Igor pro 6

The AFM instrument MFP-3D (Asylum Research, Santa Barbara, CA, USA) was used, with NCHR (NanoWorld, Neuchâtel, Switzerland) probes in tapping mode in air. These silicon probes have a nominal resonance frequency and spring constant of ~320 kHz and ~42 N/m, respectively. The tip is a pyramid with ~13 µm length, ~20 nm apex diameter, and 2:1 tip aspect ratio. For each specimen, 2 areas of 30 × 30 µm² were scanned, represented with 512² pixels. The images obtained were processed with the AFM company software Version-12, based on IgorPro 6.22 (Wavemetrics, Lake Oswego, OR, USA). The surface roughness and morphology (data-points n = 2 images/specimen × 6 specimens/sample = 12) have been described based on the distribution of step-heights RMS, i.e., S_q; the third statistical moment, i.e., skewness S_sk; and the fourth statistical moment, i.e., kurtosis S_ku [26 ].

Analysis was performed off-line using Fitmaster (Heka Elektronik) and Igor-Pro 6.0.3 (Wavemetrics, Lake Oswego, OR, USA). All results were expressed as the mean ± S.E.M. and “n” refers to a single-cell experiment from a single slice. Statistical analysis was performed with the Prism 5.0 software (GraphPad Software Inc., CA, USA) and statistical significance was assessed in Wilcoxon signed rank test using the indicated significance threshold (p).

Whole-cell patch-clamp recordings were obtained from DIV18–20 hippocampal neurons. Recordings were made with 2.0–3.5 MΩ borosilicate pipettes filled with intracellular solution (120 mM K-gluconate, 8 mM NaCl, 2 mM MgCl₂, 0.5 mM CaCl₂, 5 mM EGTA, 10 mM HEPES, 14 mM phosphocreatine, 2 mM magnesium-ATP, 0.3 mM sodium-GTP, pH adjusted to 7.3 with KOH). Patchmaster software (HEKA, Lambrecht, Germany) in combination with an EPC 9 patch-clamp amplifier (HEKA) was used for stimulation and data acquisition. Recordings were low-pass filtered at 2.9 kHz and analyzed with Fitmaster (HEKA), Igor Pro 6.03 (Wavemetrics), Mini Analysis (Synaptosoft, Decatur, GA, USA), and Excel (Microsoft). Only neurons with an access resistance <20 MΩ were evaluated. mEPSCs were recorded from hippocampal neurons of +/+ (n = 8; 1,617 events) and spastin −/− neurons (n = 14; 1,471 events) for 30 seconds and analyzed with Mini Analysis software (Synaptosoft). Recordings were made at room temperature (21–23°C) in Ringer’s solution (143 mM NaCl, 5 mM 1 KCl, 0.8 mM MgCl₂, 1 mM CaCl₂, 10 mM HEPES, 5 mM glucose, and pH adjusted to 7.3 with NaOH). mEPSCs were recorded in the presence of TTX (0.25 μM), bicuculline (10 μM), and APV (20 μM), which were added to the control solution. All substances were purchased from Sigma-Aldrich.

Off-line analysis was performed with Fitmaster (Heka Elektronik) and Igor-Pro 6.0.3 (Wavemetrics). Statistical analysis was performed with Prism 5.02 software. In all cases “n” refers to an experiment on a single cell from a single slice. All results are expressed as mean ± SEM in the text and as mean ± SD in the figures. Statistical significance was assessed in unpaired t tests, one way ANOVA, or in one-sample t tests, as appropriate, using the indicated significance threshold (p).