Ry mainly because meals deprivation was made use of in our behavioral tasks as a mild stressor [22]. Body weight was measured and recorded on the final day in the week. The rats have been sacrificed soon after the behavioral tests. The whole brains of 3 rats in every single group had been removed just after accepting heart perfusion. The brains were fixed in 4 paraformaldehyde remedy for additional histopathological assessment and immunohistochemistry. The left hippoacmpi of the remaining rats have been flash-frozen in liquid nitrogen and stored at -80 C for protein evaluation. The right hippocampi had been kept in ten volumes of RNAlater Solution (Ambion, Life technologies, Carlsbad, CA, USA) for polymerase chain reaction (PCR) evaluation. 2.four. Hormone Measurement. Blood was collected in the abdominal aorta by a DL-��-Tocopherol site puncturing needle. Cerebrospinal fluid (CSF) was extracted from the NI-42 Description foramen magnum by a 1 mL syringe. Blood samples have been placed at room temperature for 30 min ahead of centrifugation for 10 min at 3,000 . Serum adrenocorticotropic hormone (ACTH) and corticosterone (CORT) have been analyzed using an IMMULITE 2000 immunoassay program with ACTH and CORT immunoassay kits. The serum as well as the CSF levels of CRH and urocortin-2 (UCN2) had been detected by a microtiter plate reader (Victor3V , Perkin Elmer, Waltham, MA, USA) with rat CRH enzymelinked immunosorbent assay (ELISA) kit (Baoman Biotech, Shanghai) and rat UCN2 ELISA kit (Chang Yi Chemical, Chemical, Shanghai). two.five. Histopathological Assessment and Nissl’s Staining. In the histopathological examination, paraformaldehyde resolution was employed to repair the paraffin-embedded brain tissues that were reduce into serial sections (three m). The slices had been stained with hematoxylin and eosin and Nissl’s solution inside a routine process. two.six. Two-Dimensional Electrophoresis (2D) and Protein Identification. The 2D electrophoresis was performed as previously reported and repeated for three occasions [18]. Briefly, hippocampal samples containing 300 g of protein had been loaded per tube in an isoelectric focusing method (IPGphor II, GE). The samples had been isoelectrofocused and separated with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The gels have been silver-stained. The protein spots that either increased or decreased for much more than twofold have been selected for matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) identification. The3 mascot software package and also the database of SwissProt had been employed to match the mass of peptides [23]. 2.7. Western Blot Evaluation. Western blot analysis was performed as previously described [24]. Briefly, the protein lysates have been loaded onto ten SDS-PAGE for separation, electrotransferred onto PVDF membranes, and blocked in 5 nonfat milk in Tris-buffered saline Tween. The membranes were incubated overnight with principal antibodies, like anti–arrestin 2 (Cell Signaling Technologies, CST, clone C16D9, 1 : 750 dilution), anti-extracellular signal-regulated kinase (ERK, CST, Clone 137F5, 1 : 800 dilution), antiphospho-ERK (Thr202/Tyr204, CST, 1 : 800 dilution), antityrosine receptor kinase B (TrkB, Bioss, clone bs-0175R, 1 : 400 dilution), anti-BDNF (epitomics, 1 : 4000 dilution), anti-serine/threonine-protein phosphatase 2A subunit B (PP2A b, Abcam, 1 : 800 dilution), anti-PP2A c (Abcam, 1 : 750 dilution), anti-CRH receptor 1 (CRHR1, Bioss, 1 : 400 dilution), anti-CRH receptor two (CRHR2, Abcam, 1 : 800 dilution), anti-mammalian target of rapamycin (mTOR, CST, 1 : 800 dilution), anti-phospho-mTOR (phospho-S244.
