Kappa Opioid Receptors Regulate
Stress-Induced Cocaine Seeking
and Synaptic Plasticity
Nicholas M. Graziane,1,3Abigail M. Polter,1Lisa A. Briand,2R. Christopher Pierce,2and Julie A. Kauer1,*
1Brown University, Department of Molecular Pharmacology, Physiology and Biotechnology, Providence, RI 02912, USA
2Center for Neurobiology and Behavior, Department of Psychiatry, Perelman School of Medicine at the University of Pennsylvania,
Philadelphia, PA 19104, USA
3Present address: University of Pittsburgh, 900 South Trenton Avenue, Apt. 1, Pittsburgh, PA 15221, USA
Stress facilitates reinstatement of addictive drug
seeking in animals and promotes relapse in humans.
Acute stress has marked and long-lasting effects on
plasticity at both inhibitory and excitatory synapses
on dopamine neurons in the ventral tegmental
area (VTA), a key region necessary for drug rein-
forcement. Stress blocks long-term potentiation at
GABAergic synapses on dopamine neurons in the
VTA (LTPGABA), potentially removing a normal brake
on activity. Here we show that blocking kappa
opioid receptors (KORs) prior to forced-swim stress
rescues LTPGABA. In contrast, blocking KORs does
not prevent stress-induced potentiation of excitatory
synapses nor morphine-induced block of LTPGABA.
Using a kappa receptor antagonist as a selective
tool to test the role of LTPGABAin vivo, we find that
blocking KORs within the VTA prior to forced-swim
stress prevents reinstatement of cocaine seeking.
These results suggest that KORs may represent
a useful therapeutic target for treatment of stress-
triggered relapse in substance abuse.
Despite the health risks associated with drug abuse, addicted
individuals have difficulty abstaining, and relapse rates remain
high. Exposure to stress is associated with drug addiction in
humans and can induce relapse and craving (Brown et al.,
1995; Doherty et al., 1995; Sinha et al., 1999; Sinha, 2001; Sinha
and Li, 2007). Similarly, a variety of stressful stimuli can reinstate
drug seeking in animal models (Erb et al., 1996; Ahmed and
Koob, 1997; Shaham et al., 2000; Sorge and Stewart, 2005),
even when stressors are separated both in time and in context
from the drug-taking environment (Conrad et al., 2010). This
behavior relies on the mesolimbic reward system, but the
mechanisms that underlie stress-induced reinstatement remain
unclear. By understanding the influence of stress on the
mesolimbic reward system, it may be possible to develop valid
treatments addressing stress-related drug craving, a major
contributor to relapse.
Acute and sustained exposure to drugs of abuse alters
synaptic plasticity in vulnerable brain areas (Wolf, 2002; Kauer
and Malenka, 2007; Lu ¨scher and Malenka, 2011). Ventral teg-
mental area (VTA) dopamine neurons are part of the circuit
contributing to the reinforcing effects of addictive drugs (Wise,
2004; Everitt and Robbins, 2005). Furthermore, acute exposure
to either addictive drugs or stress causes parallel changes in
plasticity at excitatory and inhibitory synapses on VTA dopamine
neurons. At glutamatergic inputs, a single exposure to psychos-
timulants, morphine, nicotine, ethanol, or to acute stress in-
creases the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid receptor/N-methyl d-aspartate receptor (AMPAR/NMDAR)
ratio, a measure of long-term potentiation (LTP) (Ungless et al.,
2001; Saal et al., 2003; Kauer and Malenka, 2007). The potenti-
ation of excitatory VTA synapses following cocaine or acute
stress is blocked by NMDAR antagonists and requires insertion
of GluA1 receptors (Saal et al., 2003; Dong et al., 2004). Stress
activates the hypothalamic-pituitary-adrenal (HPA) axis leading
to an increase in circulating glucocorticoid hormone, thus
activating glucocorticoid receptors and initiating transcription.
The potentiation of VTA excitatory synapses by stress is pre-
vented when glucocorticoid receptors are blocked and can be
mimicked by direct activation of glucocorticoid receptors in
VTA slices (Saal et al., 2003; Daftary et al., 2009). At inhibitory
synapses on VTA dopamine neurons, one injection of morphine,
nicotine, cocaine, ethanol, or a single exposure to acute stress
blocks long-term potentiation (LTPGABA) at GABAA receptor
synapses on dopamine neurons (Nugent et al., 2007, 2009;
Guan and Ye, 2010; Niehaus et al., 2010). Glucocorticoid re-
ceptor activation is required for stress to block LTPGABA, but in
general much less is known about the mechanisms controlling
plasticity of inhibitory synapses in the VTA (Niehaus et al., 2010).
