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Kappa Receptors Regulate Stress-Induced Cocaine Seeking and Synaptic Plasticity

Nicholas M. Graziane,1,3 Abigail M. Polter,1 Lisa A. Briand,2 R. Christopher Pierce,2 and 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 *Correspondence: [email protected] http://dx.doi.org/10.1016/j.neuron.2012.12.034

SUMMARY mesolimbic reward system, it may be possible to develop valid treatments addressing stress-related drug craving, a major Stress facilitates reinstatement of addictive drug contributor to relapse. seeking in animals and promotes relapse in humans. Acute and sustained exposure to drugs of abuse alters Acute stress has marked and long-lasting effects on synaptic plasticity in vulnerable brain areas (Wolf, 2002; Kauer plasticity at both inhibitory and excitatory synapses and Malenka, 2007; Lu¨ scher and Malenka, 2011). Ventral teg- on dopamine neurons in the ventral tegmental mental area (VTA) dopamine neurons are part of the circuit area (VTA), a key region necessary for drug rein- contributing to the reinforcing effects of addictive drugs (Wise, 2004; Everitt and Robbins, 2005). Furthermore, acute exposure forcement. Stress blocks long-term potentiation at to either addictive drugs or stress causes parallel changes in GABAergic synapses on dopamine neurons in the plasticity at excitatory and inhibitory synapses on VTA dopamine VTA (LTPGABA), potentially removing a normal brake neurons. At glutamatergic inputs, a single exposure to psychos- on activity. Here we show that blocking kappa timulants, , nicotine, ethanol, or to acute stress in- opioid receptors (KORs) prior to forced-swim stress creases the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic rescues LTPGABA. In contrast, blocking KORs does acid receptor/N-methyl d-aspartate receptor (AMPAR/NMDAR) not prevent stress-induced potentiation of excitatory ratio, a measure of long-term potentiation (LTP) (Ungless et al., synapses nor morphine-induced block of LTPGABA. 2001; Saal et al., 2003; Kauer and Malenka, 2007). The potenti- Using a kappa as a selective ation of excitatory VTA synapses following cocaine or acute stress is blocked by NMDAR antagonists and requires insertion tool to test the role of LTPGABA in vivo, we find that blocking KORs within the VTA prior to forced-swim of GluA1 receptors (Saal et al., 2003; Dong et al., 2004). Stress activates the hypothalamic-pituitary-adrenal (HPA) axis leading stress prevents reinstatement of cocaine seeking. to an increase in circulating glucocorticoid hormone, thus These results suggest that KORs may represent activating glucocorticoid receptors and initiating transcription. a useful therapeutic target for treatment of stress- The potentiation of VTA excitatory synapses by stress is pre- triggered relapse in substance abuse. 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 INTRODUCTION synapses on VTA dopamine neurons, one injection of morphine, nicotine, cocaine, ethanol, or a single exposure to acute stress

Despite the health risks associated with drug abuse, addicted blocks long-term potentiation (LTPGABA) at GABAA receptor individuals have difficulty abstaining, and relapse rates remain synapses on dopamine neurons (Nugent et al., 2007, 2009; high. Exposure to stress is associated with drug addiction in Guan and Ye, 2010; Niehaus et al., 2010). Glucocorticoid re-

humans and can induce relapse and craving (Brown et al., ceptor activation is required for stress to block LTPGABA, but in 1995; Doherty et al., 1995; Sinha et al., 1999; Sinha, 2001; Sinha general much less is known about the mechanisms controlling and Li, 2007). Similarly, a variety of stressful stimuli can reinstate plasticity of inhibitory synapses in the VTA (Niehaus et al., 2010). drug seeking in animal models (Erb et al., 1996; Ahmed and Previous studies suggested that activation Koob, 1997; Shaham et al., 2000; Sorge and Stewart, 2005), blocks LTP at GABAergic synapses (Nugent et al., 2007; Guan even when stressors are separated both in time and in context and Ye, 2010). Here we have tested the idea that stress may from the drug-taking environment (Conrad et al., 2010). This block LTP through release of endogenous and activation behavior relies on the mesolimbic reward system, but the of opioid receptors. Stress is often accompanied by release of mechanisms that underlie stress-induced reinstatement remain the endogenous , , and subsequent unclear. By understanding the influence of stress on the activation of its receptor, the kappa opioid receptor (KOR)

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(Nabeshima et al., 1992). There is strong evidence indicating We next wanted to identify the mechanisms by which stress that the kappa opioid system regulates neuronal activity in the blocked LTPGABA. The lack of LTPGABA could reflect a stress- VTA. KORs are functionally expressed in the VTA (Speciale induced loss of the synapse’s ability to potentiate. Alternatively, et al., 1993; Arvidsson et al., 1995; Mansour et al., 1996), and if GABAergic synapses were maximally potentiated following the multiple dynorphin expressing regions innervate the VTA (Fallon stressful experience, no further potentiation would be possible et al., 1985; Meredith, 1999; Dong and Swanson, 2003; Chartoff in vitro (occlusion). We carried out three experiments to et al., 2009; Poulin et al., 2009). Pharmacological activation of determine whether acute stress blocks or occludes LTPGABA. kappa opioid receptors in the VTA causes a subset of dopamine We bath applied the adenylate cyclase activator, forskolin, to neurons to hyperpolarize and decrease their firing rate (Margolis slices from animals stressed 24 hr earlier. Forskolin potentiates et al., 2003, 2006a, 2008). KOR activation also reduces the GABAergic synapses on VTA dopamine neurons via cAMP/ somatodendritic release of dopamine in the VTA (Ford et al., PKA signaling, and this pathway intersects the NO\cGMP-

