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com
articles

Amphetamine selectively blocks

inhibitory glutamate transmission
in dopamine neurons
© 2001 Nature Publishing Group http://neurosci.nature.com

Carlos A. Paladini1, Christopher D. Fiorillo1,2, Hitoshi Morikawa1 and John T. Williams1

1 The Vollum Institute, L474, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201, USA
2 Present address: Institute for Physiology, University of Fribourg, Rue du Musèe 5, CH 1700 Fribourg, Switzerland
The first two authors contributed equally to this work
Correspondence should be addressed to J.T.W. (williamj@ohsu.edu)

Amphetamine is a highly addictive psychostimulant that promotes the release of the catecholamines
dopamine and norepinephrine. Amphetamine-induced release of dopamine in the midbrain inhibits
the activity of dopamine neurons through activation of D2 dopamine autoreceptors. Here we show
that amphetamine may also excite dopamine neurons through modulation of glutamate neurotrans-
mission. Amphetamine potently inhibits metabotropic glutamate receptor (mGluR)-mediated IPSPs
in dopamine neurons, but has no effect on ionotropic glutamate receptor-mediated EPSCs.
Amphetamine desensitizes the mGluR-mediated hyperpolarization through release of dopamine,
activation of postsynaptic α1 adrenergic receptors, and suppression of InsP3-induced calcium release
from internal stores. By selectively suppressing the inhibitory component of glutamate-mediated
transmission, amphetamine may promote burst firing of dopamine neurons. Through this
mechanism, amphetamine may enhance phasic release of dopamine, which is important in the
neural processing of reward.

The dopamine neurons of the ventral tegmental area (VTA) pro-
ject to the nucleus accumbens, limbic regions and frontal cortex.
A common feature of addictive drugs is their ability to increase
extracellular dopamine levels in these areas, particularly in the
nucleus accumbens. Amphetamine accomplishes this by acting
directly on dopamine transporters to elicit a sustained release of
dopamine, primarily through a mechanism that is independent of
action potentials and vesicular release1–3. Although the precise
mechanism of this action has not been fully determined, it is
known to depend on the reverse transport of extravesicular
dopamine out of the cytoplasm by the plasma membrane
dopamine transporter (for review, see ref. 4).
Studies have shown that amphetamine can depress dopamine
neuronal activity5, and that this occurs through D2 receptor-
mediated hyperpolarization6. However, these studies were done
either in anaesthetized animals or in vitro, where the activity of
afferents is severely altered or absent. Amphetamine may also
have excitatory influences on dopamine neurons7–10. The effect of
amphetamine on dopamine neuron activity may be of substantial
importance, as it has been suggested that phasic increases in
extracellular dopamine, dependent on afferent input, could have
consequences distinct from tonic increases11,12. Specifically, it
seems that transient, impulse-dependent release of dopamine,
on the scale of hundreds of milliseconds, is critical in the pro-
cessing of natural rewards12.
Excitatory influences of amphetamine on dopamine neurons
are thought to be mediated by glutamate-containing afferents8–10.
Although glutamate input does not necessarily increase the over-
all firing rate of dopamine neurons, it is thought to induce bursts
of activity (for review, see ref. 13). Bursts cause a greater increase
in extracellular dopamine concentration per action potential
than does the standard low-frequency activity of dopamine
cells14,15. The EPSPs mediated by ionotropic receptors that are
thought to drive bursts of activity in dopamine neurons are fol-
lowed by IPSPs mediated by metabotropic glutamate receptors
(mGluR1)16. Through the combined activation of ionotropic
and metabotropic receptors in response to synaptically released
glutamate, a burst–pause pattern would be promoted. Here we
examined the effect of amphetamine on glutamate-mediated
transmission in dopamine neurons. The primary observation
was a selective inhibition of mGluR IPSPs by postsynaptic α1
adrenergic receptors.
RESULTS
Amphetamine selectively inhibits mGluR IPSPs
Intracellular, perforated-patch or whole-cell recordings were
made to study the effect of amphetamine on the mGluR IPSP in
dopamine neurons of the VTA and substantia nigra pars com-
pacta. In the presence of antagonists for ionotropic glutamate
receptors (AMPA and NMDA), as well as for GABAA, GABAB
and dopamine D2 receptors, a train of 2–10 stimuli evoked a
mGluR-mediated slow IPSP, as previously reported16. Superfu-
sion of (+)-amphetamine, at a concentration attained during
self-administration (3 µM)17,18, inhibited the amplitude of the
mGluR IPSP by 29 ± 5% (n = 5) after 10–15 minutes. In a sepa-
rate group of cells, a higher concentration of amphetamine
(10 µM) reduced the IPSP amplitude by 52 ± 10% (n = 16; Fig. 1a,
whole cell configuration). The effect of amphetamine complete-