Previous studies suggested that opioid receptor activation
blocks LTP at GABAergic synapses (Nugent et al., 2007; Guan
and Ye, 2010). Here we have tested the idea that stress may
block LTP through release of endogenous opioids and activation
of opioid receptors. Stress is often accompanied by release of
the endogenous opioid peptide, dynorphin, and subsequent
activation of its receptor, the kappa opioid receptor (KOR)
942 Neuron 77, 942–954, March 6, 2013 ª2013 Elsevier Inc.
(Nabeshima et al., 1992). There is strong evidence indicating
that the kappa opioid system regulates neuronal activity in the
VTA. KORs are functionally expressed in the VTA (Speciale
et al., 1993; Arvidsson et al., 1995; Mansour et al., 1996), and
multiple dynorphin expressing regions innervate the VTA (Fallon
et al., 1985; Meredith, 1999; Dong and Swanson, 2003; Chartoff
et al., 2009; Poulin et al., 2009). Pharmacological activation of
kappa opioid receptors in the VTA causes a subset of dopamine
neurons to hyperpolarize and decrease their firing rate (Margolis
et al., 2003, 2006a, 2008). KOR activation also reduces the
somatodendritic release of dopamine in the VTA (Ford et al.,
The dynorphin-kappa opioid receptor system is also strongly
implicated in stress-related behaviors (Bals-Kubik et al., 1993;
Beardsley et al., 2005, 2010; Valdez et al., 2007; Land et al.,
2009; Bruchas et al., 2010). Conditioned aversion, elicited
when a place or an odor is paired with stress, is absent in mice
lacking dynorphin and in animals treated with kappa antagonists
step in the development of aversion to stressful experiences.
Kappa opioid receptors are also involved in behaviors at the
nexus between stress and drug seeking (McLaughlin et al.,
2003, 2006; Beardsley et al., 2005; Carey et al., 2007; Redila
and Chavkin, 2008; Beardsley et al., 2010; Sperling et al.,
2010). These findings suggest that the kappa opioid system
could modulate the compulsion to seek cocaine (or other drugs
of abuse) after stress (Bruchas et al., 2010).
We tested the hypothesis that kappa opioid receptor activa-
tion blocks LTPGABA in VTA dopaminergic neurons following
acute cold water swim stress. A similar stressor has recently
been shown to induce reinstatement to drug seeking in rats, an
effect that lasts for several days after stress (Conrad et al.,
2010). We report here that a KOR antagonist given prior to acute
stress rescues LTPGABA. We also find that delivery of the same
antagonist directly into the VTA prevents stress-induced rein-
statement of cocaine-seeking behavior in a self-administration
paradigm. Together our results suggest a link between the
GABAergic control of VTA neurons and cocaine seeking after
exposure to stress, and provide a rationale for the use of KOR
antagonists in therapeutic treatment of stress-induced relapse.
Stress Blocks LTPGABA
GABAergic synapses on dopamine neurons are persistently
potentiated by anincrease in postsynaptic Ca2+, which activates
nitric oxide (NO) synthase in turn generating NO. NO then travels
retrogradely to potentiate GABA release from nearby presyn-
aptic GABAergic nerve terminals through a cGMP-dependent
signaling cascade (Nugent et al., 2007), thus inducing LTPGABA
(Figure 7). Our previous work demonstrated that brief exposure
to stress blocks LTPGABAat these synapses 24 hr later (Niehaus
et al., 2010). Confirming our earlier results, after a 5 min cold
water forced swim stress (FSS), when midbrain slices were
prepared 24 hr later, LTPGABAwas blocked in VTA dopamine
neurons. In contrast, SNAP triggered robust LTPGABAin slices
from control animals that did not experience forced-swim stress
We next wanted to identify the mechanisms by which stress
blocked LTPGABA. The lack of LTPGABAcould reflect a stress-
induced loss of the synapse’s ability to potentiate. Alternatively,
if GABAergic synapses were maximally potentiated following the
stressful experience, no further potentiation would be possible
in vitro (occlusion). We carried out three experiments to
determine whether acute stress blocks or occludes LTPGABA.
We bath applied the adenylate cyclase activator, forskolin, to
slices from animals stressed 24 hr earlier. Forskolin potentiates
GABAergic synapses on VTA dopamine neurons via cAMP/
PKA signaling, and this pathway intersects the NO\cGMP-
induced potentiation of GABAA synapses as the effects of
SNAP and forskolin are not additive (Nugent et al., 2009). We
to forced swim, forskolin potentiated GABAergic synapses.
