Both Kappa and Mu Opioid Agonists Inhibit Glutamatergic Input to Ventral
Tegmental Area Neurons
Elyssa B. Margolis1, Gregory O. Hjelmstad1,2, Antonello Bonci1,2, & Howard L. Fields1,2
1. Ernest Gallo Clinic & Research Center, University of California, San Francisco, Emeryville,
California 94608 USA
2. Dept. of Neurology and Wheeler Center for the Neurobiology of Addiction, University of
California, San Francisco, California 94143-0114.
Running head: κ and µ opioid inhibition of EPSCs in the VTA
Address correspondence to:
Elyssa B. Margolis
Ernest Gallo Clinic and Research Center
5858 Horton Street, Suite #200
Emeryville, CA 94608
Tel: (510) 985-3925
Fax: (510) 985-3101
Articles in PresS. J Neurophysiol (December 22, 2004). doi:10.1152/jn.00855.2004
Copyright © 2004 by the American Physiological Society.
The ventral tegmental area (VTA) plays a critical role in motivation and reinforcement.
Kappa and mu opioid receptor (KOP-R and MOP-R) agonists microinjected into the VTA
produce powerful and largely opposing motivational actions. Glutamate transmission within the
VTA contributes to these motivational effects. Therefore, information about opioid control of
glutamate release onto VTA neurons is important. To address this issue we performed whole cell
patch-clamp recordings in VTA slices and measured excitatory postsynaptic currents (EPSCs).
There are several classes of neuron in the VTA: principal, secondary, and tertiary. The KOP-
R agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide
methane-sulfonate hydrate (U69593; 1 µM) produced a small reduction in EPSC amplitude in
principal neurons (14%), and a significantly larger inhibition in secondary (47%) and tertiary
(33%) neurons. The MOP-R agonist [D-Ala2, N-Me-Phe4, Gly-ol5]-Enkephalin (DAMGO; 3
µM) inhibited glutamate release in principal (42%), secondary (45%) and tertiary neurons (35%).
Unlike principal and tertiary neurons, in secondary neurons the magnitude of the U69593 EPSC
inhibition was positively correlated with that produced by DAMGO. Finally, DAMGO did not
occlude the U69593 effect in principal neurons, suggesting that some glutamatergic terminals are
independently controlled by KOP and MOP receptor activation. These findings demonstrate that
MOP- and KOP-R agonists regulate excitatory input onto each VTA cell type.
Keywords: EPSC, dopamine, midbrain
The ventral tegmental area (VTA) contributes to the motivational actions of natural rewards
and a variety of drugs including opioid agonists. Both κ opioid receptors (KOP-R) (Arvidsson et
al. 1995; Mansour et al. 1996; 1993) and µ opioid receptors (MOP-R) (Garzon and Pickel 2001;
Svingos et al. 2001) are present in significant density in the VTA. Further, microinjections of
both KOP-R and MOP-R agonists directly into the VTA produce robust behavioral responses.
The KOP-R agonist U50488H produces conditioned place aversion (Bals-Kubik et al. 1993).
In contrast, the MOP-R agonist [D-Ala2, N-Me-Phe4, Gly-ol5]-Enkephalin (DAMGO) produces
conditioned place preference (Bals-Kubik et al. 1993; Nader and van der Kooy 1997; Phillips
and LePiane 1980).
Glutamate transmission within the VTA is required for the motivational properties of
opioids (e.g. Cornish et al. 2001; Harris and Aston-Jones 2003; Xi and Stein 2002). In the
VTA, glutamatergic inputs are derived from neurons in the medial prefrontal cortex (mPFC),
subthalamic nucleus (STN), and the pedunculopontine nucleus (PPN) (Charara et al. 1996;
Christie et al. 1985; Groenewegen and Berendse 1990; Sesack and Pickel 1992). There is also
indirect evidence that lateral hypothalamic projections to the VTA contain glutamate (Chou et
al. 2001; Rosin et al. 2003). Under physiological conditions, glutamate can induce phasic firing
in dopaminergic neurons through the activation of N-methyl- d-aspartate (NMDA) receptors
(Chergui et al. 1993; Johnson et al. 1992; Overton and Clark 1997), and this effect is facilitated
by group 1 metabotropic glutamate receptor activation (Zheng and Johnson 2002). There is also
evidence that glutamate activation of 2-amino-3(3-hydroxy-5-methyl-4-isoxazolyl) propionic
acid (AMPA) receptors in the VTA can increase extracellular dopamine (DA) in the nucleus
accumbens (NAc) (Karreman et al. 1996; Schilstrom et al. 1998).
