y. Dong's present address: Department of Psychiatry, Stanford
University School of Medicine, Palo Alto, CA
94304-5485.
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TOP
ABSTRACT
Introduction
