Selective serotonin reuptake inhibitors potentiate the rapid antidepressant-like effects of serotonin4 receptor agonists in the rat

Abstract

Background: We have recently reported that serotonin(4) (5-HT(4)) receptor agonists have a promising potential as fast-acting antidepressants. Here, we assess the extent to which this property may be optimized by the concomitant use of conventional antidepressants.

Methodology/principal findings: We found that, in acute conditions, the 5-HT(4) agonist prucalopride was able to counteract the inhibitory effect of the selective serotonin reuptake inhibitors (SSRI) fluvoxamine and citalopram on 5-HT neuron impulse flow, in Dorsal Raphé Nucleus (DRN) cells selected for their high (>1.8 Hz) basal discharge. The co-administration of both prucalopride and RS 67333 with citalopram for 3 days elicited an enhancement of DRN 5-HT neuron average firing rate, very similar to what was observed with either 5-HT(4) agonist alone. At the postsynaptic level, this translated into the manifestation of a tonus on hippocampal postsynaptic 5-HT(1A) receptors, that was two to three times stronger when the 5-HT(4) agonist was combined with citalopram. Similarly, co-administration of citalopram synergistically potentiated the enhancing effect of RS 67333 on CREB protein phosphorylation within the hippocampus. Finally, in the Forced Swimming Test, the combination of RS 67333 with various SSRIs (fluvoxamine, citalopram and fluoxetine) was more effective to reduce time of immobility than the separate administration of each compound.

Conclusions/significance: These findings strongly suggest that the adjunction of an SSRI to a 5-HT(4) agonist may help to optimize the fast-acting antidepressant efficacy of the latter.

Figure 1. Effect of an acute administration of the 5-HT₄ agonist prucalopride (1000 μg/kg, i.v.) on the inhibition of DRN 5-HT neuron firing rate induced by the SSRIs fluvoxamine and citalopram.

A and B: Summary of the results found with fluvoxamine (FLVX; 350 µg/kg, i.v.) and citalopram (CIT; 500 µg/kg, i.v.), respectively. Bar histograms represent the mean (± S.E.M.) percentage effect, calculated for each neuron with respect to its basal firing rate (i.e., 100% level), and the value at the bottom of each column indicates the number of neurons tested. Only one single neuron was recorded per animal. * p<0.05 and *** p<0.001 vs basal, + p<0.05 vs fluvoxamine, ## p<0.01 vs citalopram, Tukey's test. Panels C and D show individual examples of integrated firing rate histograms, in the cases of a fluvoxamine and a citalopram administration, respectively.

Figure 2. Effect of an acute, intravenous administration of citalopram (500 μg/kg), alone or subsequent to an i.v. dose of prucalopride (1000 μg/kg), on CA₃ pyramidal neuron firing activity.

A and B: Bar histograms represent the mean (± S.E.M.) percentage effect, in the absence or presence of prucalopride respectively, calculated for each neuron with respect to its basal firing rate (i.e. 100% level). The value at the bottom of each column represents the number of neurons tested (one single neuron recorded per rat). *** p<0.001 vs pre-drug, # p<0.05 vs prucalopride, Tukey's test. C: A typical example (integrated firing rate histogram) of the results summarized in B. The neuron chosen to illustrate this panel is also one of the two cells in which the effect of a local, microiontophoretic application of the selective 5-HT_1A antagonist WAY 100635 was tested (see Discussion for details).

Figure 3. Effect of a 3-day co-treatment with citalopram, and either prucalopride or RS 67333, on the mean firing rate of DRN 5-HT neurons.

