Interleukin-33 (IL-33)/suppressor of tumorigenicity 2 (ST2) signaling is known to promote inflammation and the genesis and maintenance of neuropathic pain. However, it remained mostly unknown how IL-33/ST2 signaling can be enhanced by neuropathic stimulations. Here, we report that the chronic constriction nerve injury (CCI)-induced increases in the expression of IL-33 and ST2 and a decrease in microRNA (miRNA)-547-5p not only in the dorsal root ganglia (DRG) but also in spinal dorsal horn (SDH) ipsilateral to the CCI. We found that increasing endogenous miRNA-547-5p by the intrathecal (i.t.) infusion of agomir-miR-547-5p did not produce any effect in naive rats but blocked the CCI-induced increases in the IL-33 and ST2, and pain sensitivity. The reducing endogenous miRNA-547-5p by the i.t. delivering antagomir-miR-547-5p into naive rats caused significant changes in IL-33 and ST2 expressions in both the DRG and SDH, and pain sensitivity, which were similar to those induced by the CCI. Since increasing IL-33 by the i.t. infusion of recombinant IL-33 produced no change in the expression of miR-547-5p, and the CCI still reduced miR-547-5p expression in rats with the IL-33 knockdown, we conclude that the reduction of miR-547-5p can be an upstream event leading to the enhancement of IL-33/ST2 signaling induced by the CCI. The intravenous application of bone marrow stromal cells (BMSCs) reduced the depression of miR-547-5p in both the DRG and SDH, and pain hypersensitivity produced by the CCI or antagomir-miR547-5p application. However, the BMSC effect was significantly occluded by the pretreatment with miR-547-5p agomir or the IL-33 knockdown, demonstrating a novel mechanism underlying the BMSC therapy.

Src family of kinases (SFKs) has been found to play an important role in the regulation of nociception. However, how each member of this family acts in the central nervous system (CNS) structures involved in the relay and/or modulation of nociceptive signals, and thereby contributes to the formation and maintenance of pain hypersensitivity, is still a challenge. In this work, a combined study using biochemical, genetic and behavioral approaches was conducted. We found that the expression of activated SFKs in the hypothalamic arcuate nucleus (ARC) area was significantly increased following the development of inflammation induced by injection of complete freund’s adjuvant (CFA) into the hind paw of rats. Furthermore, we identified that Src, but not Fyn or Lyn in the Src family, was activated, and that Src knockdown in the ARC area blocked the inflammation-induced increases in the expression of activated SFKs, the N-Methyl-D-aspartate receptor (NMDAR) GluN2B subunit and phosphorylated GluN2B at Y1472 in this region. Moreover, the CFA injection-induced allodynia and hyperalgesia, and the analgesic effect produced by systemic application of the SFK inhibitor, SU6656, were significantly diminished. However, the Src knockdown did not induce any change in the expression of activated SFKs and the NMDAR GluN2B subunit in normal rats which were not injected with CFA. Neither the Src knockdown nor the systemic application of SU6656 affected the mechanical and thermal sensitivity of the normal rats. Thus, Src activation in the ARC may be a key event for formation and maintenance of pain hypersensitivity associated with peripheral inflammation.

The cAMP‐dependent protein kinase A family (PKAs), protein kinase C family (PKCs), and Src family kinases (SFKs) are found to play important roles in pain hypersensitivity. However, more detailed investigations are still needed in order to understand the mechanisms underlying the actions of PKAs, PKCs, and SFKs. Neurons in the hypothalamic arcuate nucleus (ARC) are found to be involved in the regulation of pain hypersensitivity. Here we report that the action potential (AP) firing activity of ARC neurons in culture was up‐regulated by application of the adenylate cyclase activator forskolin or the PKC activator PMA, and that the forskolin or PMA application‐induced up‐regulation of AP firing activity could be blocked by pre‐application of the SFK inhibitor PP2. SFK activation also up‐regulated the AP firing activity and this effect could be prevented by pre‐application of the inhibitors of PKCs, but not of PKAs. Furthermore, we identified that forskolin or PMA application caused increases in the phosphorylation not only in PKAs at T197 or PKCs at S660 and PKCα/βII at T638/641, but also in SFKs at Y416. The forskolin or PMA application‐induced increase in the phosphorylation of PKAs or PKCs was not affected by pre‐treatment with PP2. The regulations of the SFK and AP firing activities by PKCs were independent upon the translocation of either PKCα or PKCβII. Thus, it is demonstrated that PKAs may act as an upstream factor(s) to enhance SFKs while PKCs and SFKs interact reciprocally, and thereby up‐regulate the AP firing activity in hypothalamic ARC neurons.