Essential to determining how KOP and MOP receptor agonists in the VTA produce
their behavioral actions is elucidating their synaptic effects at the cellular level in all VTA
cell types. VTA neurons are classified as principal, secondary, or tertiary according to their
electrophysiological and pharmacological properties (Cameron et al. 1997). Both principal and
tertiary neurons exhibit the hyperpolarization-activated non-specific cation current (Ih) and have
relatively long duration action potentials. Secondary cells lack an Ih and have shorter duration
action potentials. They are directly hyperpolarized by MOP-R agonists and are GABA neurons.
Secondary cell inhibition by MOP-R agonists has been proposed to disinhibit principal neurons
through local circuitry (Johnson and North 1992; Margolis et al. 2003). Tertiary neurons differ
from principal cells in that they are directly hyperpolarized by MOP-R agonists and serotonin.
While most principal neurons are dopaminergic (about 80%), less than 40% of tertiary neurons
are dopaminergic. KOP-R agonists postsynaptically inhibit a subset of both principal and tertiary
neurons, an effect limited to dopaminergic neurons of each class (Margolis et al. 2003).
Despite the evidence that VTA glutamatergic transmission is critical for reward and
motivation, our understanding of presynaptic control of glutamate release by opioids is
incomplete. We therefore examined the effects of both KOP and MOP receptor agonists on
glutamate release onto each VTA cell type. We directly compared KOP and MOP effects within
and across individual neurons and neuron types. Because both KOP and MOP effects were
observed, we addressed the issue of whether KOP and MOP receptor agonists act on the same
terminals by testing whether the effects of the two agonists occluded.
Twenty to thirty six day old male Sprague-Dawley rats were anesthetized with isofluorane,
and their brains were removed. Horizontal slices (200 µm thick) containing the VTA were
prepared using a vibratome (Leica Instruments, Germany). Slices were submerged in aCSF
solution containing (in mM) 119 NaCl, 2.5 KCl, 1.3 MgSO4, 1.0 NaH2PO4, 2.5 CaCl2, 26.2
NaHCO3, and 11 glucose saturated with 95% O2-5% CO2 and allowed to recover at 32°C for at
least 1 hour.
Individual slices were visualized under a Zeiss Axioskop with differential interference
contrast optics and infrared illumination. Whole cell patch-clamp recordings were made at 31°C
using 2.5-4 MΩ pipettes containing (in mM) 123 K-gluconate, 10 HEPES, 0.2 EGTA, 8 NaCl, 2
MgATP, and 0.3 Na3GTP (pH 7.2, osmolarity adjusted to 275).
Recordings were made using an Axopatch 1-D, filtered at 2 kHz and collected at 5 kHz using
IGOR Pro (Wavemetrics, Lake Oswego, OR). Ih was recorded by voltage clamping cells and
stepping from -60 to -120 mV. Series resistance and input resistance were sampled throughout
voltage clamp experiments with 4 mV, 200 msec depolarizing steps once every 10 seconds. For
purposes of classification, every neuron was tested for postsynaptic actions of either the MOP-R
selective agonist DAMGO (3 µM) or the 5-HT1 agonist 5-carboxamidotryptamine (5-CT; 500
nM) in current clamp following the voltage clamp experiment. Cells were recorded in voltage
clamp mode (V=-70mV) while measuring EPSCs. All EPSCs were measured in the presence
of picrotoxin (100 µM). Stimulating electrodes were placed 60-150 microns rostral to the
patched cell. In neurons where paired pulses were administered, 2 pulses 50 msec apart were
delivered once every 10 seconds. The EPSC amplitude was calculated by comparing a 2 msec
period around the peak to a 2 msec interval just prior to stimulation. The paired pulse ratio was
calculated by dividing the amplitude of the second EPSC by that of the first, trial by trial, and
then averaging across trials. Spontaneous events were identified in a subset of experiments by
searching the smoothed first derivative of the data trace for values that exceeded a set threshold,
and these events were then visually confirmed. Experiments with baseline sEPSC frequencies
below 0.25 Hz were excluded from drug effect analyses because too few events were detected to
reliably measure changes in frequency.
Results are presented as means +/- SEM where appropriate. Summary comparisons were
made between the average of the 4 minutes of baseline just preceding each respective drug
application to 4 minutes of stable drug effect. The significance of drug effects was tested across
all VTA neurons and within individual cell types using the two way repeated measures ANOVA
followed by the Student-Newman-Keuls (SNK) method for multiple comparisons. Differences
in effect sizes between neuron populations were tested with one way ANOVA and the Student-
Newman-Keuls method where appropriate. The significance of effects within individual neurons
was tested with the Student’s t-test, comparing the last 4 minutes of baseline to the last 4 minutes
of drug application. Significance was defined at p < 0.05. In the case of postsynaptic drug
effects, recovery during drug washout was also required.
