D2 dopamine receptor activation facilitates endocannabinoid-mediated long-term synaptic depression of GABAergic synaptic transmission in midbrain dopamine neurons via cAMP-protein kinase A signaling.
ABSTRACT Endocannabinoid (eCB) signaling mediates short-term and long-term synaptic depression (LTD) in many brain areas. In the ventral tegmental area (VTA) and striatum, D(2) dopamine receptors cooperate with group I metabotropic glutamate receptors (mGluRs) to induce eCB-mediated LTD of glutamatergic excitatory and GABAergic inhibitory (I-LTD) synaptic transmission. Because D(2) receptors and group I mGluR agonists are capable of inducing the release of eCBs, the predominant hypothesis is that the cooperation between these receptors to induce eCB-mediated synaptic depression results from the combined activation of type I cannabinoid (CB(1)) receptors by the eCBs. By determining the downstream effectors for D(2) receptor and group I mGluR activation in VTA dopamine neurons, we show that group I mGluR activation contributes to I-LTD induction by enhancing eCB release and CB(1) receptor activation. However, D(2) receptor activation does not enhance CB(1) receptor activation, but facilitates I-LTD induction via direct inhibition of cAMP-dependent protein kinase A (PKA) signaling. We further demonstrate that cAMP/PKA signaling pathway is the downstream effector for CB(1) receptors and is required for eCB-mediated I-LTD induction. Our results suggest that D(2) receptors and CB(1) receptors target the same downstream effector cAMP/PKA signaling pathway to induce I-LTD and D(2) receptor activation facilitates eCB-mediated I-LTD in dopamine neurons not by enhancing CB(1) receptor activation, but by enhancing its downstream effects.
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ABSTRACT: Since the first reports in 2001, great advances have been made towards the understanding of endocannabinoid-mediated synaptic modulation. Electrophysiological studies have revealed that one of the two major endocannabinoids, 2-arachidonoylglycerol (2-AG), is produced from membrane lipids upon postsynaptic Ca(2+) elevation and/or activation of Gq/11-coupled receptors, and released from postsynaptic neurons. The released 2-AG then acts retrogradely onto presynaptic cannabinoid CB1 receptors and induces suppression of neurotransmitter release either transiently or persistently. These forms of 2-AG-mediated retrograde synaptic modulation are functional throughout the brain. The other major endocannabinoid, anandamide, mediates a certain form of endocannabinoid-mediated long-term depression (LTD). Anandamide also functions as an agonist for transient receptor potential vanilloid receptor type 1 (TRPV1) and mediates endocannabinoid-independent and TRPV1-dependent forms of LTD. It has also been demonstrated that the endocannabinoid system itself is plastic, which can be either up- or down-regulated by experimental or environmental conditions. In this review, I will make an overview of the mechanisms underlying endocannabinoid-mediated synaptic modulation.Proceedings of the Japan Academy Ser B Physical and Biological Sciences 01/2014; 90(7):235-50. · 2.56 Impact Factor
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ABSTRACT: Adenosine and adenosine receptors (ARs) are increasingly recognized as important therapeutic targets for controlling cognition under normal and disease conditions for its dual roles of neuromodulation as well as of homeostatic function in the brain. This chapter first presents the unique ability of adenosine, by acting on the inhibitory A1 and facilitating A2A receptor, to integrate dopamine, glutamate, and BNDF signaling and to modulate synaptic plasticity (e.g., long-term potentiation and long-term depression) in brain regions relevant to learning and memory, providing the molecular and cellular bases for adenosine receptor (AR) control of cognition. This led to the demonstration of AR modulation of social recognition memory, working memory, reference memory, reversal learning, goal-directed behavior/habit formation, Pavlovian fear conditioning, and effort-related behavior. Furthermore, human and animal studies support that AR activity can also, through cognitive enhancement and neuroprotection, reverse cognitive impairments in animal models of Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, and schizophrenia. Lastly, epidemiological evidence indicates that regular human consumption of caffeine, the most widely used psychoactive drug and nonselective AR antagonists, is associated with the reduced cognitive decline in aging and AD patients, and with the reduced risk in developing PD. Thus, there is a convergence of the molecular studies revealing AR as molecular targets for integrating neurotransmitter signaling and controlling synaptic plasticity, with animal studies demonstrating the strong procognitive impact upon AR antagonism in normal and disease brains and with epidemiological and clinical evidences in support of caffeine and AR drugs for therapeutic modulation of cognition. Since some of adenosine A2A receptor antagonists are already in phase III clinical trials for motor benefits in PD patients with remarkable safety profiles, additional animal and human studies to better understand the mechanism underlying the AR-mediated control of cognition under normal and disease conditions will provide the required rationale to stimulate the necessary clinical investigation to rapidly translate adenosine and AR drug as a novel strategy to control memory impairment in neuropsychiatric disorders.International Review of Neurobiology 01/2014; 119C:257-307. · 2.46 Impact Factor
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ABSTRACT: The endocannabinoid system (ECS) and the dopaminergic system (DAS) are two major regulators of basal ganglia function. During Huntington's disease (HD) pathogenesis, the expression of genes in both the ECS and DAS is dysregulated. The purpose of this study was to determine the changes that were consistently observed in the ECS and DAS during HD progression in the central nervous system (CNS) and in the periphery in different models of HD and human HD tissue. To do this, we conducted a meta-analysis of differential gene expression in the ECS and DAS using publicly available microarray data. The consolidated data were summarized as observed changes in gene expression (OCGE) using a weighted sum for each gene. In addition, consolidated data were compared to previously published studies that were not available in the gene expression omnibus (GEO) database. The resulting data confirm gene expression changes observed using different approaches and provide novel insights into the consistency between changes observed in human tissue and various models, as well as disease stage- and tissue-specific transcriptional dysregulation in HD. The major implication of the systems-wide data presented here is that therapeutic strategies targeting the ECS or DAS must consider the dynamic changes in gene expression over time and in different body areas, which occur during HD progression and the interconnectedness of the two systems.Pharmacology Research & Perspectives. 02/2015; 3(1).
Endocannabinoid (eCB) signaling mediates short-term and long-term synaptic depression (LTD) in many brain areas. In the ventral
tegmental area (VTA) and striatum, D2dopamine receptors cooperate with group I metabotropic glutamate receptors (mGluRs) to
induce eCB-mediated LTD of glutamatergic excitatory and GABAergic inhibitory (I-LTD) synaptic transmission. Because D2receptors
group I mGluR activation contributes to I-LTD induction by enhancing eCB release and CB1receptor activation. However, D2receptor
activation does not enhance CB1receptor activation, but facilitates I-LTD induction via direct inhibition of cAMP-dependent protein
is required for eCB-mediated I-LTD induction. Our results suggest that D2receptors and CB1receptors target the same downstream
Endocannabinoids (eCBs) are a new class of signaling molecules
that mediate short-term and long-term synaptic depression
(LTD) in many brain areas (Gerdeman and Lovinger, 2003; Al-
released “on demand” (Di Marzo et al., 1994). eCBs are released
by depolarization-induced Ca2?influx (Di Marzo et al., 1994,
1998; Stella et al., 1997) or the activation of certain G-protein-
coupled receptors (GPCRs), such as group I metabotropic gluta-
these stimuli, eCBs are released from postsynaptic neurons and
travel across the synaptic cleft to activate type I cannabinoid re-
ceptors (CB1) on presynaptic terminals, resulting in retrograde
depression of synaptic transmission.
We have shown that repetitive synaptic activation of D2do-
pamine receptors and group I mGluRs induces eCB-mediated
ation between D2receptors and group I mGluRs to induce eCB-
mediated excitatory synaptic depression has been described in the
striatum (Kreitzer and Malenka, 2005; Yin and Lovinger, 2006).
