FASEB J. Mp Biomedicals
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published as doi: 10.1096/fj.08-114462.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
fj.08-114462v1
23/1/90    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, W.-T.
Right arrow Articles by Song, X.-J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, W.-T.
Right arrow Articles by Song, X.-J.
(The FASEB Journal. 2009;23:90-98.)
© 2009 FASEB

EphB receptor signaling in mouse spinal cord contributes to physical dependence on morphine

Wen-Tao Liu*, Hao-Chuan Li*, Xue-Song Song*, Zhi-Jiang Huang* and Xue-Jun Song*,{dagger},1

* Department of Neurobiology, Parker University Research Institute, Dallas, Texas, USA; and

{dagger} Jiangsu Province Key Laboratory of Anesthesiology and Center for Pain Research and Treatment, Xuzhou Medical College, Xuzhou, Jiangsu, China

1 Correspondence: Department of Neurobiology, Parker University Research Institute, 2500 Walnut Hill Lane, Dallas, TX 75229, USA. E-mail: song{at}parkercc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cellular and molecular mechanisms underlying opioid tolerance and dependence remain elusive. We investigated roles of EphB receptor tyrosine kinases—which play important roles in synaptic connection and plasticity during development and in the matured nervous system—in development and maintenance of physical dependence on morphine in the mouse spinal cord (SC). Spinal administration of an EphB receptor blocking reagent EphB2-Fc prevents and/or suppresses behavioral responses to morphine withdrawal and associated induction of c-Fos and depletion of calcitonin gene-related peptide. Western blotting and immunohistochemical fluorescence staining demonstrates that EphB1 receptor protein is significantly up-regulated in the spinal dorsal horn following escalating morphine treatment. Chronic morphine exposure and withdrawal significantly increased phosphorylation of N-methyl-D-aspartate receptor subunit NR2B as well as the activated forms of extracellular signal-regulated kinase and the cAMP response element binding protein in SC. The increased levels of phosphorylation of these molecules, however, are significantly inhibited by the EphB receptor blocker. These findings indicate that EphB receptor signaling, probably by interacting with NR2B in SC, contributes to the development of opioid physical dependence and withdrawal effects. This novel role for EphB receptor signaling suggests that these molecules may be useful therapeutic targets for preventing, minimizing, or reversing the development of opiate dependence.—Liu, W.-T., Li, H.-C., Song, X.-S., Huang, Z.-J., Song, X.-J. EphB receptor signaling in mouse spinal cord contributes to physical dependence on morphine.


Key Words: opiate withdrawal • NR2B • ERK • CREB • c-Fos


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OPIOID DRUGS ARE USED AND ABUSED for their analgesic and rewarding properties. Repeated use of opioids such as morphine for relief of chronic pain can lead to opiate tolerance and dependence. Mechanisms of opiate tolerance and dependence are complex and involve factors at the levels of the drug receptor, the cell, and neural networks. Roles of diverse neurotransmitter and receptor systems and intracellular signaling proteins in acute and chronic opioid actions have been demonstrated (1 2 3 4 5 6) . The most intensively studied system is the glutamate/N-methyl-D-aspartate receptors (NMDAR)/nitric oxide (NO) cascade (4 , 5 , 7 8 9 10 11) . Adaptive changes following chronic opioid exposure that might underlie physical dependence by altering neuronal excitability and synaptic transmission include a withdrawal-induced rebound increase in cAMP levels and in expression of the cAMP response element binding protein (CREB) (12 13 14) and the mitogen-activated protein kinases (MAPKs) (15) . The capacity of agonists to recruit various µ-opioid receptor (MOR) regulatory events has recently been suggested to be a major determinant of their propensity to induce both tolerance and dependence (1) . Despite decades of investigation, the specific cellular and molecular mechanisms underlying opioid tolerance and withdrawal-induced pain enhancement remain elusive. One general possibility is that repetitive or prolonged MOR activation may elicit neuronal alterations that recapitulate events during development (16) , including the promotion of synapse formation.

Eph receptors constitute the largest subfamily of receptor tyrosine kinases (RTKs), and they play vital roles in transmitting external signals to the inside of many types of cells. Eph RTKs and ephrins are involved in tissue-border formation, cell migration, and axon guidance during development of the nervous system (17 18 19 20) . EphB receptors can also regulate the development and remodeling of glutamatergic synaptic connections and their plasticity in adult nervous system by interaction with NMDARs (16 , 21 22 23 24 25) . NMDARs containing both NR1 and NR2 subunits have an established role in neural plasticity and are fundamental mediators of expression, development, and maintenance of opiate tolerance, dependence, and withdrawal (1 , 26 27 28 29 30) . The opiate systems interact with NMDARs such that MOR activation results in Ca2+ influx through the NMDAR ion-channel complex. The subsequent activation of various Ca2+-dependent enzymes, such as Ca2+/calmodulin-dependent kinase (CaMK) (31 32 33) and extracellular signal-regulated kinase (ERK) (34) play a central role in the induction of persistent opioid effects (29) .

Recent studies have further demonstrated that peripheral inflammation and/or nerve injury enhances ephrinB-EphB receptor signaling (35 , 36) , and such signaling may contribute to inflammatory and neuropathic pain by altering neural excitability and synaptic transmission via interaction with NMDARs in the spinal cord (SC) (36 37 38) . Several lines of evidence indicate that the spinal dorsal horn (DH), the first central relay station for processing nociceptive information, is an important site in the development of opioid dependence and withdrawal (4 , 27 , 28 , 39 40 41 42 43 44 45) . Therefore, we hypothesized that EphB receptor signaling might be involved in development and/or maintenance of opiate physical dependence in SC. The present study provides the first evidence that activation of EphB receptor signaling may play a critical role in the development of morphine dependence and withdrawal and suggests that EphB receptor signaling may be a potential therapeutic target for preventing, minimizing, or reversing opioid tolerance and dependence.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Male CD-1 mice (25–30 g wt, n=237; Charles River Laboratories, MA, USA) were used in this study. All the experimental procedures were conducted in accordance with the regulations of the ethics committee of the International Association for the Study of Pain and approved by the Parker Research Institute Animal Care and Use Committee.

