From the Institut für Anatomie der
Charité, Humboldt Universität zu Berlin, Philippstrasse 12, 10115 Berlin, Germany, the ¶ Max-Delbrück-Centrum für
Molekulare Medizin, 13092 Berlin Buch, Germany, the
Centre for Molecular Biology and Neuroscience and Department
of Anatomy, Institute of Basic Medical Sciences, University of Oslo,
P. O. Box 1105, Blindern, N-0317 Oslo, Norway, and the
** Institut für Pharmakologie der Charité,
Humboldt Universität zu Berlin, Dorotheenstr. 94, 10117 Berlin, Germany
Received for publication, December 17, 2002, and in revised form, February 6, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Variations in the neurotransmitter content of
secretory vesicles enable neurons to adapt to network changes.
Vesicular content may be modulated by vesicle-associated
Go2, which down-regulates the activity of the
vesicular monoamine transmitter transporters VMAT1 in neuroendocrine
cells and VMAT2 in neurons. Blood platelets resemble serotonergic
neurons with respect to transmitter storage and release. In
streptolysin O-permeabilized platelets, VMAT2 activity is also
down-regulated by the G protein activator guanosine 5'-( Transport of monoamines like serotonin, catecholamines, or
histamin into the secretory vesicles of a variety of cells is mediated by vesicular monoamine transporters
(VMATs).1 In mammals two
closely related transporters, VMAT1 and VMAT2, are known (1, 2). These
two subtypes differ in their substrate specificity, pharmacological
properties, and tissue distribution (3, 4). Both transporters represent
proteins with 12 predicted transmembrane domains of different vesicle
types like large dense core vesicles, small synaptic vesicles
(SSVs), and synaptic-like microvesicles (2, 5). In contrast to the
transporters of the plasma membrane, which mainly use the
Na+ gradient across the plasma membrane to drive transport,
activity of vesicular transporters relies on the proton electrochemical gradient ( Recently, we have identified functional subsets of heterotrimeric G
proteins on secretory vesicle membranes (10, 11) and have shown that
the activity of both VMAT1 and VMAT2 is regulated by the Antibodies--
Mouse monoclonal antibodies against
G Transmitters and Receptor
Ligands--
5-Hydroxy-[3H]tryptamine trifluoroacetate
(serotonin; specific activity, 3260 Bq/mmol) and
[3H]noradrenaline (specific activity, 444 Bq/mmol) were
obtained from Amersham Biosciences. The 5HT2A receptor
antagonist spiperone, the Toxins--
Streptolysin O from Mice--
Wild type and peripheral tryptophan hydroxylase
knockout (Tph1 Preparation of Blood Platelets--
Genotyped mice of either sex
were anesthetized by intraperitoneally injecting a mixture of 50 µl
of Ketavet (Curamed Pharma, Karlsruhe, Germany) and 50 µl of Rompun
(Bayer, Leverkusen, Germany) per 10 g of body weight. Blood was
taken from the inferior caval vein in the presence of heparine (30 IU/ml). The blood was mixed with 0.5 volume of Tyrode-Hepes (TH) buffer
(134 mM NaCl, 0.34 mM
Na2HPO4, 2.9 mM KCl, 12 mM NaHCO3, 20 mM HEPES, 5 mM glucose, 1 mM MgCl2, pH 7.3) and
centrifuged for 7 min at 210 × g. The platelet-rich
plasma supernatant was taken and centrifuged for 5 min at 1090 × g. The resulting pellet was resuspended in TH buffer, and
the platelets were counted in a Neubauer chamber. Platelets from wild
type and Tph1 Serotonin Uptake--
Sedimented platelets (see above) were
washed two times with TH buffer. For vesicular uptake, platelets were
suspended in potassium glutamate buffer (KG buffer) containing 150 mM potassium glutamate, 20 mM 1,4-piperazine
diethanesulfonic acid, 4 mM EGTA, 1 mM
MgCl2, 1 mM dithiothreitol, adjusted to pH 7.0 with KOH supplemented with SLO. Incubation was performed for 5 min on
ice. Unbound toxin was removed by centrifugation (5 min, 1090 × g). The pellets were resuspended in KG buffer with 2 mM ATP added. The platelets were then divided into
individual reaction cups, and uptake was started by adding 100 µl of
KG-ATP buffer supplemented with 1 mM ascorbic acid and 80 nM [3H]serotonin. Additives such as the
nonhydrolyzable GTP-analogon GMppNp or tetrabenazine were applied
during this step. Incubation was performed for 15 min at 37 °C and
stopped by the addition of 1 ml of ice-cold KG buffer followed by rapid
centrifugation. The pellets were then lysed in 0.4% Triton-X-100 to
determine radioactivity by liquid scintillation counting and protein
content using the bicinchoninic acid method (BCA kit; Pierce).
Serotonin uptake by intact platelets was performed in TH buffer
supplemented with 80 nM [3H]serotonin and 1 mM ascorbic acid for 30 min at 37 °C. Incubation was
stopped by diluting with ice-cold TH buffer, and the samples were
processed as in the case of permeabilized platelets.
For reconstitution experiments intact blood platelets were preloaded
with 15 µM serotonin dissolved in TH buffer for 30 min at
room temperature before they were subjected to the permeabilization and
vesicular uptake procedure or analyzed by HPLC. In general, the samples
were analyzed in triplicate.
Monoamine Determination by HPLC--
The monoamine content of
wild type Tph1 Preparation of Synaptic Vesicles--
Crude synaptic vesicles
(lysis pellet 2 fraction) were prepared from mice whole brain
homogenates according to a protocol described (28). Serotonin uptake
was performed as described recently (13).