Marzo et al., 1998; Piomelli, 2003). Because D2receptor agonists
increase AEA production (Giuffrida et al., 1999; Patel et al., 2003;
Centonze et al., 2004) and group I mGluR agonists increase 2-AG
production (Jung et al., 2005), it has been hypothesized that the
cooperation between D2receptors and group I mGluRs to induce
eCB-mediated synaptic depression results from the combined ac-
tion of these two eCBs (Kreitzer and Malenka, 2005; Yin and Lov-
receptors whose activation leads to the inhibition of adenylyl
cyclase (AC), resulting in decreased cAMP accumulation and
protein kinase A (PKA) activity (Piomelli, 2003; Neve et al.,
2004). Recent studies have shown that the cAMP/PKA signaling
pathway mediates eCB-mediated I-LTD in the hippocampus
(Chevaleyre et al., 2007) and LTD in the striatum (Mato et al.,
2007). These studies raise the possibility that D2receptor activa-
tion facilitates eCB/CB1-receptor-mediated synaptic depression
through direct inhibition of cAMP/PKA signaling. In this study,
we distinguish these two possibilities by determining the down-
This work was supported by National Institutes of Health Grants DA024741 (Q.S.L) and DA09155 (C.J.H.). We
14018 • TheJournalofNeuroscience,December24,2008 • 28(52):14018–14030
stream effectors that mediate group I mGluR- and D2receptor-
induced synaptic depression in VTA dopamine neurons. We
I-LTD induction not by enhancing CB1receptor activation, but by
enhancing its downstream effects. Our study reveals a previously
unrecognized mechanism for D2receptor-induced facilitation of
Slice preparation. Midbrain slices (250 ?m) from male Sprague Dawley
rats (P18–25), CB1-knock-out (CB1?/?) mice and wild-type (CB1?/?)
mice of either sex (P16–20) were prepared as described previously (Pan
least nine generations. Slices were prepared at 4?6°C in a solution con-
7 MgSO4, 26 NaHCO3, 25 glucose, 11.6 sodium ascorbate, and 3.1 so-
dium pyruvate. The slices were incubated in artificial CSF (ACSF) con-
NaHCO3, and 10 glucose. The ACSF was saturated with 95% O2and 5%
CO2. Slices were recovered for at least 1 h at room temperature before
recordings. All recordings were performed at 32 ? 1°C by using an au-
tomatic temperature controller (Warner Instrument).
Electrophysiology. Whole-cell recordings were made from VTA dopa-
mine neurons in the midbrain slices (Pan et al., 2008). Dopamine neu-
rons were identified by the presence of large Ihcurrents, rhythmic firing
at low frequency and prominent afterhyperpolarization (Johnson and
North, 1992; Jones and Kauer, 1999; Liu et al., 2005). A recent study has
clearly demonstrated that these electrophysiological characteristics do
not exclusively belong to dopamine neurons (Margolis et al., 2006). Pu-
tative dopamine neurons in our study may contain a small number of
nondopamine neurons, which should be randomly distributed in differ-
ent experimental groups. IPSCs were evoked by a bipolar tungsten stim-
neuron. For recording of evoked IPSCs, glass pipette was filled with a
7.2 (with KOH). In experiments with postsynaptic Ca2?buffer, 10 mM
K-gluconate was replaced by 10 mM EGTA (see Fig. 3F). Cs?-based
Cs-methanesulfonate 100, CsCl 50, HEPES 10, EGTA 0.2, MgCl22,
MgATP 4, Na2GTP 0.3, and Na2-phosphocreatine 10 at pH 7.2 (with
CsOH). For recording of miniature IPSCs (mIPSCs), K-gluconate was
replaced by KCl in internal solution and tetrodotoxin (TTX) was added
in the ACSF to block action potentials. All recordings were made in the
2,3-dione (CNQX, 20 ?M) and D-2-amino-5-phosphonovaleric acid (D-
AP-5, 50 ?M). Neurons were voltage-clamped at ?70 mV unless stated
otherwise. Series resistance (15–30 M?) was monitored throughout the
recordings and data were discarded if the resistance changed by ?20%.
Tetrahydrolipstatin (THL) and CNQX were obtained from Sigma-Al-
drich; guanosine-5?-O-(2-thiodiphosphate) 3Li (GDP-?-S) was from
Biomol, EGTA-AM was from Anaspec and all other drugs were from
Statistics. Data are presented as the mean ? SEM. I-LTD (%) was
calculated as follows: 100 ? [mean amplitude of IPSCs during the final
10 min of recording/mean amplitude of baseline IPSCs]. The acute de-
pression of evoked IPSCs (%) was calculated as follows: 100 ? [mean
amplitude of 30 IPSCs after drug treatment/mean amplitude of baseline
IPSCs]. Data sets were compared with either paired or unpaired Stu-
dent’s t test, or ANOVA followed by Tukey’s post hoc analysis. Results
were considered to be significant at p ? 0.05.
We previously reported that synaptic activation of group I
mine neurons and that the presence of D2receptor agonist quin-
synaptic stimulation is required for I-LTD induction (Pan et al.,
eration between group I mGluRs and D2receptors to induce
I-LTD, we began by corroborating and extending our previous
observations. Consistent with our previous study (Pan et al.,
2008), we found that neither the application of a low concentra-
tion of cocaine (3 ?M) nor 10 Hz stimulation for 5 min signifi-
cantly affected IPSCs (cocaine, 99.2 ? 6.9% of baseline, n ? 5,
p ? 0.05; 10 Hz stimulation, 95.5 ? 6.1% of baseline, n ? 6, p ?
0.05). However, the combination of cocaine and 10 Hz stimula-
1A) (see Materials and Methods for the quantification of I-LTD
Cocaine at 3 ?M is effective in inhibiting dopamine uptake in
the VTA (Beckstead et al., 2004), whereas the repetitive synaptic
stimulation (10 Hz, 5 min) likely activates dopaminergic and
glutamatergic afferents. Indeed, it has been shown that neither
group I mGluRs nor D2dopamine receptors are activated by
ulation (Batchelor and Garthwaite, 1997; Beckstead et al., 2004).
Dopamine receptors consist of two subfamilies: D1-like (D1, D5)
2004). For simplicity, we often use D1and D2receptors to repre-
sent D1- and D2-like receptors, respectively. We determined
which subtypes of dopamine receptors mediate the induction of
Consistent with our previous study (Pan et al., 2008), we
ulation with cocaine application was not affected by D1receptor
2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH
23390) at 1 ?M (72.3 ? 7.8 of baseline, n ? 5, p ? 0.05 vs
I-LTD control) or at 10 ?M (70.8 ? 8.2% of baseline, n ? 6,
p ? 0.05 vs I-LTD control) (Fig. 1A). However, I-LTD was
SPs in VTA dopamine neurons (Cameron and Williams, 1993).
Consistent with the latter observation, we found that bath applica-
tion of D1receptor agonist SKF 38393 (10 ?M) had no significant
as supplemental material). Thus, D1receptor activation is not in-
Sulpiride is a D2-like receptor antagonist that blocks both D2
subtype receptors are expressed in the VTA (Diaz et al., 2000;
strate considerable potential for therapeutic intervention in a
subtype receptor(s) mediate I-LTD induction in dopamine neu-
rons. We found that I-LTD was significantly attenuated in the
presence of selective D2receptor antagonist L-741,626 (Pillai et
Panetal.•D2ReceptorActivationInducesI-LTDviacAMP/PKAJ.Neurosci.,December24,2008 • 28(52):14018–14030 • 14019
al., 1998) (100 nM; Fig. 1B) (89.8 ? 6.1% of baseline, n ? 8, p ?
0.05 vs I-LTD control in Fig. 1A) or D3receptor antagonist GR
7.1% of baseline, n ? 7, p ? 0.05 vs I-LTD control in Fig. 1A).
1998) had no significant effect on I-LTD induction (100 nM; Fig.
of I-LTD in dopamine neurons.
