Fig 2: PAR1 immunoreactivity co-localises with GFAP but not with MAP2 in the lumbar ventral spinal cord.A: representative images showing 50 µm transverse sections taken from the upper lumbar spinal cord of a P6 C57BL/6 mouse. Sections were stained with antibodies raised against GFAP (green) and PAR1 (red). B: representative images showing 50 µm transverse sections taken from the upper lumbar spinal cord of a mouse. Sections were stained with antibodies raised against MAP2 (green) and PAR1 (red). Arrows in Bi indicate areas of PAR1 staining between MAP2+ cells. Scale bars: 20 µm.

Fig 3: Glial stimulation results in release of adenosine and activation of neuronal A1 receptors.A: raw (top) and rectified/integrated (bottom) traces recorded from left (L) and right (R) L2 ventral roots showing the effect of the PAR1 agonist TFLLR (10 µM) applied in the presence of theophylline (20 µM). B: locomotor-burst frequency in the presence of the non-selective adenosine receptor antagonist theophylline over 3 min during a control period, immediately following TFLLR application, and following a 20-min washout period (n = 10). C: raw (top) and rectified/integrated (bottom) traces recorded from left (L) and right (R) L2 ventral roots showing the effect of the PAR1 agonist TFLLR (10 µM) applied in the presence of the A1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 50 µM). D: locomotor-burst frequency in the presence of DPCPX over 3 min during a control period, immediately following TFLLR application, and following a 20-min washout period (n = 10). E: raw (top) and rectified/integrated (bottom) traces recorded from left (L) and right (R) L2 ventral roots showing the effect of the PAR1 agonist TFLLR (10 µM) applied in the presence of SCH58261 (25 µM). F: locomotor-burst frequency in the presence of the A2A-receptor antagonist SCH58261 over 3 min during a control period, immediately following TFLLR application, and following a 20-min washout period (n = 10). Individual data points are shown in grey and means are represented by black lines. Statistically significant differences in pairwise comparisons: *p < 0.05.

Fig 4: Stimulation of glia during ongoing locomotor-related activity results in a transient reduction in burst frequency.A: raw (top) and rectified/integrated (bottom) traces recorded from left (L) and right (R) L2 ventral roots and the right L5 ventral root showing the effect of the PAR1 agonist TFLLR (10 µM). Bi: locomotor-burst frequency over 3 min during a control period, immediately following TFLLR application, and following a 20-min washout period. Individual data points are shown in grey and means are represented by black lines. Bii: time course plot of normalised data aggregated into 1-min bins showing a reduction in burst frequency upon application of TFLLR. Ci: locomotor-burst amplitude over 3 min during a control period, immediately following TFLLR application, and following a 20-min washout period. Cii: time course plot of normalised data aggregated into 1-min bins showing no change in burst amplitude upon application of TFLLR. n = 10 preparations. Statistically significant differences in pairwise comparisons: *p < 0.05, **p < 0.01.

Fig 5: The conditioned medium from Schwann cells (CM-SC) stimulated with high or low levels of thrombin (or PAR1 AP) differently affects PC12 neurite outgrowth. A) PC12 cells were left untreated, treated with the culture medium from Schwann cells (CM-SC) or treated with NGF for 7 days. B) PC12 cells were exposed for 7 days to the conditioned medium (CM-SC) from untreated Schwann cells (ctrl) or from Schwann cells treated with: 10 U/mL thrombin; 100 U/mL thrombin; 10 U/mL hirudin + 10 U/mL thrombin; 100 U/mL hirudin + 100 U/mL thrombin; 100 U/mL hirudin alone; when SC were treated with 10 U /mL of thrombin their ability to promote neurite extension of PC12 was enhanced respect to the untreated SC while it was reduced with thrombin at 100 U/mL; thrombin effect was inhibited by hirudin. C) PC12 cells were exposed for 7 days to the conditioned medium (CM-SC) from untreated Schwann cells (ctrl) or from Schwann cells treated with 10, 100 mM PAR1 AP alone or in combination with 1 mM SCH79797; When SC were treated with 10 mM PAR1 AP their ability to promote neurite extension of PC12 was enhanced respect to the untreated SC while it was reduced with PAR1 AP at 100 mM; PAR1 AP was blocked by the PAR1 inhibitor SCH79797; the percentage of neurite-bearing cells was calculated from 5 random fields per well, and the data are expressed as a mean percentage of 6 wells. Data are presented as mean ± SEM. °P=0.05; *P=0.01 respect to ctrl; **P=0.001 respect to untreated.