Intrathecal scrambled peptide (10 μg, n = 8) did not modify mechanical and thermal withdrawal responses compared to baseline in normal rats (Figure 1a, b). More interestingly, intrathecal administration of 10 μg (n = 8) of ZIP did not alter mechanical and thermal withdrawal responses compared to baseline and the control scrambled peptide group throughout the whole experiment (Figure 1a, b). Finally, neither the control scrambled peptide nor ZIP had any effect on the locomotor function of rats on the rotarod, assessed at 30 and 60 min post-injection (Figure 1c). We subsequently examined if spinal PKCζ was involved in pain following peripheral inflammation or nerve injury.

In control rats pre-treated spinally with either saline (n = 8) or the scrambled peptide (10 μg, n = 8), formalin injection induced a similar biphasic response of pain-related behaviors, as expected (Figure 1d,e; saline: 1^st phase (0-10 min), = 97 ± 8 s; 2^nd phase (10-60 min) = 328 ± 28 s; scrambled peptide: 1^st phase = 87 ± 6 s; 2^nd phase = 317 ± 13 s). In contrast, pre-treatment with ZIP, especially at the highest dose, significantly decreased pain-related behaviors during both the first and second phases of the response to formalin (Figure 1d,e; 5 μg: n = 6, 1^st phase = 59 ± 9 s, p > 0.05; 2^nd phase = 207 ± 13 s, p < 0.001; 10 μg: n = 9, 1^st phase = 46 ± 8 s, p < 0.01; 2^nd phase = 145 ± 14 s, p < 0.001) compared to the scrambled peptide group. As the high dose (10 μg) of ZIP exhibited a clear antinociceptive effect in this inflammatory pain model, this dose was tested in a model of neuropathic pain, the chronic constriction injury (CCI).

Chronic constriction injury of the sciatic nerve significantly decreased mechanical withdrawal responses and vocalisation thresholds as expected (Figure 1f,g). Intrathecal scrambled peptide (10 μg, n = 8) did not modify mechanical withdrawal responses and vocalisation thresholds compared to baseline post-CCI in CCI rats (Figure 1f,g). More interestingly, intrathecal administration of 10 μg (n = 8) of ZIP did not significantly inhibit mechanical withdrawal responses and vocalisation thresholds compared to baseline post-CCI and also the control scrambled peptide group throughout the whole experiment, indicating a lack of effect of ZIP on both mechanical allodynia and hyperalgesia following nerve injury.

Thus, PKCζ/PKMζ seems to be specifically involved in inflammatory pain and not in acute pain and, interestingly, neuropathic pain. Following these behavioral results, we have studied the effect of spinal application of ZIP on deep dorsal horn neurons activity using electrophysiology following formalin administration, and localisation, expression of PKCζ/PKMζ.

In control rats pre-treated with the scrambled peptide (10 μg, n = 8), formalin injected into the hindpaw receptive field produced the characteristic biphasic neuronal firing response of spinal WDR neurons (Figure 2a-c; 1st phase: 0-10 min, total APs = 8743 ± 1778; 2^nd phase: 10-70 min, total APs = 83358 ± 11531). Spinal pre-treatment with ZIP (10 μg, n = 8) reduced both first and second phase neuronal firing, though this effect was only statistically significant on second phase activity (Figure 2a-c; 1st phase: total APs = 4847 ± 979, p > 0.05; 2^nd phase: total APs = 29643 ± 11132, p < 0.001).

To our knowledge no specific antibody only against PKMζ is commercially available. Therefore, we used RT-PCR to identify which mRNA isoforms of PKCζ are expressed in the DRG and spinal cord of naïve rats. We found 2 bands at 481 bp and 361 bp as expected for PKMζ and PKCζ mRNA isoforms, respectively, in our spinal cord and DRG samples but also in our positive control samples (i.e. cortex, hippocampus) suggesting that both mRNA isoforms, and probably proteins, are expressed in the DRG and spinal cord (Figure 3a).

