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µ-Opioid receptor–induced synaptic plasticity in dopamine neurons mediates the rewarding properties of anabolic androgenic steroids

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Science Signaling  01 Sep 2020:
Vol. 13, Issue 647, eaba1169
DOI: 10.1126/scisignal.aba1169
  • Fig. 1 AAS strengthen excitatory synaptic transmission on putative DA neurons.

    (A to C) Representative traces (A) and analysis of mEPSC frequency (B) and amplitude (C) in putative VTA DA neurons of mice treated with a single injection of either testosterone (TS; 10 mg/kg), nandrolone (ND; 10 mg/kg), or vehicle (Veh). Cumulative distributions of amplitude and frequency were analyzed by using Kolmogorov-Smirnov test: P < 0.01 versus Veh. (D) AMPAR/NMDAR ratio of mice treated with AAS as described in (A) to (C). (E) Paired-pulse ratio obtained at 50-ms interpulse interval from mice treated as described in (A) to (C). (F) I-V (current-voltage relation) plot of pharmacological isolated AMPAR EPSC (left). All EPSCs were normalized to the EPSC amplitude measured at −70 mV. Rectification index (middle) of AMPAR EPSCs was calculated as the ratio between the amplitude of EPSC obtained at −70 and +40 mV. In (A) to (F), values are means ± SEM of n = 8 neurons recorded from four mice per condition, analyzed by one-way ANOVA with Bonferroni post hoc test: *P < 0.05, **P < 0.01 versus Veh. Representative traces are shown (right).

  • Fig. 2 Androgen receptor antagonist flutamide did not affect AAS-induced synaptic plasticity.

    (A to C) Representative traces (A) and analysis of mEPSC frequency (B) and amplitude (C) in putative VTA DA neurons of mice pretreated with flutamide (FLU; 20 mg/kg, sc) or saline (Sal) 1 hour before a single intraperitoneal injection of either testosterone (TS; 10 mg/kg), nandrolone (ND; 10 mg/kg), or vehicle. Cumulative distributions of amplitude and frequency were analyzed by using Kolmogorov-Smirnov test: P < 0.01 for the ND/TS+ conditions versus the Veh conditions. (D) AMPAR/NMDAR ratio in putative DA neuron of mice treated as described in (A) to (C). (E) Paired-pulse ratio obtained at 50-ms interpulse interval in putative DA neurons of mice treated as described in (A) to (C). (F) I-V plot of pharmacological isolated AMPAR EPSC (left). All EPSCs were normalized to the EPSC amplitude measured at −70 mV. Rectification index (middle) of AMPAR EPSCs was calculated as the ratio between the amplitude of EPSC obtained at −70 and +40 mV. Representative traces are shown (right). In (A) to (F), values are means ± SEM of n = 8 neurons recorded from four mice per condition, analyzed by one-way ANOVA with Bonferroni post hoc test: *P < 0.05; #P < 0.05; **P < 0.01; ##P < 0.01 versus Veh + Sal or Veh + FLU, respectively.

  • Fig. 3 AAS induce rapid increase of plasma and VTA β-endorphin levels.

    (A) Schematic of experiment: Mice were injected with either testosterone (TS; 10 mg/kg), nandrolone (ND; 10 mg/kg), or vehicle (Veh). After different time points (5′, 15′, 30′ and 60′), either VTA or plasma was isolated and processed for β-endorphin quantification through ELISA. (B) Quantification of β-endorphin level in the VTA (left) and plasma (right) of mice treated as described (A). Values are means ± SEM; five mice in each condition were analyzed by one-way ANOVA with Bonferroni post hoc test: *P < 0.05; **P < 0.01 versus Veh. i.p., intraperitoneally.

  • Fig. 4 AAS-induced synaptic plasticity on putative DA neurons is mediated by MOR activation.

    (A) Coronal section depicting cannula implantation in the left lateral ventricle and schematic of experiment: Mice received an ICV injection of the irreversible MOR antagonist β-FNA (0.2 μg in 0.2 μl of saline) or saline (Sal) 24 hours before injecting either testosterone (TS; 10 mg/kg), nandrolone (ND; 10 mg/kg), or vehicle (Veh). Scale bar, 1 mm. Whole-cell patch-clamp recordings were performed 24 hours after the treatment with either AAS or Veh. (B to D) Representative traces (B) and analysis of mEPSC frequency (C) and amplitude (D) recorded in putative VTA DA neurons of mice treated as described (A). Cumulative distributions of amplitude and frequency were analyzed by using Kolmogorov-Smirnov test: P < 0.01 for ND + β-FNA and TS + β-FNA versus ND + Sal, TS + Sal, Veh + Sal, and Veh + β-FNA. (E) AMPAR/NMDAR ratio in putative DA neurons of mice treated as described (A). (F) Paired-pulse ratio obtained at 50-ms interpulse interval. (G) I-V plot and rectification index in putative DA neurons of mice treated as described (A). I-V plot of pharmacological isolated AMPAR EPSC (left). All EPSCs were normalized to the EPSC amplitude measured at −70 mV. Rectification index (middle) of AMPAR EPSCs was calculated as the ratio between the amplitude of EPSC obtained at −70 and +40 mV. Representative traces (right). In (C) to (F), values are means ± SEM of n = 8 neurons recorded from four mice per condition, analyzed by one-way ANOVA with Bonferroni post hoc test: **P < 0.01 versus Veh + Sal; †P < 0.05 and ††P < 0.01 versus Veh + β-FNA; ‡P < 0.05; ‡‡P < 0.01 versus ND + β-FNA; §P < 0.05; §§P < 0.01 versus TS + β-FNA.

