SCOP/PHLPP1β in the basolateral amygdala regulates circadian expression of mouse anxiety-like behavior

While disruption of the circadian clock triggers a spectrum of affective abnormalities, how the clock regulates mammalian emotionality remains unclear. Here, we characterized the time-of-day-dependent regulation of mouse anxiety-like behaviors. We show that anxiety-like behaviors are expressed in a circadian manner in mice and demonstrate that the clock machineries in the dorsal telencephalon (dTel) are required for the time-of-day-dependent regulation of anxiety-like behaviors. We identify suprachiasmatic nucleus circadian oscillatory protein (SCOP/PHLPP1β) as an essential intracellular signaling molecule mediating this temporal regulation downstream of the clock. Using viral-mediated, basolateral amygdala (BLA)-specific knockout of Scop, we demonstrate that deletion of SCOP in the BLA exerts anxiolytic effects on the elevated plus maze at early subjective night, thereby blunting the circadian variation in the anxiety-like behavior. We conclude that the circadian expression of SCOP in the BLA plays a key role in generating circadian rhythmicity in the anxiety-like behavior. Our results demonstrate SCOP as a regulator of anxiety-like behaviors and reveal its key roles in the anxiogenic functions of the BLA.

cycle (lights on at 8 am) in temperature-and humidity-controlled compartments with food and water available ad libitum.
Upon Cre-mediated recombination, the β-geo gene is excised out, and mCherry is expressed. pCAG-iCre served as a control for recombinase activity. Transfection was carried out using a standard polyethylenimine (PEI) method. Briefly, DNA (500 µg per 100-mm culture dish) was diluted in dH₂O, mixed with PEI (100 µg/mL final), incubated for 15 min at RT, and added to the culture medium. GFP and mCherry intensities were examined 48 hours after transfection under an inverted fluorescent microscope (Keyence, Osaka, Japan). To verify that the human synapsin (hSyn) promoter induces Cre expression in neuronal cells, Neuro-2a (N2a) cells were transfected with pAAV-Cre or pAAV-GFP using Lipofectamine 3000 reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). GFP and mCherry intensities were examined as described above.
Validated pAAV vectors were then transfected to HEK293T/17 cells, together with pACG2-Y730F 6 and pHelper (Stratagene) plasmids, using a standard PEI method. Cells were harvested 72 hrs after transfection, collected in PBS, pelleted, and resuspended in 200 µl of Dulbecco's PBS per 100-mm culture dish (60 cm 2 ). After 3 freeze-thaw cycles in liquid nitrogen and 37°C water bath followed by a centrifugation, the lysate was treated with 1.25 U/µL Benzonase (Merck, Darmstadt, Germany) for 30 min at 37°C and then subjected to serial centrifugation until the supernatant was clear.
The final purified viruses were aliquoted and stored at -80°C. The titers of our purified viral vectors were as follows: AAV-Cre, 8 x 10 12 ; AAV-GFP, 1 x 10 13 genome copies/mL.
To verify the infectious ability, purified AAV viral vectors were transduced to primary hippocampal neurons. Primary hippocampal cultures were prepared as previously described 7 . Purified AAV-Cre or AAV-GFP was added to primary cultures at 20 days in vitro (DIV), and GFP signal was examined at 40 DIV as described above.

Surgery.
Male Scop fl/fl mice aged 8-10 weeks were deeply anesthetized with a mixture of ketamine (140 mg/kg) and xylazine (8.8 mg/kg) in bacteriostatic saline given intraperitoneally (20 ml/kg) and placed on a stereotactic apparatus (Narishige, Tokyo, Japan). The skull was exposed, and holes were drilled bilaterally above the basolateral amygdala. The coordinates relative to bregma were: anteroposterior, -1.65 mm; lateral, +3.30 mm; dorsoventral, -4.45 mm. Mice were bilaterally injected with 0.5 µl of either AAV-iCre::EGFP or AAV-EGFP over 5 min, and the needles were kept in place for an additional 5 min to ensure infusion.
Littermate Bmal1 fl/fl Emx1 +/+ or Scop fl/fl Emx1 +/+ mice were used for control. Prior to testing, all mice were singly housed, entrained to the LD cycle for >2 weeks, and handled daily for acclimation at random times of day for >1 week. Mice that underwent stereotactic surgeries were allowed to recover for 4 weeks before the behavioral tests. Tissue preparation. For mRNA and protein analyses, mice were sacrificed by rapid cervical dislocation. Brain sections of 1-mm thickness containing the amygdala were prepared using a brain matrix (ASI Instruments, Warren, MI, USA), and basolateral and centromedial amygdalar complexes (BLA and CeA, respectively) were dissected using surgical knives, rapidly frozen, and stored at -80°C. For profiling SCOP expression in brain tissues, the BLA and CeA were dissected as described above, posterior hippocampi were cut out from the 1-mmthick slice from which the amygdala nuclei were sampled, olfactory bulbs and striatum were dissected 8 , and a region containing the midbrain, cerebellum, pons, and medulla are combined ("posterior brain"). Bilateral tissue from each mouse were combined and treated as one sample (see below).
For the validation of Scop BLA KO, Scop fl/fl mice transduced with AAV-Cre or AAV-GFP were sacrificed, and brain sections were prepared as described above.
The slices were then mounted on a glass slide and analyzed for GFP signal under a fluorescent stereoscopic microscope (Leica, Wetzlar, Germany). GFP-positive regions of the BLA were cut out using surgical knives while mounted on the microscope, rapidly frozen, and stored at -80°C until subsequent RNA extraction.
For fluorescent microscopy, AAV-injected mice were sacrificed by rapid cervical dislocation, and the brain was removed, mounted on a brain matrix (ASI Instruments), and sliced into 1-mm thick sections. The sections were analyzed for GFP signal under a fluorescent stereoscopic microscope (Leica). Roche, Basel, Switzerland). The homogenates were kept on ice for 15 min and spun at >10,000 x g for 15 min. The supernatants were subjected to SDS-PAGE and immunoblotting as previously described 7 . Signal was visualized using a conventional enhanced chemiluminescence detection system (GE Healthcare, Waukesha, WI, USA) and quantified using ImageJ software (NIH) by densitometric analyses based on relative standards loaded on every polyacrylamide gel. Immunoblotting conditions were as follows: αCB rabbit polyclonal antibody against SCOP 10 , 1/1,500 in 3% BSA/TBST; mouse monoclonal antibody against β-actin (Sigma #A2228), 0.4 µg/mL in 3% skim milk (Difco, Detroit, MI, USA)/TBST; B1BH2 mouse monoclonal antibody against BMAL1 11 , 1 µg/mL in 1% skim milk; blocking: 1 hr at RT, primary antibody: overnight at 4°C, secondary antibody: 2 hrs at RT. Data analysis. Behavioral analyses were automated except for the elevated plus maze (EPM) test for wild-type C57BL/6J mice, for which all recorded behavioral data were shuffled into random orders, and entries into open and closed arms and the time spent on each arm were recorded by an operator blinded to time-of-day information for each subject. One-way ANOVA tests were used to examine the statistical significance of data consisting of three or more groups. Unpaired two-tail Student's t-tests were used for analysis on data with two groups. All data are presented as means with SEM. P values are presented as "P" for ANOVA tests and "p" for t-tests.