Neuropharmacology 89 (2015) 290e297

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Ht31 peptide inhibited inflammatory pain by blocking NMDA receptor-mediated nociceptive transmission in spinal dorsal horn of mice Wen-Tao Wang, Guo-Qiang Pan, Zi-Yang Zhang, Zhan-Wei Suo, Xian Yang, Xiao-Dong Hu* Department of Molecular Pharmacology, School of Pharmacy, Lanzhou University, Lanzhou, Gansu, 730000, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 June 2014 Received in revised form 10 September 2014 Accepted 30 September 2014 Available online 12 October 2014

A kinase anchoring proteins (AKAPs) assemble cAMPedependent protein kinase (PKA) into signaling complexes with a wide range of ion channels, including N-methyl-D-aspartate (NMDA)-subtype glutamate receptor (NMDAR) that is critical for the central sensitization of nociceptive behaviors. Although PKA has been widely described in the regulation of NMDAR-dependent nociceptive transmission and plasticity, the roles of AKAPs in these processes are largely unknown as yet. The present study interfered with AKAPs/PKA interaction by introducing stearated Ht31 peptide (St-Ht31) into spinal dorsal horn neurons, and investigated the possible changes of primary afferent-evoked, NMDAR-mediated excitatory postsynaptic currents (NMDAR-EPSCs). Whole-cell patch clamp recordings demonstrated that intracellular loading of St-Ht31 through the glass pipettes didn't affect NMDAR-mediated synaptic responses in the spinal cord slices from intact mice. When inflammatory pain was established by intraplantar injection of Complete Freund's Adjuvant (CFA), however, St-Ht31 significantly repressed the amplitudes of NMDAR-EPSCs by selectively removing GluN2B subunit-containing NMDAR out of synapses. With the inhibition of NMDAR-mediated nociceptive transmission, St-Ht31 effectively ameliorated CFA-induced inflammatory pain. Pharmacological manipulation of microtubule-based NMDAR transport, dynamindependent NMDAR endocytosis or actin depolymerization abolished the inhibitory effects of St-Ht31 peptide on NMDAR-EPSCs, suggesting that disruption of AKAPs/PKA interaction by St-Ht31 might disturb multiple NMDAR trafficking steps to reduce the receptor synaptic expression and spinal sensitization. © 2014 Elsevier Ltd. All rights reserved.

Keywords: A kinase anchoring proteins cAMPedependent protein kinase NMDA receptor Inflammatory pain Spinal cord

1. Introduction: Peripheral tissue injuries activate cAMPedependent protein kinase (PKA) in spinal dorsal horn, which is essential for spinal sensitization of nociceptive responses (Malmberg et al., 1997; Yang et al., 2011). PKA is a tetrameric holoenzyme composed of two regulatory (R) subunits (RIa, RIb, RIIa, RIIb), which contain the cAMP binding sites, and two catalytic (C) subunits (Ca, Cb and Cg) (Abel and Nguyen, 2008). In the absence of cAMP, the regulatory subunits keep the holoenzyme at an inactive state by interacting with the catalytic subunits. When cAMP binds to the regulatory subunits, the monomeric catalytic subunits are released from the holoenzyme, which phosphorylate the serine/threonine residues on a number of proteins involved in synaptic transmission and

* Corresponding author. Tel.: þ86 0931 8620265. E-mail address: [email protected] (X.-D. Hu). http://dx.doi.org/10.1016/j.neuropharm.2014.09.031 0028-3908/© 2014 Elsevier Ltd. All rights reserved.

plasticity (Abel and Nguyen, 2008). Inhibition of PKA activity generates an effective analgesic action against pathological pain (Malmberg et al., 1997; Yang et al., 2011). In addition to the enzymatic activity, the subcellular localization of PKA also determines its catalytic efficacy and substrate specificity (Wong and Scott, 2004). Members of A kinase anchoring proteins (AKAPs) have been identified as the scaffolding proteins that interact with RII subunits and assemble PKA into signaling complex with different substrates (Wong and Scott, 2004). One of the best-characterized AKAPs members is AKAP150 and its human ortholog AKAP79. PKA targeting by AKAP150 at the close proximity of glutamate receptors is critical for the induction of long-term potentiation (LTP) and/or long-term depression (LTD) (Colledge et al., 2000; Lu et al., 2007; Snyder et al., 2005; Tunquist et al., 2008). Because an amphipathic helix motif within the C-terminal tail of AKAPs is responsible for the binding to RII subunits of PKA (Vijayaraghavan et al., 1997), a synthetic peptide containing this

