Repeated Clozapine Increases the Level of Serotonin 5-HT1AR Heterodimerization with 5-HT2A or Dopamine D2 Receptors in the Mouse Cortex

Abstract

G-protein–coupled receptor (GPCR) heterodimers are new targets for the treatment of schizophrenia. Dopamine D2 receptors and serotonin 5-HT1A and 5-HT2A receptors play an important role in neurotransmission and have been implicated in many human psychiatric disorders, including schizophrenia. Therefore, in this study, we investigated whether antipsychotic drugs (clozapine (CLZ) and haloperidol (HAL)) affected the formation of heterodimers of D2–5-HT1A receptors as well as 5-HT1A–5-HT2A receptors. Proximity ligation assay (PLA) was used to accurately visualize, for the first time, GPCR heterodimers both at in vitro and ex vivo levels. In line with our previous behavioral studies, we used ketamine to induce cognitive deficits in mice. Our study confirmed the co-localization of D2/5-HT1A and 5-HT1A/5-HT2A receptors in the mouse cortex. Low-dose CLZ (0.3 mg/kg) administered repeatedly, but not CLZ at 1 mg/kg, increased the level of D2–5-HT1A and 5-HT1A–5-HT2A heterodimers in the mouse prefrontal and frontal cortex. On the other hand, HAL decreased the level of GPCR heterodimers. Ketamine affected the formation of 5-HT1A–5-HT2A, but not D2–5-HT1A, heterodimers.

Dimers of serotonin receptors: Impact on ligand affinity and signaling

“…For example, the prototypical atypical antipsychotic clozapine is a high affinity inverse agonist for 5-HT 2A , low affinity antagonist for D2, and a partial agonist for 5-HT 1A receptors [63]. Interestingly, D2-5-HT 1A heterodimers have been detected in-vivo by PLA experiments in the mouse prefrontal cortex [21]. A quantification of the proportion of the dimeric receptors seen by PLA would further validate these findings.…”

“…The D2-5-HT 1A complex thus possesses functional properties distinct from homodimers. Moreover, clozapine has been shown to increase the level of D2-5-HT 1A heterodimers in the mouse prefrontal cortex, while the typical antipsychotic haloperidol, a high affinity D2 antagonist, decreased it [21]. Altogether, these data indicate that D2-5-HT 1A receptor heterodimers activate specific pathways, with inositol phosphate production and ERK1/2 activation, and that the level of D2-5-HT 1A heterodimers can be differentially regulated by various antipsychotic treatments.…”

“…In-vivo, PLA-positive clusters of 5-HT 1A -5-HT 2A receptors were identified in pyramidal cells of the CA1-CA3 regions in the rat hippocampus [62]. Upon prior forced swimming test, these clusters were reduced in the mouse cortex [21], whereas they were increased by 5-HT 2A -receptor antagonist and by low dose of the antipsychotic clozapine. A quantification of the proportion of the dimeric receptors seen by PLA would be further needed to validate these findings.…”

“…Evidence for the formation of homodimers has been reported for various 5-HT receptor subtypes including 5-HT 1B [12], 5-HT 1A [13,14], 5-HT 2A [15], 5-HT 2C [16], 5-HT 4 [17], and 5-HT 7 [18] receptors in heterologous expression systems. Other evidence pointing for the possibility of different 5-HT receptor subtypes to heterodimerize was later provided for 5-HT 1B with 5-HT 1D [12,19], 5-HT 1A with 5-HT 7 [20], 5-HT 1A with 5-HT 2A [21], 5-HT 2A , with 5-HT 2B or 5-HT 2C, and 5-HT 2B , with 5-HT 2C [22] receptors. There are also observations supporting that some 5-HT receptors form heterodimeric complexes with GPCRs responding to other natural ligands than 5-HT.…”

Expression of serotonin 1A and 2A receptors in molecular- and projection-defined neurons of the mouse insular cortex

“…Guinea pig polyclonal anti-NeuN (1:500; 266004, SYSY) [35], mouse monoclonal anti-GAD67 (1:500; MAB5406, Millipore) [36][37][38], rabbit polyclonal anti-5-HT1A receptor (1:500; ADI-905-741, Enzo) [39] and rabbit polyclonal anti-5-HT2A receptor (1:250; ab66049, Abcam) [40][41][42][43][44] were used as primary antibodies. The primary antibodies used in this study were validated and widely used in previous publications [35][36][37][38][39][40][41][42][43][44]. Alexa Fluor 488 goat anti-rabbit IgG (A11034, Fisher Scientific), Alexa Fluor 555 goat anti-guinea pig IgG (A21435, Fisher Scientific) and Alexa Fluor 647 goat anti-mouse IgG (A21236, Fisher Scientific) were used as secondary antibodies.…”