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articles

Fig. 1. Amphetamine selectively inhibits the mGluR IPSP without

affecting the glutamatergic EPSC in dopamine neurons. (a) A summary a
of the inhibition of IPSPs amplitudes by amphetamine (10 µM, whole
cell configuration, n = 16). In this and other figures, the amplitudes of
the IPSPs were normalized to the average amplitude determined from a
5-min period preceding superfusion with amphetamine. Inset, example
of the inhibition of an IPSP by amphetamine. (b) The AMPA-mediated
EPSC was not affected by amphetamine. A voltage-clamp recording of
representative EPSCs (the first two in a train of ten) and a summary of
© 2001 Nature Publishing Group http://neurosci.nature.com

results obtained with whole-cell recording, showing that amphetamine

(30 µM) had no effect on the amplitude of the EPSC (n = 12).

ly reversed after washout. A similar inhibition was observed using

intracellular recordings (69 ± 9%, n = 5, Fig. 2a). b
Next, we examined the effect of amphetamine on the AMPA
receptor-mediated EPSC using whole-cell voltage clamp record-
ing (holding potential, –70 mV). The same stimulation proto-
col used for mGluR IPSPs was used to evoke fast EPSCs.
Amphetamine (30 µM) had no significant effect on the ampli-
tude of the first EPSC (Fig. 1b; a decrease of 10 ± 5%, n = 12,
p > 0.05), or on subsequent EPSCs in the train (10 pulses). The
same result was previously observed using a lower concentra-
tion of amphetamine19 (3 µM; but see ref. 20). Thus, the effect of
amphetamine was selective for the mGluR IPSP without affect-
ing the AMPA EPSC.

Amphetamine acts through α1 adrenergic receptors

We tested the possible involvement of α1 adrenergic receptors,
which are expressed on dopamine neurons21, using the selective
antagonist prazosin. Prazosin (100 nM) reversed the ampheta-
mine-induced inhibition of the mGluR IPSP to 90 ± 10% (n = 5)
of the control amplitude (Fig. 2a). Superfusion of prazosin alone
was without effect, and in the continuous presence of prazosin,
amphetamine caused an inhibition that was only 19 ± 5% (n =
5, p < 0.01 versus control inhibition; Fig. 2b). Amphetamine (10
µM) also produced a small depolarization of the membrane
a and 63 ± 8%, n = 9, respectively, Fig. 2b). In prazosin (100 nM),
potential (5 ± 1 mV, n = 5) that was sensitive to prazosin (depo-
larization reduced to 2 ± 0.5 mV, n = 5, p < 0.05).
The effects of dopamine and norepinephrine on the mGluR
IPSP were then examined, because both catecholamines are pre-
sent in the ventral midbrain and are capable of activating α1
adrenergic receptors22,23. Dopamine (100 µM) and norepineph-
rine (10 µM) produced a small depolarization of the membrane
potential (2.9 ± 0.7 mV, n = 9, and 3.7 ± 0.4 mV, n = 7, respec-
tively) and a reduction of the IPSP amplitude (64 ± 5%, n = 6
the inhibition of the IPSP caused by both dopamine and norep-
inephrine was reduced to 14 ± 7% (n = 5, p < 0.01) and 10 ± 9%
(n = 8, p < 0.01), respectively (Fig. 2b). The selective α1 recep-
tor agonist phenylephrine (1 or 10 µM) also inhibited the mGluR
IPSP by 61 ± 6% (n = 6).
The effect of dopamine was examined in the absence of eti-
clopride to determine if D2 receptors could also have a role in
the regulation of the mGluR IPSP. In the absence of eticlopride,
dopamine (100 µM) produced a small hyperpolarization rather
than depolarization of the membrane potential, but the inhibition
of the IPSP was identical (65 ± 12%, n = 4, p > 0.05). Thus, we

