a upon PAR1 activation. We have previously shown that spinal motor networks are modulated by endogenous adenosine that appears to derive from glia. We therefore investigated 5 / 17 Modulation of Spinal Motor Networks by Glia Fig 1. PAR1 immunoreactivity co-localises with GFAP but not with MAP2 in the lumbar ventral spinal cord. A: representative images showing 50 m transverse sections taken from the upper lumbar spinal cord of a P6 C57BL/6 mouse. Sections were stained with antibodies raised against GFAP and PAR1. B: representative images showing 50 m transverse sections taken from the upper lumbar spinal cord of a mouse. Sections were stained with antibodies raised against MAP2 and PAR1. Arrows in Bi indicate areas PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19754931 of PAR1 staining between MAP2+ cells. Scale bars: 20 m. doi:10.1371/journal.pone.0134488.g001 whether adenosine mediated a purchase BCTC component of the network response to PAR1 activation by applying TFLLR in the presence of the non-selective adenosine receptor antagonist theophylline. In these preparations TFLLR had no effect on the frequency of locomotor-related bursting, implying that adenosine is the dominant modulator released by glia following PAR1 activation. To investigate the adenosine receptor subtypes activated by glial cell-derived adenosine, we applied TFLLR in the presence of antagonists selective for either A1 or A2A receptors, both of which are broadly expressed high- 6 / 17 Modulation of Spinal Motor PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19756412 Networks by Glia Fig 2. Stimulation of glia during ongoing locomotor-related activity results in a transient reduction in burst frequency. A: raw and rectified/ integrated traces recorded from left and right L2 ventral roots and the right L5 ventral root showing the effect of the PAR1 agonist TFLLR. 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. doi:10.1371/journal.pone.0134488.g002 affinity adenosine receptor subtypes. Like theophylline, the A1-subtype specific antagonist DPCPX efficiently abolished the modulation of burst frequency following PAR1 activation. By contrast, in the presence of the A2Asubtype specific antagonist SCH58261, PAR1 activation caused a transient reduction Fig 3. TFLLR has no effect on locomotor-related bursting following pharmacological ablation of glia. A: raw and rectified/integrated traces recorded from left and right L2 ventral roots showing the effect of the PAR1 agonist TFLLR following glial ablation with methionine sulfoximine, which was co-applied with glutamine. B: locomotor-burst frequency in the presence of MSO and Gln 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. n = 10. doi:10.1371/journal.pone.0134488.g003 7 / 17 Modulation of Spinal Motor Networks by