Article Figures & Data
Figures
- Figure 1.
Effect of fentanyl relapse on activity in OFC and Pir cells expressing Cnr1, Drd1, and Drd2. A, Timeline of experiment 1. B, Self-administration. Number of reinforced responses (food: 5 pellets/reinforcer; fentanyl 2.5 µg/kg/infusion) during the 6-h sessions. C, Discrete choice (voluntary reduction in self-administration). Number of food-reinforced responses and fentanyl infusions earned during the 3-h choice sessions (20 trials/session). D, Relapse tests. Number of active and inactive lever presses during the 60-min test session (left) and the 15-min time course (right). E, From left to right, Number of Fos+ cells per mm2, number of Cnr1+ cells per mm2, number of Fos+Cnr1 double-labeled cells in OFC and Pir, number of Cnr1+Vgat double-labeled cells per mm2, and number of Fos+Cnr1+Vgat triple-labeled cells per mm2 in OFC. Representative images showing Fos (white), Cnr1 (green), or Vgat (red)-expressing cells (20× magnification, scale bar = 25 µm). White arrow denotes Fos-positive cell, green arrow denotes Cnr1-positive cell, and red arrow denotes Vgat-positive cells. Double-labeled cells are denoted by both a white and green arrow. Triple-labeled cells are denoted by a white, green, and red arrow. F, From left to right, Number of Fos+ cells per mm2, number of Drd1+ and Drd2+ cells per mm2, and number of Fos+Drd1 and Fos+Drd2 double-labeled cells in OFC and Pir. Representative images showing Fos (white), or Drd1 (red), Drd2 (green; 20× magnification, scale bar = 25 µm). White arrow denotes Fos-positive cell, red arrow denotes Drd1-positive cell, and green arrow denotes Drd2-positive cell. Double-labeled cells are denoted by both a white and green or red arrow (n = 6–8 per group); *p ≤ 0.05, different from the No Test group (E, F). Data are mean ± SEM. Individual data are shown separately by sex (males = circles, females = triangles) in D–F. OFC, orbitofrontal cortex; Pir, piriform cortex.
- Figure 2.
Effect of CB1 receptor blockade in OFC on relapse to fentanyl seeking. A, Timeline of experiment 2. B, Self-administration. Number of reinforced responses (food: 5 pellets/reinforcer; fentanyl 2.5 µg/kg/infusion) during the 6-h sessions. C, Discrete choice (voluntary reduction in self-administration). Number of food-reinforced responses and fentanyl infusions earned during the 3-h choice sessions (20 trials/session). D, Relapse test. Number of active and inactive lever presses during the 3-h test session (left) and 1-h time course (right) after vehicle or AM251 injections (CB1 receptor antagonist). E, Reacquisition test. Number of fentanyl infusions (2.5 µg/kg/infusion) during the 6-h session (left) and 1-h time course (right) after vehicle or AM251 injections in OFC (n = 12–20 per dose, between-subjects design). Data are mean ± SEM. Individual data are shown separately by sex (males = circles, females = triangles) in D, E. F, Images showing placement of cannula into OFC at 1.25× magnification (scale bar = 1 mm). Vehicle placements are shown with white circles, 0.3 µg AM251 with gray circles, and 1.0 µg AM251 with black circles.
- Figure 3.
Effect of CB1 receptor agonism in OFC on relapse to fentanyl seeking. A, Timeline of experiment 3. B, Self-administration. Number of reinforced responses (food: 5 pellets/reinforcer; fentanyl 2.5 µg/kg/infusion) during the 6-h sessions. C, Discrete choice (voluntary reduction in self-administration). Number of food-reinforced responses and fentanyl infusions earned during the 3-h choice sessions (20 trials/session). D, Relapse test. Number of inactive (left) and active (right) lever presses during the 3-h test session after vehicle or WIN55,212-2 OFC injections (CB1 receptor agonist). E, Reacquisition test. Number of fentanyl infusions (2.5 µg/kg/infusion) during the 6-h session after vehicle or WIN55,212-2 injections in OFC (n = 5 per group in D, n = 5–6 per group in E, mixed within/between-subjects design). Data are mean ± SEM. Individual data are shown separately by sex (males = circles, females = triangles) in D, E. F, Images showing placement of cannula into OFC at 1.25× magnification (scale bar = 1 mm). Placements are shown with white (vehicle/0.3 µg WIN55,212-2) or black (vehicle/1 µg WIN55,212-2) circles. G, Mean number of fentanyl infusions during last three sessions of training phase and four sessions of self-administration retraining. H, Number of food and fentanyl rewards during four choice sessions after fentanyl re-training.
