Primary cultures of mouse hippocampal neurons were established from E18 foetal, Eps8 KO or wild type (wt) littermates C57BL/6 mice as described by. Briefly, hippocampi were dissociated by treatment with trypsin (0.125% for 15 min at 37°C), followed by trituration with a polished Pasteur pipette. The dissociated cells were plated onto glass coverslips coated with poly-L-lysine at density of 400 cells/mm2. The cells were maintained in Neurobasal (Invitrogen, San Diego, CA) with B27 supplement and antibiotics, 2 mM glutamine and 12.5 mM glutamate (neuronal medium).
Chemical Long Term Potentiation (cLTP)
Neuronal cultures were subjected to a chemical LTP protocol consisting of an application of high doses of glycine for 3 min. In particular, for the LTP induction cultured neurons were incubated in a KRH solution devoid of Mg2+ and containing (in mM: 0.1 mM Glycine, 125 NaCl, 5 KCl, 1.2 KH2PO4, 2 CaCl2, 6 glucose, and 25 HEPES-NaOH, TTX 0.001, Strychnine 0.001 and bicuculline methiodide 0.02, pH 7.4) for 3 min followed by a wash and recovery in neuronal medium for at least 60 min. After 60 min cells were immediately fixed and stained for PSD-95, v-Glut1, and tubulin.
Transfection and Immunocytochemical staining
Mouse hippocampal neurons were transfected with pSUPER-DsRed plasmid (obtained from pSUPER-GFP, Oligoengine, Seattle, WA, USA) at DIV13 using Lipofectamine 2000 (Invitrogen). Neuronal cultures were fixed with 4% paraformaldehyde + 4% sucrose at DIV15 as described. The following antibodies were used: guinea pig anti-vGLUT1 (1:1000; Synaptic Systems), mouse anti-PSD95 (1:400; UC Davis/NIH NeuroMab Facility, CA), rabbit anti-tubulin (1:80; Sigma-Aldrich, Milan, Italy). Secondary antibodies were conjugated with Alexa-488, Alexa-555, or Alexa-633 fluorophores (Invitrogen). Images were acquired using a Zeiss LSM 510 META confocal microscope producing image stacks. Pixel size was 110 × 110 nm, and acquisition parameters (i.e., laser power, gain and offset) were kept constant among different experimental settings. Furthermore, to overcome subjective bias during result analysis, we used an automated software-based methods of analysis, ImageJ software (NIH, Bethesda, MD, USA) keeping the parameters of the analyses constant among the different groups. In particular, dendritic spines were classified according the following parameters: mushroom (length ≤ 1.2 μm, width ≥0.5 μm); filopodia (length ˃1.2 μm, width ˂0.5 μm), in line with. Colocalization of 2 or 3 selected markers was measured using the boolean function ‘AND’ for the selected channels. The resulting image was binarized and used as a colocalization mask to be subtracted to single channels. The number of the puncta resulting from colocalization mask subtraction was measured for each marker. A colocalization ratio was set as colocalizing puncta/total puncta number.
Exposure to environmental enrichment
Environmental Enrichment has been performed as described with slight modifications. 8–10 WT and Eps8 KO mice were housed together for 8 weeks starting after weaning at P21 in a large cage (60 × 60 × 40 cm) with water and food ad libitum and several different brightly coloured mouse toys and a running wheel. To stimulate active exploration of a novel environment, new toys were swapped for existing once every week. The activity of the individual mice (exploring the objects and /or running on a wheel) was monitored daily. Control littermate mice were housed in the same room in standard cages with only bedding and access to water and food pellets (SH).
Golgi staining and quantification of dendritic spines
Mice were deeply anesthetized with chloral hydrate (4%; 1 ml/100 g body weight, i.p.) and subjected to intra-cardiac perfusion with 0.9% saline solution. The brains were removed stained by modified Golgi-Cox method as described in with slight modifications. Coronal sections of 100 mm thickness from the dorsal hippocampus were obtained using a vibratome (VT1000S, Leica, Wetzlar, Germany). These sections were collected free floating in 6% sucrose solution and processed with ammonium hydroxide for 15 min, followed by 15 min in Kodak Film Fixer, and finally were rinsed with distilled water, placed on coverslips, dehydrated and mounted with a xylene-based medium. Spine density and length was measured on the secondary branches of apical dendrites of pyramidal neurons located in the CA1 subfield of the dorsal hippocampus. At least 30 neurons per animal were evaluated.
Statistical analysis was performed using Prism6 (GraphPad), data are presented as mean±SEM from the indicated number of experiments. After testing whether data were normally distributed or not, the appropriate statistical test, followed by specific multiple comparison post hoc tests, has been used as indicated in figure legends. Kolmogorov–Smirnov test was used to determine significance in cumulative distributions of mEPSC amplitudes. Differences were considered to be significant if p<0.05 and are indicated by one asterisk; those at p<0.01 are indicated by double asterisks; those at p<0.001 are indicated by triple asterisks, those at p<0.0001 are indicated by four asterisks.