 |
 Previous Article | Next Article 
The Journal of Neuroscience, August 1, 1998, 18(15):5575-5585
D2-Like Dopamine Autoreceptor Activation Reduces
Quantal Size in PC12 Cells
Emmanuel N.
Pothos1, 2, 3,
Serge
Przedborski1,
Viviana
Davila1,
Yvonne
Schmitz1, and
David
Sulzer1, 2, 3
Departments of 1 Neurology and
2 Psychiatry, Columbia University, New York, New York
10032, and 3 Department of Neuroscience, New York State
Psychiatric Institute, New York, New York 10032
 |
ABSTRACT |
D2-like dopamine autoreceptors regulate dopamine
release and are implicated in important actions of antipsychotic drugs
and rewarding behaviors. To directly observe the effects of
D2 autoreceptors on exocytic neurotransmitter release, we
measured quantal release of dopamine from pheochromocytoma PC12 cells
that express D2 and D4 autoreceptors. High
potassium-evoked secretion in PC12 cells produced a unimodal population
of quantal sizes. We found that exposures to the D2-like
agonist quinpirole that inhibited tyrosine hydroxylase activity by
~50% also reduced quantal size by ~50%. The reduced quantal size
was blocked by the D2 antagonist sulpiride and reversed by
L-DOPA. Quinpirole also decreased the frequency of
stimulation-evoked quantal release. Together, these findings indicate
effects on quantal neurotransmission by D2-like dopamine autoreceptors previously distinguished as synthesis-modulating autoreceptors that regulate tyrosine hydroxylase activity versus impulse-regulating autoreceptors that modulate membrane potential. The
results also provide an initial demonstration of a receptor-mediated mechanism that alters quantal size.
Key words:
quantal size; PC12; dopamine; synaptic vesicle; amperometry; electrochemistry; D2 receptors; quinpirole; sulpiride
| |
INTRODUCTION |
D2-like dopamine (DA)
autoreceptors that inhibit stimulation-dependent DA release are present
in mesencephalic DA dendrites (Saller and Salama, 1984 ; Nissbrandt et
al., 1985; el Mestikawy and Hamon, 1986; Strait and Kuczenski, 1986)
and presynaptic sites of mesolimbic terminals (Goldstein et al., 1990;
Tepper and Groves, 1990; Westerink et al., 1990; Chen et al., 1991;
Mercuri et al., 1992; Sesack et al., 1994). Because D2-like
autoreceptors are thought to play important roles in the action of
antipsychotics (Hetey et al., 1991), psychostimulant drugs (Brodie and
Dunwiddie, 1990; Bernardini et al., 1991; Seutin et al., 1991; Yamada
et al., 1991; Giambalvo, 1992; Zhang et al., 1992; Pitts et al., 1993;
Silvia et al., 1994; Xue et al., 1994), and sexual behavior (Hull et
al., 1990), the mechanisms by which autoreceptors mediate synaptic DA
release have received much attention. However, brain D2-like receptors are located both presynaptically on DA
afferents and postsynaptically on cortical and striatal neurons, making it difficult to isolate effects of autoreceptor activation on neurotransmitter release (Palij et al., 1990; Timmerman et al., 1990;
Santiago and Westerink, 1991). Some reports suggest that D2
autoreceptors on mesocortical DA afferents may primarily hyperpolarize membrane potential (impulse-regulating autoreceptors) (Chiodo et al.,
1984; Bean and Roth, 1991; Fedele et al., 1993), whereas mesostriatal
afferents and cell body autoreceptors may modulate both membrane
potential (Bernardini et al., 1991; Cass and Zahniser, 1991; Kennedy et
al., 1992; Tanaka et al., 1992; Cardozo, 1993) and the affinity of
tyrosine hydroxylase (TH) for tyrosine (synthesis-modulating autoreceptors) (Bohmaker et al., 1989; Onali and Olianas, 1989; Salah et al., 1989; Johnson et al., 1992; Tissari and Lillgèls, 1993).
Recently, advances in electrochemical techniques have enabled the
detection of vesicular release of monoamine transmitters in real time
from chromaffin cells (Wightman et al., 1991; Chow et al., 1992), mast
cells (Alvarez de Toledo et al., 1993), PC12 pheochromocytoma cells
(Chen et al., 1994), invertebrate neurons (Bruns and Jahn, 1995; Chen
et al., 1995), pancreatic cells (Zhou and Misler, 1996), mammalian
sympathetic neurons (Zhou and Misler, 1995), and mammalian midbrain
neurons (Pothos and Sulzer, 1998; Pothos et al., 1998). Zhou et al.
(1994) have used these techniques to demonstrate a direct
relationship between norepinephrine release and adrenergic autoreceptor
activation in isolated chromaffin cells. Because PC12 cells express
D2-like receptors that can inhibit DA release after
exposure to the agonist quinpirole (Courtney et al., 1991), we adapted
this technique to examine the effect of D2 autoreceptor
activation on quantal DA release. We report that receptor activation
reduces the amount of transmitter released per quantum, a previously
unexplored potential mechanism of synaptic plasticity.
| |
MATERIALS AND METHODS |
PC12 culture. PC12 cells were obtained from Dr. Lloyd
Greene (Columbia University) and maintained as described (Greene et al., 1991). For electrochemical experiments, 40,000 cells were plated
per culture on glass coverslips (Carolina Biological Supply, Burlington, NC), coated with 40 µg/ml poly-D-lysine
(Sigma, St. Louis, MO), and recoated with 10 µg/ml laminin
(Collaborative Research, Bedford, MA). Cultures were maintained in
media containing phenol red-free RPMI 1640 media supplemented with 10%
heat-inactivated horse serum, 5% fetal bovine serum (JRH Biologicals,
Lenexa, KS), and 50 U/ml penicillin and streptomycin (Life
Technologies, Grand Island, NY) in 7.5% CO2 atmosphere at
37°C. Experiments were performed on undifferentiated cells at least
5 d after plating, and compared cultures were derived from the
same source culture. HPLC measurements of the cultures, which are grown
in the absence of the DA  -hydroxylase cofactor ascorbic acid,
indicate that <5% of catecholamines are norepinephrine and that the
remainder is DA (Pothos et al., 1996).
RT-PCR methods. For total RNA extraction from PC12 cultures,
a 60-mm-diameter tissue culture dish (Falcon) coated with 10 µg of
Vitrogen-100 collagen/cm2 (Collagen Biomedical, Palo
Alto, CA) was plated with 2 × 106 PC12 cells.
The cells were cultured for 1 week. The monolayer was rinsed with PBS,
and 5 × 105 cells were harvested by
trituration in 1 ml of the saline. One milliliter of Trizol reagent
(Life Technologies) was added to the cell suspension. The tissue was
homogenized by trituration through a 28.5 gauge needle, 200 µl of
chloroform was added, and the RNA was precipitated from the aqueous
phase after centrifugation (12,000 × g for 15 min)
with isopropanol.
For total RNA extraction from brain tissue, 230 mg of tissue from rat
ventral striatum was dissected from postnatal day 1 pups and washed in
PBS. RNA extraction with Trizol reagent was performed as above, using 7 ml of Trizol and 1.4 ml of chloroform, and the tissue was homogenized
by trituration with an 18 gauge needle.
mRNA from the total RNA sample was isolated using the Poly ATtract mRNA
isolation system (Promega, Madison, WI) following the manufacturer's
instructions. Reverse transcriptase and reagents for cDNA preparation
from mRNA were obtained from the Superscript preamplification system
kit (Life Technologies). Twenty microliters of the reaction mix were
incubated at 42°C for 50 min, and the reaction was terminated by
heating to 70°C for 15 min. The sample was then chilled on ice for 2 min, and the remaining RNA was degraded by incubation at 37°C for 20 min with RNase H.
For PCR amplification, we used custom primers (Life Technologies) with
published sequences (Surmeier et al., 1996). For thermal cycling we
used a Perkin-Elmer (Emeryville, CA) thermal cycler. Incubation times
and temperatures were as follows: (1) 94°C for 4 min; (2) 45 cycles
of 94°C (1 min), 56°C (1 min), and 72°C (1.5 min); and (3) 72°C
for 10 min. Samples were then chilled at 4°C. Reagents were from
Promega.
Tyrosine hydroxylase assay. To estimate the effects of drug
exposure on TH activation in cultured cells, we adapted methods to
measure DOPA formation (Hayashi et al., 1990). In brief, sister cultures were rinsed twice with 1 ml of the physiological medium used
for the DA uptake assay (see below) and then incubated with DA agonists
or antagonists for 40 min at 37°C in 100 µM
L-tyrosine and 500 µM brocresine (Lederle
Laboratories, Pearl River, NY), a selective DOPA decarboxylase
inhibitor. After the incubation, medium was discarded, the cells were
covered with 1 ml of 0.1N PCA, collected, and sonicated. An aliquot (50 µl) was used to determine protein concentration (Lowry assay), and
the remainder was centrifuged at 15,000 × g at 4°C
for 10 min. A 20 µl aliquot of the supernatant was injected onto a
BAS Biophase ODS column (250 × 4.6 mm; 5 µm). Mobile phase
consisted of (in mM): 50 potassium phosphate, 0.1 EDTA, 0.2 sodium octylsulfonate, and 10% methanol, pH 2.6. L-DOPA
peaks were detected with an ESA Coulochem 5100A with a 5011 analytical
cell.