- Figure 4.
Effect of dopamine D1 receptor blockade in Pir on relapse to fentanyl seeking. A, Timeline of experiment 4. B, Self-administration. Number of reinforced responses (food: 5 pellets/reinforcer; fentanyl 2.5 µg/kg/infusion) during the 6-h sessions. C, Discrete choice (voluntary reduction in self-administration). Number of food-reinforced responses and fentanyl infusions earned during the 3-h choice sessions (20 trials/session). D, Relapse test. Number of inactive (left) and active (right) lever presses during the 3-h test session after vehicle or SCH39166 injections in Pir. E, Reacquisition test. Number of fentanyl infusions (2.5 µg/kg/infusion) during the 6-h session after vehicle or SCH39166 injections in Pir (n = 8–11 per group in D, n = 8–12 per group in E, mixed within/between-subjects design). Data are mean ± SEM. Individual data are shown separately by sex (males = circles, females = triangles) in D, E. F, Images showing placement of cannula into Pir at 1.25× magnification (scale bar = 1 mm). Placements are shown with white (vehicle/1 µg SCH39166) or black (vehicle/3 µg SCH39166) circles. G, Mean number of fentanyl infusions during last three sessions of training phase and two sessions of self-administration retraining. H, Number of food and fentanyl rewards during four choice sessions after fentanyl retraining.
Tables
Figure number Factor name F value P value Partial Eta2 Figure 1B, self-administration
Repeated-measures ANOVAWith sex as a factor
Food
Sex (male, female), between-subjects
Session (1–6), within-subjects
Sex × Session interaction
Fentanyl
Sex (male, female), between-subjects
Session (1–12), within-subjects
Sex × Session interaction
F(1,12) = 1.0
F(5,60) = 1.1
F(5,60) = 0.1
F(1,12) = 0.7
F(11,132) = 3.7
F(11,132) = 1.4
0.35
0.37
0.99
0.43
<0.001*
0.20
0.07
0.08
0.01
0.05
0.24
0.10Figure 1C, discrete choice
Repeated-measures ANOVAWith sex as a factor
Preference score
Sex (male, female), between-subjects
Session (1–12), within-subjects
Sex × Session interaction
F(1,12) = 4.8
F(11,132) = 15.4
F(11,132) = 1.0
0.05*
<0.001*
0.45
0.28
0.56
0.08Figure 1D, relapse test
Total responding
Repeated-measures ANOVAWithout sex as a factor
Lever (active, inactive), within-subjects
F(1,7) = 37.0
<0.001*
0.84Figure 1D, relapse test
Time course
Repeated-measures ANOVAWithout sex as a factor
Session Time (15, 30, 45, 60), within-subjects
Lever (active, inactive), within-subjects
Session Time × Lever interaction
F(3,21) = 11.1
F(1,7) = 37.0
F(3,21) = 9.6
<0.001*
<0.001*
<0.001*
0.61
0.84
0.58Figure 1F, Fos neuron counting
Repeated-measures ANOVAOFC Cnr1: without sex as a factor
Fos
Test Condition (Test, No Test), between-subjects
Cnr1
Test Condition (Test, No Test), between-subjects
Fos+Cnr1
Test Condition (Test, No Test), between-subjects
Pir Cnr1: without sex as a factor
