The Behavioral Effects of the Antidepressant Tianeptine Require the Mu-Opioid Receptor

Abstract

Depression is a debilitating chronic illness that affects around 350 million people worldwide. Current treatments, such as selective serotonin reuptake inhibitors, are not ideal because only a fraction of patients achieve remission. Tianeptine is an effective antidepressant with a previously unknown mechanism of action. We recently reported that tianeptine is a full agonist at the mu opioid receptor (MOR). Here we demonstrate that the acute and chronic antidepressant-like behavioral effects of tianeptine in mice require MOR. Interestingly, while tianeptine also produces many opiate-like behavioral effects such as analgesia and reward, it does not lead to tolerance or withdrawal. Furthermore, the primary metabolite of tianeptine (MC5), which has a longer half-life, mimics the behavioral effects of tianeptine in a MOR-dependent fashion. These results point to the possibility that MOR and its downstream signaling cascades may be novel targets for antidepressant drug development.

Figures

Figure 1

Dose response of tianeptine behavioral effects. (a) Timeline for (b–d). n=12–14 per group for (b and c) and 5 per group for (d). Tianeptine was administered by i.p. injection at doses of either 10 mg/kg or 30 mg/kg as indicated. (b) FST Day 1 results. Bar graph (left) shows combined immobility results of last four minutes and line graph (right) shows immobility per minute over the 6 min test. Immobility duration over the last 4 min was analyzed. One-way ANOVA: F(2,37)=3.966, p=0.0275. *p=0.0488, relative to saline; **p=0.0095, relative to saline (Fisher’s). (c) Home cage consumption over 5 min after an 18 h deprivation period was assessed as a measure of hypophagia. One-way ANOVA: F(2,37)=17.17, p<0.0001. ***p<0.0001, relative to saline. (d) Latency to jump after being placed on the hot plate was assessed. Two-way ANOVA: F(2,24)=6.063, time × treatment p=0.0074. ***p<0.0001 relative to 15 min/saline. (e) Timeline for (f). n=9–10 per group. Tianeptine was administered by i.p. injection at doses of either 10 mg/kg or 30 mg/kg as indicated. (f) Hyperactivity in an open field. Line graph (left) shows total distance traveled over 180 min (10 minute bins). Bar graph (right) shows AUC analysis of line graph. AUC analysis: one-way ANOVA: F(2,26)=4.601, p=0.0195. **p=0.0055, relative to saline (Fisher’s). Throughout the figure, all bar graphs indicate mean±SEM. For line graphs, each point indicates mean±SEM.

Figure 2

Tianeptine comparison to morphine. (a) Analgesic responses to morphine and tianeptine are shown over increasing doses as by latency to lick the hindpaw as measured in the hot-plate assay. The dose–response curve for morphine was measured 30 min post injection, and for tianeptine at 15 min post injection. The ED50s of these curves differ significantly: F(1,58)=53, p<0.0001. (b) The analgesic response to morphine and tianeptine is shown at 15 min post injection at roughly equianalgesic doses as measured by latency to jump in the hot-plate assay in a naïve group of animals. One-way ANOVA (drug): F(2, 31)=5.63, p=0.008; *p<0.05, compared to saline. (c) The percent of time spent on the drug-paired side before and after 8 days of context pairings with morphine at 5 mg/kg, tianeptine at 30 mg/kg, or saline is shown. One-way ANOVA for pre-test: F(2,27)=0.19, p=0.82; one-way ANOVA for post-test: F(2, 27)=5.01, p=0.014. *p<0.05 compared to post-test saline. (d) The preference score (total time on drug-paired side minus total time on saline-paired side) is shown for the 20 min post-pairing test. One-way ANOVA: F(2, 27)=6.47, p=0.0051. **p<0.01, compared to saline. (e) Tolerance was assessed by measuring the effect of acute drug treatment (saline, morphine at 5 mg/kg, or tianeptine at 30 mg/kg) using the hot-plate test following chronic exposure to saline, tianeptine (30 mg/kg twice daily for 34 days), or morphine (5 mg/kg twice daily, for 10 days). (f) Withdrawal was assessed through jumping behavior following acute administration of naloxone (1 mg/kg) following chronic exposure to saline, tianeptine (30 mg/kg twice daily for 34 days), or morphine (5 mg/kg twice daily, for 10 days). Two-way ANOVA: F(2, 47)=10.87, p<0.001 for drug × naloxone treatment. *p<0.05 compared to morphine-saline and tianeptine-naloxone. Throughout the figure, all bar graphs indicate mean±SEM. For line graphs, each point indicates mean±SEM.

