Chronic Opioid Potentiates Presynaptic but Impairs Postsynaptic N-Methyl-d-aspartic Acid Receptor Activity in Spinal Cords: IMPLICATIONS FOR OPIOID HYPERALGESIA AND TOLERANCE

Abstract

Background: We determined the role of presynaptic N-methyl-d-aspartate receptor (NMDAR) activity in spinal cords in opioid-induced hyperalgesia and tolerance.Results: Chronic opioid increases presynaptic NMDAR activity at primary sensory nerve terminals through protein kinase C.Conclusion: Increased presynaptic NMDAR activity potentiates nociceptive input and is responsible for opioid hyperalgesia and tolerance.Significance: Understanding mechanisms of increased NMDAR activity is important for improving opioid therapies. Background: We determined the role of presynaptic N-methyl-d-aspartate receptor (NMDAR) activity in spinal cords in opioid-induced hyperalgesia and tolerance. Results: Chronic opioid increases presynaptic NMDAR activity at primary sensory nerve terminals through protein kinase C. Conclusion: Increased presynaptic NMDAR activity potentiates nociceptive input and is responsible for opioid hyperalgesia and tolerance. Significance: Understanding mechanisms of increased NMDAR activity is important for improving opioid therapies. Opioid analgesics are the mainstay of treatment for moderate to severe pain caused by cancer or by tissue and nerve injury. However, long-term administration of μ-opioid receptor agonists for chronic pain can lead to analgesic tolerance, which is a major obstacle to adequate pain relief. Opioid analgesic tolerance is a phenomenon in which repeated exposure to an opioid agonist results in the decreased therapeutic effect of the drug or the need for a higher dose to maintain the same analgesic effect. For all μ-opioid receptor agonists, analgesic tolerance occurs with repeated use. The cellular and signaling mechanisms responsible are poorly understood. The key question is where and how opioids induce neuroplasticity in the nervous system that leads to opioid tolerance. This problem has not been adequately studied in the nociceptive circuitry in which native opioid receptors are expressed. NMDA receptors (NMDARs), 2The abbreviations used are: NMDARN-methyl-d-aspartic acid receptorAMPARα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptorAP52-amino-5-phosphonopentanoic acidEPSCexcitatory postsynaptic currentsEPSCspontaneous excitatory postsynaptic currentNMDAR-EPSCsNMDAR-mediated EPSCsAMPAR-EPSCsAMPAR-mediated EPSCsRTXresiniferatoxinTRPV1transient receptor potential vanilloid type 1(Rp)-cAMPSadenosine-3′,5′-cyclic monophosphorothioate, Rp isomer. which play an important role in opioid-induced hyperalgesia and tolerance (1.Mao J. Price D.D. Mayer D.J. Thermal hyperalgesia in association with the development of morphine tolerance in rats: roles of excitatory amino acid receptors and protein kinase C.J. Neurosci. 1994; 14: 2301-2312Crossref PubMed Google Scholar, 2.Trujillo K.A. Akil H. Inhibition of morphine tolerance and dependence by the NMDA receptor antagonist MK-801.Science. 1991; 251: 85-87Crossref PubMed Scopus (1115) Google Scholar), are located presynaptically on central terminals of primary afferent neurons as well as postsynaptically on dorsal horn neurons (3.Antal M. Fukazawa Y. Eördögh M. Muszil D. Molnár E. Itakura M. Takahashi M. Shigemoto R. Numbers, densities, and colocalization of AMPA- and NMDA-type glutamate receptors at individual synapses in the superficial spinal dorsal horn of rats.J. Neurosci. 2008; 28: 9692-9701Crossref PubMed Scopus (52) Google Scholar, 4.Bardoni R. Torsney C. Tong C.K. Prandini M. MacDermott A.B. Presynaptic NMDA receptors modulate glutamate release from primary sensory neurons in rat spinal cord dorsal horn.J. Neurosci. 2004; 24: 2774-2781Crossref PubMed Scopus (133) Google Scholar). However, it is unclear what roles these receptors play in the development of opioid analgesic tolerance. N-methyl-d-aspartic acid receptor α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor 2-amino-5-phosphonopentanoic acid excitatory postsynaptic current spontaneous excitatory postsynaptic current NMDAR-mediated EPSCs AMPAR-mediated EPSCs resiniferatoxin transient receptor potential vanilloid type 1 adenosine-3′,5′-cyclic monophosphorothioate, Rp isomer. Many studies have shown that opioids paradoxically elicit abnormal pain or hyperalgesia (i.e. enhanced pain response to noxious stimuli). Opioid-induced hyperalgesia has been demonstrated in many animal studies (1.Mao J. Price D.D. Mayer D.J. Thermal hyperalgesia in association with the development of morphine tolerance in rats: roles of excitatory amino acid receptors and protein kinase C.J. Neurosci. 