Fos
Test Condition (Test, No Test), between-subjects
Cnr1
Test Condition (Test, No Test), between-subjects
Fos+Cnr1
Test Condition (Test, No Test), between-subjects
OFC Cnr1 and Vgat: without sex as a factor
Cnr1+Vgat
Test Condition (Test, No Test), between-subjects
Fos+Cnr1+Vgat
Test Condition (Test, No Test), between-subjects
OFC Drd1 and Drd2: without sex as a factor
Fos
Test Condition (Test, No Test), between-subjects
Drd1
Test Condition (Test, No Test), between-subjects
Drd2
Test Condition (Test, No Test), between-subjects
Fos+Drd1
Test Condition (Test, No Test), between-subjects
Fos+Drd2
Test Condition (Test, No Test), between-subjects
Pir Drd1 and Drd2: without sex as a factor
Fos
Test Condition (Test, No Test), between-subjects
Drd1
Test Condition (Test, No Test), between-subjects
Drd2
Test Condition (Test, No Test), between-subjects
Fos+Drd1
Test Condition (Test, No Test), between-subjects
Fos+Drd2
Test Condition (Test, No Test), between-subjects
F(1,12) = 10.4
F(1,12) = 2.4
F(1,12) = 11.7
F(1,12) = 5.1
F(1,12) = 0.0
F(1,12) = 1.6
F(1,12) = 0.3
F(1,12) = 6.2
F(1,10) = 5.4
F(1,10) = 2.9
F(1,10) = 1.4
F(1,10) = 1.6
F(1,10) = 2.2
F(1,12) = 7.2
F(1,12) = 0.0
F(1,12) = 1.7
F(1,12) = 5.4
F(1,12) = 1.7
0.007*
0.15
0.005*
0.04*
0.89
0.23
0.57
0.03*
0.04*
0.12
0.27
0.24
0.17
0.02*
0.99
0.22
0.04*
0.22
0.47
0.17
0.49
0.30
0.00
0.12
0.03
0.34
0.35
0.22
0.12
0.14
0.18
0.37
0.00
0.12
0.31
0.13Figure 2B, self-administration
Repeated-measures ANOVAWith sex as a factor
Food
Sex (male, female), between-subjects
Session (1–6), within-subjects
Sex × Session interaction
Fentanyl
Sex (male, female), between-subjects
Session (1–12), within-subjects
Sex × Session interaction
F(1,44) = 0.2
F(5,220) = 12.4
F(5,220) = 4.6
F(1,44) = 0.8
F(11,484) = 32.0
F(11,484) = 0.7
0.69
<0.001*
<0.001*
0.38
<0.001*
0.74
0.00
0.22
0.10
0.02
0.42
0.02Figure 2C, discrete choice
Repeated-measures ANOVAWith sex as a factor
Preference score
Sex (male, female), between-subjects
Session (1–12), within-subjects
Sex × Session interaction
F(1,44) = 12.3
F(11,484) = 15.2
F(11,484) = 1.7
0.001*
<0.001*
0.07
0.22
0.26
0.04Figure 2D, relapse test
Total responding
Mixed ANOVAWith sex as a factor
Sex (male, female), between-subjects
AM251 Dose (0, 0.3, 1 µg), between-subjects
Lever (active, inactive) within-subjects
AM251 Dose × Lever interaction
Sex × AM251 Dose interaction
Sex × Lever interaction
Sex × AM251 Dose × Lever interaction
F(1,40) = 0.0
F(2,40) = 1.0
F(1,40) = 152.7
F(2,40) = 0.9
F(2,40) = 0.3
F(1,40) = 0.0
F(2,40) = 1.2
0.94
0.39
<0.001*
0.43
0.74
0.97
0.31
0.0
0.05
0.79
0.04
0.02
0.0
0.06Figure 2D, relapse test
Time course
Mixed-ANOVAWithout sex as a factor
AM251 Dose (0, 0.3, 1 µg), between-subjects
Session Hour (1–3) within-subjects
Lever (active, inactive), within-subjects
AM251 Dose × Session Hour interaction
