- Email: [email protected]
Inhibition of Protein Tyrosine Kinases Attenuated Aβ-Fiber-Evoked Synaptic Transmission in Spinal Dorsal Horn of Rats With Sciatic Nerve Transection
Journal of Pharmacological Sciences
J Pharmacol Sci 102, 64 – 71 (2006)
©2006 The Japanese Pharmacological Society
Full Paper
Inhibition of Protein Tyrosine Kinases Attenuated Aβ-Fiber-Evoked Synaptic Transmission in Spinal Dorsal Horn of Rats With Sciatic Nerve Transection Xiao-Dong Hu1,†, Neng-Wei Hu1,†, Wen-Jun Xin1, Li-Jun Zhou1, Tong Zhang1, and Xian-Guo Liu1,* 1
Department of Physiology, Zhongshan Medical School of Sun Yat-Sen University, 74 Zhongshan Rd. 2, Guangdong, China
Received May 11, 2006; Accepted July 11, 2006
Abstract. Peripheral nerve injury leads to the establishment of a novel synaptic connection between afferent Aβ-fiber and lamina II neurons in spinal dorsal horn, which is hypothesized to underlie mechanical allodynia. However, how the novel synapses transmit nociceptive information is poorly understood. In the present study, the role of protein tyrosine kinases (PTKs) in Aβ-fiber-evoked excitatory postsynaptic currents (EPSCs) recorded in lamina II neurons in transverse spinal cord slices of rats was investigated using the whole-cell patch-clamp recording technique. In the slices from sciatic nerve transection (SNT) rats, genistein (50 µM), a broadspectrum PTKs inhibitor, or PP2 (20 µM), a selective Src family tyrosine kinase inhibitor, significantly reduced the amplitude of Aβ-fiber EPSCs. In sham-operated rats, however, Aβ-fiber EPSCs were insensitive to genistein and PP2. The N-methyl-D-aspartate (NMDA) receptor antagonist AP-V (50 µM) suppressed Aβ-fiber EPSCs in slices from SNT rats but not from sham-operated rats. Following nerve injury, the slow inward currents elicited by bath application of NMDA (100 µM) significantly increased at −70 mV. In SNT rats, genistein and PP2 reduced Aβ-fiber-evoked EPSCs mediated by NMDA receptor; however, genistein produced little effect on Aβ-fiber EPSCs mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. These data suggested that PTKs, especially Src family members, participated in Aβ-fiber-evoked synaptic transmission following sciatic nerve injury via potentiation of NMDA receptor function. Keywords: neuropathic pain, allodynia, protein tyrosine kinase, Aβ-fiber, spinal dorsal horn
mechanisms underlying the Aβ-fiber-mediated pathological process are still not well understood. The lamina II of spinal dorsal horn is the first level integrating nociceptive information. In naive rats, the lamina II neurons predominantly receive the inputs from high-threshold myelinated Aδ- and unmyelinated Cafferent fibers (9, 10). It has been reported that after nerve injury, the central terminals of damaged and some intact Aβ-fibers may sprout into lamina II and this morphological change is believed to contribute to allodynia (11, 12). Although several recent studies do not support this opinion by demonstrating that nerve injury leads to only very limited Aβ-fibers sprouting into lamina II of spinal dorsal horn or even no such sprouting (13 – 15), electrophysiological data have shown that after nerve injury the majority of lamina II neurons become sensitive to stimulation of afferent Aβ-fibers,
Introduction Mechanical allodynia is one of the frequent complications of patients with neuropathic pain. Abundant evidence has shown that this sensory abnormality is transmitted by low-threshold afferent Aβ-fibers, which in physiological conditions only encode non-painful mechanical sensations (1 – 5). Several studies have demonstrated that the allodynia signaled by Aβ-fibers in nerve injury animals can be separated completely from other nociceptive disorders such as the afferent C fibermediated thermal hyperalgesia (4, 6 – 8). However, the †
The first two authors contributed equally to this work. *Corresponding author. [email protected] Published online in J-STAGE: August 26, 2006 doi: 10.1254/jphs.FP0060492
64
Role of PTK in Aβ-Fiber-Evoked EPSCs
indicating the establishment of novel functional synaptic connections between Aβ-afferents and neurons in lamina II (16 – 18). As yet, little is known about how the novel functional synapses transmit nociceptive information following nerve injury. It has been shown that the N-methyl-Daspartate (NMDA) receptor, a principal subtype of the ionic glutamate receptor, plays an important role in the maintenance of allodynia produced by peripheral nerve injury (19 – 22). Many studies have demonstrated that NMDA-receptor function can be upregulated by protein tyrosine kinases (PTKs), especially Src family kinases (SFKs) (23), and that tyrosine phosphorylation of NMDA-receptor subunit NR2B is associated with inflammatory pain (24, 25). Recently, tyrosine phosphorylation of the NR2B subunit has also been detected in postsynaptic density of superficial spinal dorsal horn neurons in neuropathic pain mice one week after transection of the L5 spinal nerve but not in naive ones, indicating that nerve injury leads to activation of SFKs in spinal dorsal horn (26). It is likely that PTKs and NMDA-receptor channels may be involved in the novel synaptic transmission produced by nerve injury. To test this, in the present study, the effects of PTKs inhibitors on Aβ-fiber-evoked excitatory postsynaptic currents (EPSCs) were investigated in intact and SNT rats, and the role of NMDA-receptor channels in the novel synaptic transmission was evaluated. Materials and Methods Sciatic nerve transection and preparation of spinal cord slices Complete sciatic nerve transaction (SNT) was performed on adult male Sprague-Dawley rats (4 – 5 weeks of age) under pentobarbital (40 mg / kg, i.p.) anesthesia. The left sciatic nerve was exposed, ligated, and severed in the popliteal fossa. As a control, agematched rats were sham-operated. At 14 – 20 days after nerve transection, the rats were re-anesthetized with urethane (20%, i.p., 1.5 g / kg), and the spinal cord was removed. A transverse spinal slice (500-µm-thick) with a L4 or L5 dorsal root ipsilateral to the transected sciatic nerve was cut in ice-cold (1°C – 4°C) Krebs solution pre-equilibrated with 95% O2 + 5% CO2 on a Vibratome stage (DTK-1000; Dosaka, Kyoto). Then the slice was perfused with oxygenated Krebs solution (4 ml / min; composition: 117 mM NaCl, 3.6 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 25 mM NaHCO3, and 11 mM glucose) at room temperature in the recording chamber for at least 1 h prior to recordings. The studies were approved by the Institutional Animal Care and Ethics Committee and all
65
procedures conformed to the guidelines set out by the International Association for the Study of Pain (27). Electrophysiological recordings Blind whole-cell patch clamp technique was used to record the dorsal root-evoked excitatory postsynaptic current (EPSC) from lamina II neurons (17, 18). Patch electrodes, made from thin-walled, glass-capillary tubings (1.0 mm OD, 0.5 mm ID; Sutter Instruments, Novato, CA, USA) with a horizontal puller (P-87, Sutter Instruments), had a tip resistance of 8 – 12 MΩ when filled with electrode solution (135 mM K-gluconate, 0.5 mM CaCl2, 2 mM MgCl2, 5 mM EGTA, 5 mM HEPES, and 5 mM Mg-ATP, pH 7.2). After a gigaseal formation (seal resistance: 2 – 50 GΩ), the membrane was ruptured to obtain the whole-cell voltage-clamp configuration. Membrane currents were recorded with an EPC10 amplifier (HEKA, Lambrecht / Pfalz, Germany). The signals were filtered at 1 kHz and digitized at 5 kHz. Data acquisition and command potentials were controlled by commercial software (Pulse, HEKA). A suction electrode was used for orthodromic stimulation (0.1-ms duration, 110% of threshold stimulus intensity) of the dorsal roots. Synaptically evoked currents were elicited by graded stimulus intensity sufficient to recruit Aβ-, Aδ-, or C-fibers. The holding potential for recording evoked EPSC was kept at −70 mV, at which inhibitory postsynaptic currents would be negligible (17, 28). Identification of Aβ-fibermediated synaptic responses was based on threshold stimulus intensity and conduction velocity of compound action potentials measured extracellularly on the dorsal root near the dorsal root entry zone as described previously (17, 18). Aβ-fiber-evoked responses were defined by conduction velocities (>15 m / s) and threshold, which was 15 ± 1 µA (range: 10 – 25 µA, n = 12) in SNT rats and 16 ± 1 µA (range: 14 – 26 µA, n = 13) in sham-operated rats. Compounds and drug application 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (CNQX), NMDA, genistein, and 2-amino-5-phosphonovaleric acid (AP-V) were purchased from Sigma (St. Louis, MO, USA). 4-Amino-5(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) was purchased from Alexis (San Diego, CA, USA). Stock solutions of NMDA, genistein, PP2, and AP-V were prepared by dissolving in saline, while CNQX was prepared in DMSO. The final concentration of DMSO used during experiments was 0.1%. All the stock solutions were diluted by Krebs solution and applied by perfusion via a three way stopcock with no alteration in either the perfusion rate or temperature.