By western immunoblotting, we investigated the expression of PKCζ and PKMζ in the DRG, spinal dorsal horn and hippocampus using a specific anti-PKCζ antibody (Figure 3b). Western blots analysis confirmed that PKCζ protein is detected in the hippocampus, DRG and importantly, spinal dorsal horn (Figure 3b). Using an antibody against PKCζ/PKMζ active form, i.e. phospho-PKCζ/p-PKMζ, for which we have characterized the specificity using the blocking peptide (Figure 4b), we observed that p-PKCζ/p-PKMζ are expressed in all categories of DRG neurons, with a highest intensity of immunostaining in the cytoplasm and membrane of small and medium DRG neurons of naïve rats (Figure 3c). We then investigated if p-PKCζ/p-PKMζ is transported to and/or expressed in primary afferent central terminals within the dorsal horn in formalin animals. Expression of p-PKCζ/p-PKMζ was observed in the superficial dorsal horn but we did not observe any obvious co-localisation of p-PKCζ/p-PKMζ with IB4 and CGRP, specific markers for non-peptidergic and peptidergic C-fibres, respectively (Figure 3d,e), even at high power magnification (Figure 4a). In addition, we used lumbar rhizotomy surgery in a separate experiment to see if p-PKCζ/p-PKMζ are expressed in primary afferent fibre terminals as we observed an almost total loss of IB4 and CGRP staining following rhizotomy, illustrating substantial deafferentation (Figure 4c,d). Interestingly, we did not observe any change in spinal p-PKCζ/p-PKMζ staining, indicating a lack of expression of p-PKCζ/p-PKMζ in primary afferent fibres terminals (Figure 4c,d) and suggesting a predominantly intrinsic spinal source.

Consequently, we attempted to identify the phenotype of p-PKCζ/p-PKMζ-expressing cells in spinal cord sections from formalin rats (Figure 5a-c). Phospho-PKCζ/p-PKMζ immunoreactivity was observed in NeuN-positive cells, marking neuronal nuclei (Figure 5c). No obvious co-localization was observed with GFAP-positive astrocytes or Iba1-positive microglial cells (Figure 5a, b). In addition, we used immunoprecipitation to reveal a physical coupling between PKCζ and NR2B subunits, which are specifically expressed in intrinsic dorsal horn sensory neurons and are also essential for spinal nociceptive plasticity (Figure 5d). PKCζ protein was co-immunoprecipitated by the anti-NR2B antibody. Normal spinal lysate was run alongside immunoprecipitation samples as positive controls, while CREB and P2X3 were used as two negative control proteins, to verify that the immunoprecipitation step had worked. The P2X3 receptor is located exclusively on the central terminals of primary afferents and CREB is a nuclear protein, therefore these two proteins should not be found in a post-synaptic, membrane-associated NR2B/PKCζ complex (Figure 5d). Indeed, both P2X3 and CREB were only found in normal spinal tissue lysates, confirming that non-specific detection of proteins in the immunoprecipitation sample had not occurred. Thus, PKCζ is physically coupled to NR2B-containing NMDA receptors in the spinal dorsal horn.

Together, these results suggest that phosphorylation of PKCζ/PKMζ following intraplantar formalin is mainly within intrinsic spinal dorsal horn neurons and could occurred in NR2B containing NMDA receptors neurons. However, phosphorylation of PKCζ/PKMζ needed to be quantified.

Intraplantar formalin injection in the saline (10 μl, n = 5) and scrambled peptide groups (10 μg, n = 5) produced a significant increase of p-PKCζ/p-PKMζ in the superficial (laminae I-II) layers of the ipsilateral dorsal horn (levels L4-L5) compared to the contralateral side (mean percentage increase = 27% ± 3.4 and 22% ± 2, respectively, p < 0.05; Figure 6a,b,d). In addition, intrathecal injection of ZIP (10 μg, n = 5) did not significantly alter formalin-induced PKCζ/PKMζ phosphorylation (mean percentage increase = 25% ± 2; Figure 6c,d).

In addition to immunohistochemistry quantification, we performed western blot analysis to assess the effect of intraplatar formalin and also PKCζ/PKMζ inhibition on the phosphorylation of PKCζ and PKMζ in L4-L5 spinal dorsal horn, 60 min after formalin injection. Scrambled-treated rats exhibited a significant increase in p-PKMζ but not p-PKCζ expression compared with the contralateral dorsal horn (p < 0.05; Figure 6e,f). Intrathecal administration of ZIP did not alter the phosphorylation of PKMζ compared with scrambled-treated rats (Figure 6e,f). As p-PKCζ expression did not significantly increase in the scrambled group following formalin, ZIP was without any effect (Figure 6e,f).

