Inhibition of mEPSCs in vlPAG neurons from the OIH rats by intro-CeLC injection of KN93.

<p>(A) mEPSCs recordings in control and OIH group after the application of KN92 or KN93. (B) Individual traces (average) of mEPSCs obtained from respective recordings. Calibration: 1 s, 20 pA. (C-D) Bar graphs showing the frequency (C) and amplitude (D) of mEPSCs from vlPAG neurons in slices from control and OIH group (12 h post induction). Results are expressed as mean ± SD; n = 8–11 for each group; *, <i>p</i> < 0.05, **, <i>p</i> < 0.01.</p

KN93 (added in the ACSF) reversed the enhanced mEPSCs in vlPAG neurons from the OIH rats.

<p>(A) mEPSCs recordings in control and OIH group at baseline and after the application of KN93. (B) Individual traces (average) of mEPSCs obtained from respective recordings. Calibration: 1 s, 20 pA. (C-D). Bar graphs showing the frequency (C) and amplitude (D) of mEPSCs in vlPAG neurons in control and OIH group (12 h post induction). Results are expressed as mean ± SD; n = 6 for each group; *, <i>p</i> < 0.05.</p

Illustration of the experimental design.

<p>Experiment 1: To investigate the effect of CeLC CaMKIIα antagonism on the level of CaMKIIα activity in CeLC, PAG, RVM and spinal cord regions and the behavioral hyperalgesia induced by fentanyl, rats were first implanted with CeLC cannulas before the induction of OIH by fentanyl. Then the Control group (n = 6) and OIH group (n = 6) received 50%DMSO (vehicle) 0.3 μl. OIH+KN92 10 nmol group (n = 6) and OIH+KN93 10 nmol group (n = 6) received equal volume (0.3 μl) of KN92 10 nmol and KN93 10 nmol respectively 6.5 h after the last injection of fentanyl. Experiment 2: To test whether spinal cord has a reverse effect on amygdala CaMKIIα signal, animals were randomly divided into four groups (n = 6). The Control group and OIH group received 50% DMSO (vehicle) 0.45 μl. OIH+KN92 45 nmol group and OIH+KN93 45 nmol group received equal volume (0.45 μl) of KN92 45 nmol and KN93 45 nmol respectively 6.5 h after the last injection of fentanyl. Experiment 3: To confirm whether <i>p-</i>CaMKIIα in vlPAG is involved in fentanyl-induced hyperalgesia and whether there is connection between PAG and other regions including CeLC, RVM and spinal cord in OIH, animals were randomly divided into four groups (n = 6). Control group and OIH group received 50% DMSO (vehicle) 0.3 μl. OIH + KN92 10 nmol group and OIH + KN93 10 nmol group received KN92 10 nmol and KN93 10 nmol respectively 6.5 h after the last injection of fentanyl. Experiment 4: To determine if there were differences between the Control rats and OIH rats in the synaptic transmission, and whether CaMKIIα modulates synaptic transmission in PAG neurons in OIH rats, the PAG slices of control rats (n = 6) and OIH rats (n = 6) were obtained 12 h after the last injection of saline or fentanyl. KN93 (10 μM) were added in the recording well 10 min after the baseline recording. Experiment 5: To further confirm whether inhibition of CeLC CaMKIIα activity has a direct effect on the enhanced synaptic transmission of vlPAG neurons in OIH rats, the Control+KN92 (n = 11) group and OIH+KN92 (n = 8) received CeLC injection of KN92 (10 nmol) and Control+KN93 group (n = 8) and OIH+KN93 (n = 8) group received CeLC injection of KN93 (10 nmol) respectively 6.5 h after the last injection of saline or fentanyl.</p

Intrathecal KN93 injection attenuated fentanyl-induced hyperalgesia and decreased the level of <i>p</i>-CaMKIIα in spinal cord but not CeLC in OIH rats.

<p>(A, B) Graphical display of the mechanical threshold of paw withdrawal (A) and thermal paw withdrawal latency (B) collected at baseline, pre-drug (6 h after the last injection of fentanyl or saline), and post-drug (0.5 h after Intrathecal injection). (C-F) Representative Immunoblots of activated CaMKII<i>α</i> (<i>p</i>-CaMKII<i>α</i>) and histogram of relative density of <i>p</i>-CaMKII<i>α</i> to GAPDH in the spinal cord (C-D) and CeLC (E-F). Control, OIH, OIH+KN92 45 nmol, and OIH+KN93 45 nmol group received i.t. injection with 50% DMSO (vehicle), 50% DMSO (vehicle), KN92 10 nmol and KN93 10 nmol respectively. Results are expressed as mean ± SD; *, compared with Control group; &, compared with OIH group; +, compared with OIH+KN92 45 nmol group, n = 6 for each group; One symbol: <i>p</i> < 0.05, Two symbols: <i>p</i> < 0.01, Three symbols: <i>p</i> < 0.001.</p