Electron Microscopy--
Sedimented platelets (see above) were
suspended in 4% paraformaldehyde, 0.1% glutaraldehyde
dissolved in PBS, and fixed for 15 min at room temperature. After
fixation the platelets were spun down again at 2000 × g for 5 min, resuspended, and incubated for 10 min at room
temperature in PBS containing 50 mM glycine and 0.1%
sodium borhydride as quenching solution. After another PBS
washing/centrifugation step, the platelets were cryoprotected in
glycerol/PBS for the following freeze substitution and embedded in
Lowicryl HM 20 (Electron Microscopy Sciences, Fort Washington, PA) as
described (29). Ultrathin sections (70 nm) were cut on a Leica
Ultratome and mounted onto Formvar-covered (Serva, Heidelberg, Germany;
0.8% in chloroform) nickel grids. The sections were subjected to
postembedding immunogold procedure as described (30). Briefly, the
sections were etched with 1% H2O2 diluted in
H2O for 30 min followed by immunocytochemistry. Unspecific
binding sites were blocked with 2% bovine serum albumin and 5% normal
goat serum in Tris-buffered saline containing 0.01% Triton X-100 (pH
7.6) for 1 h at room temperature. Then the sections were incubated with the respective primary antiserum diluted in the blocking solution
for 3 h at room temperature. After a further blocking step with
2% bovine serum albumin in Tris-buffered saline containing 0.01%
Triton X-100 (pH 7.6), the sections were incubated with goat
anti-rabbit Fab fragments coupled to 5- or 10-nm gold particles (British BioCell International, Cardiff, UK) diluted 1:20 in blocking solution for 1.5 h at room temperature. The sections were stained with uranyl acetate and lead citrate. The micrographs were taken on a
Zeiss EM 900 or a Philipps CM 10.
Immunoreplica Analysis--
Sedimented platelets or synaptic
vesicles from wild type or deletion mutants were dissolved in Laemmli
buffer, and equal amounts of protein were separated by SDS-PAGE. After
transfer to nitrocellulose the respective proteins were detected by the
indicated antibodies using the ECL detection system (Amersham
Biosciences).
Comparison of the Serotonin Uptake by Wild Type and Tph1
In Tph1
To analyze how transport is affected by G protein activation, a
Michaelis-Menten kinetic was performed using wild type platelets. The
presence of GMppNp decreased Vmax from 18.37 pmol/mg/min in control to 13.9 pmol/mg protein/min (values in a second
experiment were 26.11 and 20.05 pmol/mg protein/min, respectively) and
increased Km from 0.47 µM (control) to
0.57 µM (second experiment, 0.74 to 0.91 µM) (Fig. 2). The obtained
kinetic values for VMAT2 activity in mouse platelets are comparable
with kinetic data published for human VMAT2 (4).
Thus, GMppNp affected both Vmax and
Km of VMAT2 in platelets. Deletion of peripheral
tryptophan hydroxylase, which reduces serotonin content in the
periphery, abolished the G protein-mediated down-regulation of VMAT2
only in secretory vesicles of platelets but not in SSV.
Reconstitution of GMppNp-mediated Down-regulation of
[3H]Serotonin Uptake by Preloading Tph1
To determine whether the reconstitution of the GMppNp-mediated
down-regulation of VMAT2 in Tph1 Monoamine Content of Platelets from Wild Type and Tph1
To demonstrate that preincubation with serotonin does considerably
alter the vesicular filling in platelets, we compared the amount of
serotonin before and after a 30-min preincubation with 15 µM serotonin. In this set of experiments, the platelet
serotonin content before preincubation was 2.15 and 0.18 ng/µg
protein in wild type and Tph1 VMAT2 and G Protein Subunits in Wild Type and Tph1
To see whether a change in the occurrence of some proteins required for
proper function of vesicular serotonin storage might account for the
observed differences, we compared by immunoreplica analysis the amount
of VMAT2 and G proteins in SSV and platelet preparations from both
types of mice. An antiserum against VMAT2 detected major protein bands
at 55 kDa as well as a few minor bands in the platelets and brain of
either group, corresponding to the apparent molecular weight and
possible protein modifications of VMAT2 (32, 33). An antiserum against
G
The application of immunogold electron microscopy revealed that
platelets from Tph1 G
When we compared the GMppNp-induced inhibition of vesicular serotonin
uptake into platelets from wild type and G Down-regulation of VMAT2 by G
The G
The impaired platelet function in G G protein heterotrimers localize to secretory vesicles (11) and
nonhydrolyzable GTP analogues known to activate G proteins down-regulate the activity of VMATs (12, 13). Here we present the first
evidence that the vesicular content itself mediates the G protein
regulation. In addition we show that although VMAT2 is a downstream
target for G Vesicular Content as a Regulator for Vesicular Filling--
From
electrophysiological studies it is evident that quantal release of
neurotransmitters from a given neuron, synapse, or neuroendocrine cell
varies irrespective of the transmitter phenotype released. This leads
to the idea that vesicular content may be regulated and adapt to
environmental changes.
Overexpression of VACHT (41) or VMAT2 (42) indicates that the vesicular
content estimated by quantal release correlates with the amount of
transmitter transporter molecules per vesicle. The addition of
precursors to increase the monoamine content (43) or
Using a genetic model characterized by the depletion of Tph1, a
peripheral form of tryptophan hydroxylase (19) resulting in platelets
that are fully equipped for serotonin storage and release but that
contain only minute amounts of serotonin, we provide evidence that
vesicular content is crucial for regulation of transmitter uptake.
Preloading of Tph1
A mechanism that enables secretory vesicles to modulate their
transmitter content might prevent them from getting overloaded. A more
attractive interpretation is a general regulation performed by the
vesicles themselves that allows them to modulate their transmitter
content in correlation with their environment. Generally in the central
nervous system by such a kind of regulation fine-tuning of vesicular
concentration can be achieved in different synapses by the respective
vesicular transmitter transporters.
Regulation of vesicular content may function at the level of the
vesicle itself depending on the filling stage and does not require
direct receptor activation. Evidence came from the failure of
inhibitors of plasma membrane receptors to directly interfere with the
reconstitution of G protein-mediated regulation in Tph1 G
G
Thus, G
In summary, VMAT2 can be regulated by either Gi
-imido)triphosphate (GMppNp). Using
serotonin-depleted platelets from peripheral tryptophan hydroxylase
knockout (Tph1
/
) mice, we show here that the vesicular filling
initiates the G protein-mediated down-regulation of VMAT2 activity.
GMppNp did not influence VMAT2 activity in naive platelets from
Tph1
/
mice. GMppNp-mediated inhibition could be reconstituted,
however, when preloading Tph1
/
platelets with serotonin or
noradrenaline. G
q mediates the down-regulation of VMAT2
activity as revealed from uptake studies performed with platelets from
G
q deletion mutants. Serotonergic, noradrenergic, as
well as thromboxane A2 receptors are not directly involved
in the down-regulation of VMAT2 activity. It is concluded that in
platelets the vesicle itself regulates transmitter transporter activity
via its content and vesicle-associated G
q.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
µH+) generated by a vacuolar
H+-ATPase (6). The variability of quantal size has been
established for monoaminergic cells like PC12 cells (7, 8) or leech Retzius cells (9) using carbon fiber amperometry. These studies suggest
that secretory vesicles vary in their transmitter content and that this
variability may result from changes in vesicular volume rather than
from changes in transmitter concentration.