Finally, we evaluated the effect of ZIP on Fos expression which marks activated neurons. The number of Fos positive cells in superficial (I-II) and deep (V-VI) laminae of the lumbar dorsal horn (L4, L5 and L6) was increased at 60 min after intraplantar formalin injection in scrambled peptide-treated rats (10 μg, n = 5; Figure 7a,c). In contrast, intrathecal pre-treatment with ZIP (10 μg, n = 5) resulted in a significant reduction of Fos-positive cells in both the superficial (L4: 45.5% ± 5.8 reduction; L5: 51.3% ± 5.9 reduction; L6: 59.3% ± 1.1 reduction, all p < 0.001) and deep dorsal horn (L4: 53.2% ± 8.4 reduction; L5: 51.6% ± 9.2 reduction; L6: 52.9% ± 11.4 reduction, all p < 0.001) compared to the scrambled peptide group (Figure 7b,c).

Next, we used a more sustained model of inflammation-induced spinal nociceptive plasticity, produced by intraplantar injection of CFA. Twenty-four hours after CFA administration, we observed a significant decrease of mechanical withdrawal thresholds before treatment in all groups (Figure 8a). Intrathecal scrambled peptide administration (10 μg, n = 8) did not modify mechanical withdrawal thresholds, which remained significantly different from baseline throughout the whole experiment. In contrast, intrathecal administration of 10 μg of ZIP (n = 8) significantly increased mechanical withdrawal thresholds 30 min after its administration in comparison to the scrambled peptide-treated group (increase of 41% ± 14, p < 0.05; Figure 8a). However, mechanical withdrawal thresholds of the ipsilateral paw still differed significantly from that of the contralateral paw (p < 0.05; Figure 8a). Finally, ZIP had no effect on mechanical withdrawal thresholds at 90 and 180 min post-injection.

CFA administration also reduced thermal withdrawal latencies before treatment in all groups (Figure 8b). Intrathecal scrambled peptide administration did not alter thermal withdrawal latencies from the baseline throughout the test period (Figure 8b). In contrast, intrathecal administration of 10 μg of ZIP significantly increased thermal withdrawal latencies at 30 and 90 min post-injection compared to the scrambled peptide group (30 min: increase of 82.7% ± 31, p < 0.001; 90 min: increase of 73.5% ± 21, p < 0.05; Figure 8b). Thermal withdrawal latencies of the ipsilateral paw of ZIP-treated group still remained significantly different from those of the contralateral paw at 90 and 180 min.

Intraplantar CFA injection in the scrambled peptide group (10 μg, n = 5) produced a significant increase of p-PKCζ/p-PKMζ in the superficial (laminae I-II) layers of the ipsilateral dorsal horn (levels L4-L5) compared to the contralateral side (mean percentage increase = 19.6% ± 3.3, p < 0.05; Figure 8c,e). As in the formalin experiment, intrathecal injection of ZIP (10 μg, n = 5) did not reduce CFA-induced PKCζ/PKMζ phosphorylation (mean percentage increase = 29.4% ± 6.4; Figure 8d,e).

We also examined the effect of intrathecal administration of ZIP (10 μg, n = 5) on CFA-induced upregulation of Fos expression. In CFA animals treated with the scrambled peptide, we observed a significant increased expression of Fos in superficial (I-II) and deep (V-VI) laminae of the ipsilateral lumbar dorsal horn compared to the contralateral side (Figure 8f,h). In contrast, spinal delivery of ZIP significantly reduced CFA-induced Fos expression in superficial (47.2% ± 11.2 reduction) and deep (46.5% ± 19.4 reduction) laminae of the ipsilateral lumbar dorsal compared to the control scrambled peptide-treated group (Figure 8g,h).

All experiments were carried out using adult male Wistar rats (220-250g, Harlan, UK or Central Biological Services, University College London, UK), housed in standard laboratory conditions with free access to food and water. Experiments were approved by the UK Home Office and followed guidelines set by the International Association for the Study for Pain 42 .

Experiments were performed in a quiet room by a single blinded experimenter using the method of equal blocks with randomization of treatments in order to avoid any uncontrollable environmental influence that might introduce variability in behavioral responses.