Opiate withdrawal
Mice were injected (i.p.) with repeated pulses of morphine (Sigma, St. Louis, MO, USA) given in 7 escalating doses every 8 h (20, 40, 60, 80, 100, 100, and 100 mg/kg). Two hours after the last morphine injection, mice were injected with naloxone (Sigma; 1 mg/kg, s.c.), and withdrawal symptoms were monitored for 30 min after naloxone administration. In addition to measuring individual withdrawal signs, an overall opiate withdrawal score was calculated as (number of backward walking steps x 0.1) + (diarrhea x 2) + (number of jumps x 0.1) + (paw tremor x 0.1) + ptosis + tremor + (percentage of weight loss x 5) + number of wet-dog shakes (6) . An EphB receptor blocking reagent EphB2-Fc (1 or 2 µg, E9402; Sigma), human IgG-Fc (IgG-Fc, 1 or 2 µg, Jackson Laboratory, Bar Harbor, ME, USA), and PBS were administrated [intrathecal (i.t.) injection, all in 5 µl] in vivo in either 7 pulses accompanying morphine or 1 pulse 30 min before naloxone.

Pain threshold and morphine analgesia tests
To test possible effects of EphB2-Fc and IgG-Fc on the pain threshold and the initial analgesic response to morphine, mice were placed on a 55°C hot-plate apparatus, and the latency to lick a paw was measured. A cutoff time of 30 s was set to avoid tissue damage. Mice were tested every 30 min for 2 or 2.5 h. Data were calculated as percentage of maximal possible effect (%MPE), which was calculated by the following formula: 100% x [(drug response time – basal response time)/(30 s – basal response time)] = %MPE. Morphine was administered (10 mg/kg, s.c.) 30 min before testing. EphB2-Fc and IgG-Fc (2 µg, i.t.) were administered at time 0 in Fig. 1C and together with morphine in Fig. 1D .


Figure 1
View larger version (31K):
[in this window]
[in a new window]

  		Figure 1. EphB2-Fc attenuates behavioral signs of morphine withdrawal. A) Effects of EphB2-Fc on the behavioral signs of naloxone-precipitated morphine withdrawal. In addition to receiving morphine and naloxone, the various groups received one of the following treatments (i.t.): control 1 (Ctrl-1), no additional treatment; control 2 (Ctrl-2), PBS; 7EphB, 7 pulses of EphB2-Fc accompanied morphine; 1EphB, 1 pulse of EphB2-Fc (2 µg, 5 min after the last morphine); 7Fc, 7 pulses of IgG-Fc (1 µg accompanied morphine). B) Overall withdrawal scores of each group. C) Effects of EphB2-Fc (2 µg) and IgG-Fc (2 µg) on the pain threshold in naive mice. D) Effects of EphB2-Fc (2 µg) and IgG-Fc (2 µg) on the initial analgesic response to morphine (Mor). PBS was used as control in both C and D. *P < 0.05, **P < 0.01 vs. Ctrl-1; #P < 0.05 vs. 7EphB.

Immunohistochemical and immunofluorescence staining of c-Fos, calcitonin gene-related peptide (CGRP), and EphB1 receptor
The immunofluorescence staining was performed as previously described (46) . In brief, the lumbar segment of the SC was dissected out and postfixed, and then the embedded blocks were sectioned (10 µm thick). Sections from each group (5 mice/group) were incubated with rabbit anti-c-Fos polyclonal antibody (1:100), rabbit anti-EphB1 polyclonal antibody (1:200; both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and rabbit anti-CGRP polyclonal antibody (1:1000; Millipore, Billerica, MA, USA), respectively. Rabbit IgG (1:200; Vector Laboratories, Inc., Burlingame, CA, USA) was used as an isotype control. The morphological details of the immunofluorescence staining on SC were studied under a fluorescence microscope (Olympus BX51WI; Olympus America Inc., Melville, NY, USA). Images were randomly coded and transferred to a computer for further analysis. Fos-immunoreactive neurons were counted in blind fashion. The number of Fos-like immunoreactive neurons in spinal DH (laminae I–VI) was determined by averaging the counts made in 20 SC sections (L4–L5) for each group. To obtain quantitative measurements of CGRP immunofluorescence, 15–20 fields covering the entire DH in each group were evaluated and photographed at the same exposure time to generate the raw data. Fluorescence intensities of the different groups were analyzed using MicroSuite image analysis software (Olympus America Inc.). The average green fluorescence intensity of each pixel was normalized to the background intensity in the same image.

Western blot analysis
Western blot analysis was used to detect expression of EphB receptors, ephrinBs, and phosphorylated NR2B (pNR2B), ERK (pERK), and CREB (pCREB) proteins in the SC. In total, 108 mice were used for Western blotting experiments (SCs from 4 mice were pooled for each sample; each group consisted of 3 samples). The procedure used to quantify temporal changes in protein levels was similar to that previously described (47) . EphB1 was immunoprecipitated from 2 mg total protein/ml tissue lysate using an anti-EphB1 antibody (2 µg; Q-20, Santa Cruz Biotechnology), and ephrinBs were immunoprecipitated from 2 mg total protein/ml tissue lysate using the Protein G-Agarose (Invitrogen, Carlsbad, CA, USA) linked to agarose (50 µl of 50% slurry, 5 mg/ml binding capacity; Sigma), respectively. EphB1 and ephrin-B proteins were dissociated by heating at 100°C for 5 min in sample buffer [2% sodium dodecyl sulfate (SDS), 100 mM dithiothreitol, 10% glycerol, and 0.02% bromphenol blue] before loading on 8% or 10% SDS-polyacrylamide gels to resolve protein bands. For the pNR2B, pCREB, pERK, GAPDH Western blot analysis, whole-cell protein extract lysates were used. After transfer to nitrocellulose filters, the filters were blocked with 2% BSA and then incubated overnight at 4°C with the primary antibodies [EphB1 1:100, Q20, ephrinB1 1:100, H70, ephrinB2 1:100, C20, PY99 1:100, and pCREB (Ser-133) from Santa Cruz Biotechnology; pNR2B (Tyr 1472) 1:300 from Chemicon (Temecula, CA, USA); pERK1/2 (Thr202/Tyr204) 1:500 from Cell Signaling Technology (Danvers, MA, USA); GAPDH 1:1000 from Sigma]. The filters were developed using enhanced chemiluminescence reagents (PerkinElmer, Waltham MA, USA) with secondary antibodies from Chemicon. Data were analyzed with a molecular imager (Gel Doc XR, 170-8170) and the associated software Quantity One-4.6.5 (Bio-Rad Laboratories, Hercules, CA, USA).