All drugs were applied by bath perfusion. Stock solutions were made and diluted in
Ringer immediately prior to application. (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-
cyclohexyl] benzeneacetamide methane-sulfonate hydrate (U69593) was diluted in 50% EtOH
to a concentration of 1 mM; nor-Binaltorphimine (nor-BNI; 1 mM), 5-CT (1mM), and DAMGO
(1 mM) were diluted in H2O. Picrotoxin was diluted in DMSO (100 mM). All chemicals were
obtained from Sigma Chemical (St. Louis, MO) or Tocris (Ballwin, MO).
Whole-cell voltage-clamp recordings were made from neurons in the VTA.
Pharmacologically-isolated (100 µM picrotoxin) excitatory postsynaptic currents (EPSCs)
were electrically evoked and we confirmed that this evoked current was due to AMPA receptor
activation by blocking the response with the non-NMDA glutamate receptor antagonist 6,7-
dinitroquinoxaline-2,3-dione (DNQX; 10 µM, n=3).
To address the question of whether opioids differentially alter glutamatergic transmission
onto different cell types in the VTA, we classified neurons by their electrophysiological and
pharmacological properties as principal, secondary, or tertiary (Margolis et al. 2003). In most
neurons, the changes in stimulated EPSC amplitude following bath application of both the KOP-
R selective agonist U69593 (1 µM) and the MOP-R selective agonist DAMGO (3 µM) were
measured (Fig. 1). U69593 produced a modest reduction in EPSC amplitude in principal neurons
(14 +/- 4%), significantly smaller than the DAMGO effect in the same neurons (42 +/- 8%, n=11,
ANOVA: P<0.01). In contrast, in secondary cells, EPSCs were inhibited to a similar degree by
both U69593 (47 +/- 10%) and DAMGO (45 +/- 10%, n=10). EPSCs in tertiary neurons were
also inhibited by both U69593 (33 +/- 6%) and DAMGO (35 +/- 6%, n=9). Because inhibition
by the KOP-R agonist U69593 persisted for at least 15 minutes after washout commenced (1
µM, n=6, data not shown), we used the KOP-R selective antagonist nor-BNI (100 nM) to reverse
the KOP mediated inhibition. Application of nor-BNI prior to U69593 completely blocked the
KOP-R agonist effect (2 +/- 2%, n=3, 1 cell of each type). Because DAMGO was applied a
second time to identify the cell type while recording in current clamp mode, in most experiments
no antagonist was used to reverse the prolonged presynaptic MOP-R activation response.
However, in a separate set of cells, the application of the MOP-R selective antagonist D-Phe-
Cys-Tyr-D-Tryp-Lys-Thr-Pen-Thr-NH2 (CTAP; 500 nM) reversed the DAMGO (3 µM) effect
(n=4, data not shown).
Comparing cell types, the observed U69593 effect was significantly larger in secondary
and tertiary neurons than in principal neurons (SNK: principal vs secondary, P<0.05; principal
vs tertiary, P<0.05). The DAMGO EPSC inhibition was not significantly different among cell
To provide evidence that the observed EPSC inhibitions were presynaptic, we examined
changes in the paired pulse ratio (PPR) for each drug. A drug-induced decrease in the probability
of release is typically correlated with an increase in the paired pulse ratio (Manabe et al.
1993). We found no significant differences between the baseline PPRs of the different cell
types (principal: 0.9 +/- 0.1, n=12; secondary: 1.2 +/- 0.2, n=9; tertiary: 0.93 +/- 0.06 Hz, n=8
Although opioid-induced changes in PPR for both U69593 and DAMGO varied greatly
across cells and cell types, enhancement of PPR was observed for both receptor types. Figure
2A shows examples comparing baseline evoked EPSCs to those recorded in the presence of
either U69593 or DAMGO, and the timecourses of the EPSC amplitude and PPR. This principal
neuron exhibited a significant increase in PPR with both drugs. Overall, VTA neurons showed a
significant facilitation of paired pulse ratio in the presence of U69593 (n=29, ANOVA: P<0.05,
Fig. 2B) and DAMGO (n=28, ANOVA: P<0.05, Fig. 2C). However, when broken down by cell
type, the only significant effect observed was that produced by DAMGO in secondary neurons.
Individually, less than one third of all neurons (8/30) showed a significant increase in PPR with
U69593 (student’s t-test, P<0.05), and these neurons were distributed across all 3 cell types.
A slightly higher proportion of neurons showed a significant increase in PPR with DAMGO
(11/29), and these, too, included neurons of each type. There was overall a significant linear
correlation between the magnitude of EPSC inhibition and the change in PPR for both U69593
(Fig. 2D, principal n=12, secondary n=9, tertiary n=8, r2=0.67, P<0.05) and DAMGO (Fig. 2E,
principal n=11, secondary n=8, tertiary n=8, r2=0.22, P<0.05), but these relationships did not
hold for individual neuron types.