2007). induced potentiation of GABAA synapses as the effects of The dynorphin-kappa opioid receptor system is also strongly SNAP and forskolin are not additive (Nugent et al., 2009). We implicated in stress-related behaviors (Bals-Kubik et al., 1993; found that in slices from both naive animals and animals exposed Beardsley et al., 2005, 2010; Valdez et al., 2007; Land et al., to forced swim, forskolin potentiated GABAergic synapses. 2009; Bruchas et al., 2010). Conditioned aversion, elicited These data indicate that acute stress does not itself induce when a place or an odor is paired with stress, is absent in mice LTP, but instead blocks the ability of GABAergic synapses to lacking dynorphin and in animals treated with kappa antagonists potentiate; forskolin can overcome this block (Figures 1D–1F) (Land et al., 2008), suggesting that KOR activation is a necessary (IPSC amplitudes: naive animal, 131 ± 7% of pre-forskolin step in the development of aversion to stressful experiences. values, n = 9; stressed animal, 136 ± 5% of pre-forskolin values, Kappa opioid receptors are also involved in behaviors at the n = 5). We also recorded miniature IPSCs (mIPSCs) in dopamine nexus between stress and drug seeking (McLaughlin et al., neurons from stressed animals. Because GABAergic synapses 2003, 2006; Beardsley et al., 2005; Carey et al., 2007; Redila on VTA dopamine neurons are potentiated via increased presyn- and Chavkin, 2008; Beardsley et al., 2010; Sperling et al., aptic GABA release (Nugent et al., 2007), we examined paired 2010). These findings suggest that the kappa opioid system pulse ratios and mIPSC frequency in animals after stress. could modulate the compulsion to seek cocaine (or other drugs Because LTPGABA is associated with a decrease in the paired- of abuse) after stress (Bruchas et al., 2010). pulse ratio (PPR), a lower PPR in stressed animals versus

We tested the hypothesis that kappa opioid receptor activa- controls would indicate that LTPGABA was occluded by stress. tion blocks LTPGABA in VTA dopaminergic neurons following Instead, paired-pulse ratios in dopamine neurons from stressed acute cold water swim stress. A similar stressor has recently animals were not significantly different from those in slices from been shown to induce reinstatement to drug seeking in rats, an naive animals (PPR: nonstressed animals, 0.61 ± 0.04, n = 28; effect that lasts for several days after stress (Conrad et al., stressed animals, 0.65 ± 0.06, n = 21, p > 0.05, Student’s 2010). We report here that a KOR antagonist given prior to acute t test). Similarly, if potentiation at GABAergic synapses were stress rescues LTPGABA. We also find that delivery of the same maximally induced by stress in vivo, we would expect an antagonist directly into the VTA prevents stress-induced rein- increased mIPSC frequency. Instead, there was no significant statement of cocaine-seeking behavior in a self-administration difference in the mIPSC frequency between control and saline-in- paradigm. Together our results suggest a link between the jected stressed animals (control, 5.65 ± 1.0 Hz, n = 10; saline + GABAergic control of VTA neurons and cocaine seeking after forced swim stress, 5.49 ± 1.2 Hz, n = 10, p > 0.05, Student’s exposure to stress, and provide a rationale for the use of KOR t test) (Figure S1 available online). Together these experiments antagonists in therapeutic treatment of stress-induced relapse. support a stress-induced block of LTPGABA rather than occlusion.

RESULTS Blocking Kappa Opioid Receptors Rescues the

Stress-Induced Block of LTPGABA Stress Blocks LTPGABA How might stress block LTPGABA? Our previous work demon- GABAergic synapses on dopamine neurons are persistently strated that LTPGABA was blocked 24 hr after an in vivo injection potentiated by an increase in postsynaptic Ca2+, which activates of morphine (Nugent et al., 2007), suggesting the possibility that nitric oxide (NO) synthase in turn generating NO. NO then travels stress might act via the release of endogenous opioids. To test retrogradely to potentiate GABA release from nearby presyn- this idea, animals were pretreated with either saline or the opioid aptic GABAergic nerve terminals through a cGMP-dependent receptor antagonist, (10 mg/kg), prior to forced swim signaling cascade (Nugent et al., 2007), thus inducing LTPGABA stress, and midbrain slices were cut 24 hr later. Stress blocked (Figure 7). Our previous work demonstrated that brief exposure LTPGABA in slices from saline-treated animals (Figure 2B), but to stress blocks LTPGABA at these synapses 24 hr later (Niehaus this block was rescued in animals pretreated with naloxone (Fig- et al., 2010). Confirming our earlier results, after a 5 min cold ure 2C) (IPSC amplitudes: saline + FSS, 112 ± 5%, n = 14; water forced swim stress (FSS), when midbrain slices were naloxone + FSS, 129 ± 5%, n = 9; p < 0.05). These data support prepared 24 hr later, LTPGABA was blocked in VTA dopamine our hypothesis that opioid receptor activation is required for neurons. In contrast, SNAP triggered robust LTPGABA in slices stress-induced block of LTPGABA. from control animals that did not experience forced-swim stress Naloxone at the concentration used (10 mg/kg) blocks m, d, (Figures 1A–1C). and k opioid receptors (Lewanowitsch and Irvine, 2003). Kappa

