In vivo patch-clamp analysis of dopaminergic antinociceptive actions on substantia gelatinosa neurons in the spinal cord

To elucidate the mechanisms of antinociception mediated by the dopaminergic descending pathway in the spinal cord, we investigated the actions of dopamine (DA) on substantia gelatinosa (SG) neurons by in vivo whole-cell patch-clamp methods. In the voltage-clamp mode (V_H = −70 mV), the application of DA induced outward currents in about 70% of SG neurons tested. DA-induced outward current was observed in the presence of either Na⁺ channel blocker, tetrodotoxin (TTX) or a non-NMDA receptor antagonist, CNQX, and was inhibited by either GDP-β-S in the pipette solution or by perfusion of a non-selective K⁺ channel blocker, Ba²⁺. The DA-induced outward currents were mimicked by a selective D2-like receptor agonist, quinpirole and attenuated by a selective D2-like receptor antagonist, sulpiride, indicating that the DA-induced outward current is mediated by G-protein-activated K⁺ channels through D2-like receptors. DA significantly suppressed the frequency and amplitude of glutamatergic spontaneous excitatory postsynaptic currents (EPSCs). DA also significantly decreased the frequency of miniature EPSCs in the presence of TTX. These results suggest that DA has both presynaptic and postsynaptic inhibitory actions on synaptic transmission in SG neurons. We showed that DA produced direct inhibitory effects in SG neurons to both noxious and innocuous stimuli to the skin. Furthermore, electrical stimulation of dopaminergic diencephalic spinal neurons (A11), which project to the spinal cord, induced outward current and suppressed the frequency and amplitude of EPSCs. We conclude that the dopaminergic descending pathway has an antinociceptive effect via D2-like receptors on SG neurons in the spinal cord.     

In vivo pain-inhibitory role of nociceptin/orphanin FQ in spinal cord

Because nociceptin/orphanin FQ (N/OFQ) has both pronociceptive (hyperalgesia) and antinociceptive actions in pharmacological experiments, and there is no significant difference in the nociceptive responses between NOP(-/-) mice and their wild-type (NOP(+/+)) littermates, the physiological role of N/OFQ in pain regulation remains to be determined. Under the hypothesis that the use of molecularly distinct nociception test may reveal the pain modality-specific role of N/OFQ, we attempted to examine the physiological role of N/OFQ in pain transmission by using newly developed algogenic-induced nociceptive flexion test in NOP(-/-) and NOP(+/+) mice or NOP antagonist-treated mice. The nociceptive flexor responses upon intraplantar injection of bradykinin or substance P, which stimulates polymodal substance P-ergic fibers, were markedly potentiated in NOP(-/-) mice, compared with those in its NOP(+/+) mice. However, there were no significant changes in NOP(-/-) mice with adenosine triphosphate or prostaglandin I(2) agonist, which stimulates glutamatergic but not substance P-ergic fibers. The nocifensive responses induced by substance P (i.t.) were also potentiated in NOP(-/-) mice. On the other hand, there were no significant differences in NK1-like immunoreactivity, [(3)H]substance P binding, or NK1 gene expression in the dorsal horn of the spinal cord between NOP(-/-) and NOP(+/+) mice. In addition, NOP antagonists decreased the threshold in nociception tests driving spinal substance P neurotransmission. All these findings suggest that the N/OFQ-ergic neuron may play an in vivo inhibitory role on the second-order neurons for primary polymodal substance P-ergic fibers in the spinal cord.     

