 |
 Previous Article | Next Article 
The Journal of Neuroscience, August 15, 2000, 20(16):6013-6020
The weaver Mutation Reverses the Function of
Dopamine and GABA in Mouse Dopaminergic Neurons
Ezia
Guatteo1,
Francesca R.
Fusco1,
Patrizia
Giacomini3,
Giorgio
Bernardi1, 2, and
Nicola B.
Mercuri1, 2
1 Fondazione Santa Lucia, Istituto di Ricovero e Cura a
Carattere Scientifico, 00179 Rome, Italy, 2 Clinica
Neurologica, Università di Tor Vergata, 00173 Rome, Italy, and
3 Clinica Neurologica, Università di Roma La
Sapienza, 00161 Rome, Italy
 |
ABSTRACT |
In the present study, we characterized the intrinsic
electrophysiological properties and the membrane currents activated by dopamine (DA) D2 and GABAB receptors in
midbrain dopaminergic neurons, maintained in vitro in a
slice preparation, from wild-type and homozygous weaver
(wv/wv) mice. By using patch-clamp techniques, we found
that membrane potential, apparent input resistance, and spontaneous
firing of wv/wv dopaminergic neurons were similar to
those of dopamine-containing cells recorded from nonaffected (+/+) animals.
More interestingly, the wv/wv neurons were excited
rather than inhibited by dopamine and the GABAB agonist
baclofen. This neurotransmitter-mediated excitation was
attributable to the activation of a G-protein-gated inward
current that reversed polarity at a membrane potential of approximately
 30 mV. We suggest that the altered behavior of the receptor-operated
wv G-protein-gated inwardly rectifying
K+ channel 2 (GIRK2) might be related to the
selective degeneration of the dopaminergic neurons. In addition, the
wv GIRK2 would not only suppress the
autoreceptor-mediated feedback inhibition of DA release but could also
establish a feedforward mechanism of DA release in the terminal fields.
Key words:
substantia nigra; dopamine; baclofen; inwardly rectifying
K+ channels; weaver mouse; electrophysiology; dopamine-related disorders
| |
INTRODUCTION |
Weaver (wv) is an
autosomic recessive mutation of the mouse G-protein-gated inwardly
rectifying K+ channels (GIRK2) mainly
associated to postnatal loss of the external granuli of the cerebellum
(Rakic et al., 1973 ) and the dopaminergic cells of the ventral midbrain
(Schmidt et al., 1982; Gupta et al., 1987; Triarhou et al., 1988; Smith
et al., 1990). With regard to the dopaminergic neurons, it has been
shown that they degenerate during the first 3 weeks of life in
homozygous wv/wv mutant mice. However, the number of
tyrosine hydroxylase (TH)-positive neurons in wv/wv and
wild-type (+/+) midbrain is the same at birth (Bayer et al.,
1995; Verney et al., 1995), suggesting that the wv gene exclusively targets and impairs postmitotic dopaminergic cells. Consequently, the wv/wv mouse has a lower level of dopamine
(DA) in the brain than +/+ mice (Schmidt et al., 1982; Roffler-Tarlov and Graybiel, 1984; Triarhou et al., 1988) and represents a
genetic animal model of nigrostriatal deficiency that could mimic the pathophysiology of Parkinson's disease (Simon and Ghetti,
1994).
The wv mutation is a single amino acid substitution (glycine
156 to serine) located in the gene encoding for GIRK2 (Patil et al.,
1995). The functional GIRK channel consists of tetramers of five
subunits (Kofuji et al., 1996), and only the GIRK1-GIRK3 subunits were
found in the CNS. The GIRK channel is the functional target of
many neurotransmitters (North, 1989; Liao et al., 1996; Sodickson and
Bean, 1998) and regulates neuronal excitability, being permeable to
potassium ions (Hille, 1992; Jan and Jan, 1994). Western blotting,
in situ hybridization, and immunocytochemistry studies have
shown that a strong GIRK2 signal is present in the dopaminergic neurons
of the substantia nigra and ventral tegmental area, being the GIRK1 at
the background level (Liao et al., 1996; Murer et al., 1997; Inanobe et
al., 1999). When expressed in Xenopus oocytes, the
wv GIRK2 displays three novel properties: (1) a loss of
selectivity for potassium ions and gain of permeability for sodium
(Slesinger et al., 1996; Tong et al., 1996) and/or calcium (Silverman
et al., 1996; Tucker et al., 1996); (2) a constitutive activation that
does not require G-proteins (Navarro et al., 1996); and (3) a blockade
by a class of molecules (QX-314, MK-801, verapamil) that do not affect
the wild GIRK. Although there is general agreement on the loss of
K+ selectivity and on the pharmacology of
the wv GIRK2, the gating properties and functional effects
on native neurons appear to be very heterogeneous depending on the type
of neurons investigated. It is not yet known whether the constitutive
activation of the inwardly rectifying K+
channels is inherent to the mutated wv GIRK2 channel protein or is an indirect consequence of other cellular properties (Silverman et al., 1996). For instance, the developmental stage of
wv/wv cerebellar granule cells determines the
state of the G-protein modulation of the inwardly rectifying current
and possibly its tonic activation (Kofuji et al., 1996; Slesinger et
al., 1996, 1997; Surmeier et al., 1996; Rossi et al., 1998). On the
other hand, the wv GIRK2 is neither tonically active nor
G-protein-operated in CA3 hippocampal neurons (Jarolimek et al., 1998).
Considering that GIRK2 tetramers are almost selectively present in the
membrane of midbrain dopaminergic neurons in which they represent the
common functional target of the inhibitory inputs mediated by dopamine D2 and GABAB receptors
(Lacey et al., 1988; Kim et al., 1997; Inanobe et al., 1999), we have
been interested in studying whether the wv mutation affects
the electrical membrane properties and the response to dopamine and
GABA of these neurons recorded in slices of mouse mesencephalon.
| |
MATERIALS AND METHODS |
Preparation of the tissue. Homozygous
(wv/wv) weaver mice were obtained by breeding
heterozygous +/wv mice (B6CBA) (Jackson Laboratories, Bar
Harbor, ME). They were ~25% of littermates according to simple
mendelian segregation and were identified by their clearly visible
motor dysfunction, tremor, and cerebellar atrophy (Rakic and Sidman,
1973). Horizontal slices comprising the substantia nigra and the
ventral tegmental area were cut from the ventral mesencephalon of 16- to 20-d-old wv/wv, and +/+ mice were anesthetized with
ketamine and killed. The method used has been described previously (Mercuri et al., 1994, 1997). The brain was rapidly removed, and the
slices (200- to 250-µm-thick) were obtained by using a vibratome starting from the ventral surface of the midbrain. Slices recovered for
at least 1 hr in a holding chamber and then were transferred into a
recording chamber. They were completely submerged with a continuously
flowing (2.5 ml/min) artificial CSF solution at 34-35°C, pH
7.4. This solution contained (in mM): NaCl 126, KCl 2.5, MgCl2 1.2, NaH2PO4 1.2, CaCl2 2.4, glucose 10, NaHCO3 18, gassed with 95%
O2 and 5% CO2, pH 7.4.
