Article Figures & Data
Figures
- Figure 1.
GABAergic IPSCs in mice display NO-induced long–term potentiation (LTPGABA). A, Experimental design. Brain slices were prepared 24 h after acute stress or no stress; recordings from dopamine neurons were carried out with or without norBNI. B–D, Representative time courses and example IPSCs from electrophysiological recordings in slices from (B) control mice, (C) mice stressed 24 h previously, and (D) mice stressed 24 h previously with bath application of norBNI to the slice. All IPSCs in this and other figures were evoked with a rostrally placed electrical stimulating electrode. E, Time courses of averaged IPSC amplitudes (left) and IPSC amplitudes before and after SNAP application (right) in neurons in unstressed (n = 16 cells/10 mice, 12 cells from male mice, 4 from female mice; paired t test; p = 0.007), stressed (n = 8 cells/8 mice, 6 cells from male mice, 2 from female mice; paired t test; p = 0.61) or stressed + norBNI conditions (n = 7 cells/7 mice, 5 cells from male mice, 2 from female mice; paired t test; p = 0.04).
- Figure 2.
NAc- and PFC-projecting VTA dopamine neurons receive synapses that display LTPGABA. A, Experimental design for B and C. Neurons from NAc-projecting DA neurons were identified in slices with retrobeads. B, Representative time course and example IPSCs from NAc-projecting DA neurons before and after SNAP application. C, Time courses of averaged IPSC amplitudes (left) and IPSC amplitudes before and after SNAP application (right; NAc-projecting, n = 14 cells/12 mice, 10 cells from males, 4 from females; paired t test; p = 0.04). D, Experimental design for E and F. Neurons from PFC-projecting DA neurons were identified in slices with retrobeads. E, Representative time course and example IPSCs from PFC-projecting DA neurons before and after SNAP application. F, Time courses of averaged IPSC amplitudes (left) and IPSC amplitudes before and after SNAP application (right; PFC-projecting; n = 13 cells/10 mice, 5 cells from males, 8 from females; paired t test; p = 0.04).
- Figure 3.
Global deletion of kORs prevents stress-induced block of LTPGABA. A, Experimental design using slices from global knock-out mice (kOR−/−). B, Representative confocal images showing horizontal VTA and NAc sections from wild-type and kOR−/− mice validating global deletion of kORs. Scale bar, 30 μm. C, Representative time course and example IPSCs before and after SNAP application in a brain slice from a kOR−/− mouse. D, Time courses of averaged IPSC amplitudes (left) and IPSC amplitudes before and after SNAP application (right) in recordings from neurons from kOR−/− mice stressed 24 h previously (n = 9 cells/7 mice, 6 cells from male mice, 3 from female mice; Wilcoxon matched-pair signed–rank test; p = 0.04).
- Figure 4.
Deleting kORs from dopamine neurons does not prevent stress-induced block of LTPGABA. A, Experimental design using mice that lack kOR in dopamine neurons (kOR−/−DA). B, Representative confocal images showing horizontal VTA and NAc sections from kOR−/−DA mice validating selective deletion of kORs from dopamine neurons. Scale bar, 30 μm. C, Time courses of averaged IPSC amplitudes before and after SNAP application 24 h after stress for D and E and F and G experiments. D, Representative time course, example IPSCs and (E) IPSC amplitudes before and after SNAP application in recordings from neurons from kOR−/−DA mice 24 h after stress (n = 12 cells/11 mice, 5 cells from male mice, 6 from female mice; Wilcoxon matched-pair signed–rank test; p = 0.34). F, Representative time course, example IPSCs and (G) IPSC amplitudes before and after SNAP application in recordings in the presence of norBNI from neurons from stressed kOR−/−DA mice (n = 10 cells/9 mice, 5 cells from male mice, 5 from female mice; paired t test; p = 0.01).
- Figure 5.