DA uptake assay. We measured uptake using
3H-labeled DA (Koide et al., 1986) in a physiological
medium (in mM: 125 NaCl or choline chloride, 4.8 KCl, 1.2 potassium phosphate, 1.3 CaCl2, 1.2 MgSO4, 1 sodium ascorbate, 5.6 glucose, and 25 HEPES, pH 7.3). Sister cultures of PC12 cells were incubated for 40 min
at 37°C in the appropriate physiological medium with or without
receptor agonists and antagonists. The cultures were rinsed twice with physiological medium containing 125 mM NaCl for total
uptake and 125 mM choline chloride for nonspecific uptake
and then incubated in the same medium for 10 min at 37°C. This was
replaced with the same medium containing a mixture of
3H-labeled and unlabeled DA (0.3 nmol and 0.7 µCi/ml
solution) for 5 min at 37°C. The reaction was stopped by placing the
cultures on ice, followed by two 1 min rinses with ice-cold medium.
Then, 2 ml of 1N NaOH were added to each dish, and the dishes were
shaken overnight. An aliquot of the resulting solubilized mixture was mixed with 5 ml of scintillation cocktail, and radioactivity was counted using liquid scintillation. Specific uptake was determined by
subtracting the nonspecific count and measuring total protein by the
Lowry assay. DA uptake is expressed as picomoles per minute per
milligram of protein.
Amperometric electrodes and apparatus. Carbon fiber
electrodes were constructed by aspirating 5 µm carbon fibers (Amoco,
Greenville, SC) into 1.2 × 0.68 mm glass capillary tubes (A-M
Systems, Everett, WA) that were then pulled with a Flaming-Brown
micropipette puller (Sutter Instruments, Novato, CA). The electrode tip
was dipped into epoxy (Epo-Tek 301; Epoxy Technology, Billerica, MA)
and cured at 100°C for 15 hr. The electrodes were back-filled with 3 M KCl. Electrode tips were polished at 40° on a beveler
(World Precision Instruments, New Haven, CT). Electrode response was tested by cyclic voltammetry in a freshly prepared, nitrogen-bubbled 10 µM DA solution, and those with unstable
I-V curves were rejected. The electrode was
gently pushed against the cell body with a Huxley-style micropositioner
(Newport Instruments, Irvine, CA).
A +700 mV voltage versus Ag-AgCl ground was applied to the carbon
fiber electrode using an Axon 200A amplifier (Axon Instruments, Foster
City, CA). The output was digitized at 50 kHz and low-pass-filtered at
10 kHz using an internal four-pole Bessel filter. The traces were
digitally filtered at 2.5 kHz and analyzed using a locally written
Superscope II program (GWI Instruments, Medford, MA). The average
background current in the vicinity of the spikes was subtracted from
the signal. Spikes were identified if their amplitude was 4.5 times
greater than the rms background current, typically 0.3 pA. The number
of molecules oxidized at the electrode face was determined by the
relation N = Q/nF, where
Q is the charge of the spike, n is the number of
electrons transferred (shown to be two for catecholamines when used in
a similar experimental configuration; Ciolkowski et al., 1994),
N is the number of moles, and F is Faraday's
constant (96,485 coulombs/equivalent).
For electrochemical recordings, physiological incubation medium
contained (in mM):150 NaCl, 2 KCl, 1.2 CaCl2, 1 MgCl2, 1 NaH2PO4, 25 glucose, and 10 HEPES, pH
7.3. Drug exposures were for the times indicated at 37°C. The
drug-containing medium or control medium was then removed and replaced
with physiological incubation medium, and cells were recorded at room
temperature (~25°C). The stimulation medium was composed of (in
mM): 72 NaCl, 80 KCl, 6 CaCl2, 21 glucose, and 10 HEPES, pH 7.3. The cells were stimulated by 6 sec
superfusion of ~20 nl of stimulation medium at a distance of 18 µm
from the recorded cell (Picospritzer; General Valve, Fairfield, NJ).
Low pressure (<7-8 psi) was applied to avoid mechanical stimulation.
Five second baseline durations were recorded before stimulation to
identify spontaneously active cells. Two stimulations, spaced 20 sec
apart, were administered. Interspike intervals and the n of
events are only reported for the initial 20 sec period.
Statistics. Values are expressed as mean ± SEM.
Kolmogorov-Smirnov statistics, exponential fits, regression
analysis, and ANOVA were performed using GB-Stat (Dynamic Microsystems,
Silver Springs, MD). Box diagrams were used to display the range of all mean values for the cell populations; median values were indicated as a
point within the box, 25 and 75% values of the distribution were
indicated by the box edges, and 10 and 90% values were indicated by
the ends of the upper and lower lines. Cells that released less than
five events within the 20 sec after stimulation were excluded from
analysis.  2 values were determined by the formula
[(observed  expected)2/expected] with means as the
expected values.
| |
RESULTS |
Identification of DA autoreceptor mRNA
There are three known D2-like DA receptors, classified
as D2, D3, and D4
receptors (O'Dowd et al., 1994). To determine the expression of
D2 DA autoreceptors expressed in PC12 cells we used RT-PCR
to identify cDNA transcripts, adapting methods used in the striatum
(Surmeier et al., 1996). PC12 cells expressed message for
D2 and D4 receptors at the predicted molecular
weights (D2 long and short forms, 404 and 317 bp;
D4, 223 bp) with no detectable D3
message (Fig. 1). As a control, we also
examined D2-like mRNA in neonatal rat ventral striatum
using the same primers. The striatum displayed D3 message
(461 bp) in addition to D2 and D4 message.
View larger version (69K):
[in this window]
[in a new window]
|
Figure 1.
PCR analysis of D2-like receptors in
PC12 cells. RT-PCR analysis indicates that PC12 cells display message
for D2 (long and short forms) and D4 DA
receptors at the expected molecular weight (Materials and
Methods). No D3 message was observed. Ventral striatum
(postnatal day 1) displays message for all three D2-like
receptors. The ladders on either side indicate 100 bp intervals for
scaling. The 200 and 400 bp bands are indicated by
asterisks.
|
|
Effect of autoreceptor activation on TH activity
We recently found that the TH product L-DOPA increases
quantal size in PC12 cells by increasing cytosolic DA synthesis (Pothos et al., 1996). Interestingly, TH activity can be modulated by a
D2 receptor-mediated pathway (Chiodo et al., 1984;
Goldstein et al., 1990; Booth et al., 1994). To directly observe
modulation of TH activity by D2 activation, the TH product
L-DOPA was measured in the presence of an aromatic acid
decarboxylase inhibitor that blocked L-DOPA conversion to
DA (Arbogast et al., 1993). The D2 agonist quinpirole (0.5 µM for 40 min) decreased TH activity to 59% of control
levels (Fig. 2;
F(3,8) = 35.5; p < 0.0001 by
one-way ANOVA). The effect of quinpirole was completely blocked by
coincubation with the D2 antagonist sulpiride. We chose
this level of TH inhibition as appropriate for further study.
View larger version (54K):
[in this window]
[in a new window]
|
Figure 2.
Effect of quinpirole on TH activity. PC12 cultures
were exposed to tyrosine (100 µM for 40 min) in the
presence of the DOPA decarboxylase inhibitor brocresine (500 µM). After incubation and extraction, L-DOPA
was measured by HPLC. Quinpirole decreased L-DOPA synthesis
to 59% of controls. The inhibition was blocked by coincubation with
the D2-like antagonist sulpiride (40 µM).
n = 5 for each condition; error bars indicate SEM;
*p < 0.0001 by one-way ANOVA.
|
|
Effect of autoreceptor activation on DA uptake
DA uptake in synaptosomes (Krueger, 1990) and Xenopus
oocytes transfected with DA transporter (Zhu et al., 1995; Sonders et al., 1997) is regulated by transmembrane potential. Therefore, another
possible result of D2 activation could be to modulate uptake by the plasma membrane catecholamine uptake transporter by
altering ionic currents. We examined potential D2
modulation of DA uptake by measuring uptake of low levels (300 fmol) of
tritiated DA in the cultures; this low exposure would not saturate the
transporter or significantly affect tonic extracellular DA in the
cultures, which is ~10 nM (Pothos et al., 1996). We found
that neither quinpirole nor sulpiride significantly altered
accumulation of labeled DA (Fig. 3).
View larger version (73K):
[in this window]
[in a new window]
|
Figure 3.