Statistics
SPSS 15 (SPSS Inc., Chicago, IL, USA) was used to conduct all the statistical analyses. Alteration of expression of the proteins detected, the behavioral responses to morphine withdrawal, and the differences in latency over time among groups were tested with 1-way and 2-way ANOVA, respectively, with repeated measures followed by Bonferroni post hoc tests. All data are presented as means ± SE. Statistical results are considered significant if P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
EphB2-Fc prevents and suppresses behavioral signs of naloxone-precipitated morphine withdrawal
Naloxone-precipitated morphine withdrawal results in a characteristic morbidity, including anxiety, nausea, insomnia, hot and cold flashes, muscle aches, perspiration, and diarrhea. Intrathecal injection of a reagent that can block activation of EphB receptors, the chimeric molecule EphB2-Fc, significantly attenuated most of the morphine withdrawal signs and the overall withdrawal score in mice. As shown in Fig. 1A , repeated coapplication of EphB2-Fc (1 µg during each of the 7 morphine doses) significantly attenuated the backward walking, chewing, diarrhea, jump, tremor, wet-dog shake, and weight loss but not the paw tremor and ptosis accompanying withdrawal. Following the morphine applications, a single treatment of EphB2-Fc at a larger dose (2 µg, 30 min prior to naloxone) also significantly attenuated the withdrawal symptoms, backward walking, chewing, jump, tremor, and wet-dog shake but not diarrhea, paw tremor, ptosis, or weight loss. Consistent with these changes, the overall withdrawal score was significantly reduced in EphB2-Fc-treated mice (Fig. 1B ). Repeated coapplication of EphB2-Fc produced greater inhibition than the single, postmorphine application of the blocker on the overall withdrawal score (Fig. 1B ). IgG-Fc (2 µg, i.t.) did not significantly affect the withdrawal behaviors (Fig. 1A, B ). Neither EphB2-Fc (2 µg, i.t.) nor IgG-Fc (2 µg, i.t.) altered the pain threshold (Fig. 1C ) or the initial morphine-induced analgesia (Fig. 1D ). These results suggest that the EphB receptors may be involved in spinal mechanisms of opiate dependence and withdrawal.

EphB2-Fc suppresses induction of c-Fos and accumulation and depletion of CGRP in DH following morphine treatment and withdrawal
Induction of c-Fos protein expression following opiate withdrawal and the increase and decrease in CGRP expression in the DH following chronic morphine exposure and during withdrawal have been used as indicators of neural activity and plasticity associated with these states (43 , 48) . We used immunohistochemical and immunofluorescence staining to measure the expression of c-Fos and CGRP immunoreactivity. Representative photomicrographs and the corresponding counts of Fos-like immunoreactive neurons in DH are shown in Fig. 2A, B , respectively. As expected, c-Fos expression significantly increased during morphine withdrawal (Fig. 2Aa-c ). Interestingly, EphB2-Fc, i.t., prevented or significantly suppressed the increase in expression of c-Fos. Repeated application of EphB2-Fc (1 µg, 7 times at 8 h intervals, accompanied by morphine) completely prevented expression of c-Fos (Fig. 2Ad ), while single dose of EphB2-Fc (2 µg, 30 min prior to naloxone challenge) significantly reduced the increase in expression of c-Fos (Fig. 2Ae ). IgG-Fc (1 µg, i.t.) did not significantly affect the induction of c-Fos expression (Fig. 2Af ). Data are summarized in Fig. 2B .


Figure 2
View larger version (18K):
[in this window]
[in a new window]

  		Figure 2. EphB2-Fc reduces c-Fos expression in DH during morphine withdrawal. A) Examples of Fos-immunoreactive neurons. Six groups of mice (n=5/group) were tested and examined. Naive (a) received no treatment; Mor (b) received escalating morphine treatment; Mor + Nlx (c) received naloxone after the morphine treatment; Mor + 7EphB + Nlx (d) also received 7 pulses of EphB2-Fc (1 µg); Mor + 1EphB + Nlx (e) also received 1 treatment of EphB2-Fc (2 µg) 30 min prior to naloxone; and Mor + 7Fc + Nlx (f) also received repeated pulses of IgG-Fc (1 µg). B) Summary of data. **P < 0.01 vs. naive; #P < 0.05, ##P < 0.01 vs. Mor + Nlx; &P < 0.05 vs. Mor + 7EphB + Nlx. Scale bars = 200 µm.

Alterations of CGRP-immunoreactivity in the DH associated with chronic morphine and morphine withdrawal and the given drug treatment are shown in Fig. 3 . As expected, repeated morphine exposure greatly increased the expression of CGRP throughout the DH region of the SC (Fig. 3Aa, b ). Repeated application of EphB2-Fc but not the IgG-Fc (each administrated 1 µg during each morphine dose) significantly prevented the chronic morphine-induced increase of CGRP-like immunoreactivity (Fig. 3Ac, d ). Morphine withdrawal resulted in marked reduction of the CGRP immunoreactivity (Figure 3Ae ). A single application of EphB2-Fc but not IgG-Fc (each administrated 2 µg, i.t., 30 min prior to naloxone) significantly attenuated the withdrawal-associated reduction of CGRP immunoreactivity (Figure 3Af, g ). The corresponding measurements of fluorescence intensity are summarized in Fig. 3B .