-subunit of
Go2 (11-13). By this mechanism neurons and other secretory
cells might vary their vesicular transmitter content and therefore
alter the amount of transmitter released during exocytosis. The major
goal of the present study was to elucidate the upstream signals for the
G protein activation leading to the observed inhibition of VMAT
activity. In general, heterotrimeric G proteins are activated by
heptahelical receptors of the plasma membrane. Exceptions to this rule
are the growth cone associated protein GAP 43 (14) as well as the
amyloid precursor protein (15) that also activate G protein signaling.
Another question concerned G proteins probably involved in regulating
transporter activity in cells that store monoamines but do not express
Go2. To address these issues we examined mouse blood
platelets. Platelets are the major storage sites of serotonin apart
from the central nervous system. They take up serotonin by the
serotonin plasma membrane transporter (SERT) and, as has been shown for
human platelets, package it by VMAT2 activity (16, 17) into large dense
bodies together with other substances like ADP (for review see Ref.
18). Once activated by various substances at sites of endothelial
injury, these cell fragments become spherical, acquire surface
stickiness, aggregate, and release mediators like serotonin that act at
endothelial cells and smooth muscles. This activation cascade repairs
damaged blood vessels, maintains hemostasis, and may even lead to
diseases like thrombosis. We took advantage of mice deficient of
peripheral tryptophan hydroxylase activity (Tph1
/
), the key enzyme
in serotonin synthesis. Platelets from Tph1
/
mice contain only very
small amounts of serotonin compared with wild type ones (19). We used these platelets as an accessible model system for monoamine
transmitter-depleted vesicles that allowed us to study the impact of
the transmitter content on G protein-mediated VMAT regulation. Using
mice deficient for the
-subunit of Gq, we could identify
this G protein subunit as a regulator of VMAT2 activity in platelets.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
o2 (clone 101.1) and synaptobrevin II (clone 69.1) (20)
as well as a rabbit polyclonal antiserum against VMAT2 (13) were kindly
provided by R. Jahn (Max-Planck-Institut für
Biophysikalische Chemie, Göttingen, Germany). The following G
protein subtype-specific polyclonal antisera were used:
G
o2 (AS 371) (21), G
q/11 (AS 370) (22), G
2 (AS 36) (23), and G
5 (AS 422) (24).
All of the antisera were characterized with respect to specificity and
cross-reactivity (11, 24). A monospecific antiserum against
G
q was obtained from Calbiochem. Horseradish
peroxidase-labeled anti-rabbit or anti-mouse IgG was obtained from
Vector Laboratories (Burlingham, CA).
2A receptor antagonist BRL
44408, the D3 receptor antagonist GR 103691, as well as the
TXA2 agonist U46619 were obtained from Tocris.
-hemolytic strepococci (SLO)
(25) and
-toxin from Staphylococcus aureus were kindly
supplied by U. Weller (Institut Ray-Rocky-Weller, Baden-Baden, Germany).
/
) mice were bred as given (19). Wild type and
G
q
/
mice were kindly supplied by S. Offermanns
(Institut für Pharmakologie, Heidelberg, Germany) and bred as
previously described (26).
/
or G
q
/
mice were adjusted to an
equal number of platelets/ml prior to the experiments.
/
or G
q
/
platelets was quantitated
by HPLC using either a fluorescence detection system or an
electrochemical detection system yielding similar results. The
platelets were resuspended in 0.1 N perchloric acid dissolved in PBS and further processed with a fluorescence detection system (27) or electrochemical detection system (19) as described previously. The amounts of serotonin calculated from the standard curve
were given in nanograms and normalized to protein content or given
per platelet (see above).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
Platelets--
As a first approach we compared vesicular monoamine
uptake into platelets from wild type and Tph1
/
mutants
permeabilized by SLO. In wild type platelets a serotonin uptake of
about 50 pmol/mg protein could be detected, which was less compared
with the 65 pmol/mg protein found for Tph1
/
platelets (Fig.
1A). In both preparations
serotonin uptake was completely abolished when reserpine or
tetrabenazine was added, indicating that the observed uptake was
mediated by VMAT2 (4). A significant inhibition of uptake was observed,
when GMppNp (100 µM), an activator of trimeric G
proteins, was added to wild type platelets. In contrast, the addition
of GMppNp displayed no effect on uptake into permeabilized Tph1
/
platelets. A summary of the data obtained from five individual experiments revealed that the vesicular serotonin uptake into mutant
platelets was 40% higher compared with the wild type preparations (Fig. 1A, inset). When comparing the
GMppNp-induced inhibition of vesicular [3H]serotonin
uptake in wild type and mutant platelets (combined data from five
individual experiments, given as % of control), the uptake in the
former was inhibited by 40%, whereas no effect was seen in mutant
platelets (Fig. 1A, inset).
View larger version (20K):
[in a new window]
Fig. 1.
[3H]Serotonin uptake into blood
platelets and brain SSV from wild type and Tph1 /
mice: Effects of
GMppNp. A, SLO-permeabilized platelets were resuspended
in KG-ATP buffer containing 80 nM
[3H]serotonin with additions as indicated and incubated
for 15 min at 37 °C. The observed uptake was reserpine- and
tetrabenazine-sensitive. Both inhibitors of VMAT2 (final concentration,
5 and 10 µM, respectively) completely blocked uptake
activity in either platelet preparation. The addition of the
nonhydrolyzable GTP analogue GMppNp (100 µM) inhibited
the vesicular serotonin uptake to about 35% in wild type but not in
Tph1
/
platelets. The values represent the means of three
determinations ± S.D. Statistical significance (p < 0.05) was verified using Student's t test.
Inset, summary of the data obtained from five individual
experiments comparing vesicular uptake (upper graph, wild
type given as 100%) and GMppNp-induced inhibition (lower
graph, GMppNp-mediated inhibition given as a percentage of control
uptake) between wild type and Tph1
/
platelets. B,
[3H]serotonin uptake into brain synaptic vesicles from
wild type and Tph1
/
mice. A crude synaptic vesicle preparation from
a whole brain homogenate was incubated for 10 min with 80 nM [3H]serotonin. In both wild type and
knockout animals, the addition of GMppNp inhibited the vesicular
serotonin uptake to 25%. The significance of the GMppNp effects was
p = 0.0098 for wt and p = 0.025 for
Tph1
/
. Unspecific uptake performed in duplicates in the presence of
tetrabenazine (0.85 and 0.99 pmol/mg protein for wild type and
Tph1
/
, respectively) was subtracted. wt, wild
type.