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Figure 1. Stress Blocks LTPGABA; Forskolin Can Still Potentiate GABAergic Synapses

(A and B) Single experiment illustrating SNAP-induced LTPGABA in 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) LTPGABA in 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 LTPGABA in 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 in dopamine neurons from naive and stressed animals. Insets: IPSCs before (Control) and 30 min after 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. opioid receptors are activated following exposure to stressful was no change in mIPSC frequency between controls and nor- stimuli (Takahashi et al., 1990; Bruchas et al., 2007; Land et al., BNI-injected stressed animals (control, 5.65 ± 1.0 Hz, n = 10; 2008), so we next tested the effects of a selective kappa receptor nor-BNI + forced swim stress, 4.98 ± 0.8 Hz, n = 10, p > 0.05, antagonist on LTPGABA. Nor-BNI (10 mg/kg) was injected 24 hr Student’s t test) (Figure S2). These data suggest that kappa prior to forced swim stress. As with naloxone pretreatment, opioid receptors are required for the stress-induced block of dopamine neurons from nor-BNI pretreated animals exhibited LTPGABA, and that blocking these receptors rescues LTPGABA robust LTPGABA after stress, whereas those from saline pre- from the effects of stress. treated animals had strongly attenuated LTPGABA (Figures 2F and 2G) (saline + FSS, 100 ± 10%, n = 8; nor-BNI + FSS, 132 ± KOR Activation In Vitro Blocks LTPGABA in VTA Dopamine 8%, n = 6; p < 0.05). Animals injected with either naloxone Neurons (10 mg/kg) or nor-BNI (10 mg/kg) alone, without forced Systemic injection of naloxone and nor-BNI could influence VTA swim stress exposure, also exhibited normal potentiation of synapses either directly or via activation of other brain regions. GABAergic synapses (IPSC amplitudes: naloxone, 128 ± 2%, To test whether activation of kappa opioid receptors (KOR) n = 3; nor-BNI, 130 ± 6%, n = 4) (Figure S2). Furthermore, there blocks LTPGABA locally in the VTA, we bath-applied the KOR

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Figure 2. Blocking KORs Rescues the Stress-Induced Block of LTPGABA (A) Injection protocol for naloxone-treated animals.

(B) LTPGABA in 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 LTPGABA in 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).

(F and G) Absence of LTPGABA in a dopamine cell from an animal administered saline 24 hr prior to stress (F) and from an animal pretreated with nor-BNI 24 hr prior 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. agonist U69593 (1 mM) to midbrain slices from naive rats. The 2003; Dong et al., 2004; Daftary et al., 2009), and we hypothe-

KOR agonist entirely blocked LTPGABA in slices from naive sized that KOR activation mediates both effects. We next animals (Figures 3A–3C) (vehicle, 135 ± 7%, n = 5; U69593, tested this idea, using nor-BNI pretreatment before forced 114 ± 5%, n = 5; p < 0.05). Thus, activation of kappa opioid swim stress, and then measuring AMPA/NMDA receptor ratios receptors locally within the VTA can block LTPGABA. Moreover, in slices from these animals 24 hr later. Although excitatory because in these experiments we elicited LTP using an NO synapses from animals that had undergone forced swim donor, our data demonstrate that a KOR agonist prevents the stress were significantly potentiated compared with those potentiation of GABAergic synapses downstream of nitric oxide. from naive animals (Figures 4A–4C) (AMPA/NMDA ratio: naive

U69593 (1 mM) did not alter GABAA receptor-mediated synaptic animals, 0.43 ± 0.06, n = 10; stressed animals, 0.76 ± 0.14, currents on its own (IPSC amplitude: 103 ± 5%, n = 11) (Fig- n = 10, p < 0.05), dopamine neurons from both saline-injected ure S3). Paired-pulse ratios were also unaffected by U69593 and nor-BNI-injected animals undergoing forced swim stress (1 mM) perfusion (PPR before U69593, 0.54 ± 0.09, n = 11; PPR had similarly potentiated excitatory synapses (AMPA/NMDA after U69593, 0.49 ± 0.08, n = 11, p > 0.05, Student’s t test). ratios: saline-injected + stress, 0.84 ± 0.11, n = 19; nor-BNI- injected + stress, 0.74 ± 0.11, n = 19, p > 0.05). Thus, Kappa Opioid Receptor Activation Is Not Required for although kappa opioid receptor activation is necessary to block the Stress-Induced Potentiation of Excitatory Synapses LTP at GABAAR synapses on dopamine neurons, excitatory Stress not only blocks LTPGABA, but also triggers potentiation synapses are potentiated via a distinct mechanism (Figures of excitatory synapses on VTA dopamine neurons (Saal et al., 4D–4F).