b
Fig. 2. Amphetamine (10 µM), dopamine (100 µM) and norepinephrine
(30 µM) inhibit the mGluR IPSP through activation of α1 adrenergic
receptors. (a) The selective α1 antagonist, prazosin (100 nM) reversed
the inhibition of the IPSP caused by amphetamine (n = 5, intracellular
recording). Inset, inhibition of an IPSP by amphetamine. (b) The inhibi-
tion of IPSPs by amphetamine (amph), norepinephrine (NE) and
dopamine (DA). The effect of each agonist in the presence of prazosin
(100 nM) is shown to the right of each bar (+ praz). Numbers inside
columns indicate the number of cells. Astrisks indicate that addition of
prazosin caused a significant (p < 0.05) reduction of the inhibition
caused by each agonist.

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a aspartate-induced hyperpolarization through activation of α1 adrenergic
© 2001 Nature Publishing Group http://neurosci.nature.com
b amphetamine (53 ± 12%, n = 6) was the same as with eticlopride
c reduced the mGluR-mediated hyperpolarization. The first was
conclude that the inhibition of the mGluR IPSP by dopamine is
mediated by α1 adrenergic receptors, and it seems that D2 recep-
tors are not involved.
α1 receptors inhibit mGluR-induced hyperpolarizations
The lack of an effect of amphetamine on the AMPA EPSC togeth-
er with the expression of α1 adrenergic receptors in dopamine
neurons suggested that the inhibition of the mGluR IPSP was medi-
ated postsynaptically. To test this hypothesis, we examined the effect
of amphetamine on the hyperpolarization evoked by exogenously
applied aspartate. In the presence of AMPA and NMDA antago-
nists, iontophoresis of L-aspartate (50 ms, 50–150 nA) evoked a
hyperpolarization that was abolished by the mGluR antagonist,
MCPG (1 mM, decreased to 0.5 ± 0.5% of control, n = 3)16.
Superfusion of amphetamine (10 µM) inhibited the aspartate-
induced hyperpolarization by 47 ± 8% (n = 18; Fig. 3a). The mag-
nitude of this inhibition was significantly attenuated in the
presence of prazosin (100 nM; Fig. 3a). Similarly, dopamine (100
µM) and norepinephrine (30 µM) reduced the amplitude of the
aspartate-induced hyperpolarization (54 ± 8%, n = 11 and 54 ±
15%, n = 13, respectively) in a prazosin-sensitive fashion (Fig. 3b
and c). Finally, the aspartate-induced outward current measured
under voltage clamp was also attenuated by phenylephrine (1 µM,
inhibited by 46 ± 3%, n = 5), which is consistent with the involve-
nature neuroscience • volume 4 no 3 • march 2001
articles
Fig. 3. Amphetamine, dopamine and norepinephrine suppress the
receptors. Inhibition of the aspartate-induced hyperpolarization caused
by amphetamine (10 µM; a), dopamine (100 µM; b) and norepinephrine
(30 µM; c). This attenuation is blocked by pretreatment of the slice with
prazosin (100 nM). Insets, traces of the mGluR hyperpolarizing response
during whole-cell recordings from representative neurons.
ment of α1 adrenergic receptors. In experiments where eticlo-
pride was not included in the superfusion solution, the inhibi-
tion of the mGluR-mediated hyperpolarization caused by
(p > 0.05), further suggesting that D2 receptors were not involved.
These results indicate that activation of postsynaptic α1 adrener-
gic receptors account for the inhibition of the mGluR IPSP caused
by amphetamine.
Calcium release from internal stores
The mGluR-mediated hyperpolarization results from the release