/
mice the activity of the peripheral tryptophan hydroxylase
is abolished, whereas the respective enzyme of central neurons is
maintained, resulting in normal serotonin levels in brain. So the
GMppNp-mediated down-regulation of serotonin uptake into a crude
vesicular preparation from wild type and mutant brains was analyzed for
comparison. The tetrabenazine-sensitive uptake was unchanged in
Tph1
/
compared with wild type mice, and more importantly, the
addition of GMppNp inhibited the uptake to about 25% in either
group (Fig. 1B).
View larger version (18K):
[in a new window]
Fig. 2.
Kinetic analysis of GMppNp-mediated
regulation of VMAT2 in wild type platelets. SLO-permeabilized
platelets were resuspended in KG-ATP buffer containing 40 nM [3H]serotonin and various concentrations
of unlabeled serotonin to yield the final concentration of serotonin
(abscissa) in the absence or presence of 100 µM GMppNp. Uptake was performed for 15 min at 37 °C.
Michaelis-Menten curve yields Vmax of 18.37 or
13.9 pmol/mg of protein/min in the absence or presence of GMppNp,
respectively. Lineweaver-Burk analysis (inset) showed a
slight increase in Km from 0.47 to 0.57 µM in the presence of GMppNp. The values (nonlinear
regression) represent the means of triplicate experiments corrected for
the unspecific uptake in the presence of tetrabenazine and are
calculated using Graph Pad Prism software.
/
Platelets
with Serotonin--
Having seen that the GMppNp-mediated
down-regulation of serotonin uptake is functional in Tph1
/
brain
SSV but completely abolished in the respective platelets, we preloaded
Tph1
/
platelets with serotonin to shift the vesicular serotonin
content to a level that closely resembled the one found in platelets
from wild type animals. When treating Tph1
/
platelets with 15 µM serotonin prior to the permeabilization and uptake
procedure, GMppNp strongly inhibited uptake by about 50%, whereas
uptake in similarly treated controls was unaffected (Fig.
3A). Preincubation with
increasing serotonin concentrations between 0 and 150 µM
showed a dose-dependent reconstitution of the
GMppNp-induced down-regulation of vesicular [3H]serotonin
uptake with a maximal effect at 15 µM serotonin that caused an inhibition by 40% (Fig. 3B). At higher serotonin
concentrations the inhibition as well as the overall uptake decreased,
which might be attributed to enhanced platelet clotting.
View larger version (16K):
[in a new window]
Fig. 3.
Preincubation of Tph1 /
platelets with
serotonin reconstitutes down-regulation of vesicular
[3H]serotonin uptake by GMppNp. A, intact
Tph1
/
platelets were incubated for 30 min with buffer alone or
supplemented with 15 µM nonlabeled serotonin. After
preincubation and following SLO permeabilization, the addition of
GMppNp inhibited the vesicular [3H]serotonin uptake by
50%. No GMppNp-induced inhibition was observed in platelets receiving
just buffer during the preincubation. The values given in pmol/mg
protein represent the means of three determinations ± S.D. Statistical
significance (p = 0.022) was verified using Student's
t test. B, platelets from Tph1
/
mice were
preincubated with buffer or buffer supplemented with increasing amounts
of serotonin (concentrations given in µM on the
abscissa). A down-regulation of vesicular [3H]serotonin
uptake by GMppNp could be induced dose-dependently by
preloading the platelets with serotonin. Preincubation with 15 µM serotonin resulted in a maximum effect of 40%
inhibition of uptake because of GMppNp. The GMppNp-induced inhibition
slightly declined, when higher concentrations of serotonin were
applied. The uptake in the presence of GMppNp is expressed as a
percentage of control, representing the serotonin uptake in the absence
of GMppNp for each preincubation condition. Unspecific uptake in the
presence of tetrabenazine was subtracted before calculating values. The
data represent the means of three determinations ± S.D. C,
incubation of
-toxin permeabilized Tph1
/
platelets with 15 µM of either serotonin or noradrenaline before performing
the uptake with [3H]serotonin reconstituted
GMppNp-mediated inhibition. The ef- fects of GMppNp are expressed as a percentage of inhibition of
the respective control condition. Unspecific uptake in the presence of
tetrabenazine was subtracted before calculating the values. The data
represent the means of three determinations ± S.D.
/
platelets is also mediated by
other monoamines, such as noradrenaline, a second experimental approach
was performed. Generally, platelets take up serotonin because of the
activity of the plasma membrane transporter SERT, which less accepts
other monoamines. To directly preload secretory vesicles with
noradrenaline, pemeabilization with
-toxin instead of SLO was used.
-Toxin generates only small pores and therefore yields longer living
permeabilized preparations.
-Toxin-permeabilized Tph1
/
platelets
were preloaded with 15 µM noradrenaline or serotonin used
for comparison or just received buffer. Following preloading, the usual
uptake procedure using [3H]serotonin dissolved in uptake
buffer and supplemented with or without GMppNp was performed. Whereas
GMppNp inhibition was less than 15% in control platelets, it increased
to 42-48% because of the preloading with either noradrenaline or
serotonin, respectively (Fig. 3C). The data so far suggest
that preloading with serotonin and noradrenaline can reconstitute
GMppNp-mediated inhibition in Tph1
/
platelets.
/
Mice--
To prove that Tph1
/
platelets do not contain other
monoamines, a HPLC analysis was performed (Fig.
4A). In wild type platelets the serotonin content was calculated to be 5.31 ng/µg protein, whereas in Tph1
/
platelets it was only 0.32 ng/µg protein,
representing ~6% of the amount detected in wild type platelets (19).
Additionally, the content of noradrenaline, adrenalin, and dopamine was
examined. Both noradrenaline and adrenalin could only be detected in
minute amounts, and dopamine was below detection level.
View larger version (13K):
[in a new window]
Fig. 4.
HPLC analysis of monoamine content in wild
type and Tph1 /
platelets. A, sedimented platelets
were resuspended with 0.1 N perchloric acid, sonicated, and
centrifuged. The resulting supernatant was subjected to HPLC analysis.