Patch-clamp recordings. The recording chamber was mounted on
the stage of an upright microscope (Axioscope FS; Zeiss,
Oberkochen, Germany) equipped for an infrared video microscopy
(Hamamatsu, Hamamatsu City, Japan). Individual dopaminergic neurons
were visualized by infrared video imaging and approached by applying
positive pressure. Pipettes were made from borosilicate glass (1.5 mm; World Precision Instruments, Sarasota, FL) and pulled with a vertical PP 83 Narishige (Tokyo, Japan) puller. They had a resistance of  4
M when filled with a standard solution containing (in
mM): K+ gluconate
145, CaCl2 0.1, MgCl2 2, HEPES 10, EGTA 0.75, Mg-ATP 2, and Na3-GTP 0.3, pH 7.3. In a subset of experiments, Na3-GTP was
substituted with the nonhydrolizable analog GTP- -S (0.6 mM) or with GDP- -S (0.6 mM). Whole-cell recordings were performed with an
Axopatch 1D amplifier (Axon Instruments, Foster City, CA) and series
resistances were compensated. Hyperpolarizing voltage steps were
delivered from 60 to 120 mV (holding potential of 40 mV, 10 mV
increments) and lasted 800 msec. Activation kinetics of
Ih were calculated by fitting the
current at 120 mV with a first-order exponential decay function.
Because of the presence of the large
Ih current in the dopaminergic neurons
(Grace and Onn, 1989; Lacey et al., 1989; Johnson and North, 1992;
Mercuri et al., 1995, 1997), voltage ramps were preceded by a voltage
step of 600 msec from the holding potential of 40 to 120 mV to
fully activate the Ih current (see
Fig. 4C). The membrane voltage and current were acquired
using pClamp and Axioscope softwares (Axon Instruments); data analysis
was performed using Origin software (Microcal, Northampton, MA).
Cell-attached recordings were made after the giga seal was established,
by measuring the number of spontaneous action potentials from the
extracellular side of the membrane.
Drug application. Drugs were bath-applied by switching the
superfusing solution to one containing a known concentration of drugs.
Full exchange of the solution in the recording chamber was achieved
within 1 min. L-sulpiride was from Ravizza, CGP
55845A and R-baclofen were from Novartis Pharma Ag (Basel,
Switzerland), QX-314 was purchased from Alomone Labs (Jerusalem,
Israel), and ZD 7288 was from Tocris Cookson (Bristol, UK). Dopamine,
GTP--S, and GDP--S were from Sigma (Milano, Italy). In
voltage-clamp, experiments we used higher DA concentration to evoke
maximal current amplitudes.
Immunohystochemistry. Biocytin (free base, 5 mM; Sigma) was added to the pipette solution to
perform post hoc labeling of the recorded cells with TH.
Immediately after recording, slices containing biocytin-loaded cells
were fixed by immersion in 4% paraformaldehyde in 0.1 M PBS overnight at +4°C. The tissue was subsequently immersed in 20% sucrose-10% glycerol in 0.1 M PBS for 3 hr at room temperature for
cryoprotection. Slices were then frozen and cut to 40-µm-thick
sections by a sliding microtome. Sections were incubated with a
cocktail of avidin-conjugated fluorescein isothiocyanate (FITC) (1:200;
Sigma) and anti-mouse tyrosine hydroxylase monoclonal antibody (1:200)
in PBS containing 0.1% Triton X-100 overnight at +4°C. After 10 min
rinses in 0.1 M PBS, sections were incubated in a
mixture of avidin-conjugated FITC (1:200) and tetramethyl rhodamine
(TRITC) (Sigma) -conjugated goat anti mouse IgG 1:50 in PBS containing
0.1% Triton X-100 for 3 hr at room temperature. Sections were then
rinsed three times for 10 min in 0.1 M PBS,
mounted on slides, and coverslipped with 50% glycerol in PBS. Slides
were observed with an epi-illumination fluorescence microscope (Zeiss)
and with a confocal laser-scanning microscope (Zeiss LSM 510).
Statistical analysis. The data were presented as mean ± SEM. Statistical difference was determined by paired or unpaired
Student's t test at a significance level of 0.05.
| |
RESULTS |
Intrinsic properties of native and mutated
dopaminergic neurons
To identify the dopaminergic neurons among a heterogeneous
population of cells, we considered the electrophysiological parameters already established as "typical" for these cells (Grace and Onn, 1989; Lacey et al., 1989: Johnson and North, 1992; Mercuri et al.,
1995, 1997; Richards et al., 1997). In fact, we evaluated the presence
of a strong hyperpolarization-activated inward current (Ih), the ability to fire
spontaneously in a pacemaker manner, and the value of the resting
membrane potential of the presumed dopaminergic neurons from
both +/+ and homozygous wv/wv mice. Hyperpolarizing voltage
steps (Fig. 1A) from
60 to 120 mV (Vh of 40 mV)
evoked an inward current (Ih) in both
+/+ and wv/wv neurons. The mean current amplitudes, measured
at 120 mV, were 321 ± 46 pA (n = 10) in +/+ and
490 ± 46 pA (n = 16) (unpaired t test,
p < 0.05) in wv/wv neurons. Activation
kinetics at 120 mV were 145 ± 9 msec in +/+ (n = 10) and 116 ± 8 msec (n = 16) (unpaired
t test, p < 0.05) in wv/wv
dopaminergic neurons, respectively. Under current-clamp recordings
(Fig. 1B), depolarizing current steps elicited
regularly firing action potentials, whereas hyperpolarizing pulses
activated a characteristic "sag" potential in both +/+ (n = 10) and wv/wv (n = 16)
neurons. The mean resting membrane potential (Fig. 1C,
left) (holding potential at 0 current) was 47.6 ± 1.5 mV in +/+ (n = 17) and 47.1 ± 1.3 mV in
wv/wv (n = 18) (unpaired t test,
p = 0.81) neurons. The mean values of the spontaneous
firing (Fig. 1C, right) were 2.1 ± 0.12 Hz
(n = 7) in +/+ and 2.4 ± 0.2 Hz
(n = 15) (unpaired t test, p = 0.35) in wv/wv cells. The apparent input resistance
(measured by a small 5 mV hyperpolarizing step) was 410 ± 28 (n = 18) and 515 ± 81 (n = 7)
M (unpaired t test, p = 0.12) in +/+ and
wv/wv cells, respectively.
