ANOVA with Scheffe post-hoc test was used for multiple comparisons

onset of locomotor-related bursting at concentrations previously employed in this preparation. Any drugs 50-57-7 supplier present during the control period were also present during TFLLR application and washout. In all experiments, stable rhythmic bursting was established over a period of ~1 h prior to bath-application of the PAR1-specific agonist TFLLR. Rhythmic bursting was considered stable when the frequency, amplitude and PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19755503,22075991,18613750,22165947 duration of bursts were unchanged over several minutes. TFLLR application was limited to 5 min, consistent with methods previously employed. Data were amplified and filtered and acquired at a sampling frequency of 6 kHz with a Digidata 1440A analog-digital converter and Axoscope software. Custom-built amplifiers enabled simultaneous online rectification and integration of raw signals. 3 / 17 Modulation of Spinal Motor Networks by Glia Data analysis Data were analysed off-line with DataView software. Ventral-root bursts were identified from rectified/integrated traces and their instantaneous frequencies, peak-to-peak amplitudes, and durations were then measured from the corresponding raw traces. Amplitude was measured as a non-calibrated unit and is presented here as an arbitrary unit. For time-course plots, data were averaged across 1-min bins, or 2-min bins for disinhibited preparations, and normalised to a 10-min precontrol period to permit comparison between preparations. Duty cycle was calculated as burst duration divided by cycle period. Statistical comparisons were performed on raw data averaged over 3-min periods for PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19755095 standard preparations or 6-min periods for disinhibited preparations. Data were analysed with repeated-measures ANOVA, one-way ANOVA, or Student’s t-tests. Bonferroni post-hoc tests were applied to pairwise comparisons. Sphericity was assessed with Mauchly’s test as appropriate, and Greenhouse-Giesser corrections were applied where necessary. p values < 0.05 were considered significant. Cohen's d effect size was also determined where appropriate. Tests were performed in SPSS Statistics for Windows, Version 21.0 or Excel 2013. Immunohistochemistry P4-P11 C57BL/6 mice were dissected as above, and spinal cords were incubated in fixative containing 4% paraformaldehyde and 0.1 M phosphate buffer for 16 h at 4C, before being washed with 0.1 M phosphate-buffered saline and stored in PBS at 4C. Slices from segments L1-L3 were cut at a thickness of 50 m on a vibratome. Free-floating slices were incubated for 48 h at 4C in solution containing primary antibodies, PBS, 1% bovine serum albumin and 0.1% Triton X100. Slices were then washed with PBS and incubated for 24 h at 4C in solution containing secondary antibodies, PBS and 1% BSA. Slices were washed again with PBS and mounted with Vectashield. The stained tissue was examined with an epifluorescence microscope and structured illumination. Primary antibodies were used at the following concentrations: 1:100; chicken polyclonal anti-glial fibrillary acidic protein , 1:100; chicken polyclonal anti-microtubule-associated protein 2 , 1:200; rabbit polyclonal anti-protease activated receptor-1 . Secondary antibodies were used at the following concentrations: polyclonal anti-chicken FITC, 1:200; polyclonal anti-rabbit Cy3, 1:500. Drug and Solution Preparation The aCSF used for dissections and recordings contained 127 NaCl, 26 NaHCO3, 10 glucose, 3 KCl, 2 CaCl, 1.25 NaH2PO4, and 1 MgCl2. TFLLR, theophylline, MSO, FA and glutamine were supplied by Sigma-Aldrich; DPCPX and SCH5826