Group I mGluR regulates the polarity of spike-timing dependent plasticity in substantia gelatinosa neurons

BBRC Biochemical and Biophysical Research Communications 347 (2006) 509–516 www.elsevier.com/locate/ybbrc

Group I mGluR regulates the polarity of spike-timing dependent plasticity in substantia gelatinosa neurons Sung Jun Jung

a,1

, Sang Jeong Kim b,1, Yun Kyung Park Kwangwook Cho d, Jun Kim b,*

b,d

, Seog Bae Oh c,

a

c

Department of Physiology, Kangwon National University College of Medicine, Chuncheon 200-701, Republic of Korea b Department of Physiology, Seoul National University College of Medicine, Seoul 110-799, Republic of Korea Department of Physiology, College of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749, Republic of Korea d Department of Biomedical Science, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK Received 19 June 2006 Available online 30 June 2006

Abstract The spinal synaptic plasticity is associated with a central sensitization of nociceptive input, which accounts for the generation of hyperalgesia in chronic pain. However, how group I metabotropic glutamate receptors (mGluRs) may operate spinal plasticity remains essentially unexplored. Here, we have identified spike-timing dependent synaptic plasticity in substantia gelatinosa (SG) neurons, using perforated patch-clamp recordings of SG neuron in a spinal cord slice preparation. In the presence of bicuculline and strychnine, longterm potentiation (LTP) was blocked by AP-5 and Ca2+ chelator BAPTA-AM. The group I mGluR antagonist AIDA, PLC inhibitor U-73122, and IP3 receptor blocker 2-APB shifted LTP to long-term depression (LTD) without affecting acute synaptic transmission. These findings provide a link between postsynaptic group I mGluR/PLC/IP3-gated Ca2+ store regulating the polarity of synaptic plasticity and spinal central sensitization.  2006 Elsevier Inc. All rights reserved. Keywords: Spike-timing dependent plasticity; Long-term potentiation; Group I metabotropic glutamate receptor; Ca2+ store; Substantia gelatinosa; Central sensitization

Substantia gelatinosa (SG) neurons in the spinal dorsal horn (DH) are the first central neurons for the relay of input from primary afferent nociceptors and have an important role in chronic pain [1]. Long-term modification of primary afferent neurotransmission in the SG is believed to play an essential role in nociceptive plasticity [1,2]. Although this synaptic plasticity in spinal cord may be associated with hyperalgesia or analgesia, the underlying cellular mechanisms have yet to be elucidated [3,4]. The spinal plasticity induced by high-frequency stimulation (HFS) to afferent fibers has been studied in vitro [3,5] and in vivo [6,7]. HFS produces either long-term potentiation (LTP), long-term *

1

Corresponding author. Fax: +82 2 763 9667. E-mail address: [email protected] (J. Kim). These authors contributed equally to this work.

0006-291X/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.06.134

depression (LTD) or no change of synaptic efficacy in superficial DH, depending on the postsynaptic potential [3,6]. A similar modification of synaptic plasticity that low-frequency stimulation (LFS) coupled with postsynaptic depolarization can induce LTP has been reported in the hippocampus and different cortical areas [8–10]. In this view, it can be suggested that spinal synaptic modification depending on postsynaptic potential may be associated with relative timing of pre- and postsynaptic activity [11,12]. Group I metabotropic glutamate receptors (mGluRs) consist of mGluR1 and 5 subtypes and couple via Gq/11 proteins primarily to activation of phospholipase C (PLC), resulting in release of Ca2+ from intracellular stores and are situated at the periphery of the postsynaptic density [13]. Postsynaptic group I mGluR is required for the induction of many forms of synaptic plasticity and

510

S.J. Jung et al. / Biochemical and Biophysical Research Communications 347 (2006) 509–516