Figure 3
View larger version (37K):
[in this window]
[in a new window]

  		Figure 3. EphB2-Fc alters CGRP-immunoreactivity in DH associated with repeated, escalating morphine and morphine withdrawal. A) Six groups of mice (n=5/group) were tested and examined. Naive (a) received no treatment; Mor (b) received morphine treatment; Mor + Nlx (c) received naloxone in addition to morphine treatment; Mor + 1EphB + Nlx (d) also received 1 injection of EphB2-Fc (2 µg) 30 min prior to naloxone; Mor + 7Fc + Nlx (e) also received repeated pulses of IgG-Fc (1 µg); and Mor + 7EphB (f) received 7 pulses of EphB2-Fc (1 µg) with morphine. B) Summary of data. **P < 0.01 vs. naive; #P < 0.05, ##P < 0.01 vs. Mor; &P < 0.01 vs. Mor + Nlx. Scale bars = 200 µm.

Expression of EphB1 receptor is up-regulated by chronic morphine treatment and withdrawal in SC
Given that activation of EphB receptors is necessary for induction and/or maintenance of the behavioral and neurochemical signs associated with naloxone-precipitated morphine withdrawal, an interesting question is whether EphB receptors and their ligands are regulated/modulated by chronic morphine exposure and/or morphine withdrawal. We used protein precipitation procedures in conjunction with Western blots (47) to detect expression of EphB receptors and ephrinB proteins. Expression of EphB1 receptor protein significantly increased after escalating morphine treatment (Fig. 4A, B ). Immunohistochemical fluorescence staining further showed that the increased expression of EphB1 receptor protein was predominantly localized in the superficial laminae of the DH (Fig. 4C ). Most of the increased expression of EphB1 receptor protein was prevented or cancelled in the mice that received simultaneous treatment of escalating morphine doses and repeated pulses of the EphB receptor blocking reagent EphB2-Fc (1 µg, i.t., 7 doses; Fig. 4A, B ). Similar results were found after naloxone treatment (Fig. 4D, E ). Naloxone treatment did not significantly alter the morphine-induced increase in expression of EphB1 receptor protein or the inhibition of expression of EphB1 receptor protein caused by repeated pulses of EphB2-Fc. A single treatment of EphB2-Fc (2 µg, i.t., 30 min prior to naloxone) failed to significantly reduce the morphine-induced increase in expression of EphB1 receptor protein (Fig. 4E ). These results indicate that the EphB1 receptor is up-regulated by the escalating morphine treatment and by morphine withdrawal and suppressed by coapplication of the receptor blocker, which also attenuated the behavioral symptoms and neurochemical signs of morphine withdrawal. Our results did not show a significant change in expression of ephrinB1 or ephrinB2 or of phosphotyrosine proteins (PY99) following escalating morphine treatment or morphine withdrawal (Fig. 4F, G ).


Figure 4
View larger version (29K):
[in this window]
[in a new window]

  		Figure 4. Expression of EphB1 receptor and ephrinB protein in SC following escalating morphine treatment, morphine withdrawal and EphB2-Fc treatment. A, B) Expression of EphB1 receptor protein following escalating morphine or morphine combined with repeated pulses of EphB2-Fc: examples (A) and data summary (B). C) Example of the distribution of EphB1 receptor protein in the DH indicated by immunofluorescence staining. D, E) Expression of EphB1 receptor protein 30 min after morphine withdrawal following morphine treatment or morphine combined with single or repeated pulses of EphB2-Fc; examples (D) and data summary (E). F) Effect of morphine treatment and morphine withdrawal on expression of ephrinB1, ephrinB2, and PY99. Naive group received no treatment; 7Fc received 7 repeated pulses of IgG-Fc; Mor received morphine treatment; Mor + 7EphB received morphine plus EphB2-Fc (1 µg, 7 pulses) treatment; and Mor + 1EphB received morphine treatment followed by a single pulse of EphB2-Fc (2 µg). *P < 0.05, **P < 0.01 vs. naive; #P < 0.05, ##P < 0.01 vs. Mor.

EphB2-Fc prevents or suppresses increase in phosphorylated NR2B, ERK, and CREB associated with morphine treatment and withdrawal
NMDARs have a well-established role in opiate-related neural plasticity (26 , 28 , 30) . Chronic morphine treatment causes increases in Ca2+ levels and alterations of CaMKII (32 , 33) , ERK (34) , and CREB (13) . An interesting question is whether these signals are modulated by the activity of EphB receptors after morphine treatment and withdrawal. The results showed that escalated morphine treatment caused significant increases in the levels of pNR2B, pERK, or pCREB protein, as indicated by Western blots (Fig. 5A, B ). Repeated coapplication of EphB2-Fc (1 µg, i.t.) with morphine significantly prevented or reduced the morphine-induced increase in pNR2B, pERK, and pCREB (Fig. 5B ). Following morphine withdrawal (30 min after naloxone), there was no further increase in the levels of pNR2B, pERK, or pCREB (Fig. 5C, D ). A single coapplication of EphB2-Fc (2 µg, i.t., 30 min prior to naloxone), however, significantly reduced the increases in pNR2B and pCREB, but not pERK (Fig. 5D ). These data suggest that EphB receptor signaling may mediate morphine dependence and/or withdrawal via interaction with NMDAR subunit NR2B and further activates and modulates the intracellular signals ERK and CREB.


Figure 5
View larger version (30K):
[in this window]
[in a new window]

  		Figure 5. EphB2-Fc reduces the increased levels of pNR2B, perk, and pCREB in SC following morphine treatment and withdrawal. Groups of mice received the same treatments as in Fig. 4 . Examples of expression of the proteins tested after various treatments are shown in A and C; data are summarized in B and D. *P < 0.05, **P < 0.01 vs. corresponding naive group; #P < 0.05, ##P < 0.01 vs. corresponding morphine Mor group.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study provides the first evidence that EphB receptor signaling in the SC contributes to the development of morphine dependence and withdrawal. The principal findings are as follows: 1) escalating morphine treatment significantly up-regulates expression of EphB1 receptor protein in the SC; 2) intrathecal administration of a blocking reagent for EphB receptors prevents and/or suppresses the behavioral symptoms and neurochemical signs associated with chronic morphine treatment or morphine withdrawal, including induction of c-Fos, and accumulation and then depletion of CGRP in the DH, accompanied by recovery of EphB1 receptor protein expression; and 3) levels of the pNR2B subunit of NMDARs, as well as pERK and pCREB in the SC, are significantly increased following chronic morphine treatment and withdrawal, and these increases are prevented or reduced by coapplication of an EphB receptor blocking reagent with morphine. These findings indicate that EphB receptor signaling in the SC may play an important role in opioid physical dependence, probably by interacting with the NR2B subunit of NMDARs.

