Article Figures & Data
Figures
- Figure 1.
Differentially enhanced Fos in primary and bystander mice. A, Timeline of data collection. Blue bar represents bystander and control mice, black bar represents primary mice, with corresponding EtOH concentration at different times (% v/v); VF and orange arrows represent von Frey testing at the end of the WD period. B, Primary (n = 6) and bystander (n = 6) mice demonstrate significant decreases in mechanical thresholds compared to separately housed controls (n = 7; F(2,16) = 9.68, p = 0.002). Fos-positive cell counts in three brain regions revealed significant differences among groups, the ACC (C; bregma 1.1-0.5), INS (D; bregma 1.1-0.5), and the DMH (E; bregma -1.4 to -0.94). Brain regions are shown schematically in the top left of each panel, with representative photomicrographs of each treatment group in corresponding order on the right (bystander, black bar/top right; primary, white bar/middle right; control, striped bar/bottom right; scale bar, 100 μm). Bystander mice displayed an increase in the number of c-Fos cells in the ACC (F(2,14) = 4.8, p = 0.026), and in INS (F(2,15) = 3.8, p = 0.046) compared to the controls. By contrast, the primary group displayed enhanced Fos-ir in the DMH (F(2,16) = 4.8, p = 0.007). *p < 0.05 compared to the control group based on Fishers LSD.
- Figure 2.
Inhibition of ACC, but not somatosensory cortex reverses hyperalgesia in primary and bystander mice. A, Timeline of data collection and experimental manipulation: Sfx refers to surgery, which took place 7-14 d before beginning of experiments. Blue bar represents bystander and control mice, black bar represents primary mice, with corresponding EtOH % (v/v); VF and orange arrows represent von Frey testing at end of 24-h abstinence; WD represents withdrawal from alcohol; black syringe represents CNO injection (20 min before the second mechanical test). B, C, Overlapping pattern of viral expression (blue) for each treatment group in the ACC and SI. Representative photomicrographs of hM4Di viral expression, within the (D) ACC (orange) with DAPI in blue (E) SI (orange) and DAPI in blue. Left panels: scale bars, 100 μm; right panels: scale bars, 20 μm. F, Animals in which DREADDs were expressed in ACC and given Veh (n = 5, primary; n = 5, bystander) showed a significant decrease in threshold compared to Veh-treated separately housed control animals (n = 8). By contrast, animals given CNO before testing (n = 6, primary; n = 6, bystander) showed no hyperalgesia compared to CNO-treated controls (n = 6). According to ANOVA, this led to a significant difference of treatment (F(1,30) = 4.79, p = 0.037), as well as a significant interaction (F(2,30) = 3.37, p = 0.048). CNO groups were no longer significantly different from separately housed controls according to Fishers LSD. G, Bystander (n = 5-7/group) and primary (n = 6/group) mice bilaterally transfected with the hM4Di DREADD virus in SI demonstrated significant decreases in mechanical thresholds on the second WD session compared to separately housed controls (n = 5-6), leading to significant differences between groups (F(2,29) = 14.88, p < 0.0001), but no significant effects of CNO treatment or an interaction, indicating that inactivation of SI had no effect on hypersensitivity. Mean basal responses of all groups represented by dotted line (—). *p < 0.05 compared to controls receiving the same treatment, according to Fishers LSD.