View larger version (28K):
[in this window]
[in a new window]
|
Figure 1.
Electrophysiological and immunohistochemical
identification of midbrain dopaminergic neurons in +/+ and
wv/wv mice. A, Hyperpolarizing voltage
steps from 60 to 120 mV (10 mV increments,
Vh of 40 mV) activated the mixed cation
current (Ih) in both genotypes.
Calibration: 250 msec, 500 pA. B, The corresponding
current-clamp recording showed that hyperpolarizing pulses (0.5 and
1 nA) activated the Ih current, thus
producing a typical sag potential in both cells taken form the
two genotypes. A depolarizing current pulse (0.5 nA) elicited a
pacemaker-like sequence of action potentials in both neurons. Action
potential amplitudes are clipped because of low sampling rate of the
digital interface. Time bar: 100 msec. C,
Left, The white square indicates the mean
resting membrane potential (RMP) of +/+ dopaminergic
cells (47.6 ± 1.5 mV, n = 17), and the
black square indicates the mean resting membrane
potential of wv/wv cells (47.1 ± 1.3 mV,
n = 18); values were not significantly different
(p = 0.81). Right, The
columns indicate the mean spontaneous firing recorded in
cell-attached configuration in +/+ (white) (2.1 ± 0.12 Hz, n = 7) and wv/wv
(black) (2.4 ± 0.2 Hz, n = 15)
neurons; values were not significantly different
(p = 0.35). D, Confocal laser
scanning microscope image of a wv/wv neuron loaded with
biocytin (5 mM) through the patch pipette showing typical
features of a dopaminergic neuron (magnification, 20×):
a, biocytin staining as revealed by FITC fluorescence;
b, TH immunostaining as revealed by TRITC fluorescence
(note that many neurons, including the recorded one, resulted in being
TH-positive, within the SNc); c, merged image of the two
fluorescent stainings.
|
|
Furthermore, we loaded some wv/wv cells showing the
electrophysiological properties delineated above with biocityn (5 mM), and we immunostained them with antibodies
against TH. Nine of 10 neurons were TH-positive (Fig.
1D). Based on these observations, only neurons
showing a pronounced Ih and a
spontaneous pacemaker activity at rate of ~2 Hz were
considered to be dopaminergic and included in the present study.
Activation of D2 and GABAB receptors
mediates excitation of wv/wv dopaminergic neurons
It is well known that dopamine and GABA, by acting on
D2 and GABAB receptors,
inhibit the firing activity of dopaminergic neurons (Lacey et al.,
1988). To test the effects of dopamine on wv/wv dopaminergic
neurons without perturbing the intracellular environment with the
standard whole-cell configuration, we measured the rate of the
spontaneous action potentials in cell-attached mode, in control
condition and during the superfusion of dopamine (30 µM) (Fig.
2A). Interestingly DA
increased the firing frequency of wv/wv neurons from
2.3 ± 0.3 (n = 11) to 4.3 ± 0.4 (n = 11) Hz (paired t test,
p < 0.01), whereas it abolished the spontaneous activity of the +/+ neurons (n = 4). In whole-cell
current-clamp configuration (Fig. 2B), the
wv/wv dopaminergic neurons were depolarized by DA (30 µM), and the number of the action potentials
increased (n = 7). In contrast, DA (30 µM) hyperpolarized the membrane and abolished
the spontaneous activity of +/+ neurons (n = 4) (Lacey et al., 1988). Whole-cell voltage-clamp experiments (at 40 mV holding
potential) showed that the excitatory effect of DA on wv/wv
neurons is attributable to the activation of an inward current (58.3 ± 13 pA, n = 7) (Fig. 2C). On
the contrary, the inhibitory effect of DA in +/+ neurons is
attributable to the activation of an outward current that is caused by
the opening of GIRK channels (Lacey et al., 1988; Kim et al.,
1997).
View larger version (25K):
[in this window]
[in a new window]
|
Figure 2.
DA mediates inhibition of +/+ and excitation of
wv/wv neurons. A, Cell-attached
recordings from a +/+ (left) and wv/wv
(right) cell showing the changes of spontaneous firing
during the extracellular application of DA (30 µM). DA
clearly inhibited the firing of the +/+ neuron, whereas it increased
the activity of the wv/wv neuron. B,
Whole-cell current-clamp recordings of two dopaminergic cells in which
DA caused membrane hyperpolarization-inhibition (+/+) and
depolarization-excitation (wv/wv). C,
Voltage-clamp recordings (at Vh of 40 mV)
showing the activation of outward (+/+) and inward
(wv/wv) currents caused by DA (100 µM) in
+/+ and wv/wv dopaminergic cells, respectively.
|
|
An excitation of wv/wv dopaminergic neurons was also
observed when they were exposed to the GABAB
receptor agonist baclofen (Fig. 3). In
fact, in cell-attached configuration (Fig. 3A), baclofen (10 µM) increased the spontaneous activity of
wv/wv neurons from 2.8 ± 0.4 (n = 11)
to 5.5 ± 0.5 (n = 11) Hz (paired t
test, p < 0.01), whereas it depressed the spontaneous
firing in +/+ neurons (n = 4). In whole-cell
current-clamp mode, baclofen mimicked the effects of DA, mediating a
depolarization-excitation of wv/wv neurons
(n = 7) (Fig. 3B) and a hyperpolarization of
+/+ neurons (n = 4). In voltage-clamp recordings,
baclofen (10 µM) activated an inward current of
57.5 ± 10.8 pA (n = 7) in wv/wv
neurons (Fig. 3C). Conversely, baclofen inhibited the +/+
dopaminergic neurons, activating a classical GIRK-mediated outward
current (n = 4) (Lacey et al., 1988).
View larger version (25K):
[in this window]
[in a new window]
|
Figure 3.
The GABAB agonist baclofen mediates
inhibition of +/+ and excitation of wv/wv neurons.