The relative insensitivity of PPR in VTA neurons to U69593 and DAMGO leaves open
the possibility that the observed KOP and MOP effects on EPSC amplitude may not be due
to presynaptic mechanisms. Therefore, as an alternative method to test for a presynaptic
site of action of the observed effects, we monitored spontaneous excitatory events (sEPSCs)
simultaneously with the evoked responses in order to test for correlations between changes in
spontaneous and evoked EPSCs. Baseline sEPSC frequencies were variable across neurons, but
there were no differences in baseline frequencies between cell types (principal: 1.3 +/- 0.5 Hz,
n=8; secondary: 3.9 +/- 1.4 Hz, n=9; tertiary: 2.5 +/- 0.5 Hz, n=8, ANOVA: P>0.05). U69593
was found to inhibit sEPSCs, but did not change their amplitude (Fig. 3). Pooled data show that
the frequency of sEPSCs significantly decreased from baseline across all cell types in response
to both U69593 (ANOVA: P<0.005) and DAMGO (P<0.005, Fig. 3D). These inhibitions were
not accompanied by changes in sEPSC amplitude for either drug (ANOVA: U69593 P>0.05,
DAMGO P>0.05, Fig. 3E). The presence of a decrease in sEPSC frequency and a lack of an
effect on sEPSC amplitude are consistent with an opioid induced decrease in glutamate release
probability. Further, changes in sEPSC frequency were correlated with U69593-induced
decreases in evoked EPSC amplitudes in each cell type (Fig. 3F). Although only principal
neurons showed a significant correlation between evoked EPSC inhibition and sEPSC frequency
in the presence of DAMGO, a trend towards a relationship is evident in secondary and tertiary
neurons (Fig. 3G).
In order to ensure that the observed effects on sESPC frequencies were not due to
postsynaptic inhibitions of KOP- or MOP-sensitive spontaneously active glutamatergic neurons
in the slice that synapse onto the neurons recorded in the preceding experiments, in separate
experiments we measured changes in miniature EPSCs (mEPSCs) in the presence of TTX (500
nM). The application of TTX did not change the frequency of observed spontaneous events in
any cell type (principal: 1.8 +/- 0.6 Hz baseline, 1.6 +/- 0.6 Hz TTX, n=7; secondary: 1.6 +/- 0.4
Hz baseline, 1.5 +/- 0.5 Hz TTX, n=10; tertiary: 1.7 +/- 0.3 Hz baseline, 1.4 +/- 0.3 Hz TTX,
n=8, ANOVA: P>0.05) or amplitude (principal: 14 +/- 5 pA baseline, 13 +/- 5 pA TTX, n=7;
secondary: 13 +/- 1 pA baseline, 14 +/- 1 pA TTX, n=10; tertiary: 14 +/- 2 pA baseline, 12.8
+/- 0.6 pA TTX, n=8, ANOVA: P>0.05). The lack of an effect of TTX on either the frequency
or the amplitude of spontaneous excitatory events in these neurons suggests that the previously
observed sEPSCs are not action potential dependent.
In the presence of TTX, there was a significant inhibition of mEPSC frequency by U69593 (1
µM) among pooled VTA neurons (n=10, ANOVA: P<0.001), with the largest effect occurring in
tertiary neurons (Fig. 4A). There was no change in mEPSC amplitude observed during U69593
application (n=10, ANOVA: P>0.05, Fig. 4B). In separate experiments, DAMGO (3 µM) also
inhibited the frequency of mEPSCs in pooled VTA neurons (n=15, ANOVA: P<0.005, Fig.
4A). While as a group VTA neurons did show a significant decrease in mEPSC amplitude in
the presence of DAMGO (n=15, ANOVA: P<0.05), no individual cell type showed a significant
change (Fig. 4B). That the frequency of mEPSCs decreases with both U69593 and DAMGO
further supports a presynaptic mechanism for the observed effects.
A subset of Ih-expressing neurons are postsynaptically inhibited by KOP-R agonists
(Margolis et al. 2003). In order to confirm that this postsynaptic effect was not contributing to
our evoked presynaptic observations, we compared the evoked presynaptic effects of U69593
in Ih expressing (principal and tertiary) neurons in which the KOP-R agonist induced a positive
change in the holding current (n=6) to those recorded in neurons that exhibited no change in
holding current with U69593 (n=13, Fig. 5A). There was no difference between the KOP-R
agonist-mediated presynaptic inhibitions of EPSCs for these two groups (ANOVA: P>0.05).