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amphipathic helix domain (named Ht31) can competitively disrupt PKA interaction with endogenous AKAPs (Vijayaraghavan et al., 1997). Introduction of Ht31 into hippocampal neurons mimics PKA inhibitors by suppressing the phosphorylation of glutamate receptors and blunting synaptic plasticity (Nie et al., 2007; Snyder et al., 2005). Intraplantar injection of Ht31 peptide also attenuates the inflammatory hyperalgesia (Jeske et al., 2008; Sachs et al., 2009; Schnizler et al., 2008). Although AKAPs are expressed in spinal dorsal horn (Schnizler et al., 2008), little is known about the effect of Ht31 on central sensitization. The present study delivered Ht31 peptide into dorsal horn neurons, and performed whole-cell patch clamp recordings to investigate its possible influence on the glutamatergic transmission of nociceptive signals.

2. Materials and methods: 2.1. Animals, intrathecal injection and behavioral tests The Animal Care and Use Committee of Lanzhou University approved all the experimental procedures. Male Kunming mice (4e6 weeks) were provided by the Experimental Animal Center of Lanzhou University and acclimatized to the testing environments for at least 3 days before any experiments were conducted. To induce the inflammatory pain, Complete Freund's Adjuvant (CFA; 10 ml; Sigma, St. Louis, MO, USA) was injected into the plantar surfaces of hindpaws. Control mice received identical volume of saline. For intrathecal drug delivery, the mice were held firmly by a pelvic girdle and a 30-gauge needle attached to a 25 ml microsyringe was inserted between L5-L6 vertebrae (Fan et al., 2014). A sudden advancement of the needle accompanied by a slight flick of the tail was used as the indicator for the proper insertion into the subarachnoid space. The drugs in 5-ml volume were then injected slowly. The pain sensitivity was measured blindly as previously described (Liu et al., 2014). For Von Frey test, the mice were placed in a cage with wire mesh floor and the calibrated monofilaments were applied perpendicularly to the plantar surfaces until the filaments were bent. The pattern of positive and negative withdrawal responses was converted to 50% paw withdrawal threshold (PWT) (Fan et al., 2014). To measure the paw withdrawal latency (PWL), the mice were placed on a clear glass plate and a beam of light was focused on the plantar surfaces of hindpaws to deliver heat stimuli, with the cutoff of 10 s. The time between the onset of heat application and paw withdrawal was recorded as PWL values.

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2.4. Subcellular fractionation The mice were deeply anesthetized with sodium pentobarbital and the spinal cords were quickly removed into ice-cold ACSF. The dorsal quadrant of L4-5 spinal cord was dissected out and homogenized in Lysis Buffer [10.0 mM TriseHCl, pH 7.6, 320.0 mM sucrose, 5.0 mM EDTA and proteases/phosphatases inhibitors (10.0 mM NaF, 1.0 mM Na3VO4, 1.0 mM phenylmethylsulfonyl fluoride, 1.0 mg/ml each of aprotinin, chymostatin, leupeptin, antipain and pepstatin)]. The homogenate was centrifuged at 1000  g for 10 min at 4  C to remove the nuclei and large debris (P1). The supernatant was further centrifuged at 10, 000  g for 15 min to yield the crude synaptosomal fraction (P2). The P2 pellet was resuspended and incubated in the Lysis Buffer containing 0.5% Triton X-100 for 15 min, and then centrifuged at 32,000  g for 20 min to obtain the synaptosomal membrane fraction (P3), which is enriched with postsynaptic density marker PSD-95 (Snyder et al., 2005; Yang et al., 2009). The P3 pellet was homogenized in sodium dodecyl sulfate (SDS) sample buffer and boiled at 95  C for 5 min before processing. To assay cofilin phosphorylation (Suo et al., 2013; Yang et al., 2011), the spinal dorsal horn was homogenized in Radio-Immunoprecipitation Assay (RIPA) buffer (50.0 mM Tris$HCl, pH 8.0, 150.0 mM NaCl, 1.0 mM EDTA, 1.0% NP-40, 0.1% SDS, 0.5% sodium deoxycholate and proteases/phosphatases inhibitors). After centrifugation at 14,000  g for 10 min, the supernatant was harvested and the protein concentration was measured by using BCA protein assay kit (Pierce, Rockford, IL, USA).

