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The Journal of Neuroscience, July 15, 1998, 18(14):5180-5190
Dopamine D1-Like Receptor Activation Excites Rat
Striatal Large Aspiny Neurons In Vitro
Toshihiko
Aosaki1,
Kazutoshi
Kiuchi2, and
Yasuo
Kawaguchi1
1 Laboratory for Neural Circuits and
2 Laboratory for Genes of Motor Systems, Bio-Mimetic
Control Research Center, The Institute of Physical and Chemical
Research (RIKEN), Nagoya, Aichi 463-0003, Japan
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ABSTRACT |
The aim of this study was to elucidate electrophysiologically the
actions of dopamine and SKF38393, a D1-like dopamine
receptor agonist, on the membrane excitability of striatal large aspiny neurons (cholinergic interneurons). Whole-cell and perforated patch-clamp recordings were made of striatal cholinergic neurons in rat
brain slice preparations. Bath application of dopamine (1-100
µM) evoked a depolarization/inward current with an
increase, a decrease, or no change in membrane conductance in a
dose-dependent manner. This effect was antagonized by SCH23390, a
D1-like dopamine receptor antagonist. The
current-voltage relationships of the dopamine-induced
current determined in 23 cells suggested two conductances. In 10 cells
the current reversed at  94 mV, approximately equal to the
K+ equilibrium potential
(EK); in three cells the
I-V curves remained parallel, whereas in
10 cells the current reversed at 42 mV, which suggested an
involvement of a cation permeable channel. Change in external
K+ concentration shifted the reversal potential as
expected for Ek in low
Na+ solution. The current observed in 2 mM Ba2+-containing solution reversed at
28 mV. These actions of dopamine were mimicked by application of
SKF38393 (1-50 µM) or forskolin (10 µM),
an adenylyl cyclase activator, and were blocked by SCH23390 (10 µM) or SQ22536 (300 µM), an inhibitor of
adenylyl cyclase. These data indicate, first, that dopamine depolarizes
the striatal large aspiny neurons by a D1-mediated
suppression of resting K+ conductance and an opening
of a nonselective cation channel and, second, that both mechanisms are
mediated by an adenylyl cyclase-dependent pathway.
Key words:
dopamine; acetylcholine; striatum; basal ganglia; cholinergic neurons; giant aspiny neurons; patch clamp; slice; electrophysiology
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INTRODUCTION |
Because clinical studies conducted
in the early 1960s indicated that both muscarinic receptor antagonists
and dopaminergic receptor agonists ameliorate the symptoms of
Parkinson's disease, it was classically proposed that cholinergic and
dopaminergic tones are in balance in the extrapyramidal system
(Barbeau, 1962 ; Lehmann and Langer, 1983). With the advent of chemical
agents that act selectively on dopamine (DA) D1 receptors
and of brain microdialysis techniques, the acetylcholine (ACh)/DA
balance hypothesis has been modified to encompass the finding that
activation of DA D1 receptors stimulates the ACh release in
the striatum, whereas D2 agonists reduce it (Stoof et al.,
1992; Consolo et al., 1993; Di Chiara et al., 1994). This ACh is
thought to be supplied primarily by large aspiny neurons (cholinergic
interneurons), which account for only 1-2% of the total neuronal
population of the rat striatum (Bolam et al., 1984; Phelps et al.,
1985). In situ hybridization studies, however, have provided
evidence that only a small number of the striatal cholinergic neurons
contain D1 receptor mRNAs, whereas the majority of the
cells express D2 receptor mRNAs (Le Moine et al., 1991). It
is therefore currently hypothesized that although the inhibition of its
release by D2 DA receptor activation is direct, the ACh
release evoked by D1 DA receptor activation might be
indirect through two alternative pathways: excitation of medium-sized
spiny neurons containing D1 receptors that release substance P (Ajima et al., 1990; Anderson et al., 1994; Di Chiara et
al., 1994) and stimulation of D1 receptors in the thalamus, cerebral cortex, or substantia nigra that in turn elicits an increase in glutamate input to the striatum (Damsma et al., 1991; Consolo et
al., 1996a,b; Abercrombie and DeBoer, 1997). Recently, five DA receptor
isoforms, grouped into two subfamilies, D1-like
(D1 or D1a, and D5 or
D1b) and D2-like (D2(short),
D2(long), D3, and D4), have been defined so far. Bergson and
colleagues (1995) reported that only D5, and not
D1, receptor-specific antibodies labeled the
striatal cholinergic neurons of rhesus monkey. In addition, a
patch-clamp study using a single cell RT-PCR technique (Yan et al.,
1997) demonstrated that the majority of the neostriatal cholinergic neurons of the rat (88%) contain
D5/D1b receptor mRNAs rather than
D1a mRNAs (17%), whereas all the cells express
D2 mRNAs. Therefore, direct activation of
D5/D1b DA receptors on the striatal
cholinergic neurons might contribute to the evoked ACh release. Indeed,
Login and Harrison (1996) reported that 50 µM
(±)-SKF38393, a D1-like receptor agonist, increased the
basal rate of ACh release from dissociated striatal cholinergic cells by 50%, with this action blocked by the D1-like receptor
antagonist SCH23390. The present study was conducted to examine the
effects of D1-like receptor activation on the membrane
excitability of rat striatal giant aspiny neurons to explore the
possibility of direct regulation of ACh release by D1-like
receptors. We present evidence here that DA and (±)-SKF38393 directly
depolarize the cells, which is sufficient to generate action potentials
in some cases.
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MATERIALS AND METHODS |
Slice preparations. Rats were bred under standard
animal housing conditions, with a 12 hr light/dark cycle. Food and
water were available ad libitum. Experiments were performed
on 200-µm-thick sagittal rat brain slices containing the striatum.
