Direct pharmacological effects of the multimodal antidepressant trazodone on the serotonergic neuron activity in the dorsal raphe nucleus

Abstract: The principal research activity that characterized my PhD studies was the investigation of the electrophysiological and pharmacological direct effects of trazodone at somatodendritic 5-HT1A receptors (5-HT1AARs) of serotonergic neurons of dorsal raphe nucleus (DRN). INTRODUCTION: Multimodal antidepressant drugs form a class of heterogeneous substances that, in addition to SERT inhibition, exert a variety of receptor actions believed to participate in the therapeutic action of these medicines. Trazodone, in addition to its inhibitory activity at cell membrane serotonin transporter (SERT), displays considerable affinity for 5-HT1A 5-HT2A, 5-HT2C receptors and α1-adrenoceptors. Although these pharmacological properties have been suggested to contribute to trazodone a profile of antidepressant and sleep facilitator, the direct effects of trazodone on serotoninergic neuron activity are still not adequately known for modelling the possible pharmacological mechanisms underlying the therapeutic action(s) of the drug. Indeed, the interplay between the 5-HT1A receptor agonist activity and α1-adrenoceptor antagonist activity of trazodone at the level of serotonergic neurons likely plays a crucial role in regulating the firing of serotonergic neurons, hence the release of serotonin in projection areas during acute and chronic administration of the drug. METHODOLOGY: We designed in vitro experiments directed to quantify the agonist efficacy of trazodone at 5-HT1AARs of DRN serotonergic neurons and to establish the possible effect of α1-adrenoceptor antagonism. The activity of serotonergic neurons was recorded with loose-seal cell-attached patch-clamp (LSCAT) in brainstem slices. To reproduce in slices the noradrenergic drive which in vivo facilitates serotonergic neuron firing, ACSF was supplemented with the selective α1-adrenoceptor agonist phenylephrine (PE 10 µM, unless otherwise indicated). To quantify the intrinsic activity of trazodone, we investigated the potency and the functional effect of the drug at 5-HT1AARs using whole-cell recordings. To this purpose, we measured the changes in slope conductance of 5-HT1A receptor-coupled GIRK channels, which provided a direct measure of 5-HT1A receptor activation produced by the application of trazodone. RESULTS: In LSCAT recording of serotonergic neuron activity facilitated by 10 µM PE, the firing rate of neurons was concentration-dependently inhibited by trazodone (1 - 30 µM). The selective 5-HT1A receptor antagonist Way-100635 partially antagonized the effect of the antidepressant indicating that the inhibitory effect exerted by trazodone comprised an additional action of the drug. Both effects of the antidepressant drug were also observed in slices from Tph2-/- mice that lack synthesis of brain serotonin, showing that the activation of 5-HT1AARs and the unknown additional effect were not mediated by endogenous serotonin. As trazodone binds with relatively high affinity to α1-adrenoceptors for which it is antagonist, in conditions of α1-adrenoceptor stimulation, as those we applied in vitro, the non 5-HT1A receptor-mediated inhibitory effect of trazodone on serotonergic neuron activity could have been produced by α1-adrenoceptor block. Consistent with this hypothesis, in the absence of phenylephrine, trazodone (1 - 10 µM) concentration-dependently silenced neurons through activation of 5-HT1AARs, as the effect was fully antagonized by Way-100635. In experiments in which the 5-HT1A receptor-mediated component of trazodone action was blocked by the presence of Way-100635, trazodone (1 - 10 µM) concentration-dependently inhibited neuron firing facilitated by 1 µM phenylephrine. Parallel rightward shift of dose-response curves for trazodone recorded in higher phenylephrine concentrations (10 and 100 µM) indicated competitive antagonism at α1-adrenoceptors. On the other hand, in the presence of a high concentration of PE (100 µM, Way-100635 absent) to minimize the α1-adrenoceptor-dependent effect of trazodone, the drug dose-dependently inhibited serotonergic neuron activity. The effect was variable and often did not silence the neuron firing, indicating the possibility that trazodone is a partial agonist at 5-HT1AARs. To confirm this property, in whole-cell recordings we measured the 5-HT1AAR-coupled GIRK conductance activated by the drug. These recordings confirmed the weak partial agonism of trazodone at 5-HT1A receptors, with an efficacy of ~35 % when compared to the 5-HT1A receptor full agonist 5-carboxamidotryptamine (5-CT) in the same neurons. Consistent with its weak partial agonist activity, trazodone was able to partially antagonize the effect of 5-CT at 5-HT1AARs. CONCLUSIONS: Our data show that trazodone, at concentrations relevant to its clinical effects, exerts weak partial agonism at 5-HT1AARs and disfacilitation of firing through α1-adrenoceptor antagonism. These two actions converge in inhibiting dorsal raphe serotonergic neuron activity, albeit with varying contribution depending on the intensity of α1-adrenoceptor stimulation. These two direct effects on serotonergic neuron activity are likely to be implicated in the in the therapeutic action of trazodone. Indeed, it has been suggested that antidepressant drugs endowed of 5-HT1A receptor partial agonist property could hasten the therapeutic response during the treatment. Furthermore, antagonism at α1-adrenoceptor is likely to dampen the effects of noradrenergic system reactivation that occurs during sleep and prevent insomnia-related microarousals, which may underlie the sleep facilitating property of trazodone.