Group I mGluRs consist of mGluR1 and mGluR5 subtypes
(Conn and Pin, 1997). Both mGluR1 and mGluR5 receptor sub-
types are known to be distributed on VTA dopamine neurons
(Kane et al., 2005). Our previous study has shown that either
mGluR1 antagonist 7-(hydroxyimino)cyclopropa[b]chromen-
1a-carboxylate ethyl ester (CPCCOEt) or mGluR5 antagonist
6-methyl-2-(phenylethynyl)-pyridine (MPEP) significantly at-
study, we found that a combination of the mGluR1 antagonist
CPCCOEt (50 ?M) and mGluR5 antagonist MPEP (10 ?M)
blocked I-LTD (Fig. 1C) (93.3 ? 8.1%, n ? 7, p ? 0.05 vs I-LTD
control in Fig. 1A). In addition, I-LTD was also blocked in slices
251, 2 ?M) (Fig. 1C) (93.4 ? 8.7% of baseline, n ? 7, p ? 0.05 vs
I-LTD control in Fig. 1A). These results confirm and extend our
previous findings that I-LTD in response to cocaine and the re-
I mGluRs and CB1receptors in dopamine neurons (Pan et al.,
We next determined whether the D2-like agonist quinpirole
could mimic cocaine in facilitating I-LTD induction. Quinpirole
Barrett, 1995). Although bath application of quinpirole (1 ?M)
alone had no significant effect on evoked IPSCs (96.5 ? 6.1% of
baseline, n ? 5, p ? 0.05), the presence of quinpirole (1 ?M)
during the 10 Hz stimulation induced I-LTD (70.4 ? 7.8% of
baseline, n ? 9, p ? 0.01; Fig. 1D), which was not significantly
different from I-LTD induced in the presence of cocaine ( p ?
0.05). I-LTD induced in the presence of quinpirole was not af-
fected by D1receptor antagonist SCH 23390 (10 ?M; Fig. 1D)
(61.7 ? 7.9%, n ? 5, p ? 0.05 vs I-LTD control in Fig. 1D), but
was blocked by the D2receptor antagonist sulpiride (10 ?M; Fig.
1D) (97.8 ? 6.9%, n ? 6, p ? 0.05 vs I-LTD control in Fig. 1D),
control in Fig. 1D), and by the CB1receptor antagonist AM 251
control in Fig. 1D). Together, the above results support the hy-
pothesis that D2receptors cooperate with group I mGluRs to
induce eCB-mediated I-LTD in dopamine neurons.
14020 • J.Neurosci.,December24,2008 • 28(52):14018–14030Panetal.•D2ReceptorActivationInducesI-LTDviacAMP/PKA
To investigate the mechanism by which D2receptors cooperate
with group I mGluRs to induce eCB-mediated I-LTD in dopa-
of these receptors to I-LTD induction and determined their re-
spective downstream effectors. We first determined how the D2
receptor activation leads to the depression of inhibitory synaptic
transmission. Our previous study has shown that bath applica-
tion of higher concentrations of cocaine (10 ?M) or quinpirole
(10 ?M) to midbrain slices produced reversible depression of
IPSCs in VTA dopamine neurons, and the cocaine-induced de-
23390, but was partially blocked by D2receptor antagonist
sulpiride (Pan et al., 2008). It has been
shown that quinpirole and cocaine induce
the release of AEA in the striatum and
tivation (Giuffrida et al., 1999; Patel et al.,
2003; Centonze et al., 2004) and that
quinpirole- and cocaine-induced acute
depression of IPSCs can be partially
blocked by a CB1receptor antagonist in
the striatum (Centonze et al., 2004). If co-
caine or quinpirole depresses IPSCs in the
VTA via the release of AEA and subse-
quent activation of CB1receptors, the de-
pression should be blocked by disrupting
eCB/CB1signaling. We examined whether
CB1receptor antagonist AM 251 affected
cocaine- and quinpirole-induced depres-
sion of IPSCs. Consistent with our previ-
ous study (Pan et al., 2008), we found that
bath application of 10 ?M cocaine or 10
evoked IPSCs in rat VTA dopamine neu-
rons (cocaine, 57.8 ? 10.4% of baseline,
n ? 5, p ? 0.001, Fig. 2A) (quinpirole,
71.0 ? 7.2% of baseline, n ? 9, p ? 0.01,
Fig. 2B). However, preincubation (?1 h)
slices with CB1receptor antagonist AM
251 (2 ?M) did not significantly affect the
depression of IPSCs produced by quinpi-
role and cocaine (cocaine, 64.9 ? 7.7% of
controls) (Fig. 2A,B).
Cocaine is a local anesthetic (Grzy-
bowski, 2008) that may depress IPSCs by
blocking presynaptic Na?conductance
and action potential (AP) firing. Cocaine
tential firing by activating D2receptor-
mediated increase in K?conductance,
and this effect was completely reversed by
D2receptor antagonist sulpiride (Lacey et
al., 1990). Consistent with these findings,
we found that in the continuous presence
of sulpiride (1 ?M), bath application of 10
frequency of spontaneous action potential
firing in VTA dopamine neurons (base-
line, 1.8 ? 0.2 Hz; cocaine 1.9 ? 0.2 Hz; n ? 4, p ? 0.05). It has
EPSCs in the striatum, whereas it produces a dose-dependent
that, at the concentration used in the present study, cocaine-
induced depression of IPSCs is not mediated by suppression of
presynaptic action potential firing.
We next determined whether CB1 knock-out affects
plication of quinpirole (10 ?M) produced a similar degree of
IPSC depression in VTA dopamine neurons in midbrain slices
prepared from CB1?/?and CB1?/?mice (CB1?/?, 77.3 ? 5.8%
n ? 5 from three mice; p ? 0.05; Fig. 2C). Thus, neither CB1
cocaine produced similar depression of IPSCs (n ? 5). B, AM 251 had no significant effect on D2receptor agonist quinpirole-
Panetal.•D2ReceptorActivationInducesI-LTDviacAMP/PKA J.Neurosci.,December24,2008 • 28(52):14018–14030 • 14021
blockade nor genetic deletion affected the D2-receptor-mediated
acute depression of IPSCs in dopamine neurons.
D2autoreceptors on the somatodendritic (postsynaptic) re-
gions of midbrain dopamine neurons are associated with
G-protein-coupled, inwardly rectifying K?channels (GIRKs)
whose activation leads to an outward K?current or membrane
hyperpolarization (Lacey et al., 1987; Centonze et al., 2002b;
Beckstead et al., 2004). Indeed, we found that cocaine- and
quinpirole-induced depression of IPSCs was accompanied by an
outward shift of the baseline holding current (10 ?M cocaine,
56.7 ? 11.6 pA, n ? 5; 10 ?M quinpirole, 45.9 ? 7.4 pA, n ? 9),
presumably via the activation of GIRKs. To investigate whether
the change in the hold current contributes to cocaine- and
quinpirole-induced depression of IPSCs, we used two indepen-
dent approaches to block D2-receptor-mediated activation of
First, GDP-?-S (2 mM), an irreversible G-protein inhibitor
shown to block GIRKs (Tamae et al., 2005), was loaded into
dopamine neurons via whole-cell patch pipettes. In the presence
of GDP-?-S, quinpirole (10 ?M) induced significant depression
0.05 vs control, i.e., in the absence of GDP-?-S) (Fig. 2D). There
was no significant shift of the holding current (6.4 ? 9.9 pA, p ?
0.05), indicating that postsynaptic GIRKs were blocked by GDP-
?-S. Second, whole-cell recordings were made using Cs?-based
tion, quinpirole (10 ?M) produced significant depression of IP-
vs control group with K?-based internal solution) (Fig. 2D), al-
though it did not significantly shift the holding current (5.7 ? 11.6
pA, p ? 0.05). Together, these results suggest that D2-receptor-
mediated activation of postsynaptic GIRKs does not contribute to
Quinpirole can depress IPSCs by decreasing presynaptic
the site at which quinpirole depressed IPSCs, we examined the
effect of quinpirole on miniature spontaneous IPSCs (mIPSCs)
recorded in the presence of TTX to block action potential firing.
A change in mIPSC frequency indicates a presynaptic mecha-
postsynaptic responsiveness (Van der Kloot, 1991). We found
in the mean frequency of mIPSCs (control, 1.2 ? 0.2 Hz, quin-
the amplitude of mIPSCs, as shown in cumulative frequency
plots of amplitude distribution (Fig. 2G) (Van der Kloot, 1991).
IPSCs by activating presynaptic D2receptors.
We have shown previously that I-LTD was attenuated by D2
and D3receptor antagonists, but not by a D4receptor antagonist
IPSCs by a presynaptic mechanism (Fig. 2E–G). However, there
is no direct anatomical evidence for the presence of D2subtype
receptors on the inhibitory axonal terminals that innervate VTA
dopamine neurons. Two isoforms of the D2receptor, D2long
(D2L) and D2short (D2S), are generated from the same gene by
alternative splicing (Picetti et al., 1997). In the midbrain (VTA
and substantia nigra), the immunoreactivity of the D2S is more
prevalent, whereas D2L expression is sparse (Khan et al., 1998).