In each rat, both citalopram (or its vehicle) and the 5-HT₄ agonist (or its vehicle) were administered via osmotic minipumps inserted subcutaneously, and recordings were performed with the two minipumps still in place. A: Integrated firing rate histogram showing samples of DRN descents in different experimental groups; indicated doses refer to the total daily dosage. B: Summary of the results: bar histograms represent the mean (± S.E.M.) firing activity of 5-HT neurons, calculated on the basis of successive recording tracks performed along the DRN. Values at the bottom of each column indicates the total number of neurons recorded per group (vehicle-vehicle: n = 7 animals, citalopram-RS 67333: n = 5 animals, and n = 4 animals in the other groups). ** p<0.01 and *** p<0.001 vs vehicle/vehicle, Tukey's test.

Figure 4. Effect of the combination of citalopram with a 5-HT₄ agonist on the postsynaptic 5-HT_1A neurotransmission.

Cumulative intravenous doses of the selective 5-HT_1A antagonist WAY 100635 were performed in rats continuously treated with A, the citalopram (10 mg/kg/d) + prucalopride (2.5 mg/kg/d) or B, the citalopram + RS 67333 (1.5 mg/kg/d) combination for three days, and their effects on the firing activity of hippocampal pyramidal neurons were recorded in the CA3 sub-field. Single-cell extracellular recordings were performed in chloral-hydrate anaesthetized animals, by using multiple-barrel glass microelectrodes combined with microiontophoretic pumps, and results expressed as the mean (± S.E.M.) percentage elevation of the firing rate with respect to pre-drug values (n = 4 animals in each group). All compounds (or their vehicle) were administered through the use of osmotic minipumps, inserted subcutaneously in the region of the back. Recordings were performed with the minipumps still in place. * p<0.05, ** p<0.01 and *** p<0.001 vs respective vehicle/vehicle values; + p<0.05, ++ p<0.01 and +++ p<0.001 vs respective citalopram/vehicle values; & p<0.05 and && p<0.01 vs respective vehicle/prucalopride values; # p<0.05 vs respective vehicle/RS 67333 values (Tukey's test). The “a” symbol indicates a p<0.05 significant interaction between the citalopram “pre-treatment” and the 5-HT₄ agonist “treatment”, as revealed by the use of a two-way ANOVA.

Figure 5. Effect of the continuous co-administration of citalopram (10 mg/kg/d) and RS 67333 (1.5 mg/kg/d) for 3 days on pCREB.

The activation of CREB in hippocampal tissue was assessed by measuring phosphoCREB (pCREB) immunoreactivity. CREB phosphorylation was normalized according to the amount of protein present in each sample by expressing the data as a ratio of pCREB over total CREB immunoreactivity. Results represent mean ± SEM for the number of experiments indicated, for each treatment, by the value at the bottom of the column. Inset shows representative examples of pCREB immunoreactivity for different treatment conditions indicated on the histogram. All compounds were administered through the use of osmotic minipumps, inserted subcutaneously in the region of the back. * p<0,05 vs vehicle/vehicle, + p<0.05 vs citalopram/vehicle and # p<0.05 vs vehicle/RS 67333, Student's t-test.

Figure 6. Effect of RS 67333 (1.5 mg/kg, i.p.), in combination with various SSRIs, on the time spent immobile in the FST.

The SSRIs used were A fluvoxamine (10 mg/kg, i.p.), B citalopram (10 mg/kg, i.p.), and C fluoxetine (10 mg/kg, i.p.). All data are expressed as mean ± S.E.M. of eight animals per group (except for the vehicle/RS 67333 group which comprises 12 animals), and are from an observation of 4 min duration. Rats experienced a pre-test session (15 min) 24 h before the test session. In each animal, RS 67333 (or its vehicle) and the SSRI (or its vehicle) were administered almost concomitantly (within a 30 s interval), 30 min before the test session; the sites chosen for i.p. injections were always symmetrically opposed with respect to the abdomen midline. * p<0.05 and *** p<0.001 vs vehicle/vehicle, ++ p<0.01 vs citalopram/vehicle, ‡‡‡ p<0.001 vs fluoxetine/vehicle and # p<0.05 vs vehicle/RS 67333, Tukey's test.