A, Cell-attached recordings from a +/+
(left) and a wv/wv (right)
neuron showing the modification of the spontaneous firing caused by the
GABAB receptor agonist baclofen (10 µM). Note
the increase in firing frequency induced by this compound in the
wv/wv neuron. B, Whole-cell current-clamp
recordings showing the modification in membrane potential and firing
activity in +/+ and wv/wv neurons. C,
Corresponding voltage-clamp recordings (at
Vh of 40 mV) showing the changes in
membrane current caused by baclofen in a +/+ versus a
wv/wv neuron. Note that, like dopamine, baclofen
activated an inward rather than outward current in wv/wv
neurons.
|
|
Properties of the dopamine- and baclofen-induced
inward current
To characterize the I-V relationships of the dopamine-
and baclofen-induced inward currents, we performed voltage ramps over a
wide range of potentials, between 120 and 0 mV (Fig.
4C). The ramps were delivered
in control condition and in the presence of DA (100 µM) (Fig. 4A) or baclofen (10 µM) (Fig. 4B). The currents activated by both agonists in wv/wv neurons showed different
characteristics compared with those induced in +/+ cells. Indeed, as
expected for an increase of a pure potassium conductance (Lacey et al., 1988), the DA- and baclofen-induced currents crossed the control trace
around EK at 86 ± 6 (n = 4) and 87 ± 3 (n = 4) mV,
respectively, in +/+ neurons. Instead, the DA- and baclofen-induced
currents crossed the control trace at 27 ± 3.2 (n = 6) and 34 ± 3.2 (n = 6)
mV, respectively, in wv/wv neurons. These values of reversal potential suggest that a cation current is involved in the
neurotransmitter-mediated excitation of the wv/wv
dopaminergic neurons. Because the Ih
is a mixed cationic current, we tested the possibility that both D2 and GABAB receptors
could modulate Ih to produce
excitation. In the presence of cesium (1-3 mM),
a known blocker of Ih, the actions of
dopamine and baclofen were not modified (n = 3; data not shown). Conversely, the antiarrhythmic drug ZD 7288 (50 µM), which is also a potent inhibitor of
Ih, irreversibly depressed the
DA-induced (n = 6) and baclofen-induced
(n = 6) inward currents (Fig.
5B,D).
Furthermore, we tested a more classical cationic blocker, QX-314 (100 µM), on the neurotransmitter-induced inward currents. As already reported in Xenopus oocytes expressing
the wv GIRK2 (Kofuji et al., 1996), this drug inhibited the
excitatory effects of DA (Figs. 5A,
6C) (n = 7)
and baclofen (n = 6) (Figs. 5C,
6C) on the dopaminergic wv/wv cells without
modifying the receptor-operated outward currents in +/+ neurons (data
not shown). Furthermore, QX-314 did not change the spontaneous activity
and the membrane properties of the wv/wv dopaminergic cells
(n = 3 of 3 cells tested; data not shown). The
activation of wv GIRK2 by dopamine and baclofen was
inhibited by the D2 and
GABAB receptor antagonists sulpiride and CGP
55845A (Fig. 6B). In fact, the mean amplitudes of the
inward current activated by dopamine and baclofen at 120 mV in
control conditions, in the presence of QX-314 (100 µM), sulpiride (3-10
µM), and CGP 55845A (250-500
nM), are shown in Figure 6C. The
DA-induced current of 207 ± 33 pA (n = 7) was significantly reduced to 111 ± 13 pA (n = 7)
(paired t test, p < 0.05) in the presence
of QX-314 and to 117 ± 17 pA (n = 7) (paired t test, p < 0.05) in the presence of
sulpiride (10 µM) (Fig. 6C, top).
View larger version (18K):
[in this window]
[in a new window]
|
Figure 4.
Properties of the DA- and baclofen-induced
currents in wv/wv neurons. A, Voltage
ramps (from 120 to 0 mV) delivered in control condition and in the
presence of DA revealed that the DA-induced (100 µM)
current reversed at 86 ± 6 mV (n = 4) in
+/+ neurons (one cell is shown in the left), whereas it
reversed at 27 ± 3.2 mV (n = 6) in
wv/wv neurons (one cell is shown in the
right). B, The baclofen-activated (10 µM) current reversed at negative potentials (87 ± 3 mV, n = 4) in +/+ neurons (one cell is shown in
the left), whereas it reversed at 34.1 ± 3.2 mV
(n = 6) in wv/wv neurons (one cell
is shown in the right). The current traces of the
wv/wv neurons were recorded in the presence of TTX (0.5 µM), tetraethylammonium chloride (5 mM), and
nifedipine (10 µM) to reduce voltage-dependent sodium,
potassium, and calcium conductances. C shows the
protocol used to induce the slow depolarizing ramps.
|
|
View larger version (24K):
[in this window]
[in a new window]
|
Figure 5.
The cation channel blockers QX-314 and ZD 7288 inhibit the DA- and baclofen-induced inward currents in
wv/wv neurons. A, B, The
DA-induced (100 µM) current was strongly inhibited
by QX-314 (100 µM) and ZD 7288 (50 µM).
C, D, QX-314 (100 µM) and
ZD 7288 (50 µM) also inhibited the current induced by
baclofen (10 µM). Note that the protocol of the voltage
ramps in this and the following figures is the same shown in Figure
4C.
|
|
View larger version (20K):
[in this window]
[in a new window]
|
Figure 6.
D2 and GABAB receptor
antagonists reduce the DA- and baclofen-induced inward currents.
A, The inward current induced by both agonists in
wv/wv dopaminergic neurons was inhibited by the presence
of sulpiride (10 µM; B,
top) and CGP 55845A (250 nM;
B, bottom). C, The plots
show the mean values of DA-induced (top, black
columns) and baclofen-induced (bottom,
white columns) inward currents at the points indicated
by asterisks in A (120 mV). The
DA-induced inward current was 207 ± 33 pA (n = 7; left bar) and was significantly reduced by QX-314
(100 µM) to 111 ± 13 pA (paired t
test, p < 0.05; middle bar) and by
sulpiride (10 µM) to 117 ± 17 pA (paired
t test, p < 0.05; right
bar). The baclofen-induced inward current of 198 ± 28 pA
(n = 6; left bar) was significantly
reduced by QX-314 (100 µM) to 63 ± 15 (paired
t test, p < 0.01; middle
bar) and by CGP 55845A (250 nM) to 21 ± 8 pA
(paired t test, p < 0.01;
right bar).
|
|
The baclofen-induced current of 198 ± 28 pA (n = 6) was significantly reduced to 63 ± 15 nA (n = 6) (paired t test, p < 0.01) by QX-314 and
to 21 ± 8 pA (n = 6) (paired t test,
p < 0.01) by CGP 55845A (250 nM)
(Fig. 6C, bottom).