66
X-D Hu et al
The solution in the recording chamber (1.5 ml) was completely replaced by the perfusing solution within 30 s. Statistic analysis All data were presented as means ± S.E.M. and analyzed with one-way analysis of variance followed by Scheffe’s post hoc test. Statistical significance was determined as P<0.05. Results Fifty-four neurons with Aβ-fiber-evoked synaptic responses in SNT rats and 15 neurons in sham-operated rats were recorded. In sham-operated rats, only 18% (15 / 82) of neurons responded to a lower stimulus intensity (mean threshold: 17 ± 2 µA; range: 10 – 25 µA, n = 15). In SNT rats, however, a low stimulus intensity (mean threshold intensity: 16 ± 1 µA; range: 10 – 24 µA; n = 54) elicited Aβ-fiber-mediated EPSCs in 86% (54 / 63) of recorded neurons. In SNT rats, 80% (43 / 54) of Aβ-fiber-evoked EPSCs in lamina II neurons were polysynaptic and 20% (11 / 54) were monosynaptic. Polysynaptic responses were determined by variable synaptic latency and failure in EPSC amplitude in response to high-frequency stimulation (20 Hz) (Fig. 1A). PTKs inhibitors suppressed Aβ-fiber-evoked EPSCs in SNT rats To test the role of PTKs in Aβ-fiber-evoked synaptic transmission in SNT rats, genistein, a broad-spectrum PTKs inhibitor, was bath applied after collecting control responses. Following 5-min superfusion of genistein (50 µM), experimental data consisting of 10 consecutive responses were obtained at 0.05 Hz. Genistein significantly depressed the amplitudes of Aβ-fiber-evoked EPSCs tested in 14 neurons. There was no difference in the sensibility to genistein between polysynaptic responses and monosynaptic ones (Fig. 1A). As shown in Fig. 1B, the mean amplitude of Aβ-fiber-evoked EPSCs decreased by 32 ± 3% (n = 14, P<0.05) at 5 min after superfusion of genistein and still maintained at low level at 15 min after washout. In contrast, genistein did not affect Aβ-fiber-evoked synaptic responses in sham-operated rats (n = 5, Fig. 1B). To test the effect of SFKs on Aβ-fiber-mediated synaptic transmission, the selective SFKs inhibitor PP2 (20 µM) was bath applied to the spinal cord slices from SNT rats following recording of control responses. The result showed that the mean amplitude of Aβ-fiber responses decreased by 35 ± 3% (P<0.05, n = 5; Fig. 1C) at 5 min after PP2 and no significant recovery was
observed at 15 min after washout. The same concentration of the drug, however, produced little effect on Aβ-fiber-evoked EPSCs in sham-operated rats (n = 5, Fig. 1C). NMDA-receptor antagonist AP-V depressed Aβ-fiberevoked EPSCs in SNT rats To test whether NMDA-receptor channels were involved in Aβ-fiber-mediated synaptic transmission after sciatic nerve injury, the selective NMDA-receptor antagonist AP-V was perfused for 5 min after recordings of baseline. The results showed that AP-V (50 µM) reduced the mean amplitude of Aβ-fiber-evoked EPSCs by 42 ± 4% (P<0.05, n = 13; Fig. 2A). The remaining component could be further eliminated by the α-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist CNQX (10 µM, data not shown). In sham-operated rats, AP-V (50 µM) produced little effect on EPSC magnitudes (n = 5, Fig. 2B). Voltage-dependent sensibility of NMDA receptor responses was changed after nerve injury Our data showing that AP-V inhibited Aβ-fibermediated synaptic responses in SNT but not in shamoperated rats imply that the voltage-dependent property of NMDA-receptor channels may be changed after nerve injury. To test this, NMDA-receptor-mediated inward currents were recorded in sham-operated and SNT rats at the holding potentials of −70, −60, −50, −40, −30, and 0 mV. Bath application of NMDA (100 µM) for 30 s elicited the slow inward currents at all the mentioned holding potentials except 0 mV (Fig. 3: A and B). Compared with sham-operated rats, the amplitude of NMDA currents recorded in SNT rats was larger at the holding potentials of −70 mV (−23 ± 1 pA vs −29 ± 2 pA, P<0.05; Fig. 3C), while at the other mentioned holding potentials, no significant difference between these two groups of rats was detected. Inhibition of PTKs selectively depressed Aβ-fiberevoked NMDA-receptor components in SNT rats Next we directly examined the effect of PTKs inhibitors on NMDA-receptor-mediated synaptic responses, isolated by continuous perfusion of spinal cord slices with the AMPA-receptor antagonist CNQX (10 µM). Bath application of genistein for 5 min significantly reduced NMDA-receptor-mediated EPSCs elicited by Aβ-fiber stimulation (n = 9, P<0.05; Fig. 4A). Similarly, PP2 also reduced NMDA-receptor-mediated responses (P<0.05, n = 6; Fig. 4B). At 15-min after washout of genistein or PP2, NMDA-receptor-mediated EPSCs were still lower than controls. However, AMPAreceptor components evoked by Aβ-fiber stimulation,
Role of PTK in Aβ-Fiber-Evoked EPSCs
67
Fig. 1. Protein tyrosine kinase inhibitors depressed Aβ-fiber-evoked excitatory postsynaptic currents (EPSCs) recorded in lamina II neurons from sciatic nerve transection (SNT) rats but not from sham-operated rats. A: Typical Aβ-fiber-evoked mono- and polysynaptic EPSCs in response to 0.2 Hz (1) and 20 Hz (2) stimulation in SNT rats. Polysynaptic responses were determined by variable latencies and failure in the amplitude upon 20-Hz stimulation, while monosynaptic responses showed constant latencies and absence of failure even though the EPSC amplitudes decreased. Note that genistein (50 µM) reduced the amplitudes of both mono- and polysynaptic EPSCs (3). B and C: Summary data in histograms showed the effect of genistein (50 µM) and PP2 (20 µM) on the amplitudes of Aβ-fiber-evoked EPSCs recorded in SNT (left) and sham-operated (right) rats, respectively. Data are presented as the mean ± S.E.M. *P<0.05, compared with the control.
recorded in the presence of AP-V (50 µM), changed little 5 min after application of genistein (n = 7, Fig. 4C). Discussion Peripheral nerve injury triggers a series of morphological and molecular changes in spinal dorsal horn microglia and neurons, which combine to initiate and
maintain the tactile allodynia. Previous studies have indicated that nerve injury activates the microglia in the spinal dorsal horn by promoting the release of ATP from injuried neurons or astrocytes (29). The hyperactive phenotype of microglia displays the increased expression of ATP-receptor subtype P2X4 and active p38 mitogen-activated protein kinase (p38 MAPK), which are essential for tactile allodynia during neuropathic
68
X-D Hu et al
Fig. 2. Blockage of NMDA receptors reduced Aβ-fiber-evoked EPSCs recorded in lamina II neurons from SNT but not from sham-operated rats. A: The NMDA receptor antagonist AP-V (50 µM) inhibited Aβ-fiber-evoked EPSCs significantly 5 min after bath application in the slices from SNT rats. B: The same concentration of AP-V had no effect in sham-operated rats. Representative recordings of Aβ-fiber EPSCs were taken during control (1), 5 min after exposure to AP-V (2), and 15-min after washout (3). Data are presented as means ± S.E.M. *P<0.05, compared with the control.
