Functional study of rat 5-HT2A receptors using antisense oligonucleotides
Van Oekelen, Dirk × Megens, Anton Meert, Theo Luyten, Walter Leysen, Josée E #
Journal of Neurochemistry vol:85 issue:5 pages:1087-100
We studied the effects in rats of a 6-day intracerebroventricular (i.c.v) infusion of four different end-capped phosphorothioate-modified antisense oligonucleotides (AOs), specifically targeting different regions of the 5-hydroxytryptamine2A (5-HT2A) receptor mRNA, on central 5-HT2A receptor expression and 5-HT2A receptor-mediated behaviours. Only one of the AOs (sequence 4), directed against the 5'-untranslated region (from + 557 to + 577), specifically affected central 5-HT2A receptor expression and receptor-mediated behaviour. This AO (sequence 4) reduced binding of the 5-HT2A agonist 1-(2,5-dimethoxy-4-[125I]iodophenyl)-2-aminopropane ([125I]DOI) up to 25% in cortical areas, as measured by quantitative autoradiography. Cortical binding of the antagonist [3H]ketanserin was not affected. As the specific AO treatment presumably affects the synthesis of new receptor, we hypothesize that this newly synthesized receptor represents the major part of the functionally active, G protein coupled receptor. A 5-day infusion of AO (sequence 4) resulted in profound inhibition of the head-twitch response (HTR) to 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM). In contrast, treatment with vehicle, sense oligonucleotides (SOs) and other AOs (sequences 1, 2 and 3) caused an increased DOM-induced HTR as well as a spontaneous HTR. The latter was abolished by treatment with the 5-HT2 receptor antagonist, ritanserin. Systematic investigation of the surgical and infusion procedures revealed that the enhanced HTR already appeared following drilling of the skull. This wounding can probably damage the blood-brain barrier and cause a stress-induced increase in serotonergic transmission. AO (sequence 4) treatment also abolished the spontaneous HTR. AO (sequence 4) treatment allowed the identification of specific central 5-HT2A receptor-mediated behaviours in the complex serotonergic syndrome induced by tryptamine in rats. Only bilateral convulsions and body tremors were significantly inhibited. The backward locomotion, hunched back and Straub tail were not affected, nor was cyanosis, an index of vasoconstriction induced by peripheral 5-HT2A receptor activation. Labelling of central 5-HT2C receptors by [3H]mesulergine, and 5-HT2C receptor-mediated anxiety were not attenuated by AO or SO treatment. Rats treated with AO (sequence 4) showed increased locomotor activity and a strong reactivity towards touching. We hypothesize that the down-regulation of functional 5-HT2A receptors may shift the balance between various 5-HT receptor subtypes. Our analysis of the behavioural consequences of AO treatment and the use of different AOs and SOs has shown that specific receptor-mediated behaviour can be identified.