determines the polarity of synaptic plasticity. Recent studies established that Ca2+ releases from the endoplasmic reticulum (ER), the inositol 1,4,5-triphosphate (IP3) receptor (IP3R), and the ryanodine receptor (RyR) were found to be involved in several forms of synaptic plasticity [14]. In cerebellum, mGluR1/IP3Rs mediated Ca2+ releases are critical for the induction of LTD at parallel fiber-Purkinje cell synapses [15]. In the hippocampal CA1 region, the mechanisms and role of store-induced synaptic Ca2+ signaling are controversial [16]. Because the ER in pyramidal cell spines, in contrast to cerebellar Purkinje cells, is devoid of IP3Rs, but possesses RyRs, it is likely that Ca2+ release in CA1 pyramidal neuron occurs through different mechanisms and even may serve different functional roles. In the spinal cord, behavioral studies demonstrated that group I mGluRs play a functional role in the generation of postinjury hypersensitivity and chronic pain [17–20]. However, the cellular mechanism of group I mGluRs in spinal synaptic plasticity remains essentially unexplored. Some reports demonstrated the role of group I mGluRs in spinal synaptic plasticity, which have dual modulation—LTP or LTD—of excitatory synaptic transmission in were associated with hyperalgesia or analgesia [21–25]. Here, we investigated the role of group I mGluRs in the induction of spinal LTP and their cellular mechanism. In the present study, our data suggest that Ca2+ signaling via group I mGluR/IP3-gated Ca2+ store plays an important role in the polarity of spike-timing dependent plasticity (STDP) and may be associated with hyperalgesia for chronic pain. Materials and methods Slice preparation. Sprague–Dawley rats of both sexes aged 10–14 days were used. All efforts were made to minimize the animals used, and the experiments followed the ethical guidelines of the International Association for the Study of Pain. Before decapitation, the animals were deeply anesthetized with halothane. The spinal cord was exposed by a dorsal laminectomy and dissected out. The lumbosacral segment of spinal cord was placed into ice-cold artificial cerebrospinal fluid (aCSF) and was attached to agarose block (3% in aCSF). Transverse slices (350–400 lm thick) of the lumbar spinal cord were obtained (752H, Campden Instruments, Loughbrough, UK) and then transferred in aCSF (in mM: 130 NaCl, 3 KCl, 2.5 CaCl2, 1.5 MgSO4, 1.25 NaH2PO4, 25 NaHCO3, 1.25 Hepes, 10 glucose, 20 sucrose, and pH 7.4, 310–315 mOsm, equilibrated with 95% O2 and 5% CO2) for recovery period of at least 1 h and then maintained at room temperature (22 ± 1 C) in aCSF. Electrophysiology. The SG neurons were visually identified using a fixed-stage microscope (BX50WI, Olympus, Japan) with Nomarski optics and a 40· water-immersion objective. SG was identified as a translucent band in the outer part of the DH. The recording electrodes were fabricated from Kimax-51 borosilicate capillaries (Kimble, USA) by pulling on a microelectrode puller (PP-83, Narishige, Tokyo, Japan). Patch pipettes with resistances of 4–5 MX when filled with the pipette solutions (in mM; 126 K-gluconate, 10 NaCl, 1 MgCl2, 0.5 EGTA, 2 NaATP, 0.1 MgGTP, and pH adjusted to 7.3 with KOH) were used. The membrane currents were recorded in the nystatin-perforated patch configuration using an EPC-9 amplifier and Pulse 8.30 software (both from HEKA, Germany). Nystatin (Sigma, USA) was employed as the permeable agent in the perforated patch-clamped cells, which forms voltage-insensitive ion pores in the membrane patch that are somewhat selective for cations over anions but are

