Activation of D1R/PKA/mTOR signaling cascade in medial prefrontal cortex underlying the antidepressant effects of l-SPD

Major depressive disorder (MDD) is a common neuropsychiatric disorder characterized by diverse symptoms. Although several antidepressants can influence dopamine system in the medial prefrontal cortex (mPFC), but the role of D1R or D2R subtypes of dopamine receptor during anti-depression process is still vague in PFC region. To address this question, we investigate the antidepressant effect of levo-stepholidine (l-SPD), an antipsychotic medication with unique pharmacological profile of D1R agonism and D2R antagonism, and clarified its molecular mechanisms in the mPFC. Our results showed that l-SPD exerted antidepressant-like effects on the Sprague-Dawley rat CMS model of depression. Mechanism studies revealed that l-SPD worked as a specific D1R agonist, rather than D2 antagonist, to activate downstream signaling of PKA/mTOR pathway, which resulted in increasing synaptogenesis-related proteins, such as PSD 95 and synapsin I. In addition, l-SPD triggered long-term synaptic potentiation (LTP) in the mPFC, which was blocked by the inhibition of D1R, PKA, and mTOR, supporting that selective activation of D1R enhanced excitatory synaptic transduction in PFC. Our findings suggest a critical role of D1R/PKA/mTOR signaling cascade in the mPFC during the l-SPD mediated antidepressant process, which may also provide new insights into the role of mesocortical dopaminergic system in antidepressant effects.

cycle with 5 mice in one cage under specific pathogen free (SPF) condition. All procedures were carried out in accordance with the EU Directive 2010/63/EU on the protection of animals used for scientific purpose and the protocols were approved by the Animal Care Committees of Shanghai Institute of Materia Medica, Chinese Academy of Science. The naï ve C57BL/6 mice were divided into four groups (9-10 mice per group): 1) vehicle, 2) 5 mg· kg -1 l-SPD, 3) 10 mg· kg -1 l-SPD and 4) 10 mg· kg -1 fluoxetine administration (i.p.). All drugs were administrated once daily at 9:00 am for 10 days. The injected volume was 10 ml/kg

Tail suspension test (TST)
Tail suspension test (TST) is the most commonly used experiments to assess depressive-like behavior 1 . In this experiment, naï ve C57BL/6 mice were moved to the testing room 2 h before the behavioral tests started. The mice were suspended by fixing their tail on a TST apparatus. The test lasted 6 min, and the process was monitored online. Due to the most mice were very active at the beginning of the test and the potential effects of the treatment can be obscured during the first two minutes, the immobility of the last 4 min were measured.

Elevated plus maze (EPM)
The EPM apparatus for C57BL/6 mice consisted of two enclosed (30 x 5 x 30 cm) and two open arms (30 x 5 cm) with a height of 60 cm to the floor. The same type of arm was opposite to each other and connected by a central area (5 x 5 cm). At the beginning of the EPM test, the mice were comforted and placed in the center of maze with the head facing an enclosed arm. Each animal was allowed to perform for 6 minutes, and the activity was monitored online. Behaviors scores were calculated as the percent of the distance moved into the open arms. Arm entries were defined as entry of all four paws into the arm.

Immunohistochemistry (IHC)
The C57BL/6 mice were deeply anesthetized and perfused with 0.9% physiological saline. Next, the mice were perfused with 4% paraformaldehyde and immersed into 4% paraformaldehyde for 24 hours. The brain was dehydrated in 30% sucrose for 3 days. For the location of the PFC, we referred to the mouse brain atlas and the thickness of brain slice was 30 µm. Brain slices were treated by 0.3% Triton X-100 and 0.3% H2O2 and then blocked in 10% goat serum (Gibco) and incubated in primary antibody (anti-pmTOR (Ser 2448), 1:50, Cell Signaling; anti-PSD 95, 1:50, Invitrogen) at 4°C overnight. Brain slices were washed and incubated in secondary antibody (goat pAb to Rb IgG, 1:200, Abcam) for 2 hours. Then, the brain slices were incubated in streptavidin biotin peroxidase complex (SABC) for 1 hour and then washed and transferred to microscope slides. After being dried, the brain slices were stained using DAB substrate solution for 5 min.
Brain slices were dehydrated in gradient alcohol (70%, 95%, 100% and 100%) and cleared by xylene. Mounted brain slice by mounting solution and observed under a microscope.

Statistical analysis
The data are expressed as the mean ± SEM. The results of behavioral tests including TST and EPM in C57BL/6 mice were analyzed using one-way ANOVA, and post hoc LSD tests were performed. The western blot results were analyzed using one-way ANOVA, and post hoc LSD tests were performed.

Figure S1
Antidepressant-like and anxiolytic-like effects of l-SPD in C57BL/6 mice. (A) The schematic representation for experimental procedures. The C57BL/6 mice were treated with vehicle, l-SPD (5 mg· kg -1 or 10 mg· kg -1 ) and fluoxetine (10 mg· kg -1 ) daily for 10 days, the drugs were administrated at 9:00 am every day. Behavioral tests were performed 24 hours after the last drug injection. The brain tissues were obtained 24 hours after the last drug administration.    Figure   S2A. In Figure 3A, only red dashed boxes part were displayed.