Effects of SFKs in microglia onATP-induced long-term potentiation in spinal dorsal horn

GONG Qing-juan, CHEN Jin-sheng, HUANG Qiao-dong, LU Zhen-he

(Department of Pain Medicine, The Second Affiliated Hospital, Guangzhou Medical College, Guangzhou 510260, China. E-mail: luzhh@126.com)

EffectsofSFKsinmicrogliaonATP-inducedlong-termpotentiationinspinaldorsalhorn

GONG Qing-juan, CHEN Jin-sheng, HUANG Qiao-dong, LU Zhen-he△

(DepartmentofPainMedicine,TheSecondAffiliatedHospital,GuangzhouMedicalCollege,Guangzhou510260,China.E-mail:luzhh@126.com)

AIM: To investigate the effects of Src family kinases (SFKs) on adenosine 5'-triphosphate (ATP)-induced long-term potentiation (LTP) in the spinal dorsal horn.METHODSMale Sprague-Dawley rats (250-280 g) were used in the experiments. Western blotting, electrophysiological recording in spinal dorsal horninvivoand immunohistochemistry were used in the study. The C-fiber-evoked field potentials were recorded at the superficial layers of spinal dorsal horn at the lumbar enlargement and the phosphorylation level and location of SFKs in spinal dorsal horn were examined by Western blotting and immunohistochemistry.RESULTSThirty min and 60 min after ATP application, the levels of phosphorylated SFKs (p-SFKs) were significantly increased.The p-SFKs were expressed in microglia, but not in astrocytes or neurons. Spinal application of SFK inhibitors prevented ATP-induced LTP.CONCLUSIONMicroglial SFKs may play an important role in ATP-induced LTP of C-fiber evoked field potentials in the spinal dorsal horn.

Src-family kinases; Long-term potentiation; Adenosine triphosphate; Spinal dorsal horn; Microglia

Src family kinases (SFKs) belong to the family of non-receptor protein tyrosine kinases and are expressed in the central nerve system (CNS)[1]. In the developed CNS,some data indicate that SFKs act as a point of convergence for various signaling pathways and might be involved in the processes underlying physiological and pathological plasticity, such as learning and memory, epilepsy and neurodegeneration[1]. Recently, it has been reported that SFKs are selectively activated in spinal microglia after peripheral nerve injury, and intrathecal administration of PP2, an inhibitor of all SFK members, suppresses mechanical hypersensitivity in nerve-injured rats[2], suggesting that microglial SFKs are crucial for neuropathic pain. Long-term potentiation (LTP), a phenomenon of persistent increase in synaptic strength produced by repetitive presynaptic stimulation, was first described in hippocampus and is considered as a fundamental mechanism of memory storage[3]. Besides the hippocampus, LTP has also been found in the spinal dorsal horn[4]. Spinal LTP of C-fiber evoked field potentials can be induced by tetanic stimulation of sciatic nerve, natural noxious stimulation on peripheral tissues or nerve injury. Accordingly, it is believed to underlie pathological pain[5], manifested as allodynia, hyperalgesia and spontaneous pain. Our previous works have shown that spinal application of ATP induced LTP[6]. In the present study, the effects of SFKs on ATP-induced LTP in spinal dorsal horn were investigated by the techniques of Western blotting, immunohistochemistry and electrophysiology.

MATERIALS AND METHODS

1Animalpreparation

Experiments were performed on male Sprague-Dawley rats (250-280 g). Urethane (1.5 g/kg, ip) was used to induce and maintain anesthesia. The trachea was cannulated, and the animals breathed spontaneously. One carotid artery was cannulated to continuously monitor the mean arterial blood pressure, which was maintained from 80 to 120 mmHg. A laminectomy was performed to expose the lumbar enlargement of spinal cord. The left sciatic nerve was dissected free for bipolar electrical stimulation with platinum hook electrodes. All exposed nervous tissues were covered with warm paraffin oil except the spinal lumbar enlargement, onto which the drugs will be applied. Colorectal temperature was kept constant (37-38 ℃) by means of a feedback-controlled heating blanket. At the end of the experiments, the animals were killed with an overdose of urethane.