The EphB receptors, in addition to their important roles in tissue-border formation, cell migration, and axon guidance during development of the nervous system, can regulate the development of glutamatergic synaptic connections and their plasticity in the adult nervous system by interaction with NMDA receptors (16 , 21 22 23 24 25) . The EphB receptors are membrane proteins that initiate bidirectional signaling when the proteins aggregate (49 , 50) . The present study does not provide evidence that would allow us to decide whether EphB receptor-mediated stimulation of DH neurons occurs postsynaptically, presynaptically, or both pre- and postsynaptically. In addition, we have not examined the possibility that reverse signaling through activation of ephrinB also plays a role, either pre- or postsynaptically. However, the accumulation of EphB receptors in the DH and inhibition of CGRP expression in primary afferent fibers within the DH by a blocking reagent of EphB receptors after morphine treatment suggest that EphB receptor signaling may be important on both sides of synapse and that both forward and reverse ephrinB–EphB signaling in the SC may contribute to morphine dependence and withdrawal symptoms. Although the available blocking reagents do not identify the specific EphB receptors activated in our experiments, the specificity of the antibodies used for detection of EphB1 lends confidence to our conclusion that this specific protein is up-regulated during chronic morphine exposure and withdrawal. In addition, our unpublished observations in EphB1 knockout mice indicate that the EphB1 receptor is important to the development of morphine dependence; that is, behavioral signs of naloxone-precipitated morphine withdrawal are largely diminished in the EphB1 knockout mice.

NMDARs play important roles in neural plasticity and opiate-related alterations (13 , 51) . NMDARs are heteromeric complexes containing both NR1 and NR2 subunits. They are located at the postsynaptic side of excitatory synapses and are an important channel for Ca2+ entry (52 , 53) . The NR1 subunit is widely distributed in the CNS, whereas distribution of the NR2B subunit is more restricted but includes the superficial layers of the DH (54 , 55) . Reintroducing the deleted gene into specific brain regions by electroporation in NR2A knockout mice showed that NR2A is important for morphine dependence and withdrawal (7) . Some evidence also points to opiate-induced increases in NR1 and NR2B protein levels as a contributor to morphine tolerance responses in the amygdale (56) . The present study provides the first evidence that chronic morphine exposure and/or withdrawal increases the pNR2B subunit in the DH. Further, this increase of NR2B can be prevented and/or reduced by a blocking reagent for EphB receptors (Fig. 4) . Ligand binding causes EphB receptor clustering and reciprocal phosphorylation on multiple tyrosine residues (22) . The phosphorylated tyrosines recruit downstream signaling proteins containing SH2 domains, including Src family kinases such as Fyn (22 , 57) . Fyn is physically associated with both the EphB receptors and NMDARs as well as Fyn phosphorylates NR2B on tyrosines (58) . This might also occur in DH neurons following chronic morphine exposure and/or morphine withdrawal. Opiate systems interact with NMDARs such that activation of MOR results in Ca2+ influx through the NMDAR ion-channel complex. The subsequent activation of various Ca2+-dependent signaling pathways (29 , 59) plays a central role in morphine dependence and withdrawal. The CaMKII protein can phosphorylate (activate) CREB, which leads to increases in c-Fos mRNA and c-Fos protein expression (60) . Gene expression is thought to play an important role in many forms of plasticity, including morphine dependence and withdrawal.

Transsynaptic interactions between postsynaptic EphB receptors and presynaptic ephrinB are necessary for some forms of plasticity at mossy fiber synapses in the hippocampus (61) . We have recently found that EphB receptors and their ephrinB ligands are up-regulated pre- and postsynaptically at primary afferent synapse in the DH after nerve injury (36 , 38) . The present results show that expression of the EphB receptor protein is also significantly increased following chronic morphine exposure and withdrawal (Fig. 4A-E ). Surprisingly, expression of ephrinB1 and ephrinB2 and of proteins with phosphorylated tyrosines did not appear to be altered in the SC after morphine treatment and withdrawal (Fig. 4F, G ). In the absence of a corresponding alteration of its ligands, how does clustering and reciprocal phosphorylation of the up-regulated EphB receptors occur? One possibility is that the EphB receptors are directly modulated postsynaptically by signals such as NR2B, ERK (62) , and CREB (63 , 64) , or NO pathways (4 , 5) , following chronic morphine treatment and/or morphine withdrawal. Interaction of EphB receptors with cAMP pathway may also contribute to the postsynaptic effects of EphB receptors. For instance, expression of EphB2 is increased after exposure to forskolin, and a surge of cAMP can trigger transcriptional activity to augment expression of EphB2 receptor genes (65) .

Chronic morphine treatment induces an increase in CGRP in the terminals of primary afferent neurons, which accounts for the robust increase in CGRP immunoreactivity in DH (45) . Although the underlying mechanism is unclear, the increase in CGRP may depend on activation of a MAPK pathway and phosphorylation of CREB (63) , a transcription factor implicated in CGRP gene expression (64) . Our results show that the accumulation and depletion of CGRP in primary afferent terminals within the DH can be inhibited by an EphB receptor-blocking reagent. Such inhibition of CGRP may result from the EphB receptor blocking reagent-induced inhibition of the CREB signaling pathway (63 , 64) . Further studies are needed to show how the EphB receptors regulate expression of CGRP in presynaptic primary terminals following chronic morphine treatment.

In conclusion, our findings provide evidence for a novel mechanism contributing to opiate dependence and withdrawal symptoms and a novel role for EphB receptor signaling in the development of morphine dependence and withdrawal. This study may also provide a potential pharmacological target for preventing, minimizing, or reversing morphine dependence and thereby facilitate the use of opioid drugs in clinic.


   ACKNOWLEDGMENTS
 
We thank Dr. Edgar T. Walters for his helpful comment on the manuscript, Dr. Ronald L. Rupert for his general support as Dean of Research, and Maria Dominguez for her assistance in laboratory management. Supported by grants from Parker Research Foundation (PCCRF-BSR0703 and PCCRF-BSR0804) and National Natural Science Foundation of China (NSFC-30628027).