2.2. Preparation of spinal cord slices Mice were deeply anesthetized with sodium pentobarbital (60e90 mg/kg, i.p.). After a laminectomy, the spinal cord was quickly removed into ice-cold sucrose solution (in mM: 50.0 sucrose, 95.0 NaCl, 1.8 KCl, 0.5 CaCl2, 7.0 MgSO4, 1.2 KH2PO4, 26.0 NaHCO3, 15.0 D-glucose, bubbled with 95% O2 þ 5% CO2, pH 7.4). A transverse slice (600-mm thickness) with an intact L4 or L5 dorsal root was cut on a Vibratome stage, transferred to the recording chamber and perfused (5 ml/min) with oxygenated artificial cerebrospinal fluid (ACSF) (in mM: 119.0 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1.2 NaH2PO4, 26.0 NaHCO3, 11.0 D-glucose, bubbled with 95% O2 þ 5% CO2, pH 7.4) at room temperature for at least 1 h prior to electrophysiological recordings (Fan et al., 2014).

2.3. Electrophysiological recordings Whole-cell patch clamp recordings were performed with an Axon700B amplifier. The lamina II neurons were visually identified by using an Olympus Optical BX51WIF microscope fitted with a 40  water immersion objective (Tokyo, Japan). The glass pipettes (3e6 MU) were filled with the internal solution containing 135.0 mM potassium gluconate, 5.0 mM KCl, 2.0 mM MgCl2, 0.5 mM CaCl2, 5.0 mM HEPES, 5.0 mM EGTA, 5.0 mM Mg-ATP and 0.5 mM Na-GTP (pH 7.25; 295e300 mOsm). The attached dorsal roots were stimulated (0.1 Hz, 0.1-ms duration, 2e5 mA) through a suction electrode (Fan et al., 2014; Ikeda et al., 2006). The evoked excitatory postsynaptic currents (EPSCs) mediated by AMPA receptor (AMPAR) were recorded at 70 mV with GABAA receptor antagonist bicuculline (10.0 mM) and glycine receptor antagonist strychnine (2.0 mM) included in the perfusate. To pharmacologically isolate the NMDA receptor component of synaptic responses, the neurons were held at þ 40 mV and AMPAR antagonist CNQX (10.0 mM) was also added into the perfusate (Fan et al., 2014; Zhou et al., 2010). The monosynaptic EPSCs were identified on the basis of the constant latency and the absence of conduction failure in response to high-frequency electrical stimulation (20 Hz) (Fan et al., 2014; Zhou et al., 2010). The series and input resistances were monitored on-line throughout each experiment (Hu et al., 2007). The recordings were abandoned if any resistances changed more than 15%. The current signals were filtered at 2 kHz and sampled at 10 kHz.

Fig. 1. Intrathecal application of St-Ht31 peptide dose-dependently alleviated the inflammatory pain induced by intradermal injection of Complete Freund's Adjuvant (CFA) in mice. The St-Ht31p peptide was used as control. The changes of paw withdrawal thresholds in response to Von Frey filament stimulation (A) and paw withdrawal latencies in response to thermal stimulation (B) were plotted against time. The upward and downward arrows indicated the time points when intradermal (i.d.) and intrathecal (i.t.) injections were performed, respectively. *p < 0.05 relative to saline control. #p < 0.05 relative to St-Ht31p-treated, CFA-injected mice. n ¼ 6 mice in each group.

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2.5. Western blot The equal amounts of protein samples (20 mg) were resolved on SDS- Polyacrylamide Gel Electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk for 30 min at room temperature before incubation overnight with appropriate primary antibody at 4  C. After three washes with PBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution, goat anti-rabbit or goat anti-mouse; Jackson ImmunoResearch Laboratories, Baltimore, PA, USA) for 60 min at room temperature. The blots were visualized by Enhanced Chemiluminescence (Beyotime Institute of Biotechnology, Jiangsu, China). The primary antibodies used in the present study included the rabbit polyclonal anti-GluN2B antibody (Millipore, Temecula, CA, USA), rabbit polyclonal anti-GluN2A antibody (Millipore), mouse monoclonal anti-GluN1 antibody (BD Pharmingen, San Diego, CA, USA), mouse monoclonal anti-b-actin antibody (Sigma) and rabbit polyclonal antibody against phosphorylated cofilin at Ser3 (Anbo Biotechnology, JiangSu, China).

3.2. St-Ht31 peptide suppressed NMDA receptor-mediated synaptic transmission in spinal dorsal horn of mice with inflammatory pain Electrophysiological recordings on spinal dorsal horn neurons have illustrated that intraplantar CFA injection potentiates primary

2.6. Motor function tests The reflexes for surface righting, placing/stepping and grasping/climbing were tested to evaluate the influence of intrathecal drug application on motor functions (Suo et al., 2013). In the surface righting test, we had the mice to lie on the back and observed whether they resumed the normal upright position within 1.5 s. In the placing/stepping test, the dorsal surfaces of hindpaws were drawn over the edge of a table to see if the mice could reflexively step onto the table top. In the grasping/ climbing test, the mice were placed on a wire grid inclined at 90 . Failure to grasp and climb on the grid for 30 s indicated the motor impairment.

