The Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201
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ABSTRACT |
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Fiorillo, Christopher D. and John T. Williams. Selective Inhibition by Adenosine of mGluR IPSPs in Dopamine Neurons After Cocaine Treatment. J. Neurophysiol. 83: 1307-1314, 2000. With repeated exposure to psychostimulants such as cocaine and amphetamine, long-lasting changes occur in the mesolimbic dopamine system that are thought to underlie continued drug-seeking and relapse. One consequence of repeated cocaine treatment is an increase in extracellular adenosine in the ventral tegmental area (VTA), which results in tonic inhibition of synaptic input to dopamine neurons. The synapse specificity of this increased adenosine tone was examined on glutamate- and GABA-mediated responses using the selective A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). The slow, metabotropic glutamate receptor (mGluR)-mediated inhibitory postsynaptic potential (IPSP) was enhanced by DPCPX only in slices from psychostimulant-treated animals. Under resting conditions, DPCPX was without effect on fast excitatory postsynaptic currents (EPSCs) in slices from saline- or cocaine-treated animals. However, in the presence of amphetamine, DPCPX did augment fast EPSCs in slices from cocaine-treated rats. Although DPCPX increased GABAB IPSPs, the magnitude of the increase was not altered by cocaine pretreatment, even in the presence of amphetamine. This suggests that the elevated adenosine tone induced by cocaine treatment acts preferentially on glutamate terminals. Furthermore, the inhibition of the mGluR IPSP by endogenous adenosine may result in more effective burst firing mediated by glutamate afferents in cocaine-treated rats, a phenomenon known to enhance dopamine release.
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INTRODUCTION |
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The mesolimbic dopamine system, originating in the
ventral tegmental area (VTA), is thought to play a central role in
reward, incentive learning, and motivational processes. Consistent with this, the mesolimbic dopamine system is the target of addictive drugs,
many or all of which increase extracellular dopamine levels, particularly in the nucleus accumbens (Di Chiara and Imperato 1988). The addictive drugs best studied with respect to the
dopamine system are the psychostimulants, especially cocaine and
amphetamine (reviewed by Pierce and Kalivas 1997
).
Repeated use of psychostimulants by humans can result in the
development of profound craving for the drug, as well as sensitization
to the drug's psychotomimetic effects. These effects of drug use are
sustained even after long periods of abstinence. In rodents, repeated
administration of cocaine or amphetamine results in a long-lasting
sensitization to the locomotor stimulant effects of the drug. This
sensitization is mediated at least in part by an enhanced ability of
the stimulant to increase dopamine levels in the nucleus accumbens. The
sensitization of the mesolimbic dopamine system may render it
hypersensitive not only to psychostimulants, but also to stress and to
environmental stimuli associated with drug use, all of which are known
to promote craving and relapse to drug use (Robinson and
Berridge 1993
).
Although the behavioral consequences of repeated psychostimulant
administration are reasonably well understood, far less is known about
the underlying cellular and molecular changes. Perhaps the best
established, persistent change at the cellular level is an alteration
in signal transduction by dopamine D1 receptors, which are positively
coupled to adenylyl cyclase. This has been shown postsynaptically in
the nucleus accumbens both in vitro (Higashi et al.
1989) and in vivo (Henry and White 1991
,
1995
) and presynaptically in the VTA in vitro
(Bonci and Williams 1996
).
D1 receptors in the VTA are found on afferent terminals from the
nucleus accumbens and ventral pallidum containing GABA (Lu et
al. 1997b; Mansour et al. 1991
), and
glutamatergic afferents from the prefrontal cortex (PFC) express D1
receptor mRNA (Lu et al. 1997a
). Stimulation of D1
receptors in drug-naive animals enhances release of both GABA
(Cameron and Williams 1993
) and glutamate
(Fiorillo and Williams 1998
; Kalivas and Duffy
1995
) onto dopamine neurons. In drug-naive guinea pigs, D1
receptor agonists enhance the slow inhibitory postsynaptic potential
(IPSP). However, after 7-10 days withdrawal from repeated injections
of cocaine or morphine, D1 agonists inhibited the IPSP (Bonci
and Williams 1996
). It was found that in treated animals, the
cAMP produced by D1 activation was metabolized to extracellular
adenosine, which then inhibited transmitter release through activation
of adenosine A1 receptors. As a result, the adenosine tone was elevated in slices from drug-treated animals and was entirely dependent on tonic
activation of D1 receptors. After blocking A1 receptors or the
metabolism of cAMP, the modulation of transmitter release by D1
receptor agonists or antagonists was identical at all concentrations in
slices from saline- and drug-treated animals (Bonci and Williams 1996
). This suggests that the metabolism of cAMP to adenosine has been increased by repeated cocaine treatment, but that D1 receptors
and their coupling to adenylyl cyclase have not been changed.