Fig. 3. Voltage-dependent responses of lamina II neurons to exogenous NMDA recorded in lamina II neurons of sham-operated and SNT rats. Application of NMDA (100 µM) for 30 s induced a slow inward current at the holding potentials of −70 – −30 mV in sham-operated (A) and in SNT (B) rats. C: Pooled data in histograms showed that the mean amplitude of NMDA-induced slow currents recorded at −70 mV rather than at −60 – −30 mV was significantly higher in SNT rats, compared with that in sham-operated rats. Data are presented as means ± S.E.M. *: P<0.05.
Role of PTK in Aβ-Fiber-Evoked EPSCs
Fig. 4. Protein tyrosine kinase inhibitors selectively depressed Aβ-fiber-evoked NMDA-receptor current components in SNT rats. NMDA- or AMPA-receptor-mediated currents were isolated pharmacologically by blockage of AMPA receptor with CNQX (10 µM) or NMDA receptors with AP-V (50 µM), respectively. A and B: Genistein (50 µM) and PP2 (20 µM) attenuated Aβ-fiberevoked NMDA-receptor-mediated currents substantially. C: Aβfiber-evoked AMPA-receptor-mediated currents were unaffected by bath application of genistein. Representative recordings of Aβ-fiberevoked EPSCs were taken during control (1), 5-min after exposure to PTKs inhibitors (2), and 15-min after washout (3). Data are presented as means ± S.E.M. *P<0.05, compared with the control.
69
pain. Nerve injury also reorganizes the primary afferent input onto superficial dorsal horn neurons and dramatically changes the synaptic strength. The present work showed that 86% of the total 63 recorded neurons in lamina II of spinal dorsal horn exhibited Aβ-fiberevoked synaptic responses in SNT rats, while only 18% of tested neurons did so in sham-operated rats. These electrophysiological results support the notion that novel synaptic connection was established between the afferent Aβ-fibers and lamina II neurons following sciatic nerve transection (17, 18). In addition, we demonstrated for the first time that either a broadspectrum PTKs inhibitor (genistein) or a selective SFKs inhibitor (PP2) significantly depressed Aβ-fibermediated excitatory synaptic transmission in the slices from SNT rats but not from sham-operated rats. The NMDA-receptor antagonist AP-V selectively depressed Aβ-fiber-evoked EPSCs in SNT rats but not in shamoperated rats. In SNT rats, NMDA components but not AMPA components of Aβ-fiber-evoked EPSCs were selectively inhibited by genistein. These data suggest that PTKs, especially SFKs, may potentiate Aβ-fiberevoked synaptic transmission in lamina II following nerve injury via upregulation of postsynaptic NMDAreceptor function. Investigations with naive rats have demonstrated that the fast excitatory synaptic responses in lamina II neurons evoked by primary afferent Aβ-fibers are predominantly mediated by AMPA-receptor channels (30 – 32), while NMDA-receptor channel currents are not manifested at normal resting potential because of voltage-dependent Mg2+ blockage (33). In the present study, we showed that AP-V had no effect on Aβ-fiberevoked EPSCs in sham-operated rats, which is in complete agreement with a previous study showing that AP-V does not affect Aβ-fiber-evoked mono- and polysynaptic EPSCs in naive rats (34). We further demonstrated that AP-V did depress Aβ-fiber-evoked EPSCs in SNT rats significantly and that the amplitudes of NMDA-induced slow currents at the holding potential of −70 mV were larger in SNT rats than those in shamoperated rats. These data indicate that NMDA-receptor channels are involved in Aβ-fiber-mediated synaptic transmission following nerve injury, which may contribute to allodynia produced by nerve injury. The behavioral data showing that blockage of NMDA receptors alleviates allodynia (19 – 22) support this notion. Five types of SFKs (Src, Fyn, Yes, Lck, and Lyn) are expressed in mammalian central nervous system (35). Src and Fyn are shown to up-regulate NMDA-receptor function and synaptic plasticity in the spinal cord and other brain areas (23). Activation of SFKs phosphory-
70
X-D Hu et al
lates tyrosine residues on NMDA-receptor subunits NR2A (36) and NR2B (37) and other proteins in the NMDA-receptor complex such as the scaffold protein PSD-93 (38), which may increase the receptor channel opening probability and reduce voltage-dependent Mg2+ blocking. There is also evidence demonstrating that the induction of long-term potentiation in the CA1 region of the hippocampus in adult rats leads to rapid surface expression of NMDA receptors, which is prevented by the inhibition of either SFKs or protein kinase C (PKC) (39). Salter and Kalia (23) have suggested that SFKsmediated tyrosine phosphorylation of NMDA-receptor subunit might stabilize NMDA receptors on the cell surface and thereby increase NMDA-receptor response. These possible actions may explain our findings that both the broad-spectrum PTKs inhibitor genistein and SFKs selective inhibitor PP2 depressed NMDAreceptor-mediated synaptic transmission in adult SNT rats. It is likely that both up-regulation of NMDAreceptor gating and insertion of NMDA receptors into the postsynaptic membrane may contribute to the novel Aβ-fiber-mediated synaptic transmission after nerve injury. Genistein and PP2 produced a lasting depression (at least 15 min after washout) on NMDA-receptormediated synaptic responses in SNT rats as the drugs affected NMDA-receptor function indirectly, probably reducing NMDA-receptor gating and insertion by inhibition of SFKs. It may take time for SFKs to phosphorylate NMDA receptors and to drive them into the cell surface again following washout. Although there is evidence showing that SFKs are activated in superficial spinal dorsal horn after nerve injury (26), the mechanisms underlying the change are unclear. Nerve injury leads to multiple changes in spinal dorsal horn. Among them, increased release of neurotransmitter glutamate and neuromodulators such as brain-derived neurotrophic factor (BDNF) from primary afferent terminals in spinal dorsal horn is proved to be important for the initiation and maintenance of neuropathic pain (40). It has been shown that activation of G protein-coupled receptors can stimulate SFKs and PKC, leading to up-regulation of NMDA-receptor function (41). Guo et al. demonstrated that group I metabotropic glutamate receptors (mGluRs) are biochemically linked with SFKs and NMDA receptors in spinal dorsal horn neurons (42). Increased release of glutamate stimulates group I mGluRs that enhance src kinases activity and tyrosine phosphorylation of NMDA receptors during pain hypersensitivity. The concentration of BDNF in lumbar spinal dorsal horn also markedly increases after nerve lesion, which occludes in the timeline with behavioral signs of neuropathic pain (40). SFKs play a key role in linking BDNF signaling with downstream
NMDA receptors (43). In addition, ATP-stimulated hyperactive microglia in the spinal cord of neuropathic pain animals also releases a number of proinflammatory cyrokines such as interleukin-1β, which can increase NMDA-receptor function through activation of SFKs and play an important role in tactile allodynia (29, 44, 45). Taken together, multiple mechanisms may underlie activation of SFKs following nerve injury, and further studies are needed to elucidate the process. In conclusion, the present study demonstrated that SFKs were involved in Aβ-fiber-evoked synaptic transmission in SNT rats. The finding may be important for understanding the cellular mechanism underlying allodynia and SFKs may be a potential target for treatment of this neuropathic pain syndrome. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 30200076 and 30370474) and by China Medical Board (1998-766). References 1 Koltzenburg M, Torebjork H, Wahren L. Nociceptor modulated central sensitization causes mechanical hyperalgesia in acute chemogenic and chronic neuropathic pain. Brain. 1994;117:579– 591. 2 Woolf C, Doubell T. The pathophysiology of chronic painincreased sensitivity to low threshold A beta-fibre inputs. Curr Opin Neurobiol. 1994;4:525–534. 3 Field M, Bramwell S, Hughes J, Singh L. Detection of static and dynamic components of mechanical allodynia in rat models of neuropathic pain: are they signaled by distinct primary sensory neurons? Pain. 1999;83:303–311. 4 Ossipov M, Bian D, Malan T, Lai J, Porreca F. Lack of involvement of capsaicin-sensitive primary afferents in nerve-ligation injury induced tactile allodynia in rats. Pain. 1999;79:127–133. 5 Liu X, Eschenfelder S, Blenk K, Janig W, Habler H. Spontaneous activity of axotomized afferent neurons after L5 spinal nerve injury in rats. Pain. 2000;84:309–318. 6 Shir Y, Seltzer Z. A-fibers mediate mechanical hyperesthesia and allodynia and C-fibers mediate thermal hyperalgesia in a new model of causalgiform pain disorders in rats. Neurosci Lett. 1990;115:62–67. 7 Khan G, Chen S, Pan H. Role of primary afferent nerves in allodynia caused by diabetic neuropathy in rats. Neuroscience. 2002;114:291–299. 8 Pan H, Khan G, Alloway K, Chen S. Resiniferatoxin induces paradoxical changes in thermal and mechanical sensitivities in rats: mechanism of action. J Neurosci. 2003;23:2911–2919. 9 Ralston H, Ralston D. The distribution of dorsal root axons in laminae I, II and III of the macaque spinal cord: a quantitative electron microscope study. J Comp Neurol. 1979;184:643–684. 10 Sugiura Y, Lee C, Perl E. Central projections of identified, unmyelinated (C) afferent fibers innervating mammalian skin. Science. 1986;234:358–361.
