Cell experiment:

PC12 cells are cultured at 37℃, 5% CO2 in Dulbecco's modified Eagle's medium (DMEM), supplemented with 5% heat-inactivated fetal bovine serum (FBS),10% heat-inactivated horse serum, and 1% penicillin-streptomycin. Medium is changed two to three times a week. PC12 cells are plated onto 24- well tissue culture plates coated with poly-D-lysine/laminin. Cells are plated at relatively low density (0.25×104 cells/cm2) in DMEM medium containing 0.5% FBS,1% penicillin-streptomyc in Medium containing a minimal level of serum (0.5% FBS) is used. In this study, 2.5 ng/mL of NGF is used to study the potentiating effects of Brexpiprazole on neurite outgrowth. Twenty-four hours after plating, the medium is replaced with DMEM medium containing 0.5% FBS and 1% penicillin-streptomycin with NGF (2.5 ng/mL), with or without Brexpiprazole (0.001,0.01,0.1 or 1 μM), WAY-100,635 (5-HT1A receptor antagonist;10 μM), raclopride (dopamine D2 receptor antagonist;10 μM), DOI (5-HT2A receptor agonist;0.1,1 or 10 μM), M100,907 (5-HT2A receptor antagonist; 0.1, 1 or 10 μM), xestospongin C (IP3 receptor antagonist; 1 μM), 2-APB (IP3 receptor antagonist;100 μM), fluoxetine (5-HT transporter inhibitor: 1 μM), or paroxetine (5-HT transporter inhibitor: 1 μM). Four days after incubation with NGF (2.5ng/mL) with or without specified drugs, morphometric analysis is performed on digitized images of live cells taken under phase-contrast illumination, with an inverted microscope linked to a camera. Images of three fields per well are taken, with an average of 100 cells per field. Differentiated cells are counted by visual examination of the field; only cells that had at least one neurite with a length equal to the cell body diameter are counted, and are then expressed as a percentage of the total cells in the field. Counting is performed in ablinded manner[1].

Animal experiment:

Mice[2] Male C57BL/6NCrSlc mice aged between 4 and 5 weeks old are selected as stranger mice, while animals between 8 and 10 weeks old are used for this study. All mice are housed in groups of five percage, in a room maintained at 23±2℃ and 60±10% humidity, with a 12/12h light/dark cycle (lights on at 7:00 a.m.).The mice are given free access to food and water. Brexpiprazole is dissolved in 5% (w/v) gum Arabic and administered orally (p.o.), at 10 mL/kg, 1 h prior to sociability testing. The doses of antipsychotic drugs are selected based on doses that did not impact locomotion.

Brexpiprazole is a potent antagonist effects on 5-HT2A, α1B-, and α2C-adrenergic receptors and a partial agonist at serotonin 1A (5-HT1A) and D2 receptors. [1]
5-HT1A receptor is a G protein-coupled receptor that can mediates inhibitory neurotransmission by binding the endogenous neurotransmitter serotonin5-HT. And D2 receptor is a G protein-coupled receptor inhibits adenylyl cyclase activity.
In cloned receptor systems, brexpiprazole displayed partial agonist at h5-HT1A and hD2 receptors, and potent antagonism of h5-HT2A receptors and hα1B/2C-adrenoceptors. [2] When tested in PC12 cells, brexpiprazole increased the number of cells with neurites in a concentration-dependent manner through 5-HT1A receptors and 5-HT2A receptors.[3]
In the dizocilpine-induced social recognition deficits mice model, brexpiprazole (0.01, 0.03, 0.1mg/kg,p.o.) significantly ameliorated the recognition deficits, and had no effect on social recognition in untreated control mice. Brexpiprazole may confer a beneficial effect on social cognition deficits in patients with psychiatric disorders. [1]
References:
[1]. Noriko Yoshimi, Takashi Futamura, Kenji Hashimoto. Improvement of dizocilpine-induced social recognition deficits in mice by brexpiprazole, a novel serotonin-dopamine activity modulator. European Neuropsychopharmacology, http://dx.doi.org/10.1016/j.euroneuro.2014.12.014
[2]. Maeda K, Sugino H, Akazawa H et al. Brexpiprazole I: in vitro and in vivo characterization of a novel serotonin-dopamine activity modulator. journal of pharmacology and experimental therapeutics, 2014 Sep;350(3):589-604.
[3]. Tamaki Ishima, Takashi Futamura, Yuta Ohgi et al. Potentiation of neurite outgrowth by brexpiprazole, a novel serotonin–dopamine activity modulator: A role for serotonin 5-HT1A and 5-HT2A receptors. European Neuropsychopharmacology, http://dx.doi.org/10.1016/j.euroneuro.2015.01.014