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Figure 3. Kappa Opioid Receptor Activation In Vitro Blocks LTPGABA

(A) LTPGABA in a dopamine cell from a naive slice perfused with DMSO.

(B) SNAP does not induce LTPGABA in 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.

Morphine and Stress Block LTPGABA through Distinct VTA Kappa Receptors Regulate Stress-Induced Mechanisms Reinstatement of Drug Seeking As previously shown, a single treatment with morphine blocks Our in vitro data suggested that kappa receptors control stress-

LTPGABA in VTA dopamine neurons (Nugent et al., 2007). We induced synaptic changes in the VTA. Moreover, the kappa had not previously defined the involvement of specific opioid receptor antagonist blocked only LTP at GABAergic synapses receptors, although morphine can activate KORs directly without preventing stress-induced potentiation of excitatory (Mignat et al., 1995). To investigate which opioid receptors are synapses. Previous work has implicated kappa receptors in required, animals were administered either saline (control) or stress-induced reinstatement of drug seeking (McLaughlin nor-BNI (10 mg/kg) 24 hr prior to morphine injection. We et al., 2003, 2006; Beardsley et al., 2005, 2010; Carey et al., confirmed that morphine blocked LTPGABA both in slices from 2007; Redila and Chavkin, 2008). We therefore asked whether animals pretreated with saline (Figure 5B) (Nugent et al., 2007; blocking kappa receptors in the VTA, which we know rescues

Niehaus et al., 2010) and in animals pretreated with nor-BNI (Fig- the stress-induced loss of LTPGABA, influences the reinstatement ure 5C), indicating that the morphine-induced block of LTPGABA of stress-induced cocaine seeking. Rats were trained to is not mediated by KOR activation (morphine, 118 ± 11%, n = 12; self-administer cocaine for 19 days, and then cocaine was re- nor-BNI + morphine, 111 ± 7.4%, n = 8, ANOVA [F2,39 = 31.49, placed with saline until responding was extinguished. LTPGABA p < 0.05, Tukey’s]). Morphine binds with highest affinity to in VTA dopamine neurons was present in slices from animals m-opioid receptors, and so we next pretreated rats with cypro- at the end of the extinction period (IPSC amplitude: 126 ± dime (10 mg/kg), a m-opioid receptor antagonist, 30 min prior 10%; n = 4) (Figure S4). Either nor-BNI or saline were locally to morphine injection, and LTPGABA was tested in slices cut microinjected into the VTA (Figure S4); 24 hr later, animals 24 hr later. Cyprodime pretreatment effectively rescued LTPGABA underwent swim stress. Reinstatement responding was in animals treated with morphine (Figure 5E) (LTPGABA measured measured 24 hr after the swim stress. Robust reinstatement 15–20 min after SNAP application: morphine, 118 ± 11%, n = 12; of cocaine seeking was observed in the vehicle-stress group. cyprodime + morphine, 161 ± 11%, n = 5, ANOVA [F2,39 = 31.49, The stress-induced reinstatement response was strongly atten- p < 0.05, Tukey’s]). Together our data demonstrate that uated by pretreatment with intra-VTA nor-BNI; in fact, in these morphine’s block of LTPGABA is, in fact, mediated through the animals there was no difference between responding at the activation of m-opioid receptors and not KORs. end of the extinction period and responding 24 hr after stress We also tested for the involvement of m-opioid receptors in the (Figure 6). Total active and inactive lever press data were stress-induced block of LTPGABA. Rats were pretreated with analyzed with a two-way repeated-measures ANOVA, which cyprodime 30 min prior to forced swim stress and LTPGABA revealed a significant main effect of lever (F(1,12) = 46.42, was tested in slices cut 24 hr after stress. GABAergic synapses p < 0.0001) and treatment (F(1,12) = 19.80, p < 0.001) as well on VTA dopamine neurons from stressed animals failed to poten- as a treatment 3 lever interaction (F(1,12) = 10.23, p < 0.01). tiate despite pretreatment with cyprodime (Figures 5H and 5I) Subsequent pair wise comparisons showed that the active lever (cyprodime + stress, 102 ± 12%, n = 8). Thus, although morphine response rate in the nor-BNI treatment was significantly less and stress both inhibit synaptic potentiation of VTA GABAergic than the vehicle treatment (Bonferroni, p < .001). There was no synapses, they act through distinct mechanisms. significant difference between treatments in terms of inactive

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Figure 4. Kappa Opioid Receptor Activation Is Not Required for Long-Term Potentiation at Excitatory Synapses on DA Neurons in the VTA (A) AMPAR- (red) and NMDAR-mediated (black) EPSCs recorded from a dopamine neuron from a naive animal (naive). (B) AMPAR- and NMDAR-mediated EPSCs 24 hr after stress. (C) AMPA/NMDA ratios recorded in dopamine neurons from naive animals and animals 24 hr after stress. Asterisk, p < 0.05, t test. (D and E) AMPAR and NMDAR EPSCs from an animal injected with saline (D) or nor-BNI (E) prior to forced swim stress. (F) Summary of AMPA/NMDA ratios recorded in dopamine neurons from saline and nor-BNI pretreated animals 24 hr after swim stress. Scale bars represent 50 pA, 20 ms. Error bars represent the SEM.