BACKGROUND:Understanding molecular mechanisms underlying the induction of learning and memory impairments remains a challenge. Recent investigations have shown that the activation of group I mGluRs (mGluR1 and mGluR5) in cultured hippocampal neurons by application of (S)-3,5-Dihydroxyphenylglycine (DHPG) causes the regulated internalization of N-methyl-D-aspartate receptors (NMDARs), which subsequently activates protein kinase D1 (PKD1). Through phosphorylating the C-terminals of the NMDAR GluN2 subunits, PKD1 down-regulates the activity of remaining (non-internalized) surface NMDARs. The knockdown of PKD1 does not affect the DHPG-induced inhibition of AMPA receptor-mediated miniature excitatory post-synaptic currents (mEPSCs) but prevents the DHPG-induced inhibition of NMDAR-mediated mEPSCs in vitro. Thus, we investigated the in vivo effects of bilateral infusions of DHPG into the hippocampal CA1 area of rats in the Morris water maze (MWM) and the novel object discrimination (NOD) tests.METHODS:A total of 300 adult male Sprague Dawley rats (250-280 g) were used for behavioral tests. One hundred ninety four were used in MWM test and the other 106 rats in the NOD test. Following one week of habituation to the vivarium, rats were bilaterally implanted under deep anesthesia with cannulas aimed at the CA1 area of the hippocampus (CA1 coordinates in mm from Bregma: AP -3.14; lateral +/-2; DV -3.0). Through implanted cannulas artificial cerebrospinal fluid (ACSF), the group1 mGluR antagonist 6-Methyl-2-(phenylethynyl)pyridine (MPEP), the dynamin-dependent internalization inhibitor Dynasore, or the PKD1 inhibitor CID755673 were infused into the bilateral hippocampal CA1 areas (2 μL per side, over 5 min). The effects of these infusions and the effects of PKD1 knockdown were examined in MWM or NOD test.RESULTS:DHPG infusion increased the latency to reach the platform in the MWM test and reduced the preference for the novel object in the NOD task. We found that the DHPG effects were dose-dependent and could be maintained for up to 2 days. Notably, these effects could be prevented by pre-infusion of the group1 mGluR antagonist MPEP, the dynamin-dependent internalization inhibitor Dynasore, the PKD1 inhibitor CID755673, or by PKD1 knockdown in the hippocampal CA1 area.CONCLUSION:Altogether, these findings provide direct evidence that PKD1-mediated signaling may play a critical role in the induction of learning and memory impairments by DHPG infusion into the hippocampal CA1 area.

Cell-surface protein endocytosis is critically involved in the regulation of organismal homeostasis, immune responses, development and neurotransmission [1,2,3,4] . Mechanisms underlying the endocytosis of cell surface proteins have been extensively investigated. However, until very recently no study has reported how non-internalized cell surface proteins may behave following endocytosis of same type of proteins. Here, we highlight findings that regulated NMDA receptor (NMDAR) internalization not only reduces the amount of NMDARs expressed on neuronal surface but also through activating PKD1 pathway phosphorylates and down-regulates remaining (non-internalized) surface NMDARs. This down-regulation of remaining surface NMDARs plays a critical role in the modulation of NMDAR-mediated synaptic responses by NMDAR internalization.

Extensive studies have focused on the development and regionalization of neurons in the central nervous system (CNS). Many genes, which play crucial roles in the development of CNS neurons, have been identified. By using the technique "direct reprogramming", neurons can be produced from multiple cell sources such as fibroblasts. However, understanding the region-specific regulation of neurons in the CNS is still one of the biggest challenges in the research field of neuroscience. Neurons located in the trigeminal subnucleus caudalis (Vc) and in the spinal dorsal horn (SDH) play crucial roles in pain and sensorimotor functions in the orofacial and other somatic body regions, respectively. Anatomically, Vc represents the most caudal component of the trigeminal system, and is contiguous with SDH. This review is focused on recent data dealing with the regional specificity involved in the development of neurons in Vc and SDH.

