5-HT_1B receptors modulate release of [³H]dopamine from rat striatal synaptosomes: further evidence using 5-HT moduline, polyclonal 5-HT_1B receptor antibodies and 5-HT_1B receptor knock-out mice

Abstract.

In previous paper based on classical pharmacological tools, we identified a G_i protein-coupled presynaptic 5-hydroxytryptamine (5-HT) 1B receptor causing inhibition of dopamine (DA) release in rat striatal synaptosomes. It was the aim of the present study to further explore this receptor, using 5-HT moduline, a polyclonal antibody directed against 5-HT_1B receptors and 5-HT_1B receptor knock-out mice.

Preincubation of rat striatal synaptosomes with 5-HT moduline (0.1, 1, or 10 μM) significantly reduced the inhibitory effect of CP93,129, a selective rat 5-HT_1B receptor agonist, on K⁺-evoked overflow of [³H]DA in a non-competitive manner: 5-HT moduline did not modify the IC₅₀ of CP93,129, but concentration-dependently reduced the maximal inhibitory effect. Preincubation of rat striatal synaptosomes with a specific polyclonal 5-HT_1B receptor antibody also resulted in a significant attenuation of the inhibitory effect of CP93,129 on K⁺-evoked overflow of [³H]DA. In female 129/Sv wild-type mice, CP93,129 and 5-carboxyamidotryptamine maleate (5-CT), a non-selective 5-HT_1B receptor agonist, inhibited the K⁺-evoked [³H]DA overflow in a concentration-dependent manner. Sumatriptan, a selective rat 5-HT_1D receptor agonist, did not modify the overflow of [³H]DA. SB224289, a selective 5-HT_1B receptor antagonist, abolished the inhibitory effects of CP93,129 and 5-CT. The inhibitory effects of CP93,129 and 5-CT were absent in synaptosomes from 5-HT_1B receptor knockout mice. No compensatory inhibition effect in mutant mice was observed using sumatriptan.

In conclusion, the results show that a non-competitive antagonist of the 5-HT_1B receptor concentration-dependently decreases the maximal inhibitory effect of a 5-HT_1B receptor agonist on the synaptosomal K⁺-evoked release of [³H]DA in striatum. Moreover, a specific antibody raised against the receptor and particularly directed against a region of the receptor protein involved in signal transduction, namely the coupling with the G-protein, also antagonizes the inhibitory effect of the stimulation of 5-HT_1B receptor on the release of [³H]DA. Ultimately the disruption of 5-HT_1B receptor gene in 5-HT_1B knock-out mice leads to a total suppression of the effect of 5-HT_1B receptor agonists on [³H]DA release. These observations further support our previous observations using selective agonists/antagonists, indicating that 5-HT_1B receptors control the release of neuronal DA as presynaptic heteroreceptors.