of Ca2+ from the intracellular store and subsequent activation of
Ca 2+-sensitive K + channels 16. Production of inositol 1,4,5-
triphosphate (InsP3) is thought to mediate the release of Ca2+ by
mGluRs24. Three experiments were done to further investigate
the mechanism by which the activation of α1 adrenergic receptors
to examine the effect of activating α1 receptors on the aspartate-
evoked rise in intracellular Ca2+. Iontophoretic application of
L-aspartate evoked a rise in the fluorescence of Ca2+ indicator
dyes, Oregon Green BAPTA-2 (Kd, 600 nM; Fig. 4a) or Oregon
Green BAPTA-5N (Kd, 20 µM). This increase in fluorescence
originated near the site of iontophoretic application, peaked at
approximately 500 ms (peak ∆F/F with Oregon Green BAPTA-2,
0.66 ± 0.11; with Oregon Green BAPTA-5N, was 0.28 ± 0.06;
n = 3 for each) and decayed over 4 s (Fig. 4a). Activation of α1
receptors with norepinephrine (30 µM) inhibited the aspartate-
induced rise in fluorescence using both Oregon Green BAPTA2
by 36 ± 14% and Oregon Green BAPTA-5N by 47 ± 7% (n = 3
for each; Fig. 4b and d) and the outward current by 52 ± 4%
(n = 6). The baseline fluorescence was not changed by norepi-
nephrine (∆F/F with Oregon Green BAPTA-2, –0.02 ± 0.02; with
Oregon Green BAPTA-5N, –0.03 ± 0.02; n = 3 for each). Thus,
the inhibition of the aspartate-induced rise in calcium by nor-
epinephrine did not result from occlusion caused by a sustained
elevation of intracellular calcium. A similar inhibition of the rise
in fluorescence (Fig. 4d) and aspartate-induced outward current
(59 ± 8%, n = 6) was observed during the superfusion of a low
concentration of the mGluR agonist, DHPG (1 µM).
In the second experiment, we examined the effect of activation
of α1 receptors on the outward current induced by intracellular
application of InsP3 using photolysis of caged InsP3. Photolysis of
intracellular caged InsP3 resulted in a transient outward current
(Fig. 4c)24. Norepinephrine and phenylephrine inhibited the
InsP 3-induced outward current by 44 ± 4% and 62 ± 13%,
respectively (n = 4 for each; Fig. 4d). In addition, as was observed
in the fluorescence experiments, the InsP3-induced outward cur-
rent was reduced with a low concentration of the mGluR ago-
nist, DHPG (Fig. 4d).
In the third experiment, the mGluR-induced inward current
was recorded by buffering the intracellular Ca2+ with BAPTA
(1 mM). When using this internal solution, the activation of
mGluRs results in a MCPG-sensitive inward current25. Activa-
tion of α1 receptors (with phenylephrine; 10 µM) did not cause
277
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articles
Fig. 4. Activation of α1 adrenergic receptors cause heterologous
desensitization of the mGluR-mediated release of Ca2+ and the out-
ward current induced by InsP3. (a) Fluorescence (Oregon Green
BAPTA-2) measured before (left) and 1 s after (right) aspartate ion-
tophoresis. (b) The fluorescence within the indicated circles (a) was
measured and plotted against time. Norepinephrine (30 µM) attenu-
ated the rise in fluorescence (top) and the outward current (bottom)
during whole-cell recordings of the same cell. (c) Flash photolysis of
caged InsP3 induced an outward current that was decreased by norepi-
© 2001 Nature Publishing Group http://neurosci.nature.com
nephrine (30 µM). (d) Effects of norepinephrine (NE), phenylephrine
(PE) and DHPG (1 µM) on InsP3-induced current and aspartate-