Role of PTK in Aβ-Fiber-Evoked EPSCs 11 Woolf C, Shortland P, Coggeshall R. Peripheral nerve injury triggers central sprouting of myelinated afferents. Nature. 1992;355:75–78. 12 Doubell T, Mannion R, Woolf C. Intact sciatic myelinated primary afferent terminals collaterally sprout in the adult rat dorsal horn following section of a neighbouring peripheral nerve. J Comp Neurol. 1997;380:95–104. 13 Bao L, Wang H, Cai H, Tong Y, Jin S, Lu Y, et al. Peripheral axotomy induces only very limited sprouting of coarse myelinated afferents into inner lamina II of rat spinal cord. Eur J Neurosci. 2002;16:175–185. 14 Shehab S, Spike R, Todd A. Evidence against cholera toxin B subunit as a reliable tracer for sprouting of primary afferents following peripheral nerve injury. Brain Res. 2003;964:218– 227. 15 Hughes D, Scott D, Todd A, Riddell J. Lack of evidence for sprouting of A beta afferents into the superficial laminas of the spinal cord dorsal horn after nerve section. J Neurosci. 2003;23:9491–9499. 16 Kohama I, Ishikawa K, Kocsis J. Synaptic reorganization in the substantia gelatinosa after peripheral nerve neuroma formation: aberrant innervation of lamina II neurons by Abeta afferents. J Neurosci. 2000;20:1538–1549. 17 Okamoto M, Baba H, Goldstein P, Higashi H, Shimoji K, Yoshimura M. Functional reorganization of sensory pathways in the rat spinal dorsal horn following peripheral nerve injury. J Physiol. 2001;532:241–250. 18 Kohno T, Moore K, Baba H, Woolf C. Peripheral nerve injury alters excitatory synaptic transmission in lamina II of the rat dorsal horn. J Physiol. 2003;548:131–138. 19 Eide K, Stubhaug A, Oye I, Breivik H. Continuous subcutaneous administration of the N-methyl-D-aspartic acid (NMDA) receptor antagonist ketamine in the treatment of post-herpetic neuralgia. Pain. 1995;61:221–228. 20 Persson J, Axelsson G, Hallin R, Gustafsson L. Beneficial effects of ketamine in a chronic pain state with allodynia, possibly due to central sensitization. Pain. 1995;60:217–222. 21 Kim Y, Na H, Yoon Y, Han H, Ko K, Hong S. NMDA receptors are important for both mechanical and thermal allodynia from peripheral nerve injury in rats. Neuroreport. 1997;8:2149–2153. 22 Parsons C. NMDA receptors as targets for drug action in neuropathic pain. Eur J Pharmacol. 2001;429:71–78. 23 Salter M, Kalia L. Src kinases: a hub for NMDA receptor regulation. Nat Rev Neurosci. 2004;5:317–328. 24 Guo W, Zou S, Guan Y, Ikeda T, Tal M, Dubner R, et al. Tyrosine phosphorylation of the NR2B subunit of the NMDA receptor in the spinal cord during the development and maintenance of inflammatory hyperalgesia. J Neurosci. 2002;22: 6208–6217. 25 Sato E, Takano Y, Kuno Y, Takano M, Sato I. Involvement of spinal tyrosine kinase in inflammatory and N-methyl-Daspartate-induced hyperalgesia in rats. Eur J Pharmacol. 2003; 468:191–198. 26 Abe T, Matsumura S, Katano T, Mabuchi T, Takagi K, Xu L, et al. Fyn kinase-mediated phosphorylation of NMDA receptor NR2B subunit at Tyr1472 is essential for maintenance of neuropathic pain. Eur J Neurosci. 2005;22:1445–1454. 27 Zimmermann M. Ethical guidelines for investigation of experimental pain in conscious animals. Pain. 1983;16:109–110. 28 Yoshimura M, Nishi S. Primary afferent-evoked glycine- and GABA-mediated IPSPs in substantia gelatinosa neurones in the
71
rat spinal cord in vitro. J Physiol. 1995;482:29–38. 29 Inoue K, Tsuda M, Koizumi S. ATP- and adenosine-mediated signaling in the central nervous system: chronic pain and microglia: involvement of the ATP receptor P2X4. J Pharmacol Sci. 2004;94:112–114. 30 Morris R. Responses of spinal dorsal horn neurones evoked by myelinated primary afferent stimulation are blocked by excitatory amino acid antagonists acting at kainate /quisqualate receptors. Neurosci Lett. 1989;105:79–85. 31 Furue H, Narikawa K, Kumamoto E, Yoshimura M. Responsiveness of rat substantia gelatinosa neurones to mechanical but not thermal stimuli revealed by in vivo patch-clamp recording. J Physiol. 1999;521:529–535. 32 Li P, Wilding T, Kim S, Calejesan A, Huettner J, Zhuo M. Kainate- receptor-mediated sensory synaptic transmission in mammalian spinal cord. Nature. 1999;397:161–164. 33 Mayer M, Westbrook G, Guthrie P. Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones. Nature. 1984;309:261–263. 34 Baba H, Doubell T, Moore K, Woolf C. Silent NMDA receptormediated synapses are developmentally regulated in the dorsal horn of the rat spinal cord. J Neurophysiol. 2000;83:955–962. 35 Yu X, Salter M. Src, a molecular switch governing gain control of synaptic transmission mediated by N-methyl-D-aspartate receptors. Proc Natl Acad Sci U S A. 1999;96:7697–7704. 36 Lau L, Huganir R. Differential tyrosine phosphorylation of Nmethyl-D-aspartate receptor subunits. J Biol Chem. 1995;270: 20036–20041. 37 Moon I, Apperson M, Kennedy M. The major tyrosinephosphorylated protein in the postsynaptic density fraction is N-methyl-D-aspartate receptor subunit 2B. Proc Natl Acad Sci U S A. 1994;91:3954–3958. 38 Nada S, Shima T, Yanai H, Husi H, Grant S, Okada M, et al. Identification of PSD-93 as a substrate for the Src family tyrosine kinase Fyn. J Biol Chem. 2003;278:47610–47621. 39 Grosshans D, Clayton D, Coultrap S, Browning M. LTP leads to rapid surface expression of NMDA but not AMPA receptors in adult rat CA1. Nat Neurosci. 2002;5:27–33. 40 Miletic G, Miletic V. Increases in the concentration of brain derived neurotrophic factor in the lumbar spinal dorsal horn are associated with pain behavior following chronic constriction injury in rats. Neurosci Lett. 2002;319:137–140. 41 Lu W, Xiong Z, Lei S, Orser B, Dudek E, Browning M, et al. G-protein-coupled receptors act via protein kinase C and Src to regulate NMDA receptors. Nat Neurosci. 1999;2:331–338. 42 Guo W, Wei F, Zou S, Robbins M, Sugiyo S, Ikeda T, et al. Group I metabotropic glutamate receptor NMDA receptor coupling and signaling cascade mediate spinal dorsal horn NMDA receptor 2B tyrosine phosphorylation associated with inflammatory hyperalgesia. J Neurosci. 2004;24:9161–9173. 43 Yamada K, Nabeshima T. Interaction of BDNF /TrkB signaling with NMDA receptor in learning and memory. Drug News Perspect. 2004;17:435–438. 44 Viviani B, Bartesaghi S, Gardoni F, Vezzani A, Behrens M, Bartfai T, et al. Interleukin-1beta enhances NMDA receptormediated intracellular calcium increase through activation of the Src family of kinases. J Neurosci. 2003;23:8692–8700. 45 Inoue K. The function of microglia through purinergic receptors: Neuropathic pain and cytokine release. Pharmacol Ther. 2006;109:210–226.