Interestingly, there was a significant difference in the MOP-R agonist DAMGO inhibition of
evoked EPSCs in these two groups: Ih neurons that were postsynaptically hyperpolarized by the
KOP-R agonist showed a significantly greater EPSC inhibition during a separate application of
DAMGO (ANOVA: P<0.01). Conversely, Ih neurons that were postsynaptically inhibited by
the MOP-R agonist DAMGO (i.e. tertiary neurons, n=9) sh owed a greater EPSC inhibition by
U69593 than those that were not (i.e. principal neurons, n=12, ANOVA: P<0.05, Fig. 5B). There
was no difference in the presynaptic DAMGO effect for neurons postsynaptically inhibited by
MOP-R agonists compared to those that were not (Fig. 5B).
For both MOP and KOP receptor agonists, the largest presynaptic EPSC inhibitions were
observed in secondary neurons, and only secondary cells showed a significant correlation
between KOP and MOP receptor agonist EPSC inhibitions (Fig. 6). It is interesting to note that
the EPSCs in a subset of secondary neurons (3/9) were inhibited more than 75% by both U69593
and DAMGO, a much larger effect than that observed in any principal or tertiary neuron. In the
example traces in Fig. 1, the evoked EPSC signal appeared completely blocked, and this was
typical of all 3 secondary neurons with these large evoked EPSC inhibitions. This effect would
require that KOP and MOP receptors colocalize on glutamatergic terminals synapsing on to at
least a subpopulation of secondary neurons.
To address the question of whether MOP-Rs and KOP-Rs are segregated or are colocalized
on the same glutamate terminals synapsing onto Ih expressing neurons we carried out occlusion
experiments in principal and tertiary neurons. After a stable baseline was obtained, DAMGO
was added to the bath and the EPSC inhibition was allowed to stabilize. The addition of U69593
resulted in an additional inhibition of EPSC amplitude in all Ih neurons (n=8, Fig. 7A). Further,
while there was no difference between the magnitude of the U69593 effect in the presence (8
+/- 3%, n=4) or absence of DAMGO among principal neurons, the U69593-mediated EPSC
inhibition in tertiary neurons was significantly smaller in the presence of DAMGO (10 +/-
3%, n=4) than in aCSF alone (ANOVA: P<0.05, Fig. 7B). These data are consistent with the
hypothesis that MOP-Rs and KOP-Rs segregate to separate glutamatergic inputs to principal
neurons, but require that at least some MOP-Rs and KOP-Rs are colocalized on glutamatergic
terminals that synapse onto tertiary neurons.
Our results demonstrate that glutamate terminals onto each class of VTA neuron are inhibited
by both KOP and MOP receptor agonists. KOP EPSC inhibition is larger in secondary and
tertiary cells than it is in principal neurons. Among neurons with an Ih (principal and tertiary),
those that are postsynaptically hyperpolarized by KOP-R agonists show greater EPSC inhibition
by MOP-R agonists. In those VTA neurons without an Ih (secondary), there is a significant
correlation between the magnitudes of the KOP- and MOP-induced EPSC inhibitions.
Our results confirm and extend previous findings on opioid modulation of glutamate release
in the VTA. The results presented here are in agreement with earlier observations of presynaptic
inhibition of glutamate release by MOP-R agonists in Ih and non- Ih expressing VTA neurons
(Bonci and Malenka 1999; Manzoni and Williams 1999). However, the presynaptic KOP
effect demonstrated here was not observed in a previous investigation of glutamate release onto
Ih expressing neurons in the VTA (Manzoni and Williams 1999). Given the large variability
reported for the 7 neurons tested with U69593 in that study, an EPSC inhibition is likely to have
occurred in a subset of those neurons. The variability of KOP inhibition across all Ih neurons
reported here may have led to an average effect that was not significantly different from zero in
their study when principal and tertiary neurons were not distinguished. The small but significant
U69593 effect we observed in principal neurons was not only confirmed by sEPSC and mEPSC
measurements, but was reversed by nor-BNI, confirming its KOP-R selectivity.
We observed a previously unreported and significant relationship between the pre- and
postsynaptic effects of KOP and MOP receptor agonists in Ih neurons. The EPSC inhibition
by KOP-R agonists was larger among neurons postsynaptically hyperpolarized by MOP-R
agonists (i.e., tertiary neurons), and MOP-mediated EPSC inhibition was larger in neurons
postsynaptically inhibited by KOP-R agonists. Interestingly, while about 70% of Ih expressing
neurons in the VTA are dopaminergic, all neurons hyperpolarized by KOP are dopaminergic
(Margolis et al. 2003). Although there are also DA neurons that are not inhibited by KOP-
R agonists, the data reported here suggest that glutamatergic inputs to DA neurons are more
sensitive to MOP-R agonists than those onto non-DA, Ih expressing neurons. This conclusion
will need to be confirmed in future experiments.