Both electrophysiological and anatomical studies indicate that
the D2S isoform is the somatodendritic D2autoreceptor whose
activation hyperpolarizes dopamine neurons (Khan et al., 1998;
Centonze et al., 2002b). The D2L is strongly expressed on me-
dium spiny neurons (MSNs) in the nucleus accumbens (Khan et
innervate VTA dopamine neurons and provide the major source
of GABAergic inhibition to these neurons (Kalivas et al., 1993;
Sano and Yokoi, 2007). We speculate that the D2L is the presyn-
aptic D2subtype receptor that mediates the depression of IPSCs
in the VTA.
D2S and D2L have very similar pharmacological profiles, but
et al., 1993). This finding, together with the finding that D2S is
more prevalently expressed in the midbrain (Khan et al., 1998),
could explain why a higher concentration of quinpirole is re-
quired for depressing IPSCs (the present study), whereas a lower
pamine neurons (Centonze et al., 2002b).
It has been shown that lesions of the nucleus accumbens in-
duce large decreases in D3receptor binding in the VTA, suggest-
ing that D3receptors are distributed on the axonal terminals of
the MSNs of the nucleus accumbens that impinge on VTA neu-
rons (Diaz et al., 2000). Together, the above findings provide a
putative anatomical basis for D2/D3-receptor-mediated presyn-
aptic depression of IPSCs in VTA dopamine neurons.
Thus far, we have shown that cocaine- and quinpirole-induced
depression of IPSCs is not affected by the disruption of eCB sig-
naling. These findings suggest that increasing eCB release and
CB1receptor activation is not responsible for D2-receptor-
induced facilitation of I-LTD induction. However, an important
caveat needs to be considered. It has been shown that eCB/CB1-
mediated synaptic depression is activity-dependent, and coinci-
dent presynaptic activity exerts a powerful influence on the de-
pression (Fo ¨ldy et al., 2006; Singla et al., 2007; Heifets et al.,
2008). It is possible that D2receptor activation induces eCB re-
lease when it occurs simultaneously with repetitive presynaptic
activity. We therefore investigated whether the combination of
repetitive synaptic stimulation (10 Hz, 5 min) with cocaine or
Our previous study indicates that repetitive synaptic stimula-
tion activates group I mGluRs, and the activation of these recep-
tors could increase the release of 2-AG to activate CB1receptors
(Pan et al., 2008). We therefore used group I mGluR antagonists
CPCCOEt (50 ?M) and MPEP (10 ?M) throughout this set of
experiments to block mGluR-induced eCB release. We found
that the combination of 10 Hz stimulation with application of
cocaine (10 ?M) or quinpirole (10 ?M) induced I-LTD (cocaine,
of baseline, n ? 6; Fig. 3B; p ? 0.01). This I-LTD was not signif-
icantly affected by CB1receptor antagonist AM 251 (2 ?M) (co-
baseline, n ? 6; p ? 0.05 vs corresponding controls; Fig. 3A,B),
but was blocked by D2receptor antagonist sulpiride (10 ?M)
of baseline, n ? 5; p ? 0.01 vs corresponding controls) (Fig.
We next determined whether CB1knock-out affected the
ulation with application of cocaine (10 ?M) induced I-LTD in
VTA dopamine neurons in slices prepared from CB1-deficient
(CB1?/?) and wild-type (CB1?/?) mice. Although the magni-
14022 • J.Neurosci.,December24,2008 • 28(52):14018–14030 Panetal.•D2ReceptorActivationInducesI-LTDviacAMP/PKA
tude of I-LTD from CB1-deficient mice appeared somewhat less
than that of I-LTD from wild-type mice, the difference was not
statistically significant (CB1?/?, 63.5 ? 9.7% of baseline, n ? 5
We have shown that bath application of higher concentration
depression of IPSCs (Fig. 2A,B). Although the 10 Hz stimulation
alone has no significant effect on IPSCs, the combination of 10 Hz
stimulation with application of cocaine or
quinpirole 10 ?M induces long-lasting de-
pression of IPSCs (I-LTD) (Fig. 3A,B). But
Repetitive presynaptic stimulation in-
duces Ca2?entry into presynaptic axonal
synaptic Ca2?level is critical for the in-
at hippocampal mossy fiber synapses
mediated I-LTD at hippocampal CA1
(Heifets et al., 2008). We suspect that sim-
ilar mechanisms underlie the requirement
of the repetitive presynaptic stimulation
I-LTD in dopamine neurons. To test this
possibility, we used the membrane-
permeable Ca2?chelator EGTA-AM to
Ca2?increase. Previous studies have
shown that this approach reduces, but
does not block presynaptic transmitter re-
lease (Castillo et al., 1996; Tzounopoulos
plication of EGTA-AM (100 ?M) for 30
min produced a gradual decrease in the
amplitude of IPSCs in rat dopamine neu-
rons (61.5 ? 10.0% of baseline at 20–30
min, n ? 3, p ? 0.001) (supplemental Fig.
2, available at www.jneurosci.org as sup-
EGTA-AM blocked I-LTD induction. Slices
were preincubated with 100 ?M EGTA-AM
for 30 min and then continuously perfused
with the same concentration of EGTA-AM
and group I mGluR antagonists CPCCOEt
bination of the 10 Hz stimulation with co-
depression of IPSCs rather than I-LTD
5, p ? 0.05 vs control in Fig. 3A). However,
when EGTA (10 mM) was added into intra-
cellular solution and loaded into dopamine
neurons via whole-cell recordings, I-LTD
was induced (70.8 ? 8.9% of baseline at
tic Ca2?level is required for I-LTD induction. Together, these re-
sults suggest that CB1receptor activation is not responsible for D2
receptor-induced acute depression of IPSCs and facilitation of I-
cAMP/PKA signaling mediates D2-receptor-induced depression of
IPSCs and facilitation of I-LTD induction
The above findings raise the question of how the D2receptor
activation facilitates eCB-mediated I-LTD induction in dopa-
Panetal.•D2ReceptorActivationInducesI-LTDviacAMP/PKA J.Neurosci.,December24,2008 • 28(52):14018–14030 • 14023
mine neurons. Recent studies have shown
that cAMP/PKA signaling is required for
eCB-mediated LTD in the striatum and
I-LTD in the hippocampus (Chevaleyre et
tors and CB1receptors are Gi/o-protein-
coupled receptors whose activation leads
to the inhibition of adenylyl cyclase (AC),
resulting in a decrease in cAMP/PKA ac-
tivity (Neve et al., 2004; Howlett, 2005).
We hypothesized that D2receptor activa-
tion facilitates eCB-mediated I-LTD via
the converged inhibition of cAMP/PKA
activity. To test this hypothesis, we exam-
ined whether disruption of cAMP/PKA
induced acute depression of IPSCs and fa-
cilitation of I-LTD in dopamine neurons.
It has been shown that activating
cAMP/PKA signaling enhances neuro-
transmitter release at many excitatory and
inhibitory synapses (Chavez-Noriega and
Stevens, 1994; Capogna et al., 1995; Chen
and Regehr, 1997; Kaneko and Takahashi,
2004), whereas inhibiting cAMP/PKA sig-
naling depresses neurotransmitter release
(Marty et al., 1996; Price et al., 2005). We
first determined whether disruption of
cAMP/PKA signaling blocked D2receptor
agonist quinpirole-induced depression of
tion of forskolin (10 ?M), which activates
AC to increase intracellular cAMP levels,
significantly increased the amplitude of
IPSCs (126.7 ? 5.9% of baseline, n ? 5,
p ? 0.01). In the continuous presence of
forskolin, bath application of quinpirole
(10 ?M) for 10 min had no significant ef-
fect on IPSCs (99.3 ? 7.8% of prequinpi-
role levels, n ? 5, p ? 0.05; Fig. 4A). Be-
cause cAMP can exert its action on IPSCs
independent mechanisms (Seino and Shi-
basaki, 2005), we examined whether PKA
is involved in quinpirole-induced depres-
sion of IPSCs through the use of PKA in-
hibitors. Slices were incubated (?1 h) and
continuously superfused with PKA inhib-
itor H89 (10 ?M) or PKI 14-22 (10 ?M)
(Chevaleyre et al., 2007). In the presence of the PKA inhibitors
(Fig. 4B,C), quinpirole (10 ?M) had no significant effect on the
amplitude of IPSCs (H89, 97.0 ? 3.1% of prequinpirole levels,
0.01 vs quinpirole alone shown in Fig. 2B). Finally, we investi-
gated whether presynaptic or postsynaptic cAMP/PKA signaling
impermeable PKA inhibitor, PKI 6-22 amide (1 ?M), was loaded
into dopamine neurons via whole-cell recording for at least 15
min (Chevaleyre et al., 2007). In the presence of PKI 6-22 amide,
bath application of quinpirole (10 ?M) induced significant de-
0.01) (Fig. 4D), which is not significantly different from that in
shown earlier that postsynaptic loading of GDP-?-S did not af-
fect quinpirole-induced depression of IPSCs (Fig. 2D) and that
quinpirole decreased the mean frequency, but not the amplitude
distribution of mIPSCs (Fig. 2E–G), these results suggest that
quinpirole activates presynaptic D2receptors to decrease cAMP/
PKA activity, resulting in presynaptic depression of IPSCs.