lever responding. These data support the hypothesis that kappa rescued LTPGABA 24 hr later. A kappa receptor agonist also receptors in the VTA play a significant role in reinstatement to blocked LTPGABA in VTA slices in vitro, indicating that the kappa cocaine seeking. receptors within the VTA itself can control the induction of

LTPGABA (Figure 7). Projections from the BNST, nucleus accum- DISCUSSION bens, amygdala, and hypothalamus all contain dynorphin that

could be released in the VTA and block LTPGABA following acute This study provides three key findings. First, acute stress stress (Fallon et al., 1985; Meredith, 1999; Dong and Swanson, activates kappa opioid receptors and blocks nitric oxide- 2003; Poulin et al., 2009). The precise step at which kappa opioid triggered long-term potentiation of GABAergic synapses on receptors inhibit LTPGABA is not yet known. We favor a presyn- VTA dopamine neurons. Second, although acute stress also aptic effect on GABAergic nerve terminals (downstream of nitric potentiates excitatory synapses on dopamine neurons, this oxide production), because in our experiments, exogenously potentiation is not prevented by a kappa opioid receptor added nitric oxide fails to potentiate GABAA synapses after antagonist. Finally, blocking kappa opioid receptors in the KOR activation. ventral tegmental area prevents stress-induced reinstatement of cocaine seeking. Kappa Opioid Receptors Are Not Required for Potentiation of Glutamatergic Synapses

LTPGABA Is Blocked When Kappa Opioid Receptors Are Following a brief stressful stimulus, excitatory synapses on Activated VTA dopamine neurons are potentiated (Saal et al., 2003). In

LTPGABA is a mechanism normally available to control VTA other brain regions, e.g., the hippocampus and the amygdala, dopamine neurons, and may play an essential role in modulating decreased GABAergic inhibition increases the likelihood of LTP their firing rates (Johnson and North, 1992; Paladini and Tepper, at neighboring excitatory synapses (Wigstro¨ m and Gustafsson, 1999; Tan et al., 2012; van Zessen et al., 2012). Our results 1986; Mott et al., 1990; Shaban et al., 2006), so the loss of show that the kappa opioid receptor system mediates the LTPGABA could promote LTP at excitatory synapses on dopa- stress-induced block of LTPGABA, as systemic treatment with mine cells (Niehaus et al., 2010). Instead, we found that the naloxone or a selective kappa receptor antagonist entirely loss of LTPGABA is not required for potentiation of excitatory

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Figure 5. Morphine and Stress Block LTPGABA through Distinct Mechanisms (A) Diagram of injection protocol for (B) and (C).

(B) SNAP fails to induce LTPGABA in a dopamine neuron from an animal administered saline prior to morphine. Slices were prepared 24 hr after the animal was exposed to morphine. Insets, representative IPSCs before (Control) and 30 min after SNAP application (SNAP).

(C) SNAP also fails to induce LTPGABA in a slice from an animal treated with nor-BNI prior to morphine. (D) Injection protocol for (E) and (F).

(E) Rescue of SNAP-induced LTPGABA in a dopamine cell from an animal treated with cyprodime prior to morphine.

(F) Averaged experiments showing that LTPGABA is blocked in dopamine cells from animals treated with saline (black symbols, n = 15) or with nor-BNI (gray symbols, n = 9) before morphine; LTPGABA is rescued in dopamine cells from animals pretreated with cyprodime (open symbols, n = 6). No significant differences were found between groups of saline pretreated animals, so the data were pooled. (G) Injection protocol prior to stress (H) and (I).

(H) Lack of LTPGABA in a slice from an animal treated with cyprodime prior to stress.

(I) SNAP does not induce LTPGABA in cyprodime pretreated stressed animals (n = 8). Scale bars represent 20 ms, 100 pA. Error bars represent the SEM.

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Figure 7. Proposed Model of Signaling Molecules Involved in

LTPGABA

(A) An in vivo exposure to stress blocks LTPGABA through activation of kappa opioid receptors (KORs) with subsequent modulation of the NO signaling pathway at a point downstream of NO production. Presynaptic Figure 6. Blocking Kappa Opioid Receptors in the VTA Blocks terminals expressing LTPGABA may originate from local interneurons or Stress-Induced Reinstatement of Cocaine Seeking extrinsic GABAergic afferents. m (A) Training protocol: 19 days of intravenous cocaine self-administration (2 hr (B) In vivo exposure to morphine alters LTPGABA through activation of -opioid m per day) were followed by daily extinction sessions until lever pressing receptors ( OR), inhibiting the cGMP pathway at the level of sGC (soluble decreased to <15% of responding on the last day of cocaine self-adminis- guanylate cyclase) (Niehaus et al., 2010). tration. Rats required %8 days to achieve this criterion. Twenty-four hours (C) Forskolin potentiates GABAergic synapses by activating adenylyl cyclase following the attainment of extinction criterion, animals were given a micro- (AC), which in turn activates the cAMP/PKA pathway. This pathway is unaf- infusion of nor-BNI or vehicle into the VTA. Twenty-four hours later, they fected by in vivo manipulations with morphine or stress (Nugent et al., 2009). were exposed to a cold swim stressor and tested for reinstatement responding Error bars represent the SEM. 24 hr later. (B) Total active and inactive lever presses during the reinstatement phase are shown from animals administered vehicle (n = 7) or 2.5 mg (n = 7) nor-BNI into seeking even when excitatory synapses were allowed to poten- the ventral tegmental area prior to stress. Unlike the vehicle controls, animals tiate (discussed below). that received nor-BNI infusions into the VTA did not show significant rein- statement of cocaine responding as compared to their extinction responding. How Might the Loss of LTPGABA Influence Dopamine Error bars represent the SEM. See also Figure S4. Neuron Firing?