2Drugpreparation

ATP (Sigma) was dissolved in double-distilled water and diluted with artificial cerebrospinal fluid (ACSF) to make the final concentration of 3×10-4mol/L just prior to use. 4-Amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine(PP2, Calbiochem), 2-oxo-3-(4,5,6,7-tetrahydro-1H-indol-2-ylmethylene)-2, 3-dihydro-1H- indole-5-sulfonic acid dimenthylamide(SU6656, Calbiochem) and 4-amino-7-phenylpyrazolo [3,4-d] pyrimidine(PP3, Calbiochem) were first dissolved in dimethyl sulphoxide (DMSO) to make a stock concentration of 50 mmol/L, aliquoted in small volumes and stored at -80 ℃. The stock solution was subsequently diluted with 0.9% saline to make final concentrations immediately before administration. The final concentration of DMSO was <0.5%. Previous works have shown that spinal application of 0.5% DMSO does not affect C-fiber evoked field potentials[7].

3Methods

3.1Western blotting The dorsal quadrants of L4-L5 spinal cord on the stimulated side were isolated and put into liquid nitrogen immediately, followed by homogenization in 1.5×10-2mol/L Tris buffer, pH 7.6. The tissues were sonicated on ice, and then centrifuged at 13 000×gfor 15 min at 4 ℃ to prepare the supernatant containing protein samples. Proteins were separated by gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane (Bio-Rad). The blots were blocked with 5% W/V non-fat dry milk in TBST for 1 h at room temperature and then incubated with primary antibodies, including rabbit polyclonal anti-rat p-SFKs (Tyr416) antibody (1∶1 000, Cell Signaling Technology) and rabbit polyclonal anti-rat β-actin antibody (1∶1 000, Cell Signaling Technology), overnight at 4 ℃ with gentle shaking. The blots were washed 3 times for 15 min each with washing buffer (TBST) and then incubated with secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1∶1 000, Santa Cruz) for 2 h at room temperature. After incubated with the secondary antibody, the membrane was washed, again, as above. The immune complex was detected by ECL (Amersham) and exposed to X-ray film (Kodak). The band intensities on the film were analyzed by densitometry with a computer-assisted imaging analysis system (Kontron IBAS 2.0). To calculate the phosphorylated versus non-phosphorylated form of protein, the same membrane was stripped with stripping buffer (6.75×10-2mol/L Tris, pH 6.8, 2% SDS, and 0.7% β-mercaptoethanol) for 30 min at 50 ℃ and reprobed with rabbit polyclonal anti-rat SFKs antibody (1∶1 000, Cell Signaling Technology), and detected as above.

3.2Electrophysiological recording Recording of C-fiber-evoked field potentials in spinal dorsal horn was performed according to our previous described method[4, 6]. Briefly, following electrical stimulation of the sciatic nerve, field potentials were recorded in the spinal dorsal horn (L4 and L5 segments) at the depth of 50-500 μm from the dorsal surface with a tungsten microelectrode (World Precision Instruments; impedance 1-2 MΩ, exposed tip diameter∶1-2 μm). An A/D converter card (DT2821-F-16SE, Data Translation) was used to digitize and store data in a computer at a sampling rate of 10 kHz. Single square pulses (0.5 ms duration, in 1 min intervals) delivered to the sciatic nerve were used as test stimuli. The strength of stimulation was adjusted to 1.5-2 times the threshold for C-fiber response. Tetanic stimulation (0.5 ms duration, 40 V, 100 Hz, given in 4 trains of 1 s duration at 10 s intervals) was used to induce LTP[4, 8, 9]. The distance from the stimulation site at the sciatic nerve to the recording site in lumbar spinal dorsal horn was around 11 cm.