These data indicate that acute stress does not itself induce
LTP, but instead blocks the ability of GABAergic synapses to
potentiate; forskolin can overcome this block (Figures 1D–1F)
(IPSC amplitudes: naive animal, 131 ± 7% of pre-forskolin
values, n = 9; stressed animal, 136 ± 5% of pre-forskolin values,
n = 5). We also recorded miniature IPSCs (mIPSCs) in dopamine
neurons from stressed animals. Because GABAergic synapses
on VTA dopamine neurons are potentiated via increased presyn-
aptic GABA release (Nugent et al., 2007), we examined paired
pulse ratios and mIPSC frequency in animals after stress.
Because LTPGABAis associated with a decrease in the paired-
pulse ratio (PPR), a lower PPR in stressed animals versus
controls would indicate that LTPGABAwas occluded by stress.
Instead, paired-pulse ratios in dopamine neurons from stressed
animals were not significantly different from those in slices from
naive animals (PPR: nonstressed animals, 0.61 ± 0.04, n = 28;
stressed animals, 0.65 ± 0.06, n = 21, p > 0.05, Student’s
t test). Similarly, if potentiation at GABAergic synapses were
maximally induced by stress in vivo, we would expect an
increased mIPSC frequency. Instead, there was no significant
jected stressed animals (control, 5.65 ± 1.0 Hz, n = 10; saline +
forced swim stress, 5.49 ± 1.2 Hz, n = 10, p > 0.05, Student’s
t test) (Figure S1 available online). Together these experiments
Blocking Kappa Opioid Receptors Rescues the
Stress-Induced Block of LTPGABA
How might stress block LTPGABA? Our previous work demon-
strated that LTPGABAwas blocked 24 hr after an in vivo injection
of morphine (Nugent et al., 2007), suggesting the possibility that
stress might act via the release of endogenous opioids. To test
this idea, animals were pretreated with either saline or the opioid
receptor antagonist, naloxone (10 mg/kg), prior to forced swim
stress, and midbrain slices were cut 24 hr later. Stress blocked
LTPGABAin slices from saline-treated animals (Figure 2B), but
this block was rescued in animals pretreated with naloxone (Fig-
ure 2C) (IPSC amplitudes: saline + FSS, 112 ± 5%, n = 14;
naloxone + FSS, 129 ± 5%, n = 9; p < 0.05). These data support
our hypothesis that opioid receptor activation is required for
stress-induced block of LTPGABA.
Naloxone at the concentration used (10 mg/kg) blocks m, d,
and k opioid receptors (Lewanowitsch and Irvine, 2003). Kappa
Inhibitory VTA Plasticity and Reinstatement
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opioid receptors are activated following exposure to stressful
stimuli (Takahashi et al., 1990; Bruchas et al., 2007; Land et al.,
antagonist on LTPGABA. Nor-BNI (10 mg/kg) was injected 24 hr
prior to forced swim stress. As with naloxone pretreatment,
dopamine neurons from nor-BNI pretreated animals exhibited
robust LTPGABAafter stress, whereas those from saline pre-
treated animals had strongly attenuated LTPGABA(Figures 2F
and 2G) (saline + FSS, 100 ± 10%, n = 8; nor-BNI + FSS, 132 ±
8%, n = 6; p < 0.05). Animals injected with either naloxone
(10 mg/kg) or nor-BNI (10 mg/kg) alone, without forced
swim stress exposure, also exhibited normal potentiation of
GABAergic synapses (IPSC amplitudes: naloxone, 128 ± 2%,
n = 3; nor-BNI, 130 ± 6%, n = 4) (Figure S2). Furthermore, there
was no change in mIPSC frequency between controls and nor-
BNI-injected stressed animals (control, 5.65 ± 1.0 Hz, n = 10;
nor-BNI + forced swim stress, 4.98 ± 0.8 Hz, n = 10, p > 0.05,
Student’s t test) (Figure S2). These data suggest that kappa
opioid receptors are required for the stress-induced block of
LTPGABA, and that blocking these receptors rescues LTPGABA
from the effects of stress.
Systemic injection of naloxone and nor-BNI could influence VTA
synapses either directly or via activation of other brain regions.
To test whether activation of kappa opioid receptors (KOR)
blocks LTPGABAlocally in the VTA, we bath-applied the KOR
Figure 1. Stress Blocks LTPGABA; Forskolin Can Still Potentiate GABAergic Synapses
(A and B) Single experiment illustrating SNAP-induced LTPGABAin a dopamine cell from a naive animal (A) or from an animal exposed to 5 min cold water forced
swim (B). SNAP was bath-applied as indicated by the bar.