impermeant to multivalentons or molecules >0.8 nm in diameter. This method, therefore, minimizes dialysis of intracellular constituents with the internal pipette solution and does not suffer from the problem of whole-cell ‘wash-out’. Monosynaptic EPSCs used in the present study were identified using two criteria. Nystatin was dissolved in dimethyl sulfoxide (DMSO) at 50 mg/ml and then added to the internal solution to yield a final nystatin concentration of 200 lg/ml. The series resistances in perforated patchclamping neurons were within 30–45 MX. Signals were filtered at 1 kHz and digitized at 3 kHz. In all experiments, neurons were voltage clamped at 60 mV except during conditioning stimuli. Orthodromic stimulation of the dorsal root entry zone (DREZ; L4) was performed with a suction electrode (Theta glass, Warner Instrument Corp., USA) and a constant current stimulus isolator (WPI, USA). The DREZ was stimulated at 0.033 Hz, 0.05 ms width of stimulation, and threshold of 20–100 lA intensity. Inhibitory GABAergic and glycinergic neurotransmission were blocked by bicuculline methiodide (10 lM, Sigma) and strychnine (1 lM, Sigma), respectively. Stimulation in the DREZ-evoked monosynaptic glutamatergic responses in SGNs. Stimulus intensity was set to evoke small, single component EPSCs or multicomponent EPSC with an early component that was well separated from the rest of the response. Monosynaptic EPSCs were identified using two criteria. First, the response latency did not change with increasing intensities of electrical stimulation, and second, response latency following high-frequency stimulation (50 Hz) was not changed. Only monosynaptic EPSCs were used in the present study. The paired-pulse depression (PPD) was measured by the two subsequent stimuli that were delivered with an inter-stimulus interval of 50 ms in place of single stimuli and averaged from five consecutive sweeps obtained under control condition and after LTP induction. PPD was calculated as the ratio of the difference between the first EPSC and the second EPSC to the first EPSC. Pairing protocol. We used correlated pre- and postsynaptic activation technique to induce synaptic modification in the SG of rat spinal slices. This technique has been well established [11]. After obtaining a stable baseline of evoked synaptic responses, correlated spiking was induced by injecting depolarizing current pulses in current clamp mode (800 pA, 2 ms) into the postsynaptic neuron to fire spikes in synchrony with a train of stimuli 1 Hz (for 100 s, intensity of test stimuli) delivered to the DREZ when the onset of excitatory postsynaptic potentials (EPSPs) preceded the peak of postsynaptic action potentials by 5 ms. Pharmacology. Chemical reagents were purchased from Tocris Cookson Ltd. (Bristol, UK), unless otherwise indicated and their sources were follows: D-2-amino-5-phosphonopentanoate (D-AP5), 2-aminoethoxydiphenylborate (2-APB), 1-[[[5-(4-Nitrophenyl)-2-furanyl]methylene]amino]2,4-imidazolinedione sodium salt (dantrolene), 1H-Pyrrole-2-carboxylic acid, (3S,4R,4aR,6S,7S,8R,8aS,8bR,9S,9aS)-dodecahydro-4,6,7,8a,8b,9ahexahydroxy-3,6a,9-trimethyl-7-(1-methylethyl)-6,9-methanobenzo[1,2]pentaleno[1,6-bc]furan-8-yl ester (ryanodine), (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), ([9S-(9a,10b,11b,13a)])-2,3,10,11-12,13-Hexahydro10-methoy-9-methyl-11-(methylamino)-9,13-epoxy-1H,9H-diindolo[1,2,3gh:3 0 ,2 0 , 1 0 -1m]pyrrolo[3,4- j][1,7]bezodiazzonin-1-one (staurosporine), cyclosporin A; 1,2-bis(2-aminophenoxy)ethane-N,N,N 0 ,N 0 -tetraacetic acid-acetoxyester (BAPTA-AM, Molecular Probes, USA); 1-[6-[[(17b)-3methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrole-2,5-dione (U-73122, Sigma), 1-[6-([(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino)hexyl]2,5- pyrrolidinedione (U-73343, Sigma). Data analysis. The eEPSC was analyzed with the Pulsefit (HEKA, Germany), and eEPSC was averaged from two successive responses. Data pooled across slices are expressed as means ± SEM and statistical significance (p < 0.05) tested using Student’s paired or unpaired t-test as appropriate.

Result Spike-timing dependent plasticity in SGN In the first series of experiments we examined whether correlated pre- and postsynaptic activation can induce

S.J. Jung et al. / Biochemical and Biophysical Research Communications 347 (2006) 509–516

LTP in the SG neuron of rat spinal slice. Using a perforated patch-clamp recording, the amplitude of synaptic currents was analyzed before and after correlated lowfrequency stimulation (LFS: 100 stimuli, 1 Hz). After a stable baseline period of at least 10 min, a pairing protocol was able to induce a persistent increase in amplitude of the excitatory postsynaptic currents (EPSCs) of SG neurons, which lasted for 50 min (161 ± 4% of baseline, n = 15, p < 0.01, Fig. 1A). In contrast, an identical pairing protocol failed to induce any significant change in synaptic efficacy when the onset of EPSPs preceded the postsynaptic action potentials by 100 ms (92 ± 13% of baseline, p > 0.05, n = 4, Fig. 1B). There was no change in EPSC in the condition of postsynaptic spiking alone or presynap-

511

tic stimulation alone did not lead to any significant change in synaptic efficacy (105 ± 6% and 98 ± 12%, postsynaptic spiking alone and presynaptic stimulation alone, respectively, n = 4, Fig. 1C). Thus, correlated pairing between pre- and postsynaptic neuronal activity is required for synaptic modification. In addition, this pairing protocol has no effect on the PPD (60 ± 3%, n = 5, control; 58 ± 6%, n = 5, after LTP induction, p > 0.05, Fig. 1D), which suggests that STDP may be associated with postsynaptic mechanisms. To examine the role of postsynaptic Ca2+ signaling in spinal STDP, we loaded a rapid Ca2+ chelator, BAPTAAM (4 mM), into the SGN through the perforated recording pipette. We found that pairing protocol