Received for publication June 3, 2008. Accepted for publication August 14, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bailey, C. P., Connor, M. (2005) Opioids: cellular mechanisms of tolerance and physical dependence. Curr. Opin. Pharmacol. 5,60-68[CrossRef][Medline]
  2. Bohn, L. M., Gainetdinov, R. R., Lin, F. T., Lefkowitz, R. J., Caron, M. G. (2000) Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence. Nature 408,720-723[CrossRef][Medline]
  3. King, M., Su, W., Chang, A., Zuckerman, A., Pasternak, G. W. (2001) Transport of opioids from the brain to the periphery by P-glycoprotein: peripheral actions of central drugs. Nat. Neurosci. 4,268-274[CrossRef][Medline]
  4. Muscoli, C., Cuzzocrea, S., Ndengele, M. M., Mollace, V., Porreca, F., Fabrizi, F., Esposito, E., Masini, E., Matuschak, G. M., Salvemini, D. (2007) Therapeutic manipulation of peroxynitrite attenuates the development of opiate-induced antinociceptive tolerance in mice. J. Clin. Invest. 117,3530-3539[CrossRef][Medline]
  5. Pasternak, G. W. (2007) When it comes to opiates, just say NO. J. Clin. Invest. 117,3185-3187[CrossRef][Medline]
  6. Zachariou, V., Georgescu, D., Sanchez, N., Rahman, Z., DiLeone, R., Berton, O., Neve, R. L., Sim-Selley, L. J., Selley, D. E., Gold, S. J., Nestler, E. J. (2003) Essential role for RGS9 in opiate action. Proc. Natl. Acad. Sci. U. S. A. 100,13656-13661[Abstract/Free Full Text]
  7. Inoue, M., Mishina, M., Ueda, H. (2003) Locus-specific rescue of GluRepsilon1 NMDA receptors in mutant mice identifies the brain regions important for morphine tolerance and dependence. J. Neurosci. 23,6529-6536[Abstract/Free Full Text]
  8. Inturrisi, C. E. (2002) Clinical pharmacology of opioids for pain. Clin. J. Pain. 18,S3-S13[CrossRef][Medline]
  9. Kolesnikov, Y. A., Pick, C. G., Ciszewska, G., Pasternak, G. W. (1993) Blockade of tolerance to morphine but not to kappa opioids by a nitric oxide synthase inhibitor. Proc. Natl. Acad. Sci. U. S. A. 90,5162-5166[Abstract/Free Full Text]
  10. Pasternak, G. W., Kolesnikov, Y. (2005) The NMDA/nitric oxide synthase cascade in opioid analgesia and tolerance. Herman, B. H. eds. Contemporary Clinical Neuroscience: Glutamate and Addiction ,409-416 Humana Press Totowa, NJ, USA.
  11. Trujillo, K. A., Akil, H. (1991) Inhibition of morphine tolerance and dependence by the NMDA receptor antagonist MK-801. Science 251,85-87[Abstract/Free Full Text]
  12. Barrot, M., Olivier, J. D., Perrotti, L. I., DiLeone, R. J., Berton, O., Eisch, A. J., Impey, S., Storm, D. R., Neve, R. L., Yin, J. C., Zachariou, V., Nestler, E. J. (2002) CREB activity in the nucleus accumbens shell controls gating of behavioral responses to emotional stimuli. Proc. Natl. Acad. Sci. U. S. A. 99,11435-11440[Abstract/Free Full Text]
  13. Nestler, E. J. (2001) Molecular basis of long-term plasticity underlying addiction. Nat. Rev. Neurosci. 2,119-128[CrossRef][Medline]
  14. Shaw-Lutchman, T. Z., Barrot, M., Wallace, T., Gilden, L., Zachariou, V., Impey, S., Duman, R. S., Storm, D., Nestler, E. J. (2002) Regional and cellular mapping of cAMP response element-mediated transcription during naltrexone-precipitated morphine withdrawal. J. Neurosci. 22,3663-3672[Abstract/Free Full Text]
  15. Sweatt, J. D. (2004) Mitogen-activated protein kinases in synaptic plasticity and memory. Curr. Opin. Neurobiol. 14,311-317[CrossRef][Medline]
  16. Chen, Z. L., Yu, W. M., Strickland, S. (2007) Peripheral regeneration. Annu. Rev. Neurosci. 30,209-233[CrossRef][Medline]
  17. Krull, C. E., Lansford, R., Gale, N. W., Collazo, A., Marcelle, C., Yancopoulos, G. D., Fraser, S. E., Bronner-Fraser, M. (1997) Interactions of Eph-related receptors and ligands confer rostrocaudal pattern to trunk neural crest migration. Curr. Biol. 7,571-580[CrossRef][Medline]
  18. Wang, H. U., Anderson, D. J. (1997) Eph family transmembrane ligands can mediate repulsive guidance of trunk neural crest migration and motor axon outgrowth. Neuron 18,383-396[CrossRef][Medline]
  19. Wilkinson, D. G. (2000) Eph receptors and ephrins: regulators of guidance and assembly. Int. Rev. Cytol. 196,177-244[Medline]
  20. Wilkinson, D. G. (2001) Multiple roles of EPH receptors and ephrins in neural development. Nat. Rev. Neurosci. 2,155-164[Medline]
  21. Dalva, M. B., Takasu, M. A., Lin, M. Z., Shamah, S. M., Hu, L., Gale, N. W., Greenberg, M. E. (2000) EphB receptors interact with NMDA receptors and regulate excitatory synapse formation. Cell 103,945-956[CrossRef][Medline]
  22. Grunwald, I. C., Korte, M., Adelmann, G., Plueck, A., Kullander, K., Adams, R. H., Frotscher, M., Bonhoeffer, T., Klein, R. (2004) Hippocampal plasticity requires postsynaptic ephrinBs. Nat. Neurosci. 7,33-40[CrossRef][Medline]
  23. Grunwald, I. C., Korte, M., Wolfer, D., Wilkinson, G. A., Unsicker, K., Lipp, H. P., Bonhoeffer, T., Klein, R. (2001) Kinase-independent requirement of EphB2 receptors in hippocampal synaptic plasticity. Neuron 32,1027-1040[CrossRef][Medline]