More recently, it has been reported that the slow IPSP in dopamine
neurons of rats consists of two components, an early component mediated
by GABAB receptors, and a late component mediated
by metabotropic glutamate receptors (Fiorillo and Williams
1998). Furthermore, it is known that D1 receptors are present
on glutamate as well as GABA terminals, so the observed changes could
be present on either or both sets of terminals. It was therefore of
interest to examine the synapse specificity of the increased adenosine tone in drug-treated animals. The present study focused on adenosine tone in cocaine-treated rats. Certain experiments were also performed in rats treated with amphetamine or morphine.
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METHODS |
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Treatment protocol
Naive, male Wistar rats weighed 150-200 g. Treated rats received intraperitoneal injections of 1 ml/kg of 0.9% NaCl solution. Saline- and cocaine-treated (20 mg/kg cocaine HCl) animals were injected once daily for 14 days in their home cages. Other rats were given 2 mg/kg amphetamine sulfate every third day for 15 days (5 injections) or 10 mg/kg morphine sulfate every other day for 14 days (7 injections). They were then withdrawn for 10-20 days before killing, at which time they weighed 300-350 g. The care and killing of the rats complied with the guidelines of the National Institutes of Health.
Slice preparation
Intracellular or whole cell patch recordings were made in
horizontal slices (250-300 µm for intracellular recordings, 200 µm
for patch recordings) of ventral midbrain. Details of the method of
slice preparation and recording have been published (Williams et
al. 1984). Recordings were made from submerged slices in a chamber (0.5 ml) superfused with physiological saline at a rate of 1.5 ml/min and maintained at 35°C. The solution was equilibrated with
95% O2-5% CO2 and
contained (in mM) 126 NaCl, 2.5 KCl, 1.2 MgCl2,
2.4 CaCl2, 1.4 NaH2PO4, 25 NaHCO3, and 11 D-glucose.
Recordings
Recordings were made with an Axoclamp 2A amplifier from dopamine
neurons, identified by their electrical properties (Johnson and
North 1992), in the VTA. The VTA was defined as the area medial and rostral, but not lateral or caudal, of the medial terminal nucleus
of the accessory optic tract. Although some of these neurons are found
close to the MT in the area of dense cell bodies termed the
"compacta," they are likely to be mesolimbic (Fallon and
Loughlin 1995
). Microelectrodes (50-80 M
) were filled with
2 M KCl. The membrane potential was adjusted to between
60 and
70
mV to prevent spontaneous action potentials. For whole cell patch
recordings, cells were visualized using an upright microscope with
infrared illumination and Nomarski optics. Patch electrodes (2-5 M
)
contained (in mM) 125 KCl, 1 MgCl2, 1 EGTA, 5 HEPES, 1 ATP, and 0.3 GTP. For recordings of
N-methyl-D-aspartate (NMDA) EPSCs, electrodes contained 125 Cs-gluconate, 10 NaCl, 1 MgCl2, 10 HEPES, 1 EGTA, 0.3 CaCl2, 1 ATP, and 0.3 GTP. A
junction potential of approximately
10 mV with gluconate-containing
electrodes was not corrected. The membrane potential was clamped
between
70 and
80 mV for recording
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
excitatory postsynaptic currents (EPSCs), and at +30 mV for recording
NMDA EPSCs. The access resistance was monitored with each stimulus by
applying a 10-mV hyperpolarizing step. Membrane potential or holding
current was recorded continuously at a slower sampling frequency in all experiments.
Synaptic responses
Synaptic potentials or currents were evoked with bipolar tungsten stimulating electrodes with a tip separation of 300-1,000 µm. For all synaptic responses, a train of 8-10 stimuli of 400 µs at 0.3-1.5 mA was delivered at 66 Hz (15-ms interval) every 60 s, except for NMDA EPSCs, which were evoked at 10 Hz. Stimulating electrodes were placed within 1 mm rostral of the recording site. By stimulating rostrally, descending afferents may be preferentially activated, many of which are presumed to originate in the prefrontal cortex (PFC) and nucleus accumbens.