NAc-to-VTA but not LH-to-VTA GABAergic afferents display stress-sensitive LTPGABA, which is lost upon deleting kOR from NAc neurons. A, Experimental design for B and C. B, Representative time course and example LH-oIPSCs, and (C) time course of averaged LH-oIPSC amplitudes and LH-oIPSC amplitudes before and after SNAP application from control unstressed mice (n = 9 cells/7 mice, 4 cells from male mice, 5 from female mice; paired t test; p = 0.51). D, Experimental design for E and F. E, Representative time course, example NAc-oIPSCs, and (F) time course of averaged NAc-oIPSC amplitudes and NAc-oIPSC amplitudes before and after SNAP application from unstressed control mice (n = 20 cells/17 mice, 12 cells from male mice, 8 from female mice; Wilcoxon matched-paired signed–rank test; p = 0.03). G, Experimental design for H and I. H, Representative time course, example NAc-oIPSCs, and (I) time course of averaged NAc-oIPSC amplitudes and NAc-oIPSC amplitudes before and after SNAP application from mice stressed 24 h previously (n = 8 cells/6 mice, 5 cells from male mice, 3 from female mice; paired t test; p = 0.35). J, Experimental design for K and L using mice in which we optogenetically drove GABAergic NAc terminals from which kORs were deleted (kOR−/−NAc). K, Representative time course and example oIPSCs before and after SNAP application from stressed mice lacking kORs in NAc terminals in the VTA. L, Time course of averaged oIPSC amplitudes and oIPSC amplitudes before and after SNAP application from mice stressed 24 h previously (n = 7 cells/6 mice, 6 cells from male mice, 1 from a female mouse; Wilcoxon matched-pair signed–rank test; p = 0.03).
- Figure 6.
Chemogenetically activating dynorphin-containing neurons in the NAc mimics stress-induced block of LTPGABA. A, Experimental design. Chemogenetic activation of pdyn neurons in the NAc. B, Representative time course and example IPSC amplitudes and (C) IPSC amplitudes before and after SNAP application in slices from unstressed mice that received saline 24 h before slice preparation (n = 9 cells/7 mice, 4 cells from males, 5 from females; paired t test; p = 0.01). D, Representative time course and example IPSC amplitudes and (E) IPSC amplitudes before and after SNAP application in slices from unstressed mice that received clozapine 24 h before slice preparation (n = 7 cells/5 mice, 4 cells from males, 3 from females; paired t test; p = 0.44). F, Time courses of averaged IPSC amplitudes for C and E. G, Schemes illustrating hM3Dq injection targeting NAc from all animals.
- Figure 7.
Activation of VTA kORs reinstates cocaine-seeking behavior. A, Experimental design for cocaine subjects. B, Cannula placement verification representative image (left) and cannula placements for all animals (right). Purple dots represent U50488H injections; black dots represent control injections. C, D, Active (blue circles) and inactive (white triangles) lever presses and cocaine infusions (green squares) for Group 1 (C) and Group 2 (D) animals during 10 d of cocaine self-administration maintenance. There is no difference between groups at this point in the experiment. E, Active lever presses on the first and last day of extinction training for each group (n = 8 rats/group, 2-way RM ANOVA; main effect of day; p < 0.0001). F, Reinstatement score (calculated as active lever responses on last extinction session subtracted from active lever responses during reinstatement test) of animals given an intra-VTA injection of either U50488H or saline (n = 8 rats/group; Mann–Whitney test; U = 8; p = 0.01). G, Active lever responses per 10 min bin during the 2 h reinstatement test (n = 8 rats/group; 2-way RM ANOVA; main effect of treatment; p = 0.04). H, Experimental design for sucrose subjects. I, Cannula placements for sucrose animals. Pink dots represent U50488H injections; black dots represent control injections. J, Active lever responses for each group on Day 10 of maintenance (10), and first (F) and last (L) days of extinction for animals trained to self-administer sucrose pellets. There is no difference between groups at this point in the experiment. K, Reinstatement scores of animals trained to self-administer sucrose pellets, 10 min after receiving an intra-VTA injection of either U50488H or saline. *Indicates p < 0.05; *** indicates p < 0.0001.