J Pharmacol Sci 102, 64 – 71 (2006)
©2006 The Japanese Pharmacological Society
Full Paper
Inhibition of Protein Tyrosine Kinases Attenuated Aβ-Fiber-Evoked Synaptic Transmission in Spinal Dorsal Horn of Rats With Sciatic Nerve Transection Xiao-Dong Hu1,†, Neng-Wei Hu1,†, Wen-Jun Xin1, Li-Jun Zhou1, Tong Zhang1, and Xian-Guo Liu1,* 1
Department of Physiology, Zhongshan Medical School of Sun Yat-Sen University, 74 Zhongshan Rd. 2, Guangdong, China
Received May 11, 2006; Accepted July 11, 2006
Abstract. Peripheral nerve injury leads to the establishment of a novel synaptic connection between afferent Aβ-fiber and lamina II neurons in spinal dorsal horn, which is hypothesized to underlie mechanical allodynia. However, how the novel synapses transmit nociceptive information is poorly understood. In the present study, the role of protein tyrosine kinases (PTKs) in Aβ-fiber-evoked excitatory postsynaptic currents (EPSCs) recorded in lamina II neurons in transverse spinal cord slices of rats was investigated using the whole-cell patch-clamp recording technique. In the slices from sciatic nerve transection (SNT) rats, genistein (50 µM), a broadspectrum PTKs inhibitor, or PP2 (20 µM), a selective Src family tyrosine kinase inhibitor, significantly reduced the amplitude of Aβ-fiber EPSCs. In sham-operated rats, however, Aβ-fiber EPSCs were insensitive to genistein and PP2. The N-methyl-D-aspartate (NMDA) receptor antagonist AP-V (50 µM) suppressed Aβ-fiber EPSCs in slices from SNT rats but not from sham-operated rats. Following nerve injury, the slow inward currents elicited by bath application of NMDA (100 µM) significantly increased at −70 mV. In SNT rats, genistein and PP2 reduced Aβ-fiber-evoked EPSCs mediated by NMDA receptor; however, genistein produced little effect on Aβ-fiber EPSCs mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. These data suggested that PTKs, especially Src family members, participated in Aβ-fiber-evoked synaptic transmission following sciatic nerve injury via potentiation of NMDA receptor function. Keywords: neuropathic pain, allodynia, protein tyrosine kinase, Aβ-fiber, spinal dorsal horn
mechanisms underlying the Aβ-fiber-mediated pathological process are still not well understood. The lamina II of spinal dorsal horn is the first level integrating nociceptive information. In naive rats, the lamina II neurons predominantly receive the inputs from high-threshold myelinated Aδ- and unmyelinated Cafferent fibers (9, 10). It has been reported that after nerve injury, the central terminals of damaged and some intact Aβ-fibers may sprout into lamina II and this morphological change is believed to contribute to allodynia (11, 12). Although several recent studies do not support this opinion by demonstrating that nerve injury leads to only very limited Aβ-fibers sprouting into lamina II of spinal dorsal horn or even no such sprouting (13 – 15), electrophysiological data have shown that after nerve injury the majority of lamina II neurons become sensitive to stimulation of afferent Aβ-fibers,
Introduction Mechanical allodynia is one of the frequent complications of patients with neuropathic pain. Abundant evidence has shown that this sensory abnormality is transmitted by low-threshold afferent Aβ-fibers, which in physiological conditions only encode non-painful mechanical sensations (1 – 5). Several studies have demonstrated that the allodynia signaled by Aβ-fibers in nerve injury animals can be separated completely from other nociceptive disorders such as the afferent C fibermediated thermal hyperalgesia (4, 6 – 8). However, the †
The first two authors contributed equally to this work. *Corresponding author. [email protected] Published online in J-STAGE: August 26, 2006 doi: 10.1254/jphs.FP0060492
64
Role of PTK in Aβ-Fiber-Evoked EPSCs
indicating the establishment of novel functional synaptic connections between Aβ-afferents and neurons in lamina II (16 – 18). As yet, little is known about how the novel functional synapses transmit nociceptive information following nerve injury. It has been shown that the N-methyl-Daspartate (NMDA) receptor, a principal subtype of the ionic glutamate receptor, plays an important role in the maintenance of allodynia produced by peripheral nerve injury (19 – 22). Many studies have demonstrated that NMDA-receptor function can be upregulated by protein tyrosine kinases (PTKs), especially Src family kinases (SFKs) (23), and that tyrosine phosphorylation of NMDA-receptor subunit NR2B is associated with inflammatory pain (24, 25). Recently, tyrosine phosphorylation of the NR2B subunit has also been detected in postsynaptic density of superficial spinal dorsal horn neurons in neuropathic pain mice one week after transection of the L5 spinal nerve but not in naive ones, indicating that nerve injury leads to activation of SFKs in spinal dorsal horn (26). It is likely that PTKs and NMDA-receptor channels may be involved in the novel synaptic transmission produced by nerve injury. To test this, in the present study, the effects of PTKs inhibitors on Aβ-fiber-evoked excitatory postsynaptic currents (EPSCs) were investigated in intact and SNT rats, and the role of NMDA-receptor channels in the novel synaptic transmission was evaluated. Materials and Methods Sciatic nerve transection and preparation of spinal cord slices Complete sciatic nerve transaction (SNT) was performed on adult male Sprague-Dawley rats (4 – 5 weeks of age) under pentobarbital (40 mg / kg, i.p.) anesthesia. The left sciatic nerve was exposed, ligated, and severed in the popliteal fossa. As a control, agematched rats were sham-operated. At 14 – 20 days after nerve transection, the rats were re-anesthetized with urethane (20%, i.p., 1.5 g / kg), and the spinal cord was removed. A transverse spinal slice (500-µm-thick) with a L4 or L5 dorsal root ipsilateral to the transected sciatic nerve was cut in ice-cold (1°C – 4°C) Krebs solution pre-equilibrated with 95% O2 + 5% CO2 on a Vibratome stage (DTK-1000; Dosaka, Kyoto). Then the slice was perfused with oxygenated Krebs solution (4 ml / min; composition: 117 mM NaCl, 3.6 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 25 mM NaHCO3, and 11 mM glucose) at room temperature in the recording chamber for at least 1 h prior to recordings. The studies were approved by the Institutional Animal Care and Ethics Committee and all
65
procedures conformed to the guidelines set out by the International Association for the Study of Pain (27). Electrophysiological recordings Blind whole-cell patch clamp technique was used to record the dorsal root-evoked excitatory postsynaptic current (EPSC) from lamina II neurons (17, 18). Patch electrodes, made from thin-walled, glass-capillary tubings (1.0 mm OD, 0.5 mm ID; Sutter Instruments, Novato, CA, USA) with a horizontal puller (P-87, Sutter Instruments), had a tip resistance of 8 – 12 MΩ when filled with electrode solution (135 mM K-gluconate, 0.5 mM CaCl2, 2 mM MgCl2, 5 mM EGTA, 5 mM HEPES, and 5 mM Mg-ATP, pH 7.2). After a gigaseal formation (seal resistance: 2 – 50 GΩ), the membrane was ruptured to obtain the whole-cell voltage-clamp configuration. Membrane currents were recorded with an EPC10 amplifier (HEKA, Lambrecht / Pfalz, Germany). The signals were filtered at 1 kHz and digitized at 5 kHz. Data acquisition and command potentials were controlled by commercial software (Pulse, HEKA). A suction electrode was used for orthodromic stimulation (0.1-ms duration, 110% of threshold stimulus intensity) of the dorsal roots. Synaptically evoked currents were elicited by graded stimulus intensity sufficient to recruit Aβ-, Aδ-, or C-fibers. The holding potential for recording evoked EPSC was kept at −70 mV, at which inhibitory postsynaptic currents would be negligible (17, 28). Identification of Aβ-fibermediated synaptic responses was based on threshold stimulus intensity and conduction velocity of compound action potentials measured extracellularly on the dorsal root near the dorsal root entry zone as described previously (17, 18). Aβ-fiber-evoked responses were defined by conduction velocities (>15 m / s) and threshold, which was 15 ± 1 µA (range: 10 – 25 µA, n = 12) in SNT rats and 16 ± 1 µA (range: 14 – 26 µA, n = 13) in sham-operated rats. Compounds and drug application 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (CNQX), NMDA, genistein, and 2-amino-5-phosphonovaleric acid (AP-V) were purchased from Sigma (St. Louis, MO, USA). 4-Amino-5(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) was purchased from Alexis (San Diego, CA, USA). Stock solutions of NMDA, genistein, PP2, and AP-V were prepared by dissolving in saline, while CNQX was prepared in DMSO. The final concentration of DMSO used during experiments was 0.1%. All the stock solutions were diluted by Krebs solution and applied by perfusion via a three way stopcock with no alteration in either the perfusion rate or temperature.