D2 and GABAB receptors activate
wv GIRK channels in a G-protein-dependent manner
In heterologous expression systems, wv GIRK2 channels
not only loose the potassium selectivity but also become constitutively active, being unable to interact with the G-protein (Kofuji et al.,
1996, Slesinger et al., 1996). In cells of the CNS, different gating
properties of wv GIRK2 have been reported; in fact, some neurons had no functional wv GIRK2 channels (Surmeier et
al., 1996; Jarolimek et al., 1998), and others underwent a constitutive activation of GIRK2 (Rossi et al., 1998). To investigate the G-protein dependency of the receptor-operated wv GIRK2, we dialyzed
the cytoplasm with a nonhydrolizable GTP analog, GTP--S (0.6 mM). Before breaking the membrane patch for the
whole-cell recordings, we tested the DA and baclofen sensitivity of the
wv/wv neurons by measuring their firing rate in
cell-attached configuration. Both DA (n = 5) and
baclofen (n = 5) increased the spontaneous firing of
the wv/wv neurons (Fig.
7A). Five to 10 min after the membrane rupture, a constitutive inward current developed and reached a
plateau of 74 ± 22 pA (n = 5; holding potential
at 40 mV). The mean amplitudes of DA- and baclofen-induced currents, at 120 mV, in wv/wv cells loaded with the pipette solution
containing GTP-Na3 (control) or GTP--S were
significantly different (Fig. 7B). In fact, the DA-induced
current of 204 ± 30 pA (n = 10) in GTP-Na3-loaded neurons was reduced to 38 ± 14 pA (n = 5) (unpaired t test,
p < 0.05) in GTP--S-loaded neurons. The
baclofen-induced current of 205 ± 40 pA (n = 6)
in GTP-Na3-loaded neurons was diminished to
57 ± 10 pA (n = 5) (unpaired t test,
p < 0.05) in GTP--S-loaded cells. Moreover, we
found that the inward current tonically activated by GTP--S (Fig.
7C) was reduced in a reversible manner by QX-314 (100 µM) (n = 5) and irreversibly by
ZD 7288 (50 µM) (n = 5). These
results strongly suggest that the inward current induced by GTP--S
is the same as the one generated by DA and baclofen. On the other hand,
whereas DA (n = 5) and baclofen (n = 5)
excited the wv/wv dopaminergic neurons in cell-attached
configuration (Fig.
8A), the subsequent
intracellular dialysis with the nonhydrolizable GDP analog GDP--S
(0.6 mM) prevented DA and baclofen effects within
a few minutes after membrane rupture (Fig. 8B). In
fact, the mean amplitudes of DA-induced current (at 120 mV) of
204 ± 30 pA (n = 10) in
GTP-Na3-loaded neurons was significantly reduced to 39 ± 15 pA (n = 5) (unpaired t
test, p < 0.01) in GDP--S-loaded neurons.
Furthermore, the baclofen-induced currents (at 120 mV) of 205 ± 40 pA (n = 6) in GTP-Na3-loaded
neurons was reduced to 21 ± 19 pA (n = 5)
(unpaired t test, p < 0.01) in
GDP--S-loaded neurons.
View larger version (23K):
[in this window]
[in a new window]
|
Figure 7.
D2 and GABAB
receptors couple to wv GIRK2 in a G-protein-dependent
manner. A, Recordings of the spontaneous firing of two
wv/wv neurons in cell-attached configuration with a
pipette solution containing the GTP analog GTP--S (0.6 mM). Note that both DA (left) and baclofen
(right) induced an increase of the spontaneous firing
before the rupture of the membrane patches. B, The
black columns show the amplitude of the DA-induced
current (at 120 mV) (204 ± 30 pA, n = 10) in control conditions and during the dialysis with GTP--S
(38 ± 14 pA, n = 5)
(p < 0.05, unpaired data). The white
columns show the amplitude of the baclofen-induced current (at
120 mV) (205 ± 40 pA, n = 6) in control
condition and during the intracellular dialysis with GTP--S (57 ± 10, n = 5) (p < 0.05, unpaired data). C, GTP--S induced a tonic
inward current that was reversibly blocked by QX-314 and irreversibly
inhibited by ZD 7288. Note that, under this condition, the response to
DA application was not observed.
|
|
View larger version (21K):
[in this window]
[in a new window]
|
Figure 8.
GDP--S prevents the DA- and baclofen-induced
inward currents. A, Cell-attached recordings of two
wv/wv neurons with a pipette containing the GDP analog
GDP--S (0.6 mM) showing the increase of the spontaneous
firing caused by DA and baclofen. B,
Left, black columns, In whole-cell
configuration, the DA-induced current (at 120 mV) was 204 ± 30 pA (n = 10) and was significantly reduced by
GDP--S to 39 ± 15 pA (n = 5, p < 0.01, unpaired data). Right,
white columns, The baclofen-induced current (at 120
mV) was 205 ± 40 pA (n = 6) and was
significantly reduced by GDP--S to 21 ± 19 pA
(n = 5, p < 0.01, unpaired
data).
|
|
| |
DISCUSSION |
The present study demonstrates that the wv
mutation reverses the functional effects (inhibition into excitation)
mediated by activation of dopamine D2 and
GABAB receptors in dopaminergic neurons of the
ventral midbrain (identified physiologically and by TH
immunoreactivity) and provides the first evidence of a
D2- and GABAB-mediated
excitation in native neurons of the CNS.
The membrane properties of wv/wv dopaminergic
neurons are not affected by the mutation
There is a general agreement that the wv GIRK2 looses
selectivity for potassium ions. In fact, the permeability and gating properties of the wv GIRK2 have been extensively
investigated in heterologous expression systems in which it has been
shown that the mutated channel becomes permeable to sodium (Navarro et
al., 1996; Slesinger et al., 1996) and eventually to calcium ions
(Silverman et al., 1996; Tucker et al., 1996). In addition, this
channel appears to be constitutively opened (Kofuji et al., 1996).
Consequently, there is a leakage entry of sodium into the cells that
can be reduced by channel blockers such as QX-314, MK-801, and
verapamil. A constitutive activation of the wv GIRK2 was
also observed in cultured cerebellar granule cells (Kofuji et al.,
1996) and in putative granule cells in the postmigratory position in
wv/wv cerebellar slices (Rossi et al., 1998). A similar constitutively opened wv GIRK2 conductance, sensitive to
QX-314, has been described recently in substantia nigra
wv/wv neurons (Liss et al., 1999). In fact, the dopaminergic
cells were found to be tonically depolarized, having no pacemaker
activity. Among the surviving cells within the substantia nigra
compacta (SNc) of the wv/wv mice, we found a cell population
whose resting potential, apparent input resistance, and spontaneous
firing discharge were indistinguishable from those of the dopaminergic
neurons recorded in +/+ mice. Thus, our results strongly suggest that
the mutated GIRK2 channels do not regulate the resting properties of
native wv/wv dopaminergic neurons. This is supported by the
following arguments: (1) a depolarized resting membrane potential, a
decrease in input resistance, and a loss of spontaneous pacemaker
activity should be expected if a tonic wv inward conductance
is present; and (2) the spontaneous firing activity should be affected
by the wv GIRK2 blocker QX-314 if the channel is operative
during the resting state of the neurons.