(C) LTPGABAin slices from naive or stressed animals (IPSC amplitudes, naive rats: 128.2 ± 7.9% of control values, n = 3, 24 hr after stress, 95.4 ± 2.0%, n = 3;
p < 0.05).
(D and E) Single experiment illustrating forskolin-induced LTPGABAin slices from naive animals (D) or in slices from animals exposed to forced swim stress (E).
Forskolin (10 mM) was bath-applied as indicated by the bar.
(F) Averaged data showing that forskolin potentiates IPSCs indopamine neurons from naive and stressed animals.Insets: IPSCs before (Control) and 30minafter
drug application (SNAP, 400 mM).
Scale bars represent 20 ms, 100 pA. Insets for this and all figures are averages of ten IPCSs. Error bars represent the SEM. See also Figure S1.
Inhibitory VTA Plasticity and Reinstatement
944 Neuron 77, 942–954, March 6, 2013 ª2013 Elsevier Inc.
agonist U69593 (1 mM) to midbrain slices from naive rats. The
KOR agonist entirely blocked LTPGABA in slices from naive
animals (Figures 3A–3C) (vehicle, 135 ± 7%, n = 5; U69593,
114 ± 5%, n = 5; p < 0.05). Thus, activation of kappa opioid
receptors locally within the VTA can block LTPGABA. Moreover,
because in these experiments we elicited LTP using an NO
donor, our data demonstrate that a KOR agonist prevents the
potentiation of GABAergic synapses downstream of nitric oxide.
U69593 (1 mM) did not alter GABAAreceptor-mediated synaptic
currents on its own (IPSC amplitude: 103 ± 5%, n = 11) (Fig-
ure S3). Paired-pulse ratios were also unaffected by U69593
(1 mM) perfusion (PPR before U69593, 0.54 ± 0.09, n = 11; PPR
after U69593, 0.49 ± 0.08, n = 11, p > 0.05, Student’s t test).
Kappa Opioid Receptor Activation Is Not Required for
the Stress-Induced Potentiation of Excitatory Synapses
Stress not only blocks LTPGABA, but also triggers potentiation
of excitatory synapses on VTA dopamine neurons (Saal et al.,
2003; Dong et al., 2004; Daftary et al., 2009), and we hypothe-
sized that KOR activation mediates both effects. We next
tested this idea, using nor-BNI pretreatment before forced
swim stress, and then measuring AMPA/NMDA receptor ratios
in slices from these animals 24 hr later. Although excitatory
synapses from animals that had undergone forced swim
stress were significantly potentiated compared with those
from naive animals (Figures 4A–4C) (AMPA/NMDA ratio: naive
animals, 0.43 ± 0.06, n = 10; stressed animals, 0.76 ± 0.14,
n = 10, p < 0.05), dopamine neurons from both saline-injected
and nor-BNI-injected animals undergoing forced swim stress
had similarly potentiated excitatory synapses (AMPA/NMDA
ratios: saline-injected + stress, 0.84 ± 0.11, n = 19; nor-BNI-
injected + stress, 0.74 ± 0.11, n = 19, p > 0.05). Thus,
although kappa opioid receptor activation is necessary to block
LTP at GABAAR synapses on dopamine neurons, excitatory
synapses are potentiated via a distinct mechanism (Figures
Figure 2. Blocking KORs Rescues the Stress-Induced Block of LTPGABA
(A) Injection protocol for naloxone-treated animals.
(B) LTPGABAin a dopamine cell from an animal administered saline 15 min prior to swim stress; slices were prepared 24 hr after stress. Insets, representative
IPSCs before (Control) and 30 min after SNAP application (SNAP).
(C) Rescue of SNAP-induced LTPGABAin a slice from an animal treated with naloxone prior to stress.
(D) Averaged experiments (saline-treated rats, n = 17; naloxone-treated rats, n = 9).
(E) Injection protocol for nor-BNI-treated animals; nor-BNI is effective for over a week following injection (Endoh et al., 1992).
(Fand G)Absence of LTPGABAinadopamine cellfrom ananimal administered saline24hrpriortostress (F)and froman animalpretreatedwithnor-BNI 24hrprior
to stress (G).
(H) Averaged experiments (saline-treated rats, n = 8; nor-BNI-treated rats, n = 8).
Scale bars represent 20 ms, 100 pA. Error bars represent the SEM. See also Figure S2.