Fig. 1. Spike-timing dependent plasticity in SG. (A) Correlated spiking was induced by injecting depolarizing current pulses into the postsynaptic neuron to fire spikes in synchrony with a train of LFS. During these experiments, the series resistance of SGN remained stable (open circles, 35 MX). Synaptic traces above represent membrane currents recorded at the time points indicated (1 and 2) from SGN under voltage-clamp (60 mV). Upward arrow indicates where LFS was delivered. Postsynaptic spiking was elicited by depolarizing current pulses at 4.5 ms after onset of primary afferent-evoked monosynaptic EPSC (Dt). Scales: 50 pA and 10 ms. (B) Depolarizing current pulses in current clamp at 100 ms after onset of primary afferent-evoked monosynaptic EPSC. Scales: 100 pA and 20 ms. (C) Synaptic modification under each condition. Synchrony of both postsynaptic firing and presynaptic stimulation required for induction of LTP (*p < 0.05). These data were collected at 30 min after conditioning stimulation. (D) Analysis of paired pulse ratio in SG neurons. Fifty milliseconds interpulse-intervals induce paired-pulse depression (PPD). There was no change in PPD before and after spike timing dependent plasticity. Scales: 20 pA, 10 ms. All data points represent the mean normalized EPSC amplitude (±SEM).

512

S.J. Jung et al. / Biochemical and Biophysical Research Communications 347 (2006) 509–516

B 150 1

Normalized EPSC (%)

Normalized EPSC (%)

A 150 2

100

50

1+2

D-AP5 1

2

100

1+2

50

+BAPTA 0

0 -10

0

10 20 Time (min)

30

-10

40

0

10 20 Time (min)

30

40

Fig. 2. Requirement of NMDA R-activation and [Ca2+]i increase of SGN for STDP. (A) Summary of experiments in which the SGN was loaded with BATA-AM (4 mM, open circles). Postsynaptic inclusion of BAPTA-AM (4 mM) occluded induction of LTP. (B) Experiments similar to that in Fig. 1A, except for the presence of D-AP5 (50 lM, filled circles). Each point represents the mean normalized EPSC amplitude. In the presence of D-AP5, synaptic modification was not altered, indicating STDP was dependent on NMDAR. The percentage change in the mean normalized EPSC amplitude (at t = 30 min) did not alter. Inset traces are taken at the times indicated (1 and 2). Scales: 25 pA, 10 ms. All data points represent the mean normalized EPSC amplitude (±SEM).

20 min following the onset of BAPTA-AM loading failed to induce LTP (100 ± 4%, n = 7, Fig. 2A), indicating that STDP was dependent on the postsynaptic [Ca2+]i. Also, bath application of a NMDA antagonist D-AP5 (50 lM) also blocked LTP (92 ± 4%, n = 7, Fig. 2B). These results indicate that the change in synaptic efficacy, such as induction of LTP, requires the activation of NMDA receptors. A

At excitatory synapses, activation of group I mGluR induces PLC/IP3 mediated Ca2+ release from internal Ca2+ stores. To assess whether the activation of group I mGluRs is involved in STDP, we explored the role of group I mGluRs in STDP (Fig. 3A). In the condition of bath application of the group I mGluR antagonist AIDA

1

2

100

2

AIDA + D-AP5

50

0

AIDA -20

-10

0 10 20 Time (min)

30

40

B 200 Normalized EPSC (%)

1+2

1

AIDA (+ D-AP5)

150 Normalized EPSC (%)

The activation of group I mGluR is required for STDP of SG

1+2

1 2

150 1 U-73122 100 50 0

U-73343 -10

0

10

20

30

40

Time (min) Fig. 3. The involvement of group I mGluR in STDP. (A) Inhibition of group I mGluR by AIDA resulted in LTD (open circles), which was blocked by D-AP5 (filled circles). Inset traces are taken at the times indicated (1 and 2). Scales: AIDA, 25 pA, 25 ms; AIDA+D-AP5, 50 pA, 25 ms. (B) Inhibition of PLC-cascade induced LTD (filled circles), whereas application of inactive PLC inhibitor (U-73343) had no effect on LTP (open circles). Scales: U-73122, 10 pA, 20 ms; U-73343, 20 pA, 20 ms.

S.J. Jung et al. / Biochemical and Biophysical Research Communications 347 (2006) 509–516

Normalized EPSC (%)

A 200

Dan or Ry

1

1+2

2 150 1

Dan

100 50 0

Ry -10

B 150 Normalized EPSC (%)

513

0

10 20 Time (min)

30

40

1

2-APB (ext.)

1+2

1 100

2-APB (int.)