脊髓组织工程的体外构建

目的:观察神经干细胞在聚乳酸-羟基乙酸支架上的生长和分化行为,探讨组织工程体外构建人工脊髓修复脊髓损伤。方法:实验于2005-10/2006-06在南京医科大学第一附属医院中心实验室完成。①在聚乳酸-羟基乙酸支架(孔径200～300μm,孔隙率90%,聚乳酸/聚羟基乙酸=75∶25;山东医疗器械研究所)上种植孕14d的胚胎大鼠脑细胞,培养7d。②将培养7d的聚乳酸-羟基乙酸支架置入含体积分数为0.05的胎牛血清和含脑源性神经营养因子20μg/L的DMEM/F12培养基中继续培养5d,诱导其向神经元分化。③用倒置相差显微镜和扫描电镜观察细胞在聚乳酸-羟基乙酸支架上的生长和分化行为;应用免疫组织化学鉴定聚乳酸-羟基乙酸支架上培养的细胞。结果:①神经干细胞生长形态:神经干细胞克隆球充满聚乳酸-羟基乙酸支架孔隙,其形状近似孔隙的不规则几何形状。②神经干细胞超微结构:诱导分化后,大量神经干细胞长出的神经突触跨越支架孔隙的三维空间结构,布满支架的表面和孔隙,并彼此建立了突触联系。③诱导分化前后的聚乳酸-羟基乙酸石蜡切片免疫组织化学鉴定结果:诱导分化前后免疫组织化学鉴定分别为巢蛋白和微管相关蛋白2抗体阳性,说明诱导分化前是神经干细胞,诱导分化后有神经元形成。结论:聚乳酸-羟基乙酸适合神经干细胞的生长和分化,其孔隙结构规范调节了神经干细胞生长增殖的空间排列,而脑源性神经营养因子调控了神经干细胞的分化方向,可以利用该支架的空间结构和细胞因子适度调控神经干细胞在聚乳酸-羟基乙酸上的生长和分化构建脊髓组织。     

Peripheral inflammation induces tumor necrosis factor dependent AMPA receptor trafficking and Akt phosphorylation in spinal cord in addition to pain behavior

In the present study, intraplantar carrageenan induced increased mechanical allodynia, phosphorylation of PKB/Akt and GluR1 ser 845 (PKA site) as well as GluR1, but not GluR2 movement into neuronal membranes. This change in membrane GluR1/GluR2 ratio is indicative of Ca²⁺ permeable AMPA receptor insertion. Pain behavior was reduced and biochemical changes blocked by spinal pretreatment, but not post-treatment, with a tumor necrosis factor (TNF) antagonist, Etanercept (100 μg). Pain behavior was also reduced by spinal inhibition of phosphatidylinositol 3-kinase (PI-3K) (wortmannin; 1 and 5 μg) and LY294002; 50 and 100 μg) and Akt (Akt inhibitor IV; 3 μg). Phosphorylated Akt was found exclusively in neurons in grey matter and in oligodendrocytes in white matter. Interestingly, this increase was seen first in superficial dorsal horn and α-motor neurons (peak 45 min) and later (peak 2 h post-injection) in deep dorsal horn neurons. Akt and GluR1 phosphorylation, AMPA receptor trafficking and mechanical allodynia were all TNF dependent. Whether phosphorylation of Akt and of GluR1 are in series or in parallel or upstream of pain behavior remains to be determined. Certainly, TNF-mediated GluR1 trafficking appears to play a major role in inflammatory pain and TNF-mediated effects such as these could represent a path by which glia contribute to neuronal sensitization (spinal LTP) and pathological pain.     

In vivo imaging of inhibitory, GABAergic neurons by MRI

The unambiguous detection of specific neuronal subtypes is up to now only possible with invasive techniques or optical imaging after genetic modification. High field magnetic resonance imaging (MRI) has the ability to visualize the brain structure and anatomy noninvasively, with high resolution--but missing the cell specific and functional information. Here we present a new tool for neuroimaging with MRI, enabling the selective detection of GABAergic neurons under in vivo conditions. The specific imaging contrast is achieved by a novel paramagnetic contrast agent, which responds to the activity of the enzyme glutamic acid decarboxylase--expressed solely by inhibitory neurons. The relaxivity of the complex is increased upon decarboxylation of two glutamic acid moieties, thus allowing increased water access to the inner and outer coordination spheres of the paramagnetic ion. The mechanism and specificity of activation were proven with tissue lysates and further applied to a differentiation protocol for murine embryonic stem cells. The relaxation enhancement was studied quantitatively and revealed decreased longitudinal relaxation times in the inhibitory neuron samples compared to the na?ve stem cells in vitro and in vivo. Furthermore, this approach offers not only the discrimination of inhibitory, GABAergic neurons in the brain but also may expand the usefulness of MRI for functional imaging on a cellular level.     