目的 研究代谢型谷氨酸受体激动剂3,5-二羟基苯甘氨酸(DHPG)对SD大鼠学习记忆行为的影响.方法 选用雄性SD大鼠,随机分为DHPG组和人工脑脊液(ACSF)组,利用大鼠脑立体定位方法分别于两侧海马CA1区微量注射DHPG和ACSF.应用Morris水迷宫进行定位航行实验和空间探索实验测定两组大鼠DHPG和ACSF注射前后的学习记忆能力.结果 DHPG组海马CA1区注射DHPG后,与ACSF组比较,大鼠定位航行实验中逃避潜伏期延长(P＜0.05);空间探索实验中穿越原平台次数、目标象限路程均减少(P＜0.05).结论 DHPG可能与大鼠学习记忆障碍有关.

Neurons in the trigeminal subnucleus caudalis (Vc) and spinal cord dorsal horn (SDH) play important roles in modulating and relaying pain signals to the higher centers of the central nervous system (CNS). Morphologically, many aspects including a laminated structure, cytoarchitecture and cellular elements in these two central regions are very similar [1,2,3,4] . Most nociceptive afferents with a small-diameter terminate in the superficial laminae of Vc and SDH. Functionally, sensory neurons in Vc and SDH respectively receive nociceptive inputs from the orofacial and other somatic regions, and convey the inputs to higher brain centers [1,2,3,4,5] . According to the mechanoreceptive properties, sensory neurons in both the Vc and SDH are classified into three types. They are low-threshold mechanoreceptive (LTM), wide-dynamic range (WDR) and nociceptive-specific neurons (NS) [1,2,3,4,5] . Because of these similarities Vc has been considered as analogous to SDH [4,6] . However, some differences between Vc and SDH have also been reported. For example, the distribution pattern of substance P (SP) and calcitonin gene related peptide (CGRP) in Vc of adult animals is different from that in SDH. In SDH there is no dual representation of peripheral regions, which can be found in rostral and caudal Vc. A transitional zone found between Vc and trigeminal subnucleus interpolaris plays a special role in sensorimotor function. However, no such region is found in the spinal system [5,7,8] . CNS neurons are regionalized. They are organized as groups through their circuitry. Although it is known that differences in functions among these groups in the adult CNS are predetermined during the development of the neuroepithelium [9,10,11,12,13] , understanding how the regional specificity is developed in the CNS is still a big challenge in neuroscience research. It has recently been found that ninhydrin-reacting small molecules released locally are involved in the region-specific regulation of neuronal development in cultured Vc and SDH neurons [14,15] . In this review we focused on present knowledge about the region-specific regulation of neuronal development in these two CNS regions associated with the transmission of nociceptive signals.

BACKGROUND:Constitutive and regulated internalization of cell surface proteins has been extensively investigated. The regulated internalization has been characterized as a principal mechanism for removing cell-surface receptors from the plasma membrane, and signaling to downstream targets of receptors. However, so far it is still not known whether the functional properties of remaining (non-internalized) receptor/channels may be regulated by internalization of the same class of receptor/channels. The N-methyl-D-aspartate receptor (NMDAR) is a principal subtype of glutamate-gated ion channel and plays key roles in neuronal plasticity and memory functions. NMDARs are well-known to undergo two types of regulated internalization - homologous and heterologous, which can be induced by high NMDA/glycine and DHPG, respectively. In the present work, we investigated effects of regulated NMDAR internalization on the activity of residual cell-surface NMDARs and neuronal functions.RESULTS:In electrophysiological experiments we discovered that the regulated internalization of NMDARs not only reduced the number of cell surface NMDARs but also caused an inhibition of the activity of remaining (non-internalized) surface NMDARs. In biochemical experiments we identified that this functional inhibition of remaining surface NMDARs was mediated by increased serine phosphorylation of surface NMDARs, resulting from the activation of protein kinase D1 (PKD1). Knockdown of PKD1 did not affect NMDAR internalization but prevented the phosphorylation and inhibition of remaining surface NMDARs and NMDAR-mediated synaptic functions.CONCLUSION:These data demonstrate a novel concept that regulated internalization of cell surface NMDARs not only reduces the number of NMDARs on the cell surface but also causes an inhibition of the activity of remaining surface NMDARs through intracellular signaling pathway(s). Furthermore, modulating the activity of remaining surface receptors may be an effective approach for treating receptor internalization-induced changes in neuronal functions of the CNS.