The following antagonists were used to isolate the desired synaptic response. Picrotoxin (100 µM, GABAA), strychnine (1 µM, glycine), and eticlopride (100 nM, D2) were present in all experiments. 1,2,3,4-Tetrahydro-6-nitro-2,3,-dioxo-benzo[f]quinoxaline (NBQX) (5 µM, AMPA) and MK-801 (50-100 µM, NMDA, pretreatment only) were used to isolate all slow IPSPs. The GABAB antagonist CGP 35348 (100-300 µM) or CGP 56999a (100-1,000 nM) was used to isolate mGluR IPSPs. GABAB IPSPs were studied after treating the slice with apamin (100 nM, SK channels) or in cases in which a GABAB antagonist completely blocked the slow IPSP. Pretreatment with MK-801 was performed before studying AMPA EPSCs, and NBQX was present in experiments on the NMDA EPSC.
An mGluR-mediated slow excitatory postsynaptic potential (EPSP) can
also be evoked in dopamine neurons and overlaps with the IPSP
(Shen and Johnson 1997). The slow EPSP is infrequently
observed with microelectrode recording and, when present, requires more stimuli than the IPSP (Fiorillo and Williams 1998
). The
depolarizing response to mGluR activation requires a more prolonged
activation of the receptor than does the hyperpolarizing response,
whereas the hyperpolarizing response desensitizes with prolonged
receptor activation. It is therefore possible that a drug that
increases glutamate release might enhance the EPSP and thereby mask or
desensitize the IPSP. For this reason, after
1,3-dipropyl-8-cyclopentylxanthine (DPCPX) was applied to mGluR IPSPs,
apamin was superfused to reveal a slow EPSP if present. In the few
cases in which an EPSP was present, the data were discarded.
Drugs
All drugs were applied to the slice by superfusion. The majority of drugs used, including DPCPX, had no effect on membrane potential or holding current. N6-cyclopentyladenosine (CPA) at high concentrations sometimes caused a hyperpolarization of up to 5 mV. Amphetamine (3 µM, in the presence of eticlopride) consistently produced a small depolarization (~3 mV). CGP 35348 (200 µM) and CGP 56999a (100-1,000 nM) consistently caused depolarizations of several millivolts, suggesting tonic activation of postsynaptic GABAB receptors.
Adenosine, adenosine trisphosphate (ATP), S(+)-amphetamine sulfate,
apamin, guanosine trisphosphate (GTP), picrotoxin, and strychnine were
from Sigma (St. Louis, MO). CPA, DPCPX, S()-eticlopride, and MK-801
were from Research Biochemicals International (Natick, MA). NBQX was
from Tocris Cookson (St. Louis, MO). Cocaine HCl and morphine sulfate
were from the National Institute on Drug Abuse (Rockville, MD). CGP
35348 and CGP 56999a were a gift from Novartis Pharmaceuticals (Basel, Switzerland).
Data analysis
Each specific experiment was performed only once per rat. Values are given as arithmetic means ± SE. The percent change produced by a drug was calculated as the mean amplitude of 5-10 synaptic responses after equilibrium had been reached (7-20 min) relative to the mean of five responses before drug superfusion. To construct concentration-response curves, two concentrations of CPA were superfused sequentially before reversal with DPCPX. To estimate the EC50 and maximal response, concentration-response curves were fit with a least-squares regression using the logistic equation. Statview software was used for performing statistical tests; P < 0.05 was considered as a significant difference. One- and two-way ANOVAs were performed with Fisher's post hoc test. Unpaired comparisons between two groups were made with a Mann-Whitney U test, whereas paired comparisons were made using a Wilcoxan signed rank test.
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RESULTS |
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Identification of dopamine neurons
Dopamine neurons in the VTA of horizontal slices were identified
by their electrical properties (Johnson and North 1992). With intracellular, KCl-containing electrodes, these cells exhibited slow, spontaneous firing (~1-3 Hz), broad action potentials followed by large after hyperpolarizations, and a depolarizing "sag" in response to hyperpolarizing current injection. With patch electrodes, dopamine neurons were identified by their firing pattern in the cell-attached mode, and the presence of a large time- and
voltage-dependent inward rectification in the current-voltage relation
(H current) measured in the whole cell mode. As in previous studies,
these neurons were found to respond to electrical field stimulation of
the slice with both fast and slow synaptic responses mediated by both
glutamate and GABA.
