Dysfunction of dopamine neurotransmission is considered a central feature of schizophrenia, with antipsychotic drugs (APDs) targeting dopamine D2 (D2) receptors to alleviate positive psychotic symptoms in about one half of patients. However the therapeutic window of D2 receptor blockade (65-78%) within which most APDs achieve optimal clinical utility does not extend to the atypical APD clozapine, despite its superior efficacy in treatment resistant schizophrenia. Sub-threshold levels of D2 receptor blockade exerted by clozapine argues for a mechanism of action not solely reliant on D2 receptor antagonism. In accord with this, we have reported that clozapine signals to the mitogen-activated protein kinase-extracellular signal regulated kinase (MAPK-ERK) cascade via G-protein coupled receptor (GPCR) transactivation of the epidermal growth factor (EGF) receptor (EGFR or ErbB1) [1–3]. This was typified by clozapine induced early inhibition and delayed activation of the ERK response in prefrontal cortex (PFC) and striatum dependent on EGFR signaling in vitro  and in vivo[2, 3] unlike olanzapine or haloperidol. The convergence of the ERK cascade with GPCR and growth factor signaling systems upon activation by APDs is noteworthy since signal transduction from the cell surface to the nucleus can regulate cortical neurogenesis, synaptogenesis and neurotransmitter release, processes affected in schizophrenia [4–6]. Whether these cell signaling effects observed with clozapine extend to the second generation atypical APDs aripiprazole and quetiapine has not been tested. This is relevant given that aripiprazole and quetiapine display agonist and antagonist properties in animal models of dopaminergic hypoactivity and hyperactivity, respectively, share clozapine’s low D2 receptor binding profile (quetiapine) and are predicted to stabilize dopaminergic transmission.
Aripiprazole differs from other atypical APDs in that it acts by partial agonism at D2 and serotonin (5HT) 1A receptors . The drug modulates dopaminergic activity in areas where dopamine may be increased (mesolimbic regions) or diminished (mesocortical regions) in the brains of people with schizophrenia. Like other atypical APDs, aripiprazole antagonizes 5HT2A receptors and has moderate affinity for histamine and α-adrenergic receptors. Quetiapine is a multiple receptor antagonist with low affinity for D2 and higher affinity for 5HT2A, 5HT1A, α-1 and α-2 adrenergic and histamine H1 receptors . Positron emission topography studies indicate that quetiapine rapidly disassociates from the D2 receptor producing normal physiological surges of dopamine in the nigrostriatal and tuberoinfundibular tracts of the brain, thus minimizing the risk of extrapyramidal side effects (EPS) and elevations in prolactin. For both aripiprazole and quetiapine, however, effects on downstream ERK signaling that can regulate transcription factors such as Elk1 or CREB to shape gene expression, protein synthesis and receptor function is less well characterized. In this regard, acute aripiprazole treatment in mice reduced PFC ERK phosphorylation levels in one recorded study to date . By contrast, single time point experiments in Chinese Hamster Ovary (CHO) cells stably transfected with D2 short and 5HT1A  and D3 receptors  indicated that aripiprazole stimulated ERK phosphorylation via agonist activities at these receptors. Moreover, Urban et al.  reported that aripiprazole exerted only partial activation of the ERK pathway in CHO cells expressing D2 long receptors, whereas in PC12 cells aripiprazole promoted neurite outgrowth through activation at 5HT1A rather than D2 receptors and by Ca2+, inositol 1,4,5-triphosphate receptor and ERK signaling . Given the cell-dependent differences in the intrinsic activity of aripiprazole, a primary mechanism of action attributable to its functional selectivity at the D2 receptor and/or combined action at non-dopamine receptor systems rather than simple partial agonism has also been argued . Quetiapine too induces ERK mediated neurite sprouting via Gi/o coupled receptors in PC12 cells , and activated ERK2 after acute treatment in C6 glioma cells thought to be related to the putative antidepressant efficacy of the drug . In vivo, quetiapine facilitated oligodendrocyte development and prevented cuprizone induced cognitive impairments via ERK in mouse cortex  and upregulated ERK gene expression in rat cortex and striatum when administered acutely in conjunction with the mood stabilizer valproate . Whilst cortical and/or striatal ERK phosphorylation by aripiprazole and quetiapine integrate multiple signaling pathways to regulate neuronal processes relevant to the symptom domains of schizophrenia, there remains a paucity of data on the effects of these APDs on the expression of downstream proteins such as 90 kDa ribosomal s6 protein kinase (p90RSK) or c-fos, which potentially define their distinct clinical profiles.
p90RSK comprising the isoforms RSK1, RSK2 and RSK3 are a family of broadly expressed serine/threonine kinases activated by ERK. As a regulator of transcription, p90RSK phosphorylates the transcription factor cyclic AMP response element binding (CREB), which leads to the recruitment of transcriptional co-activators CREB binding protein and the induction of immediate-early genes such as c-Fos . ERK1 knock-out mice exhibit reduced phosphorylation of RSK1 in PFC and striatum, but not in hippocampus or cerebellum indicating ERK signaling deficits that are isoform and region-specific . However there is limited data on the effects of APDs on p90RSK levels, with aripiprazole or quetiapine treatment effects not documented. Similarly there is limited data for aripiprazole and quetiapine in relation to c-Fos which signals a genomic response to a variety of stimuli including growth factors and neurotransmitters, with regulation via the phosphorylation of transcription factors Elk-1 and CREB by ERK and RSK respectively . When compared with other D2 receptor partial agonists, aripiprazole caused less rotation in nigrostriatal lesioned rats (hypo-dopaminergic model) but clear Fos induction in the nucleus accumbens shell, indicative of low intrinsic activity despite functional antagonism, a purported marker of its antipsychotic efficacy . For quetiapine, elevated c-Fos expression in limbic but not motor related brain regions [8, 21] with a greater increase in Fos immunoreactivity in rat nucleus accumbens shell than dorsolateral striatum is in keeping with its atypical index and reduced EPS propensity . Apart from these data, the effects of aripiprazole and quetiapine on p90RSK and c-Fos signaling via the ERK pathway and the interrelated EGFR system and how these may differ from clozapine are yet to be profiled.
From this standpoint and to examine whether ERK pathway signaling and transactivation of the EGFR is a mechanism that applies to atypical APDs other than clozapine, the studies undertaken here expand our earlier in vitro neuronal culture and in vivo animal experiments [1–3] to (i) determine whether acute aripiprazole or quetiapine treatment differentially regulates ERK1/2 phosphorylation over a 24 hr period in mouse PFC or striatum (ii) assess whether changes in ERK1/2 phosphorylation parallel changes in expression of the transcriptional regulators p90RSK and c-Fos across 24 hrs in PFC or striatum and (iii) establish if variations in ERK1/2, p90RSK or c-Fos levels following aripiprazole or quetiapine treatment in cortex or striatum are dependent on EGFR signaling. The PFC and striatum are examined given their relevance to APD signaling and innervation by the major dopamine tracts of the brain, the mesocortical and nigrostriatal pathways, respectively, and also by glutamatergic and gamma-amino-butyric acid (GABA)-ergic neurons . Both regions are important in the pathology of schizophrenia, since the PFC is linked with cognitive, negative and deficit syndrome symptoms and the striatum with motor control, reward and decision making and the EPS triggered by some APDs.