LTPGABA represents a mechanism to increase the magnitude of VTA synapses, as nor-BNI blocked the former but not the latter. GABAergic inhibition of dopamine neurons, so its loss after stress

Thus, kappa opioid receptor activation is not required for stress- is expected to increase cell firing or bursting. Changes in GABAA induced LTP at excitatory synapses. inhibition have particularly significant consequences in dopa- Previous work demonstrated that glucocorticoid receptor mine neurons, which are relatively depolarized and therefore at (GR) activation is required both for stress-induced LTP of excit- or very close to the threshold for firing action potentials (Johnson atory synapses and for the block of LTPGABA. A GR antagonist and North, 1992). Optogenetic activation of VTA GABA neurons blocks both forms of plasticity (Saal et al., 2003; Dong et al., drastically decreases the firing rate of dopamine neurons (Tan 2004; Daftary et al., 2009; Niehaus et al., 2010), and an in vivo et al., 2012; van Zessen et al., 2012), and local pressure applica- treatment or an in vitro bath application of a glucocorticoid tion of GABAA receptor antagonists powerfully shifts the firing receptor agonist increases the AMPA/NMDA ratio (Daftary pattern of dopamine neurons from tonic to bursting (Paladini et al., 2009), similarly to what is seen after exposure to stress and Tepper, 1999). Because burst firing activity in dopamine (Saal et al., 2003; Dong et al., 2004; Daftary et al., 2009). It is neurons is more effective than tonic firing at releasing dopamine reasonable then to speculate that glucocorticoids released after in target sites, stress-induced loss of LTPGABA also is expected to exposure to stress set into motion plasticity at both inhibitory and increase dopamine release in target sites (Gonon, 1988; Manley excitatory VTA synapses, but ultimately act through independent et al., 1992). Consistent with this idea, in vivo recordings show downstream mechanisms. Our ability to use nor-BNI as a selec- that burst firing of VTA dopamine neurons increases with acute tive tool to rescue stress effects on LTPGABA allowed us to test restraint stress and persists for at least 24 hr (Anstrom and Wood- the role of LTPGABA in stress-induced reinstatement of drug ward, 2005), and administration of the GABAA receptor agonist,

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muscimol, into the VTA blocks footshock-induced reinstatement the relevant site of kappa opioid action was unknown, as these (McFarland et al., 2004). We speculate that the dynorphin/KOR studies utilized systemic drug administration. Our results indi- system could regulate dopamine neuron firing by potently cate that the kappa receptors within the VTA critically control reducing GABAA-mediated inhibition of dopamine cells over stress-induced drug seeking in animals trained to self-admin- many hours, increasing drug-seeking behavior. ister cocaine. It is worth noting that nor-BNI’s effect on drug reinstatement is Blocking Kappa Receptors within the VTA Attenuates not likely to result from a reduction in the perceived desirability of Cocaine Reinstatement cocaine, as nor-BNI does not affect acquisition or maintenance