The ages of the rats (Wistar) ranged from 12- to 20-d-old. The animals
were deeply anesthetized with ether and decapitated. The brains were quickly removed and submerged in ice-cold physiological Ringer's solution oxygenated with a mixture of 95% O2 and 5%
CO2. Slices were superfused at 3 ml/min with physiological
saline at 33°C. The solution comprised (in mM): NaCl 124, KCl 3, CaCl2 2.4, MgCl2 1.2, NaH2PO4 1, NaHCO3 26, glucose 10, gassed with 95% O2 and 5% CO2.
Whole-cell and perforated patch-clamp recordings. Whole-cell
recordings were made with glass pipettes (3-4 M ) that contained (in
mM): potassium methylsulfate 120, KCl 6, NaCl 6, EGTA 0.6, HEPES 10, MgCl2 2, ATP 4, GTP 0.3, biocytin 20, pH 7.2. Tetrodotoxin (TTX) was added to the external solution at 0.5 µM when required. For ion substitution experiments, NaCl
was replaced with choline chloride in low-sodium external solutions.
When CdCl2 was added at 200 µM,
NaH2PO4 was omitted. To isolate the
nonselective cation current, tetraethylammonium chloride (TEA-Cl) 30 mM, 4-aminopyridine (4-AP) 10 mM, CsCl 2 mM, MgCl2 3.6 mM, no
Ca2+, and TTX 0.5 µM were added to the
external solution, and cesium-methanesulfonate was substituted for
potassium methylsulfate in the pipette solution. Membrane potentials
and currents were recorded with an EPC-7 amplifier (List). Series
resistance was partially compensated for by the amplifier. The given
membrane potentials have been corrected for the junction potential
between the pipette solution (potassium methylsulfate) and the bath
solution before making a gigaseal (usually the pipette 7 mV negative to
the bath). Membrane conductance was measured from the slope of the
chord in 10 mV step pulses.
Perforated patch recordings using nystatin were also made to confirm
the results obtained by the whole-cell recordings. Nystatin (5 mg) was
dissolved in a methanol solution containing
N-methyl-D-glucamine (Sigma, St. Louis, MO) (0.1 M) and methanesulfonate (0.1 M) adjusted to pH
7.0 immediately before use. After the nystatin solution (50-100 µl)
was dried by nitrogen gas, the internal solution was added and shaken
well at a final concentration of 250-500 µg/ml.
Electrophysiological identification of the neostriatal
neurons. Neurons in the neostriatum were visualized using a 40×
water immersion objective. Particular attention was paid to
preferentially recording the largest neurons in a slice. At the
beginning of the recording, a set of depolarizing and hyperpolarizing
step current pulses were routinely given in the current-clamp mode to
identify the neuronal types. Cells whose resting membrane potentials were over 50 mV were discarded. Electrophysiological criteria for
identification of the neostriatal neurons of the rat have been
described in detail previously (Kawaguchi, 1992, 1993). Briefly, the
cells that had a resting potential of approximately 60 mV and
displayed long-lasting afterhyperpolarizations and strong time-dependent hyperpolarizing rectification were classified as long-lasting afterhyperpolarization (LA) cells. Even when recordings were made with a Cs+-containing pipette, the voltage
traces obtained immediately after the rupture of the patch membranes
usually displayed, very briefly (a few seconds), firing patterns
similar to those obtained with K+-patch pipettes,
which suggested the neuronal types. Immunohistochemical evidence has
indicated that LA cells are large aspiny cholinergic interneurons
(Kawaguchi, 1992, 1993).
Histochemical procedures. To identify the recorded cells
morphologically, 20 mM biocytin was included in the pipette
solution to fill them by diffusion (Horikawa and Armstrong, 1988).
Slices (200 µm) containing biocytin-filled cells were fixed by
immersion in 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (PB) overnight in at 4°C, rinsed in PB
for 30 min, and incubated in PB containing 0.5%
H2O2 for 30 min to suppress endogenous peroxidase activity. They were then incubated in 10 and 20% sucrose for 30 min and 1 hr, respectively, frozen, and stored in a freezer until histochemistry was performed. The slices, without resectioning, were then washed with Tris-buffered saline (TBS) containing 0.5% Triton X-100 and avidin-biotin peroxidase complex (Vector
Laboratories, Burlingame, CA) at a dilution of 1:100 for 4-6 hr at
room temperature. After rinsing they were reacted with 3, 3'-diaminobenzidine tetrahydrochloride (DAB; 0.05%) and
H2O2 (0.003%) in TBS, post-fixed in 0.1%
osmium tetroxide in 0.1 M PB for 5 min, and mounted on
slides.
Drugs. DA and other agents such as CsCl,
CdCl2, TTX, and nifedipine were applied by changing
the solution superfusing the slice to one that contained the drug. Time
taken to reach the neurons was usually <1 min. Drugs used were DA,
(±)-sulpiride (Sigma), (±)-SKF38393 hydrochloride, ()-quinpirole
hydrochloride, R(+)-SCH23390 hydrochloride, (+)-bromocriptine
methanesulfonate (RBI, Natick, MA), tetrodotoxin (Sankyo),
4-aminopyridine (Sigma), tetraethylammonium chloride (Nakarai tesque),
nifedipine (Sigma), 9-(tetrahydro-2-furyl) adenine (SQ22536, Sigma),
and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA, Sigma). DA was dissolved in the external solution containing 0.1% L(+)-ascorbic acid or 50 µM sodium
metabisulfite immediately before use to prevent its oxidative
degradation. L(+)-ascorbic acid or sodium metabisulfite
alone did not exert any effects on the excitability of the neurons. All
the experiments were performed in a dark room to slow the light-induced
degradation of some drugs.