We showed in Figure 3 that in the presence of group I mGluR
(10 ?M) or cocaine (10 ?M) with the 10 Hz stimulation induced
I-LTD that was not affected by the CB1receptor antagonist AM
251. To determine whether cAMP/PKA signaling mediates this
I-LTD, we examined the effects of PKA inhibitor H89 on I-LTD
induction in dopamine neurons. We found that H89 blocked
I-LTD induced in the presence of either cocaine (93.8 ? 7.8% of
A, Bath application of AC activator forskolin (FSK, 10 ?M) increased the amplitude of IPSCs and blocked quinpirole-induced
(n ? 5). C, Bath application of PKA inhibitor PKI 14-22 (10 ?M) blocked quinpirole-induced depression of IPSCs (n ? 6). D,
14024 • J.Neurosci.,December24,2008 • 28(52):14018–14030Panetal.•D2ReceptorActivationInducesI-LTDviacAMP/PKA
absence of H89; Fig. 4E) or quinpirole (92.7 ? 8.3% of baseline,
of H89; Fig. 4F). Together, the above results indicate that D2
receptor activation facilitates I-LTD induction via direct inhibi-
tion of cAMP/PKA activity.
CB1-receptor-mediated acute depression of IPSCs and I-LTD
depend on cAMP/PKA signaling
We hypothesized that D2receptors and CB1receptors target the
same downstream effector cAMP/PKA signaling pathway to in-
duce I-LTD in dopamine neurons. Having shown that D2recep-
investigated whether eCB-mediated I-LTD in VTA dopamine
neurons is mediated by the same downstream signaling mecha-
nism. It has been shown that CB1receptor agonist (R)-(?)-[2,3-
1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55,212-2)
depresses EPSCs in the striatum and IPSCs in the hippocampus
by inhibiting cAMP/PKA signaling (Huang et al., 2002; Cheva-
leyre et al., 2007). We first examined whether CB1receptor ago-
nist WIN 55,212-2 depressed IPSCs via cAMP/PKA signaling in
dopamine neurons. Consistent with previous studies (Szabo et
al., 2002; Pan et al., 2008), we found that bath application of CB1
receptor agonist WIN 55,212-2 (2 ?M) produced a gradual de-
pression of IPSCs (72.3 ? 8.9% of baseline, n ? 5, p ? 0.01),
which was reversed by subsequent addition of the CB1receptor
antagonist AM 251 (4 ?M, Fig. 5A). Bath application of the AC
activator forskolin (10 ?M) increased the amplitude of evoked
IPSCs (132.3 ? 7.1%, n ? 5, p ? 0.01) and prevented WIN
55,212-2 levels, n ? 5, p ? 0.05) (Fig. 5B). In the continuous
presence of PKA inhibitor H89 (10 ?M), WIN55,212-2 had no
significant effect on evoked IPSCs (94.9 ? 8.8% of baseline con-
trol, n ? 5, p ? 0.05) (Fig. 5C). These results suggest that CB1
receptor agonist WIN 55,212-2 depresses IPSCs in dopamine
neurons by inhibiting cAMP/PKA activity.
We have shown that the combination of cocaine (3 ?M) or
quinpirole (1 ?M) with the 10 Hz stimulation induced eCB-
Panetal.•D2ReceptorActivationInducesI-LTDviacAMP/PKA J.Neurosci.,December24,2008 • 28(52):14018–14030 • 14025
mediated I-LTD induction (Fig. 1). We
first examined whether I-LTD induced in
the presence of cocaine and the 10 Hz
stimulation was blocked by disrupting
cAMP/PKA signaling. As shown before,
the combination of cocaine (3 ?M) with
the 10 Hz stimulation induced I-LTD in
dopamine neurons (68.7 ? 5.5% of base-
of IPSCs (140.8 ? 8.2%, n ? 5, p ? 0.01)
and prevented I-LTD induction (95.3 ?
6.5% of pre-10 Hz stimulation levels, n ?
5, p ? 0.05; Fig. 5E). This I-LTD was
blocked by PKA inhibitor H89 (10 ?M,
97.5 ? 7.9% of baseline, n ? 5, p ? 0.05;
Fig. 5F). We next examined whether
I-LTD induced in the presence of quinpi-
role and the 10 Hz stimulation was
blocked by disrupting cAMP/PKA signal-
(70.4 ? 7.8% of baseline, n ? 9, p ? 0.01)
(Fig. 5G). Forskolin (10 ?M) increased the
amplitude of IPSCs (137.0 ? 6.2%, n ? 5,
p ? 0.01) and prevented I-LTD induction
els, n ? 5, p ? 0.05; Fig. 5H). This I-LTD
?M, 94.3 ? 7.2% of baseline, n ? 5, p ?
0.05; Fig. 5I). These results indicate that
eCB-mediated I-LTD in the VTA also re-
quires cAMP/PKA signaling. Together,
these data support the hypothesis that D2
mediated I-LTD through converged inhi-
bition of cAMP/PKA signaling.
We have shown that D2receptors cooper-
ate with group I mGluRs to induce I-LTD
in dopamine neurons and this I-LTD was
blocked by CB1receptor antagonist AM
251 (Fig. 1). Because D2receptor activa-
tion does not induce eCB release in dopa-
mine neurons (Fig. 2), it is very likely that
group I mGluR activation induces eCB re-
ical manipulations to enhance group I
mGluR activation and examined whether
group I mGluR-induced synaptic depres-
sion could be blocked by disrupting eCB
signaling. Group I mGluR activation was
enhanced by glutamate uptake inhibitor
(TBOA). We found that bath application
of TBOA (40 ?M) had no significant effect
on the amplitude of evoked IPSCs (87.5 ? 7.5% of baseline, n ?
6, p ? 0.05) (Fig. 6A), suggesting that glutamate accumulation
during low-frequency synaptic stimulation does not induce sig-
nificant synaptic depression. Although the 10 Hz stimulation
alone did not have a significant effect on IPSCs (95.5 ? 6.1% of
with the 10 Hz stimulation resulted in I-LTD induction (68.2 ?
7.4% of baseline, n ? 7, p ? 0.01) (Fig. 6B). The TBOA-enabled
6) blocked I-LTD induction. F, Bath application of PKA inhibitor H89 (10 ?M) blocked I-LTD induction (n ? 7). G, H, Bath
14026 • J.Neurosci.,December24,2008 • 28(52):14018–14030Panetal.•D2ReceptorActivationInducesI-LTDviacAMP/PKA
?M) and MPEP (10 ?M) (92.1 ? 8.4% of baseline, n ? 6, p ?
0.05) (Fig. 6C). Thus, when glutamate uptake is compromised,
repetitive synaptic stimulation at 10 Hz induces significant in-
to induce I-LTD. We also examined the effect of D2receptor
sulpiride (10 ?M) had no significant effect on I-LTD induction
(68.9 ? 8.9% of baseline, n ? 7; p ? 0.05 vs I-LTD in Fig. 6B).