1994; 14: 2301-2312Crossref PubMed Google Scholar, 5.Yaksh T.L. Harty G.J. Onofrio B.M. High dose of spinal morphine produce a nonopiate receptor-mediated hyperesthesia: clinical and theoretic implications.Anesthesiology. 1986; 64: 590-597Crossref PubMed Scopus (228) Google Scholar, 6.Yaksh T.L. Harty G.J. Pharmacology of the allodynia in rats evoked by high dose intrathecal morphine.J. Pharmacol. Exp. Ther. 1988; 244: 501-507PubMed Google Scholar, 7.Larcher A. Laulin J.P. Celerier E. Le Moal M. Simonnet G. Acute tolerance associated with a single opiate administration: involvement of N-methyl-d-aspartate-dependent pain facilitatory systems.Neuroscience. 1998; 84: 583-589Crossref PubMed Scopus (170) Google Scholar, 8.Célèrier E. Rivat C. Jun Y. Laulin J.P. Larcher A. Reynier P. Simonnet G. Long-lasting hyperalgesia induced by fentanyl in rats: preventive effect of ketamine.Anesthesiology. 2000; 92: 465-472Crossref PubMed Scopus (482) Google Scholar), and even brief exposure to fentanyl or morphine can induce long-lasting hyperalgesia (8.Célèrier E. Rivat C. Jun Y. Laulin J.P. Larcher A. Reynier P. Simonnet G. Long-lasting hyperalgesia induced by fentanyl in rats: preventive effect of ketamine.Anesthesiology. 2000; 92: 465-472Crossref PubMed Scopus (482) Google Scholar, 9.Van Elstraete A.C. Sitbon P. Trabold F. Mazoit J.X. Benhamou D. A single dose of intrathecal morphine in rats induces long-lasting hyperalgesia: the protective effect of prior administration of ketamine.Anesth. Analg. 2005; 101: 1750-1756Crossref PubMed Scopus (69) Google Scholar). Hyperalgesia also occurs in patients after administration of various opioids (10.Chia Y.Y. Liu K. Wang J.J. Kuo M.C. Ho S.T. Intraoperative high dose fentanyl induces postoperative fentanyl tolerance.Can. J. Anaesth. 1999; 46: 872-877Crossref PubMed Scopus (257) Google Scholar, 11.Devulder J. Hyperalgesia induced by high-dose intrathecal sufentanil in neuropathic pain.J. Neurosurg. Anesthesiol. 1997; 9: 146-148Crossref PubMed Scopus (72) Google Scholar, 12.Stubhaug A. Breivik H. Eide P.K. Kreunen M. Foss A. Mapping of punctate hyperalgesia around a surgical incision demonstrates that ketamine is a powerful suppressor of central sensitization to pain following surgery.Acta Anaesthesiol. Scand. 1997; 41: 1124-1132Crossref PubMed Scopus (442) Google Scholar). The increased pain sensitivity requires the use of additional opioids to sustain the adequate analgesic effects and, consequently, could be interpreted as analgesic tolerance. The μ-opioid receptors at the spinal level are essential for the analgesic effect of opioids (13.Chen S.R. Pan H.L. Blocking mu opioid receptors in the spinal cord prevents the analgesic action by subsequent systemic opioids.Brain Res. 2006; 1081: 119-125Crossref PubMed Scopus (60) Google Scholar). We recently showed that in spinal lamina I and II neurons, brief opioid exposure can cause a long-lasting increase in glutamate release from nociceptive primary afferents (i.e. opioid-induced primary afferent hyperactivity) through activation of presynaptic NMDARs (14.Zhou H.Y. Chen S.R. Chen H. Pan H.L. Opioid-induced long-term potentiation in the spinal cord is a presynaptic event.J. Neurosci. 2010; 30: 4460-4466Crossref PubMed Scopus (109) Google Scholar). It is not clear, however, how pre- and postsynaptic NMDARs help regulate glutamatergic synaptic input to spinal dorsal horn neurons after chronic opioid treatment. Identifying the origin of NMDAR activity associated with chronic opioid treatment is critical because it will influence whether research should focus on primary sensory neurons or spinal cord second-order neurons to improve the efficacy of opioid therapies. In this study, we determined how chronic opioid treatment affects the function of pre- and postsynaptic NMDARs in the spinal cord. We provide new evidence showing that presynaptic NMDAR activity at primary afferent terminals is increased by chronic morphine and potentiates glutamatergic input to spinal dorsal