Results are expressed as mean ± SD values, and comparisons
between groups were made using the Student's t test.
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RESULTS |
Physiological and morphological identification of the large
aspiny neurons
The results presented here were obtained with a total of 186 rat
neostriatal neurons identified as large aspiny cholinergic interneurons
(LA cells). For this neuron sample, resting membrane potential was
60.1 ± 5.5 mV (n = 42), and membrane input
conductance was 3.5 ± 1.7 nS (n = 41). Injection
of depolarizing current pulses elicited action potentials (amplitude
66.0 ± 11.6 mV, width at half-amplitude 1.6 ± 0.3 msec)
that were followed by a longer-duration and larger-amplitude
afterhyperpolarization (16.5 ± 8.2 mV; n = 40)
than the other classes of neurons (Fig.
1A). During
hyperpolarizing current pulses, they showed prominent sag, which was
demonstrated to be caused by activation of hyperpolarization-activated
cation currents (Ih) (Jiang and North,
1991; Kawaguchi, 1993). Subsequent staining with biocytin revealed
large somata with aspiny or sparsely spiny dendrites.
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Figure 1.
Effects of DA on neostriatal large aspiny neurons.
A, Membrane properties in response to constant-current
pulses applied intracellularly. The resting membrane potentials (~ 60 mV) and input resistances (~430 M), as well as the
long-duration, large-amplitude afterhyperpolarization and the prominent
sag during hyperpolarizing current pulses all fit well with the
physiological properties of large aspiny neurons (LA cells).
B, Dose-response relation for DA in neostriatal large
aspiny cells. The holding potential was 60 mV. DA was bath-applied
with 50 µM sodium metabisulfite or 0.1% ascorbic acid to
prevent its oxidative degradation. Vertical lines
represent SD values. Numbers in parentheses refer to
numbers of tested cells. C, DA depolarization of a
striatal large aspiny neuron. The external solution contained
L(+)-ascorbic acid (0.1%). DA (50 µM),
bath-applied at 3 ml/min, caused a transient depolarization and a train
of action potentials. Haloperidol (100 µM) completely
blocked the DA response. D, The effects of DA were
analyzed under a whole-cell voltage clamp (holding potential 60 mV).
Da, Suppression of the 100 µM DA-induced
current by pretreatment with SCH23390 (10 µM), an
antagonist of the D1-like DA receptor. TTX was present at
0.5 µM in the external solution. Note that the amplitude
of DA-induced current gradually increased during washout of the
antagonist. Db, SCH23390 (10 µM)) alone
evoked an outward shift of the holding current. Application of DA (100 µM) elicited a small inward current. TTX was 0.5 µM. Membrane conductances were monitored periodically
with hyperpolarizing voltage steps of 10 mV. Holding potential was 60
mV. Calibration bars apply to both a and
b.
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Dopamine depolarized the LA cells by activation of
D1-like DA receptor in a dose-dependent manner
Figure 1C shows the effects of DA on the excitability
of a striatal large aspiny neuron. The whole-cell patch technique was used under a current clamp without TTX. DA was bath-applied in external
solution containing 0.1% L(+)-ascorbic acid or 50 µM sodium metabisulfite. In all cells tested (8/8 cells),
application of DA (1-100 µM) elicited a slow transient
depolarization (9.4 ± 5.0 mV). This was sufficient to initiate a
train of action potentials in three cells. The depolarization was
completely blocked by haloperidol (1 µM), a nonselective
DA receptor antagonist.
The effects of DA were analyzed under whole-cell voltage clamp (holding
potential 60 mV) with TTX (0.5 µM) in the external solution (Fig. 1D). DA invariably induced an inward
current, and it was suppressed by application of SCH23390 (10 µM), an antagonist of the D1-like DA
receptor. Combined administration of DA and SCH23390 evoked an outward
current (7/19 cells, 12.0 ± 8.6 pA), no change (1/19 cells), or a
small inward current (11/19 cells, 12.3 ± 9.3 pA), so that the
average amplitude of the DA-induced current (60.6 ± 32.5 pA,
n = 17) was significantly larger than that obtained
with SCH23390, an antagonist of the D1-like DA receptor (2.7 ± 14.6 pA; n = 19; p < 0.01) (see Fig. 6). As shown in Figure 1Da, combined
application of DA (100 µM) plus SCH23390 (10 µM) produced almost no appreciable change in the holding
current (+0.7 pA) in this cell. However, application of DA (100 µM) alone produced a small decrease in membrane
conductance (from 6.9 to 6.8 nS) along with an inward shift of the
holding current (23.7 pA). The suppression of the DA-induced current
by the antagonist was gradually relieved during washing, reaching
35.0 pA at the end of the experiment. In all cells tested
(n = 6), the DA-induced current in the presence of
SCH23390 (8.35 ± 17.4 pA) was significantly smaller than that
obtained 6 min after washout of the antagonist (23.2 ± 11.8 pA;
p < 0.01). Notably, application of SCH23390 itself
elicited an outward shift of the holding current in 12 of 19 cells as
exemplified in Figure 1Db. The average
SCH23390-induced current was 11.5 ± 15.0 pA (n = 19). This indicates that some cells might be continuously slightly
depolarized by D1-like receptor activation, possibly by
spontaneous release of DA from terminals in the vicinity. These results
suggest that the majority of the DA-induced current is mediated by
D1-like DA receptor activation.