2 50

0

2-APB (ext.) -10

0

10

20

30

40

Time (min) 2+

2+

Fig. 4. The role of Ca release from Ca stores in STDP in SG neurons. (A) Ryanodine receptor blockers (Dan, dantrolene and Ry, ryanodine; filled circles and open circles, respectively) had no effect on spike time dependent LTP. Scales: dantrolene, 50 pA, 25 ms; ryanodine, 100 pA, 25 ms. (B) Bath application of IP3Rs blocker (2-APB) resulted in LTD instead of LTP (open circles). For 2-APB loading to SGN, the conversion of LTP to LTD was observed (filled circles). Scales: postsynaptic inclusion of 2-APB, 50 pA, 25 ms; bath application of 2-APB, 25 pA, 25 ms.

(100 lM), pairing protocol induced LTD rather than LTP (68 ± 5%, n = 5, open circles, Fig. 3B). In addition, when both AIDA and AP5 were bath applied, any synaptic changes by pairing protocol were not observed (93 ± 8%, n = 5, filled circles, Fig. 3B). Therefore, we analyzed the role of the PLC signaling, which is considered as downstream cascades of group I mGluR, in STDP. Bath application of U-73122 (10 lM), an inhibitor of PLC, resulted in LTD (53 ± 6%, n = 7, filled circles; Fig. 3B), whereas U-73343 (10 lM, an inactive isoform) had no effect on the induction of LTP (136 ± 3%, n = 7, open circles, Fig. 3B). This result was consistent with that of AIDA. Taken together, it suggests that the activation of group I mGluR is necessary to induce LTP, and modulation of group I mGluRs has an important role in changes in the polarity of STDP. The role of intracellular Ca2+ stores in STDP of SG In the next series of experiments, we investigated whether Ca2+ mobilization from intracellular Ca2+ stores is required for LTP. Bath application of dantrolene (10 lM) or ryanodine (100 lM), blockers of RyR-mediated Ca2+ signaling, had no effect on LTP (149 ± 5%, n = 7, dantrolene; 133 ± 5%, n = 7, ryanodine; Fig. 4A). However, blocking IP3R-mediated Ca2+ mobilization with IP3R blocker 2-APB (20 lM) induced LTD rather than LTP

(54 ± 4%, n = 7, open circles, Fig. 4B). Moreover, inclusion of 100 lM 2-APB in the pipette solution also resulted in LTD (57 ± 5%, n = 7; filled circles; Fig. 4B). Taken together, these data indicate that PLC induced formation of IP3 and subsequent release of Ca2+ from IP3-gated Ca2+ store is responsible for the LTP observed and the polarity of STDP is dependent on the activation of IP3-gated Ca2+ store. Different mechanisms are involved in the regulation of the polarity of STDP Following demonstration of above results, we asked how the same protocol induced a wide variety of synaptic plasticity. One possible reason is that a rise of postsynaptic [Ca2+]i by pairing protocol may regulate the polarity of STDP. This is the previous proposal that the bi-directional control of synaptic plasticity relies on increase in postsynaptic [Ca2+]i to different level, which can activate different induction mechanisms, such as protein kinases and phosphatase [9,10]. To confirm this possibility, we investigated whether LTP or LTD was blocked by the protein kinase inhibitor, staurosporine (2 lM) or the protein phosphatase 2B (PP2B) inhibitor, cyclosporine A (20 lM). In Fig. 5A, LTP was blocked by staurosporine (101 ± 1%, n = 7, filled circles), not by cyclosporine A (144 ± 8%, n = 7, open circles), indicating that LTP is mediated by the activation of

514

S.J. Jung et al. / Biochemical and Biophysical Research Communications 347 (2006) 509–516

A

B

2 150

1 100 50

1+2 Cyclosporin

0

+ AIDA 150

Staurosporine Normalized EPSC (%)

Normalized EPSC (%)

200

1+2

1+2 Staurosporine

100 1 50

2

1+2

Cyclosporin 0

-10

0

10

20

30

40

Time (min)

-10

0

10

20

30

40

Time (min)

Fig. 5. The polarity of STDP is regulated by different mechanisms. Staurosporine, a PKC inhibitor, loading to SGN blocked LTP (filled circles), while cyclosporin A, a phosphatase 2B inhibitor, did not affect LTP (open circles). (B) LTD in the presence of AIDA is blocked by cyclosporin A (open circles), but not staurosporine (filled circles). Each point represents the mean normalized EPSC amplitude. Scales: (A) staurosporine, 200 pA, 10 ms; cyclosporin A, 100 pA, 10 ms, (B) staurosporine, 200 pA, 10 ms; cyclosporin A, 200 pA, 10 ms.