Neurophysiological characterization of the anterolateral spinal cord neurons contributing to pain perception in man

These studies have examined threshold, frequency, and refractory period characteristics of a neural population in the anterolateral quadrant (ALQ) of the spinal cord of man, stimulation of which produces pain. Subjects were 18 conscious humans undergoing percutaneous anterolateral cordotomy for relief of intractable pain. Pain could be produced by ALQ stimulation in all subjects. Pain thresholds ranged from 120 to 1000 μA (at 50 pulses/sec; 0.2 msec pulses), but the majority of thresholds were below 300 μA. A linear relationship was found between stimulation frequency and percentage of subjects reporting pain. This relationship ranged from 5 to 25 pulses/sec with 100% reporting pain at 25/sec and 0% at 5/sec. In 2 of 3 subjects, increases in stimulation frequency up to 500/sec did not produce pain when stimulation intensity was below threshold at 50/sec. The neuronal refractory period for pain in these subjects ranged between 1.0 and 2.0 msec, but the majority of relative refractory periods fell between 1.0 and 1.5 msec. The threshold, frequency and refractory period data obtained in this study are similar to those found for wide dynamic range cells in the ventral half of the dorsal horn in the monkey and suggest that activation of these cells is a sufficient condition to produce pain in man.     

In vivo DTI of the healthy and injured cat spinal cord at high spatial and angular resolution

Spinal cord diffusion tensor imaging (DTI) is challenging in many ways: the small size of the cord, physiological motion and susceptibility artifacts pose daunting obstacles to the acquisition of high-quality data. Here, we present DTI results computed from in vivo studies of the healthy and injured spinal cord of five cats. Both high spatial (1.1 mm3) and angular (55 directions) resolutions were used to optimise modelling of the diffusion process. Also, particular effort was directed towards a strategy that limits susceptibility artifacts. For validation purposes, acquisitions were repeated in two cats before and after making a spinal lesion. As a result, various axonal trajectories were identified by tractography including dorsal and ventral columns as well as lateral tracts. Also, fibre bundles showed robust disruption at the site of spinal cord injuries (partial and complete) via tractography, accompanied with significantly lower fractional anisotropy values at the site of lesions. Important outcomes of this work are (i) tractography-based localisation of anatomical tracts in the thoraco-lumbar spinal cord and (ii) in vivo assessment of axonal integrity following experimental spinal cord injury.     

Epidural spinal cord stimulation for the treatment of painful legs and moving toes syndrome

A case of 51-year-old male with painful legs and moving toes syndrome was successfully treated with epidural spinal cord stimulation. He was previously treated with varieties of medication, epidural block, transcutaneous nerve stimulation, lumbar sympathetic block, with no or only a transient effect. Epidural spinal cord stimulation was applied by means of percutaneously inserted epidural electrodes connected to a percutaneous extension for 2-week test stimulation period and later to a permanent device (ITREL). Pain and involuntary movement were relieved almost completely during the stimulation and the effect was still evident 6 months later.     

Phosphorylation of spinal N‐methyl‐d‐aspartate receptor NR1 subunits by extracellular signal‐regulated kinase in dorsal horn neurons and microglia contributes to diabetes‐induced painful neuropathy

The N‐methyl‐d ‐aspartate receptor (NMDAR) contributes to central sensitization in the spinal cord, a phenomenon which comprises various pathophysiological mechanisms responsible for neuropathic pain‐like signs in animal models. NMDAR function is modulated by post‐translational modifications including phosphorylation, and this is proposed to underlie its involvement in the production of pain hypersensitivity. As in diabetic patients, streptozotocin‐induced diabetic rats exhibit or not somatic mechanical hyperalgesia; these rats were named DH and DNH respectively. At three weeks of diabetes, we present evidence that somatic mechanical hyperalgesia was correlated with an enhanced phosphorylation of the NMDAR NR1 subunit (pNR1) in the rat spinal cord. This increase was not found in normal and DNH rats, suggesting that this regulation was specific to hyperalgesia. Double immunofluorescence studies revealed that the numbers of pNR1‐immunoreactive neurons and microglial cells were significantly increased in all laminae (I—II and III—VI) of the dorsal horn from DH animals. Western‐blots analysis showed no change in NR1 protein levels, whatever the behavioural and glycemic status of the animals. Chronic intrathecal treatment (5μg/rat/day for 7 days) by U0126 and MK801, which blocked MEK (an upstream kinase of extracellular signal‐regulated protein kinase: ERK) and the NMDAR respectively, simultaneously suppressed somatic mechanical hyperalgesia developed by diabetic rats and decreased pNR1. These results indicate for the first time that increased expression of pNR1 is regulated by ERK and the NMDAR via a feedforward mechanism in spinal neurons and microglia and represents one mechanism involved in central sensitization and somatic mechanical hyperalgesia after diabetes.     