Voltage-gated sodium (Na+) and potassium (K+)channels have been found to be regulated by Src family kinases(SFKs).However, how these channels are regulated by SFKs in cochlear spiral ganglion neurons (SGNs) remains unknown.Here, we report that altering the activity of endogenous SFKs modulated voltage-gated Na+, but not K+, currents recorded in embryonic SGNs in culture. Voltage-gated Na+ current was suppressed by inhibition of endogenous SFKs or just Src and potentiated by the activation of these enzymes. Detailed investigations showed that under basal conditions, SFK inhibitor application did not significantly affect the voltage-dependent activation, but shifted the steady-state inactivation curves of Na+ currents and delayed the recovery of Na+ currents from inactivation. Application of Src specific inhibitor, Src40–58,not only shifted the inactivation curve but also delayed the recovery of Na+ currents and moved the voltage-dependent activation curve towards the left. The pre-inhibition of SFKs occluded all the effects induced by Src40–58 application, except the left shift of the activation curve. The activation of SFKs did not change either steady-state inactivation or recovery of Na+ currents, but caused the left shift of the activation curve.SFK inhibitor application effectively prevented all the effects induced by SFK activation, suggesting that both the voltage-dependent activation and steady-state inactivation of Na+ current are subjects of SFK regulation. The different effects induced by activation versus inhibition of SFKs implied that under basal conditions, endogenously active and inactive SFKs might be differentially involved in the regulation of voltage-gated Na+ channels in SGNs.

J. Neurochem. (2012) 122 , 605–618. Abstract Neurons located in the trigeminal subnucleus caudalis (Vc) play crucial roles in pain and sensorimotor functions in the orofacial region. Because of many anatomical and functional similarities with the spinal dorsal horn (SDH), Vc has been termed the medullary dorsal horn ‐ analogous to the SDH. Here, we report that when compared with embryonic SDH neurons in culture, neurons isolated from the Vc region showed significantly slower growth, lower glutamate receptor activity, and more cells undergoing cell death. SDH neuron development was inhibited in co‐cultures of SDH and Vc tissues while Vc neuron development was promoted by co‐culture with SDH tissues. Furthermore, we identified that small (non‐protein) ninhydrin‐reacting molecules purified from either embryonic or post‐natal Vc‐conditioned medium inhibited neuronal growth whereas ninhydrin‐reacting molecules from SDH‐conditioned medium promoted neuronal growth. These findings suggest the involvement of locally released factors in the region‐specific regulation of neuronal development in Vc and SDH, central nervous system regions playing critical roles in pain, and point to novel avenues for investigating central nervous system regionalization and for designing therapeutic approaches to manage neurodegenerative diseases and pain.

Src family kinases (SFKs) play critical roles in the regulation of many cellular functions by growth factors, G‐protein‐coupled receptors and ligand‐gated ion channels. Recent data have shown that SFKs serve as a convergent point of multiple signaling pathways regulating N‐methyl‐d‐aspartate (NMDA) receptors in the central nervous system. Multiple SFK molecules, such as Src and Fyn, closely associate with their substrate, NMDA receptors, via indirect and direct binding mechanisms. The NMDA receptor is associated with an SFK signaling complex consisting of SFKs; the SFK‐activating phosphatase, protein tyrosine phosphatase α; and the SFK‐inactivating kinase, C‐terminal Src kinase. Early studies have demonstrated that intramolecular interactions with the SH2 or SH3 domain lock SFKs in a closed conformation. Disruption of the interdomain interactions can induce the activation of SFKs with multiple signaling pathways involved in regulation of this process. The enzyme activity of SFKs appears ‘graded’, exhibiting different levels coinciding with activation states. It has also been proposed that the SH2 and SH3 domains may stimulate catalytic activity of protein tyrosine kinases, such as Abl. Recently, it has been found that the enzyme activity of neuronal Src protein is associated with its stability, and that the SH2 and SH3 domain interactions may act not only to constrain the activation of neuronal Src, but also to regulate the enzyme activity of active neuronal Src. Collectively, these findings demonstrate novel mechanisms underlying the regulation of SFKs.