AM251 Dose × Lever interaction
Session Hour × Lever interaction
AM251 Dose × Session Hour × Lever interaction
F(2,43) = 1.1
F(2,86) = 144.2
F(1,43) = 160.4
F(4,86) = 1.8
F(2,43) = 1.0
F(2,86) = 131.5
F(4,86) = 1.4
0.34
<0.001*
<0.001*
0.14
0.39
<0.001*
0.24
0.05
0.77
0.79
0.08
0.04
0.75
0.06Figure 2E, reacquisition
Mixed-ANOVAWith sex as a factor
Sex (male, female), between-subjects
AM251 Dose (0, 0.3, 1 µg), between-subjects
Session Hour (1–6) within-subjects
AM251 Dose × Session Hour interaction
Sex × AM251 Dose interaction
Sex × Session Hour interaction
Sex × AM251 Dose × Session Hour interaction
F(1,40) = 1.9
F(2,40) = 1.2
F(5,200) = 8.7
F(10,200) = 1.3
F(2,40) = 2.9
F(5,200) = 1.9
F(10,200) = 1.2
0.18
0.30
<0.001*
0.26
0.07
0.10
0.32
0.04
0.06
0.18
0.06
0.13
0.04
0.06Figure 3B, self-administration
Repeated-measures ANOVAWithout sex as a factor
Food
Session (1–6), within-subjects
Fentanyl
Session (1–12), within-subjects
F(5,50) = 1.5
F(11,110) = 5.3
0.22
<0.001*
0.13
0.35Figure 3C, discrete choice
Repeated-measures ANOVAWithout sex as a factor
Preference score
Session (1–12), within-subjects
F(11,110) = 2.7
0.004*
0.22Figure 3D, relapse test
Total responding
Repeated-measures ANOVAWithout sex as a factor (without statistical outlier)
WIN55,212-2 Injection (vehicle, WIN55,212-2), within-subjects
WIN55,212-2 Dose (0.3, 1 µg), between-subjects
Lever (active, inactive) within-subjects
WIN55,212-2 Injection × Dose interaction
WIN55,212-2 Injection × Lever interaction
WIN55,212-2 Dose × Lever interaction
WIN55,212-2 Injection × Dose × Lever interaction
F(1,8) = 0.4
F(1,8) = 0.0
F(1,8) = 38.4
F(1,8) = 0.0
F(1,8) = 0.6
F(1,8) = 0.0
F(1,8) = 0.0
0.57
0.87
<0.001*
0.86
0.46
0.94
0.85
0.04
0.00
0.83
0.00
0.07
0.00
0.00Figure 3D, relapse test
Total responding
Repeated-measures ANOVAWithout sex as a factor (with statistical outlier)
WIN55,212-2 Injection (vehicle, WIN55,212-2), within-subjects
WIN55,212-2 Dose (0.3, 1 µg), between-subjects
Lever (active, inactive) within-subjects
WIN55,212-2 Injection × Dose interaction
WIN55,212-2 Injection × Lever interaction
WIN55,212-2 Dose × Lever interaction
WIN55,212-2 Injection × Dose × Lever interaction
F(1,9) = 0.1
F(1,9) = 0.6
F(1,9) = 26.5
F(1,9) = 0.7
F(1,9) = 0.1
F(1,9) = 0.5
F(1,9) = 0.8
0.75
0.45
<0.001*
0.42
0.73
0.48
0.40
0.01
0.07
0.75
0.07
0.01
0.06
0.08Figure 3E, reacquisition
Repeated-measures ANOVAWithout sex as a factor
WIN55,212-2 Injection (vehicle, WIN55,212-2), within-subjects
WIN55,212-2 Dose (0.3, 1 µg), between-subjects
WIN55,212-2 Injection × Dose interaction
F(1,9) = 0.6
F(1,9) = 4.5
F(1,9) = 0.3
0.44
0.06
0.61
0.07
0.33
0.03Figure 3G, re-training
Repeated-measures ANOVAWithout sex as a factor
Fentanyl
Session (1–4), within-subjects
F(3,30) = 0.2
0.92
0.02Figure 3H, discrete choice
Repeated-measures ANOVAWithout sex as a factor