We reported earlier that LTPGABA is blocked in slices 24 hr after of cocaine self-administration (Glick et al., 1995; Negus et al., in vivo exposure to morphine (Nugent et al., 2007; Niehaus et al., 1997; Negus, 2004) or place preference (Carlezon et al., 1998). 2010); indeed, this result first suggested that endogenous KOR antagonists also fail to attenuate cocaine-primed reinstate- opioids could in theory mediate the effects of stress exposure ment, suggesting that KORs exert a selective effect on stress- on synaptic function. One hypothesis we entertained was that induced behavior (Beardsley et al., 2005; Carey et al., 2007; both addictive drugs and stress might block LTPGABA through Redila and Chavkin, 2008). a common mechanism. Instead, however, we report here that morphine and stress block LTPGABA through distinct signaling Stress Alters Synaptic Plasticity in the VTA, Contributing pathways: whereas stress acts via the kappa opioid receptor, to Drug Seeking morphine acts instead via the m-opioid receptor. Consistent Our in vitro results demonstrate that pretreatment with nor- with this model, blocking m-receptors fails to rescue the stress- BNI rescued LTPGABA after stress, with no effect on LTP at induced block of LTPGABA, whereas blocking KORs does not glutamatergic synapses in stressed animals. Yet in animals rescue the morphine-induced block. Similarly, others have that had learned and then extinguished responding for cocaine, shown that ethanol inhibits LTPGABA, and this inhibition is delivery of nor-BNI into the VTA blocked cocaine seeking after rescued by prior naloxone and was attributed to m-opioid stress; there was no significant difference between lever receptor block (although at a concentration not entirely selective pressing for cocaine after extinction versus after stress in the for m receptors) (Guan and Ye, 2010). Much like an acute nor-BNI-treated animals. These data are consistent with a model stressor, drug administration can robustly trigger reinstatement in which the rescue of LTPGABA in the VTA blunts stress-induced of drug seeking (Stewart, 2000). However, the mechanisms reinstatement even when excitatory synapses on dopamine appear to be different. In fact, m-opioid receptors may be re- neurons are potentiated. Previous work has suggested that quired for drug-triggered reinstatement of drug seeking (Comer NMDAR-dependent processes in the VTA play an important et al., 1993; Shaham and Stewart, 1996; Leˆ et al., 1999; Cicco- role in reinforcement. Administration of an NMDAR antagonist cioppo et al., 2002; Redila and Chavkin, 2008; Simmons and directly into the VTA (that would be expected to block LTP at Self, 2009). Moreover, kappa receptor agonists appear to reduce glutamatergic synapses) (Bonci and Malenka, 1999) decreases cocaine-primed reinstatement, with no observed effect of nor- the total number of cocaine reinforcements on a progressive BNI, although age and cannula placements may also contribute ratio schedule following social defeat stress, and also signifi- to differences (Sun et al., 2010). Although our data cannot cantly reduces the effect of social defeat stress on cocaine directly address drug-primed reinstatement, it will be important intake during a 24 hr binge access period (Covington et al., in future work to compare differences in corticotrophin releasing 2008). However, excitatory synapses remain potentiated in factor and noradrenergic systems, which may be selectively animals that have self-administered cocaine even after an activated in stress-induced reinstatement (Stewart, 2000; Briand extinction period, suggesting that further potentiation of these and Blendy, 2010). synapses is unlikely to contribute to the reinstatement observed We found that stress-induced reinstatement of cocaine after stress (Chen et al., 2008). Taken together, the evidence seeking was significantly reduced in animals that had received suggests that the block of LTP at GABAergic synapses contrib- intra-VTA injections of nor-BNI. Previous work showed that utes to stress-induced reinstatement. Our experiments cannot kappa receptor antagonists block stress-induced reinstatement rule out other effects of KORs in the VTA, such as transient of cocaine or ethanol conditioned place preference as well as dopamine neuron hyperpolarization and inhibition of glutama- stress-induced reinstatement of cocaine or ethanol self-admin- tergic transmission (Margolis et al., 2003, 2006a). However, istration (Beardsley et al., 2005; Carey et al., 2007; Redila and these effects are reversed rapidly upon removal of the agonist,

Chavkin, 2008; Sperling et al., 2010). Additionally, mice lacking whereas the long time course over which LTPGABA is blocked either kappa opioid receptors or fail to exhibit following stress is consistent with the persistent increase in footshock stress-induced reinstatement of cocaine place pref- cocaine seeking reported after an acute stressor (Conrad erence (Redila and Chavkin, 2008), and prodynorphin knockout et al., 2010). mice fail to exhibit swim stress-induced reinstatement of ethanol Exposure to either an aversive stimulus, (such as stress or place preference (Sperling et al., 2010). Conversely, pharmaco- KOR activation), or to an appetitive stimulus such as a priming logical activation of KORs is sufficient to induce robust rein- dose of drug robustly induces reinstatement (Piazza and Le statement of place preference to drugs of abuse in unstressed Moal, 1998; McLaughlin et al., 2006; Carey et al., 2007; Redila animals (Redila and Chavkin, 2008; Sperling et al., 2010). Taken and Chavkin, 2008; Conrad et al., 2010). Both stimuli also block together, these studies support the involvement of kappa LTPGABA on VTA dopamine neurons (Niehaus et al., 2010; opioid receptors in animal models of drug relapse. However, Lu¨ scher and Malenka, 2011). The similar synaptic responses to

950 Neuron 77, 942–954, March 6, 2013 ª2013 Elsevier Inc. Neuron Inhibitory VTA Plasticity and Reinstatement

both stress and drugs are compelling, suggesting that the each experiment (n = number of animals). The acute stress was a modified synaptic responses may make an important contribution to Porsolt forced cold-water swim task (Niehaus et al., 2010). Naloxone was relapse to drug seeking. Several studies suggest that kappa administered 15 min prior to forced swim except as noted; cyprodime 30 min prior to cold swim or morphine, and 24 hr prior receptor agonists do not reverse cocaine-primed reinstatement to forced swim stress or morphine injection; at this time point nor-BNI is of drug seeking or conditioned place preference, suggesting that selective for KORs (Endoh et al., 1992; Bruchas et al., 2007; Redila and KORs exert a selective effect on stress-induced behavior Chavkin, 2008). Nor-BNI was given 15 min prior to forced swim stress (Beardsley et al., 2005; Carey et al., 2007; Redila and Chavkin, when testing AMPA/NMDA ratios, to block m- and k-opioid receptors (Endoh 2008), or perhaps that additional mechanisms are involved in et al., 1992). Midbrain slices were prepared 24 hr after drug administration or drug-primed reinstatement. stress exposure.