induced increase in fluorescence. The results for the two calcium indi-
cators are shown separately. B-2, Oregon Green BAPTA-2; B-5N,
Oregon Green BAPTA-5N.
a significant change in the amplitude of the mGluR-mediated
inward current in response to iontophoretic application of aspar-
tate (inhibition of 10 ± 9%, n = 6, p > 0.05). Assuming that the
depolarizing and hyperpolarizing responses are mediated by the
same receptor, the inability of α1 receptor activation to change
the depolarizing response to mGluRs suggests that the inhibition
of the hyperpolarizing mGluR response occurs downstream from
the receptor.
Taken together, the results suggest that the activation of α1
adrenergic receptors, as well as mGluRs, inhibits the mGluR-
mediated hyperpolarization by desensitizing the InsP3-induced
release of Ca2+. This desensitization could result from depletion
of Ca2+ stores, or by affecting the InsP3 receptor directly.
a one group of 8 cells, GBR 12909 (100 nM) decreased the effect
b mGluRs. These experiments do not rule out the potential role
278
a b
c
d
Amphetamine actions depend on dopamine reuptake
As amphetamine is equally effective in releasing dopamine and
norepinephrine, and both neurotransmitters inhibited the mGluR
IPSP, we examined the identity of the transmitter that mediated
this effect. Amphetamine is a substrate for each of the cate-
cholamine transporters, and amphetamine-induced release of
transmitter is dependent on functional transporters3,26. There-
fore, we tested the effect of selective blockade of each cate-
cholamine transporter by using nisoxetine for the norepinephrine
transporter, and GBR 12909 for the dopamine transporter. In
of amphetamine on the aspartate-induced hyperpolarization
from 51 ± 10% to 15 ± 11% (p < 0.05; Fig. 5a). In a second group
of 5 cells, the inhibition caused by amphetamine was 46 ± 13% in
control, and was not significantly different in the presence of
nisoxetine (30 ± 16%, p > 0.05; Fig. 5b). Similarly, GBR 12909
(100 nM) dramatically attenuated the inhibition of the mGluR
IPSP by amphetamine (13 ± 10%, n = 3; compared to 45 ± 9%,
n = 11), whereas nisoxetine (100 nM) did not affect the inhibition
induced by amphetamine (45 ± 12%, p > 0.05, n = 4). Neither
transporter blocker had an effect on the aspartate-induced hyper-
polarization or the mGluR IPSP by itself (Fig. 5a and b).
The results suggest that under the conditions of these exper-
iments, amphetamine increases extracellular dopamine via a
transporter-dependent mechanism, and that dopamine acts on
α1 adrenergic receptors to inhibit the hyperpolarization caused by
for norepinephrine. Under different experimental conditions,
particularly in vivo, endogenous norephinephrine affects the fir-
ing of dopamine neurons9,27.
Fig. 5. The amphetamine-induced inhibition of the hyperpolarization
evoked by iontophoretically applied aspartate was blocked by the
dopamine transporter blocker, GBR 12909. (a) Amphetamine-induced
(10 µM) inhibition of the mGluR response was attenuated in
GBR 12909 (100 nM, n = 8, whole cell recording). Inset, traces of the
aspartate induced hyperpolarization in control, during amphetamine
and in the presence of both amphetamine and GBR 12909.
(b) Amphetamine-induced inhibition is still present in the
norephinephrine transport inhibitor, nisoxetine (100 nM, n = 5).
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articles