2.7. Drugs The stearated (St) form of Ht31 (St-Ht31), its control peptide St-Ht31p (Promega, Madison, WI), phalloidin and 8-Br-cAMP (Sigma) were dissolved in ACSF or internal solution. Ifenprodil, nocodazole and dynasore (Sigma) were dissolved in dimethyl sulfoxide (DMSO), which were diluted with internal solution or ACSF just before use (the final DMSO concentration was 0.1%).

2.8. Statistical analysis All the data were expressed as mean ± SEM. The peak amplitudes of synaptic responses were analyzed with Clampfit 9.0 software. For western blot analysis, the scanned digital images were quantified by Image J software. The relative immunoreactive densities of phosphorylated cofilin and each NMDA receptor subunit were determined by the ratio of their signals to b-actin signals. The statistically significant interaction between the effects of independent variables (intradermal injection, drug treatment or time) on the dependent variable was first determined by Twoway or Three-way ANOVA, followed by Student t test to compare the differences between groups. Statistical significance was set at p < 0.05.

3. Results: 3.1. Intrathecal application of St-Ht31 peptide alleviated the inflammatory pain Intraplantar injection of Complete Freund's Adjuvant (CFA) reduced the paw withdrawal thresholds (PWT) of mice in response to Von Frey filament stimuli (Fig. 1A) and the paw withdrawal latencies (PWL) in response to thermal stimuli (Fig. 1B). One day after CFA injection, a stearated (St) form of Ht31 peptide (St-Ht31) was used to evaluate the possible role of AKAPs/PKA interaction in inflammatory pain. Our data showed that St-Ht31, when intrathecally given at 3.5, 7.0 and 14.0 mg, dose-dependently elevated the PWT and PWL values of CFA-injected mice (Fig. 1AeB). The stearated control peptide St-Ht31p (7.0 mg) had no effects on the inflammatory allodynia and hyperalgesia (Fig. 1AeB). At each St-Ht31 dose tested, the mice displayed normal reflexes for surface righting, placing/stepping and grasping/climbing, suggesting that disruption of AKAPs/PKA interaction didn't impair the motor functions. Although the inflammatory pain was alleviated, the basal pain thresholds of intact mice underwent no significant changes after StHt31 peptide application (data not shown).

Fig. 2. St-Ht31 repressed NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) in spinal dorsal horn neurons of CFA-injected mice. (A) The dorsal root-evoked EPSCs mediated by AMPA receptor (AMPAR) and NMDA receptor (NMDAR) were recorded in lamina II neurons one day after intradermal saline or CFA injection. St-Ht31 (50.0 mM) or its control peptide St-Ht31p (50.0 mM) was introduced into neurons through the recording pipettes. The graph showed the percentage changes in the ratios of AMPAR-EPSCs amplitudes to NMDAR-EPSCs amplitudes (AMPAR/NMDAR ratios). * p < 0.05 relative to saline control. #p < 0.05 relative to CFA-injected mice. n ¼ 6 neurons in each group. (B) Postsynaptic loading of St-Ht31 into lamina II neurons of CFA-injected mice reduced the amplitudes of NMDAR-EPSCs. St-Ht31p peptide was used as control. Sample traces were obtained at the time points indicated by 1 and 2. The horizontal bar indicated the period of intracellular St-Ht31 or St-Ht31p perfusion. The graph summarized the percentage changes of NMDAR-EPSCs amplitudes. *p < 0.05 relative to St-Ht31p control. n ¼ 6 neurons in each group.