Ethanol directly depresses AMPA and NMDA glutamate currents in spinal cord motor neurons independent of actions on GABAA or glycine receptors.

Ethanol is a general anesthetic agent as defined by abolition of movement in response to noxious stimulation. This anesthetic endpoint is due to spinal anesthetic actions. This study was designed to test the hypothesis that ethanol acts directly on motor neurons to inhibit excitatory synaptic transmission at glutamate receptors. Whole cell recordings were made in visually identified motor neurons in spinal cord slices from 14- to 23-day-old rats. Currents were evoked by stimulating a dorsal root fragment or by brief pulses of glutamate. Ethanol at general anesthetic concentrations (50-200 mM) depressed both responses. Ethanol also depressed glutamate-evoked responses in the presence of tetrodotoxin (300 nM), showing that its actions are postsynaptic. Block of inhibitory gamma-aminobutyric acidA and glycine receptors by bicuculline (50 microM) and strychnine (5 microM), respectively, did not significantly reduce the effects of ethanol on glutamate currents. Ethanol also depressed glutamate-evoked currents when the inhibitory receptors were blocked and either D, L-2-amino-5-phosphonopentanoic acid (40 microM) or 6-cyano-7-nitroquinoxaline-2,3-dione disodium (10 microM) were applied to block N-methyl-D-aspartate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors, respectively. The results show that ethanol exerts direct depressant effects on both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate glutamate currents in motor neurons. Enhancement of gamma-aminobutyric acidA and glycine inhibition is not required for this effect. Direct depression of glutamatergic excitatory transmission by a postsynaptic action on motor neurons thus may contribute to general anesthesia as defined by immobility in response to a noxious stimulus.     

Neuronal inhibitory effects of methadone are predominantly opioid receptor mediated in the rat spinal cord in vivo.

This study aims to assess whether the antinociceptive actions of methadone are mediated solely through opioid mechanisms, or whether its reported affinity for NMDA receptors has physiological relevance in vivo. Methadone is a mu-opioid receptor agonist reported to relieve pain unresponsive to other opioids. It is a racemic mixture comprising d- and l-optical isomers; the d-isomer has a lower affinity for opioid receptors, and both also exhibit NMDA receptor binding, likely to indicate antagonist activity. d -Methadone is antinociceptive in behavioural studies via non-opioid mechanisms, which could include functional NMDA receptor-blocking activity. Here we investigate the ability of d - and dl -methadone to inhibit noxious and innocuous electrically-evoked responses of dorsal horn neurones in the anaesthetized rat.Racemic methadone (5, 25, 50, 250 microg) applied spinally, dose-relatedly inhibited the C-fibre evoked response, input and wind-up of the neurones, with a profile resembling that of morphine. d-Methadone (5, 25, 50, 250, 500 microg) was also inhibitory, although less potent by a factor of between 13 and 48 depending on the neuronal measure; its profile of inhibition resembled that of the racemic mixture rather than an NMDA receptor antagonist. Both compounds had minimal effects on Abeta-fibre-evoked activity. The inhibitory effects of both d - and dl -methadone on noxious-evoked activity were naloxone reversible. The naloxone reversibility of d -methadone inhibitions is best interpreted as indicative of a purely opioid mechanism of action. However, the ability of naloxone to reverse the effects of d -methadone may also reflect a degree of synergy between weak NMDA antagonist and opioid agonist activity.     

Activation of lamina I spinal cord neurons that express the substance P receptor in visceral nociception and hyperalgesia.