Aim: To investigate the role of glutamate and N -methyl- D -aspartate (NMDA) receptors in central sensitization following peripheral inflammation in the arcuate nucleus (ARC) of the mediobasal hypothalamus. Methods: Mediobasal hypothalamic slices were prepared from rats undergoing peripheral inflammation, which was induced by a unilateral injection of complete Freund's adjuvant (CFA) into hind paw. Neuronal activation levels in the ARC were monitored by recording extracellular unit discharges. The NMDA receptor NR1 subunit (NR1) was measured using Western blot analysis. Results: Enhanced NR1 phosphorylation was observed in the ARC of CFA-inflamed rats. Compared with the control rats, the firing rate of spontaneous discharges in ARC neurons of inflamed rats was significantly higher, and it was significantly reduced both by an NMDA receptor antagonist (MK-801, 300 μmol/L) and by a non-NMDA receptor antagonist (CNQX, 30 μmol/L). Application of exogenous glutamate (200 μmol/L) or NMDA (25 μmol/L) resulted in increased neuronal discharges for ARC neurons, which was enhanced to a greater extent in inflamed rats than in control rats. Conclusion: Glutamate receptor activation in the hypothalamic ARC plays a crucial role in central sensitization associated with peripheral inflammation.

Previous studies demonstrated that intra-domain interactions between Src family kinases (SFKs), stabilized by binding of the phosphorylated C-terminus to the SH2 domain and/or binding of the SH2 kinase linker to the SH3 domain, lock the molecules in a closed conformation, disrupt the kinase active site, and inactivate SFKs. Here we report that the up-regulation of N-methyl-D-aspartate receptors (NMDARs) induced by expression of constitutively active neuronal Src (n-Src), in which the C-terminus tyrosine is mutated to phenylalanine (n-Src/Y535F), is significantly reduced by dysfunctions of the SH2 and/or SH3 domains of the protein. Furthermore, we found that dysfunctions of SH2 and/or SH3 domains reduce autophosphorylation of the kinase activation loop, depress kinase activity, and decrease NMDAR phosphorylation. The SH2 domain plays a greater regulatory role than the SH3 domain. Our data also show that n-Src binds directly to the C-terminus of the NMDAR NR2A subunit in vitro, with a K-D of 108.2 +/- 13.3 nM. This binding is not Src kinase activity-dependent, and dysfunctions of the SH2 and/or SH3 domains do not significantly affect the binding. These data indicate that the SH2 and SH3 domains may function to promote the catalytic activity of active n-Src, which is important in the regulation of NMDAR functions.

Src family kinases (SFKs) are key factors in the process of coupling signals from the cell surface to intracellular machinery and critically involved in the regulation of many neural functions mediated through growth factors, G-protein-coupled receptors or ligand-gated ion channels. The three minireviews here focus on recent findings dealing with the regulation of N-methyl-d-aspartate (NMDA) receptors by SFKs.

Previous studies have shown that Csk plays critical roles in the regulation of neural development, differentiation and glutamate-mediated synaptic plasticity. It has been found that Csk associates with the NR2A and 2B subunits of N-methyl-D-aspartate receptors (NMDARs) in a Src activity-dependent manner and serves as an intrinsic mechanism to provide a “brake” on the induction of long-term synaptic potentiation (LTP) mediated by NMDARs. In contrast to the NR2A and 2B subunits, no apparent tyrosine phosphorylation is found in the NR1 subunit of NMDARs. Here, we report that Csk can also associate with the NR1 subunit in a Src activity-dependent manner. The truncation of the NR1 subunit C-tail which contains only one tyrosine (Y837) significantly reduced the Csk association with the NR1-1a/NR2A receptor complex. Furthermore, we found that either the truncation of NR2A C-tail at aa 857 or the mutation of Y837 in the NR1-1a subunit to phenylalanine blocked the inhibition of NR1-1a/NR2A receptors induced by intracellular application of Csk. Thus, both the NR1 and NR2 subunits are required for the regulation of NMDAR activity by Csk.