protein kinase, not PP2B. In the next series of experiments, we found that cyclosporine A prevented the induction of LTD in the presence of AIDA (102 ± 6%, n = 7, open circles, Fig. 5B), while staurosporine had no effect on LTD (78 ± 3%, n = 7, filled circles, Fig. 5B,). Taking the staurosporine and cyclosporine A results together suggest that this bi-directional synaptic plasticity was induced by different cellular mechanisms. Discussion The present study addressed the role of group I mGluRs signal transduction in the induction of LTP in the SG of the rat spinal cord. We demonstrated here that group I mGluR/PLC/IP3-gated Ca2+ store regulates the polarity of STDP in the spinal cord. Presynaptic stimulation paired with single postsynaptic action potential induced spinal LTP, and this plastic change was shifted to LTD by the block of group I mGluR/PLC/IP3 cascade. Thus, the pairing protocol can induce LTP through the activation of protein kinases, whereas the magnitude of [Ca2+]i by blocking group I mGluR/IP3-gated Ca2+ store is sufficient for only LTD induction via activation of phosphatases. Synaptic modification of excitatory connections between primary afferents and SG neurons is likely to play an important role in the plasticity of nociceptive spinal synaptic transmission [1,2]. Evidence that the spinal LTP is associated with hyperalgesia has been accumulating. Most of such experiments used HFS for induction LTP. However, this HFS has been criticized because of the physiological features of DH neurons. Generally, C-fibers in the spinal cord do not fire more than a few spikes at HFS required experimentally to induce the LTP [2]. Thus, various responses of DH neuron to HFS of a primary afferent might be explained by the complexity in coupling between presynaptic inputs and postsynaptic spikes. Because DH neurons have the convergent inputs from neighboring thin myelinated Ad- and

unmyelinated C-fiber, an appropriate cooperativity between residual postsynaptic depolarization by Ad-input and activation of C-fiber is necessary for the successful induction of LTP. Recent studies have shown that preand postsynaptic correlated activation of synaptic connections can lead to LTP or LTD of excitatory synaptic transmission in a variety of brain area [11,12]. In our previous in vivo study [6], it was proposed that coincident depolarization of the postsynaptic DH neurons during peripheral conditioning stimuli may be necessary for successful induction of LTP. Like in vivo experiments, this result suggests that LTP depends on the correlation of pre- and postsynaptic activities. This finding is also associated with the role of postsynaptic potential in the polarity of synaptic plasticity, suggesting that the cooperation of the back propagation of action potentials generated by fast Ad-fiber inputs (postsynaptic spiking) and the slow C-fiber input (presynaptic stimulation) is involved in spinal STDP. Therefore we hypothesize that spike-timing in Ad- and C-fibers is crucial and is a possible physiological scenario for the production of activity-dependent synaptic plasticity. What is the cellular mechanism underlying the spinal STDP? We found that a group I mGluR/PLC/IP3-Ca2+ signaling is involved in spinal STDP. Group I mGluRs are coupled via Gq/11 proteins primarily to activation of PLC, resulting in phosphoinositide hydrolysis, Ca2+ release from Ca2+ stores, and activation of PKC [26]. Thus, Group I mGluRs have been postulated to play a role in many forms of synaptic plasticity in a various brain areas [26]. Previous studies suggest an important role of group I mGluRs at the level of the spinal cord in the modulation of nociceptive transmission and plasticity [2,21,27]. Group I mGluRs are crucial in inflammation-induced hyperexcitability of spinal cord neurons [28], and its agonists induce nociceptive behaviors, thermal hyperalgesia, and mechanical allodynia [18]. Also, intrathecal group I mGluR antagonists reduce neuropathic pain [19]. Although behavioral study demonstrated that group I mGluR was associated

S.J. Jung et al. / Biochemical and Biophysical Research Communications 347 (2006) 509–516