The serotonin content in wild type platelets was 5.31 ng/µg protein,
whereas in Tph1
/
platelets only 0.32 ng of serotonin/µg of
protein were detected. Noradrenaline and adrenalin were detected at
very low levels, and dopamine was below detection level (not shown).
The values represent the means of three determinations ± S.D.
B, the serotonin content in wt or Tph1
/
platelets before
and after a 30-min preincubation with 15 µM serotonin was
compared by HPLC analysis. In the wild type, the amount of serotonin
slightly increased by 20% from 2.15 to 2.66 ng serotonin/µg protein
after preincubation. In Tph1
/
platelets, an approximately 8-fold
increase from 0.18 to 1.51 ng serotonin/µg protein could be detected.
The values represent the means of three determinations ± S.D.
wt, wild type.
/
platelets, respectively (Fig.
4B). After preincubation with serotonin, the serotonin
content was increased by about 20% (2.66 ng/µg) in wild type
platelets. In Tph1
/
platelets the increase was much more
pronounced, reaching 8-fold the amount of untreated platelets (1.51 ng/µg). The resulting serotonin content approximates serotonin levels
found in wild type platelets (Fig. 4B).
/
Mice--
The ultrastructure of the platelets was examined by electron
microscopy to see whether the deficiency in serotonin synthesis in
Tph1
/
mice causes morphological changes of the vesicular storage
organelles. However, the comparison with platelets of wild type mice
revealed no differences in the ultrastructural morphology (Fig.
5A). All platelets contained
some few vesicular elements with an average diameter of ~120 nm and a
sharp membraneous envelope, which identified them as dense bodies,
although most of them had lost their dense core because of the
histological preparation for electron microscopy. The dense bodies
could be easily discriminated from profiles of the open
canalicular system of the platelet plasma membrane that is
characterized by a fuzzy glycocalyx coat on its luminal appearing
membrane. In addition to the dense bodies, two other types of opaque
vesicular elements could be observed in platelets of both, Tph1
/
and wild type mice. These comprise large
-granules with an
approximate diameter of 100-250 nm. Some of them contained vesicular
structures inside and may therefore represent an immature stage of
-granules, also called multivesicular bodies (31). Also, small dense
granules with a diameter of 30 nm were observed throughout in the
platelet cytoplasm.
View larger version (102K):
[in a new window]
Fig. 5.
Detection of VMAT2 and G protein subunits in
wt and Tph1 /
platelets. A, platelets isolated from
wt and Tph1
/
mice show no ultrastructural differences. Three
different types of vesicular organelles could be identified within the
platelets of both preparations: dense bodies (black
asterisk),
-granules (white asterisk), and small
dense vesicles (arrowheads). Scale bars, 200 nm.
B, brain postnuclear supernatants and platelets from wild
type and Tph1
/
mice were subjected to SDS-PAGE and subsequent
immunoreplica analysis. Note that G
o2 showed a strong
immunoreaction in brain, but only minute amounts were detectable in
platelets. G
5 exhibited a strong signal in brain, but no
immunoreactivity was found in platelets. C, application of
antisera against VMAT2, G
q/11 or synaptobrevin
(syb) revealed a membrane-associated immunogold labeling of
dense bodies as well as of small dense vesicles and
-granules from
Tph1
/
platelets. Scale bars, 100 nm. wt, wild
type.
o2 exhibited a strong immunosignal in brain, but
virtually no signal could be detected in platelets of either type.
Antisera against G
q/11 and G
2 showed strong signals in platelets and brain of either group, whereas G
5 was only detected in brain preparations. Protein
bands in the wild type appeared to be a little stronger than in
knockout animals, but no significant differences were seen in the
general pattern (Fig. 5B).
/
and wild type mice also do not differ with
respect to the subcellular distribution of the vesicular proteins
synaptobrevin, VMAT2, as well as the G protein G
q
subunit. Immunogold signals for all three proteins were found to be
associated with membranes of the three vesicular organelles described
above (Fig. 5C). Interestingly, the immunoreactivities for
VMAT2 and synaptobrevin were more pronounced on
-granules and small
dense vesicles compared with dense bodies. On the other hand dense
bodies have been described to be the only serotonin storage sites in human platelets (18, 34). Probably, mouse platelets for which no
ultrastructural data were found in the literature differ from human
platelets in this respect.
q Regulates Vesicular Monoamine
Transport in Platelets--
In contrast to the situation in neurons
and neuroendocrine cells, the absence of G
o2 in
platelets (Ref. 35 and this work) excludes its involvement in the
inhibition of VMAT2 activity. In platelets G
q is one of
the most important G proteins (35). In addition to its localization on
the plasma membrane, it also sediments with the dense granule fraction
in human platelets (36) and localized together with VMAT2 on secretory
vesicles (Fig. 5C). Therefore, G
q might be
well suited as a regulator of vesicular monoamine transport in
platelets. Consequently, we performed uptake experiments on mice
lacking the gene for the
-subunit of Gq. G
q
/
mice have increased bleeding times, because of a
defective platelet activation mediated by G
q signaling
(26). They also suffer from several deficits like motor
discoordination, probably because of a reduced developmental regression
of surplus cerebellar climbing fibers innervating Purkinje cells
(37).
q
/
mice, no significant down-regulation of vesicular serotonin uptake could be
detected in G
q
/
platelets (Fig.
6A). To rule out the
possibility that the lack of GMppNp-mediated modulation of VMAT2 simply
results from monoamine depleted granules in the G
q
/
platelets (comparable with the situation in Tph1
/
platelets), we
analyzed the platelet serotonin content. The HPLC analysis (Fig.
6B) demonstrated that there was no significant difference in
the amount of serotonin stored in wild type and G
q
/
platelets. In addition, no significant difference was detected in the
duodenum where serotonin secreting enterochromaffin cells provide the
serotonin taken up by the platelets during their passage through the
capillary net of the duodenal mucosal villi. Thus, G
q is
responsible for the regulation of VMAT2 in mouse platelets.
View larger version (15K):
[in a new window]
Fig. 6.
G q-deficient mice exhibit no
GMppNp-mediated inhibition of vesicular serotonin uptake.
A, platelets from wild type and G
q
/
mice
were incubated for 15 min at 37 °C with 80 nM
[3H]serotonin in the absence or presence of 100 µM GMppNp. In platelets obtained from wild type animals,
the addition of GMppNp inhibited the serotonin uptake to about 40%.