3.3Immunohistochemistry Following electrophysiological recordings, the rats were perfused with 300 mL saline followed by 350 mL of cold 0.1 mol/L phosphate-buffer saline (PBS) containing 4% paraformaldehyde. The L4-L5 of spinal cord were removed, post-fixed for 3 h, and then replaced with 30% sucrose for 2 d at 4 ℃. Transverse spinal sections (free-floating, 25 μm) were cut in a cryostat (Leica CM3050S) and processed for immunofluorescence, according to the methods as described previously[6]. Briefly, the sections were blocked with 3% donkey serum in 0.3% Triton for 1 h at room temperature and incubated over night at 4 ℃ with anti-p-SFKs antibody (1∶500). The sections were then incubated for 1 h at room temperature with Cy3-conjugated secondary antibody (1∶300, Jackson immunolab). For double immunofluorescence, spinal sections were incubated with a mixture of anti-p-SFKs receptors antibody and monoclonal neuronal-specific nuclear protein (NeuN, neuronal marker, 1∶500, Chemicon), glial fibrillary acidic protein (GFAP, astrocyte marker, 1∶500, Chemicon), Iba-1 (microglia marker, 1∶500, Serotec) over night at 4 ℃, followed by a mixture of FITC- (1∶200; Jackson ImmunoResearch) and CY3-conjugated secondary antibodies for 1 h at room temperature. The stained sections were examined with an Olympus IX71 fluorescence microscope (Olympus Optical) and the images were captured with a CCD spot camera. The area of p-SFKs-immunoreactive (IR) per section was measured in the spinal dorsal horn using a computerized image analysis system (NIH ImageJ). A density threshold was set above background level firstly to identify positively stained structure. In each rat, 4 to 6 sections of the spinal cord at each time point were selected randomly. An average percentage of area of p-SFKs-IR, relative to the total area of the spinal dorsal horn of the sections, was obtained for each animal across the different tissue sections. Six rats were included in each group for quantification of the immunohistochemical results[10, 11]. The measurement of the area of p-SFKs-IR was performed by a person who did not know the experiment design.

4Statisticalanalysis

RESULTS

1SpinalapplicationofATPactivatedSFKsinmicroglia

Evidence has shown that activation of SFKs in spinal dorsal horn plays an important role in the development of neuropathic pain[2]. We therefore examined whether SFKs are activated after LTP induction by ATP. The results showed that the levels of p-SFKs in spinal dorsal horn were significantly increased 30 min and 60 min after ATP application, compared with the ACSF group (Figure 1, Figure 2A-D). To determine the cell types that expressed the activated SFKs, we performed double immunofluorescence staining 30 min after spinal application of ATP and found that p-SFKs was co-localized exclusively with Iba-1, but not with GFAP or NeuN (Figure 3A-C), indicating that ATP may activate SFKs only in microglia but not in astrocyte or neuron.

Figure3.p-SFKsexpressedinmicrogliaofspinaldorsalhornafterLTPinductionbyATP.Theresultsofdoubleimmunofluorescencestainingofp-SFKs(red,A,B,C)withIba-1,amicrogliamarker(green,A),NeuN,aneuronalmarker(green,B),andGFAP,anastrocytemarker(green,C)showedthatp-SFKsexclusivelyco-localizedwithIba-1(yellowinA).Scalebar:200μm.

2InhibitionofSFKspreventedATP-inducedspinalLTP

To evaluate the role of activation of SFKs in spinal LTP induction, PP2, a specific inhibitor of SFKs was applied onto spinal dorsal surface at recording segments 30 min before ATP. We found that ATP (3×10-4mol/L) induced spinal LTP of C-fiber-evoked field potentials (Figure 4C) and PP2 (1×10-4mol/L) completely blocked LTP induced by ATP (Figure 4A), whereas PP3 (1×10-4mol/L), an inactive form of

PP2, had no influence on LTP induction (Figure 4B). To confirm the role of SFKs in ATP-induced spinal LTP, another SFKs inhibitor SU6656 was tested with the same protocol. We found that in the rats pretreated with SU6656 (2×10-4mol/L), ATP did not induce spinal LTP (Figure 4A). PP2 and SU6656 did not affect the baseline of C-fiber response[12]. The results indicate that the activation of SFKs contributes to ATP-induced spinal LTP.

