R washing in PBS, cells were imaged right away or mounted on two ll glycerol. Epifluorescence images had been digitized with an AxioCam MRm-CCD camera (Zeiss) in the focal plane from the axonal network with a 25and 100objective (Zeiss). Photos had been acquired using the application Axiovision 4.5 (Zeiss) and analyzed with Metamorph computer software (Universal Imaging Inc.). Immunopositive spots had been determined utilizing a threshold-based detection routine, with all the threshold adjusted to the background signal of the dendrite. Immunosignals had been quantified as meanfluorescent intensity. For the evaluation of synaptic density, synaptophysin-positive puncta were counted along 50 lm dendrite length (Guzman et al, 2010). Histochemistry and immunostainings in brain slices Experimentally naive 14-week-old mice had been anesthetized with isoflurane and transcardially perfused with PBS at the same time as four paraformaldehyde (PFA; RotiHistofix four , Roth). Prior to and just after the postfixation with PFA in PBS (4 ) for 1.five h, brains had been washed in PBS then embedded in 2 agarose (in PBS). Coronal sections (one hundred lm) had been reduce on a vibratome (Leica VT1000S) and kept in PBS. For the immunostaining, they have been initially pretreated with 0.5 H2O2 (Fluka Analytical) in PBS for 15 mins to quench endogenous peroxidase activity, followed by Answer D1 (1 albumin from bovine serum (BSA; Sigma), 0.three Triton X-100 (Sigma) in PBS) supplemented with two standard goat serum (NGS; Phenoxyacetic acid Biological Activity Invitrogen) to block unspecific protein binding sites. Numerous washing actions with PBS have been performed in involving. Soon after 1 h, brain slices have been incubated with primary antibodies overnight. The dilutions were prepared in D1 and consisted of rabbit antibody targeting either GluA1 (1:1,000; abcam, glial fibrillary acidic protein (GFAP; 1:500; DakoCytomation), or somatostatin (1:400; abcam or mouse antibody targeting NeuN (1:1,000; Millipore. Around the subsequent day, soon after rinsing with D2 (0.33 BSA (Sigma), 0.1 Triton X-100 (Sigma) in PBS), the sections had been incubated for 1 h with peroxidase-labeled anti-rabbit IgG secondary antibody (Vector), diluted 1:600 in D2, followed by washing measures. Slices had been then stained with diaminobenzidine remedy (0.four mg/ml DAB in 20 mM Tris (pH 7.six) and 30 H2O2). The reaction was stopped with PBS. Stained sections had been mounted onto glass slides making use of ten mM Tris (pH 7.six). Soon after drying, slices had been embedded in xylene (Merck) with Eukittquick-hardening mounting medium (Fluka Analytical). All staining processes have been performed at space temperature. For Nissl staining, brains were taken from experimentally naive 14-week-old mice, immediately frozen on dry ice, and stored at 0 . Transverse sections (12 lm) have been cut at 0 on a cryostat (Leica CM3050 S) and mounted onto microscope slides, previously coated with poly-L-lysine. Slides have been kept at 0 . Brain sections have been dried at space temperature for two h ahead of they had been incubated for 140 s in thionine resolution (0.1 thionine in 0.1 M acetic acid and 0.1 M Na-acetate, filtered) and rinsed with distilled water. Right after drying, slices had been embedded in xylene with Eukitt Vibrant field images of DAB- and Nissl-stained slices were taken together with the Axio Imager M1 (10magnifying objective, Zeiss) utilizing the Zeiss Axiovision application. Behavioral assays Animals were housed in an animal facility having a frequent 12-h light/dark cycle (light on at 7:00 A.M.). Meals and water was supplied ad libitum, unless the mice had been kept on a food-restricted diet program through an appetite-motivated finding out task.