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afferent fiber-evoked excitatory postsynaptic currents (EPSCs) mediated by NMDA receptor (NMDAR), but not by AMPA receptor (AMPAR) (Fan et al., 2014; Kopach et al., 2011; Park et al., 2009). Consistent with these results, the ratios of AMPAR-EPSCs amplitudes to NMDAR-EPSCs amplitudes (AMPAR/NMDAR ratios) were significantly reduced one day after CFA injection (Fig. 2A). To investigate the possible regulation by AKAPs-bound PKA of nociceptive synaptic transmission, we prepared spinal cord slices one day after CFA injection, and St-Ht31 or St-Ht31p peptide (50.0 mM) was introduced postsynaptically into lamina II neurons through the recording pipettes (Schnizler et al., 2008; Snyder et al., 2005). Our data showed that intracellular perfusion of St-Ht31 significantly enhanced the AMPAR/NMDAR ratios of CFA-injected mice, which became indistinguishable from those in saline-injected control mice (Fig. 2A). By contrast, the inactive St-Ht31p peptide produced no significant changes in the ratio values of inflamed mice (Fig. 2A). We also examined the effects of St-Ht31 on the basal synaptic transmission in intact mice, finding that the AMPAR/NMDAR ratios after intracellular St-Ht31 perfusion remained at 98.3 ± 17.9% of control (p > 0.05, n ¼ 6 neurons in each group). These data implicated that St-Ht31 peptide generated a pronounced inhibition of NMDAR-mediated nociceptive transmission in CFA-injected mice. To confirm these results, we monitored the time-dependent changes of NMDAR-EPSCs amplitudes, finding that postsynaptic loading of St-Ht31, but not St-Ht31p control peptide, reduced the amplitudes of NMDAR-EPSCs in inflamed mice (Fig. 2B).

central sensitization. Inhibition of GluN2B receptor greatly reduces NMDAR-mediated nociceptive transmission (Fan et al., 2014; Wu et al., 2005) and thus, alleviates inflammatory pain (Zhuo, 2009). To investigate whether AKAPs-bound PKA regulated the GluN2B component of synaptic responses in inflamed mice, we incubated the slices with St-Ht31 or St-Ht31p peptide before examining the effect of GluN2B-selective antagonist ifenprodil on NMDAR currents. In St-Ht31p-treated slices, extracellular perfusion of ifenprodil (3.0 mM) for 25 min decreased the amplitudes of NMDAR synaptic responses to 46.9 ± 6.9% of baseline (p < 0.05, n ¼ 6 neurons; Fig. 3A). Pretreatment with St-Ht31 peptide, however, suppressed the magnitudes of reduction in NMDAR-EPSCs amplitudes induced by ifenprodil (Fig. 3A), suggesting that St-Ht31 occluded the inhibitory effect of GluN2B receptor antagonist on NMDAR currents. In another experiment, we isolated the synaptosomal membrane fraction (P3) from spinal dorsal horn one day after CFA injection, and probed the protein expression of each NMDAR subunit by immunoblotting. Consistent with previous reports (Yang et al., 2009), CFA significantly enhanced the band density of NMDA receptor GluN1 and GluN2B subunit at P3 fraction, with that of GluN2A subunit unaltered (Fig. 3B). Intrathecal application of St-Ht31 (7.0 mg), but not St-Ht31p (7.0 mg), repressed the band densities of GluN2B and GluN1 subunits in CFA-injected mice to control values (Fig. 3B), providing additional evidence that the synaptic inhibition by St-Ht31 peptide might result from the removal of GluN2B receptor out of synapses.

3.3. St-Ht31 suppressed the synaptic expression of GluN2B subunitcontaining NMDAR in spinal dorsal horn of inflamed mice

3.4. St-Ht31 peptide regulated the synaptic trafficking of NMDAR

Previous studies have illustrated that peripheral inflammation specifically recruits GluN2B subunit-containing NMDAR (GluN2B receptor) at postsynaptic membrane (Fan et al., 2014; Wu et al., 2005; Zhuo, 2009), conferring GluN2B receptor a critical role in

The microtubule network has been implicated in the neuronal activity-dependent transport of GluN2B receptor to dendritic plasma membrane (Setou et al., 2000; Yuen et al., 2005b). This process was subjected to the regulation by PKA. Direct PKA activation by intracellular loading of 8-Br-cAMP (100.0 mM)

Fig. 3. St-Ht31 peptide reduced the synaptic expression of GluN2B receptors in spinal dorsal horn of CFA-injected mice. (A) The spinal cord slices were prepared one day after CFA injection and pretreated with St-Ht31 or St-Ht31p peptide (50.0 mM) for 0.5e2 h before whole-cell recording of NMDA receptor-mediated excitatory postsynaptic currents (NMDAREPSCs) in lamina II neurons. Note that extracellular perfusion of GluN2B receptor antagonist ifenprodil (3.0 mM) reduced the amplitudes of NMDAR-EPSCs in slices pretreated with St-Ht31p, but not with St-Ht31. The sample traces were obtained at the time points indicated by 1 and 2. The horizontal bar indicated the period of ifenprodil perfusion. The graph summarized the percentage changes of NMDAR-EPSCs amplitudes. *p < 0.05 relative to St-Ht31 group. n ¼ 6 neurons in each group. (B) Intrathecal application of St-Ht31 (7.0 mg), but not St-Ht31p, suppressed the contents of NMDA receptor GluN1 and GluN2B subunits at synaptosomal membrane fraction (P3) of spinal dorsal horn one day after CFA injection. CFA, St-Ht31 and St-Ht31p didn't influence the content of NMDA receptor GluN2A subunit at P3 fraction. Equal protein loadings were indicated by b-actin signals (left). The graph showed the percentage changes in the immunoreactivity of each NMDAR subunit (right). *p < 0.05 relative saline-injected control mice. #p < 0.05 relative to CFA-injected mice. n ¼ 6 experiments for each NMDAR subunit.