66
X-D Hu et al
The solution in the recording chamber (1.5 ml) was completely replaced by the perfusing solution within 30 s. Statistic analysis All data were presented as means ± S.E.M. and analyzed with one-way analysis of variance followed by Scheffe’s post hoc test. Statistical significance was determined as P<0.05. Results Fifty-four neurons with Aβ-fiber-evoked synaptic responses in SNT rats and 15 neurons in sham-operated rats were recorded. In sham-operated rats, only 18% (15 / 82) of neurons responded to a lower stimulus intensity (mean threshold: 17 ± 2 µA; range: 10 – 25 µA, n = 15). In SNT rats, however, a low stimulus intensity (mean threshold intensity: 16 ± 1 µA; range: 10 – 24 µA; n = 54) elicited Aβ-fiber-mediated EPSCs in 86% (54 / 63) of recorded neurons. In SNT rats, 80% (43 / 54) of Aβ-fiber-evoked EPSCs in lamina II neurons were polysynaptic and 20% (11 / 54) were monosynaptic. Polysynaptic responses were determined by variable synaptic latency and failure in EPSC amplitude in response to high-frequency stimulation (20 Hz) (Fig. 1A). PTKs inhibitors suppressed Aβ-fiber-evoked EPSCs in SNT rats To test the role of PTKs in Aβ-fiber-evoked synaptic transmission in SNT rats, genistein, a broad-spectrum PTKs inhibitor, was bath applied after collecting control responses. Following 5-min superfusion of genistein (50 µM), experimental data consisting of 10 consecutive responses were obtained at 0.05 Hz. Genistein significantly depressed the amplitudes of Aβ-fiber-evoked EPSCs tested in 14 neurons. There was no difference in the sensibility to genistein between polysynaptic responses and monosynaptic ones (Fig. 1A). As shown in Fig. 1B, the mean amplitude of Aβ-fiber-evoked EPSCs decreased by 32 ± 3% (n = 14, P<0.05) at 5 min after superfusion of genistein and still maintained at low level at 15 min after washout. In contrast, genistein did not affect Aβ-fiber-evoked synaptic responses in sham-operated rats (n = 5, Fig. 1B). To test the effect of SFKs on Aβ-fiber-mediated synaptic transmission, the selective SFKs inhibitor PP2 (20 µM) was bath applied to the spinal cord slices from SNT rats following recording of control responses. The result showed that the mean amplitude of Aβ-fiber responses decreased by 35 ± 3% (P<0.05, n = 5; Fig. 1C) at 5 min after PP2 and no significant recovery was
observed at 15 min after washout. The same concentration of the drug, however, produced little effect on Aβ-fiber-evoked EPSCs in sham-operated rats (n = 5, Fig. 1C). NMDA-receptor antagonist AP-V depressed Aβ-fiberevoked EPSCs in SNT rats To test whether NMDA-receptor channels were involved in Aβ-fiber-mediated synaptic transmission after sciatic nerve injury, the selective NMDA-receptor antagonist AP-V was perfused for 5 min after recordings of baseline. The results showed that AP-V (50 µM) reduced the mean amplitude of Aβ-fiber-evoked EPSCs by 42 ± 4% (P<0.05, n = 13; Fig. 2A). The remaining component could be further eliminated by the α-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist CNQX (10 µM, data not shown). In sham-operated rats, AP-V (50 µM) produced little effect on EPSC magnitudes (n = 5, Fig. 2B). Voltage-dependent sensibility of NMDA receptor responses was changed after nerve injury Our data showing that AP-V inhibited Aβ-fibermediated synaptic responses in SNT but not in shamoperated rats imply that the voltage-dependent property of NMDA-receptor channels may be changed after nerve injury. To test this, NMDA-receptor-mediated inward currents were recorded in sham-operated and SNT rats at the holding potentials of −70, −60, −50, −40, −30, and 0 mV. Bath application of NMDA (100 µM) for 30 s elicited the slow inward currents at all the mentioned holding potentials except 0 mV (Fig. 3: A and B). Compared with sham-operated rats, the amplitude of NMDA currents recorded in SNT rats was larger at the holding potentials of −70 mV (−23 ± 1 pA vs −29 ± 2 pA, P<0.05; Fig. 3C), while at the other mentioned holding potentials, no significant difference between these two groups of rats was detected. Inhibition of PTKs selectively depressed Aβ-fiberevoked NMDA-receptor components in SNT rats Next we directly examined the effect of PTKs inhibitors on NMDA-receptor-mediated synaptic responses, isolated by continuous perfusion of spinal cord slices with the AMPA-receptor antagonist CNQX (10 µM). Bath application of genistein for 5 min significantly reduced NMDA-receptor-mediated EPSCs elicited by Aβ-fiber stimulation (n = 9, P<0.05; Fig. 4A). Similarly, PP2 also reduced NMDA-receptor-mediated responses (P<0.05, n = 6; Fig. 4B). At 15-min after washout of genistein or PP2, NMDA-receptor-mediated EPSCs were still lower than controls. However, AMPAreceptor components evoked by Aβ-fiber stimulation,
Role of PTK in Aβ-Fiber-Evoked EPSCs
67
Fig. 1. Protein tyrosine kinase inhibitors depressed Aβ-fiber-evoked excitatory postsynaptic currents (EPSCs) recorded in lamina II neurons from sciatic nerve transection (SNT) rats but not from sham-operated rats. A: Typical Aβ-fiber-evoked mono- and polysynaptic EPSCs in response to 0.2 Hz (1) and 20 Hz (2) stimulation in SNT rats. Polysynaptic responses were determined by variable latencies and failure in the amplitude upon 20-Hz stimulation, while monosynaptic responses showed constant latencies and absence of failure even though the EPSC amplitudes decreased. Note that genistein (50 µM) reduced the amplitudes of both mono- and polysynaptic EPSCs (3). B and C: Summary data in histograms showed the effect of genistein (50 µM) and PP2 (20 µM) on the amplitudes of Aβ-fiber-evoked EPSCs recorded in SNT (left) and sham-operated (right) rats, respectively. Data are presented as the mean ± S.E.M. *P<0.05, compared with the control.
recorded in the presence of AP-V (50 µM), changed little 5 min after application of genistein (n = 7, Fig. 4C). Discussion Peripheral nerve injury triggers a series of morphological and molecular changes in spinal dorsal horn microglia and neurons, which combine to initiate and
maintain the tactile allodynia. Previous studies have indicated that nerve injury activates the microglia in the spinal dorsal horn by promoting the release of ATP from injuried neurons or astrocytes (29). The hyperactive phenotype of microglia displays the increased expression of ATP-receptor subtype P2X4 and active p38 mitogen-activated protein kinase (p38 MAPK), which are essential for tactile allodynia during neuropathic
68
X-D Hu et al
Fig. 2. Blockage of NMDA receptors reduced Aβ-fiber-evoked EPSCs recorded in lamina II neurons from SNT but not from sham-operated rats. A: The NMDA receptor antagonist AP-V (50 µM) inhibited Aβ-fiber-evoked EPSCs significantly 5 min after bath application in the slices from SNT rats. B: The same concentration of AP-V had no effect in sham-operated rats. Representative recordings of Aβ-fiber EPSCs were taken during control (1), 5 min after exposure to AP-V (2), and 15-min after washout (3). Data are presented as means ± S.E.M. *P<0.05, compared with the control.
Fig. 3. Voltage-dependent responses of lamina II neurons to exogenous NMDA recorded in lamina II neurons of sham-operated and SNT rats. Application of NMDA (100 µM) for 30 s induced a slow inward current at the holding potentials of −70 – −30 mV in sham-operated (A) and in SNT (B) rats. C: Pooled data in histograms showed that the mean amplitude of NMDA-induced slow currents recorded at −70 mV rather than at −60 – −30 mV was significantly higher in SNT rats, compared with that in sham-operated rats. Data are presented as means ± S.E.M. *: P<0.05.