Inhibitory VTA Plasticity and Reinstatement
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Morphine and Stress Block LTPGABAthrough Distinct
As previously shown, a single treatment with morphine blocks
LTPGABAin VTA dopamine neurons (Nugent et al., 2007). We
had not previously defined the involvement of specific opioid
receptors, although morphine can activate KORs directly
(Mignat et al., 1995). To investigate which opioid receptors are
required, animals were administered either saline (control) or
nor-BNI (10 mg/kg) 24 hr prior to morphine injection. We
confirmed that morphine blocked LTPGABAboth in slices from
animals pretreated with saline (Figure 5B) (Nugent et al., 2007;
Niehaus et al., 2010) and in animals pretreated with nor-BNI (Fig-
ure 5C), indicating that the morphine-induced block of LTPGABA
is notmediated byKOR activation (morphine, 118 ±11%,n =12;
nor-BNI + morphine, 111 ± 7.4%, n = 8, ANOVA [F2,39= 31.49,
p < 0.05, Tukey’s]). Morphine binds with highest affinity to
m-opioid receptors, and so we next pretreated rats with cypro-
dime (10 mg/kg), a m-opioid receptor antagonist, 30 min prior
to morphine injection, and LTPGABAwas tested in slices cut
24 hrlater.Cyprodime pretreatment effectivelyrescued LTPGABA
in animals treated with morphine (Figure 5E) (LTPGABAmeasured
15–20 min after SNAP application: morphine, 118 ± 11%, n = 12;
cyprodime + morphine, 161 ± 11%, n = 5, ANOVA [F2,39= 31.49,
p < 0.05, Tukey’s]). Together our data demonstrate that
morphine’s block of LTPGABAis, in fact, mediated through the
activation of m-opioid receptors and not KORs.
We also tested for the involvement of m-opioid receptors in the
stress-induced block of LTPGABA. Rats were pretreated with
cyprodime 30 min prior to forced swim stress and LTPGABA
was tested in slices cut 24 hr after stress. GABAergic synapses
tiate despite pretreatment with cyprodime (Figures 5H and 5I)
(cyprodime +stress,102 ±12%,n=8). Thus, although morphine
and stress both inhibit synaptic potentiation of VTA GABAergic
synapses, they act through distinct mechanisms.
VTA Kappa Receptors Regulate Stress-Induced
Reinstatement of Drug Seeking
Our in vitro data suggested that kappa receptors control stress-
induced synaptic changes in the VTA. Moreover, the kappa
receptor antagonist blocked only LTP at GABAergic synapses
without preventing stress-induced potentiation of excitatory
synapses. Previous work has implicated kappa receptors in
stress-induced reinstatement of drug seeking (McLaughlin
et al., 2003, 2006; Beardsley et al., 2005, 2010; Carey et al.,
2007; Redila and Chavkin, 2008). We therefore asked whether
blocking kappa receptors in the VTA, which we know rescues
of stress-induced cocaine seeking. Rats were trained to
self-administer cocaine for 19 days, and then cocaine was re-
placed with saline until responding was extinguished. LTPGABA
in VTA dopamine neurons was present in slices from animals
at the end of the extinction period (IPSC amplitude: 126 ±
10%; n = 4) (Figure S4). Either nor-BNI or saline were locally
microinjected into the VTA (Figure S4); 24 hr later, animals
measured 24 hr after the swim stress. Robust reinstatement
of cocaine seeking was observed in the vehicle-stress group.
The stress-induced reinstatement response was strongly atten-
uated by pretreatment with intra-VTA nor-BNI; in fact, in these
animals there was no difference between responding at the
end of the extinction period and responding 24 hr after stress
(Figure 6). Total active and inactive lever press data were
analyzed with a two-way repeated-measures ANOVA, which
revealed a significant main effect of lever (F(1,12) = 46.42,
p < 0.0001) and treatment (F(1,12) = 19.80, p < 0.001) as well
as a treatment 3 lever interaction (F(1,12) = 10.23, p < 0.01).
Subsequent pair wise comparisons showed that the active lever
response rate in the nor-BNI treatment was significantly less
than the vehicle treatment (Bonferroni, p < .001). There was no
significant difference between treatments in terms of inactive
Figure 3. Kappa Opioid Receptor Activation In Vitro Blocks LTPGABA
(A) LTPGABAin a dopamine cell from a naive slice perfused with DMSO.
(B) SNAP does not induce LTPGABAin a dopamine neuron perfused with the kappa agonist, U69593 (1 mM) 10 min prior to SNAP application.
(C) Averaged experiments; U69593 (n = 6), DMSO (n = 5). Insets are representative IPSCs before (Control) and 30 min after SNAP (SNAP). Scale bars represent
20 ms, 100 pA.
Inhibitory VTA Plasticity and Reinstatement
946 Neuron 77, 942–954, March 6, 2013 ª2013 Elsevier Inc.