Tables
Data structure Type of test CI Data structure Type of test CI Table 2, row 1; Fig. 1C Automated quantification of Fos+ cells in CG1 from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA with significant main effect of group followed by Fishers LSD n = 5-7 *p < 0.05 CTRL: (119.9-193.3) primary: (147.1-294.4) bystander: (195.9-308.0) Table 2, row 14 Manual quantification of Fos+ cells in posteromedial cortical amygdaloid from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA n = 5-6 p > 0.05 CTRL: (5.040-14.10) primary: (4.533-10.61) bystander: (7.170-10.27) Table 2, row 2 Automated quantification of Fos+ cells in CG2 from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA n = 5-7 p > 0.05 CTRL: (62.64-86.59) primary: (23.61-126.7) bystander: 41.03-113.6) Table 2, row 15 Manual quantification of Fos+ cells in basolateral amygdala from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA n = 5-6 p > 0.05 CTRL: (6.156-19.07) primary: (5.469-18.79) bystander: (5.186-30.38) Table 2, row 3 Automated quantification of Fos+ cells in GI from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA n = 5-7 p > 0.05 CTRL: (12.35-25.82) primary: (16.69-30.81) bystander: (8.518-40.40) Table 2, row 16 Manual quantification of Fos+ cells in central nucleus of the amygdala from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA n = 5-6 p > 0.05 CTRL: (5.992-12.13) primary: (2.869-8.797) bystander: (3.828-16.21) Table 2, row 4; Fig. 1D Automated quantification of Fos+ cells in INS from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA with significant main effect of group followed by Fishers LSD n = 5-6 *p < 0.05 CTRL: (25.97-45.22) primary: (27.64-74.08) bystander: (29.72-116.3) Table 2, row 17 Manual quantification of Fos+ cells in paraventricular nucleus from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA n = 5-6 p > 0.05 CTRL: (1.755-8.645) primary: (7.872-12.30) bystander: (0.972-25.83) Table 2, row 5 Automated quantification of Fos+ cells in S1 from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA n = 5-6 p > 0.05 CTRL: (432.5-651.8) primary: (191.4-1128) bystander: (262.4-636.9) Table 2, row 18 Quantification of Fos+ cells in the DMH from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA with significant main effect followed by Fishers LSD n = 6-7 **p = 0.007 CTRL: (10.64-21.66) primary: (17.37-43.81) bystander: (15.25-26.22) Table 2, row 6 Manual quantification of Fos+ cells in dorsal lateral septum from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA n = 5-6 p > 0.05 CTRL: (3.120-10.67) primary: (1.763-12.15) bystander: (3.195-9.805) Table 2, row 19 Manual quantification of Fos+ cells in the centrally projecting Edinger Westphal from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA n = 5-6 p > 0.05 CTRL: (4.866-11.11) primary: (4.261-20.06) bystander: (6.974-15.78) Table 2, row 7 Manual quantification of Fos+ cells in intermediate lateral septum from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA n = 5-6 p > 0.05 CTRL: (18.13-29.51) primary: (12.99-25.89) bystander: (20.04-27.38) Table 2, row 20 Manual quantification of Fos+ cells in periaqueductal gray from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA n = 5-6 p > 0.05 CTRL: (27.73-62.22) primary: (30.75-129.2) bystander: (42.31-85.50) Table 2, row 8 Manual quantification of Fos+ cells in ventral lateral septum from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA n = 5-6 p > 0.05 CTRL: (11.23-16.07) primary: (5.770-22.95) bystander: (5.559-18.98) Table 2, row 21 Manual quantification of Fos+ cells in substantia nigra from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA n = 5-6 p > 0.05 CTRL: (-0.945-10.30) primary: (1.372-14.46) bystander: (2.184-10.03) Table 2, row 9 Manual quantification of Fos+ cells in nucleus accumbens from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA n = 5-6 p > 0.05 CTRL: (5.400-37.91) primary: (10.44-41.08) bystander: (15.03-31.01) Table 2, row 22 Manual quantification of Fos+ cells in ventral tegmental Area from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA n = 5-6 p > 0.05 CTRL: (-1.562-8.729) primary: (-4.473-20.07) bystander: (-9.419-31.42) Table 2, row 10 