  • Fig. 5 AAS-induced locomotor activity required VTA MOR activation.

    (A) Locomotor activity in mice treated with a single intraperitoneal injection with either testosterone (TS; 10 mg/kg), nandrolone (ND; 10 mg/kg), or vehicle (Veh). Values are means ± SEM (nine mice per group), analyzed by one-way ANOVA with Bonferroni post hoc test: P < 0.01 ND versus Veh and P < 0.001 TS versus Veh at 15 min; P < 0.01 ND versus Veh and P < 0.001 TS versus Veh at 30 min. (B) Coronal section depicting intra-VTA cannula implantation. (C) Locomotor activity in mice pretreated with an intra-VTA injection of β-FNA (0.2 μg in 0.2 μl of saline) or saline (0.2 μl; Sal), 24 hours before a single intraperitoneal injection with either TS, ND, or Veh as described in (A). Values are means ± SEM of seven mice per group, analyzed by one-way ANOVA with Bonferroni post hoc test: P < 0.01 for ND + Sal versus TS + β-FNA, TS + Sal versus Veh + Sal, and TS + Sal versus Veh + β-FNA; P < 0.001 for ND + Sal versus Veh + β-FNA and P < 0.0001 for ND + Sal versus Veh + Sal at 15 min; P < 0.05 for TS + Sal versus TS + β-FNA; P < 0.01 for ND + Sal versus Veh + β-FNA; P < 0.001 for ND + Sal versus Veh + Sal, ND + Sal versus ND + β-FNA, and TS + Sal versus Veh + β-FNA; P < 0.0001 for TS + Sal versus Veh + Sal and TS + Sal versus ND + β-FNA at 30 min.

  • Fig. 6 Irreversible blockade of VTA MOR counteracted AAS-induced place preference.

    (A) Schematic of experiment: During the pretest, mice were allowed to explore all compartments and were monitored for 15 min to assess any preference for a certain compartment. During conditioning, mice were injected in the morning during days 2, 4, and 6 and in the afternoon during days 3 and 5 with either testosterone (TS; 10 mg/kg) or nandrolone (ND; 10 mg/kg) and were confined to the not preferred compartment. In the afternoon of days 2, 4, and 6 or in the morning of days 3 and 5, mice were treated with vehicle (Veh) and were confined to the preferred side. The test day occurred in the middle of the day, and the mice were allowed to explore all compartments for 15 min. (B) Conditioned place preference (CPP) in mice conditioned as described (A). CPP score was calculated, subtracting the time spent in the drug-paired side to the Veh-paired side. Values are means ± SEM (10 mice per group). Two-tailed paired Student’s t test; ***P < 0.001, ****P < 0.0001. (C) Coronal section depicting intra-VTA cannula implantation (scale bar: 2mm). (D) Schematic of experiment: The experiment was performed as shown previously (A), except for the fact that β-FNA (0.2 μg in 0.2 μl of Sal) was injected in the VTA every 48 hours to maintain VTA MOR blocked for the whole duration of the experiment. The injection occurred on day 1 after the pretest and on days 3 and 5 after the conditioning session to not allow the association between any possible aversive effect of β-FNA with any compartment of the CPP apparatus. (E and F) CPP in mice conditioned as described (D). Values are means ± SEM (10 mice per group); two-tailed paired t test comparing the time spent on the drug-paired side before and after conditioning. ***P < 0.001, ****P < 0.0001.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/13/647/eaba1169/DC1

    Fig. S1. Intrinsic properties of putative DA neurons were not affected by AAS treatment.

    Fig. S2. Treatment with AAS increased AMPAR EPSCs, but not NMDAR EPSCs, in putative DA neurons.

    Fig. S3. GABAergic synaptic transmission in putative DA neurons was not affected 24 hours after AAS treatment.

    Fig. S4. Blockade of VTA MOR 60 min after AAS injection did not affect AAS-induced synaptic plasticity in putative DA neurons.

    Fig. S5. Histological verification of VTA cannula placement used for locomotor experiments.

    Fig. S6. β-FNA irreversibly blocked VTA MOR activity for 48 hours.

    Fig. S7. Histological verification of VTA cannula placement used for CPP experiments.

    Fig. S8. Histological verification of VTA cannula placement used for CPP experiments with inverted AAS and vehicle conditioning sessions.

    Fig. S9. β-FNA blocked AAS-induced place preference also when the daily AAS and vehicle conditioning sessions were inverted.

  • This PDF file includes:

    • Fig. S1. Intrinsic properties of putative DA neurons were not affected by AAS treatment.
    • Fig. S2. Treatment with AAS increased AMPAR EPSCs, but not NMDAR EPSCs, in putative DA neurons.
    • Fig. S3. GABAergic synaptic transmission in putative DA neurons was not affected 24 hours after AAS treatment.
    • Fig. S4. Blockade of VTA MOR 60 min after AAS injection did not affect AAS-induced synaptic plasticity in putative DA neurons.
    • Fig. S5. Histological verification of VTA cannula placement used for locomotor experiments.
    • Fig. S6. β-FNA irreversibly blocked VTA MOR activity for 48 hours.
    • Fig. S7. Histological verification of VTA cannula placement used for CPP experiments.
    • Fig. S8. Histological verification of VTA cannula placement used for CPP experiments with inverted AAS and vehicle conditioning sessions.
    • Fig. S9. β-FNA blocked AAS-induced place preference also when the daily AAS and vehicle conditioning sessions were inverted.

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