Effect of quinpirole on DA uptake. PC12 cultures
were exposed to a combination of unlabeled and
[3H]DA (300 fmol) in physiological medium for 5 min. DA uptake was halted by replacing with ice-cold physiological
medium, and the cells were solubilized with PCA. Background DA uptake
was determined in saline in which choline replaced sodium. Exposure to
either sulpiride or quinpirole did not alter specific DA uptake.
n = 5 for each condition; error bars indicate
SEM.
|
|
Quantal release from PC12 cells
To directly observe quantal release from undifferentiated PC12
cells, we used amperometric recording after low-pressure superfusion of
high-K+ medium. This resulted in current spikes
(Fig. 4A) that were
clearly resolved as individual events at an expanded time scale (Fig. 4B). Spikes were characterized by the number of
molecules represented (molecules × 103),
amplitude (Imax), width at half-height
(t1/2), and the interval between
successive events (intspike) for those spikes within
20 sec after stimulation (Table 1). A
low-amplitude "foot" was occasionally observed to precede the full
current spike, particularly for higher-amplitude events (Fig.
4C), presumably because the events were not disguised in
baseline noise. However, in all cases, the detectable feet contributed
to <1% of release (Table 1). Such events have been observed in
recordings of chromaffin, mast cell, and pancreatic -cell exocytosis
(Chow et al., 1992; Alvarez de Toledo et al., 1993; Zhou and Misler,
1996) and are suggested to represent transmitter release from an
intermediate stage of fusion pore formation.
View larger version (22K):
[in this window]
[in a new window]
|
Figure 4.
Depolarization by high potassium elicits quantal
release in PC12 cells. A, Amperometric spikes after PC12
stimulation with 80 mM KCl for 6 sec (applied at
arrow). This representative control trace represents 15 spikes (Imax > 4.5 rms noise), ranging from
1.25 to 14.0 pA with a mean amplitude of 4.8 ± 1.0 pA and quantal
size of 201,000 ± 39,400 molecules. B, The portion
above the broken line in A is displayed
with an expanded time resolution. The quantal size
(#molecules), peak amplitudes
(Imax; in picoamperes), and width at
half height (t1/2; in milliseconds)
are displayed under four corresponding events. C, For
the sample event indicated with the asterisk in
B, the maximal amplitude is indicated by the top
broken line, and the half height is indicated by the
middle broken line. The foot, filled by an array of
points, is estimated by integrating the portion of the event preceding
the slope (broken line) between the 60 and 90%
rise-times (Chow and von Ruden, 1995). D, For control
cells (events in Table 1), the interspike intervals
(intspike; in milliseconds) are displayed in 100 msec bins on the x-axis. The percentage of intervals
within each bin (n/N) is labeled
on the y-axis. The decreased representation at longer
interval values is approximated by an exponential decay (see Materials
and Methods).
|
|
As expected, if release events were independent (Alvarez de Toledo and
Fernandez, 1990), there was a good fit of intspike values
during the first 1500 msec after stimulation to an exponential decay
[Fig. 4D; y = (0.256)e( 0.0027/t);
r2 = 0.96]. Given that the average spike
width at half-height (t1/2) in controls
is 6.7 msec, the probability that two or more independent events would
occur within this period can be estimated as:
where t = 6.7 msec; k = rate constant = 0.0027; and  = 1/k = 370 msec. Therefore, randomly
overlapping spikes are rare.
Effect of D2 autoreceptor activation on
quantal release
In preliminary liquid chromatography experiments that confirmed
the previous findings of Courtney et al. (1991), we found that
incubation of PC12 cultures with 0.5 µM quinpirole for 25 min reduced DA release in high-potassium saline to 54 ± 13% of control levels, and that this effect was completely inhibited by 40 µM sulpiride (data not shown).
As an initial approach, we analyzed quantal release by examining the
response of individual cells to successive stimulations (80 mM K+, 6 sec) at 0, 5, 15, 25, and 35 min in the presence of normal recording medium or 0.5 µM
quinpirole. Although >90% of PC12 cells displayed quantal release
after the first stimulation, only 19% (4 of 21 cells recorded) of the
control cells that responded to the first stimulation responded to a
second stimulation 5 min later. This was further reduced at three
subsequent 10 min intervals to 14% (3 of 21) of control levels at 15 min after the first release, to 10% (2 of 21) at 25 min, and to 5% (1 of 21) at 35 min; the decrease was significant at p < 0.0001; 2 = 44.97. Moreover, the number of events per
stimulation decreased in successive stimulations (mean, 40 events per
cell at 5 min, 7 events at 15 min, 4 events at 25 min, and 3 events at
35 min; the decrease was significant by ANOVA with repeated measures
(F(4,54) = 4.597; p < 0.01).
Similar results were found for quinpirole-treated cells (data not
shown). We conclude that although the recording configuration is
stable, refill of the releasable vesicle pool in PC12 cells is very
slow. This contrasts with bovine adrenal chromaffin cells, which, as we
and others have observed, continue to exhibit stable release after
numerous stimulations (Finnegan et al., 1996). This appears to be
because bovine chromaffin cells are densely filled with granules that
provide rapid replacement of docked vesicles (Steyer et al., 1997),
whereas PC12 cells have a smaller presence of mobile granules,
requiring increased time for replenishment of the releasable pool
(Burke et al., 1997).
An alternate successful approach was to compare bins of large groups of
quanta from multiple cells after single applications of secretagogue,
particularly because the quantal size distribution was found to fit a
lognormal distribution. To determine the population distribution of
quantal parameters in PC12 cells, we examined 2316 quantal events
elicited from 48 control cells (Table 1). As reported previously,
untransformed PC12 quanta and other amperometrically detected quanta do
not show a normal size distribution but are skewed to the right (Fig.
5H, left). Earlier
studies have shown that either a cubed root or log transformation of
the quantal amplitudes of postsynaptically recorded quanta (Van der
Kloot, 1991) or amperometrically recorded quantal sizes (Finnegan et al., 1996) results in a normal distribution. The suggested rationales are that (1) vesicle diameters are distributed normally; therefore, if
transmitter filling is proportional to volume, the population distribution should produce a cubed root normal distribution (Bekkers et al., 1990); and (2) if multiplicative deviations from the mean occur, this would produce a lognormal distribution (Van der Kloot, 1991); an example would be if integral differences in the number of
uptake transporters per vesicle resulted in multiplicative transmitter
accumulation rates. In the present case, we found that the lognormal
transformation resulted in a closer fit of the individual values to a
normal probability function than the cubed root transformation (Table
2). Therefore, the quantal size populations are lognormal and unimodal. Although a small number of
values at the extreme edges are not well described by this distribution, they are typically <1% of the events. In the case of
larger events that deviate from the lognormal distribution, this may be
attributable to overlapping multiple events. In a second statistical
test of the normal distribution of quantal sizes, the
Kolmogorov-Smirnov (K-S) test of normality showed a close fit
of the lognormal distribution to a Gaussian distribution (K-S
Z score = 2.0216; p < 0.05).
View larger version (33K):
[in this window]
[in a new window]
|
Figure 5.
Effects of quinpirole on quantal release. Examples
of traces recorded from (A) a control cell and
(B) a cell exposed to 0.5 µM
quinpirole for 40 min. Cells were stimulated by 6 sec applications of
80 mM KCl at the arrow. Quinpirole decreased
the mean quantal size of individual release events (Table 1).
C, Distribution of mean quantal size values for all
cells that displayed more than five events within 20 sec of stimulation
(controls, n = 34; quinpirole,
n = 18; sulpiride, n = 30;
quinpirole/sulpiride, n = 13).
D-G, The cumulative frequencies of
interspike intervals are compared between treatments. Quinpirole
shifted interspike values to longer durations. The effect was blocked
by sulpiride (B).
H-K, For each group, the distribution of
quantal sizes (#molecules/1000) is indicated as a
histogram of the untransformed quantal sizes (left
column) and the log values of those quantal sizes (right
column). Quinpirole shifted the frequency distribution of
quantal sizes to the left. The lower limit of each bin size is
displayed on the x-axis. The distribution of log
transformations in each case is closely approximated by a normal
distribution (r2  0.987; Table
2).
|
|
By comparing binned quantal sizes from multiple cells, we found that
quinpirole reduced the average quantal size to 51% of control values
(K-S Z = 2.886; p < 0.0001; Fig.
5B, Table 1), close to the inhibition of TH reported above.
The distribution of the mean quantal sizes from each cell measured is
indicated (Fig. 5C). Quinpirole was also observed to
decrease the maximum amplitude, width at half-height, and number of
molecules represented by the foot when detected (Table 1). We
reproduced this experiment four additional times and observed
quinpirole-induced reduction of quantal size to 49, 54, 62, and 74% of
control levels. In an experiment in which cells were exposed to 0.5 µM quinpirole for 150 min rather than 40 min, quantal
size was reduced to 31% of control levels (K-S Z = 4.499; p < 0.0001; n = 144 events from 13 control cells; n = 169 events from 25 quinpirole-treated cells).