Figure4.TheeffectofSFKsinATP-inducedspinalLTP.A:spinalapplicationofATPdidnotinduceLTPofC-fiber-evokedfieldpotentialsintheratspretreatedwithSFKsinhibitor(PP2orSU6656).B:spinalapplicationofPP3hadnoeffectonATP-inducedLTP.C:spinalapplicationofATPinducedLTP.

DISCUSSION

In this study, we found that spinal application of ATP significantly increased the level of p-SFKs, and p-SFKs were only localized in spinal microglia. Spinal application of SFKs inhibitors (PP2 and SU6656) prevented ATP-induced LTP. These results indicate that SFKs may play a key role in ATP-induced LTP of C-fiber evoked field potentials in the spinal dorsal horn.

Src, Lyn, Fyn, Lck and Yes are 5 members of the SFK family and express in the CNS. As non-receptor protein tyrosine kinases, SFKs are involved in neuronal development and synaptic plasticity[1], and play a key role in regulating the functions of glial cells, including microglia, a type of immune cell that resides in the CNS. Accumulating evidence has shown that the activation of glial cells is crucial for the pathogenesis of pathological pain produced by nerve injury[13, 14]. The activated microglia may contribute to pain hypersensitivity by releasing the pro-inflammatory cytokines such as TNF-α and IL-1β. Furthermore, the activation of glial cells is necessary for the spinal LTP inductioninvivo[15]andinvitro[16]. Recent data indicate that the level of p-SFKs in spinal microglia is increased after LTP induced by high frequent stimulation[12]. Similar results were also found in our study. It has been demonstrated that SFKs are activated in spinal microglia[2]and can induce upregulation of P2X4receptors after peripheral nerve injury[17]. Our previous study has shown that ATP bound to intrinsic P2X4receptors and enhanced the expression of P2X4receptors, contributing to LTP in the spinal dorsal horn[6]. Therefore, it is possible that SFKs may play an important role in ATP-induced spinal LTP by upregulating the expression of P2X4receptors, and further studies are needed to confirm how the activation of microglial SFKs regulates synaptic plasticity in the spinal dorsal horn.

ACKNOWLEDGEMENTS

The authors are very grateful to Professor LIU Xian-guo (Department of Physiology, Zhongshan School of Medicine, Sun Yat-sen University) for his support and help in our study.

[1] Salter MW, Kalia LV. Src kinases: A hub for NMDA receptor regulation[J]. Nat Rev Neurosci, 2004, 5(4):317-328.

[2] Katsura H, Obata K, Mizushima T, et al. Activation of Src-family kinases in spinal microglia contributes to mechanical hypersensitivity after nerve injury[J]. J Neurosci, 2006, 26(34):8680-8690.

[3] Bliss TV, Collingridge GL. A synaptic model of memory: long term potentiation in the hippocampus[J]. Nature, 1993, 361(6407):31-39.

[4] Liu XG, Sandkühler J. Long-term potentiation of C-fiber-evoked potentials in the rat spinal dorsal horn is prevented by spinalN-methyl-D-aspartic acid receptor blockage[J]. Neurosci Lett, 1995, 191(1-2):43-46.

[5] Sandkühler J. Understanding LTP in pain pathways[J]. Mol Pain, 2007, 3:9.

[6] Gong QJ, Li YY, Xin WJ, et al. ATP induces LTP of C-fiber evoked field potentials in spinal dorsal horn: the roles of P2X4receptors and p38 MAPK in microglia[J]. Glia, 2009, 57(6):583-591.

[7] Yang HW, Hu XD, Zhang HM, et al. Roles of CaMKII, PKA, and PKC in the induction and maintenance of LTP of C-fiber-evoked field potentials in rat spinal dorsal horn[J]. J Neurophysiol, 2004, 91(3):1122-1133.