horn neurons through protein kinase C (PKC). Strikingly, chronic morphine administration leads to a large reduction in postsynaptic NMDAR activity, which results from increased glutamate release from transient receptor potential vanilloid type 1 (TRPV1)-expressing primary afferents. This new information is important for understanding the mechanisms involved in NMDAR plasticity at the spinal level and their roles in opioid-induced hyperalgesia and analgesic tolerance. Male Sprague-Dawley rats (280–320 g; Harlan, Indianapolis, IN) were used in this study. Opioid hyperalgesia and analgesic tolerance were induced with daily intraperitoneal injections of morphine (10 mg/kg) for 8 consecutive days (15.Chen S.R. Prunean A. Pan H.M. Welker K.L. Pan H.L. Resistance to morphine analgesic tolerance in rats with deleted transient receptor potential vanilloid type 1-expressing sensory neurons.Neuroscience. 2007; 145: 676-685Crossref PubMed Scopus (35) Google Scholar). Rats in the control group received daily intraperitoneal injections of the vehicle (saline). For behavioral experiments with live rats, intrathecal catheters were implanted during isoflurane-induced anesthesia. Briefly, each animal was placed prone on a stereotaxic frame, and a small incision was made at the back of the neck of the animal. A small puncture was made in the atlantooccipital membrane of the cisterna magna, and a catheter was then inserted such that the caudal tip reached the lumbar enlargement of the spinal cord (13.Chen S.R. Pan H.L. Blocking mu opioid receptors in the spinal cord prevents the analgesic action by subsequent systemic opioids.Brain Res. 2006; 1081: 119-125Crossref PubMed Scopus (60) Google Scholar, 15.Chen S.R. Prunean A. Pan H.M. Welker K.L. Pan H.L. Resistance to morphine analgesic tolerance in rats with deleted transient receptor potential vanilloid type 1-expressing sensory neurons.Neuroscience. 2007; 145: 676-685Crossref PubMed Scopus (35) Google Scholar). We then exteriorized the rostral end of the catheter and closed the wound with sutures. The animals were allowed to recover for 5 days before intrathecal injections, and rats displaying signs of motor or neurological dysfunction were excluded from the study. Drugs were injected intrathecally in a volume of 10 μl followed by a 10-μl flush with normal saline. All surgical preparation and experimental protocols were approved by the Animal Care and Use Committee of The University of Texas M. D. Anderson Cancer Center and conformed to National Institutes of Health guidelines on the ethical use of animals. The mechanical nociception of rats was assessed by the paw pressure test using an Ugo Basile analgesimeter (Varese, Italy). Noxious pressure was gradually applied to hind paws by pressing the device pedal to increase the force at a constant rate. The pedal was immediately released when the animal displayed pain by withdrawing the paw or vocalizing, and the nociceptive threshold of the animal was read on the scale. To avoid potential tissue injury, a maximum of 400 g was used as a cutoff (15.Chen S.R. Prunean A. Pan H.M. Welker K.L. Pan H.L. Resistance to morphine analgesic tolerance in rats with deleted transient receptor potential vanilloid type 1-expressing sensory neurons.Neuroscience. 2007; 145: 676-685Crossref PubMed Scopus (35) Google Scholar, 16.Chen S.R. Pan H.L. Antinociceptive effect of morphine, but not μ-opioid receptor number, is attenuated in the spinal cord of diabetic rats.Anesthesiology. 2003; 99: 1409-1414Crossref PubMed Scopus (80) Google Scholar). Both hind paws were tested for each rat, and the mean value was used as the pressure nociceptive withdrawal threshold for that animal. To quantitatively assess the thermal nociceptive threshold, rats were tested with a thermal testing apparatus (IITC Inc., Woodland Hills, CA). The rats were allowed to acclimate for 30 min on a glass surface with the temperature maintained at 30 °C. A mobile radiant heat source was focused onto the plantar surface of the hind paw. The paw withdrawal latency was recorded using a timer. A cutoff of 30 s was used to prevent potential tissue damage (15.Chen S.R. Prunean A. Pan H.M. Welker K.L. Pan H.L. Resistance to morphine analgesic tolerance in rats with deleted transient receptor potential vanilloid type 1-expressing sensory neurons.Neuroscience. 