Overall, this DA-induced inward current was obtained in all of the
striatal LA cells tested (43 cells), and the effect was concentration-dependent. A dose-response curve for the DA-induced inward current is shown in Figure 1B. The
EC50 value was determined to be ~15 µM. The
DA-induced current reached steady-state values with a delay of ~1 min
without showing any desensitization. Usually the current stayed
constant during DA application and longer treatment (>1 min), often
preventing return of the holding current to the pretreatment level
(Fig. 2B). The
DA-induced inward current persisted even in perfusion medium containing
200 µM Cd2+, 0 mM
Ca2+, 3.6 mM Mg2+,
and 0.5 µM TTX (n = 5), suggesting
mediation by a direct effect of DA on recorded cells (data not shown;
also see Fig. 5). In a small population of the cells (3/36 cells) that
were mostly recorded with the nystatin method, the inward current was
preceded by a small transient outward current as shown in Figure
4B. This was not studied further in detail.
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Figure 2.
Two conductances mediate the DA-induced inward
current. Shown are typical examples expressing a reversal potential
close to the potassium equilibrium potential
(EK, 100.6 mV)
(A), or a reversal close to 40 mV
(B), or no reversal potential within the tested
voltage range (C). Voltage ramps (10 mV/sec) were
used to construct I-V curves for
A and B. Data for
I-V plots in C were taken
at the end of the voltage pulse. C2, open
circles indicate before DA application; filled
circles indicate after DA application. There was no significant
difference between measurements taken at the beginning and at the end
of the voltage pulse. A3, B3,
and C3 indicate the net DA-induced currents,
calculated as differences between I-V
plots obtained before and during the peak of the DA responses.
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Two separate conductances are involved in the DA responses in
LA cells
Detailed comparison of the current-voltage relationships of the
DA-induced current suggested that at least two separate conductances mediated the DA response. In 10 of 23 cells, the response to DA reversed at 93.7 ± 15.7 mV, approximately equal to the
estimated K+ equilibrium potential
(EK, 100.6 mV), with a slight reduction in membrane conductance from 7.4 ± 2.1 to 7.0 ± 1.7 nS, as
shown in Figure 2A. The net current was 35.5 ± 18.4 pA. In contrast, 10 of 23 cells showed an increase in membrane
conductance from 5.9 ± 3.4 to 8.3 ± 3.6 nS. These cells
showed a linear current-voltage relation with a positive slope and a
reversal potential of 42.1 ± 10.1 mV (Fig.
2B). The amplitude of the net current was 48.0 ± 47.1 pA. In the remaining three cells, the DA-induced inward current
was associated with almost no change in membrane conductance from
4.5 ± 1.3 to 4.5 ± 0.8 nS (Fig. 2C). The net
current was 22.6 pA and the I-V relations did
not cross within the voltage range tested (120 to 40 mV).
Because these results suggested an involvement of multiple separate
conductances in the generation of DA responses, cells were first tested
in perfusion medium containing low Na+ (27 mM) and 0.5 µM TTX to maximize the
contribution of a K+ current component to the DA
response (Fig. 3). The reversal
potentials of the DA-induced current in this particular experiment were
88.5 ± 17.8, 56.1 ± 19.5, and 45.0 ± 16.3 mV
with external K+ concentrations of 3, 9, and 16 mM, respectively. The values were rather more positive than
estimated with the Nernst equation, probably because they contained
effects of Na+-permeable conductance and
insufficient space clamping. However, the reversal potentials shifted
to less negative values in solutions of elevated K+
as expected and gave a Nernst slope of 53.3 ± 5.4 mV per
10-fold change in K+ concentration. These results
indicate that K+ channels were closed during flow of
the DA-mediated inward current.
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Figure 3.
One of the inward currents caused by DA is
K+ dependent. A Nernst plot of reversal potentials
against three K+ concentrations.
Na+ concentration in the external solutions was
reduced to 27 mM to maximize the K+
component. TTX was present at 0.5 µM. The
continuous line plots the mean values of the reversal
potentials obtained at each concentration. The dashed
line was calculated from the Nernst equation for a
K+-selective electrode. Numbers in
parentheses refer to numbers of tested cells.
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Figure 4 shows an example in which the
same cell was exposed first to 100 µM DA in a low
Na+ solution and then in normal Ringer's solution.
It showed linear current-voltage relations within a voltage range
tested with a reversal potential near the equilibrium potential of
K+ channel in a low Na+ solution.
In the control solution (151 mM Na+),
however, the I-V relations did not cross within
the voltage range between 120 and 60 mV. Importantly, the net
current in this cell was 33.2 pA in the low Na+
solution and 95.8 pA in the control solution. On average, the DA-induced inward current at the holding potential of 60 mV in the
low Na+ solution was 27.6 ± 14.6 pA
(n = 7), which was significantly smaller than that in
the control solution (60.6 ± 32.5 pA; n = 17;
p < 0.05) (see Fig. 6). These results suggest that at
least in some cells, activation of Na+-permeable
cation conductance is involved in the DA-induced current, together with
a block of resting K+ conductance.
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Figure 4.
Lowering external Na+ ion
concentration reduces the amplitude of the DA-induced inward current in
a cell. Inward currents caused by DA (100 µM) in 27 mM (A1) and 151 mM
(B1)
[Na+]o, respectively. Voltage
ramps (125 to 60 mV, 9.3 mV/sec) were applied before (1,
2) and during (3, 4) the inward shift of
the holding current. A2,
B2, Two superimposed
I-V plots, one before (1,
3) and the other during (2, 4) the peak
DA response. A3, B3,
I-V plots of the net DA-induced
currents. Note that the reversal potential was close to
EK in 27 mM
[Na+]o, but no reversal
potential was obtained within the voltage range tested in 151 mM [Na+]o. In a small
population of the cells, DA elicited an early outward current followed
by an inward current as observed in B1.
