Article Figures & Data
Figures
- Figure 1.
DREADD expression in VTA dopamine cells. A, Representative image of TH (green) and mCherry (red) expression in the VTA. Scale bar, 200 μm. B, Four line drawings of coronal sections indicating the representative spread of mCherry expression through the rostral (−4.56 mm from bregma), middle (−5.28 and −5.64 mm from bregma) and caudal (−6.00 mm from bregma) subregions of the VTA in all (red) and the majority (gray) of animals. C–K, Representative images of TH (C, F, I), mCherry (D, G, J), and merged TH+mCherry (E, H, K) immunoreactivity in the IF (C–E) and PBP (F–H) subregions of the VTA and in substantia nigra (I–K). Arrows illustrate an example of a VTA TH cell without detectable mCherry expression, while all other TH cells coexpress mCherry. Scale bar, 25 μm. L–N, Higher magnification of representative TH (L), mCherry (M), and merged TH+mCherry (N) immunoreactivity in the PBP subregion of the VTA. Scale bar, 25 μm. SN, Substantia nigra; aq, cerebral aqueduct; ml, medial lemniscus; PLH, peduncular part of lateral hypothalamus; fr, fasciculus retroflexus; MM, mammillary nuclei; tth, trigeminothalamic tract.
- Figure 2.
CNO did not affect sexual behavior during acquisition of sexual experience. A, B, Quantitative analysis of latencies (in seconds) to mount (A) and ejaculation (B) during 5 consecutive days of sexual behavior in saline-pretreated (SAL; black line, closed markers; n = 10) and CNO-pretreated (blue line, open markers; n = 11) TH::Cre transgenic rats expressing Gi DREADD in VTA TH cells. Data represent mean ± SEM *, Significant difference compared with day 1 of both groups combined.
- Figure 3.
CNO blocked mating-induced cFos in VTA dopamine cells. A–D, Four representative rostral-caudal coronal VTA sections (−4.80, −5.28, −5.64, −6.00 mm from bregma), with boxes indicating the areas of analysis (IF, 200 × 600 μm; PBP, 1000 × 800 μm). E, F, Representative images of cells immunopositive for cFos and TH (cFos/TH+; arrows) or cFos only (cFos/TH−; arrowhead) cells in the IF (E) and PBP (F) subregions of the VTA. Scale bar, 50 μm. MM, Mammillary nuclei; SN, Substantia nigra; cp, cerebral peduncle; fr, fasciculus retroflexus; ml, medial lemniscus; IP, interpeduncular nucleus; tth, trigeminothalamic tract. G–L, Quantitative analysis of the number of cFos/TH+ cells (per mm2; G, H), percentage of TH cells that are also cFos-positive (I, J), and number of cFos/TH− cells (per mm2) in rostral-middle-caudal levels (K, L) through the medial (G, I, K) and lateral (H, J, L) VTA subregions. Groups: saline + no mating (SAL+NO MATING), white bars, n = 6; CNO + no mating (CNO+NO MATING), light blue bars, n = 6; saline + mating (SAL+MATING), black bars, n = 10; CNO + mating (CNO+MATING), dark blue bars, n = 10. Data represent mean ± SEM. * with line, Significant difference, compared with no-mating control groups. *, Significant difference compared with the saline + no-mating control group. #, Significant difference compared with saline + mating group.
- Figure 4.
CNO did not affect mating-induced cFos in VTA projection areas. A, B, Quantitative analysis of cFos immunoreactivity (per mm2) in NAc core and shell, BLA (A), and ACA, PL, and IL subregions of mPFC (B) in saline + no mating (SAL+NO MATING; white bars; n = 6) or CNO + no mating (CNO+NO MATING; light blue bars; n = 6) and saline + mating (SAL+MATING; black bars; n = 10) or CNO + mating (CNO+MATING; dark blue bars; n = 11) groups. Data represent mean ± SEM. C–F, Representative images of cFos expression in NAc of SAL+NO MATING (C), CNO+NO MATING (D), SAL+NO MATING (E) and CNO+MATING (F) groups. Scale bar,100 μm. ac, Anterior commissure. *, Significant differences to no-mating controls.
- Figure 5.
Inhibition of hM4D-expressing VTA dopamine neurons following CNO application. A, Sample traces of spontaneous (top) and evoked action potentials (bottom) in hM4D-expressing dopamine neurons before and after CNO (10 μm) application. B–D, Group data for the effect of CNO on membrane potential (B), input resistance (C), and firing rate of the spontaneous action potentials (D; n = 7) in hM4D-expressing VTA dopamine neurons. Data represent mean ± SEM. *, Significant difference compared with the baseline.
- Figure 6.
