Article Figures & Data
Figures
- Figure 1.
Heroin self-administration reduced NAc SCN1b, and AAV-shSCN1b mimicked this effect. A, Experimental timeline for tissue collection. B, Heroin versus saline infusions and (C) paired (solid lines) versus unpaired lever presses (dotted lines) from self-administering rats. D, Left, Immunoblotting showed that heroin self-administration reduced NAc SCN1b protein. D, Right, In contrast, immunoblotting showed that experimenter-administered heroin had no effect on NAc SCN1b protein. E, A diagram representation of the NAc stereotaxic injections and an (F) experimental timeline for AAV characterization. G, Quantitative RT-PCR results demonstrated that AAV-shSCN1b reduced NAc SCN1b mRNA. H, Representative images of AAV-shSCN1b expression in the NAc following IHC for coexpressed GFP. Scale bar, 150 um. The circled region is the anterior commissure. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01.
- Figure 2.
SCN1b knockdown increased intrinsic excitability in NAc MSNs. A, Experimental timeline. B, Top, Representative traces of control (green) versus AAV-shSCN1b NAc neurons (black) at 100 pA and B, bottom, at 300 pA. C, AAV-shSCN1b reduced the rheobase of NAc MSNs, but (D) no overall effect on excitability was observed when currents were increased. E, Representative traces of an action potential from control versus AAV-shSCN1b infected neurons. F, AAV-shSCN1b increased the amplitude, (G) rise time, and (H) decay of action potentials. I, As shown by a representative image, (J) AAV-shSCN1b did not alter the AMPA/NMDA ratio. K, As shown by a representative image, (L) AAV-shSCN1b did not alter paired-pulse ratio (50 ms interstimulus interval). Data are expressed as mean ± SEM. #p < 0.10, *p < 0.05, **p < 0.001, and ***p < 0.0001.
- Figure 3.
NAc SCN1b regulates cue-induced heroin-seeking behavior. A, Experimental timeline. B, AAV-shSCN1b had no effect on the number of infusions, (C) paired (solid lines) or unpaired lever presses (dotted lines), (D) the discrimination ratio between paired and unpaired lever presses, or (E) time-out responding on the paired lever during heroin self-administration. F, During extinction training, AAV-shSCN1b had no effect on the number of paired (solid lines) or unpaired lever presses (dotted lines). G, AAV-shSCN1b in the NAc led to increased cued-reinstatement of heroin seeking. H, Heroin versus saline infusions and (I) paired versus unpaired lever presses from self-administering rats. J, Paired versus unpaired lever presses during extinction from the same self-administering rats. K, Immunoblotting showed that abstinence and extinction following heroin self-administration returned NAc SCN1b protein to baseline levels. Data are expressed as mean ± SEM. **p < 0.01.
Tables
Test Groups N (rats) p-value Grubbs’ test Figure 1 B RM two-way ANOVA (interaction) Saline versus heroin (infusions) (7,7) <0.0001 F(11,132) = 7.969 B RM two-way ANOVA (time) Saline versus heroin (infusions) (7,7) 0.1029 F(4.358,52.30) = 1.998 B RM two-way ANOVA (drug) Saline versus heroin (infusions) (7,7) 0.6352 F(1,12) = 0.2370 C RM two-way ANOVA (interaction) Saline versus heroin (paired lever presses) (7,7) <0.0001 F(11,132) = 5.900 C RM two-way ANOVA (time) Saline versus heroin (paired lever presses) (7,7) 0.1536 F(2.948,35.38) = 1.8688 C RM two-way ANOVA (drug) Saline versus heroin (paired lever presses) (7,7) 0.0146 F(1,12) = 8.128 D, left t test Self-administered saline versus heroin (SCN1b/beta-tubulin protein) (7,7) 0.0113 t = 2.987, df = 12 D, right t test Experimenter-administered saline versus heroin (SCN1b/beta-tubulin) (8,8) 0.9861 t = 0.01771, df = 14 G t test AAV-shLUC versus AAV-shSCN1b (3,3) 0.0096 t = 4.659, df = 4 Figure 2 C t test AAV-shLUC versus AAV-shSCN1b—rheobase (19/8, 16/6) 0.0002 t = 4.153, df = 33 One outlier removed D RM two-way ANOVA (interaction) AAV-shLUC versus AAV-shSCN1b—spiking (19/8, 17/6) 0.1783 F(14,476) = 1.342 D RM two-ay ANOVA (time) AAV-shLUC versus AAV-shSCN1b—spiking (19/8, 17/6) <0.0001 F(2.653,90.22) = 23.20 D RM two-way ANOVA (virus) AAV-shLUC versus AAV-shSCN1b—spiking (19/8, 17/6) 0.4492 F(1,34) = 0.5862 F t test AAV-shLUC versus AAV-shSCN1b—amplitude (19/8, 17/6) 0.0149 t = 2.565, df = 34 G t test AAV-shLUC versus AAV-shSCN1b—rise