Role of PTK in Aβ-Fiber-Evoked EPSCs
Fig. 4. Protein tyrosine kinase inhibitors selectively depressed Aβ-fiber-evoked NMDA-receptor current components in SNT rats. NMDA- or AMPA-receptor-mediated currents were isolated pharmacologically by blockage of AMPA receptor with CNQX (10 µM) or NMDA receptors with AP-V (50 µM), respectively. A and B: Genistein (50 µM) and PP2 (20 µM) attenuated Aβ-fiberevoked NMDA-receptor-mediated currents substantially. C: Aβfiber-evoked AMPA-receptor-mediated currents were unaffected by bath application of genistein. Representative recordings of Aβ-fiberevoked EPSCs were taken during control (1), 5-min after exposure to PTKs inhibitors (2), and 15-min after washout (3). Data are presented as means ± S.E.M. *P<0.05, compared with the control.
69
pain. Nerve injury also reorganizes the primary afferent input onto superficial dorsal horn neurons and dramatically changes the synaptic strength. The present work showed that 86% of the total 63 recorded neurons in lamina II of spinal dorsal horn exhibited Aβ-fiberevoked synaptic responses in SNT rats, while only 18% of tested neurons did so in sham-operated rats. These electrophysiological results support the notion that novel synaptic connection was established between the afferent Aβ-fibers and lamina II neurons following sciatic nerve transection (17, 18). In addition, we demonstrated for the first time that either a broadspectrum PTKs inhibitor (genistein) or a selective SFKs inhibitor (PP2) significantly depressed Aβ-fibermediated excitatory synaptic transmission in the slices from SNT rats but not from sham-operated rats. The NMDA-receptor antagonist AP-V selectively depressed Aβ-fiber-evoked EPSCs in SNT rats but not in shamoperated rats. In SNT rats, NMDA components but not AMPA components of Aβ-fiber-evoked EPSCs were selectively inhibited by genistein. These data suggest that PTKs, especially SFKs, may potentiate Aβ-fiberevoked synaptic transmission in lamina II following nerve injury via upregulation of postsynaptic NMDAreceptor function. Investigations with naive rats have demonstrated that the fast excitatory synaptic responses in lamina II neurons evoked by primary afferent Aβ-fibers are predominantly mediated by AMPA-receptor channels (30 – 32), while NMDA-receptor channel currents are not manifested at normal resting potential because of voltage-dependent Mg2+ blockage (33). In the present study, we showed that AP-V had no effect on Aβ-fiberevoked EPSCs in sham-operated rats, which is in complete agreement with a previous study showing that AP-V does not affect Aβ-fiber-evoked mono- and polysynaptic EPSCs in naive rats (34). We further demonstrated that AP-V did depress Aβ-fiber-evoked EPSCs in SNT rats significantly and that the amplitudes of NMDA-induced slow currents at the holding potential of −70 mV were larger in SNT rats than those in shamoperated rats. These data indicate that NMDA-receptor channels are involved in Aβ-fiber-mediated synaptic transmission following nerve injury, which may contribute to allodynia produced by nerve injury. The behavioral data showing that blockage of NMDA receptors alleviates allodynia (19 – 22) support this notion. Five types of SFKs (Src, Fyn, Yes, Lck, and Lyn) are expressed in mammalian central nervous system (35). Src and Fyn are shown to up-regulate NMDA-receptor function and synaptic plasticity in the spinal cord and other brain areas (23). Activation of SFKs phosphory-
70
X-D Hu et al
lates tyrosine residues on NMDA-receptor subunits NR2A (36) and NR2B (37) and other proteins in the NMDA-receptor complex such as the scaffold protein PSD-93 (38), which may increase the receptor channel opening probability and reduce voltage-dependent Mg2+ blocking. There is also evidence demonstrating that the induction of long-term potentiation in the CA1 region of the hippocampus in adult rats leads to rapid surface expression of NMDA receptors, which is prevented by the inhibition of either SFKs or protein kinase C (PKC) (39). Salter and Kalia (23) have suggested that SFKsmediated tyrosine phosphorylation of NMDA-receptor subunit might stabilize NMDA receptors on the cell surface and thereby increase NMDA-receptor response. These possible actions may explain our findings that both the broad-spectrum PTKs inhibitor genistein and SFKs selective inhibitor PP2 depressed NMDAreceptor-mediated synaptic transmission in adult SNT rats. It is likely that both up-regulation of NMDAreceptor gating and insertion of NMDA receptors into the postsynaptic membrane may contribute to the novel Aβ-fiber-mediated synaptic transmission after nerve injury. Genistein and PP2 produced a lasting depression (at least 15 min after washout) on NMDA-receptormediated synaptic responses in SNT rats as the drugs affected NMDA-receptor function indirectly, probably reducing NMDA-receptor gating and insertion by inhibition of SFKs. It may take time for SFKs to phosphorylate NMDA receptors and to drive them into the cell surface again following washout. Although there is evidence showing that SFKs are activated in superficial spinal dorsal horn after nerve injury (26), the mechanisms underlying the change are unclear. Nerve injury leads to multiple changes in spinal dorsal horn. Among them, increased release of neurotransmitter glutamate and neuromodulators such as brain-derived neurotrophic factor (BDNF) from primary afferent terminals in spinal dorsal horn is proved to be important for the initiation and maintenance of neuropathic pain (40). It has been shown that activation of G protein-coupled receptors can stimulate SFKs and PKC, leading to up-regulation of NMDA-receptor function (41). Guo et al. demonstrated that group I metabotropic glutamate receptors (mGluRs) are biochemically linked with SFKs and NMDA receptors in spinal dorsal horn neurons (42). Increased release of glutamate stimulates group I mGluRs that enhance src kinases activity and tyrosine phosphorylation of NMDA receptors during pain hypersensitivity. The concentration of BDNF in lumbar spinal dorsal horn also markedly increases after nerve lesion, which occludes in the timeline with behavioral signs of neuropathic pain (40). SFKs play a key role in linking BDNF signaling with downstream
NMDA receptors (43). In addition, ATP-stimulated hyperactive microglia in the spinal cord of neuropathic pain animals also releases a number of proinflammatory cyrokines such as interleukin-1β, which can increase NMDA-receptor function through activation of SFKs and play an important role in tactile allodynia (29, 44, 45). Taken together, multiple mechanisms may underlie activation of SFKs following nerve injury, and further studies are needed to elucidate the process. In conclusion, the present study demonstrated that SFKs were involved in Aβ-fiber-evoked synaptic transmission in SNT rats. The finding may be important for understanding the cellular mechanism underlying allodynia and SFKs may be a potential target for treatment of this neuropathic pain syndrome. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 30200076 and 30370474) and by China Medical Board (1998-766). References 1 Koltzenburg M, Torebjork H, Wahren L. Nociceptor modulated central sensitization causes mechanical hyperalgesia in acute chemogenic and chronic neuropathic pain. Brain. 1994;117:579– 591. 2 Woolf C, Doubell T. The pathophysiology of chronic painincreased sensitivity to low threshold A beta-fibre inputs. Curr Opin Neurobiol. 1994;4:525–534. 3 Field M, Bramwell S, Hughes J, Singh L. Detection of static and dynamic components of mechanical allodynia in rat models of neuropathic pain: are they signaled by distinct primary sensory neurons? Pain. 1999;83:303–311. 4 Ossipov M, Bian D, Malan T, Lai J, Porreca F. Lack of involvement of capsaicin-sensitive primary afferents in nerve-ligation injury induced tactile allodynia in rats. Pain. 1999;79:127–133. 5 Liu X, Eschenfelder S, Blenk K, Janig W, Habler H. Spontaneous activity of axotomized afferent neurons after L5 spinal nerve injury in rats. Pain. 2000;84:309–318. 6 Shir Y, Seltzer Z. A-fibers mediate mechanical hyperesthesia and allodynia and C-fibers mediate thermal hyperalgesia in a new model of causalgiform pain disorders in rats. Neurosci Lett. 1990;115:62–67. 7 Khan G, Chen S, Pan H. Role of primary afferent nerves in allodynia caused by diabetic neuropathy in rats. Neuroscience. 2002;114:291–299. 8 Pan H, Khan G, Alloway K, Chen S. Resiniferatoxin induces paradoxical changes in thermal and mechanical sensitivities in rats: mechanism of action. J Neurosci. 2003;23:2911–2919. 9 Ralston H, Ralston D. The distribution of dorsal root axons in laminae I, II and III of the macaque spinal cord: a quantitative electron microscope study. J Comp Neurol. 1979;184:643–684. 10 Sugiura Y, Lee C, Perl E. Central projections of identified, unmyelinated (C) afferent fibers innervating mammalian skin. Science. 1986;234:358–361.