To examine whether the effect of D2-like activation on
quantal size could be blocked, we used the D2-like
antagonist sulpiride. Sulpiride (40 µM) alone had no
effect on quantal size but blocked the effects of quinpirole (K-S
Z = 3.619; p < 0.0001); the
combination of quinpirole and sulpiride was not different from controls
(Table 1). In preliminary experiments, we found that lower
concentrations of sulpiride (2 and 10 µM) produced only
partial blockade (data not shown) (the affinity of quinpirole is 3.9 nM for D2 and ~12 nM for
D4; the affinity for sulpiride is 14.7 nM for D2 and 52 nM for
D4; O'Dowd et al., 1994).
Quinpirole also had a significant (K-S Z = 2.258;
p < 0.0001) effect on the frequency of release events
as measured by the interspike intervals (Fig. 5D; Table 1).
The reduction in frequency was also blocked by sulpiride (K-S
Z = 2.756; p < 0.0001; Fig. 5E,G). Sulpiride and
sulpiride-quinpirole treatments were not significantly different from
controls, although sulpiride exposure induced spontaneous release in a
fraction of PC12 cells, nearly the only instances of this observed. Of
the cells recorded after sulpiride exposure, 23% (9 of 39) showed more
than one release event in a 5 sec period before application of the
secretagogue; these were excluded from the analysis of interspike
intervals (Fig. 5, Table 1). The effect of sulpiride on release
suggests that low tonic levels of DA in these cultures may serve to
block spontaneous release by D2-like receptor
activation.
Effects of L-DOPA on quinpirole action
If quinpirole reduces quantal size by inhibiting TH, the effect
should be reversed by the TH product L-DOPA. We exposed
PC12 cultures to 50 µM L-DOPA for 30 min,
which elevated the average quantal size by 250% (Fig.
6G-J; the total
population was increased, K-S Z = 14.628;
p < 0.0001) as observed in a previous study (Pothos et
al., 1996). The L-DOPA and L-DOPA-quinpirole
treatments did not produce different effects. The combination of
L-DOPA and quinpirole increased the average quantal size
over controls by 213% (K-S Z = 12.631;
p < 0.0001) and the average quantal size over
quinpirole-only treatments by 416% (K-S Z = 5.508, p < 0.0001). This effect is consistent with inhibition
of L-DOPA synthesis by D2 activation and also
demonstrates that quinpirole does not reduce quantal size by blocking
vesicular uptake of cytosolic transmitter. Surprisingly, L-DOPA blocked the decreased frequency of release elicited
by quinpirole (p < 0.0001; K-S
Z = 3.3015; L-DOPA-quinpirole was not
different from controls; Fig.
6D-F).
View larger version (26K):
[in this window]
[in a new window]
|
Figure 6.
Effect of L-DOPA on quinpirole action.
Examples of traces recorded from a cell exposed to L-DOPA
(50 µM for 40 min) (A) and a cell
that has undergone identical L-DOPA exposure in the
presence of quinpirole (0.5 µM for 40 min)
(B). Cells were stimulated by 6 sec applications
of 80 mM KCl at the arrow. C,
Distribution of mean quantal size values for cells that displayed more
than five events within 20 sec of stimulation (controls,
n = 34; quinpirole, n = 18;
L-DOPA, n = 25;
L-DOPA-quinpirole, n = 19). The
control and quinpirole-only groups are the same as those represented in
Figure 5. L-DOPA increased the mean quantal size of both
control and quinpirole-treated release events to a similar degree
(Table 1). D-F, Cumulative frequencies
of interspike intervals are compared between treatments.
L-DOPA blocked the effect of quinpirole on interspike
intervals. G-J, Distribution of quantal
sizes indicated as histograms of log transforms of the quantal size as
in Figure 5. For comparison, the control and quinpirole-treated groups
are also shown. L-DOPA shifted the frequency distribution
of quantal sizes to the right.
|
|
Although it might be assumed that DA itself would have quinpirole-like
effects, we found that 50 µM DA actually elevated quantal release (425,400 ± 18,300 molecules; n = 1691 events recorded from 49 cells), to levels similar to 50 µM L-DOPA. We presume that whereas DA itself
is an effective agonist at D2 receptors, it has multiple
actions including activation of D1-like receptors, which
are also present in PC12 cells (Inoue et al., 1992). In addition, it is
a substrate for the plasma membrane catecholamine uptake transporter of
these cells (Ramachandran et al., 1993) and the vesicular monoamine
transporter VMAT1 (Liu et al., 1994) and thus would be loaded
into the vesicles. Therefore, DA activation of D2-like
receptors does not provide a straightforward approach to analyzing
D2-mediated alteration of quantal size, mostly because of
the elevation of cytosolic stores of DA. We also found that including
sulpiride (40 µM) with DA had no effect on the DA
response (460,100 ± 20,400 molecules; n = 1365 events from 30 cells) and did not unmask D2-like mediated
effects on quantal size.
| |
DISCUSSION |
Modulation of synaptic neurotransmitter release can be
attributable either to effects on the number of quanta released or the
quantal size. The activation of a variety of neurotransmitter receptors
is known to alter the frequency of quantal release events (Thompson et
al., 1993). Although autoreceptors inhibit stimulation-dependent neurotransmitter release from a range of secretory cells, it is unclear
whether such effects are attributable solely to a reduction in the
number of quanta released, or if reduced quantal size might also play a
role. Here we report clear evidence indicating that autoreceptor
activation can reduce quantal size.
We are aware of four mechanisms by which D2-like
autoreceptors might mediate apparent quantal size: (1) D2
activation could decrease the occurrence of simultaneous release events
that are misidentified as single quanta; (2) D2 activation
could induce morphological changes resulting in an increased distance
of release from the electrode, causing the spikes to appear smaller
because of diffusion; (3) D2 activation could promote
release of a subpopulation of vesicles with lower levels of
transmitter; and (4) by inhibiting DA synthesis or vesicular
accumulation, autoreceptors could reduce the total amount of
neurotransmitter sequestered within vesicles and hence the number of
molecules released during the expression of the fusion pore.
Our results suggest that overlapping events do not explain altered
quantal size, because the exponential decay of the distribution of the
interspike intervals suggests that <2% of spikes in the control
preparations would overlap. The results are also inconsistent with
release from more distal sites. In the protocol used in this study, the
electrode was placed directly on the cell surface. If amperometric
current spikes were not directly under the electrode, they would have
shorter amplitudes and increased durations because of DA diffusion
(Schroeder et al., 1992); however, quantal release from
quinpirole-treated cells exhibited shorter amplitudes and decreased
duration. Release of a smaller subpopulation of vesicles is
inconsistent with the unimodal lognormal distribution of the populations. Although PC12 cells have both small (~50 nm diameter) and large (~150 nm diameter) vesicles, the small vesicles typically contain acetylcholine rather than DA (Schubert et al., 1980; Bauerfeind et al., 1993), which would not be detected. Our data support the hypothesis that quinpirole decreases quantal size by reducing the
amount of DA packaged and released per vesicle. A difference in
transmitter packaging is suggested by the leftward shift of the cubed
roots of quantal sizes, which is consistent with a reduction of the
amount of DA per vesicle volume. Moreover, this finding is congruent
with decreased release from an entire culture, as previously reported
(Courtney et al., 1991), confirming a genuine reduction of DA
release.
Mechanism of altered quantal size
A potential mechanism by which D2-like receptor
activation might alter the amount of DA packaged per vesicle is by
modulation of the activity of VMAT1. VMAT1 has been reported to be
inhibited by cAMP-dependent phosphorylation (Nakanishi et al., 1995);
however, the effect we observed is in the opposite direction of that
which would be predicted after D2 activation, which
decreases intracellular cAMP (Goldstein et al., 1990).
An alternate possibility stems from the idea that, because vesicular
transmitter accumulation is dependent on the transvesicular electrochemical gradient and substrate concentration gradient, D2 receptor activation might reduce the available pool of
cytosolic DA. We examined two mechanisms that could underlie altered
cytosolic pools, decreased reuptake and decreased synthesis. We found
that although D2 activation has been found to modulate DA
reuptake (McElvain and Schenk, 1992; Meiergerd et al., 1993; Cass and
Gerhardt, 1994; Wieczorek and Kruk, 1994), under the conditions used,
quinpirole did not inhibit the uptake of labeled extracellular DA by
the PC12 catecholamine transporter.
The second mechanism addressed was suggested by a large body of
evidence suggesting that D2 receptor activation inhibits DA synthesis by blocking cAMP-dependent PKA-mediated phosphorylation of
TH. PKA-mediated phosphorylation of the serine 40 residue activates TH,
whereas reduced PKA activity after D2 autoreceptor
activation causes a reduction in ser40 phosphorylation (Harada et al.,
1996). The reduced phosphorylation of ser40 in turn increases binding affinity of catecholamines to the iron moiety of TH resulting in a
feedback inhibition of enzymatic activity (Griffith and Schulman, 1988;
Haycock and Haycock, 1991; Daubner et al., 1992; Kumer and Vrana,
1996). Consistently, D2 autoreceptor effects have long been
known to inhibit DA synthesis (Chiodo et al., 1984; Goldstein et al.,
1990; Booth et al., 1994). The quinpirole exposures we used inhibited
TH activity to nearly the same level as the reduction in quantal size.