- Figure 8.
VTA dopamine neurons are controlled by different inhibitory afferents that exhibit distinct synaptic plasticity properties. (1) Stress causes the release of dynorphin into the VTA. (2) Dynorphin binds to kORs on GABAergic terminals from the NAc to block LTPGABA. Local VTA GABAergic neurons are similarly affected (Polter et al., 2018). (3) Dopamine neurons express kORs, but these do not control LTPGABA. (4) Exogenously delivered U50488 mimics stress-induced release of dynorphin, reinstating cocaine-seeking behavior.
Tables
Figure Sample size (cells) Male (mice/cells) Female (mice/cells) Mean SEM Normality test Test performed p value Coefficient 1E Electrical LTP control 16 6/12 4/4 −330.5 46.4 Passed Paired t test 0.007 t = 3.102; df = 15 −479.8 75.8 Electrical LTP stress 8 6/6 2/2 −652.1 193.1 Passed Paired t test 0.610 t = 0.5336; df = 7 −617.2 170.4 Electrical LTP norBNI 9 7/7 2/2 −291.8 71.5 Passed Paired t test 0.036 t = 2.509; df = 8 −351.7 76.3 2C NAc projecting 14 8/10 4/4 −419.4 56.4 Passed Paired t test 0.041 t = 2.269; df = 13 −492.6 78.5 2F PFC projecting 13 3/5 7/8 −258.3 40.57 Passed Paired t test 0.045 t = 2.243; df = 12 −286.3 43.1 3D KOR−/− 9 5/6 2/3 −305.6 28.9 Not passed Wilcoxon matched-pair signed–rank test 0.039 W = −35 −390.5 51.2 4E KOR−/− DA 12 5/5 7/7 −388.1 78.8 Not passed Wilcoxon matched-pair signed–rank test 0.339 W = 26 −371.6 82.0 4G KOR−/− DA norBNI 10 4/5 5/5 −181.7 28.6 Not passed Wilcoxon matched-pair signed–rank test 0.010 W = −49 −259.4 39.7 5C LH opto 9 4/4 3/5 −364.7 54.6 Passed Paired t test 0.517 t = 0.6777; df = 8 −337.7 63.7 5F NAc opto control 20 9/12 8/8 −262.7 29.0 Not passed Wilcoxon matched-pair signed–rank test 0.030 W = −116 −314 40.2 5I NAc opto stress 8 3/5 3/3 −472.7 57.9 Passed Paired t test 0.347 t = 1.009; df = 7 −415.6 54.0 5L NAc KO stress 7 5/6 1/1 −210.3 15.8 Not passed Wilcoxon matched-pair signed–rank test 0.031 W = −26 −284.9 46.8 6C Chemogenetic saline 9 4/4 3/5 −416.2 48.1 Passed Paired t test 0.007 t = 3.485; df = 9 −540.9 62.5 6E Chemogenetic clozapine 7 3/4 2/3 −288.6 49.7 Passed Paired t test 0.436 t = 0.8349; df = 6 −269.4 47.1 7F Cocaine extinction (last day) n/a 16 0 4.375 1.1 Passed Two-way repeated–measure ANOVA 0.000 F = 47.24; df = 1, 14 5.375 0.9 7G Cocaine self-administration n/a 16 0 6 4.9 Not passed Mann–Whitney test 0.010 U = 8 20 3.8 7J Sucrose self-administration n/a 11 0 2.4 7.6 Not passed Mann–Whitney test 0.530 U = 13 4.8 8.6 For each dataset, the number and sex of mice, number of cells analyzed, the mean ± standard error of the mean (SEM), and references to the corresponding figures are provided. The outcome of normality testing is indicated (passed/not passed), and the statistical test used to assess significance is listed along with its relevant parameters (p values, coefficients).
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