Clozapine functions through the prefrontal cortex serotonin 1A receptor to heighten neuronal activity via calmodulin kinase II-NMDA receptor interactions

Aberrant dopamine release in the prefrontal cortex (PFC) is believed to underlie schizophrenia, but the mechanistic pathway through which a widely used antipsychotic, clozapine (Clz), evokes neurotransmitter-releasing electrical stimulation is unclear. We analyzed Clz-evoked regulation of neuronal activity in the PFC by stimulating axons in layers IV and V and recording the electrical effect in the post-synaptic pyramidal cells of layers II and III. We observed a Clz-evoked increase in population spike (PS), which was mediated by serotonin 1A receptor (5-HT _1A-R), phospholipase Cβ, and Ca ²⁺/calmodulin-dependent protein kinase II (CaMKII). Immunoblotting demonstrated that the Clz-activation of CaMKII was 5-HT _1A-R-mediated. Intriguingly, the NMDA receptor (NMDA-R) antagonist (±)2-amino-5- phosphonovaleric acid (APV) eliminated the Clz-mediated increase in PS, suggesting that the 5-HT _1A-R, NMDA-R and CaMKII form a synergistic triad, which boosts excitatory post-synaptic potential (EPSP), thereby enhancing PS. In corroboration, Clz as well as NMDA augmented field EPSP (fEPSP), and WAY100635 (a 5-HT _1A-R antagonist), APV, and a CaMKII inhibitor eliminated this increase. As previously shown, CaMKII binds to the NMDA-R 2B (NR2B) subunit to become constitutively active, thereby inducing α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor recruitment to the post-synaptic membrane and an increase in fEPSP. Co-immunoprecipitation demonstrated that Clz potentiates interactions among CaMKII, NR2B, and 5-HT _1A-R, possibly in the membrane rafts of the post-synaptic density (PSD), because pretreatment with methyl-β- cyclodextrin (MCD), an agent that disrupts rafts, inhibited both co-immunoprecipitation as well as fEPSP. In summary, Clz functions in the PFC by orchestrating a synergism among 5-HT _1A-R, CaMKII, and NMDA-R, which augments excitability in the PFC neurons of layers II/III. How clozapine worksNerve signals in the analytical center of the brain prefrontal cortex (PFC) are impaired in Schizophrenia but how the therapeutic agent clozapine normalizes these signals is unknown. This study analyzes nerve signals and molecular interactions in the PFC to explain how clozapine works. Our findings unify multiple prior hypotheses into one mechanistic model for the action of clozapine.