The inhibition of excitatory input to DA neurons by MOP-R agonists seems counter to the
observation that MOP-R agonists in the VTA excite DA neurons. However, it is possible that the
function of MOP-R activation in the VTA is to increase firing rate without producing bursting
in DA neurons. Glutamate input to DA neurons tends to shift the tonic, spontaneous activity of
DA neurons to a bursting pattern, often without changing the overall firing rate of the neuron
(Chergui et al. 1993; Connelly and Shepard 1997; Floresco et al. 2003; Johnson et al. 1992;
Overton and Clark 1992). The combination of indirect disinhibition of DA neurons by MOP-R
agonists (through the inhibition of local GABAergic neurons) with presynaptic EPSC inhibition
could produce greater neuron activity without shifting the firing pattern to bursting. Such a
change could increase a temporally broad DA signal, which is likely to carry very different
information from the pulsed release associated with bursting of VTA DA neurons (Phillips et al.
Presynaptic inhibition of glutamate release in the VTA provides an important mechanism
by which inputs to the VTA can be differentially controlled by KOP and MOP receptor
agonists. The occlusion experiments reported here provide evidence that MOP-Rs and KOP-Rs
differentially regulate glutamatergic inputs onto both principal and tertiary neurons. The lack
of a difference among principal neurons in EPSC inhibition by U69593 applied in the presence
of DAMGO compared to that observed in aCSF is consistent with KOP-Rs and MOP-Rs being
segregated to separate terminals. However, secondary and tertiary neurons must have at least
partial overlap of receptor expression on individual glutamatergic terminals. In tertiary neurons,
this conclusion is supported by the finding that the KOP-R agonist mediated EPSC inhibition
was diminished in the presence of the MOP-R agonist. In many secondary cells U69593 and
DAMGO each inhibit EPSC amplitude by over 50%. Together with the correlation between
U69593 and DAMGO EPSC inhibitions in these neurons, these data support the hypothesis
that KOP-Rs and MOP-Rs are on the same glutamatergic terminals onto secondary neurons.
Interestingly, glutamate excitation of secondary neurons in the VTA increases firing rates
without causing bursting. Therefore, not only does glutamate appear to have a very different
postsynaptic function in secondary neurons compared to that in principal and tertiary cells, but
the opioid regulation of these inputs seems to be fundamentally different.
Unlike the combinations of postsynaptic KOP-Rs and MOP-Rs that are differentially
expressed in different VTA cell classes, presynaptic KOP and MOP receptor activation does
inhibit glutamate release onto all VTA neurons. This provides a broad functional range of
opioid modulation of VTA neuronal activity. Similar presynaptic KOP inhibition of glutamate
release onto cell types having different postsynaptic opioid responses has also been observed
in the nucleus raphe magnus (Bie and Pan 2003). Functional roles for these multiple opioid
receptor sites in the VTA may be related to the synaptic location and timing of release of
endogenous KOP and MOP receptor ligands. Projections to the VTA of neurons containing
enkephalin, a MOP and δ opioid receptor agonist peptide, arise from the ventral pallidum, and
those immunoreactive for endomorphin, a MOP-R selective agonist peptide, arise from the
hypothalamus. It is possible then that endogenous ligands acting at the MOP-R in the VTA
could be released by different events and at different times from those that lead to the release of
the endogenous KOP-R selective ligand dynorphin, which is released from terminals of neurons
located in the nucleus accumbens, lateral hypothalamus, and amygdala (Chou et al. 2001; Fallon
et al. 1985; Greenwell et al. 2002; Kalivas et al. 1993).
Postsynaptic inhibitions by both KOP-R and MOP-R agonists in the VTA have previously
been examined (Cameron et al. 1997; Margolis et al. 2003). Postsynaptically, only subsets
of dopaminergic principal and tertiary neurons are inhibited by KOP-R agonists, while, by
definition, all secondary and tertiary cells are directly inhibited by MOP-R agonists. Thus,
concurrent KOP- or MOP-induced presynaptic inhibition of glutamate release and postsynaptic
hyperpolarization would be synergistic in secondary and tertiary neurons. It is also important
to point out that principal and tertiary neurons fire in the absence of synaptic input. Therefore,
opioids can modify the output of these VTA neurons in the absence of an excitatory input, and
this modulation may have very different consequences from inhibiting excitatory inputs to the
same neuron. There may also be an anatomical difference between the different actions of
opioids on these signals. Endogenous opioids may have a limited radius of effect when released
and therefore, depending on their precise location, pre- and postsynaptic KOP-Rs and MOP-
Rs could be activated independently in vivo. Such mechanisms could account for the seeming
contradiction that MOP-R agonists both inhibit excitatory input and indirectly disinhibit principal
The sources of the differences in the responses to opioids reported here and their possible
functional implications are unclear. Since in most cases, especially in the principal neurons,
inhibition of glutamatergic inputs by KOP-R ligands was only partial, it is likely that not all
glutamatergic terminals bear the KOP-R. Thus, it is tempting to hypothesize that variation in
EPSC modulation by opioids across cell types depends on the source of glutamatergic afferents.