Naïve rats (180-200g, Harlan, UK) were injected intrathecally with 10 μg of myristoylated PKCζ pseudosubstrate inhibitor, ZIP (Myr-SIYRRGARRWRKL-OH; Biosource International Inc., USA) previously used as a specific inhibitor of PKCζ/PKMζ in several studies 15 24 36 43 or myristoylated control scrambled peptide (Myr-RLYRKRIWRSAGR-OH; Genscript, USA). Thermal and mechanical thresholds were assessed 30, 60, 90 and 120 min post-injection using the thermal plantar test and automatic Von Frey test, respectively (for details see below).

Locomotor function and motor effects of drugs were assessed using an accelerating rotarod device (Bioseb, France). Naïve rats (180-200 g) underwent a training period over 2 consecutive days (1 trial/day) prior to testing of drug effects in order to ensure that they could remain on the rotarod at 4 revolutions per minute (rpm) for 5 min. On the day of drug administration, the apparatus was set to accelerate from 4 to 40 rpm over 5 min and the latency to fall was monitored. Locomotor function in the accelerating rotarod was assessed 30 and 60 min following intrathecal injection of ZIP or the scrambled peptide (both 10 μg/rat).

Prior to being placed in a Plexiglas box for 30 min to acclimatize, rats received a direct intrathecal (i.t.) injection of 5 or 10 μg of myristoylated PKCζ pseudosubstrate inhibitor, ZIP, (Biosource International Inc., USA), control scrambled peptide (Genscript, USA) or 0.9% saline (n = 8 in each group). Thirty minutes later rats received 50 μl of 5% formalin injected subcutaneously (s.c.) into the plantar surface of the right hindpaw. Lifting, shaking, biting and licking of the injected paw were recorded by measuring the total duration of the response in seconds during the 60 min period following formalin administration. Data are presented in 5 min time bins. At the end of the test, animals were terminally anaesthetized and perfused (for details see below).

Unilateral peripheral mononeuropathy was induced according to the method previously described by 44 . Briefly, rats (150-175 g) were anesthetized with sodium pentobarbital (6%, 1 ml/kg, i.p.). After skin incision, the right sciatic nerve was exposed and 4 polyester sutures (MERSUTURE^® 3-0, Ethicon, Johnson & Johnson, France) were tied loosely around at 1 mm intervals, so that the nerve was constricted but the circulation was not interrupted. The skin was then sutured (MONOCRYL^® 5-0, Ethicon, Johnson & Johnson, Issy-les-Moulineaux, France). The animals were allowed to recover and were routinely monitored to assure good health. To examine mechanical allodynia (i.e. Von Frey test) and hyperalgesia (i.e. paw pressure test), we used two separate groups of animals. The right hindpaw was tested and measurements were taken on two separate days prior CCI surgery (baseline), 14 days after CCI induction (baseline post-CCI) and 30, 90 and 180 min or 30, 45, 120, 180 and 240 min for mechanical allodynia and hyperalgesia, respectively, after administration of a single direct intrathecal injection of ZIP or the scrambled peptide (both 10 μg/rat, n = 8 in each group).

Rats were injected with 50 μl of Complete Freund's Adjuvant (CFA; 1mg/ml Mycobacterium Tuberculosis; Sigma, UK) into the plantar surface of the right hindpaw to induce inflammation. The CFA-treated paw showed marked swelling within 2-3 hrs which persisted for several days. To examine mechanical and thermal hypersensitivity, the left and right hindpaws were tested alternatively and measurements were taken on two separate days prior to CFA administration, 24 hrs after CFA injection (baseline post-CFA) and 30, 90 and 180 min after administration of a single direct intrathecal injection of ZIP or the scrambled peptide (both 10 μg/rat, n = 8 in each group). At the end of the experiment, animals were terminally anaesthetized and perfused (for details see below).

Each animal was placed in a clear acrylic cubicle (22 × 16.5 × 14 cm) on top of a glass floor in a temperature controlled room (~22°C) and allowed to acclimatize for 15 min before testing. Thermal responses were tested using the method described by 45 . Withdrawal latencies were averaged over three consecutive tests, at least 5 min apart, in response to the thermal challenge from a calibrated radiant light source (output of 190 mW/cm2). A cut-off of 20 s was imposed to prevent tissue damage.