Therefore, we suggest that D2 activation decreases quantal
size by inhibiting TH activity and reducing cytosolic DA available for
vesicle accumulation.
We also noted a decreased quantal release frequency after
D2 activation that was blocked by sulpiride. Surprisingly,
L-DOPA also blocked the quinpirole-mediated decrease in
frequency. The mechanism by which L-DOPA produces this
response is not clear. It is possible that, rather than a reduction in
the rate of exocytosis, vesicle fusion after quinpirole proceeds at the
normal rate but includes quanta with small amounts of transmitter that
are below detection. These empty vesicles might then be filled if
elevated cytosolic DA were available after L-DOPA. However,
the ability of sulpiride to promote spontaneous release without a
change in quantal size is consistent with a D2 influence on
vesicle exocytosis. Additional potential explanations for the
L-DOPA reversal of the effects of quinpirole on frequency
are that very high levels of L-DOPA act as an antagonist at
D2-like receptors or that upregulated DA after
L-DOPA provides additional effects. An elegant approach to
this in the future may be provided by simultaneous patch capacitance and amperometric recording of monoquantal release (Albillos et al.,
1997).
Cellular and behavioral effects
Recent work indicates that the quantal size of catecholamine
release can be regulated by various pharmacological interventions. (1)
Transmitter accumulation is dependent on the magnitude of the vesicular
electrochemical gradient. Amphetamine reduces the quantal size of DA
release by collapsing the vesicular proton gradient (Sulzer et al.,
1995). (2) Quantal size can be increased by elevating cytosolic
neurotransmitter levels, and L-DOPA increases the quantal
size of DA release (Pothos et al., 1996, 1998). (3) We now provide
evidence that autoreceptor-mediated inhibition of neurotransmitter
synthesis decreases the quantal size of DA release. More generally,
although quantal size is often modeled as invariant (cf. Van der Kloot,
1991; Frerking et al., 1995) and is rarely invoked as a mechanism of
synaptic plasticity, we and others have found that regulation of
vesicular transporters (Song et al., 1997) and exposure to neurotrophic
factors (Wang et al., 1995; Pothos et al., 1998) alters quantal size.
These findings may provide insights into the synaptic changes induced
by psychostimulant and antipsychotic drugs. The psychostimulants cocaine and amphetamine elevate extracellular DA levels and activate mesolimbic and mesocortical D2 autoreceptors. Therefore,
psychostimulants may reduce appropriate stimulation-dependent
perisynaptic DA input to postsynaptic targets by decreasing quantal
size (Pothos and Sulzer, 1998) while increasing stimulation-independent
overflow of extrasynaptic DA. This has important implications for the
signal-to-background ratio of DA input; indeed, a precise level of DA
input is required for maintenance of working memory at mesocortical
connections, and it is striking that a feature of amphetamine psychosis
is an impairment in working memory (Williams and Goldman-Rakic,
1995).
Classically, D2-like autoreceptors have been classified as
displaying impulse-regulating or synthesis-modulating responses. The
data in this study are consistent with the possibility that both
impulse-regulating and synthesis-modulating responses occur in the same
cell population. D2 autoreceptors of mesolimbic DA neurons,
perhaps including D3 autoreceptors (Meller et al., 1993; Rivet et al., 1994), inhibit firing by hyperpolarization and increasing input resistance (Grace and Bunney, 1984a) in addition to inhibiting DA
synthesis. In the high-affinity state, D2 receptors have a binding constant of ~7.5 nM (O'Dowd et al., 1994).
Because background extracellular striatal DA levels are ~4
nM (Parsons and Justice, 1992), there may be significant
inhibition of DA release at tonic levels, and indeed the D2
antagonist sulpiride alone elevates DA release in awake, behaving
animals (Tanaka et al., 1992). Antipsychotic drugs that act as
D2-like antagonists elevate extracellular striatal DA
levels and increase DA cell firing rates after acute administration but
reduce extracellular DA and firing rate after longer term exposure. A
decreased quantal size would be expected to reduce stimulation-dependent diffusional overflow of DA from the release site.
Because monoamine synapses provide extrasynaptic transmitter overflow
(Garris et al., 1994), it is likely that alteration of quantal size
plays a more important role for monoamine release than fast-acting
transmitters.
In summary, these findings directly demonstrate that activation of
neurotransmitter receptors alters quantal size; this apparently occurs
by inhibition of transmitter synthesis. If a similar mechanism exists
in neurons, alteration of quantal size by autoreceptor activation would
appear to play a role in modulating synaptic plasticity.
| |
FOOTNOTES |
Received Feb. 10, 1998; revised May 8, 1998; accepted May 12, 1998.
This work was funded by National Institute on Drug Abuse (NIDA) Grants
07418 and 10154 (D.S.), an NIDA Shannon Award (D.S.), the Aaron Diamond
Foundation (E.N.P., D.S.), National Alliance for Research on
Schizophrenia and Depression (NARSAD) (E.N.P., D.S.), and the
Parkinson's Disease Foundation. E.N.P. is an Aaron Diamond Foundation
Fellow and recipient of a 1995 NARSAD Young Investigator Award. We are
grateful to Dr. Lloyd Greene for providing parent PC12 cultures, Dr.
Tjoeben Nygaard for use of his thermal cycler, Drs. James Surmeier and
Zhen Yan for advice on RT-PCR techniques, Dr. Michael Neystat for
advice on molecular techniques, and Dr. Steven Siegelbaum for
discussion of this manuscript.
Correspondence should be addressed to David Sulzer, Black Building 305, 650 West 168th Street, Columbia University, New York, NY 10032.
| |
REFERENCES |
-
Albillos A,
Dernick G,
Hostmann H,
Almers W,
Alvarez de Toledo G,
Lindau M
(1997)
The exocytic event in chromaffin cells revealed by patch amperometry.
Nature
389:509-512[Medline].
-
Alvarez de Toledo G,
Fernandez JM
(1990)
Compound versus multigranular exocytosis in peritoneal mast cells.
J Gen Physiol
95:397-409[Abstract/Free Full Text].
-
Alvarez de Toledo G,
Fernandez-Chacon R,
Fernandez JM
(1993)
Release of secretory products during transient vesicle fusion.
Nature
363:554-558[Medline].
-
Arbogast LA,
Soares MJ,
Robertson MC,
Voogt JL
(1993)
A factor(s) from a trophoblast cell line increases tyrosine hydroxylase activity in fetal hypothalamic cell cultures.
Endocrinology
133:111-120[Abstract/Free Full Text].
-
Bauerfeind R,
Regnier-Vigouroux A,
Flatmark T,
Huttner WB
(1993)
Selective storage of acetylcholine, but not catecholamines, in neuroendocrine synaptic-like microvesicles of early endosomal origin.
Neuron
11:105-121[Web of Science][Medline].
-
Bean AJ,
Roth RH
(1991)
Extracellular dopamine and neurotensin in rat prefrontal cortex in vivo: effects of median forebrain bundle stimulation frequency, stimulation pattern, and dopamine autoreceptors.
J Neurosci
11:2694-2702[Abstract].
-
Bekkers JM,
Richerson GB,
Stevens CF
(1990)
Origin of variability in quantal size in cultured hippocampal neurons and hippocampal slices.
Proc Natl Acad Sci USA
87:5359-5362[Abstract/Free Full Text].
-
Bernardini GL,
Gu X,
Viscardi E,
German DC
(1991)
Amphetamine-induced and spontaneous release of dopamine from A9 and A10 cell dendrites: an in vitro electrophysiological study in the mouse.
J Neural Transm
84:183-193[Web of Science][Medline].
-
Bohmaker K,
Puza T,
Goldstein M,
Meller E
(1989)
Absence of spare autoreceptors regulating dopamine agonist inhibition of tyrosine hydroxylation in slices of rat striatum.
J Pharmacol Exp Ther
248:97-103[Abstract/Free Full Text].
-
Booth RG,
Baldessarini RJ,
Marsh E,
Owens CE
(1994)
Actions of (±)-7-hydroxy-N,N-dipropylaminotetralin (7-OH-DPAT) on dopamine synthesis in limbic and extrapyramidal regions of rat brain.
Brain Res
662:283-288[Web of Science][Medline].
-
Brodie MS,
Dunwiddie TV
(1990)
Cocaine effects in the ventral tegmental area: Evidence for an indirect dopaminergic mechanism of action.
Naunyn Schmiedebergs Arch Pharmacol
342:660-665[Web of Science][Medline].
-
Bruns D,
Jahn R
(1995)
Real-time measurement of transmitter release from single synaptic vesicles.
Nature
377:62-65[Medline].
-
Burke NV,
Han W,
Li D,
Takimoto K,
Watkins SC,
Levitan ES
(1997)
Neuronal peptide release is limited by secretory granule mobility.