Figure 3

Pharmacokinetics of tianeptine and the MC5 metabolite. (a) Chemical structures of tianeptine and its MC5 metabolite. (b) In vitro activities of tianeptine and MC5 in G protein BRET assays measuring activation of mouse and human MOR and DOR, data points represent mean EC50±SEM (μM); Plasma (c) and brain (d) concentrations±STD of tianeptine and MC5 in C57BL/6 mice following a single administration of tianeptine (30 mg/kg).

Figure 4

Behavioral effects of tianeptine and its MC5 metabolite in MOR-deficient mice. (a) Timeline for (b–d). n=7–9 per group. Tianeptine (30 mg/kg) or MC5 (30 mg/kg) were administered by i.p. injection. (b) FST Day 1 results. Bar graph (left) shows combined immobility results of last 4 min, and line graph (right) shows immobility per minute over the 6 min test. Immobility duration over the last 4 min was analyzed. Two-way ANOVA: F(2,46)=9.756, genotype × treatment p=0.0003. ***p<0.0001, relative to WT/saline. (c) Home cage consumption over 5 min after an 18-h deprivation period was assessed as a measure of hypophagia. Two-way ANOVA: F(2,46)=7.726, genotype × treatment p=0.0013. *p=0.0101, relative to WT/saline; ***p=0.0007, relative to WT/saline (Fisher’s). (d) Latency to jump after being placed on the hot plate was assessed. Two-way ANOVA: F(2,46)=4.652, genotype × treatment p=0.0145. ***p=0.0005, relative to WT/saline; *p=0.0240 relative to WT/saline (Fisher’s). (e) Timeline for (f). n=7–9 per group. Tianeptine (30 mg/kg) or MC5 (30 mg/kg) were administered by i.p. injection. (f) Hyperactivity in an open field. Line graph (left) shows total distance traveled over 180 min (10 min bins). Bar graph (right) shows AUC analysis of line graph. AUC analysis: Two-way ANOVA: F(2,46)=4.701, p=0.0139. ***p=0.0006, relative to WT/saline; **p=0.0038, relative to WT/saline (Fisher’s). Throughout the figure, all bar graphs indicate mean±SEM. For line graphs, each point indicates mean±SEM.

Figure 5

Chronic effects of tianeptine in WT and MOR-deficient mice. (a) Latency to feed in the novel arena is shown from the novelty suppressed feeding assay 18 h post tianeptine injection, following chronic treatment with tianeptine (30 mg/kg twice daily for 21 days) in chronic corticosterone treated mice. Each dot represents an individual mouse. Logrank (Mantel–Cox Survival): X3=7.57, p=0.056, saline vs tianeptine—*p=0.035 for WT; p=0.12 for MOR KO. (b) A control measure of latency to feed in the home cage is also shown measured immediately following the novelty suppressed feeding assay. Each dot represents an individual mouse. Logrank (Mantel–Cox): X3=4.41, p=0.22, planned comparisons: saline vs tianeptine—p=0.20, for WT; p=0.75 for MOR KO. (c) Total distance traveled in the open-field arena measured 16–18 h post tianeptine injection shows no significant effect of chronic tianeptine on locomotion. Two-way ANOVA: F(1,44)=0.11, p=0.74 treatment × genotype. (d) Tolerance was assessed by measuring the effect of acute tianeptine using the hot-plate test following chronic exposure. Three-way ANOVA (genotype × acute × chronic): main effect of genotype F(1,38)=18.16, **p<0.001. (e) Withdrawal was assessed through jumping behavior following acute administration of naloxone (1 mg/kg) following chronic exposure to saline or tianeptine. Three-way ANOVA (genotype × chronic × naloxone): F(1,38)=0.14, p=0.71. For (c–e), all bar graphs indicate mean±SEM.