Utility of organotypic raphe slice cultures to investigate the effects of sustained exposure to selective 5-HT reuptake inhibitors on 5-HT release

Abstract

Background and purpose: Selective 5-hydroxytryptamine (5-HT, serotonin) reuptake inhibitors (SSRIs) are widely used antidepressants and their therapeutic effect requires several weeks of drug administration. The delayed onset of SSRI efficacy is due to the slow neuroadaptive changes of the 5-hydroxytryptaminergic (5-HTergic) system. In this study, we examined the acute and chronic effects of SSRIs on the 5-HTergic system using rat raphe slice cultures.

Experimental approach: For organotypic raphe slice cultures, mesencephalic coronal sections containing dorsal and median raphe nuclei were prepared from neonatal Wistar rats and cultured for 14-16 days.

Key results: Acute treatment with citalopram, paroxetine or fluoxetine (0.1-10 µM) in the slice cultures slightly increased extracellular 5-HT levels, while sustained exposure for 4 days augmented the elevation of 5-HT level in a time-dependent manner. Sustained exposure to citalopram had no effect on tissue contents of 5-HT and its metabolite, expression of tryptophan hydroxylase or the membrane expression of 5-HT transporters. The augmented 5-HT release was attenuated by Ca(2+) -free incubation medium or treatment with tetrodotoxin. Experiments with 5-HT(1A/B) receptor agonists and antagonists revealed that desensitization of 5-HT(1) autoreceptors was not involved in the augmentation of 5-HT release. Finally, co-treatment with an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate, but not an N-methyl-d-aspartate, receptor antagonist, suppressed this augmentation.

Conclusion and implications: These results suggest that sustained exposure to SSRIs induces the augmentation of exocytotic 5-HT release, which is caused, at least in part, by the activation of AMPA/kainate receptors in the raphe slice cultures.

Figure 1

Effects of acute treatment or sustained exposure to citalopram (A, D), paroxetine (B, E) and fluoxetine (C, F) on the extracellular 5-HT levels in raphe slice cultures. (A–C) At Day 0, slice cultures were treated with citalopram, paroxetine, fluoxetine (0.1, 1, 10 µM) or vehicle [phosphate-buffered saline (PBS) for citalopram and fluoxetine; 1% dimethyl sulfoxide for paroxetine] in Krebs–Ringer–Henseleit for 30 min, and extracellular 5-HT levels were then determined. (D–F) Following sustained exposure to selective 5-HT (serotonin) reuptake inhibitor (SSRIs) in culture medium for 4 days, slice cultures were treated with 10 µM of the corresponding SSRI for 30 min on Day 4, and extracellular 5-HT levels determined. Values represent the means of the 5-HT concentration ± SEM. *P < 0.05, ***P < 0.001 versus vehicle-exposed cultures. n = 3–8.

Figure 2

Time course of the increase in extracellular 5-HT levels following sustained exposure to citalopram. Slice cultures were exposed to citalopram (1 µM) for 4 days. Prior to sustained exposure (Day 0), 1 (Day 1), 2 (Day 2) and 4 (Day 4) days after the initiation of sustained exposure, slice cultures were treated with citalopram (1 µM) for 30 min, and then the extracellular 5-HT levels were determined. Values represent the means of the 5-HT concentration ± SEM. **P < 0.01, ***P < 0.001 versus Day 0. n = 7.

Figure 3

Effects of sustained exposure to citalopram on 5-HT (A) and 5-hydroxyindolacetic acid (5-HIAA) (B) content of slices, and on the expression of tryptophan hydroxylase (TPH) (C). Slice cultures were exposed to citalopram (1 µM) for 4 days. Slices were collected on Day 4 with no subsequent treatment, and 5-HT (A) and its metabolite 5-HIAA (B) contents were measured. Values represent means of 5-HT or 5-HIAA levels (nmol·mg⁻¹·protein) ± SEM. n = 12-13. (C) The expression of TPH protein was determined by Western blot analysis. Actin was used as a loading control. Upper panel shows representative blots of TPH and actin. Lower graph shows densitometric analysis of TPH expression. TPH levels were normalized against actin. The values of citalopram-exposed slices are expressed relative to those of phosphate-buffered saline (PBS)-exposed slices. n = 4–5.