  24. Henderson, J. T., Georgiou, J., Jia, Z., Robertson, J., Elowe, S., Roder, J. C., Pawson, T. (2001) The receptor tyrosine kinase EphB2 regulates NMDA-dependent synaptic function. Neuron 32,1041-1056[CrossRef][Medline]
  25. Takasu, M. A., Dalva, M. B., Zigmond, R. E., Greenberg, M. E. (2002) Modulation of NMDA receptor-dependent calcium influx and gene expression through EphB receptors. Science 295,491-495[Abstract/Free Full Text]
  26. Mao, J. (1999) NMDA and opioid receptors: their interactions in antinociception, tolerance and neuroplasticity. Brain Res. Brain Res. Rev. 30,289-304[CrossRef][Medline]
  27. Mao, J., Sung, B., Ji, R. R., Lim, G. (2002) Chronic morphine induces downregulation of spinal glutamate transporters: implications in morphine tolerance and abnormal pain sensitivity. J. Neurosci. 22,8312-8323[Abstract/Free Full Text]
  28. Mayer, D. J., Mao, J., Holt, J., Price, D. D. (1999) Cellular mechanisms of neuropathic pain, morphine tolerance, and their interactions. Proc. Natl. Acad. Sci. U. S. A. 96,7731-7736[Abstract/Free Full Text]
  29. Noda, Y., Nabeshima, T. (2004) Opiate physical dependence and N-methyl-D-aspartate receptors. Eur. J. Pharmacol. 500,121-128[CrossRef][Medline]
  30. Zhu, H., Barr, G. A. (2001) Inhibition of morphine withdrawal by the NMDA receptor antagonist MK-801 in rat is age-dependent. Synapse 40,282-293[CrossRef][Medline]
  31. Hamdy, M. M., Noda, Y., Miyazaki, M., Mamiya, T., Nozaki, A., Nitta, A., Sayed, M., Assi, A. A., Gomaa, A., Nabeshima, T. (2004) Molecular mechanisms in dizocilpine-induced attenuation of development of morphine dependence: an association with cortical Ca2+/calmodulin-dependent signal cascade. Behav. Brain Res. 152,263-270[CrossRef][Medline]
  32. Liang, D., Li, X., Clark, J. D. (2004) Increased expression of Ca2+/calmodulin-dependent protein kinase II alpha during chronic morphine exposure. Neuroscience 123,769-775[CrossRef][Medline]
  33. Lu, L., Zeng, S., Liu, D., Ceng, X. (2000) Inhibition of the amygdala and hippocampal calcium/calmodulin-dependent protein kinase II attenuates the dependence and relapse to morphine differently in rats. Neurosci. Lett. 291,191-195[CrossRef][Medline]
  34. Ren, X., Noda, Y., Mamiya, T., Nagai, T., Nabeshima, T. (2004) A neuroactive steroid, dehydroepiandrosterone sulfate, prevents the development of morphine dependence and tolerance via c-fos expression linked to the extracellular signal-regulated protein kinase. Behav. Brain Res. 152,243-250[CrossRef][Medline]
  35. Kobayashi, H., Kitamura, T., Sekiguchi, M., Homma, M. K., Kabuyama, Y., Konno, S., Kikuchi, S., Homma, Y. (2007) Involvement of EphB1 receptor/EphrinB2 ligand in neuropathic pain. Spine 32,1592-1598[CrossRef][Medline]
  36. Song, X. J., Cao, J. L., Li, H. C., Zheng, J. H., Song, X. S., Xiong, L. Z. (2008) Upregulation and redistribution of ephrinB and EphB receptor in dorsal root ganglion and spinal dorsal horn neurons after peripheral nerve injury and dorsal rhizotomy. Eur. J. Pain 12,1031-1039[CrossRef][Medline]
  37. Battaglia, A. A., Sehayek, K., Grist, J., McMahon, S. B., Gavazzi, I. (2003) EphB receptors and ephrin-B ligands regulate spinal sensory connectivity and modulate pain processing. Nat. Neurosci. 6,339-340[CrossRef][Medline]
  38. Song, X. J., Zheng, J. H., Cao, J. L., Liu, W. T., Song, X. S., Huang, Z. J. (2008) Ephrin-EphB receptor signaling contributes to neuropathic pain by regulating neural excitability and spinal plasticity in rats. Pain 139,168-180[CrossRef][Medline]
  39. Cao, J. L., He, J. H., Ding, H. L., Zeng, Y. M. (2005) Activation of the spinal ERK signaling pathway contributes naloxone-precipitated withdrawal in morphine-dependent rats. Pain 118,336-349[CrossRef][Medline]
  40. Jhamandas, K. H., Marsala, M., Ibuki, T., Yaksh, T. L. (1996) Spinal amino acid release and precipitated withdrawal in rats chronically infused with spinal morphine. J. Neurosci. 16,2758-2766[Abstract/Free Full Text]
  41. Marshall, D. C., Buccafusco, J. J. (1985) Spontaneous morphine withdrawal from the rat spinal cord. Experientia 41,1151-1152[CrossRef][Medline]
  42. Rohde, D. S., Basbaum, A. I. (1998) Activation of coeruleospinal noradrenergic inhibitory controls during withdrawal from morphine in the rat. J. Neurosci. 18,4393-4402[Abstract/Free Full Text]
  43. Rohde, D. S., Detweiler, D. J., Basbaum, A. I. (1996) Spinal cord mechanisms of opioid tolerance and dependence: Fos-like immunoreactivity increases in subpopulations of spinal cord neurons during withdrawal. Neuroscience 72,233-242[CrossRef][Medline]
  44. Rohde, D. S., McKay, W. R., Abbadie, C., Basbaum, A. I. (1997) Contribution of sacral spinal cord neurons to the autonomic and somatic consequences of withdrawal from morphine in the rat. Brain Res. 745,83-95[CrossRef][Medline]
  45. Trang, T., Sutak, M., Quirion, R., Jhamandas, K. (2003) Spinal administration of lipoxygenase inhibitors suppresses behavioural and neurochemical manifestations of naloxone-precipitated opioid withdrawal. Br. J. Pharmacol. 140,295-304[CrossRef][Medline]
