Fig 2: P2Y13 knock-out increases ATP-evoked membrane current changes but not tonic activity of THIK-1 K+ channels. (a) Specimen current responses of microglia in WT and P2Y13 KO hippocampal slices to voltage steps between -124 and +56 mV, from a holding potential of -34 mV. (b) Mean steady-state I–V relations from data as in (a) from 10 WT and 12 KO cells. (c) Peak outward current evoked at 0 mV in WT and KO microglia by puffing 100 µM ADP above the slice. (d) Mean ADP-evoked current density (number of cells on bars). (e–g) Mean cell capacitance (e) resting potential (f), tetrapentylammonium-suppressed tonic THIK-1 current density (g), and input resistance (h) in WT and KO cells (numbers of cells on bars). All data from P60-120 littermate-matched mice

Fig 3: Intrathecal injection of SB203580 (50 µM) or PDTC (5 µg/10 ml) suppressed the increased expression of P2Y12 and P2Y13R in the dorsal spinal cord of CCI rats. a Comparison of thermal stimulation pain threshold in the ipsilateral hind paw of the rats (n = 8). Data are presented as TWL. CCI reduced TWL from days 1 to 7 after nerve injury. *P < 0.05 compared with control group. Pretreated with SB203580 (50 µM) or PDTC (5 µg/10 ml) significantly inhibited the reduced withdrawal threshold days 1 to 7 in CCI rats, respectively (compared with vehicle-treated CCI group, SB203580: + P < 0.05; PDTC: + P < 0.05). b RT-PCR results show the expression of P2Y12 mRNA expression in the rat dorsal spinal cord (n = 8 per group). *P < 0.05 means comparison with the control group; + P < 0.05 means comparison with the vehicle-treated CCI group. c RT-PCR results show the expression of P2Y13 mRNA expression in the rat dorsal spinal cord (n = 8 per group). *P < 0.05 means comparison with the control group; + P < 0.05 means comparison with the vehicle-treated CCI group. d Western blotting image of P2Y12R and P2Y13R expression. The top panel was the target band, and the bottom one was for the loading control beta-actin. e Western blotting quantitative analysis of the P2Y12R expression in the rat dorsal spinal cord. *P < 0.05 means comparison with the sham group; + P < 0.05 means comparison with the vehicle-treated CCI group. f Western blotting quantitative analysis of the P2Y13R expression in the rat dorsal spinal cord. *P < 0.05 means comparison with the sham group; + P < 0.05 means comparison with the vehicle-treated CCI group

Fig 4: The effect of minocycline on the expression of P2Y12 and P2Y13 receptors. All values represent mean ± standard deviation. a RT-PCR results show the expression of P2Y12 mRNA expression in the rat dorsal spinal cord (n = 8 per group). *P < 0.05 means comparison with the sham group; + P < 0.05 means comparison with the CCI rats. b RT-PCR results show the expression of P2Y13 mRNA expression in the rat dorsal spinal cord (n = 8 per group). *P < 0.05 means comparison with the sham group; + P < 0.05 means comparison with the CCI rats. c Western blotting quantitative analysis of the P2Y12R expression in the rat dorsal spinal cord. *P < 0.05 means comparison with the control; + P < 0.05 means comparison with the ADPbetaS-treated rats. d Western blotting quantitative analysis of the P2Y13R expression in the rat dorsal spinal cord. *P < 0.05 means comparison with the control; + P < 0.05 means comparison with the ADPbetaS-treated rats. e Western blotting image of P2Y12R expression. The top panel was the target band, P2Y12R, and the bottom one was for the loading control beta-actin. f Western blotting image of P2Y13R expression. The top panel was the target band, P2Y13R, and the bottom one was for the loading control beta-actin

Fig 5: Effect of P2Y13 receptor knock-out on microglial directed motility in hippocampal slices. (a-b) Directed motility to an ATP-filled pipette. (a) Time course of directed motility quantified as reduction of the “clear area” not occupied by microglia around the 1 mM ADP-filled pipette tip in 6 WT and 5 KO mice. (b) Time course of directed motility quantified as reduction of the mean distance (µm, averaged over sectors) from the microglial processes to the ADP-filled pipette tip in 6 WT and 5 KO mice. (c,d) Directed motility to a laser ablation. (c) Time course of directed motility quantified as reduction of the “clear area” not occupied by microglia around the ablation site in 3 WT and 3 KO mice. (d) Time course of directed motility quantified as reduction of the mean distance (µm, averaged over sectors) from the microglial processes to the ablation site in three WT and three KO mice. (e) Time course of directed motility quantified as reduction of the normalized “clear area” around the ADP-filled pipette in 6 WT and 5 KO mice and around the laser ablation site in 3 WT and 3 KO mice. The values were normalized to the initial “clear area” values of each recording. (f) Quantification of the time needed for the plots in (e) to drop to the 1/e value, that is, the time needed for the “clear area” to drop 36.7% of the initial area. (g) Quantification of the speed of convergence of the WT and KO microglial processes onto the tip of the pipette and the laser ablation site, calculated as the distance (µm) moved per 30 sec, averaged over the period needed to converge on the pipette tip or the ablation site, and then averaged over all the slices. Number of slices are shown on bars. Age for pipette experiments (a, b, e–g) was P55–P121 for WT and P56–P136 for KO and for laser ablation experiments (c, d, e–g) was around P60