A, pCREB immunohistochemistry in rACC slices representative of different treatments. Low right, double immunofluorescence reveals that NMDA-induced pERK (green) largely colocalizes with pCREB (red), and pCREB (red) colocalizes with NeuN (green). Arrowheads indicate double-labeled cells. B, numbers of pCREB positive nuclei significantly increase after exposure to NMDA (50 μM), forskolin (50 μM), and Sp-cAMP (50 μM) for 10 min. Pre-incubation of APv (50 μM), SQ22536 (50 μM), Rp-cAMP (50 μM), or PD98059 (50 μM) blocks pCREB increase induced by NMDA, forskolin, or Sp-cAMP. * p<0.05 and ** p<0.01 compared with control; ^## p<0.01 compared with NMDA-treated; ⁺⁺ p<0.01 compared with forskolin-treated; ^^ p<0.01 compared with Sp-cAMP-treated (n=5–9).

A, Schematic demonstration of behavioral testing. B, Intra-rACC microinjection of MEK inhibitor U0126 (2 nmol) before test blocks F-CPA expression. ** p<0.01 compared with vehicle (35% DMSO) control (n=8–11). C, Schematic of experimental design used in the following studies. D–I, Time course of F-CPA retrieval-induced pERK and pCREB increases in the rACC. Immunohistochemistry for pERK (D and E) and western blot for pERK1 and pERK2 (F and H) as well as pCREB (G and I) from the rACC show significant increases in the levels of pERK and pCREB following F-CPA retrieval, and all the increases are blocked by intra-rACC injection of MEK inhibitor U0126, given 30 min prior to retrieval. * p<0.05, ** p<0.01 compared with non-retrieval; ## p<0.01 compared with vehicle (35 % DMSO) control (n=5).

A, pCREB immunohistochemistry in the contralateral rACC from coronal brain sections representative of normal saline (NS) injection at 10 min and formalin injection at 10 min, 30 min, 2 hrs, and 24 hrs. Low right, double immunofluroescence reveals that formalin-induced pERK (green) colocalizes with pCREB (red) in vivo. Arrowheads indicate double-labeled cells. B, numbers of pCREB-positive neurons in the bilateral rACC are significantly greater in formalin-treated rats than that of NS controls. C, Western blot analysis reveals an increase in the pCREB levels in the rACC after formalin injection, which is blocked by intra-rACC pre-administration of PD98059 (10 nmol). * p<0.05, ** p<0.01 compared with control; ⁺⁺ p<0.01 compared with DMSO pre-treatment (n=5).

A, pERK immunohistochemistry in rACC slices representative of different treatments. B, Numbers of pERK positive neurons increase after exposure to NMDA (20, 50, and 100μM), forskolin (50 μM), and Sp-cAMP (50 μM) for 10 min. Pre-incubation of APv (50 μM), SQ22536 (50 μM), Rp-cAMP (50 μM), or PD98059 (50 μM) significantly reduces pERK induced by NMDA (50 μM), forskolin, or Sp-cAMP. * p<0.05 and ** p<0.01 compared with control; ^## p<0.01 compared with NMDA-treated; ⁺⁺ p<0.01 compared with forskolin-treated; ^^ p<0.01 compared with Sp-cAMP treated (n=6–12). C, D, Formalin-induced increases in levels of pERK1 and pERK2 are blocked by intra-rACC pre-administration of APv (10 nmol), SQ22536 (0.01, and 1 nmol), and Rp-cAMP (1, and 10 nmol). Both ipsilateral and contralateral sides of the rACC were pooled together in this experiment. ** p<0.01 compared with vehicle (10 % DMSO) control (n=4–5). E, Intra-rACC microinjection of AC inhibitor SQ22536 (0.01, 0.1, and 1 nmol) or PKA inhibitor Rp-cAMP (1, and 10 nmol) before conditioning blocks F-CPA induction. ** p<0.01 compared with vehicle (NS) control (n=6–9). F, Reconstruction of sites for the microinjections of SQ22536 and Rp-cAMP in the bilateral rACC.

A, Histograms showing formalin-induced CPA, as indicated by time in treatment (i.pl. NS or formalin)-paired compartment on pre- and post-conditioning days and CPA scores (indicated by the difference of time (seconds) spent on the pre-conditioning day and time spent on the post-conditioning day in the treatment-paired compartment). ** p<0.01. n=10. B, Schematic of the protocol for behavioral testing. C, Intra-rACC microinjection of MEK inhibitors PD98059 (0.1, 1, and 10 nmol) and U0126 (0.2, and 2 nmol) before conditioning dose-dependently reduces F-CPA scores. ** p<0.01 compared with vehicle control (n=8–12). D, Coronal brain section showing reconstruction of microinjection sites and cannula placement in the bilateral rACC. E,F, Formalin-induced increases in levels of pERK1 and pERK2 are blocked by intra-rACC pre-administration of PD98059 (1, and 10 nmol). Both ipsilateral and contralateral sides of the rACC were pooled together in this experiment. ** p<0.01 compared with vehicle (10 % DMSO) control (n=4–5).