For instance, Carr and Sesack (2000) demonstrated that glutamate afferents from the mPFC
to the VTA synapse selectively on DA neurons that project back to the mPFC and GABAergic
neurons that project to the NAc, but not DA neurons that project to the NAc or GABA neurons
that project to the mPFC. Therefore, some combination of PPN, STN, and hypothalamus
afferents to the VTA likely provides major glutamate input to the non-dopaminergic neurons that
comprise 60% of the VTA projection to the mPFC, the dopaminergic neurons that comprise 80%
of the VTA projection to the NAc, and possibly the VTA neurons that project to other targets
such as the amygdala and hippocampus (Swanson 1982). However, since in other brain regions
a single axon can give rise to multiple excitatory synapses with significantly different properties
(Maccaferri et al. 1998; Markram et al. 1998; Scanziani et al. 1998), we cannot conclude that
the observed differences are due to differential anatomical origins of the glutamatergic afferents.
Further work needs to be done to discern if there is indeed an anatomical correlate to the effects
In conclusion, we demonstrate that KOP and MOP receptor agonists inhibit glutamatergic
input onto all neuron types in the VTA. Presynaptic regulation of synaptic transmission by
opioids in the VTA provides a mechanism for selective control of specific inputs to VTA neurons.
How these presynaptic effects interact with the postsynaptic inhibitions through MOP and KOP
receptor activation in the VTA not only depends on whether the glutamatergic afferents are
active when opioid ligands are present, but may also depend on the site of origin of the neuron
giving rise to the glutamatergic terminal, or the projection target of the postsynaptic neuron.
The modulation of glutamate release by KOP and MOP receptor agonists reported here provides
important information for understanding signal modulation in the VTA, and is an important key
to elucidating the influence on motivation and reward of endogenous opioids acting in the VTA.
This research was supported by funds provided by the National Institutes of Health/National
Institute of Drug Abuse, award number 01949, and the State of California for medical research
on alcohol and substance abuse through the University of California, San Francisco. The project
or effort depicted was also sponsored by the Department of the Army, award number DAMD17-
03-1-0059. The U.S. Army Medical Research Acquisition Activity, 820 Chandler Street, Fort
Detrick, MD 21702-5014 is the awarding and administering acquisition office. The content of
the information does not necessarily reflect the position or the policy of the Government, and no
official endorsement should be inferred.
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Figure 1. EPSCs in VTA neurons are inhibited by both KOP and MOP receptor agonists. Evoked
EPSCs in principal (n=11), secondary (n=10), and tertiary (n=9) neurons are inhibited by the
KOP-R agonist U69593 (1 µM). This inhibition is reversed by the KOP-R selective antagonist
nor-BNI (100 nM). DAMGO (3 µM), a MOP-R selective agonist, inhibits EPSC amplitude in
the same cells of each cell type.
Figure 2. Paired pulse ratio in VTA neurons increases due to KOP and MOP receptor agonists.
A, This example neuron shows a significant paired pulse facilitation in response to both U69593
(1 µM) and DAMGO (3 µM). Recording traces show example baseline (light) and drug (dark)
evoked EPSCs. Timecourses show both inhibition of evoked EPSC amplitude and facilitation of
PPR during drug applications. B, There is a significant paired pulse facilitation in pooled VTA
neurons in the presence of U69593, however no cell type alone exhibits a significant change in
PPR (principal n=12, secondary n=9, tertiary n=8, ANOVA: P<0.05, SNK: P>0.05 principal,
secondary, and tertiary). C, In addition to the overall paired pulse facilitation observed in the
pooled VTA neurons during DAMGO application, there was also a significant increase among
secondary neurons (principal n=11, secondary n=8, tertiary n=8, ANOVA: P<0.05, SNK: P<0.05
secondary, P>0.05 principal and tertiary). D, There is a significant linear correlation (P<0.05)
between the magnitude of EPSC inhibition by U69593 and the change in the paired pulse ratio
caused by the drug in pooled VTA neurons. E, Similarly, there is a significant linear correlation
in pooled VTA neurons (P<0.05) between DAMGO-induced EPSC inhibition and paired pulse
Figure 3. The frequency, but not amplitude, of sEPSCs is diminished by the KOP-R agonist
U69593. A, Sample traces of spontaneous activity recordings in a secondary neuron during
baseline and U69593 application (1 µM). B, In the same neuron, the cumulative plot shows
no difference between sEPSC amplitudes during baseline and in the presence of U69593. C,
U69593 application shifts the cumulative plot of the inter-event interval to the right of baseline.