Adenosine tone on slow, mGluR IPSPs
We first measured the increase in slow IPSP amplitude in response
to superfusion of the adenosine A1 receptor selective antagonist DPCPX
(Fig. 1). In saline-treated animals,
DPCPX (200 nM) did not cause a significant increase in IPSP amplitude
(7.6 ± 6.6%, mean ± SE, n = 9). However,
in cocaine- or amphetamine-treated rats, DPCPX caused a 52.1 ± 12.7% (n = 16) and 91.0 ± 24.0%
(n = 6) increase (Fig. 1B), respectively
(1-way ANOVA, main effect of treatment, F[2,29] = 6.2, P = 0.006; effect of cocaine, P = 0.028, and amphetamine, P = 0.002). This confirms in
rats previous results obtained in guinea pigs (Bonci and
Williams 1996), showing that cocaine pretreatment potentiates
the augmentation of slow IPSPs by A1 receptor antagonists.
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In the above analysis of "slow IPSPs," responses were selected in which the mGluR-mediated IPSP was present, as determined pharmacologically or by visual inspection. A GABAB-mediated IPSP was present in most but not all cases. The increase produced by DPCPX was observed to be primarily due to an increase in the late (mGluR) component of the IPSP (Fig. 1A), although it was not possible under these conditions to accurately separate and measure the effect of DPCPX on the two synaptic components individually.
Adenosine tone on fast, glutamate-mediated EPSCs
Because it appeared that DPCPX had an effect on the mGluR IPSP,
the adenosine tone was next examined on glutamate-mediated fast EPSCs.
AMPA-mediated EPSCs were evoked using the same protocol as used to
evoke slow IPSPs (10 stimuli at 66 Hz, as shown in Fig. 5A).
The amplitudes of the first peak were 113 ± 19 pA
(n = 22) and 120 ± 13 pA (n = 53)
in slices from saline- and cocaine-pretreated animals, respectively.
Cocaine pretreatment did not alter the paired-pulse ratio of the third
(saline, 0.72 ± 0.07; cocaine, 0.70 ± 0.05); or the 10th
EPSC (saline, 0.44 ± 0.06; cocaine, 0.42 ± 0.04) relative
to the first EPSC. The paired-pulse ratios measured here are very
similar to those previously published for EPSCs in dopamine neurons
from naive rats (Bonci and Malenka 1999). DPCPX was
without effect on EPSCs in slices from either saline- or
cocaine-treated rats (Fig. 2; 0.3 ± 4.5%, n = 10, and 0.9 ± 6.9%, n = 10, respectively, percent increase in amplitude of the 1st EPSC in
the train). Another recent study also found a lack of adenosine tone on
AMPA EPSCs in dopamine neurons of the VTA, in both naive and acutely
morphine-withdrawn rats (Manzoni and Williams 1999
).
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It is generally thought that the same terminals release glutamate onto
both AMPA and NMDA subtypes of glutamate receptor. However, because of
the unique role that NMDA receptors are thought to play in the burst
firing of dopamine neurons (Johnson et al. 1992b;
reviewed by Overton and Clark 1997
), as well as the
possible presence of "NMDA-only" synapses, adenosine tone was also
examined on NMDA-mediated EPSCs, studied at +30 mV to relieve
Mg2+ blockade. DPCPX had no effect or caused a
small decrease in the amplitude of NMDA EPSCs in both saline- and
cocaine-treated rats (
9.7 ± 2.4%, n = 6, and
9.7 ± 4.5%, n = 7, respectively). Therefore while adenosine tone is present on mGluR synaptic responses in cocaine-treated rats, it is absent on synaptic responses mediated by
ionotropic glutamate receptors (iGluRs).
Having examined adenosine tone under resting conditions, we next
investigated adenosine tone in the presence of D-
amphetamine at a concentration known to be self-administered by rats (3 µM) (Clausing et al. 1995; Yokel and Pickens
1974
). By releasing dopamine and activating D1 receptors,
amphetamine may increase adenosine tone, as implied by Bonci and
Williams (1996)
. Amphetamine (3 µM) itself did not have a
clear effect on AMPA EPSCs in slices from saline- or cocaine-treated
rats after 10-15 min of superfusion (
0.6 ± 13%,
n = 4 and
1.5 ± 5.0%, n = 12, respectively).
