Constitutive activation of kappa opioid receptors at ventral tegmental area inhibitory synapses following acute stress

Abstract

Stressful experiences potently activate kappa opioid receptors (κORs). κORs in the ventral tegmental area regulate multiple aspects of dopaminergic and non-dopaminergic cell function. Here we show that at GABAergic synapses on rat VTA dopamine neurons, a single exposure to a brief cold-water swim stress induces prolonged activation of κORs. This is mediated by activation of the receptor during the stressor followed by a persistent, ligand-independent constitutive activation of the κOR itself. This lasting change in function is not seen at κORs at neighboring excitatory synapses, suggesting distinct time courses and mechanisms of regulation of different subsets of κORs. We also provide evidence that constitutive activity of κORs governs the prolonged reinstatement to cocaine-seeking observed after cold water swim stress. Together, our studies indicate that stress-induced constitutive activation is a novel mechanism of κOR regulation that plays a critical role in reinstatement of drug seeking.

Keywords: GABA; Kappa Opioid Receptor; Stress; Synaptic Plasticity; VTA; dopamine; neuroscience; rat.

Figure 1.. norBNI rescues LTP_GABA through activation of JNK.

(A) Summary data showing the blockade of LTP_GABA after stress. (B) Comparison of the magnitude of LTP_GABA10–15 min after SNAP application. (IPSC amplitudes, control: 140 ± 5% of baseline values, n = 13; stress: 94 ± 11% of baseline values, n = 6; unpaired t-test, *p=0.0005. (C) Schematic of norBNI’s competitive and non-competitive inhibition of κOR signaling. (D) Experimental design. (E) Representative single experiment showing that bath application of norBNI (100 nM) rescues LTP_GABA in a slice prepared 24 hr after stress. (F) Representative single experiment from a slice prepared 24 hr after stress showing that norBNI does not rescue LTP_GABA in the presence of the JNK inhibitor SP600125 (20 µM). (G) Summary data from both groups. (H) Comparison of the magnitude of LTP_GABA10–15 min after SNAP application. (IPSC amplitudes, norBNI only: 139 ± 7% of baseline values, n = 6; norBNI+SP600125: 106 ± 9% of baseline values, n = 11; unpaired t-test, *p=0.029.) Insets for this and all figures: IPSCs before (black trace, control) and 15 min after drug application (red trace, SNAP, 400 µM). Scale bars: 20 ms, 100 pA. Insets are averages of 12 IPSCs. DOI: http://dx.doi.org/10.7554/eLife.23785.003

Figure 1—figure supplement 1.. Inhibition of JNK does not affect LTP_GABA or its block by stress in the absence of norBNI.

(A) Summary data showing that LTP_GABA is expressed in slices from naïve animals in the presence of the JNK inhibitor SP600125 (20 µM). Average magnitude of LTP_GABA10–15 min after SNAP = 144 ± 13% of baseline values, n = 5; one-sample t-test, p=0.0257. (B) Summary data showing that LTP_GABA remains blocked in slices from stressed animals in the presence of the JNK inhibitor SP600125 (20 µM). Average magnitude of LTP_GABA10–15 min after SNAP = 111 ± 11% of baseline values, n = 6; one-sample t-test, p=0.38. DOI: http://dx.doi.org/10.7554/eLife.23785.004

Figure 2.. The neutral antagonist 6β-naltrexol fails to rescue LTP_GABA in slices from stressed animals.

(A) Schematic of norBNI and 6β-naltrexol inhibition of κOR signaling. (B) Experimental design. (C) Representative experiment showing that bath application of norBNI (100 nM) rescues LTP_GABA in a slice prepared 24 hr after stress. (D) Representative experiment from a cell 24 hr after stress showing that 6β-naltrexol (10 µM) fails to rescue LTP_GABA. (E) Summary data from both groups. (F) Comparison of the magnitude of LTP_GABA10–15 min after SNAP application. (IPSC amplitudes, norBNI: 141 ± 20% of baseline values, n = 10; 6β-naltrexol: 100 ± 8% of baseline values, n = 10; unpaired t-test, *p=0.048). DOI: http://dx.doi.org/10.7554/eLife.23785.005

Figure 2—figure supplement 1.. 6β-naltrexol does not affect basal inhibitory synaptic transmission but does block κORs.

(A) Summary data showing that 6β-naltrexol (10 µM) does not affect basal inhibitory transmission in cells from control or stressed rats. Normalized IPSC amplitude 5–10 min after 6β-naltrexol: control = 97 ± 6% of baseline values, n = 6 (4 animals); stress = 92 ± 17% of baseline values, n = 4 (2 animals); unpaired t-test p=0.79. (B) Summary data showing that U50488 depresses EPSC amplitudes from VTA dopamine neurons and that 6β-naltrexol (10 µM) prevents this depression. Normalized EPSC amplitude 5–10 min after U50488: U50488 alone = 75 ± 2% of baseline values, n = 3; U50488 + 6β-naltrexol = 95 ± 1% of baseline values, n = 4; unpaired t-test *p=0.0003. DOI: http://dx.doi.org/10.7554/eLife.23785.006

Figure 3.. 6β-naltrexol rescues LTP_GABA when administered pre-stress, but not post-stress.