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To characterize further the role of the cation conductances, barium
ions (2 mM) were included in the external solution to block
K+ conductance. Barium ions are known to inhibit
various K+ channels such as the delayed rectifier,
inward rectifier, leak K+, and M-channels. In all
cells (n = 8), switching to the
Ba2+-containing medium evoked an inward shift of the
holding current of 108.4 ± 48.6 pA with a reduction of membrane
conductance from 10.6 ± 5.0 to 5.1 ± 1.8 nS. The reversal
potential of the Ba2+-induced current was
86.2 ± 6.4 mV, suggesting that the K+
channels were indeed blocked. Application of DA (100 µM)
under these conditions induced a further shift of the holding current (16.8 ± 9.2 pA; n = 8) (see Fig. 6) accompanied
by an increase in membrane conductance from 4.7 ± 1.5 nS to
5.1 ± 1.7 nS in six of eight cells tested (Fig.
5A). The current-voltage
relationships of the net DA-induced inward current showed a slightly
positive curve within a voltage range between 110 and 35 mV and a
negative curve in more depolarized regions (35 to +20 mV). To test
the possibility that the latter component might be attributable to opening of voltage-sensitive Ca2+ channels, 200 µM Cd2+ and 5 µM
nifedipine were added to the external solution. Cs+
(2 mM) was also included to block the
hyperpolarization-induced cation channel
(Ih). In 10 of 11 cells tested, DA (100 µM) evoked a small inward current (21.7 ± 7.3 pA;
n = 10) (Fig. 6) with a
small increase in membrane conductance from 4.9 ± 1.8 nS to 5.1 ± 2.1 nS in Ba2+-containing solution. The
I-V curves obtained before and during DA
exposure again crossed or almost crossed at potentials around 40 mV
in 10 cells (Fig. 5Ba). Also, as the membrane potential was
further depolarized to +20 mV, these cells showed a decrease in
membrane conductance, indicating that this component might be not
caused by activation of voltage-dependent Ca2+
channels. Rather, it might be caused by either the voltage dependency of the channel or simply by a space clamp error. In one cell, the net
DA current reversed the sign at 28 mV (Fig. 5Bb),
suggesting that cations permeated nonselectively through the channel.
Similar findings were obtained when the recording was made with a
Cs+-containing patch pipette in the presence of TTX
(n = 13; data not shown).
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Figure 5.
The Ba2+-resistant component of
the DA-induced current was evoked by DA (100 µM) in a
solution containing 2 mM Ba2+
(A) and a solution containing
Ba2+ (2 mM), Cs+ (2 mM), and Cd2+ (200 µM)
(B). A2,
Ba2, Bb2, Two
superimposed I-V plots, one before and
the other during the peak DA response.
A3, The net DA-induced current showed an
atypical I-V relation. Addition of
Cd2+ did not eliminate the net DA inward current
seen at more depolarized potentials than 35 mV
(Ba3). In another cell the current reversed the
sign at 28 mV (Bb3).
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Figure 6.
Summary histograms representing mean ± SD of
the amplitude of DA-induced inward currents. SDs are shown with
bars. Numbers in parentheses refer to
numbers of tested cells. From the left, the current
induced by DA (100 µM) as a control (open
bar), that with DA plus SCH 23390 (10 µM)
(filled bar) in saline with TTX (0.5 µM), that with DA alone in a solution containing 27 mM Na+ (gray bar),
that in a solution containing Ba2+, that in a
solution containing Ba2+, Cs+,
Cd2+, and nifedipine (5 µM), that
recorded with a Cs+-filled pipette in a saline, and
that in a low Na+ (27 mM) solution.
Comparisons were made with the Student's t test against
the control group of neurons tested in the saline solution (*
p < 0.05; ** p < 0.01).
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The Na+ permeability was also tested with a
Cs+-containing patch pipette by lowering the
external Na+ concentration. Overall, the DA-induced
inward current in 26 mM Na+ was
10.7 ± 13.2 pA (n = 5), whereas that under high
Na+ conditions was 34.7 ± 18.1 pA
(n = 15) (Fig. 6), suggesting that a TTX-insensitive
Na+-permeable conductance may contribute partly to
the inward current. A similar cation conductance has been reported for
neurons of the prefrontal cortex of the rat (Shi et al., 1997) and
characterized as a nonspecific mechanism in that it was not sensitive
to classical DA agonists and antagonists. However, application of
(±)-SKF38393 (5 µM), an agonist of D1-like
DA receptor, elicited a small DA-induced inward current in
Ba2+-containing solution in one of four cells
tested, suggesting that this possibility is unlikely in this cell.
Effects of a D5/D1 DA receptor
agonist on striatal LA cells
The effects of (±)-SKF38393 were tested in 108 striatal LA cells
to allow comparison with the DA-induced membrane depolarization. Four
cells were also analyzed for the effects of a mixture of DA (100 µM) plus sulpiride (10 µM), a
D2-like receptor antagonist, but the results were similar
to those obtained with (±)-SKF38393 (data not shown). In the current
clamp mode, (±)-SKF38393 (10 µM) depolarized 10 of 11 cells (9.2 ± 4.9 mV; n = 10) and elicited action
potentials in four cells (Fig.
7A). (±)-SKF38393 (1-50 µM) evoked an inward current in the whole-cell clamp
configuration in a dose-dependent manner. The amplitude clamped at the
resting membrane potential (60 mV) was 17.3 ± 31.3 pA
(n = 12), 19.1 ± 30.2 pA (n = 8), 41.7 ± 52.1 pA (n = 40), and 53.1 ± 39.4 pA (n = 6) at 1, 3, 10, and 50 µM,
respectively. The current-voltage relations were also similar to those
with DA. The I-V curves of the net
SKF38393-induced current of 10 of 14 cells reversed the sign at
88.26 ± 24.7 mV with a negative slope conductance (Fig. 7B). On the other hand, those of the remaining four cells
did not cross the voltage axis within the voltage range tested (120 to 40 mV) (Fig. 7C). Also, the I-V
curve of the net outward current elicited by administration of SCH23390
was just the opposite of the SKF38393-induced current (data not shown).