Neuron
19:1095-1102[Web of Science][Medline].
-
Cardozo DL
(1993)
Midbrain dopaminergic neurons from postnatal rat in long-term primary culture.
Neuroscience
56:409-421[Web of Science][Medline].
-
Cass WA,
Gerhardt GA
(1994)
Direct in vivo evidence that D2 dopamine receptors can modulate dopamine uptake.
Neurosci Lett
176:259-263[Web of Science][Medline].
-
Cass WA,
Zahniser NR
(1991)
Potassium channel blockers inhibit D2 dopamine, but not A1 adenosine, receptor-mediated inhibition of striatal dopamine release.
J Neurochem
57:147-152[Web of Science][Medline].
-
Chen JF,
Qin ZH,
Szele F,
Bai G,
Weiss B
(1991)
Neuronal localization and modulation of the D2 dopamine receptor mRNA in brain of normal mice and mice lesioned with 6-hydroxydopamine.
Neuropharmacology
30:927-941[Web of Science][Medline].
-
Chen TK,
Luo G,
Ewing AG
(1994)
Amperometric monitoring of stimulated catecholamine release from rat pheochromocytoma (PC12) cells at the zeptomole level.
Anal Chem
66:3031-3035[Medline].
-
Chen G,
Gavin PF,
Luo G,
Ewing AG
(1995)
Observation and quantitation of exocytosis from the cell body of a fully developed neuron in Planorbis corneus.
J Neurosci
15:7747-7755[Abstract].
-
Chiodo LA,
Bannon MJ,
Grace AA,
Roth RH,
Bunney BS
(1984)
Evidence for the absence of impulse-regulating somatodendritic and synthesis-modulating nerve terminal autoreceptors on subpopulations of mesocortical dopamine neurons.
J Neurosci
12:1-16[Abstract].
-
Chow RH,
von Ruden L
(1995)
Electrochemical detection of secretion from single cells.
In: Single-channel recording (Sakmann B,
Neher E,
eds), pp 245-276. New York: Plenum.
-
Chow RH,
Von Rueden L,
Neher E
(1992)
Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells.
Nature
356:60-63[Medline].
-
Ciolkowski EL,
Maness KM,
Cahill PS,
Wightman RM,
Evans DH,
Fosset B,
Amatore C
(1994)
Disproportionation during electrooxidation of catecholamines at carbon-fiber microelectrodes.
Anal Chem
66:3611-3617.
-
Courtney ND,
Howlett AC,
Westfall TC
(1991)
Dopaminergic regulation of dopamine release from PC12 cells via a pertussis toxin-sensitive G-protein.
Neurosci Lett
122:261-264[Web of Science][Medline].
-
Daubner SC,
Lauriano C,
Haycock JW,
Fitzpatrick PF
(1992)
Site-directed mutagenesis of serine 40 of rat tyrosine hydroxylase: effects of dopamine and cAMP-dependent phosphorylation on enzyme activity.
J Biol Chem
267:12639-12646[Abstract/Free Full Text].
-
el Mestikawy S,
Hamon M
(1986)
Is dopamine-induced inhibition of adenylate cyclase involved in the autoreceptor-mediated negative control of tyrosine hydroxylase in striatal dopaminergic terminals?
J Neurochem
47:1425-1433[Web of Science][Medline].
-
Fedele E,
Andrioli GC,
Ruelle A,
Raiteri M
(1993)
Release-regulating dopamine autoreceptors in human cerebral cortex.
Br J Pharmacol
110:20-22[Web of Science][Medline].
-
Finnegan JM,
Pihel K,
Cahill PS,
Haung L,
Zerby SE,
Ewing AG,
Kennedy RT,
Wightman RM
(1996)
Vesicular quantal size measured by amperometry at chromaffin, mast, pheochromocytoma, and pancreatic beta cells.
J Neurochem
66:1914-1923[Web of Science][Medline].
-
Frerking M,
Borges S,
Wilson M
(1995)
Variation in GABA mini amplitude is the consequence of variation in transmitter concentration.
Neuron
15:885-895[Web of Science][Medline].
-
Garris PA,
Ciolkowski EL,
Pastore P,
Wightman RM
(1994)
Efflux of dopamine from the synaptic cleft in the nucleus accumbens of the rat brain.
J Neurosci
14:6084-6093[Abstract].
-
Giambalvo CT
(1992)
Protein kinase C and dopamine transport-1. Effects of amphetamine in vivo.
Neuropharmacology
31:1201-1210[Web of Science][Medline].
-
Goldstein M,
Harada K,
Meller E,
Schalling M,
Hokfelt T
(1990)
Dopamine autoreceptors: biochemical, pharmacological, and morphological studies.
Ann NY Acad Sci
604:169-175[Web of Science][Medline].
-
Grace AA,
Bunney BS
(1984)
The control of firing pattern in nigral dopamine neurons: single spike firing.
J Neurosci
4:2866-2876[Abstract].
-
Greene LA,
Sobeih MM,
Teng KK
(1991)
Methodologies for the culture and experimental use of the PC12 rat pheochromocytoma cell line.
In: Cell culture of neocortex and basal forebrain from postnatal rats (Banker G,
Goslin K,
eds), pp 208-226. Cambridge: MIT.
-
Griffith LC,
Schulman H
(1988)
The multifunctional Ca2+/calmodulin-dependent protein kinase mediates Ca2+-dependent phosphorylation of tyrosine hydroxylase.
J Biol Chem
263:9542-9549[Abstract/Free Full Text].
-
Harada K,
Wu J,
Haycock JW,
Goldstein M
(1996)
Regulation of L-DOPA biosynthesis by site-specific phosphorylation of tyrosine hydroxylase in AtT-20 cells expressing wild-type and serine 40-substituted enzyme.
J Neurochem
67:629-635[Web of Science][Medline].
-
Hayashi Y,
Miwa S,
Lee K,
Koshimura K,
Hamahata K,
Hasewaga H,
Fujiwara M,
Watanabe Y
(1990)
Enhancement of in vivo tyrosine hydroxylation in the rat adrenal gland under hypoxic conditions.
J Neurochem
54:1115-1121[Web of Science][Medline].
-
Haycock JW,
Haycock DA
(1991)
Tyrosine hydroxylase in rat brain dopaminergic nerve terminals. Multiple-site phosphorylation in vivo and in synaptosomes.
J Biol Chem
266:5650-5657[Abstract/Free Full Text].
-
Hetey L,
Schwitzkowsky R,
Ott T,
Barz H
(1991)
Diminished synaptosomal dopamine (DA) release and DA autoreceptor supersensitivity in schizophrenia.
J Neural Transm
83:25-35[Web of Science][Medline].
-
Hull EM,
Bazzett TJ,
Warner RK,
Eaton RC,
Thompson JT
(1990)
Dopamine receptors in the ventral tegmental area modulate male sexual behavior in rats.
Brain Res
512:1-6[Web of Science][Medline].
-
Inoue K,
Nakazawa K,
Watano T,
Ohara-Imaizumi M,
Fujimori K,
Takanaka A
(1992)
Dopamine receptor agonists and antagonists enhance ATP-activated currents.
Eur J Pharmacol
215:321-324[Web of Science][Medline].
-
Johnson EA,
Tsai CE,
Lucci J,
Harrison-Shahan Y,
Azzaro AJ
(1992)
Dopamine D2 synthesis-modulating receptors are present in the striatum of the guinea pig.
Neuropharmacology
31:95-101[Web of Science][Medline].
-
Kennedy RT,
Jones SR,
Wightman RM
(1992)
Dynamic observation of dopamine autoreceptor effects in rat striatal slices.
J Neurochem
59:449-455[Web of Science][Medline].
-
Koide M,
Cho AK,
Howard BD
(1986)
Characterization of xylamine binding to proteins of PC12 pheochromocytoma cells.
J Neurochem
47:1277-1285[Web of Science][Medline].
-
Krueger BK
(1990)
Kinetics and block of dopamine uptake in synaptosomes from rat caudate nucleus.
J Neurochem
55:260-267[Web of Science][Medline].
-
Kumer SC,
Vrana KE
(1996)
Intricate regulation of tyrosine hydroxylase activity and gene expression.
J Neurochem
67:443-462[Web of Science][Medline].
-
Liu Y,
Schweitzer ES,
Nirenberg MJ,
Pickel VM,
Evans CJ,
Edwards RH
(1994)
Preferential localization of a vesicular monoamine transporter to dense core vesicles in PC12 cells.
J Cell Biol
127:1419-1433[Abstract/Free Full Text].
-
McElvain JS,
Schenk JO
(1992)
Blockade of dopamine autoreceptors by haloperidol and the apparent dynamics of potassium-stimulated endogenous release of dopamine from and reuptake into striatal suspensions in the rat.
Neuropharmacology
31:649-659[Web of Science][Medline].