Role of PTK in Aβ-Fiber-Evoked EPSCs 11 Woolf C, Shortland P, Coggeshall R. Peripheral nerve injury triggers central sprouting of myelinated afferents. Nature. 1992;355:75–78. 12 Doubell T, Mannion R, Woolf C. Intact sciatic myelinated primary afferent terminals collaterally sprout in the adult rat dorsal horn following section of a neighbouring peripheral nerve. J Comp Neurol. 1997;380:95–104. 13 Bao L, Wang H, Cai H, Tong Y, Jin S, Lu Y, et al. Peripheral axotomy induces only very limited sprouting of coarse myelinated afferents into inner lamina II of rat spinal cord. Eur J Neurosci. 2002;16:175–185. 14 Shehab S, Spike R, Todd A. Evidence against cholera toxin B subunit as a reliable tracer for sprouting of primary afferents following peripheral nerve injury. Brain Res. 2003;964:218– 227. 15 Hughes D, Scott D, Todd A, Riddell J. Lack of evidence for sprouting of A beta afferents into the superficial laminas of the spinal cord dorsal horn after nerve section. J Neurosci. 2003;23:9491–9499. 16 Kohama I, Ishikawa K, Kocsis J. Synaptic reorganization in the substantia gelatinosa after peripheral nerve neuroma formation: aberrant innervation of lamina II neurons by Abeta afferents. J Neurosci. 2000;20:1538–1549. 17 Okamoto M, Baba H, Goldstein P, Higashi H, Shimoji K, Yoshimura M. Functional reorganization of sensory pathways in the rat spinal dorsal horn following peripheral nerve injury. J Physiol. 2001;532:241–250. 18 Kohno T, Moore K, Baba H, Woolf C. Peripheral nerve injury alters excitatory synaptic transmission in lamina II of the rat dorsal horn. J Physiol. 2003;548:131–138. 19 Eide K, Stubhaug A, Oye I, Breivik H. Continuous subcutaneous administration of the N-methyl-D-aspartic acid (NMDA) receptor antagonist ketamine in the treatment of post-herpetic neuralgia. Pain. 1995;61:221–228. 20 Persson J, Axelsson G, Hallin R, Gustafsson L. Beneficial effects of ketamine in a chronic pain state with allodynia, possibly due to central sensitization. Pain. 1995;60:217–222. 21 Kim Y, Na H, Yoon Y, Han H, Ko K, Hong S. NMDA receptors are important for both mechanical and thermal allodynia from peripheral nerve injury in rats. Neuroreport. 1997;8:2149–2153. 22 Parsons C. NMDA receptors as targets for drug action in neuropathic pain. Eur J Pharmacol. 2001;429:71–78. 23 Salter M, Kalia L. Src kinases: a hub for NMDA receptor regulation. Nat Rev Neurosci. 2004;5:317–328. 24 Guo W, Zou S, Guan Y, Ikeda T, Tal M, Dubner R, et al. Tyrosine phosphorylation of the NR2B subunit of the NMDA receptor in the spinal cord during the development and maintenance of inflammatory hyperalgesia. J Neurosci. 2002;22: 6208–6217. 25 Sato E, Takano Y, Kuno Y, Takano M, Sato I. Involvement of spinal tyrosine kinase in inflammatory and N-methyl-Daspartate-induced hyperalgesia in rats. Eur J Pharmacol. 2003; 468:191–198. 26 Abe T, Matsumura S, Katano T, Mabuchi T, Takagi K, Xu L, et al. Fyn kinase-mediated phosphorylation of NMDA receptor NR2B subunit at Tyr1472 is essential for maintenance of neuropathic pain. Eur J Neurosci. 2005;22:1445–1454. 27 Zimmermann M. Ethical guidelines for investigation of experimental pain in conscious animals. Pain. 1983;16:109–110. 28 Yoshimura M, Nishi S. Primary afferent-evoked glycine- and GABA-mediated IPSPs in substantia gelatinosa neurones in the
71
rat spinal cord in vitro. J Physiol. 1995;482:29–38. 29 Inoue K, Tsuda M, Koizumi S. ATP- and adenosine-mediated signaling in the central nervous system: chronic pain and microglia: involvement of the ATP receptor P2X4. J Pharmacol Sci. 2004;94:112–114. 30 Morris R. Responses of spinal dorsal horn neurones evoked by myelinated primary afferent stimulation are blocked by excitatory amino acid antagonists acting at kainate /quisqualate receptors. Neurosci Lett. 1989;105:79–85. 31 Furue H, Narikawa K, Kumamoto E, Yoshimura M. Responsiveness of rat substantia gelatinosa neurones to mechanical but not thermal stimuli revealed by in vivo patch-clamp recording. J Physiol. 1999;521:529–535. 32 Li P, Wilding T, Kim S, Calejesan A, Huettner J, Zhuo M. Kainate- receptor-mediated sensory synaptic transmission in mammalian spinal cord. Nature. 1999;397:161–164. 33 Mayer M, Westbrook G, Guthrie P. Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones. Nature. 1984;309:261–263. 34 Baba H, Doubell T, Moore K, Woolf C. Silent NMDA receptormediated synapses are developmentally regulated in the dorsal horn of the rat spinal cord. J Neurophysiol. 2000;83:955–962. 35 Yu X, Salter M. Src, a molecular switch governing gain control of synaptic transmission mediated by N-methyl-D-aspartate receptors. Proc Natl Acad Sci U S A. 1999;96:7697–7704. 36 Lau L, Huganir R. Differential tyrosine phosphorylation of Nmethyl-D-aspartate receptor subunits. J Biol Chem. 1995;270: 20036–20041. 37 Moon I, Apperson M, Kennedy M. The major tyrosinephosphorylated protein in the postsynaptic density fraction is N-methyl-D-aspartate receptor subunit 2B. Proc Natl Acad Sci U S A. 1994;91:3954–3958. 38 Nada S, Shima T, Yanai H, Husi H, Grant S, Okada M, et al. Identification of PSD-93 as a substrate for the Src family tyrosine kinase Fyn. J Biol Chem. 2003;278:47610–47621. 39 Grosshans D, Clayton D, Coultrap S, Browning M. LTP leads to rapid surface expression of NMDA but not AMPA receptors in adult rat CA1. Nat Neurosci. 2002;5:27–33. 40 Miletic G, Miletic V. Increases in the concentration of brain derived neurotrophic factor in the lumbar spinal dorsal horn are associated with pain behavior following chronic constriction injury in rats. Neurosci Lett. 2002;319:137–140. 41 Lu W, Xiong Z, Lei S, Orser B, Dudek E, Browning M, et al. G-protein-coupled receptors act via protein kinase C and Src to regulate NMDA receptors. Nat Neurosci. 1999;2:331–338. 42 Guo W, Wei F, Zou S, Robbins M, Sugiyo S, Ikeda T, et al. Group I metabotropic glutamate receptor NMDA receptor coupling and signaling cascade mediate spinal dorsal horn NMDA receptor 2B tyrosine phosphorylation associated with inflammatory hyperalgesia. J Neurosci. 2004;24:9161–9173. 43 Yamada K, Nabeshima T. Interaction of BDNF /TrkB signaling with NMDA receptor in learning and memory. Drug News Perspect. 2004;17:435–438. 44 Viviani B, Bartesaghi S, Gardoni F, Vezzani A, Behrens M, Bartfai T, et al. Interleukin-1beta enhances NMDA receptormediated intracellular calcium increase through activation of the Src family of kinases. J Neurosci. 2003;23:8692–8700. 45 Inoue K. The function of microglia through purinergic receptors: Neuropathic pain and cytokine release. Pharmacol Ther. 2006;109:210–226.