time (18/8, 17/6) 0.0059 t = 2.944, df = 33 One outlier removed H t test AAV-shLUC versus AAV-shSCN1b—decay (18/8, 17/6) 0.0950 t = 1.719, df = 33 One outlier removed J t test AAV-shLUC versus AAV-shSCN1b—AMPA/NMDA ratio (15/5,18/5) 0.9820 t = 0.02268, df=31 One outlier removed L t test AAV-shLUC versus AAV-shSCN1b—paired-pulse ratio (7/3,12/3) 0.3048 t = 1.058, df = 17 One outlier removed Figure 3 B RM two-way ANOVA (interaction) AAV-shLUC versus AAV-shSCN1b—infusions (8,9) 0.8629 F(11,165) = 0.5548 B RM two-way ANOVA (time) AAV-shLUC versus AAV-shSCN1b—infusions (8,9) 0.0001 F(11,165) = 21.78 B RM two-way ANOVA (virus) AAV-shLUC versus AAV-shSCN1b—infusions (8,9) 0.8987 F(1,15) = 0.01676 C RM two-way ANOVA (interaction) AAV-shLUC versus AAV-shSCN1b—paired lever presses (8,9) 0.8693 F(11,165) = 0.5462 C RM two-way ANOVA (time) AAV-shLUC versus AAV-shSCN1b—paired lever presses (8,9) 0.0001 F(11,165) = 7.469 C RM two-way ANOVA (virus) AAV-shLUC versus AAV-shSCN1b—paired lever presses (8,9) 0.6782 F(1,15) = 0.1790 C RM two-way ANOVA (interaction) AAV-shLUC versus AAV-shSCN1b—unpaired lever presses (8,9) 0.6192 F(11,165) = 0.8210 C RM two-way ANOVA (time) AAV-shLUC versus AAV-shSCN1b—unpaired lever presses (8,9) 0.7658 F(11,165) = 0.6696 C RM two-way ANOVA (virus) AAV-shLUC versus AAV-shSCN1b—unpaired lever presses (8,9) 0.9014 F(1,15) = 0.01587 D RM two-way ANOVA (interaction) AAV-shLUC versus AAV-shSCN1b—discrimination ratio (8,9) 0.7076 F(11,165) = 0.7310 D RM two-way ANOVA (time) AAV-shLUC versus AAV-shSCN1b—discrimination ratio (8,9) <0.0001 F(11,165) = 5.414 D RM two-way ANOVA (virus) AAV-shLUC versus AAV-shSCN1b—discrimination ratio (8,9) 0.6216 F(1,15) = 0.2539 E RM two-way ANOVA (interaction) AAV-shLUC versus AAV-shSCN1b—time-out responding (8,9) 0.8836 F(11,165) = 0.5262 E RM two-way ANOVA (time) AAV-shLUC versus AAV-shSCN1b—time-out responding (8,9) 0.0001 F(11,165) = 5.920 E RM two-way ANOVA (virus) AAV-shLUC versus AAV-shSCN1b—time-out responding (8,9) 0.6500 F(1,15) = 0.2144 F RM two-way ANOVA (interaction) AAV-shLUC versus AAV-shSCN1b—extinction paired lever presses (8,9) 0.9450 F(5,75) = 0.2370 F RM two-way ANOVA (time) AAV-shLUC versus AAV-shSCN1b—extinction paired lever presses (8,9) 0.0001 F(5,75) = 21.05 F RM two-way ANOVA (virus) AAV-shLUC versus AAV-shSCN1b—extinction paired lever presses (8,9) 0.5487 F(1,15) = 0.3765 F RM two-way ANOVA (interaction) AAV-shLUC versus AAV-shSCN1b—extinction unpaired lever presses (8,9) 0.9883 F(5,75) = 0.1170 F RM two-way ANOVA (time) AAV-shLUC versus AAV-shSCN1b—extinction unpaired lever presses (8,9) 0.0001 F(5,75) = 12.19 F RM two-way ANOVA (virus) AAV-shLUC versus AAV-shSCN1b—extinction unpaired lever presses (8,9) 0.5620 F(1,15) = 0.3517 G RM two-way ANOVA (interaction) AAV-shLUC versus AAV-shSCN1b—cue-induced reinstatement (8,9) 0.0471 F(1,15) = 4.680 G RM two-way ANOVA (time) AAV-shLUC versus AAV-shSCN1b—cue-induced reinstatement (8,9) 0.0002 F(1,15) = 23.85 G RM two-way ANOVA (virus) AAV-shLUC versus AAV-shSCN1b—cue-induced reinstatement (8,9) 0.0957 F(1,15) = 3.160 G RM two-way ANOVA (virus) Post hoc test following a significant interaction (8,9) 0.0096 H RM two-way ANOVA (interaction) Saline versus heroin (infusions) (6,10) <0.0001 F(11,154) = 10.25 H RM two-way ANOVA (time) Saline versus heroin (infusions) (6,10) 0.3044 F(2.653,37.15) = 1.249 H RM two-way ANOVA (drug) Saline versus heroin (infusions) (6,10) 0.2161 F(1,14) = 1.678 I RM two-way ANOVA (interaction) Saline versus heroin (paired lever presses) (6,10) 0.0001 F(11,154) = 3.646 I RM two-way ANOVA (time) Saline versus heroin (paired lever presses) (6,10) 0.5600 F(2.326,32.56) = 0.6337 I RM two-way ANOVA (drug) Saline versus heroin (paired lever presses) (6,10) 0.1985 F(1,14) = 1.822 J RM two-way ANOVA (interaction) Saline versus heroin—extinction paired lever presses (6,10) <0.0001 F(5,70) = 7.239 J RM two-way ANOVA (time) Saline versus heroin—extinction paired lever presses (6,10) <0.0001 F(2.047,28.66) = 12.77 J RM two-way ANOVA (drug) Saline versus heroin—extinction paired lever presses (6,10) 0.0019 F(1,14) = 14.45 K t test Saline versus heroin plus extinction (SCN1b/beta-tubulin protein) (6,10) 0.2763 t = 1.148, df = 14
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