SCDH atypical PKC expression, and effects of PKMζ/PKC inhibition on capsaicin-induced allodynia. (a) Western blots and (b) histograms (% change) of atypical PKC showing i.pl. capsaicin increased PKMζ, but not PKCζ or PKCι/λ, in SCDH from 2-24 h (*P < 0.05, n = 7/12 vehicle/capsaicin). c) Primary/secondary allodynia test sites in capsaicin-injected rats. d) Paw-withdrawal thresholds (PWTs) 24 h post-capsaicin after anesthesia (n = 5) or no-anesthesia (n = 5) of the primary site. Primary, but not secondary, allodynia was attenuated by anesthesia (*P < 0.05, from no-anesthesia). (e,f) Pre- and post-capsaicin PWTs (g) with ZIP, scr-ZIP, NPC-15437 or vehicle (n = 15,4,6,6, respectively) given 24 h post-capsaicin. ZIP (*P < 0.05), but neither scr-ZIP nor NPC-15437, significantly reversed capsaicin-induced allodynia 40-120 min post-drug. (g,h) Effect of ZIP given 20 min before testing on paw-withdrawal latencies, PWLs (s) and PWTs (g) 24 h after capsaicin or vehicle (n = 6,4, respectively). g) Heat hyperalgesia present at 2 h post-capsaicin (*P < 0.05, from vehicle), abated by 24 h, and PWLs were unaffected by ZIP. h) Mechanical allodynia 24 h post-capsaicin (*P < 0.05 from vehicle) was reversed by ZIP. (i,j) Effect of ZIP or scr-ZIP (n = 11, 8, respectively) 100 min post-capsaicin on PWLs and PWTs 20 min, 2 and 24 h post-capsaicin. i) Heat hyperalgesia (20 min-2 h, †P < 0.05 from baseline), abated by 24 h, and was unaffected by ZIP at 2-24 h post-capsaicin. j) Mechanical allodynia was significantly attenuated at 2 h, and reversed at 24 h, post-capsaicin by ZIP (*P < 0.05 from scr-ZIP).

Effects of PKMζ inhibition on capsaicin-induced sensitization of spinal WDR neurons. (a,b) Spike rate (spikes/sec) of evoked responses of 2 WDR neurons to von Frey filaments pre- and post-capsaicin. ZIP (90 min post-capsaicin) reduced the capsaicin-induced sensitization of mechanically-evoked responses of the neuron in (a). The neuron in (b) was not sensitized by capsaicin, and ZIP did not reduce evoked responses to the mechanical stimuli. (c-e) Mechanically-evoked WDR neuron responses to 1 (c), 5 (d) or 20 (e) g stimuli pre- and post-capsaicin (% of baseline action potentials evoked/20 s). ZIP, but not scr-ZIP (n = 6, 5, respectively), significantly reversed capsaicin-enhanced evoked responses to 5-20 g stimuli at 30-60 min post-drug (*P < 0.05). (f-h) Mechanically-evoked WDR neuron responses to 1 (f), 5 (g) or 20 (h) g stimuli pre- (baseline, BL) and post-vehicle (before (Veh) and after ZIP (30-60 min) applied 90 min post-vehicle) (n = 5). Neither i.pl. vehicle, nor i.t. ZIP, significantly affected evoked responses of WDR neurons. i) Background activity and evoked responses to heat pre- and post-capsaicin. Although capsaicin significantly increased the background activity of WDR neurons (** P < 0.01 from Pre-capsaicin), it failed to increase their heat-evoked responses (P >0.05, n = 8). j) Background activity of WDR neurons pre- (baseline, BL) and post-capsaicin (before (Cap) and after ZIP (30-60 min) applied 90 min post-capsaicin, n = 6). Capsaicin significantly increased background activity (*P < 0.05), and ZIP failed to significantly reduce the capsaicin-induced increase in the background activity of WDR neurons (P > 0.05).

SCDH atypical PKC expression, and effects of PKMζ/PKC inhibition on formalin nociception. (a) Western blots and (b) histograms (% change) of atypical PKC showing i.pl. formalin increased PKMζ, PKCζ and PKCι/λ (n = 5/6 for vehicle/formalin) in SCDH (*P < 0.05). (c) Effect of ZIP on rotarod latencies, which significantly increased over training (*p < 0.05 from day 1). Latencies on day 6 (after ZIP) did not differ from day 5 (n.s.: non-significant difference). (d,e) Dose-dependent effects of pretreatment with ZIP (n = 5-7/dose) vs. scr-ZIP (n = 7) (d) or NPC-15437 (n = 5-7/dose) vs. vehicle (n = 8) (e) on formalin pain. ZIP dose-dependently reduced late phase (9-45 min) scores (†P < 0.05, *P < 0.01 red dash-boxed or individual symbols from scr-ZIP), but not early phase scores (0-6 min). NPC-15437 also significantly reduced late phase scores (†P < 0.05, *P < 0.01 for red dash-boxed or individual symbols), but not early phase scores. (f,g) Effect of ZIP (n = 6) or scr-ZIP (n = 4) pretreatment on von Frey paw-withdrawal thresholds (PWT, f) or plantar test paw-withdrawal latencies (PWL, g) in naïve rats. Neither ZIP nor scr-ZIP significantly affected either PWTs or PWLs. (h,i) Painful responses induced by formalin before and after ZIP (n = 5) or scr-ZIP (n = 5) (h), and NPC-15437 (n = 5) or vehicle (n = 7) (i), administered 25 min post-formalin. ZIP (*P <.05), but not NPC-15437, significantly attenuated persistent formalin pain. For these and subsequent graphs ZIP, scr-ZIP, NPC-15437 and vehicle were given i.t., and data is expressed as means ± s.e.m. unless otherwise stated.

SCDH atypical PKC expression, and effects of PKMζ/PKC inhibition on spinal DHPG-induced allodynia. (a) Spike rate (spikes/sec) of evoked responses of a WDR neuron to 20 and 50 g von Frey filaments before and 5 or 25 min after DHPG. Note the increased evoked responses to both mechanical stimuli, and the prolonged after-discharges to the 50 g stimulus, both 5 and 25 min post-DHPG. Scale bar (20 sec). (b) Western blots and (c) histograms of % change in atypical PKC levels compared to vehicle illustrating that i.t. DHPG significantly increased PKMζ (n = 12), but not PKCζ (n = 12) or PKCι/λ (n = 8) in SCDH (vehicle, n = 11), 2 and 24 h post-DHPG combined (*P < 0.05). (d,e) Pre- and post-DHPG paw withdrawal thresholds (PWTs, g) for rats given i.t. ZIP or scr-ZIP (d), i.t. NPC-15437 or vehicle (e), 30 min prior to testing at 24 h post-DHPG, with additional testing 48 h post-drug. ZIP (*P < 0.05), but not scr-ZIP or NPC-15437, reversed DHPG-induced mechanical allodynia at both time-points (n = 5 for each drug). f) Pre-drug (base) and post-drug PWTs of CCI rats (n = 8), early CPIP rats (n = 9) and late CPIP rats treated with i.t. ZIP (n = 7), or late CPIP rats treated with i.t. scr-ZIP (n = 8). ZIP, but not scr-ZIP, reversed allodynia in late CPIP rats, while ZIP had no effects in CCI or early CPIP rats (* P < 0.01; ** P < 0.001 from scr-ZIP; † P < 0.05; †† P < 0.01 from pre-drug baseline).