Mechanical withdrawal thresholds were tested using a Dynamic Plantar Aesthesiometer (Ugo Basile, Italy). The stimulus was applied via an actuator filament (0.5 mm diameter) which, under computer control, applied a linearly increasing force ramp (2.5 g/sec, maximum force 50 g) to the plantar surface of the hindpaw. The force necessary to elicit paw withdrawal was recorded. The withdrawal threshold was calculated as the average of three consecutive tests, with at least 5 min between each test. A cut-off of 50 g which is the maximum force applied by the apparatus was used.

In order to assess mechanical hyperalgesia, a group of CCI rats were submitted to the paw pressure test previously described by 46 . Nociceptive thresholds, expressed in grams, were measured using an Ugo Basile analgesimeter (Bioseb, France) which applied an increasing pressure to the right hind paw until a squeak (vocalisation threshold) was elicited. The vocalisation threshold was measured 2 or 3 times before (baseline) and after ligatures (baseline post-CCI) in order to obtain two consecutive values that differed no more than 10%, and respecting an interval of at least 10 min between two measures. The maximal pressure (cut-off) applied was 750 g.

Intrathecal (i.t.) injections were performed under isoflurane anaesthesia (4% induction, 2% maintenance) according to the method previously described 47 . The anaesthetised rat was held firmly at the pelvic girdle and drug was delivered (10 μl/rat) using a 25-gauge × 1-inch needle connected to a 25 μl Hamilton syringe inserted into the subarachnoidal space between lumbar vertebrae L5 and L6, eliciting a tail flick. The syringe was held in position for a few seconds after the injection.

Lumbar rhizotomy was carried out as previously described by 37 and 38 . In brief, animals were deeply anaesthetized by intraperitoneal (i.p.) injection of a mixture of medetomidine (0.25 mg/kg) and ketamine (60 mg/kg). Skin and muscle incisions were made to expose the vertebral laminae. The intervertebral foraminae were enlarged and L4, L5 and L6 dorsal roots were exposed and crushed for 10s each. The muscle and skin were then closed with 4.0 sutures. Seven days after surgery, animals were deeply anaesthetised and perfused (see below). Tissue processing (lumbar spinal cord) and immunostaining were conducted as described below.

At the end of the formalin and CFA behavioral experiments, animals were terminally anaesthetised using pentobarbitone and quickly perfused transcardially with saline followed by 4% paraformaldehyde (PFA) with 15% of a saturated solution of picric acid to reduce substantial dephosphorylation. After perfusion, the lumbar spinal cord was excised, post-fixed for 4 hrs in the perfusion fixative, cryoprotected in 20% sucrose in 0.1 M phosphate buffer (PB) overnight at 4°C, and then frozen in O.C.T compound. Transverse sections (30 μm) were cut on a cryostat and thaw-mounted onto glass slides. Sections were stained for p-PKCζ immunohistochemistry as follows: after 3 washes in PBS, sections were incubated for 48 hrs at 4°C with a rabbit primary antibody for anti-phospho-PKCζ (anti-p-PKCζ, Thr 410 sc-12894-R; 1:100; Santa Cruz Biotechnology, USA) followed by a secondary antibody solution for 4 hrs (goat anti-rabbit IgG-conjugated Alexa Fluor 488™; 1:1000; Molecular Probes, USA), as previously described 22 . Slides were washed in PBS and cover-slipped with Vectashield mounting medium (Vector Laboratories, CA, USA).

Sections were visualized under a Zeiss Axioplan 2 fluorescent microscope running Axiovision 3.1 image analysis software and classified according to spinal level (L4, L5 and L6). Quantitative assessment of p-PKCζ/p-PKMζ staining was carried out by determining the immunofluorescence intensity using grey scale within a fixed area of the dorsal horn of the spinal cord (ipsilateral and contralateral to injury), as described by 19 . A box measuring 10⁴ μm² (100 × 100) was placed over the medial part of the superficial dorsal horn and the mean intensity of each area recorded. The measurement protocol was carried out on L4-L6 spinal sections of each animal (at least 5 sections per animal). The background fluorescence intensity of each tissue section was also determined and subtracted from recorded values. Data are presented as mean ± s.e.m. of immunofluorescence intensity (arbitrary unit) of p-PKCζ/p-PKMζ staining in ipsilateral and contralateral dorsal horn for each group.