In the presence of amphetamine, DPCPX was without effect on AMPA-mediated EPSCs in slices from saline-treated animals (0.0 ± 3.2%, n = 10), but augmented EPSCs by 31 ± 9.7% (n = 10) in slices from cocaine-treated animals (Fig. 2; 2-way ANOVA, effect of pretreatment, F[1,36] = 5.6, P = 0.024; amphetamine, F[1,36] = 4.9, P = 0.033; and a pretreatment X amphetamine interaction, F[1,36] = 4.7, P = 0.037). Although the first EPSC in the train was significantly augmented, the third (Fig. 2, inset) and 10th EPSCs were not (14 ± 11%, n = 10, and 14 ± 14%, n = 8, respectively), consistent with a presynaptic effect on glutamate release. Amphetamine thus reveals an increased adenosine tone on glutamate terminals caused by cocaine pretreatment.
One concern was the lack of effect of amphetamine on EPCS amplitude.
This observation may suggest that D1 receptors were not activated.
Multiple effects of amphetamine in the VTA have recently been
described. At slightly higher concentrations amphetamine decreases EPSC
amplitude in the VTA through 5-HT-mediated presynaptic inhibition
(Jones and Kauer 1999). In addition, amphetamine results in the activation of postsynaptic alpha-1-adrenoceptors on dopamine cells to cause a potent inhibition of the mGluR IPSP (C. D. Fiorillo, C. Paladini, and J. T. Williams, unpublished
observations). Also note that amphetamine decreased the amplitude of
the GABAB IPSP (Fig. 4). Thus the lack of effect of
amphetamine on the EPCS amplitude may be complicated by multiple effects.
GABAB receptors may be necessary for adenosine tone
As illustrated by in Fig. 1, DPCPX significantly augmented mGluR IPSPs under conditions where both GABAB and mGluR IPSPs were present. The effect of DPCPX on pharmacologically isolated mGluR IPSPs were examined in the presence of a GABAB antagonist (CGP 35348, 100-300 µM or CGP 56999a, 0.1-1 µM). In these experiments the augmentation of the mGluR IPSP by DPCPX was quite variable, and a significant augmentation was not observed in either saline- or cocaine-treated rats (7.0 ± 14.0%, n = 5 and 20.6 ± 13.4%, n = 8, respectively, Fig. 3). It appears that GABAB antagonists prevent or reduce the augmentation of glutamate release by DPCPX.
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We therefore examined the effect of a GABAB
antagonist (CGP 56999a, 0.3-1.0 µM) on amphetamine-induced adenosine
tone on AMPA EPSCs in slices from cocaine-treated rats. CGP 56999a was
itself without significant effect on the amplitude of the AMPA EPSCs in
naive or cocaine-treated rats, similar to previous results in naive and
morphine-treated rats (Manzoni and Williams 1999). In
the presence of CGP 56999a, DPCPX was without effect on the amplitude
of EPSCs in cocaine-treated animals (0.4 ± 5.8%,
n = 7), even in the presence of amphetamine (2.4 ± 4.0%, n = 10, not shown). This suggests that tonic
activation of GABAB receptors is necessary for
adenosine tone on glutamate terminals.
The mechanism by which CGP 56999a blocks adenosine tone was further
investigated by measuring EPSC inhibition by exogenous adenosine in
slices from cocaine-treated rats. Initially adenosine (50 µM)
produced an inhibition of 20 ± 7.0% (n = 5).
After superfusion of CGP 56999a (0.5-1.0 µM), adenosine caused a
similar inhibition of
28 ± 4.5% in the same cells. This
indicates that blockade of GABAB receptors does
not decrease the sensitivity of presynaptic A1 receptors. It may
therefore be the case that tonic activation of
GABAB receptors is necessary for adenosine
production, although it is not clear through what mechanism this could occur.
Adenosine tone on GABAB IPSPs
Isolated GABAB IPSPs were studied after
exposure of the slice to apamin (100 nM), or in cases in which the
entire IPSP could be blocked by CGP 35348 (100-300 µM). DPCPX
augmented the isolated GABAB IPSP by 19.3 ± 3.8% (n = 8, Wilcoxon signed-rank test,
P = 0.008, Fig. 4).
However, the facilitation of GABAB IPSPs by DPCPX
was unchanged by cocaine pretreatment (20.4 ± 2.8%,
n = 10, Fig. 4). This is in apparent contradiction to
previously published results in cocaine-treated guinea pigs
(Bonci and Williams 1996). However, in the present study
it was found that in slices taken from rats 10 or more days withdrawn
from repeated morphine injections, DPCPX produced a substantial
increase in the amplitude of GABAB IPSPs
(47.9 ± 6.9%, n = 5). This is significantly
greater than its effect in slices from saline-treated rats
(P = 0.008, Mann-Whitney U test) and is
similar to results from guinea pigs (Bonci and Williams
1996
).