Group 1 mGluR activation induces the release of 2-AG, an
eCB, in hippocampal slices (Jung et al., 2005). To test whether
this I-LTD induction. In slices were preincubated (?1 h) and
continuously superfused with CB1receptor AM 251 (2 ?M),
7, p ? 0.05 vs I-LTD control in Fig. 6B; see Fig. 6E). We next
examined whether disruption of 2-AG synthesis blocked I-LTD
induction. Group 1 mGluRs are positively coupled to phospho-
lipase C, which cleaves phosphatidylinositol 1,4,5-bisphosphate
into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate, and
DAG is subsequently converted into 2-AG by DAG lipase (Di
Marzo et al., 1998; Piomelli, 2003). The DAG lipase inhibitor
tetrahydrolipstatin (THL, 10 ?M) was loaded into dopamine
neurons via whole-cell recordings and its effect on I-LTD was
examined. In the presence of THL, the combination of TBOA
6.9% of baseline, n ? 6, p ? 0.05 vs I-LTD control in Fig. 6B; see
Fig. 6E). These results suggest that synaptic activation of group I
mGluRs induces the release of 2-AG, which subsequently acti-
vates CB1receptors to induce I-LTD.
Because CB1receptor agonist WIN 55,212-2 induces synaptic
depression via the inhibition of cAMP/PKA activity (Fig. 5), we
examined whether PKA inhibitor H89 blocked group I mGluR-
mediated I-LTD. As shown in Figure 6F, H89 (10 ?M) blocked
n ? 7, p ? 0.05 vs I-LTD control in Fig.
group I mGluRs initiates a cascade of
events to induce I-LTD, which include the
release of 2-AG, activation of CB1recep-
tors and inhibition of cAMP/PKA activity.
Thus far we have used a number of an-
and group I mGluRs converge on cAMP/
dopamine neurons. In addition to the
pharmacological blockade, occlusion is
another powerful means to demonstrate
specific signaling pathway. We have
shown previously that I-LTD induced by
the combination of 10 Hz stimulation and
I mGluR agonist DHPG (Pan et al., 2008).
In the present study, we determined
whether I-LTD induced by the combina-
tion of 10 Hz stimulation and TBOA ap-
pirole. Bath application of cocaine or
quinpirole (10 ?M) induced rapid depression of IPSCs [cocaine,
50.7 ? 4.3% of baseline at 20–25 min, n ? 5, p ? 0.001 vs
baseline, (Fig. 6G); quinpirole, 68.3 ? 4.4% of baseline at 20–25
min, n ? 6, p ? 0.001 vs baseline (Fig. 6H)]. In the continuous
presence of cocaine or quinpirole, the combination of 10 Hz
stimulation and TBOA application did not induce further de-
pression [cocaine, 45. 5 ? 7.1% of baseline at 40–50 min, n ? 5,
p ? 0.05 vs prestimulation level at 20–25 min (Fig. 6G); quinpi-
role, 60.8 ? 7.6% of baseline at 40–50 min, n ? 6, p ? 0.05 vs
prestimulation level at 20–25 min (Fig. 6H)]. These results pro-
verge on a common signaling pathway to induce I-LTD in dopa-
D2receptors cooperate with group I mGluRs to induce eCB-
mediated synaptic depression in the striatum and VTA (Kreitzer
and Malenka, 2005; Yin and Lovinger, 2006; Pan et al., 2008).
Based on findings that D2receptor activation induces the release
of AEA (Giuffrida et al., 1999) and group I mGluR activation
induces the release of 2-AG (Jung et al., 2005), we and others
hypothesized that coactivation of CB1receptors underlies the
eCB-mediated synaptic depression (Kreitzer and Malenka, 2005;
Yin and Lovinger, 2006; Pan et al., 2008). Although this hypoth-
esis (Fig. 7A) provides an explanation for the mechanisms re-
sponsible, experimental data supporting this hypothesis have
D2receptors cooperate with group I mGluRs to induce eCB-
I mGluR activation facilitates I-LTD induction via the enhance-
ment of eCB signaling and subsequent inhibition of cAMP/PKA
activity, whereas D2receptor activation facilitates I-LTD induc-
tion via direct inhibition of cAMP/PKA activity. Based on these
results, we propose a refined model to explain the cooperation
between group I mGluRs and D2receptors to induce eCB-
mediated synaptic depression (Fig. 7B).
release of 2-AG from dopamine neurons. These two eCBs travel across the synapses and activate presynaptic CB1receptors,
Signaling mechanisms underlying the cooperation between D2receptors and group I mGluRs to induce I-LTD in
Panetal.•D2ReceptorActivationInducesI-LTDviacAMP/PKA J.Neurosci.,December24,2008 • 28(52):14018–14030 • 14027
CB1receptors are not a downstream effector for D2
Gas chromatography/mass spectrometry studies have revealed
cocaine-induced acute depression of IPSCs was partially blocked
by a CB1receptor antagonist in the striatum (Centonze et al.,
2004). It is temping to hypothesize that D2receptor activation
facilitates I-LTD induction via eCB signaling in VTA dopamine
neurons. However, we show that the D2receptor-mediated de-
pression of IPSCs induced by bath application of quinpirole and
cocaine was not affected by CB1receptor blockade or knock-out.
It has been shown that coincident presynaptic activity exerts a
of D2receptor activation with repetitive synaptic stimulation at
10 Hz induced a form of I-LTD that was also insensitive to CB1
blockade or knock-out if group I mGluR antagonists were
present (Fig. 3). Thus, eCB signaling is not involved in D2
receptor-induced depression of IPSCs and facilitation of I-LTD
induction in dopamine neurons.
D2receptors could recruit different downstream effectors to
of AEAinvolves thehydrolysis
pholipase D (NAPE-PLD) (Okamoto et al., 2007). A potential
mechanism for D2-receptor-induced release of AEA is via the
activation of NAPE-PLD (Senogles, 2000). Neuroanatomical
studies indicate region-specific distribution of NAPE-PLD. For
ule cells in the hippocampus, but is absent in several other brain
areas(Egertova ´ etal.,2008;Nyilasetal.,2008).ThelackofNAPE-
PLD or the uncoupling between D2receptor and NAPE-PLD in
the VTA could explain why eCB signaling is not involved in D2
receptor-mediated depression of IPSCs and facilitation of I-LTD
eCB-mediated I-LTD and D2-receptor-induced facilitation of I-
LTD induction require cAMP/PKA signaling
Both D2and CB1receptors are coupled to Gi/oproteins whose
activation leads to the inhibition of cAMP/PKA activity (Neve et
I-LTD and D2receptor-induced facilitation of I-LTD induction
cAMP/PKA signaling with either the AC activator forskolin or
PKA inhibitors H89 and PKI 14-22 abrogated both D2and CB1
agonist-induced depression of IPSCs and eCB-mediated I-LTD
in dopamine neurons (Figs. 4, 5). The latter findings are consis-
tent with recent studies showing that cAMP/PKA signaling is
required for eCB-mediated I-LTD in the hippocampus and LTD
in the striatum (Chevaleyre et al., 2007; Mato et al., 2007).
Interestingly, the cAMP/PKA signaling pathway is also in-
the activation of mGluR II/III induces LTD through the inhibi-
los et al., 1998; Robbe et al., 2002; Huang et al., 2007). We find
that, when group I mGluRs were blocked, the combination of 10
tion (10 ?M) induced a form of I-LTD that was not affected by
CB1receptor antagonist or CB1knock-out, but was blocked by
ity appears to be a common signal transduction mechanism by
which eCB- and non-eCB-mediated LTD can be induced.
D2receptors cooperate with group I mGluRs to induce I-LTD
We show that group I mGluR activation facilitates I-LTD induc-
tion via the enhancement of eCB release and CB1R-mediated
inhibition of cAMP/PKA signaling represents a mechanism by
which D2receptors cooperate with group I mGluRs to induce
I-LTD. Our data support a model in which a threshold level of
cAMP/PKA activity controls I-LTD induction. When group I
mGluRs or D2receptors are modestly activated (i.e., with 3 ?M
of both receptors is required to decrease cAMP/PKA activity to
the threshold for induction of I-LTD. However, stronger activa-
tion of either group I mGluRs (i.e., with TBOA, Fig. 6) or D2
can decrease cAMP/PKA sufficiently to induce I-LTD. Previous
studies have shown that 10 ?M cocaine produces greater D2
receptor-mediated responses than 3 ?M cocaine (Lacey et al.,
1990; Beckstead et al., 2004). Therefore, it is likely that 10 ?M
cocaine produces a significantly greater decrease in cAMP/PKA
10 ?M cocaine or quinpirole alone without the participation of
group I mGluRs and CB1receptors. Nevertheless, the coopera-
tion between D2receptors and group I mGluRs allows I-LTD
cocaine exposure (Pan et al., 2008).
