Ry because food deprivation was made use of in our behavioral tasks as a mild stressor [22]. Body weight was measured and recorded on the final day on the week. The rats have been sacrificed immediately after the behavioral tests. The entire brains of 3 rats in each and every group had been removed soon after accepting heart perfusion. The brains were fixed in 4 paraformaldehyde answer for additional histopathological assessment and immunohistochemistry. The left hippoacmpi with 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 10 volumes of RNAlater Option (Ambion, Life technologies, Carlsbad, CA, USA) for polymerase chain reaction (PCR) evaluation. two.4. Hormone Measurement. Blood was collected in the abdominal aorta by a puncturing needle. Cerebrospinal fluid (CSF) was extracted in the foramen magnum by a 1 mL syringe. Blood samples have been placed at room temperature for 30 min just before centrifugation for ten min at three,000 . Serum adrenocorticotropic hormone (ACTH) and corticosterone (CORT) were analyzed working with an IMMULITE 2000 immunoassay system with ACTH and CORT immunoassay kits. The serum and also the CSF levels of CRH and urocortin-2 (UCN2) have 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). 2.five. Histopathological Assessment and Nissl’s Staining. Within the histopathological examination, paraformaldehyde remedy was used to repair the paraffin-embedded brain tissues that have been reduce into serial sections (three m). The slices have been stained with hematoxylin and eosin and Nissl’s answer within a routine procedure. 2.6. Two-Dimensional Electrophoresis (2D) and Protein Fluroxypyr-meptyl References identification. The 2D electrophoresis was performed as previously reported and repeated for three occasions [18]. Briefly, hippocampal samples containing 300 g of protein have been loaded per tube in an Nalfurafine Technical Information isoelectric focusing method (IPGphor II, GE). The samples were isoelectrofocused and separated with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The gels were silver-stained. The protein spots that either improved or decreased for far more than twofold were selected for matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) identification. The3 mascot application package and the database of SwissProt were 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 were loaded onto 10 SDS-PAGE for separation, electrotransferred onto PVDF membranes, and blocked in 5 nonfat milk in Tris-buffered saline Tween. The membranes had been incubated overnight with principal antibodies, for instance 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.