The membrane responses of dopaminergic neurons to dopamine and GABA
are modified by the wv mutation
Among the characteristics that identify the dopaminergic neurons
in the ventral midbrain, an inhibitory response to dopamine is an
important criterion (Mercuri et al., 1992; White, 1996). In
wv/wv, mice we found neurons displaying spontaneous firing, resting membrane potential, apparent input resistance, and firing rate
comparable with the ones recorded in wild animals. A more pronounced
Ih (Mercuri et al., 1995) was also
found in wv/wv cells. This might depend on either
compensatory changes of the hyperpolarization-activated channels or a
reduced apparent inhibition caused by DA and GABA (Watts et al., 1996).
Interestingly, the cellular responses to dopamine and the
GABAB agonist baclofen were opposite to those observed in control animals. In fact, both dopamine and baclofen, instead of inhibiting the wv/wv dopaminergic cells by
activating a G-protein-dependent outward current (Lacey et al., 1988;
Kim et al., 1997), caused an excitation that was generated by an inward current. The obtained null potential (approximately 30 mV) and the
sensitivity of this current to cationic channel blockers support the
hypothesis that DA and baclofen activate wv GIRK2 channels, which are permeable to sodium and potassium ions (Kofuji et al., 1996).
The loss of selectivity of the wv GIRK2 to potassium ions has been already demonstrated in other systems expressing the homomeric
wv GIRK2 (Kofuji et al., 1996; Navarro et al., 1996; Slesinger et al., 1996). Nevertheless, it is worth mentioning that the
responses to baclofen were lost in wv/wv hippocampal (Jarolimek et al., 1998) and cerebellar granule cells (Slesinger et
al., 1997). The lack of neurotransmitter action in these cells without
a gain of function might depend on the compensatory effect of other
GIRK family members (Hou et al., 1999).
It is noteworthy that the neurotransmitter-operated wv GIRK2
is not only sensitive to QX-314 but also to the bradycardic agent ZD
7288, a blocker of the mixed sodium/potassium current
Ih. This observation rises the
possibility that dopamine and baclofen might modulate the
Ih in the wv/wv
dopaminergic cells. However, this is not the case because, when the
Ih was blocked by extracellular cesium
(Mercuri et al., 1995), dopamine and baclofen still induced an inward
current in the wv/wv dopaminergic cells. Moreover, it appears that the inward current caused by dopamine and baclofen is
attributable to the activation of D2 and
GABAB receptors, respectively. In fact,
antagonists such as sulpiride (D2) and CGP
55845A, (GABAB) specifically inhibited the
effects of these neurotransmitters. The observation that the DA-induced
inward current was not completely blocked by sulpiride and QX 314 suggests that this catecholamine might have additional non-D2,
non-GIRK2 actions. Further experiments will be necessary to address
this problem.
An additional important conclusion of our results is that the
neurotransmitter activation of the mutated GIRK2 channel uses, as in
control condition, a G-protein. Indeed, intracellular dialysis with
GTP--S or GDP--S prevented the neurotransmitter-mediated inward
currents. In agreement with the involvement of a G-protein in the
activation of the wv GIRK2, the intracellular dialysis of
the nonhydrolizable analog of GTP, GTP--S, caused a sustained inward
current, which determined membrane depolarization, loss of spontaneous
activity, and occluded DA- and baclofen-induced responses. The fact
that the inward current caused by GTP--S was reversibly inhibited by
QX-314 and irreversibly blocked by ZD 7288 also suggests that it is
produced by G-protein-regulated opening of the wv GIRK2 channels.
Conclusions
In the present paper, we provide evidence that wv GIRK2
are specifically operated by D2 and
GABAB receptors in a G-protein-dependent manner
to induce depolarization of native dopaminergic neurons. It is likely
that the aberrant receptor-activated cationic entry throughout the
wv GIRK2 could result in activity-dependent cell death,
attributable to severe depolarization and sodium/calcium accumulation.
Moreover, the presence of synaptic excitation instead of a
K+-dependent inhibition failing to
counterbalance the depolarizing drive sustained by the release of
excitatory amino acids (Jensen et al., 1999) certainly contributes to
initiate sodium/calcium-dependent intracellular processes, which lead
to degeneration of wv/wv dopaminergic cells (Bertolino and
Llinas, 1992; Choi, 1995; Verney et al., 1995; Oo et al., 1996).
Although the mechanisms producing neuronal degeneration are not known
yet, our results suggest that they can be related to the tone of
dopamine and GABA in the ventral mesencephalon that regulates a deviate
function of the wv GIRK2 channels (Liao et al., 1996). In
addition, the mutation of the GIRK2 channel, by reversing the
functional effects of dopamine, would not only impair the
autoreceptor-mediated control of neurotransmitter release but also
enhance, with a feedforward mechanism, the level of this catecholamine
in the terminal fields. The altered behavior of the spared dopaminergic
cells, as a consequence of DA autoreceptors stimulation, could account
for the enhanced fractional release of dopamine in the striatum of
wv/wv mice (Richter et al., 1995). Thus, the observed
hyperactivity of the wv/wv mice could, at least in part,
depend on the dysfunction of the autoreceptor-mediated control of the
dopaminergic neurons.
| |
FOOTNOTES |
Received April 3, 2000; revised May 22, 2000; accepted May 25, 2000.
This work was supported by Telethon Grant E.747 to N.B.M. We thank Dr.
Marco Molinari and Dr. Maria Teresa Viscomi for their advice in
hystochemistry experiments, Dr. Nicola Berretta for help in the
discussion, and Mauro Federici for technical assistance.
Correspondence should be addressed to Dr. Nicola B. Mercuri, Laboratory
of Experimental Neurology, Fondazione Santa Lucia, Istituto di
Ricorvero e Cura a Carattere Scientifico, Via Ardeatina 306, 00179 Rome, Italy. E-mail: mercurin{at}med.uniroma2.it.
| |
REFERENCES |
-
Bayer SA,
Willis KV,
Triarhou LC,
Verina T,
Thomas JD,
Ghetti B
(1995)
Selective vulnerability of late-generated dopaminergic neurones of the substantia nigra in weaver mutant mice.