A–D, Intra-rACC administration of PD98059 has no effect on formalin-induced biphasic nociceptive responses. A is an example of automatic recording of 5 % formalin-induced two-phase nociceptive agitation responses. Bin=500 ms. B and C indicate the number of agitation events (automatic recording, B) and pain scores (manual observation, C). n=6–11. D is the reconstruction of microinjection sites in the bilateral rACC for formalin and CCI tests. E-H, Intra-rACC injection of PD98059 (10 nmol) has no effect on CCI-induced thermal hyperalgesia (E) and mechanical allodynia (G) in the ipsilateral hindpaw. Neither does PD98059 alter the paw withdrawal latencies (PWLs) and paw withdrawal thresholds (PWTs) in the contralateral hindpaw (F and H) of CCI rats and in sham surgery rats. PD98059 or vehicle (10 % DMSO) was given 3 days after CCI. ** p<0.05 compared with sham CCI. n=8.

A–C, Intra-rACC PD98059 has no significant effect on electric foot-shock (0.5 mA, 2s)-induced CPA (S-CPA). A shows the protocol for behavioral testing. B shows CPA scores of low intensity S-CPA. C is reconstruction of microinjection sites of in the bilateral rACC. ** p<0.01 compared with non-treatment control (n=5–11). D–F, Intra-rACC infusion of PD98059 dose not inhibit U69,593-induced CPA. D is the protocol for behavioral testing. E shows CPA scores of s.c. injection of U69,593-induced CPA. F shows reconstruction of single (left) and double (right) microinjection sites per side of the rACC. PD98059 was given at a dose of 10 nmol for two sites (5 nmol each site) and 20 nmol for four sites (5 nmol each site) for both sides of the rACC).** p<0.01 compared with vehicle control (n=5–11). G–J, Intra-rACC injection of PD98059 does not impair spatial learning and memory in the Morris water maze, G is the protocol for behavioral testing. H shows animal’s escape latencies to find the submerged platform. Cutoff time was 60 s. PD98059 (10 nmol) or vehicle (10 % DMSO) was given 30 min before training in the Morris water maze with two training sessions of six trials each, and a 30 min resting period between the two sessions. Memory retention was tested 48 hrs after training. The retention values are calculated as the mean of three-trial retention test. Inset, representative swimming traces of a PD98059- and a DMSO-treated rat at 48-h retention test. I, animal’s escape latencies to find the visible platform. The platform was raised above the turbid liqid surface to be visible. n=8–11. J is reconstruction of microinjection sites of in the bilateral rACC.

A, Immunohistochemistry for pERK in the contralateral rostral ACC (rACC) from coronal brain sections representative of normal saline (NS) injection at 10 min and formalin injection at 10 min, 2 hrs, and 24 hrs. Right, a high magnification inset showing pERK immunoreactivity in the nucleus (arrowhead), cytoplasm (large arrow) and dendrites (small arrow). B, Numbers of pERK positive cells in both sides of rACC at all time points after formalin injection are significantly higher than that of NS controls. * p<0.05 and ** p<0.01 compared with respective NS control (n=5). C. Left, schematic drawing indicating different laminae of the rACC. Right, a low-magnification section showing pERK immunoreactivity in laminae II–VI of the rACC at 3 min after formalin injection. D, Histograms showing the distribution of pERK-positive cells in all layers of the bilateral rACC. ⁺p<0.05, ⁺⁺p<0.01 compared with the number of pERK-positive cells in laminae II–III. E, pERK expression in the caudal ACC (cACC) 10 min after formalin injection. F, Double immunofluorescence reveals that pERK co-localizes with NeuN, and MAP-2, but does not co-localize with GFAP and OX-42 in the rACC. Arrowheads indicate double-labeled cells. G, Western blot for pERK1/2 and total ERK1/2 from the ipsilateral and contralateral side of rACC after formalin injection. Tubulin serves as loading control. H, Densitometry analysis showing a similar increase in the levels of both pERK1 and pERK2 in the rACC after formalin injection. * p<0.05 and ** p<0.01 compared with NS control (n=5). Cg1, cingulate cortex, area 1; Cg2, cingulate cortex, area 2; CPu, caudate putamen; M1, primary motor cortex; M2, secondary motor cortex; Prl, prelimbic cortex; SIFL, primary somatosensory cortex, forelimb region; VO, ventral orbital cortex. Values are expressed as mean ± SEM