Figure 4

Effects of sustained exposure to citalopram on the membrane expression of 5-HT (serotonin) transporter (SERT). Slice cultures were exposed to citalopram (1 µM) for 4 days. Slices were collected without treatment on Day 4, and homogenized. Membrane fractions of homogenates were prepared as described in Materials and Methods. The expression of SERT in the membrane fraction was determined by Western blot analysis. Na⁺/K⁺-ATPase was used as a loading control. A representative blot is shown above the graph. SERT levels were normalized against the Na⁺/K⁺-ATPase signal. The values of citalopram-exposed slices are expressed relative to those of phosphate-buffered saline (PBS)-exposed slices. n.s., not significant; n = 7.

Figure 5

Effects of Ca²⁺-free external solution and tetrodotoxin (TTX) treatment on the increase in extracellular 5-HT levels. Following sustained exposure to citalopram (1 µM) in culture medium for 4 days, slices were treated with citalopram (1 µM) in Ca²⁺-free Krebs–Ringer–Henseleit (KRH) (A) or in the presence of TTX (1 µM) in KRH (B) for 30 min on Day 4, and the extracellular 5-HT levels were determined. Values represent the means of 5-HT concentration ± SEM. *P < 0.05, ***P < 0.001. n = 7–11 (A), n = 4 (B).

Figure 6

Effects of the 5-HT_1A or 5-HT_1B receptor agonists on [³⁵S]GTPγS binding (A, B) and on the augmentation of 5-HT release (C, D) following sustained exposure of slice cultures to citalopram. (A, B) Following sustained exposure to citalopram (1 µM) in culture medium for 4 days, slices were collected without citalopram treatment on Day 4 and membrane suspensions were prepared as described in Methods. 8-OH-DPAT-stimulated (A) and CGS12066A-stimulated (B) [³⁵S]GTPγS binding was measured. Values represent the means of percent stimulation of [³⁵S]GTPγS binding normalized to basal binding. n.s., not significant, *P < 0.05. n = 4–5. (C, D) Following sustained exposure to citalopram (1 µM) in culture medium for 4 days, slices were treated with citalopram (1 µM) in the presence of 8-OH-DPAT (10 µM) or CGS12066A (1 µM) in Krebs-Ringer-Henseleit for 30 min on Day 4, and the extracellular 5-HT levels were determined. Values represent the means of the 5-HT concentration ± SEM. n.s., not significant, ***P < 0.001. n = 4–6 (C), n = 5–6 (D).

Figure 7

Effects of sustained co-treatment with the 5-HT_1A or 5-HT_1B antagonist and citalopram on the augmentation of 5-HT release. Following sustained exposure to citalopram (1 µM) with or without WAY100635 (1 µM; A) or SB224289 (10 µM; B) in culture medium for 4 days, slices were treated with citalopram (1 µM) in the presence or absence of WAY100635 (1 µM; A) or SB224289 (10 µM; B) in Krebs-Ringer-Henseleit for 30 min on Day 4, and the extracellular 5-HT levels were determined. Values represent the means of the 5-HT concentration ± SEM. n.s., not significant, *P < 0.05, ***P < 0.001. n = 3.

Figure 8

Effects of sustained exposure to glutamate receptor antagonists on the augmentation of 5-HT release. Following sustained exposure to citalopram (1 µM) with or without MK-801 (10 µM) (A) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 µM) (B) in culture medium for 4 days, slices were treated with citalopram (1 µM) in the presence or absence of MK-801 (10 µM) or CNQX (10 µM), respectively, in Krebs-Ringer-Henseleit for 30 min on Day 4, and extracellular 5-HT levels were determined. Values represent the means of the 5-HT concentration ± SEM. n.s., not significant, *P < 0.05, ***P < 0.001. n = 4–5 (A), n = 6 (B).