Self-Administration Procedures Brain Circuitry Underlying Stress-Induced Relapse Procedures for self-administration are identical to those described previously Stress is strongly associated with relapse to compulsive drug (Famous et al., 2008). Rats were anesthetized with (80 mg/kg, intra- use in drug addiction (Sinha, 2001; Cleck and Blendy, 2008; peritoneally [i.p.]; Sigma-Aldrich, St. Louis, MO) and xylazine (12 mg/kg, i.p.; Koob, 2009). The kappa opioid system in the VTA is likely to Sigma-Aldrich). An indwelling silastic catheter was inserted into the right be component of a larger circuit, including the bed nucleus of jugular vein and was sutured in place. Two stainless steel guide cannulae for the stria terminalis (BNST), dorsal raphe and amygdala (Erb microinjections were implanted bilaterally 2 mm dorsal to the ventral tegmental area in each animal (À5.6 mm A/P, ± 0.6 mm M/L and À6.2 mm D/V; Paxinos et al., 2001; Leri et al., 2002; Wang et al., 2007; Land et al., and Watson). 2009; Bruchas et al., 2011; Nawata et al., 2012; Smith et al., Cocaine Self-Administration 2012). Swim stress triggers c-fos activation in neurons that Rats (200–225 g) were allowed to lever press for cocaine 6 days a week on project to the VTA and this activation in many regions is depen- a FR1 schedule with a time-out of 20 s and a maximum of 30 infusions per dent upon CREB (Briand and Blendy, 2010). CREB regulates session. Once an animal achieved at least 20 infusions in a single daily session, dynorphin expression, and overexpression of CREB increases the subject was switched to a fixed-ratio 5 (FR5) schedule with the same maximum. dynorphin mRNA (Cole et al., 1995; Carlezon et al., 1998). One region that is both activated by stress and necessary for swim Extinction and Reinstatement stress-induced reinstatement of conditioned place preference Following 19 days of cocaine self-administration, cocaine was replaced is the BNST (Briand and Blendy, 2010). Dynorphin-containing with saline, and extinction phase continued until responding on the active neurons in the BNST project to the VTA (Dong and Swanson, lever was <15% of the response rate maintained by cocaine self-adminis- 2003), and it will be of interest to test whether this projection tration under the FR5 schedule (8 days). Next, an additional extinction is required for the observed block of LTP seen in our session was given and 1 hr later animals were exposed to a 3 min cold-water GABA swim stress. The reinstatement session occurred 24 hr later (Conrad et al., experiments. 2010).

Kappa Receptor Antagonists in Treating Substance Microinjections Abuse Rats were bilaterally microinjected with nor-BNI (2.5 mg) or vehicle in the VTA Kappa opioid receptors have been implicated previously in over a 120 s time period 24 hr prior to the stress exposure. human substance abuse. Polymorphisms in the prodynorphin Preparation of Brain Slices gene promoter region are associated with the risk of cocaine Midbrain slices (250 mm) were prepared as previously described from dependence and abuse (Chen et al., 2002; Dahl et al., 2005; Wil- deeply anesthetized Sprague-Dawley rats (Nugent et al., 2007; Niehaus liams et al., 2007). Prodynorphin gene expression levels are et al., 2010). Slices were stored in oxygenated artificial cerebrospinal fluid higher in individuals with a history of psychostimulant abuse, (Nugent et al., 2007; Niehaus et al., 2010), or (in mM): 86 NaCl, 2.5 KCl, although this may have been true even before the drug abuse 1.2 NaH2PO4, 35 NaHCO3, 20 HEPES, 25 glucose, 5 sodium ascorbate, 3 3 (Hurd and Herkenham, 1993). Our data show that kappa recep- 2 thiourea, 3 sodium pyruvate, 1 MgSO4 7H2O, and 2 CaCl2 2H2O (Zhao et al., 2011). tors in the VTA modulate stress-induced reinstatement, and as discussed above, there is a growing preclinical literature sup- Analysis porting this hypothesis. There is also considerable evidence Data are presented as means ± SEM of the percent change in EPSC amplitude. that KORs contribute to depression, and perhaps most strongly Significance was determined using a two-tailed Student’s t test or a one-way to depression resulting from repeated stress exposure (Knoll and ANOVA with a significance level of p < 0.05. Carlezon, 2010). Kappa opioid receptors are an important regu- latory control on dopamine neurotransmission during stress, and SUPPLEMENTAL INFORMATION may provide a useful strategy for the prevention of stress- Supplemental Information includes four figures and Supplemental Experi- induced relapse (Kreek et al., 2009; Shippenberg, 2009; Bruchas mental Procedures and can be found with this article online at http://dx.doi. et al., 2010; Wee and Koob, 2010). org/10.1016/j.neuron.2012.12.034.

EXPERIMENTAL PROCEDURES ACKNOWLEDGMENTS

Animals for In Vitro Experiments We would like to thank Drs. Jane Sullivan, Jonathan Ting, Pavel Ortinski, and All procedures were carried out in accordance with the guidelines of the Pierce and Kauer lab members for their help and suggestions. This research National Institutes of Health for animal care and use, and were approved by was supported by DA011289 (J.A.K.), MH019118 (N.M.G. and A.M.P.), the Brown University Institutional Animal Care and Use Committee. For AA007459 (A.M.P.) DA026660 (L.A.B.), DA15214 (R.C.P.), and DA18678 morphine and stress experiments, one brain slice per rat was used for (R.C.P.).

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