CNO blocked effects of sexual experience on amphetamine CPP and ΔFosB expression. A, CPP scores (time spent in amphetamine-paired chamber, post-test–pretest; in seconds) of sexually naive (white bars; saline-treated and CNO-treated groups were combined into one saline/CNO + naive (SAL/CNO+NAIVE) group; n = 8 (including saline, n = 3 and CNO, n = 5), saline + experienced (SAL+EXP; black bars; n = 8), CNO + experienced (CNO+EXP; dark blue bars; n = 9). *, Significant difference, compared with SAL/CNO+NAIVE. B–E, Representative images of ΔFosB immunoreactivity in NAc of SAL+NAIVE (B), CNO+NAIVE (C), SAL+EXP (D), and CNO+EXP (E) groups. Scale bar, 100 μm. ac, Anterior commissure. F, G, Quantitative analysis of numbers of ΔFosB-positive cells (per mm2) in VTA projection areas in SAL+NAIVE (white bars; n = 6), CNO+NAIVE (light blue bars; n = 6), SAL+EXP (black bars; n = 10), and CNO+EXP (dark blue bars; n = 9) groups. Data represent mean ± SEM. *, Significant difference from naive group within same treatment: compared with SAL+NAIVE or CNO+NAIVE. #, Significant difference from SAL+EXP.
- Figure 7.
CNO blocked effects of sexual experience on morphology of VTA dopamine cells. A, B, Quantitative analyses of VTA dopamine soma perimeter (A) and area (B) in saline + naive (SAL+NAIVE; white bars; n = 6), CNO + naive (CNO+NAIVE; light blue bars; n = 6; 97% perimeter, 97% area compared with SAL+NAIVE), saline + experienced (SAL+EXP; black bars; n = 9; 92% perimeter, 87% area compared with SAL+NAIVE), and CNO + experienced (CNO+EXP; dark blue bars; n = 10; 95% perimeter, 92% area compared with SAL+NAIVE) groups. Data represent mean ± SEM. *, Significant difference from SAL+NAIVE or CNO+NAIVE. #, Significant difference from SAL+EXP. C–F, Representative images of TH-immunoreactive cells in the PBP of SAL+NAIVE (C), CNO+NAIVE (D), SAL+EXP (E), and CNO+EXP (F) groups. Scale bar, 10 μm.
Tables
Group Day 1 Day 2 Day 3 Day 4 Day 5 Intromission latency Saline 716 ± 236 s 440 ± 161 s 842 ± 310 s 365 ± 162 s 42 ± 12 sb CNO 434 ± 117 s 175 ± 64 s 315 ± 115 s 154 ± 49 s 42 ± 13 sb Copulation efficiency [intromissions/( mounts + intromissions)] Saline 0.8 ± 0.1 0.5 ± 0.1 0.6 ± 0.1 0.6 ± 0.0 0.7 ± 0.0 CNO 0.6 ± 0.0 0.7 ± 0.1 0.6 ± 0.1 0.6 ± 0.0 0.7 ± 0.1 Mounts (% of males) Saline 70 80 90 90 91 CNO 91 91 73 100 91 Intromissions (% of males) Saline 90 80 100 90 100 CNO 91 100 73 100 100 Ejaculations (% of males) Saline 70 80 90 90 100 CNO 64 91 73 100 100 ↵aCNO did not affect sexual behavior during acquisition of sexual experience. Quantitative analysis of intromission latencies (in seconds), copulation efficiency, percentages of males that display mounts, intromissions, and ejaculations, during 5 consecutive days of sexual behavior in saline-pretreated (n = 10) and CNO-pretreated (n = 11) TH::Cre transgenic rats expressing Gi DREADD in VTA TH cells. Data represent mean ± SEM.
↵bSignificant difference compared to day 1.
Group Day 1 Day 5 Numbers Saline 7.33 ± 1.26 3.17 ± 1.85b CNO 6.00 ± 0.55 4.22 ± 1.18b Latencies Saline 14.33 ± 3.70 s 17.25 ± 8.72 s CNO 23.50 ± 1.26 s 12.71 ± 3.88 s % Males Saline 100.00 50.00 CNO 100.00 77.78 ↵aCNO did not affect anogenital investigations during acquisition of sexual experience. Quantitative analysis of numbers of anogenital investigations, latencies (seconds) to first anogenital investigation, and percentages of males that display anogenital investigations, during the first 2 min of day 1 and day 5 of 5 consecutive days of sexual behavior in saline-treated (n = 6) and CNO-treated (n = 9) TH::Cre transgenic rats expressing Gi DREADD in VTA TH cells. Data represent mean ± SEM.
↵bSignificant difference compared to day 1. No significant differences between saline and CNO groups were detected.
Group RLi IF CLi PBP Rostral Middle Caudal Saline + no mating 34 ± 7 38 ± 6 53 ± 7 55 ± 12 67 ± 9 122 ± 13 CNO + no mating 27 ± 2 35 ± 2 43 ± 3 53 ± 7 76 ± 10 123 ± 18 Saline + mating 30 ± 3 34 ± 2 46 ± 4 52 ± 10 67 ± 13 109 ± 15 CNO + mating 29 ± 4 35 ± 3 52 ± 3 62 ± 9 68 ± 10 101 ± 9 ↵aCNO did not affect TH immunoreactivity in the VTA. Quantitative analysis of numbers of TH-positive cells in the RLi, IF, CLi, and rostral, middle, and caudal VTA subregions. Data represent mean ± SEM.