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significantly enhanced NMDAR-EPSCs amplitudes in intact mice (Fig. 4A), which could be blocked by nocodazole (10.0 mM), a microtubule destabilizer (Yuen et al., 2005a, 2005b). Given that peripheral inflammation naturally activated spinal PKA, it was conceivable that microtubule-based GluN2B transport might be necessary for GluN2B synaptic accumulation during inflammatory pain. Indeed, postsynaptic loading of nocodazole dramatically decreased the amplitudes of NMDAR-EPSCs in spinal cord slices from CFA-injected mice (Fig. 4B). Notably, if St-Ht31 peptide was used to pretreat the slices for 0.5e2 h, the inhibitory effect of nocodazole on NMDAR-EPSCs was significantly attenuated (Fig. 4B), suggesting that AKAPs-bound PKA might be involved in the regulation of microtubule-based NMDAR trafficking during inflammatory pain. To confirm this, we pretreated the slices with nocodazole for 0.5e2 h, and found that intracellular perfusion of St-Ht31 failed to induce a significant inhibition of synaptic responses in the presence of nocodazole (Fig. 4C). In addition to the anterograde transport of NMDAR along microtubules, the clathrin-mediated, dynamin-dependent endocytosis is another step for the dynamic regulation of NMDAR surface expression (Washbourne et al., 2004; Wild et al., 2014). We tested whether St-Ht31 influenced NMDAR endocytosis process by using dynasore (80.0 mM), a cell-permeable dynamin GTPase activity inhibitor (Muir et al., 2010). Postsynaptic introduction of dynasore alone didn't change the synaptic responses in inflamed mice (Fig. 5A). However, when dynasore was used to pretreat slices for 0.5e2 h prior to recordings, it blocked the decrease of NMDAREPSCs amplitudes induced by St-Ht31 (Fig. 5B). More recent studies have demonstrated that NMDAR on the plasma membrane can diffuse from extrasynaptic to postsynaptic sites, where F-actin cytoskeleton is required for synaptic NMDAR stabilization (Morishita et al., 2005; Rosenmund and Westbrook, 1993). Interestingly, PKA can prevent actin depolymerization by phosphorylating and inhibiting cofilin (Nadella et al., 2009; Parisiadou et al., 2014), an important actin-severing protein

involved in synaptic plasticity (Morishita et al., 2005; Zhou et al., 2004). In spinal dorsal horn, there was a marked increase of cofilin phosphorylation at Ser3 one day after CFA injection when compared to saline control (Fig. 6A). Intrathecal application of StHt31 (7.0 mg), but not St-Ht31p (7.0 mg), repressed the phosphorylation levels of cofilin in inflamed mice to control values (Fig. 6A), suggesting that AKAPs-bound PKA was likely to block cofilinmediated actin depolymerization during inflammatory pain. To evaluate the possible role of F-actin cytoskeleton in the regulation of synaptic NMDAR by St-Ht31, we pretreated the slices from inflamed mice with phalloidin (100.0 mM), an actin cytoskeleton stabilizer (Morishita et al., 2005). Our data showed that phalloidin clearly prevented St-Ht31 peptide from repressing NMDAR-EPSCs (Fig. 6B), although postsynaptic loading of phalloidin per se didn't influence NMDAR synaptic currents in CFA-injected mice (Fig. 6C). Taken together, these data suggested that St-Ht31 might repress NMDAR-mediated nociceptive transmission in inflamed mice by disturbing multiple steps involved in the receptor trafficking process.