Spinal lamina I neurons expressing the substance P receptor (SPR) have been shown to play a role in the transmission of somatic inflammatory and neuropathic pain. To evaluate their involvement in visceral nociception in both the noninflamed and inflamed colon, we examined the expression and ligand-induced internalization of the SPR in the rat spinal cord after distention of the noninflamed colon and in rats with inflammation induced by intracolonic instillation of zymosan (3 hours). In the noninflamed animal, acute noxious but not non-noxious colorectal distention induced SPR internalization in lamina I neurons at the thoracolumbar (T13) and lumbosacral (S1) spinal levels, whereas SPR internalization was not detected in lamina I neurons at spinal lumbar segment L4. Although zymosan-induced colorectal inflammation alone did not induce SPR internalization in lamina I neurons, there was an increased number of SPR-expressing lamina I neurons showing SPR internalization in segments T12 through S2 of the spinal cord after colorectal distention. These results show that acute noxious visceral stimuli induce activation of spinal lamina I neurons expressing the SPR and, that after visceral inflammation, there is a marked increase in both the number and rostrocaudal extent of lamina I SPR neurons activated in response to both normally non-noxious and noxious distention of the colon.     

In vivo regulation of plasminogen function by plasma carboxypeptidase B

The major functions of plasminogen (Plg) in fibrinolysis and cell migration depend on its binding to carboxy-terminal lysyl residues. The ability of plasma carboxypeptidase B (pCPB) to remove these residues suggests that it may act as a suppressor of these Plg functions. To evaluate this role of pCPB in vivo, homozygote pCPB-deficient mice were generated by homologous recombination, and the resulting pCPB(-/-) mice, which were viable and healthy, were mated to Plg(-/-) mice. Plg(+/-) mice show intermediate levels of fibrinolysis and cell migration compared with Plg wild-type and deficient mice, reflecting the intermediate levels of the Plg antigen in their plasma. Differences in Plg-dependent functions between pCPB(+/+), pCPB(+/-), and pCPB(-/-) mice were then analyzed in a Plg(+/-) background. In a pulmonary clot lysis model, fibrinolysis was significantly increased in mice with partial (pCPB(+/-)) or total absence (pCPB(-/-)) of pCPB compared with their wild-type counterparts (pCPB(+/+)). In a thioglycollate model of peritoneal inflammation, leukocyte migration at 72 hours increased significantly in Plg(+/-)/pCPB(+/-) and Plg(+/-)/pCPB(-/-) compared with their wild-type counterparts. These studies demonstrate a definitive role of pCPB as a modulator of the pivotal functions of Plg in fibrinolysis and cell migration in vivo.     

脊髓损伤大鼠远端运动神经元继发性损害的变化特点

目的探讨脊髓损伤大鼠远端运动神经元继发性损害的变化特点。 方法成年雌性SD大鼠40只,采用改良Allen’s撞击法制作T10不完全性脊髓损伤模型。分别在损伤前、损伤后第1周、2周、3周、4周、5周时采用斜板试验、改良Tarlov评分、BBB评分进行运动功能评定;在各时间点取5只大鼠用4%多聚甲醛心脏灌注,取损伤局部T10节段及损伤远端L5节段脊髓进行形态学检测。损伤前时间点检测结果作为正常对照。 结果①损伤远端运动神经元病理变化：损伤后第1周时,损伤远端前角运动神经元突起和尼氏体有所减少,胞核和核仁无明显改变;损伤后第2周时,损伤远端前角运动神经元突起和尼氏体进一步减少,核仁模糊、偏位;损伤后第3周时,损伤远端前角运动神经元突起和尼氏体较前无进一步减少,组织内可见萎缩及凋亡神经元;损伤后第4周时,损伤远端前角运动神经元突起和尼氏体较第3周时有所增加,仍可见少数凋亡神经元存留;损伤后第5周时,损伤远端前角运动神经元突起和尼氏体较前明显增多。②损伤局部病理变化：损伤后第1周时,损伤局部灰、白质界限不清,可见出血、炎症反应,灰质部分神经纤维及组织坏死、液化,白质空泡化;损伤后第2周时,损伤局部出血、血肿及炎症反应减退,可见胶质细胞、神经纤维增生和血管新生;损伤后第3周时,损伤局部胶质细胞增生显著,神经纤维增生较多;损伤后第4周、第5周时,损伤局部胶质细胞、神经纤维较前无明显增生。 ③运动功能变化：损伤后第1周、2周、3周、4周、5周时的斜板角度、改良Tarlov评分、BBB评分均较损伤前显著降低（P<0.05）;损伤后第2周、3周、4周、5周时各项评分均较第1周时显著增加（P<0.05）;损伤后第3周、4周、5周时呈增加趋势,但与第2周时比较差异无统计学意义（P&rt;0.05）。 结论脊髓损伤后早期远端脊髓运动神经元即会发生继发性损害,包括突起和尼氏体减少、细胞萎缩与凋亡,并且与损伤局部病理变化及损伤时程相关。     