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Although cocaine-pretreatment did not elevate adenosine tone on
GABA terminals in slices under resting conditions, there may be
elevated adenosine tone in the presence of amphetamine. Unexpectedly, amphetamine reduced the augmentation produced by DPCPX in both treatment groups (Fig. 4). This inhibition was not mediated by an
increase in adenosine synthesis because DPCPX had no additional effect.
In addition, in the presence of amphetamine the effect of DPCPX was not
different between slices from saline- and cocaine-pretreated rats
(6.1 ± 2.0%, n = 4 and 9.6 ± 3.7%,
n = 10, respectively, 2-way ANOVA, effect of
amphetamine only, F[1,28] = 10.2, P = 0.004). The inhibition by amphetamine may result from the release of
5-HT, which causes a potent presynaptic inhibition of the
GABAB IPSP (Cameron and Williams
1994; Johnson et al. 1992a
). Although the interpretation of the present result is complicated by the inhibition by amphetamine, it suggests that the increased adenosine in the VTA
caused by repeated cocaine treatment may be restricted to glutamate terminals.
Sensitivity of synaptic responses to A1 receptor inhibition
It has been reported that repeated cocaine treatment does
not alter the inhibition by CPA (a metabolically stable, A1
receptor-selective analogue of adenosine) of either
GABAB IPSPs in the VTA (Bonci and Williams
1996) or glutamate EPSPs in the nucleus accumbens (Manzoni et al. 1998
). However, the potency of adenosine
in the nucleus accumbens (at least at room temperature) is decreased following cocaine withdrawal due to enhanced uptake (Manzoni et al. 1998
). In the present study an approximately half-maximal concentration of adenosine (50 µM) caused an inhibition of the AMPA
EPSC of
32 ± 9.8% (n = 6) in slices from
saline-treated rats,
35 ± 2.4% (n = 5) in
cocaine-treated rats, and
33 ± 4.9% (n = 7) in
amphetamine-treated rats (Fig.
5C). This suggests that in the
VTA, repeated psychostimulant administration does not produce a
long-lasting change in the uptake of adenosine or in the sensitivity of
glutamate terminals to adenosine inhibition.
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The inhibition by CPA of the various synaptic inputs to dopamine
neurons was measured in slices from naive rats. The sensitivity and
efficacy of CPA in inhibiting AMPA, mGluR, and
GABAB synaptic responses was similar. The potency
and maximal inhibition were, respectively, 96 ± 13 nM and
65 ± 2% for the GABAB IPSP, 79 ± 45 nM and
55 ± 6% for the AMPA EPSC, and 19 ± 6 nM and
54 ± 4% for the mGluR IPSP (Fig. 5B). If we
consider the presence of endogenous adenosine tone on the
GABAB IPSP that was not corrected for in these
experiments, it appears that the efficacy of A1 receptor-mediated inhibition is greatest on the GABAB IPSP, as
previously suggested (Wu et al. 1995
). However, the
mGluR IPSP may be the most sensitive to A1 inhibition (Fig.
5B). The disparity between the A1 inhibition of AMPA and
mGluR synaptic responses may be particularly large considering that,
although the inhibition by CPA of the first AMPA EPSC in the train is
similar to the inhibition of the mGluR IPSP (Fig. 5B), CPA
did not cause consistent inhibition of either the third (
6 ± 21%, n = 7, 1 µM) or the 10th EPSC (
15 ± 13%, n = 7, 1 µM) at any dose (Fig. 5A).
However, the latter stimuli in the train do make a significant
contribution to the mGluR IPSP (Fig. 6).
The lack of efficacy of A1 receptors in inhibiting the latter EPSCs in
a train is similar to previous studies of other inhibitory presynaptic
receptors, particularly the GABAB receptor, which
can in fact augment the latter EPSCs in a train of sufficiently high
frequency (Brenowitz et al. 1998
).
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DISCUSSION |
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Increased adenosine tone on glutamate terminals
This study confirms previous work showing that repeated cocaine
treatment increases adenosine tone in the VTA (Bonci and
Williams 1996). The present results indicate that the increase
in adenosine tone selectively inhibits glutamate release, whereas
cocaine pretreatment does not alter adenosine tone on
GABAB IPSPs. Although there was no adenosine tone
on fast or slow glutamate-mediated synaptic responses in control
animals, cocaine or amphetamine treatment resulted in substantial
adenosine tone on slow, mGluR-mediated IPSPs. In the presence of
amphetamine, at a concentration known to be self-administered (3 µM)
(Clausing et al. 1995
; Yokel and Pickens
1974
), adenosine tone was also present on fast EPSCs in cocaine- but not saline-treated animals.