Conversely, only a very weak GMppNp effect was observed in
G
q
/
mice. Data from five individual experiments,
each performed with material obtained from eight mice, were summarized.
The values given in percentages inhibition (controls in the absence of
GMppNp set as 100%) are expressed as the means of the five
experiments ± S.D. Unspecific uptake in the presence of
tetrabenazine was subtracted prior to calculations. Statistical
significance (p = 0.011) was verified using Student's
t test. B, an HPLC analysis demonstrates that
there is no significant difference in the amount of serotonin stored in
platelets or EC cells of the duodenum between wild type and
G
q
/
mice. The values represent the means of three
determinations ± S.D. wt, wild type.
q Does Not Involve
Plasma Membrane Receptor Activation--
The data so far indicate that
the vesicular content and G
q are crucial for the
regulation of platelet VMAT2 activity. However, preloading of Tph1
/
platelets with serotonin could cause activation of receptors that in
turn may mediate the change in the modulation of VMAT2 by GMppNp. Even
under permeabilization such an involvement of receptors may not be
ruled out with certainty. To distinguish whether G protein regulation
of VMAT2 is mediated by plasma membrane receptors or directly by
vesicular properties, a variety of control experiments were performed
using antagonists or agonists of receptors known to be expressed in platelets.
q-coupled 5HT2A receptor is one of the
key elements in Ca2+ mobilization during platelet
activation and aggregation (38). Activation of the
G
s-coupled
2A adrenoreceptor is reported
to inhibit platelet serotonin release (39). The expression of dopamine receptors (D3 type) in platelets was recently published
(40). Preloading of intact Tph1
/
platelets was performed with
either serotonin or noradrenaline in the absence or presence of
spiperone, which blocks 5HT2A and D2-like
receptors, BRL 44408, which interferes with
2A
receptors, and GR 103691, antagonizing D3 receptor-mediated effects at 100 nM each. Fig.
7A illustrates that neither
spiperone alone nor a combination of all three antagonists
significantly affected the GMppNp-mediated inhibition of vesicular
serotonin uptake into cells preincubated with serotonin.
GMppNp-mediated down-regulation of VMAT2 could also be reconstituted by
noradrenaline using permeabilized platelets (Fig. 3C). For
preincubation with noradrenaline, intact platelets had to be incubated
with relatively high concentrations (100 µM) to
circumvent substrate specificity of SERT. In platelets pretreated with
noradrenaline (Fig. 7B), the addition of GMppNp inhibited
the [3H]serotonin uptake by 20% comparable with the
situation in permeabilized platelets. About the same effect was
observed, when receptor antagonists were added during
preincubation.
View larger version (24K):
[in a new window]
Fig. 7.
Receptor activation is not required for G
protein regulation of VMAT2. A and B, during
preloading with serotonin (A) or noradrenaline
(B), intact Tph1 /
platelets were incubated with or
without the monoamine receptor antagonists spiperone
(5HT2A, D2-like), BRL44408 (
A2),
and GR 103601 (D3) at a concentration of 100 nM
each. Neither spiperone nor a combination of all three ligands affected
the GMppNp-mediated inhibition of [3H]serotonin uptake
observed after preloading with 15 µM serotonin
(A) or 100 µM noradrenaline (B).
C, incubation of permeabilized wild type platelets with 1 µM of the TXA2 receptor agonist U46619 had no
effect on vesicular [3H]serotonin uptake.
q deletion mutants is
due to a defective activation by several physiological activators that
all signal via G
q (26), the most important in this
respect being the thromboxane A2 (TXA2)
receptor. To exclude the possibility that the
G
q-mediated modulation of VMAT2 is mainly due to an activation of G
q-coupled receptors even under
permeabilized conditions, we performed uptake experiments in the
presence of the TXA2 receptor agonist U46619. The addition
of 1 µM U46619 to permeabilized wild type platelets had
no effect on vesicular serotonin uptake (Fig. 7C). These
data suggest that G
q-mediated down-regulation of VMAT2
is independent from plasma membrane receptor activation and sustained
by vesicular properties.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
o2 in brain (13), it is regulated by
G
q in platelets.
-vinyl-GABA to
inhibit degradation of GABA by GABA transaminases (44) led to an
increased amplitude of quantal transmitter release. These findings
suggest that secretory vesicles can accept more neurotransmitter if
available. Although these manipulations increased the transmitter
content over a given amount, the opposite model characterized by empty
vesicles is more difficult to find. In the case of monoaminergic
vesicles reducing transmitter content by directly interfering with
inhibitors of tyrosine or tryptophan hydroxylases for
catecholamine or serotonin synthesis, respectively, proved to be
difficult because the inhibitors may also interfere with VMAT itself.
Direct inhibition of VMAT by reserpine expectedly reduced vesicular
content but resulted in secretory granules of reduced volume at least
in PC12 cells (8). Although these data suggest that vesicular volume is
critical in regulating transmitter content, further analysis of
putative regulatory mechanisms focusing on vesicular content is
hindered by the irreversible inhibition of VMAT caused by reserpine
treatment. Less filled or probably empty secretory vesicles have been
generated at the frog neuromuscular junction by repetitive stimulation
in combination with an impaired uptake because of hemicholinium,
vesamicol, and NH
/
platelets with serotonin fully reconstitutes
the G protein-mediated inhibition. In this respect it is noteworthy
that reconstitution of the GMppNp inhibition is not serotonin-specific
and works also with noradrenaline, which is transported by VMAT at a
comparable Km. These data indicate that signal
transduction starts from the luminal site of the vesicle. As required
for a general regulation, other monoamines in addition to serotonin do
the same job. So far we do not know the "receptor" working from the
luminal site that senses the transmitter content. VMAT2 by one of its
intravesicular loops may be a candidate.
/
platelets.
