Repeated swim impairs serotonin clearance via a corticosterone-sensitive mechanism: organic cation transporter 3, the smoking gun

Abstract

Activation of the hypothalamic-pituitary-adrenal (HPA) axis is associated with increased extracellular serotonin (5-HT) in limbic brain regions. The mechanism through which this occurs remains unclear. One way could be via HPA axis-dependent impairment of serotonin transporter (SERT) function, the high-affinity uptake mechanism for 5-HT. Consistent with this idea, we found that 5-HT clearance rate in hippocampus was dramatically reduced in mice exposed to repeated swim, a stimulus known to activate the HPA axis. However, this phenomenon also occurred in mice lacking SERT, ruling out SERT as a mechanism. The organic cation transporter 3 (OCT3) is emerging as an important regulator of brain 5-HT. Moreover, corticosterone, which is released upon HPA axis activation, blocks 5-HT uptake by OCT3. Repeated swim produced a persistent elevation in plasma corticosterone, and, consistent with prolonged blockade by corticosterone, we found that OCT3 expression and function were reduced in these mice. Importantly, this effect of repeated swim to reduce 5-HT clearance rate was corticosterone dependent, as evidenced by its absence in adrenalectomized mice, in which plasma corticosterone levels were essentially undetectable. Behaviorally, mice subjected to repeated swim spent less time immobile in the tail suspension test than control mice, but responded similarly to SERT- and norepinephrine transporter-selective antidepressants. Together, these results show that reduced 5-HT clearance following HPA axis activation is likely mediated, at least in part, by the corticosterone-sensitive OCT3, and that drugs developed to selectively target OCT3 (unlike corticosterone) may be candidates for the development of novel antidepressant medications.

Figure 1.

Serotonin clearance is impaired in the CA3 region of the hippocampus of mice subjected to repeated swim. A, Representative oxidation currents (converted to 5-HT concentration, μm) produced by locally applied 5-HT (barrel concentration 200 μm) in the CA3 region of the hippocampus of mice exposed to 0 d (black line) or 14 d (gray line) of swim. Raw traces are superimposed for ease of comparison. Time of clearance is T₈₀ (s), defined as the amount of time it takes for the signal to decline by 80% of its peak amplitude. The Tc is defined here as the slope of the most linear portion of the descending signal ranging from T₂₀ to T₆₀. B, C, Compared with controls, 5-HT clearance rate (nm/s) is slower (B) and time (s) is longer (C) in hippocampus of mice swum for 14 d (2-way ANOVA with Bonferroni post hoc comparison, *p < 0.05; **p < 0.01; ★p < 0.01; n = 5–9/group/pmol amount). D, For a given amount of 5-HT (pmol) administered, 5-HT signal amplitudes (μm) were equivalent in both groups of mice.

Figure 2.

SERT function and expression is the same in mice subjected to 0 or 14 d of repeated swim. A, B, As expected, fluvoxamine inhibited 5-HT clearance time (A) and rate (B) in hippocampus of control mice (no swim); however, these effects of fluvoxamine were equivalent in mice exposed to repeated swim. Compared with PBS vehicle, fluvoxamine significantly slowed 5-HT clearance time and rate in both groups of mice (2-way ANOVA with Bonferroni post hoc test, *p < 0.05; **p < 0.01, ★p < 0.01, n = 5–9/group/dose). C, In a behavioral readout of SERT and NET function, repeated swim did not affect the response to SERT and NET blockers in the tail suspension test. Immobility was sampled every 5 s over a 6 min test. Compared with saline, fluvoxamine (30 mg/kg), fluoxetine (15 mg/kg), and DMI (15 mg/kg) reduced immobility time in the TST in both groups of mice equally, regardless of swim. Data are expressed as a percentage of respective saline control (2-way ANOVA with Bonferroni post hoc test, ★p < 0.01).

Figure 3.

Serotonin clearance is impaired in SERT KO mice exposed to repeated swim. A, As expected, in mice not exposed to swim, 5-HT clearance rate was slower in SERT KO (C57BL/6J background) mice than in C57BL/6J mice. Serotonin clearance rate in C57BL/6J mice from Figure 1 is replotted here for ease of comparison. In SERT KO mice, 5-HT clearance rate was slower in mice exposed to 14 d of swim, compared with unswum SERT KO mice (2-way ANOVA with Bonferroni post hoc test, *p < 0.05; **p < 0.01; ★p < 0.01, n = 5–9/group/dose). B, Summary of clearance rates for 20 pmol of 5-HT highlighting the dramatic effect of repeated swim to decrease the rate of 5-HT clearance in both C57BL/6J and SERT KO mice. C, For a given amount of 5-HT (pmol) administered, 5-HT signal amplitudes (μm) were equivalent in all groups of mice. Serotonin signal amplitudes obtained in C57BL/6J mice from Figure 1 are replotted here for ease of comparison.

Figure 4.

Repeated swim reduces OCT3 expression and function in hippocampus. A, Hippocampal OCT3 density was reduced in mice exposed to 14 d of swim, compared with no-swim counterparts (percentage normalized to 0 d controls; t test, *p = 0.023, n = 10/group). B, C, Histamine clearance time (*p < 0.05) (B) and rate (*p < 0.01) (C) was slower in hippocampus of mice exposed to 14 d of swim.

Figure 5.

The ability of corticosterone to inhibit 5-HT clearance in hippocampus is dependent on exposure to repeated swim. Change in 5-HT clearance rate (expressed as percentage of baseline) 2 min after hippocampal application of PBS control, EtOH vehicle (0.8 nmol), or corticosterone (272 pmol) in mice exposed to 0 or 14 d of swim. In both groups of mice, corticosterone inhibited 5-HT clearance rate, compared with PBS vehicle, but this effect of corticosterone was blunted in mice exposed to repeated swim (2-way ANOVA with Bonferroni post hoc test, *p < 0.05; ★p < 0.01). EtOH significantly reduced 5-HT clearance rate only in mice that were not exposed to swim. Predrug/vehicle signal amplitudes did not vary among groups. Pooled values were 3.59 ± 0.35 μm (n = 17) and 3.20 ± 0.18 μm (n = 18) for 0 and 14 d swim, respectively. Predrug/vehicle clearance rate was slower in mice exposed to 14 d swim (31.82 ± 5.56 nm/s) compared with control mice (56.58 ± 8.4 nm/s) (t₍₃₃₎ = 2.595, p = 0.015).

Figure 6.

Adrenalectomy abolishes the swim-induced decrease in 5-HT clearance. A, Maximal velocity for 5-HT clearance was slower in hippocampus of sham-operated mice exposed to 14 d of swim (2-way ANOVA with Bonferroni post hoc test, ★p < 0.001), compared with sham-operated mice that were not exposed to swim and ADX mice swum for 0 or 14 d. B, Plasma corticosterone concentration (ng/ml) was essentially undetectable in ADX mice, regardless of swim, compared with sham-operated mice (★p < 0.01). Corticosterone levels were elevated in sham-operated mice exposed to 14 d of swim, compared with 0 d counterparts (**p < 0.01).