Modified cold pressor test by cold application to the foot after spinal cord injury: suggestion of hemodynamic control by the spinal cord

OBJECTIVE: Study hemodynamic responses to cold application to the foot (CAF) to explore the autonomic cardiovascular control by the spinal cord. DESIGN: Controlled experimental study. Hemodynamic variables were measured or calculated for 13 healthy subjects, 10 patients with traumatic T4-T6 paraplegia, and 11 patients with traumatic C4-C7 tetraplegia. Subjects were continuously monitored for heart rate (HR), blood pressure (BP), and cerebral blood-flow velocity (CBFV) from 5 mins before to 5 mins after 40-120 secs of CAF by ice water foot immersion. The recorded signals were digitized online and analyzed offline in the time and frequency domains. RESULTS: During CAF, HR increased in the control group but decreased in patients (P < 0.001). BP increased significantly in the control and tetraplegia groups (P < 0.001) and nonsignificantly in the paraplegia group. HR and BP spectral components (LF, HF, LF/HF) did not change significantly. CBFV increased significantly in the patient groups (P < 0.05) but not in the control subjects. The cerebrovascular resistance increased significantly in the control and tetraplegia groups (P < 0.001), but not in the paraplegia group. CONCLUSIONS: The findings support the presence of hemodynamic autonomic control by the spinal cord and show that responses to CAF can be used to assess its integrity.     

胚胎脊髓对损伤脊髓的修复作用研究

目的探讨不同条件下胚胎脊髓移值修复成鼠急性脊髓损伤(SCI)的能力。方法成鼠胸髓损伤后分别移植孕14d胚胎脊髓(FSC组)、游离正中神经加胚胎脊髓(P＋F组)、带血管蒂正中神经加胚胎脊髓(V＋F组),术后8周行组织学检查。结果V＋F组胚胎脊髓与受体融合佳,体积增长速度、神经纤维和神经元数目显著高于P＋F、FSC组（P<0.01）,细胞分化比较好,突触较成熟,界面区也无明显的胶质增生。结论带血管蒂周围胚胎脊髓联合移植,对FSC的生长发育、对损伤神经元的再生能力均有一定的促进作用。     

Oxidative stress generated by hemorrhagic shock recruits Toll-like receptor 4 to the plasma membrane in macrophages

Oxidative stress generated by ischemia/reperfusion is known to prime inflammatory cells for increased responsiveness to subsequent stimuli, such as lipopolysaccharide (LPS). The mechanism(s) underlying this effect remains poorly elucidated. These studies show that alveolar macrophages recovered from rodents subjected to hemorrhagic shock/resuscitation expressed increased surface levels of Toll-like receptor 4 (TLR4), an effect inhibited by adding the antioxidant N-acetylcysteine to the resuscitation fluid. Consistent with a role for oxidative stress in this effect, in vitro H2O2 treatment of RAW 264.7 macrophages similarly caused an increase in surface TLR4. The H2O2-induced increase in surface TLR4 was prevented by depleting intracellular calcium or disrupting the cytoskeleton, suggesting the involvement of receptor exocytosis. Further, fluorescent resonance energy transfer between TLR4 and the raft marker GM1 as well as biochemical analysis of the raft components demonstrated that oxidative stress redistributes TLR4 to lipid rafts in the plasma membrane. Preventing the oxidant-induced movement of TLR4 to lipid rafts using methyl-beta-cyclodextrin precluded the increased responsiveness of cells to LPS after H2O2 treatment. Collectively, these studies suggest a novel mechanism whereby oxidative stress might prime the responsiveness of cells of the innate immune system.     