These include serotonergic (5HT2A), noradrenergic, or
dopaminergic receptors relevant for platelet activation. This does not,
however, exclude cross-talk between membrane receptors and vesicles,
which would enable neurons, neuroendocrine cells, or even platelets to
adapt their vesicular transmitter content to the requirements of the
network or their respective environment. Reduction of vesicular content
has been achieved by activating D2 receptors using
quinpirol, which inhibited tyrosine hydroxylase and reduced quantal
size in PC12 cells (43). The reduction in quantal size may be directly
linked to the reduced catecholamine content. Alternatively, activation
of D2 receptors may turn on G
o (46) present in
chromaffin granules (11) and in PC12 cells by which VMAT activity can
be down-regulated (12). Future investigations will define the signal
cascade between plasma membrane receptors and vesicular content.
q as a Regulator of VMAT2 in
Platelets--
Platelets resemble neurons with respect to transmitter
storage and release and thus are sometimes regarded as a peripheral indicator of functional aspects of central serotonergic and other monoaminergic neurons. Platelets, however, do not contain G
o, which
is mainly present in neurons and neuroendocrine cells where it is
involved in VMAT regulation (12, 13). Using a G
q
deletion mutant, we show here that VMAT2 of platelets is regulated by
G
q. G
q-mediated regulation of VMAT2
appears to be restricted to platelets because VMAT2 activity in the
brain-derived SSV was inhibited by G
o2 (13) and not
affected in G
q
/
mice.2 Failure of
G
q
/
platelets to respond to a GMppNp stimulus is not
due to a reduced monoamine content as revealed by HPLC analysis. This
indicates that VMAT2 may be regulated by different G proteins depending
on the respective tissue. Whether such promiscuity also applies to
VMAT1 or other vesicular transmitter transporters is not known so far.
q deletion causes a severe phenotype in platelets
characterized by a complete failure of aggregation. Receptors including the one for TXA2, which is the most important receptor for
platelet activation (35), are linked to this G protein. So regulation of VMAT2 might at least partially be obtained by activation of G
q-linked receptors. However, the addition of a
TXA2 agonist to permeabilized wild type platelets has no
effect, excluding the possibility that under permeabilized conditions
an activation of TXA2 (or other G
q-linked
receptors) may activate G
q, which in turn down-regulates VMAT2.
q appears to work at the vesicular membrane. This
assumption is substantiated by electron microscopic studies revealing that VMAT2 and G
q occur on vesicular structures
including dense bodies,
-granules, and a third vesicular population
we called small dense vesicles. From human platelets it is well known
that especially dense bodies are the main source of serotonin storage (18, 34). The small dense vesicles we observed in mouse platelets appeared to be absent in human platelets. The fact that these small
dense vesicles also contain, in addition to VMAT2 and
G
q, synaptobrevin immunoreactivity suggests that they
are important storage organelles for serotonin at least in mouse platelets.
q (this
work) or G
o2 (13). The vesicular filling mediates the G
protein effects, which by an as yet unknown signal cascade, probably
depending on the G protein involved, modulates VMAT2.
![]() |
ACKNOWLEDGEMENTS |
---|
We are indebted to Stefan Offermanns for
generously providing the Gq-deficient mice. We also
thank Ursel Tofote for expert technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
To whom correspondence should be addressed.
E-mail: gudrun.ahnert@charite.de.
Published, JBC Papers in Press, February 25, 2003, DOI 10.1074/jbc.M212816200
2 S. Winter, D. Walther, M. Höltje, I. Pahner, and G. Ahnert-Hilger, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
VMAT, vesicular
monoamine transporter;
SSV, small synaptic vesicle;
AS, antiserum;
SLO, streptolysin O;
TH, Tyrode-Hepes;
HPLC, high pressure liquid
chromatography;
PBS, phosphate-buffered saline;
TXA2, thromboxane A2;
GABA, -aminobutyric acid;
GMppNp, guanosine 5'-(
i
-imido)triphosphate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Liu, Y., Peter, D., Roghani, A., Schuldiner, S., Prive, G. G., Eisenberg, D., Brecha, N., and Edwards, R. H. (1992) Cell 70, 539-551[Medline] [Order article via Infotrieve] |
2. | Erickson, J.-D., Eiden, L.-E., and Hoffman, B.-J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10993-10997[Abstract] |
3. |
Peter, D.,
Jimenez, J.,
Liu, Y.,
Kim, J.,
and Edwards, R. H.
(1994)
J. Biol. Chem.
269,
7231-7237 |
4. |
Erickson, J. D.,
Schäfer, M. K.,
Bonner, T. I.,
Eiden, L. E.,
and Weihe, E.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
5166-5171 |
5. | Nirenberg, M. J., Liu, Y., Peter, D., Edwards, R. H., and Pickel, V. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8773-8777[Abstract] |
6. | Kanner, B. I., and Schuldiner, S. (1987) CRC Crit. Rev. Biochem. 22, 1-38[Medline] [Order article via Infotrieve] |
7. |
Pothos, E. N.,
Davila, V.,
and Sulzer, D.
(1998)
J. Neurosci.
18,
4106-4118 |
8. |
Colliver, T. L.,
Pyott, S. J.,
Achalabun, M.,
and Ewing, A. G.
(2000)
J. Neurosci.
20,
5276-5282 |
9. | Bruns, D., Riedel, D., Klingauf, J., and Jahn, R. (2000) Neuron 28, 205-220[Medline] [Order article via Infotrieve] |
10. | Ahnert-Hilger, G., Schäfer, T., Spicher, K., Grund, C., Schultz, G., and Wiedenmann, B. (1994) Eur. J. Cell Biol. 65, 26-38[Medline] [Order article via Infotrieve] |
11. | Pahner, I., Höltje, M., Winter, S., Nürnberg, B., Ottersen, O. P., and Ahnert-Hilger, G. (2002) Eur. J. Cell Biol. 81, 449-456[Medline] [Order article via Infotrieve] |
12. |
Ahnert-Hilger, G.,
Nürnberg, B.,
Exner, T.,
Schäfer, T.,
and Jahn, R.
(1998)
EMBO J.
17,
406-413 |
13. |
Höltje, M.,
von Jagow, B.,
Pahner, I.,
Lautenschlager, M.,
Hörtnagl, H.,
Nürnberg, B.,
Jahn, R.,
and Ahnert-Hilger, G.
(2000)
J. Neurosci.
20,
2131-2141 |
14. |
Gasman, S.,
Chasserot Golaz, S.,
Hubert, P.,
Aunis, D.,
and Bader, M. F.
(1998)
J. Biol. Chem.
273,
16913-16920 |
15. |
Brouillet, E.,
Trembleau, A.,
Galanaud, D.,
Volovitch, M.,
Bouillot, C.,
Valenza, C.,
Prochiantz, A.,
and Allinquant, B.