-
Meiergerd SM,
Patterson TA,
Schenk JO
(1993)
D2 receptors may modulate the function of the striatal transporter for dopamine: kinetic evidence from studies in vitro and in vivo.
J Neurochem
61:764-767[Web of Science][Medline].
-
Meller E,
Bohmaker K,
Goldstein M,
Basham DA
(1993)
Evidence that striatal synthesis-inhibiting autoreceptors are dopamine D3 receptors.
Eur J Pharmacol
249:R5-R6[Web of Science][Medline].
-
Mercuri NB,
Calabresi P,
Bernardi G
(1992)
The electrophysiological actions of dopamine and dopaminergic drugs on neurons of the substantia nigra pars compacta and ventral tegmental area.
Life Sci
51:711-718[Web of Science][Medline].
-
Nakanishi N,
Onozawa S,
Matsumoto R,
Hasegawa H,
Yamada S
(1995)
Cyclic AMP-dependent modulation of vesicular monoamine transport in pheochromocytoma cells.
J Neurochem
64:600-607[Web of Science][Medline].
-
Nissbrandt H,
Pileblad E,
Carlsson A
(1985)
Evidence for dopamine release and metabolism beyond the control of nerve impulses and dopamine receptors in rat substantia nigra.
J Pharm Pharmacol
37:884-889[Web of Science][Medline].
-
O'Dowd BF,
Seeman P,
George SR
(1994)
Dopamine receptors.
In: Handbook of receptors and channels (Peroutka SJ,
ed), pp 95-124. Boca Raton, FL: CRC.
-
Onali P,
Olianas MC
(1989)
Involvement of adenylate cyclase inhibition in dopamine autoreceptor regulation of tyrosine hydroxylase in rat nucleus accumbens.
Neurosci Lett
17:91-96.
-
Palij P,
Bull DR,
Sheehan MJ,
Millar J,
Stamford J,
Kruk ZL,
Humphrey PP
(1990)
Presynaptic regulation of dopamine release in corpus striatum monitored in vitro in real time by fast cyclic voltammetry.
Brain Res
509:172-174[Web of Science][Medline].
-
Parsons LH,
Justice Jr JB
(1992)
Extracellular concentration and in vivo recovery of dopamine in the nucleus accumbens using microdialysis.
J Neurochem
58:212-218[Web of Science][Medline].
-
Pitts DK,
Kelland MD,
Freeman AS,
Chiodo LA
(1993)
Repeated amphetamine administration: Role of forebrain in reduced responsiveness of nigrostriatal dopamine neurons to dopamine agonists.
J Pharmacol Exp Ther
264:616-621[Abstract/Free Full Text].
-
Pothos EN,
Sulzer D
(1998)
Modulation of quantal dopamine release by psychostimulants.
Adv Pharmacol
42:198-202.
-
Pothos EN,
Desmond M,
Sulzer D
(1996)
L-3,4-Dihydroxyphenylalanine increases the quantal size of exocytic dopamine release in vitro.
J Neurochem
66:629-636[Web of Science][Medline].
-
Pothos EN,
Davila V,
Sulzer D
(1998)
Presynaptic recording of quanta from midbrain dopamine neurons and modulation of the quantal size.
J Neurosci
18:4106-4118[Abstract/Free Full Text].
-
Ramachandran B,
Houben K,
Rozenberg YY,
Haigh JR,
Varpetian A,
Howard BD
(1993)
Differential expression of transporters for norepinephrine and glutamate in wild type, variant, and WNT1-expressing PC12 cells.
J Biol Chem
268:23891-23897[Abstract/Free Full Text].
-
Rivet JM,
Audinot V,
Gobert A,
Peglion JL,
Millan MJ
(1994)
Modulation of mesolimbic dopamine release by the selective dopamine D3 receptor antagonist, (+)-S 14297.
Eur J Pharmacol
265:175-177[Web of Science][Medline].
-
Salah RS,
Kuhn DM,
Galloway MP
(1989)
Dopamine autoreceptors modulate the phosphorylation of tyrosine hydroxylase in rat striatal slices.
J Neurochem
52:1517-1522[Web of Science][Medline].
-
Saller CF,
Salama AI
(1984)
Dopamine synthesis in synaptosomes: relation of autoreceptor functioning to pH, membrane depolarization, and intrasynaptosomal dopamine content.
J Neurochem
43:675-688[Web of Science][Medline].
-
Santiago M,
Westerink BHC
(1991)
Characterization and pharmacological responsiveness of dopamine release recorded by microdialysis in the substantia nigra of conscious rats.
J Neurochem
57:738-747[Web of Science][Medline].
-
Schroeder TJ,
Jankowski JA,
Kawagoe KT,
Wightman RM,
Lefrou C,
Amatore C
(1992)
Analysis of diffusional broadening of vesicular packets of catecholamines released from biological cells during exocytosis.
Anal Chem
64:3077-3083[Medline].
-
Schubert D,
LaCorbiere M,
Klier FG,
Steinbach JH
(1980)
The modulation of neurotransmitter synthesis by steroid hormones and insulin.
Brain Res
190:67-79[Web of Science][Medline].
-
Sesack SR,
Aoki C,
Pickel VM
(1994)
Ultrastructural localization of D2 receptor-like immunoreactivity in midbrain dopamine neurons and their striatal targets.
J Neurosci
14:88-106[Abstract].
-
Seutin V,
Verbanck P,
Massotte L,
Dresse A
(1991)
Acute amphetamine-induced subsensitivity of A10 dopamine autoreceptors in vitro.
Brain Res
558:141-144[Web of Science][Medline].
-
Silvia CP,
King GR,
Lee TH,
Zy X,
Caron MG,
Ellinwood EH
(1994)
Intranigral administration of D2 dopamine receptor antisense oligodeoxynucleotides establishes a role for nigrostriatal D2 autoreceptors in the motor actions of cocaine.
Mol Pharmacol
46:51-57[Abstract].
-
Sonders MS,
Zhu SJ,
Zahniser NR,
Kavanaugh MP,
Amara SG
(1997)
Multiple ionic conductances of the human dopamine transporter: the actions of dopamine and psychostimulants.
J Neurosci
17:960-974[Abstract/Free Full Text].
-
Song HJ,
Ming GL,
Fon E,
Bellocchio E,
Edwards RH,
Poo MM
(1997)
Expression of a putative vesicular acetylcholine transporter facilitates quantal transmitter packaging.
Neuron
18:815-826[Web of Science][Medline].
-
Steyer JA, Horstmann H, Almers W (1997) Transport, docking
and exocytosis of single secretory granules in live chromaffin cells.
Nature 474-478.
-
Strait KA,
Kuczenski R
(1986)
Dopamine autoreceptor regulation of the kinetic state of striatal tyrosine hydroxylase.
Mol Pharmacol
29:561-569[Abstract].
-
Sulzer D,
Chen TK,
Lau YY,
Kristensen H,
Rayport S,
Ewing A
(1995)
Amphetamine redistributes dopamine from synaptic vesicles to the cytosol and promotes reverse transport.
J Neurosci
15:4102-4108[Abstract].
-
Surmeier DJ,
Song WJ,
Yan Z
(1996)
Coordinated expression of dopamine receptors in neostriatal medium spiny neurons.
J Neurosci
16:6579-6591[Abstract/Free Full Text].
-
Tanaka T,
Vincent SR,
Nomikos GG,
Fibiger HC
(1992)
Effect of quinine on autoreceptor-regulated dopamine release in the rat striatum.
J Neurochem
59:1640-1645[Web of Science][Medline].
-
Tepper JM,
Groves PM
(1990)
In vivo electrophysiology of central nervous system terminal autoreceptors.
Ann NY Acad Sci
604:470-487[Web of Science][Medline].
-
Thompson SM,
Capogna M,
Scanziani M
(1993)
Presynaptic inhibition in the hippocampus.
Trends Neurosci
16:222-227[Web of Science][Medline].
-
Timmerman W,
De Vries JB,
Westerink BHC
(1990)
Effects of D-2 agonists on the release of dopamine: localization of the mechanism of action.
Naunyn Schmiedebergs Arch Pharmacol
342:650-654[Web of Science][Medline].
-
Tissari AH,
Lillgèls MS
(1993)
Reduction of dopamine synthesis inhibition by dopamine autoreceptor activation in striatal synaptosomes with in vivo reserpine administration.
J Neurochem
61:231-238[Web of Science][Medline].
-
Van der Kloot W
(1991)
The regulation of quantal size.
Prog Neurobiol
36:93-130[Web of Science][Medline].
-
Wang T,
Xie K,
Lu B
(1995)
Neurotrophins promote maturation of developing neuromuscular synapses.
J Neurosci
15:4796-4805[Abstract].
-
Westerink BHC,
De Boer P,
Timmerman W,
De Vries JB
(1990)
In vivo evidence for the existence of autoreceptors on dopaminergic, serotonergic, and cholinergic neurons in the brain.
Ann NY Acad Sci
604:492-504[Web of Science][Medline].