(A) Experimental design. (B) Representative experiment showing that a cell from a vehicle-treated stressed animal does not exhibit LTP_GABA. (C) Representative experiment showing that a cell from an animal treated with 6β-naltrexol (10 mg/kg) 30 min pre-stress exhibits LTP_GABA. (D) Representative experiment showing that a cell from an animal treated with 6β-naltrexol 24 hr post-stress does not exhibit LTP_GABA. (E) Summary data showing compiled data from all groups. (F) Comparison of the magnitude of LTP_GABA 10–15 min after SNAP application. (1-way ANOVA followed by Dunnett’s multiple comparison test. F_{2, 30}=4.231,p=0.024. IPSC amplitudes, 6β-naltrexol pre-stress: 136 ± 12% of baseline values, n = 12, p<0.05 from vehicle; 6β-naltrexol post-stress: 100 ± 9% of baseline values, n = 11, n.s. from vehicle; vehicle+stress: 102 ± 8% of baseline values, n = 10). DOI: http://dx.doi.org/10.7554/eLife.23785.007

Figure 4.. Single treatment with a κOR agonist leads to prolonged blockade of LTP_GABA.

(A) Experimental design. (B) Representative experiment showing that a cell from a saline-treated animal exhibits LTP_GABA. (C) Representative single experiment showing a cell prepared 24 hr after a single treatment with U50488 (5 mg/kg) does not exhibit LTP_GABA. (D) Representative experiment showing that a cell prepared five days after a single treatment with U50488 does not exhibit LTP_GABA. (E) Summary data from all groups. (F) Comparison of the magnitude of LTP_GABA10–15 min after SNAP application. (1-way ANOVA followed by Dunnett’s multiple comparison test. F_{2, 27}=12.21, p=0.0002. IPSC amplitudes, Saline: 137 ± 6% of baseline values, n = 11; U50488 1 day: 108 ± 6% of baseline values, n = 9, p<0.05 vs. saline; U50488 5 days: 100 ± 5% of baseline values, n = 10, p<0.05 vs. saline). DOI: http://dx.doi.org/10.7554/eLife.23785.008

Figure 5.. κORs at VTA excitatory synapses are not constitutively activated by stress.

(A) Representative experiment showing that norBNI (100 nM) does not potentiate excitatory synapses on I_h+ VTA neurons in a slice prepared from a control animal. (B) Representative experiment showing that norBNI does not potentiate excitatory synapses on I_h+ VTA neurons in a slice prepared from a stressed animal. (C) Summary data from I_h+ neurons. No significant difference in IPSC amplitude 10–15 min after norBNI application (t-test p=0.81 IPSC amplitudes, control: 94 ± 2% of baseline values, n = 5; stressed: 92 ± 6% of baseline values, n = 6). (D) Representative experiment showing that norBNI does not potentiate excitatory synapses on I_h− VTA neurons in a slice prepared from a control animal. (E) Representative single experiment showing that norBNI does not potentiate excitatory synapses on I_h− VTA neurons in a slice prepared from a stressed animals. (F) Summary data from I_h− neurons. No significant difference in IPSC amplitude 10–15 min after norBNI application (t-test p=0.49 IPSC amplitudes, control: 112 ± 2% of baseline values, n = 5; stressed: 110 ± 4% of baseline values, n = 5). DOI: http://dx.doi.org/10.7554/eLife.23785.009

Figure 6.. Post-stress rescue of reinstatement by norBNI but not 6β-naltrexol.

(A) Experimental design. (B) Lever pressing during the final extinction session (white bar) and reinstatement session (colored bar). Saline (black): last extinction session: 6.4 ± 1.7 lever presses; reinstatement session: 13.8 ± 2.4 lever presses; n = 8, *p=0.011, paired t-test. norBNI (green): last extinction session: 4.8 ± 1.4 lever presses; reinstatement session: 5.2 ± 1.1 lever presses; n = 6, p=0.76, paired t-test. 6β-naltrexol (blue): last extinction session: 5.7 ± 1.8 lever presses; reinstatement session: 11.2 ± 3.4 lever presses; n = 11, *p=0.033, paired t-test. DOI: http://dx.doi.org/10.7554/eLife.23785.010

Figure 7.. Constitutive activation of κORs by stress.

(A) During stress, dynorphin binding to the κOR triggers a shift to a constitutively active state. By blocking dynorphin binding, both norBNI and 6β-naltrexol prevent the loss of LTP_GABA during this time. (B) After stress, the block of LTP_GABA is maintained by constitutive activity of κORs and is no longer dependent on dynorphin binding. (C) norBNI reverses the stress-induced block of LTP_GABA by activating the JNK signaling pathway which non-competitively reduces κOR activity. DOI: http://dx.doi.org/10.7554/eLife.23785.011