The lack of increase in adenosine tone on GABAB
IPSPs resulting from cocaine treatment in the present study is in
apparent contradiction with previously published results (Bonci
and Williams 1996). Because the presence of an mGluR IPSP was
not yet known at the time of the earlier study, it is possible that the
effects observed on the slow IPSP were due to changes in release of
glutamate rather than GABA. However, under the conditions of the
previous study, the mGluR IPSP would have been less prominent than in
the present study (unpublished observations). A species difference in
adenosine tone could therefore account for the disparate results. For
instance, if adenosine is produced in glutamate terminals, it could
diffuse far enough to reach GABA terminals in slices from guinea pigs
but not rats.
The present study did not address the mechanism by which adenosine tone
is increased. Presumably, it is the result of dopamine activation of D1
receptors, production of cAMP, and subsequent metabolism of cAMP to
adenosine (Bonci and Williams 1996; Shoji et al.
1999
). In support of such a mechanism, adenosine tone on fast
EPSCs in cocaine-pretreated rats was increased by amphetamine, which
enhances dopamine release. Furthermore, inhibition of EPSCs by
exogenous adenosine was not altered by cocaine or amphetamine pretreatment, suggesting that increased adenosine tone must be dependent on enhanced adenosine production (Bonci and Williams 1996
).
D1 receptors are thought to be present on glutamate terminals of
afferents from the prefrontal cortex (Lu et al. 1997a),
as well as GABA terminals of afferents from the nucleus accumbens and
ventral pallidum (Lu et al. 1997b
; Mansour et al.
1991
). In light of the present results, it is likely
that the increased adenosine produced after cocaine pretreatment
derives from D1 receptor activation on glutamate-containing terminals
from the prefrontal cortex. Although D1 receptors on GABA terminals
produced more adenosine than those on glutamate terminals in slices
from control rats, this transduction mechanism appeared unaltered by repeated cocaine treatment. The present results also indicate that
extracellular adenosine can be localized to specific synapses, because
adenosine tone inhibited only GABA release in slices from control
animals and was increased only on glutamate terminals by cocaine pretreatment.
Selective inhibition of mGluR IPSPs
Repeated cocaine treatment resulted in adenosine tone on the mGluR
IPSP, but not on the AMPA or NMDA receptor-mediated EPSCs. Similarly,
adenosine tone is enhanced on mGluR IPSPs (Williams, unpublished
observations) but not EPSCs (Manzoni and Williams 1999)
during acute morphine withdrawal. The reason for this selectivity of
adenosine action is not known. Concentration-response curves to the
metabolically stable A1 receptor agonist CPA suggest that the mGluR
IPSP may be slightly more sensitive than the AMPA EPSC to A1
inhibition. It is possible that the difference in glutamate concentration and kinetics necessary for receptor activation, or the
very different postsynaptic transduction mechanisms, could account for
the slightly different sensitivities to A1 inhibition. However, the
relatively small difference in sensitivity to A1 inhibition is unable
to account for the substantial difference in adenosine tone on iGluR
and mGluR synaptic responses.
There are a number of mechanisms through which the mGluR IPSP could be
modulated postsynaptically (Fiorillo and Williams 1998). A postsynaptic effect of A1 receptors on the mGluR IPSP is possible but
unlikely, because the maximal inhibition by CPA was the same for AMPA
and mGluR synaptic responses. Furthermore, in guinea pigs acutely
withdrawn from morphine, DPCPX enhanced mGluR IPSPs, but not
mGluR-mediated hyperpolarizations by aspartate, in dopamine neurons of
VTA (Williams, unpublished observations). It is therefore concluded
that the inhibition by A1 receptors of the mGluR IPSP occurs
presynaptically, with little effect postsynaptically.
One explanation for the difference in adenosine tone is that different
populations of terminals mediate the iGluR and mGluR synaptic
responses. Although there is no evidence for "mGluR only" synapses,
it is possible that a large proportion of terminals releasing glutamate
onto AMPA and NMDA receptors do not release glutamate onto mGluRs. It
appears that an analogous situation exists with respect to GABA
terminals on dopamine neurons of the VTA. The vast majority of
terminals releasing GABA onto GABAA receptors in
response to local stimulation of the VTA do not activate postsynaptic
GABAB receptors, because the
GABAB IPSP, but not the
GABAA IPSP, is sensitive to presynaptic
regulation by D1 (Cameron and Williams 1993) and 5-HT1
agonists (Johnson et al. 1992a
; Sugita et al.