4. Discussion: AKAPs family members are present at both peripheral DRG neurons and spinal dorsal horn neurons (Schnizler et al., 2008). Intradermal Ht31 injection reduces PKA-mediated phosphorylation of transient receptor potential family V type 1 (TRPV1) in DRG neurons and generates an effective antinociception (Sachs et al., 2009; Schnizler et al., 2008). The present study found that intrathecal St-Ht31 injection also attenuated CFA-induced inflammatory pain in a dose-dependent manner. In addition to act at peripheral sites, St-Ht31 might achieve this analgesic effect through spinal mechanisms. Selective introduction of St-Ht31 into spinal dorsal horn neurons blunted NMDAR synaptic currents in CFA-injected mice, suggesting that the inhibition of NMDAR-mediated

Fig. 4. AKAPs-bound PKA regulated NMDA receptor-mediated excitatory postsynaptic currents (NMDAR-EPSCs) through microtubule cytoskeleton. (A) Direct PKA activation by postsynaptic perfusion of 8-Br-cAMP (100.0 mM) enhanced NMDAR-EPSCs amplitudes in spinal dorsal horn neurons of intact mice, which, however, could be blocked if nocodazole (10.0 mM) was used to pretreat the slices for 0.5e2 h. The horizontal bar indicated the period of intracellular perfusion. The sample traces were obtained at the time points indicated by 1 and 2. The graph summarized the percentage changes of NMDAR-EPSCs amplitudes. *p < 0.05 relative to control group. n ¼ 6 neurons in each group. (B) Postsynaptic loading of nocodazole (10.0 mM) through the glass pipettes reduced the amplitudes of NMDAR-EPSCs in spinal cord slices prepared one day after CFA injection (n ¼ 6 neurons). Note that pretreatment of slices with St-Ht31 peptide (50.0 mM) attenuated the inhibitory effect of nocodazole on NMDAR-EPSCs (n ¼ 6 neurons). *p < 0.05 relative to DMSO group (n ¼ 3 neurons). (C) Pretreatment of slices with nocodazole also attenuated the inhibitory effect of intracellular St-Ht31 (50.0 mM) perfusion on NMDAR-EPSCs. *p < 0.05 relative to nocodazole group. n ¼ 6 neurons in each group.

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Fig. 5. Dynamin GTPase activity inhibitor dynasore blocked the inhibitory effect of St-Ht31 peptide on NMDA receptor-mediated excitatory postsynaptic currents (NMDAR-EPSCs) in spinal dorsal horn of CFA-injected mice. (A) Postsynaptic perfusion of dynasore (80.0 mM) through the glass pipettes didn't influence the amplitudes NMDAR-EPSCs in spinal cord slices prepared one day after CFA injection. The horizontal bar indicated the period of intracellular drug perfusion. The sample traces were obtained at the time points indicated by 1 and 2. The graph summarized the percentage changes of NMDAR-EPSCs amplitudes. n ¼ 6 neurons in each group. (B) Intracellular loading of St-Ht31 peptide (50.0 mM) suppressed NMDAR-EPSCs in slices from CFA-injected mice, which, however, could be blocked when dynasore (80.0 mM) was used to pretreat the slices for 0.5e2 h *p < 0.05 relative to dynasore group. n ¼ 6 neurons in each group.

Fig. 6. Role of actin depolymerization in the suppression by St-Ht31 of NMDA receptor-mediated excitatory postsynaptic currents (NMDAR-EPSCs) during inflammatory pain. (A) The spinal dorsal horns of mice were isolated one day after CFA injection for immunoblotting analysis of cofilin phosphorylation at Ser3 (p-Cofilin). Note that intrathecal application of St-Ht31 (7.0 mg), but not St-Ht31p (7.0 mg), repressed CFA-induced cofilin phosphorylation. Equal protein loadings were indicated by b-actin signals. The graph showed the percentage changes of cofilin phosphorylation levels. *p < 0.05 relative saline-injected control mice. #p < 0.05 relative to CFA-injected mice. n ¼ 6 experiments. (B) Intracellular loading of St-Ht31 peptide (50.0 mM) suppressed NMDAR-EPSCs in spinal cord slices from CFA-injected mice, which, however, could be blocked if actin cytoskeleton stabilizer phalloidin (100.0 mM) was used to pretreat the slices for 0.5e2 h. The horizontal bar indicated the period of intracellular St-Ht31 perfusion. The sample traces were obtained at the time points indicated by 1 and 2. The graph summarized the percentage changes of NMDAR-EPSCs amplitudes. *p < 0.05 relative to phalloidin group. n ¼ 6 neurons in each group. (C) Intracellular loading of phalloidin (100.0 mM) per se didn't change the amplitudes of NMDAR-EPSCs in the spinal cord slices prepared one day after CFA injection (n ¼ 6 neurons in each group).