Manual quantification of Fos+ cells in anterior bed nucleus of the stria terminalis from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA n = 5-6 p > 0.05 CTRL: (25.51-36.28) primary: (19.48-76.52) bystander: (25.76-52.53) Fig. 1B Comparison of mechanical thresholds of each group over 3 test sessions Two-way RM ANOVA, significant for test session n = 6-7 *p < 0.05 CTRL: (1.521-2.143) primary: (-0.255-2.787) bystander: (0.031-2.950) Table 2, row 11 Manual quantification of Fos+ cells in posterior bed nucleus of the stria terminalis from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA n = 5-6 p > 0.05 CTRL: (-1.066-17.96) primary: (0.4374-5.382) bystander: (1.829-7.948) Fig. 2F Comparison of mechanical thresholds of each group following treatment with Veh or CNO on the second test session (inhibition of the ACC) Two-way ANOVA, significant main effect of treatment and significant interaction followed by Fishers LSD n = 5-7 *p < 0.05 CTRL/Veh: (0.76-1.68) CTRL/CNO: (0.70-1.54) bystander/VEH: (0.31-0.76) bystander/CNO: (0.39-1.85) primary/Veh: (0.16-0.65) primary/CNO:(0.36-2.14) Table 2, row 12 Manual quantification of Fos+ cells in dentate gyrus from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA n = 5-6 p > 0.05 CTRL: (24.09-29.40) primary: (22.46-55.44) bystander: (28.60-38.16) Fig. 2G Comparison of mechanical thresholds of each group following treatment with Veh or CNO on the second test session (inhibition of S1) Two-way ANOVA, significant main effect of group, no interaction n = 5-7 *p < 0.0001 CTRL/Veh: (-7.73-11.46) CTRL/CNO: (0.368-2.32) bystander/VEH: (-0.088-1.23) bystander/CNO: (-4.63-6.18) primary/Veh: (-0.62-1.91) primary/CNO:(-0.80-3.36) Table 2, row 13 Manual quantification of Fos+ cells in posterolateral cortical amygdaloid from two separate reactions. Counts were averaged between the 2 runs, with 2-4 slices for each region per mouse per batch (average of 4-8 slices per mouse) One-way ANOVA n = 5-6 p > 0.05 CTRL: (18.99-30.39) primary: (8.949-25.65) bystander: (13.75-19.45) Brain area Control Primary Bystander ANOVA Anterior cingulate (CG1) 156.6 ± 14.28 220.8 ± 28.66 251.9 ± 20.18* F(2,14) = 4.77 Anterior cingulate (CG2) 73.56 ± 5.65 75.14 ± 20.05 77.33 ± 14.12 F(2,15) = 0.017 GranularInsula (GI) 19.08 ± 2.752 23.89 ± 2.695 24.46 ± 6.201 F(2,16) = 0.547 Agranular insula (INS) 36.1 ± 3.73 50.86 ± 9.032 73.02 ± 15.59* F(2,15) = 3.779 Somatosensory 542.1 ± 44.79 659.8 ± 168.7 449.6 ± 76.52 F(2,16) = 1.147 Dorsal lateral septum 6.893 ± 1.542 6.958 ± 2.021 6.5 ± 1.351 F(2,17) = 0.023 Intermediate lateral septum 23.82 ± 2.326 23.71 ± 1.429 19.44 ± 2.637 F(2,17) = 1.250 Ventral lateral septum 13.65 ± 0.991 14.36 ± 3.342 12.27 ± 2.743 F(2,17) = 0.183 Nucleus accumbens 21.65 ± 6.323 25.76 ± 5.959 23.02 ± 3.265 F(2,16) = 0.155 Bed nucleus of the stria terminalis (anterior) 30.89 ± 2.2 48 ± 11.1 39.14 ± 5.47 F(2,17) = 1.561 Bed nucleus of the stria terminalis (posterior) 20.53 ± 8.444 25.45 ± 2.91 23.33 ± 4.889 F(2,15) = 1.472 Dentate gyrus 36.19 ± 5.337 39.14 ± 6.744 31.27 ± 6.609 F(2,17) = 0.4011 Posterolateral cortical amygdaloid 24.69 ± 2.328 17.3 ± 3.412 16.6 ± 1.027 F(2,16) = 2.845 Posteromedial cortical amygdaloid 9.571 ± 1.852 7.571 ± 1.242 8.722 ± 0.604 F(2,17) = 0.5452 Basolateral amygdala 12.61 ± 12.61 12.13 ± 2.592 17.78 ± 4.901 F(2,15) = 0.7956 Central nucleus of the amygdala 9.063 ± 1.194 5.833 ± 1.153 10.02 ± 2.409 F(2,15) = 1.687 Paraventricular nucleus 3.4 ± 0.75 11.17 ± 3.55 12.13 ± 3.29 F(2,12) = 2.584 Dorsal medial hypothalamus 16.15 ± 2.251 30.59 ± 5.143** 20.73 ± 2.242 F(2,16) = 4.842 Centrally projecting Edinger Westphal 7.988 ± 1.276 12.16 ± 3.073 11.38 ± 1.799 F(2,17) = 1.150 Periaqueductal gray 44.98 ± 7.049 79.96 ± 19.14 63.9 ± 8.827 F(2,17) = 2.073 Substantia nigra 4.679 ± 2.298 7.917 ± 2.546 6.107 ± 1.603 F(2,17) = 0.5495 Ventral tegmental area 2.93 ± 1.02 4.88 ± 2.54 9.43 ± 4.66 F(2,15) = 1.115 Mean (±SEM) c-Fos-positive cell counts for experimental each group per brain area examined. ANOVA values are presented in the right column, with significant main effects of group in bold. *p < 0.05 contrast to controls, Fishers LSD.
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