These results suggest that, as in the DA case, SKF38393 evokes an
inward current by two separate ionic mechanisms: blockade of
K+ conductance and opening of nonselective cation
conductances.
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Figure 7.
Effects of the D1-like agonist,
SKF38393, on striatal LA neurons. A, A whole-cell
current-clamp recording with a resting membrane potential of 65 mV
illustrates a slowly rising, prolonged, and reversible membrane
depolarization with action potentials occurring during the peak of the
response. B1, C1,
Voltage-clamp traces (holding potential 60 mV) recorded from another
LA cell in saline containing TTX (0.5 µM) illustrate a
slow inward current induced by SKF38393. Two superimposed
I-V plots before (open
circles in B2 and 1 in
C2) and during the peak of the response
(filled circles in B2 and
2 in C2) show a reversal
potential close to EK
(B2) and no reversal potential within the tested
voltage range (C2). B3,
C3, I-V plots of
the net SKF38393-induced currents (filled
triangles in B3 and 2-1
in C3).
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Forskolin mimics and occludes the (±)-SKF38393-induced
inward current
Because D1-class DA receptors are known to positively
couple with G-proteins and an adenylyl cyclase-cAMP cascade, the
effects of (±)-SKF38393 on the membrane excitability of LA cells might also be mediated by this signaling pathway. To test this possibility, we bath-applied forskolin, a lipophilic adenylyl cyclase activator, to
the cells at 10 µM (Fig.
8A). A large inward
current was evoked in all cells tested (98.1 ± 82.3 pA;
n = 13), whereas dideoxyforskolin, an inactive
enantiomer of the forskolin, caused no response at 10 µM
(1.2 ± 2.2 pA; n = 3). The average
I-V curve of the net forskolin-induced current
was qualitatively similar to those obtained with DA and (±)-SKF38393
(data not shown). The current decreased with membrane depolarization,
but no reversal of polarity was seen within the tested voltage range.
Furthermore, it was found that the effects of (±)-SKF38393 were no
longer evident after the forskolin current reached a steady state (Fig.
8B). These results suggest that adenylyl cyclase
transduces D1-like DA receptor-mediated effects in striatal
LA cells.
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Figure 8.
Forskolin, an activator of adenylyl cyclase,
mimics and occludes the effects of SKF38393 on the striatal LA cells.
A, Dideoxyforskolin (10 µM,
stippled bar), an inactive forskolin analog, evoked no
response, whereas forskolin (10 µM, filled
bar) caused a slow and reversible inward current. Holding
potential was 60 mV. B, Application of SKF38393
(filled bars) resulted in a slow inward current
(left trace). Fifteen minutes after washout of SKF38393
(10 µM), treatment with forskolin (10 µM,
stippled bar) elicited a large slow inward current and
occluded the effect of the second application of SKF38393.
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SQ22536, an adenylyl cyclase inhibitor, reduces
D5/D1 DA receptor-mediated inward
currents
If adenylyl cyclase mediates the effects of (±)-SKF38393,
its inhibition should reduce the actions of (±)-SKF38393. In fact, application of 9-(tetrahydro-2-furyl) adenine (SQ22536), an agent that
is known to decrease the activity of adenylyl cyclase, reduced the
amplitude of the (±)-SKF38393-induced inward current. Neostriatal slices were preincubated in an oxygenated saline containing SQ22536 (300 µM) for 30 min to 6 hr. Control slices were also
taken from the same animals and incubated in the saline in the same
manner. Recordings were made alternately. The amplitudes of the
(±)-SKF38393-induced currents in SQ22536-treated cells were 9.6 ± 13.9 pA (n = 5), 8.9 ± 13.9 pA
(n = 5), and 10.9 ± 14.2 pA (n = 6) at 3, 10, and 50 µM, respectively. In contrast,
those in control cells were 3.1 ± 6.0 pA (n = 6), 38.9 ± 65.0 pA (n = 6), and 53.1 ± 39.4 pA (n = 6; p < 0.05) at 3, 10, and 50 µM, respectively. These results provide further
evidence that activation of adenylyl cyclase is required to
elicit the actions of (±)-SKF38393.
BAPTA has no effects on D5/D1 DA
receptor-mediated inward currents
Dopamine D1-like receptors have been observed to
increase the production of inositol triphosphates and mobilize
intracellular Ca2+ in oocytes from Xenopus
laevis with messenger RNA isolated from rat striatum (Mahan et
al., 1990). In addition, activation of D1-like receptors
has been reported to be associated with the facilitation of L-type
Ca2+ channels, an effect mediated through a
cAMP-dependent protein kinase in chromaffin cells (Artalejo et al.,
1990) and in a subset of rat neostriatal neurons (Surmeier et al.,
1995). Therefore, to test whether a rise in intracellular
Ca2+ mediated the effects of D1-like
receptor stimulation in the cells, BAPTA, an agent that is known to
chelate cytosolic Ca2+ ions, was included in patch
pipettes at 10 or 20 mM, and the actions of (±)-SKF38393
(10 µM) were compared with those of forskolin (10 µM), and substance P (0.5 µM) as a control,
which is known to elicit an inward current in LA cells (Aosaki and
Kawaguchi, 1996). As shown in Figure 9,
our data clearly showed no signs of suppression of the
(±)-SKF38393-induced currents evoked by (±)-SKF38393 in all cells
tested [100.7 ± 78.5 pA at 10 mM BAPTA (n = 5), 56.7 ± 32.2 pA at 20 mM
BAPTA (n = 5)]. These results suggest that a rise in
cytosolic Ca2+ ions is not associated with the
observed actions of (±)-SKF38393 in LA cells.