Proc Natl Acad Sci USA
92:9137-9140[Abstract/Free Full Text].
-
Bertolino M,
Llinas RR
(1992)
The central role of voltage-activated and receptor-operated calcium channels in neuronal cells.
Annu Rev Pharmacol Toxicol
32:399-421[ISI][Medline].
-
Choi DW
(1995)
Calcium: still center-stage in hypoxic-ischemic neuronal death.
Trends Neurosci
18:58-60[ISI][Medline].
-
Grace A,
Onn SP
(1989)
Morphology and electrophysiological properties of immunocytochemically identified rat dopamine neurones recorded in vitro.
J Neurosci
9:3463-3481[Abstract].
-
Gupta AM,
Felten DL,
Ghetti B
(1987)
Selective loss of monoaminergic neurones in the weaver mutant mice: an immunocytochemical study.
Brain Res
402:379-382[ISI][Medline].
-
Hille B
(1992)
In: Ionic channels of excitable membranes. Ed 2. Sunderland, MA: Sinauer.
-
Hou P,
Yan S,
Tang W,
Nelson DJ
(1999)
The inwardly rectifying K+ channel submit GIRK1 rescues the GIRK2 weaver phenotype.
J Neurosci
19:8327-8336[Abstract/Free Full Text].
-
Inanobe A,
Yoshimoto Y,
Horio Y,
Morishige KI,
Hibino H,
Matsumoto S,
Tokunaga Y,
Maeda T,
Hata Y,
Takai Y,
Kurachi Y
(1999)
Characterization of G-protein-gated K+ channels composed of Kir3.2 subunits in dopaminergic neurons of the substantia nigra.
J Neurosci
19:1006-1017[Abstract/Free Full Text].
-
Jan LY,
Jan YN
(1994)
Potassium channels and their evolving gates.
Nature
371:119-122[Medline].
-
Jarolimek W,
Bäurle J,
Misgeld U
(1998)
Pore mutation in a G-protein-gated inwardly rectifying K+ channel subunit causes loss of K+-dependent inhibition in weaver hippocampus.
J Neurosci
18:4001-4007[Abstract/Free Full Text].
-
Jensen P,
Surmeier DJ,
Goldowitz D
(1999)
Rescue of cerebellar granule cells from death in weaver NR1 double mutants.
J Neurosci
19:7991-7998[Abstract/Free Full Text].
-
Johnson SW,
North RA
(1992)
Two types of neurone in the rat ventral tegmental area and their synaptic inputs.
J Physiol (Lond)
450:455-468[Abstract/Free Full Text].
-
Kim KM,
Nakajima S,
Nakajima Y
(1997)
Dopamine and GABA receptors in cultured substantia nigra neurons: correlation of electrophysiology and immunocytochemistry.
Neuroscience
78:759-769[Medline].
-
Kofuji P,
Hofer M,
Millen KJ,
Millonig JH,
Davidson N,
Lester HA,
Hatten ME
(1996)
Functional analysis of the weaver mutant GIRK2 K+ channel and rescue of weaver granule cells.
Neuron
16:941-952[ISI][Medline].
-
Lacey MG,
Mercuri NB,
North RA
(1988)
On the potassium conductance increase activated by GABAB and D2 receptors in rat substantia nigra neurones.
J Physiol (Lond)
401:437-453[Abstract/Free Full Text].
-
Lacey MG,
Mercuri NB,
North RA
(1989)
Two cell types in rat substantia nigra zona compacta distinguished by membrane properties and the actions of dopamine and opioids.
J Neurosci
9:1233-1241[Abstract].
-
Liao YJ,
Jan YN,
Jan LJ
(1996)
Heteromultimerization of G-protein-gated inwardly rectifying K+ channel proteins GIRK1 and GIRK2 and their altered expression in weaver brain.
J Neurosci
16:7137-7150[Abstract/Free Full Text].
-
Liss B,
Neu A,
Roeper J
(1999)
The weaver mouse gain-of-function phenotype of dopaminergic neurons is determined by coactivation of wvGIRK2 and K-ATP channels.
J Neurosci
19:8839-8848[Abstract/Free Full Text].
-
Mercuri NB,
Calabresi P,
Bernardi G
(1992)
The electrophysiological actions of dopamine and dopaminergic drugs on neurones of the substantia nigra pars compacta and ventral tegmental area.
Life Sci
51:711-718[ISI][Medline].
-
Mercuri NB,
Bonci A,
Johnson SW,
Stratta F,
Calabresi P,
Bernardi G
(1994)
Effects of anoxia on rat midbrain dopamine neurons.
J Neurophysiol
71:1165-1173[Abstract/Free Full Text].
-
Mercuri NB,
Bonci A,
Calabresi P,
Stefani A,
Bernardi G
(1995)
Properties of the hyperpolarization-activated cation current (Ih) in rat midbrain dopaminergic neurons.
Eur J Neurosci
7:462-469[ISI][Medline].
-
Mercuri NB,
Saiardi A,
Bonci A,
Picetti R,
Calabresi P,
Bernardi G,
Borrelli E
(1997)
Loss of autoreceptor function in dopaminergic neurons from dopamine D2 receptor deficient mice.
Neuroscience
79:323-327[ISI][Medline].
-
Murer G,
Adelbrecht C,
Lauritzen I,
Lesage F,
Lazdunski M,
Agid Y,
Raisman-Vozari R
(1997)
An immunocytochemical study on the distribution of two G-protein-gated inward rectifier potassium channels (GIRK2 and GIRK4) in the adult rat brain.
Neuroscience
80:345-357[ISI][Medline].
-
Navarro B,
Kennedy ME,
Velimirovic B,
Bhat D,
Peterson AS,
Clapham DE
(1996)
Nonselective and G
  -insensitive weaver K+ channels.
Science
272:1950-1953[Abstract]. -
North RA
(1989)
Drug receptors and the inhibition of nerve cells. Twelfth Gaddum memorial lecture.
Br J Pharmacol
98:13-28[ISI][Medline].
-
Oo TF,
Blazeski R,
Harrison SM,
Henchcliffe C,
Mason CA,
Roffler-Tarlov SK,
Burke RE
(1996)
Neuron death in the substantia nigra of weaver mouse occurs late in development and is not apoptotic.
J Neurosci
16:6134-6145[Abstract/Free Full Text].
-
Patil N,
Cox DR,
Bhat D,
Faham M,
Myers RM,
Peterson AS
(1995)
A potassium channel mutation in weaver mice implicates membrane excitability in granule cell differentiation.
Nat Genet
11:126-129[ISI][Medline].