nociceptive transmission might represent another important way for St-Ht31 to alleviate inflammatory pain. Pharmacological activation of PKA is well known to increase the synaptic abundance of NMDAR (Cerne et al., 1993; Yang et al., 2009). Nevertheless, the effects of AKAPs-bound PKA on NMDAR synaptic responses remain to be elucidated. Previously, RNAi knockdown of AKAP150 expression in hippocampal neurons has been shown to increase basal synaptic transmission mediated by AMPAR, but not by NMDAR (Jurado et al., 2010). This result is likely

attributed to the altered compartmentalization of calcineurin (PP2B), another AKAP150-binding partner that tonically inhibits basal AMPAR synaptic responses (Jurado et al., 2010). In spinal dorsal horn, PKA phosphorylation of AMPAR only exhibits a transient increase after CFA injection (Kopach et al., 2011; Lu et al., 2008; Park et al., 2009). The present study found that St-Ht31 noticeably reversed the reduction of AMPAR/NMDAR ratios in CFA-injected mice, suggesting that disruption of AKAPs/PKA interaction during inflammatory pain might, at least in part, reduce

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NMDAR synaptic responses to a more extent than AMPAR synaptic responses. NMDAR is a hetero-oligomeric protein consisting of combination of GluN1, GluN2 (GluN2A-GluN2D) and GluN3 (GluN3AGluN3B) subunit. The most prevalent NMDAR subtypes in spinal dorsal horn are composed of the obligatory GluN1 and regulatory GluN2A or GluN2B subunits. After peripheral inflammation, the specific accumulation of GluN2B receptors at synapses has been described as an important way to cause spinal NMDAR hyperfunction (Tan et al., 2005; Yang et al., 2009; Zhuo, 2009). We found that the analgesic action of St-Ht31 correlated closely with the inhibition of synaptic GluN2B receptor. With respect to the mechanisms underlying GluN2B receptor trafficking, the kinesin proteins (such as KIF17) have been identified for the anterograde transport of GluN2B receptor-containing vesicles along microtubules from endoplasmic reticulum (ER)-Golgi networks to dendritic plasma membrane (Guillaud et al., 2003; Setou et al., 2000; Yuen et al., 2005b). A large proportion of GluN2B receptors that have been incorporated onto plasma membrane can be internalized in response to the altered neuronal activity, which also governs the surface expression of NMDAR (Lau and Zukin, 2007; Lavezzari et al., 2004; Washbourne et al., 2004; Wild et al., 2014). Notably, PKA signaling can be utilized by multiple extracellular stimuli to modulate both the microtubule-based transport and endocytosis process of NMDAR (Liu et al., 2006; Yang et al., 2011; Yuen et al., 2005). We found that interference with microtubule dynamics and dynamin-dependent endocytosis blocked the inhibitory effect of St-Ht31 on NMDAR-EPSCs, suggesting that disruption of AKAPs/ PKA interaction might act at several NMDAR trafficking steps to reduce the nociceptive transmission during inflammatory pain. On the plasma membrane, NMDAR diffuses laterally between extrasynaptic and synaptic sites (Lau and Zukin, 2007). The C-terminal region of NMDAR interacts with actin cytoskeleton at postsynaptic density (PSD) (Wyszynski et al., 1997). Actin depolymerization can promote the diffusion of NMDAR from postsynaptic to extrasynaptic membrane, leading to the longlasting depression of NMDAR-mediated synaptic transmission (Morishita et al., 2005). As an F-actin-severing protein, cofilin plays an important role in LTD of hippocampal synapses through actin depolymerization (Morishita et al., 2005; Zhou et al., 2004). Previous studies have indicated that cofilin activity can be inhibited by LIM kinases-mediated phosphorylation of Ser-3 residue, while Slingshot family protein phosphatases (SSHs) can dephosphorylate and reactivate Ser-3-phosphorylated cofilin (Mizuno, 2013). In the spinal cord, cofilin phosphorylation correlated well with inflammatory hyperalgesia (Zulauf et al., 2009). Our data also illustrated a significant increase of cofilin phosphorylation after CFA injection, which might result from LIM kinases activation and/or SSHs inhibition (Nadella et al., 2009; Parisiadou et al., 2014). Intrathecal StHt31 application suppressed CFA-induced cofilin phosphorylation, a process that might promote actin depolymerization and contribute to the synaptic inhibition in inflamed mice. Taken together, the present study demonstrated that intrathecal application of St-Ht31 suppressed NMDAR-mediated nociceptive transmission and ameliorated the inflammatory pain. These data provided an important clue as to how AKAPs-bound PKA regulated the synaptic trafficking of NMDAR. However, which AKAPs family member in dorsal horn neurons is utilized by peripheral inflammation to induce NMDAR-dependent pain sensitization requires further identification and detailed investigation. Acknowledgement This work was supported by the National Natural Science Foundation of China (N. 31271186). We declare that we have no

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