Fig. 6. Amphetamine selectively decreased the mGluR-mediated

hyperpolarization. Glutamate-mediated synaptic transmission was
examined in the presence of GABA receptor antagonists (picrotoxin
and CGP56999). A train of electrical stimuli (30 at 20 Hz) was used to
evoke a sustained release of glutamate. The resting membrane poten-
tial before the onset of each stimulation was set at –65 mV. Top, the
rapid AMPA- and NMDA-mediated EPSPs superimposed on the mGluR
IPSP. The mGluR IPSP is not sustained throughout the period of stimu-
lation. Middle, same sequence of stimuli where the mGluR IPSP is
© 2001 Nature Publishing Group http://neurosci.nature.com

reduced by the presence of amphetamine (10 µM). Each trace is the

average of 3 taken at 1-min intervals. Bottom, the two traces (above)
superimposed in an expanded scale to show the effect of amphetamine
on the synaptic potentials evoked by the first 9 stimuli.

DISCUSSION
Amphetamine selectively inhibited the mGluR-mediated slow
inhibitory component of glutamate transmission without affect-
ing the fast excitatory component. The postsynaptic blockade of
mGluR-mediated inhibition would have very different conse-
quences from the presynaptic inhibition of glutamate release that
has been reported previously20,28–30. By selectively blocking the
inhibitory mGluR component, excitation caused by AMPA and
NMDA receptors should be enhanced.
The mGluR IPSP depends on release of Ca2+ from intracellular
stores and activation of K+ channels16. It is readily desensitized by
a postsynaptic action of receptors coupled to phosphoinositide
hydrolysis, including mGluR116 and M1 muscarinic receptors31.
Here we show that activation of α1 adrenergic receptors suppress-
es the release of Ca2+ evoked either by the rapid activation of
mGluR1 or by intracellular application of InsP3. The mGluR-medi-
ated depolarization, or inward current, did not desensitize under
these conditions and is independent of the rise in intracellular Ca2+
(ref. 25). Thus, the heterologous desensitization is receptor inde-
pendent and most likely occurs through depletion of Ca2+ stores or
desensitization of the InsP3 receptor by InsP3 (ref. 32).
The overall effect of glutamate-mediated transmission was
examined in experiments in which the membrane potential was
measured in the absence of glutamate receptor antagonists (Fig. 6).
With repetitive stimulation (20 Hz), the first 3–4 stimuli often
resulted in summating AMPA and NMDA receptor-mediated
EPSPs (Fig. 6). After the first few stimuli, the membrane potential
hyperpolarized (Fig. 6). This hyperpolarization resulted from the
activation of mGluRs. Superfusion with amphetamine or phenyle-
phrine selectively decreased the mGluR component (Fig. 6). The
time course of the early excitatory component of glutamate affer-
ents (100–160 ms, Fig. 6) followed by a long-lasting hyperpolar-
ization is consistent with descriptions of kinetics of the burst/pause
recorded in vivo. Bursts of dopamine cells typically consist of 2–4
action potentials with an interspike interval of about 75 ms that is
followed by a pause of about 200 ms (ref. 33, 34).
Although it is thought that glutamate-containing afferents
control burst firing of dopamine neurons in vivo, the activity pat-
tern of the glutamate input is not known. Furthermore, no pro-
tocol has been developed to mimic burst firing in vitro through
endogenous glutamate (for review, see ref. 13). Therefore, it has
not been possible to address directly the function of the mGluR
IPSP and amphetamine in burst firing. Nonetheless, the kinet-
ics and magnitude of the mGluR IPSP suggest that it may cur-
tail burst activity. The pattern of depolarization and
hyperpolarization illustrated in Fig. 6 during sustained activa-
tion of glutamate-containing afferents could be important in the
pattern of burst firing of dopamine cells. The inhibition of the
mGluR component of glutamate transmission through activa-
tion of α1-receptors would be expected to extend the depolar-
ization induced by ionotropic receptor activation.
A long-term depression of the AMPA EPSC can be induced
following sufficient depolarization of dopamine neurons10,35.
This phenomenon, which is dependent on the postsynaptic entry
of Ca2+, is blocked by amphetamine acting through dopamine
D2 receptors10. Thus, in addition to blocking the inhibitory com-
ponent of glutamate transmission, amphetamine may also
enhance the excitatory component under certain conditions.
It is well established that α1 adrenergic receptor antagonists
suppress dopamine-mediated behaviors stimulated by amphet-
amine8,36–41. There is evidence that amphetamine can increase
impulse-dependent release of dopamine in the nucleus accum-
bens through activation of α1 adrenergic receptors7,8. These
effects of amphetamine on the dopamine system may be
explained in part by the observation that amphetamine promotes
burst firing of dopamine neurons in vivo through a mechanism
that requires activation of α1 adrenergic receptors9. The present
results suggest how these actions of amphetamine might occur
at the cellular level. Systemic administration of the α1 receptor
antagonist prazosin reduced spontaneous burst firing in
dopamine cells9,42, whereas electrical stimulation of the locus
coeruleus increased firing of dopamine neurons27. These obser-
vations may be explained by the present results, and suggest that
α1 adrenergic receptors dynamically regulate the mGluR IPSP
under normal physiological conditions. In addition, this work
suggests an alternative mechanism by which psychostimulants
could exert their dopamine-dependent behavioral effects. Beyond
modulating behavior by enhancing impulse-independent
dopamine release in projection areas, amphetamine may enhance
glutamate-driven bursts of activity in dopamine neurons. This
would bring the effect of amphetamine on dopamine release
under the control of contextual environmental stimuli.