To determine the cellular distribution of p-PKCζ/p-PKMζ, sections from formalin rats were counterstained with primary antibodies against markers for neurons [mouse anti-neuronal nuclei (anti-NeuN); 1:500; Chemicon, Hampshire, UK], astrocytes [mouse anti-glial fibrillary acidic protein (anti-GFAP); 1:1000; Abcam, Cambridge, UK], microglial cells [rabbit anti-ionized calcium binding adaptor molecule 1 (anti-Iba1); 1:100; Wako Pure Chemical Industries Ltd, Japan], peptidergic primary afferent fibres [sheep anti-calcitonin gene related peptide (anti-CGRP); 1:800; Biomol International LP, USA], and non-peptidergic primary afferent fibres, [biotinylated lectin IB4 (anti-IB4); 1:1000; Sigma, UK], followed by the appropriate secondary antibody solution [goat anti-rabbit IgG-conjugated Alexa Fluor 546™; (1:1000; Molecular Probes, USA) for NeuN, GFAP and Iba1 or anti-sheep Cy3 (1:400; Stratech Scientific Ltd, UK) and extra avidin-TRICT [1:200; Sigma, UK; for CGRP and IB4].

Double immunostaining for p-PKCζ/p-PKMζ with CGRP or IB4 (see protocol above for CGRP or IB4 staining), was also conducted on lumbar spinal cord sections from a group of animals which had rhizotomy surgery (see 2.5). Following CGRP or IB4 staining, sections were incubated with rabbit anti-p-PKCζ (Thr 410, sc-12894-R; 1:100; Santa Cruz Biotechnology, USA) followed by secondary antibody solution (goat anti-rabbit IgG-conjugated Alexa Fluor 488™; 1:1000; Molecular Probes, USA). Slides were washed in PBS, cover-slipped and visualised under a Zeiss Axioplan 2 Microscope (Zeiss, Hertfordshire, UK).

The specificity of p-PKCζ/p-PKMζ immunostaining was verified by preabsorption of the primary antibody (Thr 410, sc-12894-R; 1:100; Santa Cruz Biotechnology, USA) with a 5 times excess (weight/weight) of the peptide used to generate the antibody (sc-12891 P, blocking peptide) overnight at 4°C. The pre-absorbed peptide was then centrifuged 20 min at 10 000 rpm and the top half of the liquid was collected and applied on tissue sections. A positive control (antibody alone at the same dilution) and a negative control (lack of primary antibody) were run in parallel and applied on sister sections. The antibody against IB4 was added to all tubes include in this specificity test. Immunostaining was revealed by incubation with secondary antibody (goat anti-rabbit IgG-conjugated Alexa Fluor 546™; 1:1000; Molecular Probes, USA) for 2 hours.

Sections were processed for Fos staining as follows: following 3 washes in PBS, sections were incubated overnight, at room temperature with the primary rabbit anti-Fos antiserum (1:2500 in PBST-Azide, AB5; Oncogene Science, Uniondale, NY, USA). Next, sections were incubated for 4 hrs with secondary antibody (AlexaFluor™ 488 goat anti-rabbit IgG; 1:1000; Molecular Probes, USA). Slides were washed in PBS and cover-slipped with Vectashield mounting medium (Vector Laboratories, CA, USA). From each animal 4-6 sections (L4-L6) were randomly selected for counting Fos positive cells by a blinded investigator and an average of these counts was taken.

Naïve rats anaesthetized with urethane were sacrificed by decapitation and fresh dorsal roots ganglia (DRG), spinal cord (dorsal horn only) and hippocampus were dissected out and snap frozen. Tissue samples were subsequently homogenized in RIPA (Radioimmunoprecipitation assay) buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS) + 0.5% DOC (Deoxycholic acid) + Complete protease inhibitor cocktail) using a glass homogenizer. Homogenate was then centrifuged at 14000 rpm for 10 min at 4°C and supernatant was collected. DRG, spinal dorsal horn and hippocampus whole cell lysates were next titrated to determine their protein concentrations using a BCA Protein Assay kit (Pierce, UK).

In a separate experiment, ipsilateral and contralateral sides of superficial spinal dorsal horn from scrambled peptide-treated animals (n = 4) and ZIP-treated animals (n = 4) were dissected out 60 min after intraplantar formalin 5%. Following the dissection, tissues samples followed the same procedure as above.