with chronic pain, the cellular mechanism for group I mGluRs in spinal plasticity was controversial. Recently, it was reported that the activation of mGluR5 may contribute to the induction of LTP in trigeminal synapses [23] and that immunopositive reaction for mGluR5 was confined to lamina I and II [29], which were consistent with ours. This discrepancy between studies might be caused by whether cell types located at lamina I and II or not and by what kind of conditioning stimuli was applied. Interestingly, an identical pairing protocol can induce LTD under some conditions (in the presence of AIDA, 2-APB, and U-73122). We can implicate possibility that this shift of LTP to LTD may be due to the magnitude of [Ca2+]i depending on the recruitment of Ca2+ source, such as NMDAR, Ca2+ channel, and Ca2+ stores. It is generally believed that protein kinases and phosphatases differ in their affinity for Ca2+ and postsynaptic Ca2+ elevation determines whether LTP or LTD is observed [9]. The phosphatase calcineurin B has a higher affinity for Ca2+ (0.3 lM) than either PKC (5 lM). Thus, sustained lowlevel Ca2+ elevation may selectively activate phosphatases and result in LTD, whereas transient high-level Ca2+ elevation may lead to activation of certain protein kinases and LTP [30]. Our results suggest that each LTP and LTD possess their unique zone of [Ca2+]i and different cellular mechanism depending on [Ca2+]i. Block of group I mGluR, which inhibited the release Ca2+ from IP3-gated Ca2+ stores, induced LTD through phosphatase activation. Furthermore, the block of LTP by staurosporine did not result in the uncovering of LTD. Therefore, high-level [Ca2+]i can produce LTP by phosphorylation via PKC/CaMKII, while sustained low-level [Ca2+]i can induce LTD by activation of phosphatase. The previous study for pain processing reported this possibility. Animals with central sensitization have enhanced [Ca2+]i in spinal neurons, and require activation of Ca2+-dependent protein kinase II [31], which may lead to phosphorylation of GluR1 subunit of AMPA receptor and enhance glutamatergic synaptic transmission [32]. On the other hand, spinal LTD is associated with the activation of Ca2+-dependent phosphatases [33]. Importantly, our study found a fundamental role for group I mGluRs in the polarity of STDP. Here we observed that spike-timing dependent LTP is mediated through the activation of group I mGluRs, which induced a rise in [Ca2+]i through PLC/IP3-gated Ca2+ stores, but induced LTD when mGuRs are inactivated. This underlying role of group I mGluRs in the polarity of STDP has shed new light on the mechanisms of chronic pain and suggests possible therapeutic strategies for the future. Acknowledgments This study was supported by the Brain Research Center of the 21st Century Frontier Research Program Grant funded by the Korean Ministry of Science and Technology (Grant No. M103KV010009-04K2201-00920) and by the Korea Research Foundation Grant funded by the Korean

515

Government (MOEHRD, Basic Research Promotion Fund) (KRF-2003-015-E00146). References [1] C.J. Woolf, M.W. Salter, Neuronal plasticity: increasing the gain in pain, Science 288 (2000) 1765–1769. [2] R.R. Ji, T. Kohno, K.A. Moore, C.J. Woolf, Central sensitization and LTP: do pain and memory share similar mechanisms? Trends Neurosci. 26 (2003) 696–705. [3] M. Randic, M.C. Jiang, R. Cerne, Long-term potentiation and longterm depression of primary afferent neurotransmission in the rat spinal cord, J. Neurosci. 13 (1993) 5228–5241. [4] W.D. Willis Jr., Is central sensitization of nociceptive transmission in the spinal cord a variety of long-term potentiation? Neuroreport 8 (1997) iii. [5] H. Ikeda, B. Heinke, R. Ruscheweyh, J. Sandkuhler, Synaptic plasticity in spinal lamina I projection neurons that mediate hyperalgesia, Science 299 (2003) 1237–1240. [6] D.K. Kim, S.J. Jung, S.J. Kim, J. Kwak, J. Kim, Dependence of longterm potentiation on the interval between A- and C-responses of the spinal dorsal horn neurons in rats, Neurosci. Lett. 348 (2003) 33–36. [7] F. Svendsen, A. Tjolsen, J. Gjerstad, K. Hole, Long term potentiation of single WDR neurons in spinalized rats, Brain Res. 816 (1999) 487– 492. [8] A. Artola, S. Brocher, W. Singer, Different voltage-dependent thresholds for inducing long-term depression and long-term potentiation in slices of rat visual cortex, Nature 347 (1990) 69–72. [9] A. Artola, W. Singer, Long-term depression of excitatory synaptic transmission and its relationship to long-term potentiation, Trends Neurosci. 16 (1993) 480–487. [10] K. Cho, J.P. Aggleton, M.W. Brown, Z.I. Bashir, An experimental test of the role of postsynaptic calcium levels in determining synaptic strength using perirhinal cortex of rat, J. Physiol. 532 (2001) 459–466. [11] G. Bi, M. Poo, Synaptic modification by correlated activity: Hebb’s postulate revisited, Annu. Rev. Neurosci. 24 (2001) 139–166. [12] P.J. Sjostrom, S.B. Nelson, Spike timing, calcium signals and synaptic plasticity, Curr. Opin. Neurobiol. 12 (2002) 305–314. [13] R. Lujan, J.D. Roberts, R. Shigemoto, H. Ohishi, P. Somogyi, Differential plasma membrane distribution of metabotropic glutamate receptors mGluR1 a, mGluR2 and mGluR5, relative to neurotransmitter release sites, J. Chem. Neuroanat. 13 (1997) 219–241. [14] C.R. Rose, A. Konnerth, Stores not just for storage. Intracellular calcium release and synaptic plasticity, Neuron 31 (2001) 519–522. [15] E.A. Finch, G.J. Augustine, Local calcium signalling by inositol-1,4,5trisphosphate in Purkinje cell dendrites, Nature 396 (1998) 753–756. [16] M. Nishiyama, K. Hong, K. Mikoshiba, M.M. Poo, K. Kato, Calcium stores regulate the polarity and input specificity of synaptic modification, Nature 408 (2000) 584–588. [17] K. Fisher, T.J. Coderre, The contribution of metabotropic glutamate receptors (mGluRs) to formalin-induced nociception, Pain 68 (1996) 255–263. [18] K. Fisher, T.J. Coderre, Hyperalgesia and allodynia induced by intrathecal (RS)-dihydroxyphenylglycine in rats, Neuroreport 9 (1998) 1169–1172. [19] K. Fisher, M.E. Fundytus, C.M. Cahill, T.J. Coderre, Intrathecal administration of the mGluR compound, (S)-4CPG, attenuates hyperalgesia and allodynia associated with sciatic nerve constriction injury in rats, Pain 77 (1998) 59–66. [20] C.D. Mills, K.M. Johnson, C.E. Hulsebosch, Group I metabotropic glutamate receptors in spinal cord injury: roles in neuroprotection and the development of chronic central pain, J. Neurotrauma 19 (2002) 23–42. [21] J. Zhong, G. Gerber, L. Kojic, M. Randic, Dual modulation of excitatory synaptic transmission by agonists at group I metabotropic glutamate receptors in the rat spinal dorsal horn, Brain Res. 887 (2000) 359–377.