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METHODS
Recordings. Midbrain horizontal slices (200–300 µm) were prepared
from adult male Wistar rats (150–200 g), as described previously43. Hor-
izontal slices were placed in a chamber (0.5 ml) superfused with physio-
logical saline (35°C) at a rate of 1.5 ml/min. The solution contained
126 mM NaCl, 2.5 mM KCl, 1.2 mM MgCl2, 2.4 mM CaCl2, 1.4 mM
NaH2PO4, 25 mM NaHCO3 and 11 mM D-glucose equilibrated with 95%
O2/5% CO2. Dopamine neurons of the VTA and substantia nigra pars
© 2001 Nature Publishing Group http://neurosci.nature.com
compacta were identified by their electrical properties44. Dopamine neu-
rons of the VTA constituted the majority of recorded cells (∼70%). For
current-clamp recordings, the membrane potential was adjusted to
between –60 and –70 mV to prevent spontaneous action potentials.
Intracellular electrodes (50–70 MΩ) were filled with 2 M KCl. Perfo-
rated-patch electrodes (3–4 MΩ) were filled with 30 µg/ml gramicidin
in a 115-mM K-HEPES solution. The internal solution used for whole-
cell voltage clamp recordings of the mGluR-induced outward currents
contained 115 mM K-methyl sulfate, 20 mM KCl, 1 mM MgCl2, 10 mM
HEPES, 0.1 mM EGTA, 2 mM ATP, 0.3 mM GTP and 10 mM creatine
phosphate. In experiments in which the inward current induced by
mGluRs was studied, BAPTA (1 mM) was substituted for EGTA in the
internal solution. Voltage-clamp recordings of fast EPSCs were done with
whole-cell patch electrodes (3–4 MΩ) containing 125 mM KCl, 1 mM
MgCl2, 1 mM EGTA, 5 mM HEPES, 1 mM ATP and 0.3 mM GTP.
Evoked responses. Synaptic potentials or currents were evoked with bipo-
lar tungsten stimulating electrodes with a tip separation of 300–600 µm.
A train of 2–10 stimuli of 400 µs at 0.3–1.5 mA was delivered at 66 Hz
every minute. Stimulating electrodes were placed within 1 mm rostral
or caudal of the recording site.
Iontophoretic pipettes (20–50 MΩ) were filled with L-aspartate (1 M,
pH 7.5) and placed within 10 µm of the soma or proximal dendrite. Ion-
tophoretic pulses were applied once per minute.
Experiments were done in the presence of the dopamine D2 receptor
antagonist eticlopride (100–300 nM), unless otherwise stated. Picrotox-
in (100 µM) and strychnine (1 µM) were used to block GABAA and
glycine receptors, respectively. The mGluR IPSP was isolated using NBQX
(5 µM), MK-801 (50–100 µM) and CGP 56999a (100–300 nM) to block
AMPA, NMDA and GABAB receptors, respectively.
Ca2+ imaging. Fluorescence imaging was done with whole-cell voltage
clamp recordings using patch electrodes containing 115 mM K-methyl