D, sEPSC frequency was significantly decreased by both the KOP-R agonist U69593 (1 µM;
principal n=8, secondary n=9, tertiary n=8) and the MOP-R agonist DAMGO (3 µM; principal
n=6, secondary n=7, tertiary n=7). E, sEPSC amplitude was not affected by U69593 (1 µM)
or DAMGO (3 µM). F, There is a significant correlation between the effect of U69593 on the
amplitude of evoked EPSCs and the inhibition of sEPSC frequency in principal, secondary, and
tertiary neurons. G, While there is a trend towards a relationship between the effect of DAMGO
on the amplitude of evoked EPSCs and the inhibition of sEPSC frequency in secondary and
tertiary neurons, only principal neurons show a significant relationship between these measures.
Figure 4. Opioids decrease the frequency of miniature excitatory events. A, mEPSC frequency
among pooled VTA neurons is significantly diminished by U69593 (1 µM, ANOVA: P<0.05).
Further, tertiary neurons (n=2), but not principal (n=4) or secondary (n=4) neurons, show a
decrease in mEPSC frequency during U69593 application (SNK: P<0.05 tertiary, P>0.05
principal and secondary). The MOP-R agonist DAMGO (3 µM) significantly decreased the
frequency of mEPSCs in pooled (ANOVA: P<0.05), principal (n=3) and secondary (n=6),
but not tertiary (n=6) neurons (SNK: P<0.05 principal and secondary, P>0.05 tertiary). B,
U69593 did not significantly affect mEPSC amplitude in pooled data or any cell type. DAMGO
significantly decreased mEPSC amplitude in the pooled data, but not in any individual neuron
Figure 5. Presynaptic KOP and MOP actions in Ih neurons partially segregate according to
postsynaptic KOP and MOP actions. A, Compared to Ih neurons not postsynaptically inhibited
by KOP-R agonist U69593 (1 µM, n=13), those that are exhibit a greater EPSC inhibition by
DAMGO (3 µM, n=6). There is no difference in EPSC inhibition by U69593 (1 µM) between
neurons postsynaptically hyperpolarized by U69593 and those with no postsynaptic sensitivity to
U69593. B, Neurons that were postsynaptically hyperpolarized by the MOP-R agonist DAMGO
(3 µM, tertiary neurons, n=9) showed a greater EPSC inhibition by U69593 than those that were
not (principal neurons, n=12). There was no difference in EPSC inhibition by DAMGO between
those neurons that were postsynaptically inhibited by DAMGO and those that were not. *P<0.05,
Figure 6. EPSC inhibition by KOP and MOP in secondary neurons is correlated. There is
no apparent relationship between the magnitude of EPSC inhibition by U69593 (1 µM) and
DAMGO (3 µM) in principal (n=12) or tertiary (n=8) neurons. There is a significant linear
correlation between EPSC inhibition by U69593 and DAMGO in secondary neurons (n=9,
P<0.05). The greatest inhibitions by both U69593 and DAMGO were also observed in
Figure 7. MOP inhibition of glutamate release does not occlude the KOP-R mediated EPSC
inhibition. A, Ih neurons exhibit further EPSC inhibition when U69593 (1 µM) is applied in the
presence of DAMGO (3 µM, n=8). B, Among principal neurons, the magnitude of the additional
EPSC inhibition due to U69593 in the presence (n=4) or absence (n=11) of DAMGO was
not different. EPSC inhibition by U69593 in tertiary neurons was significantly smaller in the
presence of DAMGO (n=4) than in control aCSF (n=9). *P<0.05
% PPR Change
108060 40 200
% EPSC Inhibition
% PPR Change
10080 6040 200
% EPSC Inhibition
EPSC Ampl. (%)
Paired Pulse Ratio
All I II III
All I II III
Paired Pulse Ratio
Cumulative Rel. Freq. (%)
2520 1510 50
Inter-event Interval (sec)
Cumulative Rel. Freq. (%)
% sEPSC Inhibition
100 80 6040200
% sEPSC Inhibition
% Evoked EPSC Inhibition
80 6040 200
100 8060 40 200
100 806040 200
% sEPSC Frequency Inhibition
Principal Secondary Tertiary
sEPSC Amplitude (pA)
All I II III
All I II III
All I II III
All I II III
mEPSC Amplitude (pA) 25
All I II III
% mEPSC Frequency Inhibition
All I II III
% EPSC Inhibition
% EPSC Inhibition
% U69593 Inhibition
1008060 40 200
% DAMGO Inhibition
100 8060 40200
% DAMGO Inhibition
% DAMGO Inhibition
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% Inhibition by U69593