In addition to a decrease in cAMP/PKA level, repetitive pre-
synaptic activity is also required for I-LTD induction. We find
permeable Ca2?chelator EGTA-AM, but was not affected by
EGTA loading into postsynaptic neurons (Fig. 3). Thus, activity-
induction in dopamine neurons. Such changes in presynaptic
Ca2?levels are also required for LTD and I-LTD in the hip-
pocampus (Tzounopoulos et al., 1998; Heifets et al., 2008). Im-
get that is activated by presynaptic Ca2?increase during I-LTD
induction (Heifets et al., 2008). Similar mechanisms may under-
lie the requirement of the change of presynaptic Ca2?levels for
D2receptor-mediated I-LTD induction in VTA dopamine
It remains unclear whether the mechanism discovered here
underlies the cooperation between D2receptors and group I
mGluRs to induce eCB-mediated frequency-specific depression
and LTD of EPSCs in the striatum (Kreitzer and Malenka, 2005;
Yin and Lovinger, 2006). D2receptor and group I mGluR ago-
et al., 1999; Jung et al., 2005). However, there is evidence that
AEA and 2-AG do not always act cooperatively to enhance eCB-
mediated synaptic depression. AEA is also an endogenous ligand
for transient receptor potential vanilloid 1 (TRPV1) channels
(Starowicz et al., 2007). A recent study has shown that the group
I mGluR agonist DHPG depressed IPSCs in the striatum via the
application of AEA or a stable AEA analog attenuated 2-AG pro-
duction and DHPG-induced depression of IPSCs via the activa-
tion of TRPV1 (Maccarrone et al., 2008). Whether these two
eCBs also have similar antagonistic effects on EPSCs in the stria-
possibility that the converged inhibition of cAMP/PKA signaling
also participates in the cooperation between D2receptors and
14028 • J.Neurosci.,December24,2008 • 28(52):14018–14030 Panetal.•D2ReceptorActivationInducesI-LTDviacAMP/PKA
group I mGluRs to induce eCB-mediated depression in the
In summary, the present study reveals a novel mechanism by
which D2receptor activation facilitates eCB-mediated I-LTD in
that D2receptor activation facilitates I-LTD by enhancing eCB
release and subsequent CB1receptor activation, our study has
shown that D2receptors and CB1receptors inhibit the same
downstream effector adenylyl cyclase, and this combined inhibi-
leading to I-LTD induction.
Alger BE (2005) Endocannabinoid identification in the brain: studies of
breakdown lead to breakthrough, and there may be NO hope. Sci STKE
Batchelor AM, Garthwaite J (1997) Frequency detection and temporally
dispersed synaptic signal association through a metabotropic glutamate
receptor pathway. Nature 385:74–77.
Beckstead MJ, Grandy DK, Wickman K, Williams JT (2004) Vesicular do-
pamine release elicits an inhibitory postsynaptic current in midbrain do-
pamine neurons. Neuron 42:939–946.
Cameron DL, Williams JT (1993) Dopamine D1 receptors facilitate trans-
mitter release. Nature 366:344–347.
Capogna M, Ga ¨hwiler BH, Thompson SM (1995) Presynaptic enhance-
rat hippocampus in vitro. J Neurosci 15:1249–1260.
Castillo PE, Salin PA, Weisskopf MG, Nicoll RA (1996) Characterizing the
site and mode of action of dynorphin at hippocampal mossy fiber syn-
apses in the guinea pig. J Neurosci 16:5942–5950.
Centonze D, Picconi B, Baunez C, Borrelli E, Pisani A, Bernardi G, Calabresi
P (2002a) CocaineandamphetaminedepressstriatalGABAergicsynap-
tic transmission through D2 dopamine receptors. Neuropsychopharma-
P (2002b) DopamineD2receptor-mediatedinhibitionofdopaminergic
neurons in mice lacking D2L receptors. Neuropsychopharmacology
Centonze D, Battista N, Rossi S, Mercuri NB, Finazzi-Agro ` A, Bernardi G,
Calabresi P, Maccarrone M (2004) A critical interaction between dopa-
mine D2 receptors and endocannabinoids mediates the effects of cocaine
on striatal gabaergic Transmission. Neuropsychopharmacology 29:
Chavez-Noriega LE, Stevens CF (1994) Increased transmitter release at ex-
citatory synapses produced by direct activation of adenylate cyclase in rat
hippocampal slices. J Neurosci 14:310–317.
Chen C, Regehr WG (1997) The mechanism of cAMP-mediated enhance-
ment at a cerebellar synapse. J Neurosci 17:8687–8694.
Chevaleyre V, Takahashi KA, Castillo PE (2006) Endocannabinoid-
mediated synaptic plasticity in the CNS. Annu Rev Neurosci 29:37–76.
Chevaleyre V, Heifets BD, Kaeser PS, Su ¨dhof TC, Purpura DP, Castillo PE
PKA signaling and RIM1alpha. Neuron 54:801–812.
Conn PJ, Pin JP (1997) Pharmacology and functions of metabotropic glu-
C,LeFollB,GrosC,TrillerA,SchwartzJC,SokoloffP (2000) Dopamine
D3 receptors expressed by all mesencephalic dopamine neurons. J Neu-
Di Marzo V, Fontana A, Cadas H, Schinelli S, Cimino G, Schwartz JC, Pio-
melli D (1994) Formation and inactivation of endogenous cannabinoid
anandamide in central neurons. Nature 372:686–691.
Di Marzo V, Melck D, Bisogno T, De Petrocellis L (1998) Endocannabi-
noids: endogenous cannabinoid receptor ligands with neuromodulatory
action. Trends Neurosci 21:521–528.
Egertova ´ M, Simon GM, Cravatt BF, Elphick MR (2008) Localization of
N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) ex-
pression in mouse brain: A new perspective on N-acylethanolamines as
neural signaling molecules. J Comp Neurol 506:604–615.
Fo ¨ldyC,NeuA,JonesMV,SolteszI (2006) Presynaptic,activity-dependent
release. J Neurosci 26:1465–1469.
Gerdeman GL, Lovinger DM (2003) Emerging roles for endocannabinoids
in long-term synaptic plasticity. Br J Pharmacol 140:781–789.
Giuffrida A, Parsons LH, Kerr TM, Rodríguez de Fonseca F, Navarro M,
Piomelli D (1999) Dopamine activation of endogenous cannabinoid
signaling in dorsal striatum. Nat Neurosci 2:358–363.
Grzybowski A (2008) Cocaine and the eye: a historical overview. Ophthal-
Heifets BD, Chevaleyre V, Castillo PE (2008) Interneuron activity controls
endocannabinoid-mediated presynaptic plasticity through calcineurin.
Proc Natl Acad Sci U S A 105:10250–10255.
HowlettAC (2005) Cannabinoidreceptorsignaling.HandbExpPharmacol
Huang CC, Chen YL, Lo SW, Hsu KS (2002) Activation of cAMP-
tion of glutamatergic transmission at corticostriatal synapses. Mol Phar-
Huang CC, Yang PC, Lin HJ, Hsu KS (2007) Repeated cocaine administra-
tion impairs group II metabotropic glutamate receptor-mediated long-
TW, Lai J, Porreca F, Makriyannis A, Malan TP Jr (2003) Activation of
CB2 cannabinoid receptors by AM1241 inhibits experimental neuro-
pathic pain: pain inhibition by receptors not present in the CNS. Proc
Natl Acad Sci U S A 100:10529–10533.
Johnson SW, North RA (1992) Two types of neurone in the rat ventral
tegmental area and their synaptic inputs. J Physiol 450:455–468.
JonesS,KauerJA (1999) Amphetaminedepressesexcitatorysynaptictrans-
mission via serotonin receptors in the ventral tegmental area. J Neurosci
Jung KM, Mangieri R, Stapleton C, Kim J, Fegley D, Wallace M, Mackie K,
Piomelli D (2005) Stimulation of endocannabinoid formation in brain
slice cultures through activation of group I metabotropic glutamate re-
ceptors. Mol Pharmacol 68:1196–1202.