[8] Yang HW, Zhang HM, Hu XD, et al. Protein kinase C is involved in the induction and early-phase maintenance of long-term potentiation of C-fiber evoked field potentials in spinal dorsal horn of adult rats[J]. Chin J Pathophysiol, 2005, 21(2):252-255.

[9] Yang HW, Zhang HM, Hu XD, et al. PKA is required in the induction and early-phase maintenance of LTP of C-fiber evoked field potentials in spinal dorsal horn of adult rats[J]. Chin J Pathophysiol, 2004, 20(4): 513-517.

[10]George A, Marziniak M, Schäfers M, et al. Thalidomide treatment in chronic constrictive neuropathy decreases endoneurial tumor necrosis factor-α, increases interleukin-10 and has long-term effects on spinal cord dorsal horn met-enkephalin[J]. Pain, 2000, 88(3):267-275.

[11]Ji RR, Samad TA, Jin SX, et al. p38 MAPK activation by NGF in primary sensory neurons after inflammation increases TRPV1 levels and maintains heat hyperalgesia[J]. Neuron, 2002, 36(1):57-68.

[12]Zhong Y, Zhou LJ, Ren WJ, et al. The direction of synaptic plasticity mediated by C-fibers in spinal dorsal horn is decided by Src-family kinases in microglia: The role of tumor necrosis factor-α[J]. Brain Behav Immun, 2010, 24(6): 874-880.

[13]Tsuda M, Shigemoto-Mogami Y, Koizumi S, et al. P2X4receptors induced in spinal microglia gate tactile allodynia after nerve injury[J]. Nature, 2003, 424 (6950):778-783.

[14]Watkins LR, Maier SF. Beyond neurons: Evidence that immune and glial cells contribute to pathological pain states[J]. Physiol Rev, 2002, 82(4):981-1011.

[15]Ma JY, Zhao ZQ. The involvement of glia in long-term plasticity in the spinal dorsal horn of the rat[J]. Neuroreport, 2002, 13(14):1781-1784.

[16]Ikeda H, Tsuda M, Inoue K, et al. Long-term potentiation of neuronal excitation by neuron-glia interactions in the rat spinal dorsal horn[J]. Eur J Neurosci, 2007, 25(5):1297-1306.

[17]Tsuda M, Tozaki-Saitoh H, Masuda T, et al. Lyn tyrosine kinase is required for P2X4receptor upregulation and neuropathic pain after peripheral nerve injury[J]. Glia, 2008, 56(1):50-58.

小胶质细胞SFKs在ATP诱导的脊髓背角LTP中的作用

宫庆娟， 陈金生， 黄乔东， 卢振和

(广州医学院第二附属医院疼痛科，广东 广州 510260)

目的研究Src家族激酶(SFKs) 在5'-三磷酸腺苷(ATP)诱导的脊髓背角长时程增强(LTP)中的作用。方法雄性SD大鼠(250-280 g)。在体电生理记录脊髓腰膨大部背角浅层神经元C-纤维诱发电位，Western blotting和免疫组织化学观察脊髓背角SFKs的磷酸化水平和表达部位。结果ATP应用后30 min和60 min，磷酸化的Src-家族激酶(p-SFKs)的水平明显升高；p-SFKs只表达于小胶质细胞中，星型胶质细胞和神经元中没有表达。脊髓表面应用SFKs抑制剂阻断ATP诱导的LTP。结论小胶质细胞SFKs可能在ATP诱导的脊髓背角C-纤维诱发电位LTP中发挥重要作用。

Src家族激酶类； 长时程增强； 腺苷三磷酸； 脊髓背角； 小神经胶质细胞

R338.2

A

R338.2 [Documentcode] A

date] 2011-02-11Accepted2011-06-15

△ Corresponding author Tel: 020-34153611; E-mail: luzhh@126.com

1000-4718(2011)08-1563-06

10.3969/j.issn.1000-4718.2011.08.020