516

S.J. Jung et al. / Biochemical and Biophysical Research Communications 347 (2006) 509–516

[22] Y.K. Park, J. Galik, P.D. Ryu, M. Randic, Activation of presynaptic group I metabotropic glutamate receptors enhances glutamate release in the rat spinal cord substantia gelatinosa, Neurosci. Lett. 361 (2004) 220–224. [23] Y.C. Liang, C.C. Huang, K.S. Hsu, Characterization of long-term potentiation of primary afferent transmission at trigeminal synapses of juvenile rats: essential role of subtype 5 metabotropic glutamate receptors, Pain 114 (2005) 417–428. [24] J.J. Azkue, X.G. Liu, M. Zimmermann, J. Sandkuhler, Induction of long-term potentiation of C fibre-evoked spinal field potentials requires recruitment of group I, but not group II/III metabotropic glutamate receptors, Pain 106 (2003) 373–379. [25] J. Chen, B. Heinke, J. Sandkuhler, Activation of group I metabotropic glutamate receptors induces long-term depression at sensory synapses in superficial spinal dorsal horn, Neuropharmacology 39 (2000) 2231–2243. [26] R. Anwyl, Metabotropic glutamate receptors: electrophysiological properties and role in plasticity, Brain Res. Brain Res. Rev. 29 (1999) 83–120. [27] G. Gerber, D.H. Youn, C.H. Hsu, D. Isaev, M. Randic, Spinal dorsal horn synaptic plasticity: involvement of group I metabotropic glutamate receptors, Prog. Brain Res. 129 (2000) 115–134.

[28] V. Neugebauer, T. Lucke, H.G. Schaible, Requirement of metabotropic glutamate receptors for the generation of inflammation-evoked hyperexcitability in rat spinal cord neurons, Eur. J. Neurosci. 6 (1994) 1179–1186. [29] F.J. Alvarez, R.M. Villalba, P.A. Carr, P. Grandes, P.M. Somohano, Differential distribution of metabotropic glutamate receptors 1a, 1b, and 5 in the rat spinal cord, J. Comp. Neurol. 422 (2000) 464– 487. [30] C.M. Coussens, T.J. Teyler, Protein kinase and phosphatase activity regulate the form of synaptic plasticity expressed, Synapse 24 (1996) 97–103. [31] L. Fang, J. Wu, Q. Lin, W.D. Willis, Calcium-calmodulin-dependent protein kinase II contributes to spinal cord central sensitization, J. Neurosci. 22 (2002) 4196–4204. [32] L. Fang, J. Wu, X. Zhang, Q. Lin, W.D. Willis, Increased phosphorylation of the GluR1 subunit of spinal cord a-amino-3hydroxy-5-methyl-4-isoxazole propionate receptor in rats following intradermal injection of capsaicin, Neuroscience 122 (2003) 237–245. [33] G. Cheng, M. Randic, Involvement of intracellular calcium and protein phosphatases in long-term depression of A-fiber-mediated primary afferent neurotransmission, Brain Res. Dev. Brain Res. 144 (2003) 73–82.