Kalivas PW, Churchill L, Klitenick MA (1993) GABA and enkephalin pro-
jection from the nucleus accumbens and ventral pallidum to the ventral
tegmental area. Neuroscience 57:1047–1060.
Kane JK, Hwang Y, Konu O, Loughlin SE, Leslie FM, Li MD (2005) Regu-
lation of Homer and group I metabotropic glutamate receptors by nico-
tine. Eur J Neurosci 21:1145–1154.
Kaneko M, Takahashi T (2004) Presynaptic mechanism underlying cAMP-
dependent synaptic potentiation. J Neurosci 24:5202–5208.
Khan ZU, Mrzljak L, Gutierrez A, de la Calle A, Goldman-Rakic PS (1998)
Prominence of the dopamine D2 short isoform in dopaminergic path-
ways. Proc Natl Acad Sci U S A 95:7731–7736.
KreitzerAC,MalenkaRC (2005) Dopaminemodulationofstate-dependent
endocannabinoid release and long-term depression in the striatum.
J Neurosci 25:10537–10545.
Lacey MG, Mercuri NB, North RA (1987) Dopamine acts on D2 receptors
zona compacta. J Physiol 392:397–416.
Lacey MG, Mercuri NB, North RA (1990) Actions of cocaine on rat dopa-
minergic neurones in vitro. Br J Pharmacol 99:731–735.
Le Foll B, Goldberg SR, Sokoloff P (2005) The dopamine D3 receptor and
drug dependence: effects on reward or beyond? Neuropharmacology
Liu QS, Pu L, Poo MM (2005) Repeated cocaine exposure in vivo facilitates
LTP induction in midbrain dopamine neurons. Nature 437:1027–1031.
Maccarrone M, Rossi S, Bari M, De Chiara V, Fezza F, Musella A, Gasperi V,
Prosperetti C, Bernardi G, Finazzi-Agro ` A, Cravatt BF, Centonze D
(2008) Anandamide inhibits metabolism and physiological actions of
2-arachidonoylglycerol in the striatum. Nat Neurosci 11:152–159.
Maejima T, Hashimoto K, Yoshida T, Aiba A, Kano M (2001) Presynaptic
inhibition caused by retrograde signal from metabotropic glutamate to
cannabinoid receptors. Neuron 31:463–475.
MargolisEB,LockH,HjelmstadGO,FieldsHL (2006) Theventraltegmen-
tal area revisited: is there an electrophysiological marker for dopaminer-
gic neurons? J Physiol 577:907–924.
Marty A, Glitsch M, Kondo S, Llano I (1996) Cyclic AMP-regulated GABA
release at inhibitory synapses in rat cerebellar slices. J Physiol Paris
Mato S, Lafourcade M, Robbe D, Bakiri Y, Manzoni OJ (2008) Role of the
cyclic-AMP/PKA cascade andof P/Q-type Ca2?
Panetal.•D2ReceptorActivationInducesI-LTDviacAMP/PKA J.Neurosci.,December24,2008 • 28(52):14018–14030 • 14029
endocannabinoid-mediated long-term depression in the nucleus accum-
bens. Neuropharmacology 54:87–94.
Montmayeur JP, Guiramand J, Borrelli E (1993) Preferential coupling be-
tween dopamine D2 receptors and G-proteins. Mol Endocrinol
Neve KA, Seamans JK, Trantham-Davidson H (2004) Dopamine receptor
signaling. J Recept Signal Transduct Res 24:165–205.
Nyilas R, Dudok B, Urba ´n GM, Mackie K, Watanabe M, Cravatt BF, Freund
TF,KatonaI (2008) Enzymaticmachineryforendocannabinoidbiosyn-
thesis associated with calcium stores in glutamatergic axon terminals.
J Neurosci 28:1058–1063.
Okamoto Y, Wang J, Morishita J, Ueda N (2007) Biosynthetic pathways of
the endocannabinoid anandamide. Chem Biodivers 4:1842–1857.
Pan B, Hillard CJ, Liu QS (2008) Endocannabinoid signaling mediates
cocaine-induced inhibitory synaptic plasticity in midbrain dopamine
neurons. J Neurosci 28:1385–1397.
Patel S, Rademacher DJ, Hillard CJ (2003) Differential regulation of the
endocannabinoids anandamide and 2-arachidonylglycerol within the
limbic forebrain by dopamine receptor activity. J Pharmacol Exp Ther
Picetti R, Saiardi A, Abdel Samad T, Bozzi Y, Baik JH, Borrelli E (1997)
Dopamine D2 receptors in signal transduction and behavior. Crit Rev
Pillai G, Brown NA, McAllister G, Milligan G, Seabrook GR (1998) Human
D2 and D4 dopamine receptors couple through betagamma G-protein
expression system: selective antagonism by L-741,626 and L-745,870 re-
spectively. Neuropharmacology 37:983–987.
Piomelli D (2003) The molecular logic of endocannabinoid signalling. Nat
Rev Neurosci 4:873–884.
Pollack A (2004) Coactivation of D1 and D2 dopamine receptors: in mar-
riage, a case of his, hers, and theirs. Sci STKE 2004:pe50.
Price CJ, Karayannis T, Pa ´l BZ, Capogna M (2005) Group II and III
mGluRs-mediated presynaptic inhibition of EPSCs recorded from hip-
pocampal interneurons of CA1 stratum lacunosum moleculare. Neuro-
pharmacology 49 [Suppl 1]:45–56.
Robbe D, Bockaert J, Manzoni OJ (2002) Metabotropic glutamate receptor
in morphine withdrawn mice. Eur J Neurosci 16:2231–2235.
Rosenzweig-Lipson S, Barrett JE (1995) K-channel blockers attenuate the
col Biochem Behav 51:843–848.
Sano H, Yokoi M (2007) Striatal medium spiny neurons terminate in a dis-
orexin/hypocretin- or melanin-concentrating hormone-containing neu-
rons. J Neurosci 27:6948–6955.
Seeman P, Van Tol HH (1994) Dopamine receptor pharmacology. Trends
Pharmacol Sci 15:264–270.
Seino S, Shibasaki T (2005) PKA-dependent and PKA-independent path-
ways for cAMP-regulated exocytosis. Physiol Rev 85:1303–1342.
SenoglesSE (2000) TheD2sdopaminereceptorstimulatesphospholipaseD
activity: a novel signaling pathway for dopamine. Mol Pharmacol
SinglaS,KreitzerAC,MalenkaRC (2007) Mechanismsforsynapsespecific-
ity during striatal long-term depression. J Neurosci 27:5260–5264.
Starowicz K, Nigam S, Di Marzo V (2007) Biochemistry and pharmacology
of endovanilloids. Pharmacol Ther 114:13–33.
Stella N, Schweitzer P, Piomelli D (1997) A second endogenous cannabi-
noid that modulates long-term potentiation. Nature 388:773–778.
Szabo B, Siemes S, Wallmichrath I (2002) Inhibition of GABAergic neuro-
(2005) Direct inhibition of substantia gelatinosa neurones in the rat spi-
nal cord by activation of dopamine D2-like receptors. J Physiol (Lond)
Tzounopoulos T, Janz R, Su ¨dhof TC, Nicoll RA, Malenka RC (1998) A role
for cAMP in long-term depression at hippocampal mossy fiber synapses.
Van der Kloot W (1991) The regulation of quantal size. Prog Neurobiol
XiZX,GardnerEL (2007) PharmacologicalactionsofNGB2904,aselective
dopamine D3 receptor antagonist, in animal models of drug addiction.
CNS Drug Rev 13:240–259.
Yin HH, Lovinger DM (2006) Frequency-specific and D2 receptor-
mediated inhibition of glutamate release by retrograde endocannabinoid
signaling. Proc Natl Acad Sci U S A 103:8251–8256.
Zapata A, Shippenberg TS (2002) D(3) receptor ligands modulate extracel-
lular dopamine clearance in the nucleus accumbens. J Neurochem 81:
14030 • J.Neurosci.,December24,2008 • 28(52):14018–14030Panetal.•D2ReceptorActivationInducesI-LTDviacAMP/PKA