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Figure 9.
Slow inward currents evoked by SKF38393 (10 µM), substance P (0.5 µM), and forskolin
(10 µM) are unaffected by intracellular application of
BAPTA (20 mM), a potent Ca2+
chelator.
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It is thought that activation of D2 DA receptor inhibits
ACh release in the striatum. We therefore examined the actions of two
kinds of D2-like DA receptor agonists, such as quinpirole (1-20 µM) and bromocriptine (10 µM), to
study whether the D2-like DA receptor activation causes any
effect on the membrane excitability of striatal cholinergic neurons.
Unexpectedly, however, quinpirole elicited mixed results such as an
inward current (7/17 cells), no response (3/17 cells), an inward
current followed by an outward current (1/17 cells), and an outward
current (6/17 cells), whereas bromocriptine caused no response in all
cells tested (four cells). Moreover, combined application of quinpirole
and sulpiride produced inconsistent results (data no shown). These
results imply that the site of action of D2-like agents
might be primarily at the presynaptic axon terminals of the LA cells.
Further investigation is needed on this matter.
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DISCUSSION |
The present study demonstrates that DA induces a membrane
depolarization/inward current in striatal cholinergic interneurons via
activation of postsynaptic D1-like DA receptors. Another
novel finding is that a depolarization/inward current evoked by
D1 class receptor activation is caused by closing a resting
K+ conductance and opening a nonselective cation
conductance via an adenylyl cyclase-cAMP-dependent pathway. These
results support the idea that D1-class receptors located on
the neostriatal cholinergic interneurons might participate in
enhancement of membrane excitability, thereby facilitating ACh
release.
D1-class DA receptors (D1,
D5) are known to activate adenylyl cyclase, whereas
D2-like receptors (D2,
D3, D4) inhibit, or are not
coupled to, adenylyl cyclase. Our conclusion that DA acts on
D1-like receptors on the LA neurons is based on the
following observations. First, the effect of DA was blocked by the
D1-like antagonist SCH23390 and mimicked by the
D1-like agonist SKF38393 or DA plus sulpiride, a
D2-like antagonist. Second, SCH23390 itself elicited
an outward current with an opposite I-V relation
to that obtained with DA or SKF38393 (data not shown). Third, our
finding that forskolin, which stimulates adenylyl cyclase activity and increases cAMP levels, mimicked the effects of DA and SKF38393 and the
inactive congener 1,9-dideoxyforskolin had no effects, provides further
support for the involvement of D1-like receptors. Requirement of adenylyl cyclase activation for expression of the effects of SKF38393 was also confirmed by the fact that SQ22536, which
decreases the adenylyl cyclase activity, reduced the SKF38393-induced current. In addition, D2-like agonists such as quinpirole
and bromocriptine elicited inconsistent effects on the membrane
excitability, suggesting that the site of action of the
D2-like agents might be primarily at the presynaptic axon
terminals of the striatal cholinergic interneurons.
The present study showed that membrane depolarization might involve at
least two ionic mechanisms: blockade of resting K+
conductance and activation of nonselective cationic conductances. The
former appears to predominate in striatal LA cells, because the
Ba2+-resistant component of the DA-induced current
was only around 20 pA, whereas the control DA current was
approximately 60 pA (Fig. 6). Reduction of Na+
concentration in the external solution revealed that (1) the current
was decreased with hyperpolarizing potentials and reversed at a
potential close to the estimated EK: (2) this
current was associated with a decrease in membrane conductance; and (3)
the reversal potential shifted to less negative potentials in solutions of elevated K+, as expected for
K+ conductances. A comparable enhancement of cell
excitability, mediated by suppression of a K+
conductance, has been suggested previously from experiments with cultured substantia nigra pars reticulata neurons (Kim et al., 1995),
in which a D1 receptor agonist reduced an inwardly
rectifying K+ conductance. However, this mechanism
cannot apply to the data presented here because the
I-V relationships did not show any anomalous
rectification in the striatal LA cells and these have no detectable
inward rectifier (Jiang and North, 1991; Aosaki and Kawaguchi,
1996).
Our data revealed an additional Ba2+-resistant
component, considered to be a nonselective cation conductance because
(1) the DA-induced current showed a linear I-V
relation with a positive slope conductance and a reversal potential of
around 40 mV in 10 of 23 cells and the
Ba2+-resistant current reversed at 28 mV in one
cell, and (2) the current component was associated with an increase in
membrane conductance. However, in 10 of 11 cells the component
displayed an atypical I-V relationship: between
35 and +20 mV, the current remained inward and showed a negative
slope conductance. This cannot be attributed to activation of
voltage-dependent Ca2+ channels because it was
observed even in the presence of Cd2+ in the
external solution. Therefore, this might be attributable to a space
clamp error or to its own voltage-dependent property, because
activation of the similar nonselective cation conductance by DA has
been reported in LB-cluster neurons of Aplysia (Matsumoto et
al., 1988). In this latter case, D1 receptor stimulation
produced an increase in permeability, mainly to Na+,
to elicit a cAMP-dependent inward current. This depolarizing response
was also associated with negative slope conductance at more depolarized
potentials. In addition, a similar atypical I-V relationship was reported for the ionic mechanism of an inward current
elicited by muscarinic receptor activation (Haj-Dahmane and Andrade,
1996) and a DA-induced inward current (Shi et al., 1997) in prefrontal
cortical neurons. It is noteworthy, however, that, unlike the striatal
LA neurons, the DA-induced inward current observed in the prefrontal
neurons was not sensitive to any of the classical DA agonists and
antagonists (Shi et al., 1997). The mechanism proposed here (block of
resting K+ conductance and activation of
nonselective cationic conductances) has been proposed for other
neurotransmitters (Benson et al., 1988; Larkman and Kelly, 1992;
Guérineau et al., 1994, 1995; Dong et al., 1996; Kolaj et al.,
1997). Taking into consideration recent studies showing that the
striatal cholinergic interneurons possess D5 and, to a
lesser extent, D1 receptors (Bergson et al., 1995; Yan et
al., 1997), the specific findings described here, compared with those
reported for actions of conventional D1 receptors, might
stem from the unique properties of D5 receptors.