1992
). For glutamate terminals, however, the degree of modulation by maximally effective concentrations of A1, µ-opioid, 5-HT1, and D1 receptor agonists is similar for both AMPA and mGluR synaptic responses (present results; Fiorillo and Williams
1998
; Manzoni and Williams 1999
; unpublished
observations); so at present there is little evidence for a difference
in the pool of glutamate terminals responsible for the iGluR and mGluR
synaptic responses.
Regardless of whether the iGluR and mGluR synaptic responses are mediated by identical or distinct glutamate terminals, it is anticipated that adenosine would cause greater inhibition of the mGluR response than of the iGluR response. Although A1 receptors caused substantial inhibition of the first EPSP in a train, subsequent EPSPs were inhibited little or not at all (Figs. 2A and 5A). Thus with a short train of presynaptic action potentials, adenosine would cause a greater inhibition of the mGluR IPSP than of the sum of the EPSPs. The net result would be a greater excitation of dopamine neurons for a given presynaptic fiber volley in cocaine-treated rats.
Implications for the mesolimbic dopamine system
Because of the difference in kinetics of the iGluR and mGluR
synaptic potentials, it is predicted that the mGluR IPSP acts to limit
the duration of the burst of action potentials driven by the iGluR EPSP
(particularly the NMDA component of the EPSP). A selective inhibition
of the mGluR IPSP would therefore be expected to prolong the duration
of burst events. It has been reported that at 10 days withdrawal from
repeated amphetamine treatment of rats, stimulation of prefrontal
cortex more reliably elicits bursts in dopamine neurons of the VTA
(Tong et al. 1995). The present results, with both
cocaine and amphetamine treatment, suggest a mechanism that may
account, at least in part, for the enhanced excitation of dopamine
neurons after psychostimulant treatment.
A recent study has shown that systemic cocaine evokes greater glutamate
release in the VTA of rats 21 days withdrawn from daily cocaine
treatment (Kalivas and Duffy 1998). The glutamate release was blocked by prior infusion of a D1 antagonist into the VTA.
However, the enhanced increase in glutamate release in response to
cocaine was very transient and may have been due to a conditioned
response of the behaving animal rather than a pharmacological action of cocaine.
It is well established that conditioned cues are a primary trigger of
relapse to drug use in both human addicts and other species. It has
been hypothesized that sensitization of the mesolimbic dopamine system
may underlie the powerful craving elicited by external stimuli
associated with drug effects (Robinson and Berridge 1993). The potential role of dopamine neurons in such a process has been advanced by studies of dopamine cell activity during the
learning of cues predicting reward in monkeys (Ljungberg et al.
1992
; Schultz et al. 1993
; reviewed by
Schultz 1998
). Initially, dopamine cells are unaffected
by a neutral stimulus, but respond to an unpredicted natural reward,
such as juice, with a burst of action potentials. With repeated
pairings of the neutral stimulus and reward, the response of dopamine
neurons is conditioned such that the formerly neutral stimulus now
elicits a response, but the primary reward is no longer effective.
Dopamine neurons therefore respond to errors in the prediction of
reward. This finding promotes the theory that an increased activity of
dopamine neurons underlies the craving and anticipation of rewards and
not necessarily the response to rewards themselves (Robinson and
Berridge 1993
). It is not known which input(s) to dopamine
cells mediates this excitation, but the glutamatergic inputs from the
PFC are a likely candidate. If this is the case, then selective
inhibition of the mGluR IPSP by adenosine might be one mechanism by
which a conditioned cue could elicit a greater dopamine response, and
presumably greater craving for drug, in a cocaine-experienced animal.
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ACKNOWLEDGMENTS |
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We thank Drs. James Brundege and Gary Westbrook for helpful comments on the manuscript.
This work was supported by National Institute on Drug Abuse Grants DA-04523 and DA-05793.
Present address of C. D. Fiorillo: Institute of Physiology, University of Fribourg, Rue de Musee 5, CH 1700 Fribourg, Switzerland.
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FOOTNOTES |
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Address for reprint requests: J. T. Williams, The Vollum Institute, L474, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd., Portland, OR 97201.
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.
Received 27 August 1999; accepted in final form 29 October 1999.
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REFERENCES |
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