Preference score
Session (1–4), within-subjects
F(3,30) = 1.5
0.25
0.13Figure 4B, self-administration
Repeated-measures ANOVAWith sex as a factor
Food
Sex (male, female), between-subjects
Session (1–6), within-subjects
Sex × Session interaction
Fentanyl
Sex (male, female), between-subjects
Session (1–12), within-subjects
Sex × Session interaction
F(1,18) = 1.3
F(5,90) = 3.9
F(5,90) = 5.8
F(1,18) = 0.0
F(11,198) = 2.9
F(11,198) = 0.5
0.27
0.003*
<0.001*
0.97
0.001*
0.89
0.07
0.18
0.24
0.00
0.14
0.03Figure 4C, discrete choice
Repeated-measures ANOVAWith sex as a factor
Preference score
Sex (male, female), between-subjects
Session (1–12), within-subjects
Sex × Session interaction
F(1,18) = 0.2
F(11,198) = 5.9
F(11,198) = 1.5
0.66
<0.001*
0.12
0.01
0.25
0.08Figure 4D, relapse test
Total responding
Repeated-measures ANOVAWithout sex as a factor (without statistical outlier)
SCH39166 Injection (vehicle, SCH39166), within-subjects
SCH39166 Dose (1, 3 µg), between-subjects
Lever (active, inactive) within-subjects
SCH39166 Injection × Dose interaction
SCH39166 Injection × Lever interaction
SCH39166 Dose × Lever interaction
SCH39166 Injection × Dose × Lever interaction
F(1,17) = 0.0
F(1,17) = 0.9
F(1,17) = 130.4
F(1,17) = 0.2
F(1,17) = 0.1
F(1,17) = 4.6
F(1,17) = 0.0
0.86
0.35
<0.001*
0.65
0.82
0.05*
0.93
0.00
0.05
0.89
0.01
0.00
0.21
0.00Figure 4D, relapse test
Total responding
Repeated-measures ANOVAWithout sex as a factor (with statistical outlier)
SCH39166 Injection (vehicle, SCH39166), within-subjects
SCH39166 Dose (1, 3 µg), between-subjects
Lever (active, inactive) within-subjects
SCH39166 Injection × Dose interaction
SCH39166 Injection × Lever interaction
SCH39166 Dose × Lever interaction
SCH39166 Injection × Dose × Lever interaction
F(1,18) = 0.6
F(1,18) = 0.0
F(1,18) = 44.2
F(1,18) = 0.9
F(1,18) = 0.3
F(1,18) = 0.2
F(1,18) = 0.5
0.46
0.99
<0.001*
0.36
0.61
0.68
0.48
0.03
0.00
0.71
0.05
0.02
0.01
0.03Figure 4E, reacquisition
Repeated-measures ANOVAWithout sex as a factor
SCH39166 Injection (vehicle, SCH39166), within-subjects
SCH39166 Dose (1, 3 µg), between-subjects
SCH39166 Injection × Dose interaction
F(1,18) = 0.2
F(1,18) = 1.8
F(1,18) = 0.1
0.63
0.20
0.77
0.01
0.09
0.01Figure 4G, re-training
repeated-measures ANOVAWith sex as a factor
Fentanyl
Sex (male, female), between-subjects
Session (1–2), within-subjects
Sex × Session interaction
F(1,18) = 2.2
F(1,18) = 1.9
F(1,18) = 0.4
0.15
0.19
0.55
0.11
0.09
0.02Figure 4H, discrete choice
Repeated-measures ANOVAWith sex as a factor
Preference score
Sex (male, female), between-subjects
Session (1–4), within-subjects
Sex × Session interaction
F(1,18) = 3.2
F(3,54) = 0.1
F(3,54) = 0.1
0.09
0.93
0.94
0.15
0.01
0.01Partial Eta2 = proportion of explained variance.
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