Organic cation transporter 3: Keeping the brake on extracellular serotonin in serotonin-transporter-deficient mice

Abstract

Mood disorders cause much suffering and are the single greatest cause of lost productivity worldwide. Although multiple medications, along with behavioral therapies, have proven effective for some individuals, millions of people lack an effective therapeutic option. A common serotonin (5-HT) transporter (5-HTT/SERT, SLC6A4) polymorphism is believed to confer lower 5-HTT expression in vivo and elevates risk for multiple mood disorders including anxiety, alcoholism, and major depression. Importantly, this variant is also associated with reduced responsiveness to selective 5-HT reuptake inhibitor antidepressants. We hypothesized that a reduced antidepressant response in individuals with a constitutive reduction in 5-HTT expression could arise because of the compensatory expression of other genes that inactivate 5-HT in the brain. A functionally upregulated alternate transporter for 5-HT may prevent extracellular 5-HT from rising to levels sufficiently high enough to trigger the adaptive neurochemical events necessary for therapeutic benefit. Here we demonstrate that expression of the organic cation transporter type 3 (OCT3, SLC22A3), which also transports 5-HT, is upregulated in the brains of mice with constitutively reduced 5-HTT expression. Moreover, the OCT blocker decynium-22 diminishes 5-HT clearance and exerts antidepressant-like effects in these mice but not in WT animals. OCT3 may be an important transporter mediating serotonergic signaling when 5-HTT expression or function is compromised.

Fig. 1.

OCT3 protein expression is increased in the hippocampi of 5-HTT^+/− and 5-HTT^−/− mice compared with those of 5-HTT^+/+ mice. (A) (Upper) OCT3 expression is increased by 27% and 36% in 5-HTT^+/− (★, P = 0.0276) and 5-HTT^−/− (★★, P = 0.0063) mice compared with their WT counterpart (1-way ANOVA with Tukey's post hoc comparison). (Lower) Representative Western blot showing OCT band at 70 kDa using an OCT3-specific antibody and β-actin loading control at 40 kDa. (B) Reciprocal expression of 5-HTT and OCT3 in 5-HTT mutant mice. Expression for each transporter was normalized to the expression level in WT mice. The values used to calculate 5-HTT expression were derived from our published [³H]cyanoimipramine binding to the 5-HTT (14). Values for OCT3 were derived from the data shown in A. (**, P < 0.001, ★, P < 0.01, *, P < 0.05 compared with 5-HTT^+/+; #, P < 0.01 compared with 5-HTT^+/−, 1-way ANOVA with Tukey's post hoc comparison). (C) OCT3 immunostaining in hippocampus using the same OCT3-specific antibody as for Western blots. OCT3 was detected in the CA1, CA3, and dentate gyrus regions of the hippocampus. Staining was most pronounced in the CA3 region of the hippocampus, as well as in the polymorph region of the dentate gyrus, the granular layer of the dentate gyrus, and the pyramidal cell layer. A section of the CA3 region is enlarged to illustrate specific details of these regions.

Fig. 2.

OCT blockade inhibits 5-HT clearance in the hippocampi of 5-HTT mutant mice but not WT mice. (A) Representative oxidation currents (converted to micromolar values) produced by pressure ejection of 5-HT into the CA3 region of hippocampi before (gray line) and 2 min after (black line) intrahippocampal administration of D-22 (1.4 pmol) in 5-HTT^+/+, 5-HTT^+/−, and 5-HTT^−/− mice, respectively. Raw tracings are superimposed for ease of comparison. Note the marked increase in T₈₀ after D-22 in 5-HTT mutant mice compared with 5-HTT^+/+ mice. (B) Serotonin-clearance-inhibiting effects of hippocampally applied D-22 are evident only in 5-HTT mutant mice. Increasing concentrations of D-22 caused a significant increase in T₈₀ values (expressed as a percent of baseline values) in hippocampi of 5-HTT^−/− and 5-HTT^+/− mice but was without effect in 5-HTT^+/+ mice. ★, P < 0.001; *, P < 0.05 vs. 5-HTT^+/+. Vehicle ejection did not influence T₈₀ values. The percent change from prevehicle baseline was 103 ± 5 (n = 8), 107 ± 5 (n = 8) and 107 ± 14 (n = 4) for 5-HTT^+/+, 5-HTT^+/−, and 5-HTT^−/− mice, respectively. (C) Corticosterone inhibits 5-HT clearance in the hippocampi of 5-HTT mutant mice. The OCT antagonist, corticosterone (55 pmol), inhibited 5-HT clearance in hippocampi of 5-HTT^+/− (*, P < 0.05) and 5-HTT^−/− (**, P < 0.001) mice, compared with predrug baseline (paired t test). Compared to 5-HTT^+/+ mice, corticosterone inhibited 5-HT clearance in hippocampi of 5-HTT^−/− mice. ★, P < 0.01, 1-way ANOVA with Tukey's post hoc comparisons. Data are expressed as mean and SEM.

Fig. 3.

Histamine clearance is faster and inhibited more profoundly by OCT blockade in 5-HTT mutant mice than in WT mice. (A) Basal histamine T₈₀ values were significantly faster in 5-HTT^−/− and 5-HTT^+/− mice than in 5-HTT^+/+ mice. Shown is the reciprocal relationship between basal histamine and 5-HT T₈₀ values, where 5-HT clearance is significantly longer in 5-HTT^−/− mice than 5-HTT^+/+ or 5-HTT^+/− mice. ★, P < 0.05 and ★★, P < 0.01 vs. 5-HTT^+/+ (1-way ANOVA with Tukey's post hoc comparisons). (B) Intrahippocampally applied D-22 (1.4 pmol) caused a robust percent increase in T₈₀ values for histamine clearance over pre-D-22 baseline values in hippocampi of 5-HTT^−/− and 5-HTT^+/− mice but was without effect in 5-HTT^+/+ mice. Data shown are mean and SEM for 2 min after injection of D-22. *, P < 0.05 compared to predrug baseline value (paired t test); ★, P < 0.05 compared with 5-HTT^+/+ mice (1-way ANOVA with Tukey's post hoc comparisons).

Fig. 4.

Blockade of OCT3 elicits antidepressant-like effects in the tail suspension test in 5-HTT mutant mice but not in WT mice. Data shown are for the TST carried out 60 min after injection of D-22 or saline solution. Data are expressed as a percent of immobility time in saline-treated mice within genotype. All data are expressed as mean and SEM. ★, P < 0.05 vs. 5-HTT^+/+, ANOVA with Tukey's post hoc.