  46. Kerr, B. J., Cafferty, W. B., Gupta, Y. K., Bacon, A., Wynick, D., McMahon, S. B., Thompson, S. W. (2000) Galanin knockout mice reveal nociceptive deficits following peripheral nerve injury. Eur. J. Neurosci. 12,793-802[CrossRef][Medline]
  47. Bundesen, L. Q., Scheel, T. A., Bregman, B. S., Kromer, L. F. (2003) Ephrin-B2 and EphB2 regulation of astrocyte-meningeal fibroblast interactions in response to spinal cord lesions in adult rats. J. Neurosci. 23,7789-7800[Abstract/Free Full Text]
  48. Trang, T., Ma, W., Chabot, J. G., Quirion, R., Jhamandas, K. (2006) Spinal modulation of calcitonin gene-related peptide by endocannabinoids in the development of opioid physical dependence. Pain 126,256-271[CrossRef][Medline]
  49. Kullander, K., Klein, R. (2002) Mechanisms and functions of Eph and ephrin signalling. Nat. Rev. Mol. Cell Biol. 3,475-486[CrossRef][Medline]
  50. Palmer, A., Klein, R. (2003) Multiple roles of ephrins in morphogenesis, neuronal networking, and brain function. Genes Dev. 17,1429-1450[Free Full Text]
  51. Mao, J., Mayer, D. J. (2001) Spinal cord neuroplasticity following repeated opioid exposure and its relation to pathological pain. Ann. N. Y. Acad. Sci. 933,175-184[Medline]
  52. Cull-Candy, S., Brickley, S., Farrant, M. (2001) NMDA receptor subunits: diversity, development and disease. Curr. Opin. Neurobiol. 11,327-335[CrossRef][Medline]
  53. Prybylowski, K., Wenthold, R. J. (2004) N-Methyl-D-aspartate receptors: subunit assembly and trafficking to the synapse. J. Biol. Chem. 279,9673-9676[Free Full Text]
  54. Boyce, S., Wyatt, A., Webb, J. K., O'Donnell, R., Mason, G., Rigby, M., Sirinathsinghji, D., Hill, R. G., Rupniak, N. M. (1999) Selective NMDA NR2B antagonists induce antinociception without motor dysfunction: correlation with restricted localisation of NR2B subunit in dorsal horn. Neuropharmacology 38,611-623[CrossRef][Medline]
  55. Nagy, G. G., Watanabe, M., Fukaya, M., Todd, A. J. (2004) Synaptic distribution of the NR1, NR2A and NR2B subunits of the N-methyl-d-aspartate receptor in the rat lumbar spinal cord revealed with an antigen-unmasking technique. Eur. J. Neurosci. 20,3301-3312[CrossRef][Medline]
  56. Bajo, M., Crawford, E. F., Roberto, M., Madamba, S. G., Siggins, G. R. (2006) Chronic morphine treatment alters expression of N-methyl-D-aspartate receptor subunits in the extended amygdala. J. Neurosci. Res. 83,532-537[CrossRef][Medline]
  57. Murai, K. K., Pasquale, E. B. (2004) Eph receptors, ephrins, and synaptic function. Neuroscientist 10,304-314[Abstract/Free Full Text]
  58. Nakazawa, T., Komai, S., Tezuka, T., Hisatsune, C., Umemori, H., Semba, K., Mishina, M., Manabe, T., Yamamoto, T. (2001) Characterization of Fyn-mediated tyrosine phosphorylation sites on GluR epsilon 2 (NR2B) subunit of the N-methyl-D-aspartate receptor. J. Biol. Chem. 276,693-699[Abstract/Free Full Text]
  59. Fan, G. H., Wang, L. Z., Qiu, H. C., Ma, L., Pei, G. (1999) Inhibition of calcium/calmodulin-dependent protein kinase II in rat hippocampus attenuates morphine tolerance and dependence. Mol. Pharmacol. 56,39-45[Abstract/Free Full Text]
  60. Sheng, M., Thompson, M. A., Greenberg, M. E. (1991) CREB: a Ca(2+)-regulated transcription factor phosphorylated by calmodulin-dependent kinases. Science 252,1427-1430[Abstract/Free Full Text]
  61. Contractor, A., Rogers, C., Maron, C., Henkemeyer, M., Swanson, G. T., Heinemann, S. F. (2002) Trans-synaptic Eph receptor-ephrin signaling in hippocampal mossy fiber LTP. Science 296,1864-1869[Abstract/Free Full Text]
  62. Nateri, A. S., Raivich, G., Gebhardt, C., Da Costa, C., Naumann, H., Vreugdenhil, M., Makwana, M., Brandner, S., Adams, R. H., Jefferys, J. G., Kann, O., Behrens, A. (2007) ERK activation causes epilepsy by stimulating NMDA receptor activity. EMBO J. 26,4891-4901[CrossRef][Medline]
  63. Ma, W., Zheng, W. H., Powell, K., Jhamandas, K., Quirion, R. (2001) Chronic morphine exposure increases the phosphorylation of MAP kinases and the transcription factor CREB in dorsal root ganglion neurons: an in vitro and in vivo study. Eur. J. Neurosci. 14,1091-1104[CrossRef][Medline]
  64. Watson, A., Latchman, D. (1995) The cyclic AMP response element in the calcitonin/calcitonin gene-related peptide gene promoter is necessary but not sufficient for its activation by nerve growth factor. J. Biol. Chem. 270,9655-9660[Abstract/Free Full Text]
  65. Jassen, A. K., Yang, H., Miller, G. M., Calder, E., Madras, B. K. (2006) Receptor regulation of gene expression of axon guidance molecules: implications for adaptation. Mol. Pharmacol. 70,71-77[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
fj.08-114462v1
23/1/90    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, W.-T.
Right arrow Articles by Song, X.-J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, W.-T.
Right arrow Articles by Song, X.-J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS