rotenone Based on the observation that antipsychotic
Based on the observation that antipsychotic drugs increase 5-HT1A–D2 heteromerization level (Łukasiewicz et al., 2016), we set out to compare the effect of paroxetine to that of risperidone. Paroxetine, a potent SSRI, is very effective in the treatment of depression and anxiety disorders, e.g., generalized anxiety disorder, panic disorder, social anxiety disorder and obsessive–compulsive disorder (Zohar and Judge, 1996, Ballenger et al., 1998, Stein et al., 1998, Snyderman et al., 2004, Manassis, 2005, Sanchez et al., 2014). Risperidone is an atypical antipsychotic drug, but there are many clinical proofs that it is effective as an adjunctive or in monotherapy in the treatment of anxiety-related disorders, i.e., panic attacks, generalized anxiety disorder, obsessive–compulsive disorder and post-traumatic stress disorder (Jacobsen, 1995, Saxena et al., 1996, Bartzokis et al., 2005, Brawman-Mintzer et al., 2005, Erzegovesi et al., 2005, Galynker et al., 2005, Gao et al., 2006; Padala et al., 2006, Simon et al., 2006). What is important, relief in anxiety symptoms might occur with very low doses of risperidone, far below those used in treating psychosis, what implies a concomitant reduction of adverse effects (Prosser et al., 2009), but the molecular mechanisms underlying this effect are not clear. The interaction of 5-HT1A–D2 receptors upon risperidone administration is an interesting possible target, since risperidone has affinity toward both receptors types (Leysen et al., 1994). To evaluate the level of 5-HT1A–D2 heteromerization in mouse rotenone and primary neuronal cells, we used the in situ proximity ligation assay (PLA) (Sӧderberg et al., 2006, Trifilieff et al., 2011), which has been recently successfully applied to studies of other pairs of GPCRs (Borroto-Escuela et al., 2013, Rico et al., 2016). PLA is gaining constantly growing appreciation because it enables studying protein interactions in the natural brain microenvironment. While PLA can detect two proteins (here 5-HT1A and D2 receptors) located in very close physical proximity (i.e., <17 nm) (Sӧderberg et al., 2006, Weibrecht et al., 2010, Trifilieff et al., 2011), it does not provide any indication on the total number of 5-HT1A and D2 receptors, when considered separately. To overcome this limitation, the expression of 5-HT1A and D2 receptors was measured by mRNA in situ hybridization and ligand autoradiography. Additionally, we performed counterstaining with neuronal and astrocytal markers in adult primary brain cell culture after PLA to indicate the exact cellular localization of 5-HT1A–D2 receptor heteromers. Furthermore, we decided to confirm and extend the obtained results with in vitro homogenous time-resolved FRET (HTRF) technology applied to human 5-HT1A and D2 receptors.
Discussion An ever increasing number of studies, performed both in vitro and ex vivo with a number of techniques, e.g., co-immunoprecipitation, fluorescence resonance energy transfer (FRET), and competition binding assays, have reported heteromerization between GPCRs and its functional significance in receptors trafficking, internalization and the modulation of signaling pathways (Devi, 2001, Milligan, 2004, Albizu et al., 2011, Fribourg et al., 2011, González-Maeso, 2011). The direct interaction of 5-HT1A and D2 receptors has been recently confirmed in vitro with two FRET-based techniques, i.e., time-resolved fluorescence lifetime imaging microscopy and HTRF, and completed with second messenger assays (Łukasiewicz et al., 2016). Most detected heteromers, including 5-HT1A–D2 receptor heteromers, have exhibited properties distinct from those of each component receptor (protomers), when considered individually, i.e., unique pharmacology, signaling, or trafficking. The above-mentioned in vitro study has shown that human 5-HT1A and D2 receptors are able to form heteromers in HEK293 cells, and antipsychotic medications are able to induce the increase in the number of 5-HT1A–D2 receptor heterodimers. Moreover, 5-HT1A–D2 receptor heteromerization has potent functional consequences, such as increased ERK1/2 signaling under the presence of antipsychotics (Łukasiewicz et al., 2016).