Objective To study the activation of hypothalamic arcuate nucleus neurons in inflammatory hyperagesia and the role of NMDA receptor.Methods The inflammatory hyperalgesia model was induced by complete Freund's adjuvant in rats.Pain threshold was measured by radiant heat-withdrawal method.The neuronal activation in arcuate nucleus was evaluated by c-fos expression.The immunoreactive positive cells were counted using unbiased stereological method.The role of NMDA receptor was tested by using its antagonist MK-801.Results 7,14 and 21 days after model made the pain threshold decreased significantly(P0.05),at the same time the number of Fos-positive neurons in arcuate nucleus was up-regulated significantly(P0.05).Intracerebroventricular microinjection of NMDA receptor antagonist MK-801 could reverse the decrease of pain threshold,as well as the up-regulation of Fos-positive neurons in arcuate nucleus.No significant changes were observed in pain threshold as well as in number of Fos-positive cells in the saline control group.Conclusion Neuronal activation in arcuate nucleus plays an important role in the production and maintenance of inflammatory hyperalgesia.This activation is mediated by NMDA receptor.Central sensitization may be produced as well in the supraspinal higher pain-modulatory centers.

Objective To study the mechanism of central sensitization of hyperalgesia at supraspinal level,by observing the effects of microinjection of NMDA receptor antagonists and protein kinase inhibitors injected into arcuate nucleus on the hyperalgesia induced by partial sciatic nerve ligature.Methods The changes of mechanical pain threshold and thermal pain threshold were measured using mechanical withdrawal and thermal withdrawal methods,respectively,after microinjecting MK801(non-competitive NMDA receptor antagonist),APV(competitive NMDA receptor antagonist),PP2(Src protein tyrosine kinase inhibitor),GF109203X(protein kinase C inhibitor) into arcuate nucleus in the period of 70 minutes.Results The pain threshold of rats treated with PSL decreased significantly several hours after operation,mechanical and thermal hyperalgesia appeared at the same time.With microinjecting of MK801(5 nmol) and APV(1.5 nmol) into arcuate nucleus,the pain threshold increased significantly,mechanical and thermal hyperalgesia were inhibited;with microinjecting of PP2(5 nmol) and GF109203X(0.04 nmol),the pain threshold increased more significantly and hyperalgesia was also inhibited.Conclusion NMDA receptor,Src protein tyrosine kinase and protein kinase C in the hypothalamic arcuate nucleus are involved in the process of central sensitization of neuropathic hyperalgesia.

Neuronal Src (n-Src) is an alternative isoform of Src kinase containing a 6-amino acid insert in the SH3 domain that is highly expressed in neurons of the central nervous system (CNS). To investigate the function of n-Src, wild-type n-Src, constitutively active n-Src in which the C-tail tyrosine 535 was mutated to phenylalanine (n-Src/Y535F) and inactive n-Src in which the lysine 303 was mutated to arginine in addition to the mutation of Y535F (n-Src/K303R/Y535F), were expressed and purified from Escherichia coli BL21(DE3) cells. We found that all three types of n-Src constructs expressed at very high yields (∼500 mg/L) at 37°C, but formed inclusion bodies. In the presence of 8M urea these proteins could be solubilized, purified under denaturing conditions, and subsequently refolded in the presence of arginine (0.5M). These Src proteins were enzymatically active except for the n-Src/K303R/Y535F mutant. n-Src proteins expressed at 18°C were soluble, albeit at lower yields (∼10-20 mg/L). The lowest yields were for n-Src/Y535F (∼10 mg/L) and the highest for n-Src/K303R/Y535F (∼20 mg/L). We characterized the purified n-Src proteins expressed at 18°C. We found that altering n-Src enzyme activity either pharmacologically (e.g., application of ATP or a Src inhibitor) or genetically (mutation of Y535 or K303) was consistently associated with changes in n-Src stability: an increase in n-Src activity was coupled with a decrease in n-Src stability and vice versa. These findings, therefore, indicate that n-Src activity and stability are interdependent. Finally, the successful production of functionally active n-Src in this study indicates that the bacterial expression system may be a useful protein source in future investigations of n-Src regulation and function.