For western immunoblotting, lamaelli loading buffer was added to DRG, dorsal horn and hippocampus protein lysates (30 μg) from naïve animals and samples were incubated at 70°C for 30 min. Samples were then loaded onto 8% gels and separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). After protein transfer to nitrocellulose membranes, membranes were incubated with rabbit anti-PKCζ primary antibody (1:500, ab59364, Abcam, UK) and rabbit anti-neuronal ßIII-tubulin (1:3000, ab18207, Abcam, UK), which served as a loading control, overnight at 4°C, in order to identify PKCζ/PKMζ expression in either DRG, lumbar spinal cord or hippocampus in naïve rats.

Samples from ipsilateral and contralateral sides of superficial spinal dorsal horn from formalin rats were submitted to the same procedure as above and finally transfer to nitrocellulose membranes. Membranes were then incubated with rabbit anti-pPKCζ primary antibody (Thr 410 sc-12894-R; 1:500; Santa Cruz Biotechnology, USA) overnight at 4°C, in order to quantify p-PKCζ and p-PKMζ expression between ipsilateral and contralateral dorsal horn of formalin animals.

Following incubation with dye-linked donkey anti-rabbit IR800 or goat anti-mouse IR600 secondary antibody, proteins were revealed using the Odyssey fluorescence detection system (Licor, UK). For p-PKCζ and p-PKMζ expression levels, bands were quantified by densitometric analysis using Image J software. Results are expressed as mean ± s.e.m. of the densitometric analysis (arbitrary unit) of the phosphorylation of p-PKCζ and p-PKMζ expression levels in the ipsilateral and contralateral dorsal horn for each group (both ipsilateral and contralateral samples were run on the same gel).

For immunoprecipitation of NR2B subunits, Dynabeads^® Protein G (Invitrogen Ltd, UK) were washed and coupled with 5 μg of rabbit anti-NR2B antibody (06-600, Upstate, USA). Next 500 μg of spinal dorsal horn lysate from naïve animals was added to the antibody-dynabeads complex and allowed to incubate overnight for capture of target antigen. Captured protein was eluted from the dynabeads by resuspension in 40 μl of Lamaelli loading buffer and heating at 70°C for 30 min. Samples were then separated onto 8% gels by SDS-PAGE. Normal spinal lysates samples were run alongside IP samples as positive controls. Following electrophoresis and protein transfer to nitrocellulose membranes, membranes were incubated with primary antibody overnight at 4°C. Antibodies used were mouse anti-NR2B (1:500, 75-101, Neuromab, USA), rabbit anti-PKCζ (1:500, ab59364, Abcam, UK). Membranes were also probed with rabbit anti-CREB (1:500, ab5803, Abcam, UK) and rabbit anti-P₂X₃ (1:500, ab10269, Abcam, UK) antibodies used as negative controls. Following incubation with dye-linked donkey anti-rabbit IR800 or goat anti-mouse IR600 secondary antibody, proteins were revealed using the Odyssey fluorescence detection system (Licor, UK).

Total RNA from cortex, hippocampus, spinal cord and DRG from naïve rats was used to synthesize cDNA with the SuperScript™ II Reverserve Transcriptase kit (Invitrogen). 1 μg of cDNA was used for PCR (50 μl final reaction volume) and amplified according to 11 . Cycle parameters were set as 34 cycles at 94°C for 30 s, 60°C for 1 min and 72°C for 1 min, with a final step of 72°C for 10 min. For amplification, F 5'-CCATGCCCAGCAGGACCACC-3' and 5'-CCTTCTATTAGATGCCTGCTCTCC-3' were used as specific forward primers for PKCζ and PKMζ, respectively and R 5'-TGAAGGAAGGTCTACACCATCGTTC-3' was the reverse primer for both.

All data are presented as mean ± s.e.m. Cell characteristics from PKCζ/PKMζ pseudosubstrate inhibitor or scrambled peptide groups were compared by one-way analysis of variance (ANOVA), followed by Bonferroni's post-test. Formalin neuronal and behavioral response time-course data for formalin and CFA rats were compared between treatment groups by two-way repeated measures (RM) ANOVA, followed by Bonferroni's post-test. Total activity in the first and second phases was compared between treatment groups by one-way ANOVA, followed by Bonferroni's post-test. Immunostaining and western immunoblotting of p-PKCζ/p-PKMζ and Fos was analyzed by one-way and two-way ANOVA, respectively, followed by Bonferroni post-tests. Statistical analyses were carried out using GraphPad Prism v.4 software (GraphPad Software Inc., San Diego, CA, USA).