-
Rakic R,
Sidman RL
(1973)
Sequence of developmental abnormalities leading to granule cell deficit in cerebellar cortex of weaver mutant mice.
J Comp Neurol
152:103-132[ISI][Medline].
-
Richards CD,
Shiroyama T,
Kitai ST
(1997)
Electrophysiological and immunocytochemical characterization of GABA and dopamine neurons in the substantia nigra of the rat.
Neuroscience
80:545-557[ISI][Medline].
-
Richter JA,
Bare DJ,
Yu H,
Ghetti B,
Simon JR
(1995)
Dopamine transporter-dependent and -independent endogenous dopamine release from weaver mouse striatum in vitro.
J Neurochem
64:191-198[Medline].
-
Roffler-Tarlov S,
Graybiel AM
(1984)
Weaver mutant has differential effects on the dopamine-containing innervation of the limbic and non-limbic striatum.
Nature
307:62-66[Medline].
-
Rossi P,
De Filippi G,
Armano S,
Taglietti V,
D'Angelo E
(1998)
The weaver mutation causes a loss of inward rectifier current regulation in premigratory granule cells of the mouse cerebellum.
J Neurosci
18:3537-3547[Abstract/Free Full Text].
-
Schmidt MJ,
Sawyer BD,
Perry KW,
Foreman MM,
Ghetti B
(1982)
Dopamine deficiency in the weaver mutant mouse.
J Neurosci
2:376-380[Abstract].
-
Silverman SK,
Kofuji P,
Dougherty DA,
Davidson N,
Lester HA
(1996)
A rigenerative link in the ionic fluxes through the weaver potassium channel underlies the pathophysiology of the mutation.
Proc Natl Acad Sci USA
93:15429-15434[Abstract/Free Full Text].
-
Simon JR,
Ghetti B
(1994)
The weaver mutant mouse as a model of nigrostriatal dysfunction.
Mol Neurobiol
9:183-189[ISI][Medline].
-
Slesinger PA,
Patil N,
Liao YJ,
Jan YN,
Jan LY,
Cox DR
(1996)
Functional effects of the mouse weaver mutation on G-protein-gated inwardly rectifying K+ channels.
Neuron
16:321-331[ISI][Medline].
-
Slesinger PA,
Stoffel M,
Jan YN,
Jan LY
(1997)
Defective gamma-aminobutyric acid type B receptor-activated inwardly rectifying K+ currents in cerebellar granule cells isolated from weaver and Girk2 null mutant mice.
Proc Natl Acad Sci USA
94:12210-12217[Abstract/Free Full Text].
-
Smith III MW,
Cooper TR,
Joh TH,
Smith DE
(1990)
Cell loss and class distribution of TH-I cells in the substantia nigra of the neurological mutant, weaver.
Brain Res
510:242-250[ISI][Medline].
-
Sodickson DL,
Bean BP
(1998)
Neurotransmitter activation of inwardly rectifying potassium current in dissociated hippocampal CA3 neurons: interactions among multiple receptors.
J Neurosci
18:8153-8162[Abstract/Free Full Text].
-
Surmeier DJ,
Merlmestein PG,
Goldowitz D
(1996)
The weaver mutation of GIRK2 results in a loss of inwardly rectifying K+ current in cerebellar granule cells.
Proc Natl Acad Sci USA
93:11191-11195[Abstract/Free Full Text].
-
Tong Y,
Wei J,
Zhang S,
Strong JA,
Dlouhy SR,
Hodes ME,
Ghetti B,
Yu L
(1996)
The weaver mutation changes the ion selectivity of the affected inwardly rectifying potassium channel GIRK2.
FEBS Lett
390:63-68[Medline].
-
Triarhou LC,
Norton J,
Ghetti B
(1988)
Mesencephalic dopamine cell deficit involves areas A8, A9 and A10 in weaver mice.
Exp Brain Res
70:256-265[ISI][Medline].
-
Tucker SJ,
Pessia M,
Moorhouse AJ,
Gribble F,
Ashcroft FM,
Maylie J,
Adelman JP
(1996)
Heteromeric channel formation and Ca2+-free media reduce the toxic effect of the weaver Kir 3.2 allele.
FEBS Lett
390:253-257[ISI][Medline].
-
Verney C,
Febvret-Muzerelle A,
Gaspar P
(1995)
Early postnatal changes of the dopaminergic mesencephalic neurons in the weaver mutant mouse.
Brain Res Dev Brain Res
89:115-119[Medline].
-
Watts AE,
Williams JT,
Henderson G
(1996)
Baclofen inhibition of the hyperpolarization-activated cation current, Ih, in rat substantia nigra zona compacta neurons may be secondary to potassium current activation.
J Neurophysiol
76:2262-2270[Abstract/Free Full Text].
-
White FJ
(1996)
Synaptic regulation of mesocorticolimbic dopamine neurons.
Annu Rev Neurosci
19:405-436[ISI][Medline].
Copyright © 2000 Society for Neuroscience 0270-6474/00/20166013-08$05.00/0
This article has been cited by other articles:
|
|
|
|
 
M. Giustizieri, G. Bernardi, N. B. Mercuri, and N. Berretta
Distinct Mechanisms of Presynaptic Inhibition at GABAergic Synapses of the Rat Substantia Nigra Pars Compacta
J Neurophysiol,
September 1, 2005;
94(3):
1992 - 2003.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Mendez, R. Sanchez-Pernaute, O. Cooper, A. Vinuela, D. Ferrari, L. Bjorklund, A. Dagher, and O. Isacson
Cell type analysis of functional fetal dopamine cell suspension transplants in the striatum and substantia nigra of patients with Parkinson's disease
Brain,
July 1, 2005;
128(7):
1498 - 1510.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Guatteo, C. P. Bengtson, G. Bernardi, and N. B. Mercuri
Voltage-Gated Calcium Channels Mediate Intracellular Calcium Increase in Weaver Dopaminergic Neurons During Stimulation of D2 and GABAB Receptors
J Neurophysiol,
December 1, 2004;
92(6):
3368 - 3374.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Bettler, K. Kaupmann, J. Mosbacher, and M. Gassmann
Molecular Structure and Physiological Functions of GABAB Receptors
Physiol Rev,
July 1, 2004;
84(3):
835 - 867.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Neuhoff, A. Neu, B. Liss, and J. Roeper
Ih Channels Contribute to the Different Functional Properties of Identified Dopaminergic Subpopulations in the Midbrain
J. Neurosci.,
February 15, 2002;
22(4):
1290 - 1302.
[Abstract]
[Full Text]
| |
TOP
ABSTRACT
INTRODUCTION