Recent microdialysis studies have consistently indicated that
application of DA, whether in vivo or in vitro,
increases net ACh release and that this is mediated by activation of
D1-like receptors (Ajima et al., 1990; Bertorelli and
Consolo, 1990; Consolo et al., 1992; Zocchi and Pert, 1993). However,
there are several possible mechanisms of D1-like
receptor-mediated control of ACh release. First, several in
vivo studies have provided evidence of the importance of glutamate
released from extrastriatal pathways such as in the parafascicular
nucleus of the thalamus, cerebral cortex, and substantia nigra pars
reticulata (Damsma et al., 1991; Consolo et al., 1996a,b; Abercrombie
and DeBoer, 1997). Second, it has been demonstrated that local release
of substance P from the terminals of medium spiny neurons, which
contain D1-like receptors, increases the ACh release
(Arenas et al., 1991; Guevara Guzman et al., 1993; Anderson et al.,
1994; Steinberg et al., 1995; Khan et al., 1996). Finally, direct
activation of the D1-like receptors on the cholinergic
neurons might directly facilitate the ACh release (Di Chiara et al.,
1994), as suggested in this paper. However, in vitro studies
using striatal slice preparations similar to ours have generated
conflicting results. Although SKF38393 stimulated K+-evoked ACh release in neostriatal slice
preparations at concentrations of 1 µM (Stoof and
Kebabian, 1982) and 10 µM (Gorell and Czarnecki, 1986),
it was found to be without effect, up to 100 µM, by
Scatton (1982). Electrically evoked release of ACh has also been found to be reduced by D1-like antagonists (Consolo et al., 1987)
or remain unchanged by D1-like selective agents (Dolezal et
al., 1992; Tedford et al., 1992). These discrepancies might be
attributable to differences in the experimental conditions applied and
difficulties in controlling various related factors. In this regard,
the studies of Login and colleagues (1995, 1996) are noteworthy. They
developed a dissociated striatal neuron preparation to monitor
fractional ACh efflux, with isolation of the activity of cholinergic
neurons per se, and found that 50 µM (±)-SKF38393
increased the basal rate of release by 50% and that this was inhibited
by a D1-like antagonist. Our observations, therefore,
provide further support for their conclusion that ACh is secreted via
direct activation of D1-like receptors on cholinergic cells
and provide an explanation of how it might occur. The question now is
whether this mechanism is feasible under physiological conditions.
Evidence has accumulated that most D1 and D5 DA
receptors are located outside synaptic contacts formed by dopaminergic
terminals (Bergson et al., 1995; Hersch et al., 1995; Caillé et
al., 1996), suggesting that a nonsynaptic dopaminergic neuromodulation
predominates in the striatum. Recent electrochemical measurements of DA
efflux from synaptic clefts have shown that DA released by bursts of action potentials diffuses up to 12 µm from release sites reaching a
homogeneous DA concentration of 0.2 to 1 µM in the
extrasynaptic extracellular space before elimination by reuptake
(Garris et al., 1994; Gonon, 1997). Because it is known that DA binds
to D5/D1b receptors with an affinity
that is highest among DA receptor subtypes (for example, 3- to 10-fold
higher than that of DA binding to D1 receptors) (Seeman and
Van Tol, 1994), this concentration range would be high enough to
depolarize striatal cholinergic neurons. These are characterized by
shallow resting membrane potentials (approximately 60 mV) and slow
irregular but tonic (3-10 Hz) spontaneous activity, and therefore
small (0.5-5 mV) EPSPs could easily trigger their action potentials
(Wilson et al., 1990). Furthermore, activation of D1-like
receptors has been reported to potentiate the NMDA component in
striatal neurons (Cepeda et al., 1993), which might further stimulate
ACh release, because an NMDA component of the EPSPs is already fully
operative at rest in these neurons (Kawaguchi, 1992). Taking all the
available data together, we propose that DA directly activates
D1-like receptors (mainly D5), thus
depolarizing the striatal cholinergic neurons by blocking resting
K+ and opening nonselective cation conductances and
making them ready to respond to EPSPs driven by thalamic, cortical, and
nigral input.
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FOOTNOTES |
Received March 10, 1998; revised April 27, 1998; accepted April 28, 1998.
This work was supported by the Frontier Research Program and
Grants-in-Aid 07458217 (B) and 07279250 for Scientific Research in the
Priority Area of "Functional Development of Neural Circuits" of the
Ministry of Education, Science, Sports and Culture of Japan. We thank
Ms. Naoko Wada for technical assistance.
Correspondence should be addressed to Dr. Toshihiko Aosaki, Laboratory
for Neural Circuits, Bio-Mimetic Control Research Center, The Institute
of Physical and Chemical Research (RIKEN), 2271-130, Anagahora,
Shimoshidami, Moriyama-ku, Nagoya, Aichi 463-0003, Japan.
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Abstract
Introduction