In vivo imaging reveals PKA regulation of ERK activity during neutrophil recruitment to inflamed intestines

Many chemical mediators regulate neutrophil recruitment to inflammatory sites. Although the actions of each chemical mediator have been demonstrated with neutrophils in vitro, how such chemical mediators act cooperatively or counteractively in vivo remains largely unknown. Here, by in vivo two-photon excitation microscopy with transgenic mice expressing biosensors based on Förster resonance energy transfer, we time-lapse–imaged the activities of extracellular signal–regulated kinase (ERK) and protein kinase A (PKA) in neutrophils in inflamed intestinal tissue. ERK activity in neutrophils rapidly increased during spreading on the endothelial cells and showed positive correlation with the migration velocity on endothelial cells or in interstitial tissue. Meanwhile, in the neutrophils migrating in the interstitial tissue, high PKA activity correlated negatively with migration velocity. In contradiction to previous in vitro studies that showed ERK activation by prostaglandin E₂ (PGE₂) engagement with prostaglandin receptor EP4, intravenous administration of EP4 agonist activated PKA, inhibited ERK, and suppressed migration of neutrophils. The opposite results were obtained using nonsteroidal antiinflammatory drugs (NSAIDs). Therefore, NSAID-induced enteritis may be caused at least partially by the inhibition of EP4 receptor signaling of neutrophils. Our results demonstrate that ERK positively regulates the neutrophil recruitment cascade by promoting adhesion and migration steps.Once inflammation occurs in tissues, in response to the gradient of chemoattractants such as leukotriene B₄ (LTB₄), IL-8, and formyl-methionyl-leucyl-phenylalanine (fMLP), neutrophils begin to emigrate from inside the venules to the inflammatory sites (Phillipson and Kubes, 2011). Using various in vivo microscopy approaches, it has been demonstrated that the neutrophil recruitment involves four steps: rolling, adhesion, crawling, and transmigration (Borregaard, 2010; Megens et al., 2011; Germain et al., 2012; Sanz and Kubes, 2012; Kolaczkowska and Kubes, 2013). Thereafter, neutrophils that have emigrated into the interstitial tissue migrate toward the inflammatory sites by the gradient of chemoattractants. Most chemoattractant receptors expressed on neutrophils are coupled with the heterotrimeric G_i protein, which inhibits protein kinase A (PKA) and activates p42/44 extracellular signal-regulated kinase (ERK) through both the α and βγ subunits of G_i (Alblas et al., 1993; Howe and Marshall, 1993; Winitz et al., 1993). The G_i-mediated ERK activation is required for adhesion and migration of neutrophils upon the engagement of the chemoattractants with the cognate receptors (Pillinger et al., 1996; Zarbock et al., 2007). However, this model was recently challenged by Liu et al. (2012), who proposed that fMLP-stimulated neutrophil migration is regulated negatively by ERK.Prostaglandins at the inflammatory sites play pleiotropic roles in inflammation (Hata and Breyer, 2004; Narumiya, 2009). For example, prostaglandin E₂ (PGE₂), which is a major cyclooxygenase product in several physiological settings, regulates multiple functions of different immune cells (Ricciotti and FitzGerald, 2011; Kalinski, 2012). The main signal transduction of the four PGE₂-sensitive (EP) receptors, EP1 to EP4, consists of a rise in intracellular cAMP concentration and subsequent PKA activation via G_s in EP2 and EP4, a rise in intracellular free calcium ion concentration in EP1, and a decrease in intracellular cAMP concentration and ERK activation via G_i in EP3 (Narumiya et al., 1999). Further complexity arises from the strength of the coupling to G_s and sensitivity to the metabolic inactivation: Although both EP2 and EP4 receptors couple to G_s, the EP2 receptor transduces signals primarily through PKA, whereas the EP4 receptor primarily utilizes phosphatidylinositol 3-kinase (PI3K) and ERK (Fujino et al., 2003). EP4 signaling is rapidly desensitized after its PGE₂ interaction, whereas EP2 is resistant to ligand-induced desensitization (Nishigaki et al., 1996). Reflecting these differences in molecular properties, EP2 and EP4 are regarded as pro- and antiinflammatory receptors, respectively (Kabashima et al., 2002; Hata and Breyer, 2004).Upon activation of G_s-coupled receptors in many cell types, PKA suppresses ERK mitogen-activated protein kinase (MAPK) via phosphorylation and inhibition of c-Raf, a MAPK kinase (Häfner et al., 1994; Pillinger et al., 1996). In neutrophils, for example, PKA has been shown to suppress respiratory burst by inhibition of the ERK signaling (Bengis-Garber and Gruener, 1996). However, in neuronal cells, an increase in cytoplasmic cAMP can activate ERK in a Rap1-dependent manner (Vossler et al., 1997). Therefore, the regulation of ERK activity by G_s-coupled receptors is dependent on the cell context.In the inflammatory tissues, neutrophils perceive several extracellular signals, which activate or inactivate ERK and PKA. Under this circumstance, it is hardly predictable which signaling pathway will be dominant in neutrophils during the course of inflammation. Although the recent advent of in vivo microscopy has enabled us to visualize the neutrophil recruitment to inflammatory sites (Megens et al., 2011; Germain et al., 2012; Sanz and Kubes, 2012), the activity change of signaling molecules has not been examined because of technical constraints. To overcome this problem, we generated transgenic mice expressing functional Förster resonance energy transfer (FRET) biosensors for ERK and showed that ERK activity correlated with migration velocity in the neutrophils of inflamed subcutaneous tissue (Kamioka et al., 2012). However, we failed to observe extravasation of neutrophils or activity change of PKA because of technical difficulty. Here, in vivo observation of the intestines of the transgenic mice enabled us to examine the role of ERK and PKA in the neutrophil recruitment to the inflammatory sites by two-photon excitation microscopy (2PM). In contradiction to a previous study that showed activation of ERK by EP4 engagement to PGE₂ (Fujino et al., 2003), we show that PGE₂ stimulates PKA in an EP4-dependent manner and suppresses neutrophil migration via down-regulation of ERK in the inflamed intestine.     