sulfate, 20 mM KCl, 1 mM MgCl2, 10 mM HEPES, 2 mM ATP, 0.3 mM
GTP, 10 mM creatine phosphate and the Ca2+ indicator dye Oregon
Green BAPTA-2 (100 µM) or Oregon Green BAPTA-5N (500 µM).
Images were taken every 130 ms for 4 s (35 images) using a confocal
microscope (Odyssey, Noran Instruments, Madison, Wisconsin). A region
of interest was identified in the cell. Baseline fluorescence was determined
before the aspartate iontophoresis. The change in fluorescence was quan-
tified and presented as the fluorescence at any time minus the baseline,
divided by the baseline (∆F/F).
Flash photolysis. Whole-cell voltage clamp recordings were made with
the same intracellular solution, except that caged InsP3 (100 µM; gift
from K. Khodakhah) was added. A xenon arc lamp (Cairn Research,
Faversham, UK) was used to produce UV pulses (∼1 ms). The intensity of
UV pulses was adjusted to evoke a near-maximal K+ current in each cell.
Drugs. Drugs were applied to the slice by superfusion. Adenosine trispho-
sphate (ATP) (+)-amphetamine sulfate, dopamine HCl, BAPTA, guano-
sine trisphosphate (GTP), norepinephrine HCl, picrotoxin, L-aspartate,
prazosin, phenylephrine, gramicidin and strychnine were from Sigma
(St. Louis, Missouri). S(–)-eticlopride and MK-801 were from Research
Biochemicals International (Natick, Massachusetts). 1,2,3,4-tetrahydro-6-
nitro-2,3-dioxo-benzo[f]quinoxaline (NBQX), GBR 12909 dihydrochloride
(S)-α-methyl-4-carboxyphenylglycine (MCPG) (S)-3,5-dihydro-
xyphenylglycine (DHPG) and nisoxetine hydrochloride were from Tocris
Cookson (St. Louis, Missouri). CGP 56999a was a gift from Novartis Phar-
maceuticals (Basel, Switzerland). Oregon Green BAPTA-2 and Oregon Green
BAPTA-5N were obtained from Molecular Probes (Eugene, Oregon).
280
Data analysis. Values are given as means ± standard error of the mean.
The percent change produced by a drug was calculated from mean ampli-
tude of three to five responses before and after equilibrium had been
reached (5–15 min). Unpaired comparisons between two groups were
made with a Mann–Whitney U test, whereas paired comparisons were
made using a Wilcoxon signed-rank test; p < 0.05 was considered a sig-
nificant difference.
ACKNOWLEDGEMENTS
We thank K. Khodakhah for instruction and guidance in the flash photolysis
experiments, and O. Manzoni for comments on the work and manuscript. This
work was supported by NIH grants DA04523 (J.T.W.), DA05793 (C.D.F.) and
DA07262 (C.A.P.).
RECEIVED 27 DECEMBER 2000; ACCEPTED 18 JANUARY 2001
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