(1999)
J. Neurosci.
19,
1717-1727 |
16. | Cesura, A. M., Bertocci, B., and Da Prada, M. (1990) Eur. J. Pharmacol. 186, 95-104[CrossRef][Medline] [Order article via Infotrieve] |
17. | Zucker, M., Weizman, A., and Rehavi, M. (2001) Life Sci. 69, 2311-2317[CrossRef][Medline] [Order article via Infotrieve] |
18. | Wurzinger, L. J. (1990) Adv. Anat. Embryol. Cell Biol. 120, 1-96[Medline] [Order article via Infotrieve] |
19. | Walther, D. J., Peter, J.-U., Bashammakh, S., Hörtnagl, H., Voits, M., Fink, H., and Bader, M. (2002) Science 299, 5603, 76 |
20. | Edelmann, L., Hanson, P. I., Chapman, E. R., and Jahn, R. (1995) EMBO J. 14, 224-231[Abstract] |
21. |
Laugwitz, K. L.,
Allgeier, A.,
Offermanns, S.,
Spicher, K.,
Van, S.,
Dumont, J. E.,
and Schultz, G.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
116-120 |
22. | Spicher, K., Kalkbrenner, F., Zobel, A., Harhammer, R., Nurnberg, B., Soling, A., and Schultz, G. (1994) Biochem. Biophys. Res. Commun. 198, 906-914[CrossRef][Medline] [Order article via Infotrieve] |
23. | Hinsch, K.-D., Tychowiecka, I., Gausepohl, H., Frank, R., Rosenthal, W., and Schultz, G. (1989) Biochim. Biophys. Acta 1013, 60-67[Medline] [Order article via Infotrieve] |
24. | Brunk, I., Pahner, I., Maier, U., Jenner, B., Veh, R. W., Nürnberg, B., and Ahnert Hilger, G. (1999) Eur. J. Cell Biol. 78, 311-322[Medline] [Order article via Infotrieve] |
25. | Weller, U., Muller, L., Messner, M., Palmer, M., Valeva, A., Jensen, J., Agrawal, P., Biermann, C., Dobereiner, A., Kehoe, M. A., and Bhakdi, S. (1996) Eur. J. Biochem. 236, 34-39[Abstract] |
26. | Offermanns, S., Toombs, C. F., Hu, Y. H., and Simon, M. I. (1997) Nature 389, 183-186[CrossRef][Medline] [Order article via Infotrieve] |
27. | Walther, D. J., and Bader, M. (1999) Mol. Brain Res. 68, 55-63[Medline] [Order article via Infotrieve] |
28. | Huttner, W. B., Schiebler, W., Greengard, P., and De Camilli, P. (1983) J. Cell Biol. 96, 1374-1388[Abstract] |
29. | Takumi, Y., Ramirez Leon, V., Laake, P., Rinvik, E., and Ottersen, O. P. (1999) Nat. Neurosci. 2, 618-624[CrossRef][Medline] [Order article via Infotrieve] |
30. | Wenzel, H. J., Buckmaster, P. S., Anderson, N. L., Wenzel, M. E., and Schwartzkroin, P. A. (1997) Hippocampus 7, 559-570[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Youssefian, T.,
Massé, J.-M.,
Rendu, F.,
Guichard, J.,
and Cramer, E. M.
(1997)
Blood
89,
4047-4057 |
32. |
Krantz, D. E.,
Peter, D.,
Liu, Y.,
and Edwards, R. H.
(1997)
J. Biol. Chem.
272,
6752-6759 |
33. | Miller, G. W., Erickson, J. D., Perez, J. T., Penland, S. N., Mash, D. C., Rye, D. B., and Levey, A. I. (1999) Exp. Neurol. 156, 138-148[CrossRef][Medline] [Order article via Infotrieve] |
34. | Morgenstern, E. (1995) Eur. J. Cell Biol. 68, 183-190[Medline] [Order article via Infotrieve] |
35. | Offermanns, S. (2000) Biol. Chem. 381, 389-396[Medline] [Order article via Infotrieve] |
36. | Giesberts, A. N., van Ginneken, M., Gorter, G., Lapetina, E. G., Akkerman, J. W., and van Willigen, G. (1997) Biochem. Biophys. Res. Commun. 234, 439-444[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Offermanns, S.,
Hashimoto, K.,
Watanabe, M.,
Sun, W.,
Kurihara, H.,
Thompson, R. F.,
Inoue, Y.,
Kano, M.,
and Simon, M. I.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
14089-14094 |
38. | Mendelson, S. D. (2000) J. Affect. Disord. 57, 13-24[CrossRef][Medline] [Order article via Infotrieve] |
39. | Berk, M., Plein, H., Ferreira, D., and Jersky, B. (2001) Eur. Neuropsychopharmacol. 11, 183-186[CrossRef][Medline] [Order article via Infotrieve] |
40. | Ricci, A., Bronzetti, E., Mannino, F., Mignini, F., Morosetti, C., Tayebati, S. K., and Amenta, F. (2001) Naunyn-Schmiedeberg's Arch. Pharmacol. 363, 376-382[CrossRef][Medline] [Order article via Infotrieve] |
41. | Song, H., Ming, G., Fon, E., Bellocchio, E., Edwards, R. H., and Poo, M. (1997) Neuron 18, 815-826[Medline] [Order article via Infotrieve] |
42. |
Pothos, E. N.,
Larsen, K. E.,
Krantz, D. E.,
Liu, Y.,
Haycock, J. W.,
Setlik, W.,
Gershon, M. D.,
Edwards, R. H.,
and Sulzer, D.
(2000)
J. Neurosci.
20,
7297-7306 |
43. |
Pothos, E. N.,
Przedborski, S.,
Davila, V.,
Schmitz, Y.,
and Sulzer, D.
(1998)
J. Neurosci.
18,
5575-5585 |
44. |
Engel, D.,
Pahner, I.,
Schulze, K.,
Frahm, C.,
Jarry, H.,
Ahnert-Hilger, G.,
and Draguhn, A.
(2001)
J. Physiol. (Lond.)
535,
473-482 |
45. |
Van der Kloot, W.,
Colasante, C.,
Cameron, R.,
and Molgo, J.
(2000)
J. Physiol. (Lond.)
523,
247-258 |
46. |
Watts, V. J.,
Wiens, B. L.,
Cumbay, M. G.,
Vu, M. N.,
Neve, R. L.,
and Neve, K. A.
(1998)
J. Neurosci.
18,
8692-8699 |