Adenosine inhibits excitatory transmission to substantia gelatinosa neurons of the adult rat spinal cord through the activation of presynaptic A(1) adenosine receptor.

Although intrathecal administration of adenosine analogues or A(1) adenosine receptor agonists is known to result in antinociception, this has not been examined yet at the cellular level. In the present study, we examined in pharmacology an action of adenosine on glutamatergic miniature excitatory postsynaptic currents (mEPSCs) in substantia gelatinosa (SG) neurons of an adult rat spinal cord slice; this was done under the condition where a postsynaptic action of adenosine was blocked. In 65% of the neurons examined (n=72), adenosine at a concentration of 100 microM depressed the frequency of mEPSC in a reversible manner; the remaining neurons exhibited an inhibition followed by potentiation of the frequency. When examined quantitatively in extent in some cells (n=25), the inhibition was 40+/-3% (n=25) while the potentiation was 42+/-8% (n=6). These actions were not accompanied by a change in mEPSC amplitude. The inhibitory action on mEPSC frequency was dose-dependent in a range of 10-500 microM with an EC(50) value of 277 microM. The inhibitory action of adenosine was mimicked by a selective A(1) adenosine receptor agonist, CPA (1 microM; depression: 54+/-9%, n=4); this action of adenosine (100 microM) was not observed in the presence of a specific A(1) adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (1 microM; 94+/-4% of control, n=3). The facilitatory action of adenosine (100 microM) was unaffected by an A(2a) antagonist, ZM 241385 (0.1 microM, n=3); an A(2a) agonist, CGS 21680 (0.1-10 microM; n=6), was without actions on mEPSC frequency. It is concluded that adenosine inhibits excitatory transmission to SG neurons through the activation of presynaptic A(1) adenosine receptor and that some of the inhibition is followed by a potentiation of the transmission. It remains to be examined which subtypes of adenosine receptors except for the A(1)- and A(2a)-subtypes are involved in the potentiating action. Considering that adenosine-like immunoreactivity and adenosine receptors are expressed at a high density in the SG, which is thought to play an important role in modulating nociceptive transmission from the periphery to the central nervous system, this inhibitory action of adenosine could contribute to a negative modulation of pain transmission.