-
Wieczorek WJ,
Kruk ZL
(1994)
A quantitative comparison on the effects of benztropine, cocaine and nomifensine on electrically evoked dopamine overflow and rate of re-uptake in the caudate putamen and nucleus accumbens in the rat brain slice.
Brain Res
657:42-50[Web of Science][Medline].
-
Wightman RM,
Jankowski JA,
Kennedy RT,
Kawagoe KT,
Schroeder TJ,
Leszczyszyn DJ,
Near JA,
Diliberto Jr EJ,
Viveros OH
(1991)
Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells.
Proc Natl Acad Sci USA
88:10754-10758[Abstract/Free Full Text].
-
Williams GW,
Goldman-Rakic PS
(1995)
Modulation of memory fields by dopamine D1 receptors in prefrontal cortex.
Nature
376:572-575[Medline].
-
Xue ZY,
Caron MG,
Ellinwood EH
(1994)
Intranigral administration of D2 dopamine receptor antisense oligodeoxynucleotides establishes a role for nigrostriatal D2 autoreceptors in the motor actions of cocaine.
Mol Pharmacol
46:51-57.
-
Yamada S,
Yokoo H,
Nishi S
(1991)
Changes in sensitivity of dopamine autoreceptors in rat striatum after subchronic treatment with methamphetamine.
Eur J Pharmacol
205:43-47[Web of Science][Medline].
-
Zhang H,
Lee TH,
Ellinwood Jr EH
(1992)
The progressive changes of neuronal activities of the nigral dopaminergic neurons upon withdrawal from continuous infusion of cocaine.
Brain Res
594:315-318[Web of Science][Medline].
-
Zhou Z,
Misler S
(1995)
Detection of quantal release of catecholamines from developing neurons in culture by amperometry.
Biophys J
39A:398.
-
Zhou Z,
Misler S
(1996)
Amperometric detection of quantal secretion from rat pancreatic beta-cells.
J Biol Chem
270:270-277.
-
Zhou R,
Luo G,
Ewing AG
(1994)
Direct observation of the effect of autoreceptors on stimulated release of catecholamines from adrenal cells.
J Neurosci
14:2402-2407[Abstract].
-
Zhu HJ,
Sonders MS,
Amara SG,
Kavanaugh MP,
Zahniser NR
(1995)
Uptake of dopamine by the cloned human dopamine transporter is voltage-dependent in Xenopus oocytes.
Soc Neurosci Abstr
21:375.
Copyright © 1998 Society for Neuroscience 0270-6474/98/18155575-11$05.00/0
This article has been cited by other articles:
|
|
|
|
 
S. J. Farnsworth, T. J. Volz, G. R. Hanson, and A. E. Fleckenstein
Cocaine Alters Vesicular Dopamine Sequestration and Potassium-Stimulated Dopamine Release: The Role of D2 Receptor Activation
J. Pharmacol. Exp. Ther.,
March 1, 2009;
328(3):
807 - 812.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. J. Volz, S. J. Farnsworth, S. D. Rowley, G. R. Hanson, and A. E. Fleckenstein
Methylphenidate-Induced Increases in Vesicular Dopamine Sequestration and Dopamine Release in the Striatum: The Role of Muscarinic and Dopamine D2 Receptors
J. Pharmacol. Exp. Ther.,
October 1, 2008;
327(1):
161 - 167.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. L. Brody, M. A. Mandelkern, R. E. Olmstead, D. Scheibal, E. Hahn, S. Shiraga, E. Zamora-Paja, J. Farahi, S. Saxena, E. D. London, et al.
Gene Variants of Brain Dopamine Pathways and Smoking-Induced Dopamine Release in the Ventral Caudate/Nucleus Accumbens.
Arch Gen Psychiatry,
July 1, 2006;
63(7):
808 - 816.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Xu, K. S. Tang, V. B. Lu, C. P. Weerasinghe, A. Tse, and F. W. Tse
Maintenance of quantal size and immediately releasable granules in rat chromaffin cells by glucocorticoid
Am J Physiol Cell Physiol,
November 1, 2005;
289(5):
C1122 - C1133.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Wang and P. S. Goldman-Rakic
D2 receptor regulation of synaptic burst firing in prefrontal cortical pyramidal neurons
PNAS,
April 6, 2004;
101(14):
5093 - 5098.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. H. V. Schapira and C. W. Olanow
Neuroprotection in Parkinson Disease: Mysteries, Myths, and Misconceptions
JAMA,
January 21, 2004;
291(3):
358 - 364.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. A. Sombers, H. J. Hanchar, T. L. Colliver, N. Wittenberg, A. Cans, S. Arbault, C. Amatore, and A. G. Ewing
The Effects of Vesicular Volume on Secretion through the Fusion Pore in Exocytotic Release from PC12 Cells
J. Neurosci.,
January 14, 2004;
24(2):
303 - 309.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Axmacher, M. Stemmler, D. Engel, A. Draguhn, and R. Ritz
Transmitter Metabolism as a Mechanism of Synaptic Plasticity: A Modeling Study
J Neurophysiol,
January 1, 2004;
91(1):
25 - 39.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
H. Zhang and D. Sulzer
Glutamate Spillover in the Striatum Depresses Dopaminergic Transmission by Activating Group I Metabotropic Glutamate Receptors
J. Neurosci.,
November 19, 2003;
23(33):
10585 - 10592.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Holtje, S. Winter, D. Walther, I. Pahner, H. Hortnagl, O. P. Ottersen, M. Bader, and G. Ahnert-Hilger
The Vesicular Monoamine Content Regulates VMAT2 Activity through Galpha q in Mouse Platelets. EVIDENCE FOR AUTOREGULATION OF VESICULAR TRANSMITTER UPTAKE
J. Biol. Chem.,
April 25, 2003;
278(18):
15850 - 15858.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. E. Ahlskog
Slowing Parkinson's disease progression: Recent dopamine agonist trials
Neurology,
February 11, 2003;
60(3):
381 - 389.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Schmitz, C. Schmauss, and D. Sulzer
Altered Dopamine Release and Uptake Kinetics in Mice Lacking D2 Receptors
J. Neurosci.,
September 15, 2002;
22(18):
8002 - 8009.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. A. Serra, L. Sciola, M. R. Delogu, A. Spano, G. Monaco, E. Miele, G. Rocchitta, M. Miele, R. Migheli, and M. S. Desole
The Neurotoxin 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine Induces Apoptosis in Mouse Nigrostriatal Glia. RELEVANCE TO NIGRAL NEURONAL DEATH AND STRIATAL NEUROCHEMICAL CHANGES
J. Biol. Chem.,
September 6, 2002;
277(37):
34451 - 34461.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. M. Prasad and S. G. Amara
The Dopamine Transporter in Mesencephalic Cultures Is Refractory to Physiological Changes in Membrane Voltage
J. Neurosci.,
October 1, 2001;
21(19):
7561 - 7567.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. M. Brown, G. R. Hanson, and A. E. Fleckenstein
Cocaine-Induced Increases in Vesicular Dopamine Uptake: Role of Dopamine Receptors
J. Pharmacol. Exp. Ther.,
September 1, 2001;
298(3):
1150 - 1153.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. D. Machado, A. Morales, J. F. Gomez, and R. Borges
cAMP Modulates Exocytotic Kinetics and Increases Quantal Size in Chromaffin Cells
Mol. Pharmacol.,
September 1, 2001;
60(3):
514 - 520.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. N. Pothos, K. E. Larsen, D. E. Krantz, Y.-j. Liu, J. W. Haycock, W. Setlik, M. D. Gershon, R. H. Edwards, and D. Sulzer
Synaptic Vesicle Transporter Expression Regulates Vesicle Phenotype and Quantal Size
J. Neurosci.,
October 1, 2000;
20(19):
7297 - 7306.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. L. Colliver, S. J. Pyott, M. Achalabun, and A. G. Ewing
VMAT-Mediated Changes in Quantal Size and Vesicular Volume
J. Neurosci.,
July 15, 2000;
20(14):
5276 - 5282.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Holtje, B. von Jagow, I. Pahner, M. Lautenschlager, H. Hortnagl, B. Nurnberg, R. Jahn, and G. Ahnert-Hilger
The Neuronal Monoamine Transporter VMAT2 Is Regulated by the Trimeric GTPase Go2
J. Neurosci.,
March 15, 2000;
20(6):
2131 - 2141.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Xu and F. W. Tse
Brefeldin A Increases the Quantal Size and Alters the Kinetics of Catecholamine Release from Rat Adrenal Chromaffin Cells
J. Biol. Chem.,
July 2, 1999;
274(27):
19095 - 19102.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. K. Park, S. A. Sedore, C. Cronmiller, and J. Hirsh
Type II cAMP-dependent Protein Kinase-deficient Drosophila Are Viable but Show Developmental, Circadian, and Drug Response Phenotypes
J. Biol. Chem.,
June 30, 2000;
275(27):
20588 - 20596.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
Top
Abstract
Introduction
Compound via MeSH