2007; 145: 676-685Crossref PubMed Scopus (35) Google Scholar, 16.Chen S.R. Pan H.L. Antinociceptive effect of morphine, but not μ-opioid receptor number, is attenuated in the spinal cord of diabetic rats.Anesthesiology. 2003; 99: 1409-1414Crossref PubMed Scopus (80) Google Scholar). Both of the hind paws were tested in each animal, and the mean value was used as the thermal nociceptive withdrawal threshold. Originally isolated from the cactus-like plant Euphorbia resinifera, resiniferatoxin (RTX) is an ultrapotent TRPV1 agonist (17.Szallasi A. Blumberg P.M. Annicelli L.L. Krause J.E. Cortright D.N. The cloned rat vanilloid receptor VR1 mediates both R-type binding and C-type calcium response in dorsal root ganglion neurons.Mol. Pharmacol. 1999; 56: 581-587Crossref PubMed Scopus (122) Google Scholar). Systemic injection of RTX selectively kills TRPV1-expressing primary afferent neurons and their central terminals in adult rats (14.Zhou H.Y. Chen S.R. Chen H. Pan H.L. Opioid-induced long-term potentiation in the spinal cord is a presynaptic event.J. Neurosci. 2010; 30: 4460-4466Crossref PubMed Scopus (109) Google Scholar, 18.Chen S.R. Pan H.L. Loss of TRPV1-expressing sensory neurons reduces spinal μ-opioid receptors but paradoxically potentiates opioid analgesia.J. Neurophysiol. 2006; 95: 3086-3096Crossref PubMed Scopus (69) Google Scholar, 19.Zhou H.Y. Chen S.R. Chen H. Pan H.L. The glutamatergic nature of TRPV1-expressing neurons in the spinal dorsal horn.J. Neurochem. 2009; 108: 305-318Crossref PubMed Scopus (44) Google Scholar). Rats were anesthetized with isoflurane before receiving a single intraperitoneal injection of RTX (200 μg/kg, LC Laboratories, Woburn, MA) dissolved in a mixture of 10% ethanol and 10% Tween 80 in normal saline. Rats in the control group received a single intraperitoneal injection of the vehicle. Morphine treatment was conducted 7 days after the RTX and vehicle injections. The effect of RTX on TRPV1-expressing dorsal root ganglion neurons and central terminals was confirmed by using double labeling of TRPV1 and Griffonia simplicifolia isolectin B4 (IB4, a marker for unmyelinated afferent neurons and fibers) in the dorsal root ganglion and spinal dorsal horn in rats 7 days after RTX or vehicle injection, as described previously (13.Chen S.R. Pan H.L. Blocking mu opioid receptors in the spinal cord prevents the analgesic action by subsequent systemic opioids.Brain Res. 2006; 1081: 119-125Crossref PubMed Scopus (60) Google Scholar, 19.Zhou H.Y. Chen S.R. Chen H. Pan H.L. The glutamatergic nature of TRPV1-expressing neurons in the spinal dorsal horn.J. Neurochem. 2009; 108: 305-318Crossref PubMed Scopus (44) Google Scholar). The lumbar segment of the spinal cord was rapidly removed through laminectomy after the rats were anesthetized with 2% isoflurane. The lumbar spinal cord tissues were immediately placed in ice-cold sucrose artificial cerebrospinal fluid containing (in mm) 234 sucrose, 3.6 KCl, 1.2 MgCl2, 2.5 CaCl2, 1.2 NaH2PO4, 25.0 NaHCO3, and 12.0 glucose. The spinal cord was placed in a gelatin block and glued onto the stage of a vibratome. Transverse spinal cord slices were cut (400 μm in thickness) in ice-cold sucrose artificial cerebrospinal fluid and then transferred in Krebs solution presaturated with 95% O2 and 5% CO2 at 34 °C for at least 1 h before being transferred to the recording chamber. Neurons in the lamina II outer zone were visualized on a fixed stage microscope (BX50WI; Olympus, Tokyo, Japan) with differential interference contrast/infrared illumination. Lamina II of the spinal cord is the central site of termination of the majority of unmyelinated C-fibers carrying nociceptive information (20.Pan H.L. Khan G.M. Alloway K.D. Chen S.R. Resiniferatoxin induces paradoxical changes in thermal and mechanical sensitivities in rats: mechanism of action.J. Neurosci. 2003; 23: 2911-2919Crossref PubMed Google Scholar, 21.Pan Y.Z. Pan H.L. Primary afferent stimulation differentially potentiates excitatory and inhibitory inputs to spinal lamina II outer and inner neurons.J. Neurophysiol. 2004; 91: 2413-2421Crossref PubMed Scopus (62) Google Scholar, 22.Woodbury C.J. Ritter A.M. Koerber H.R. On the problem of lamination in the superficial dorsal horn of mammals: a reappraisal of the substantia gelatinosa in postnatal life.J. Comp. Neurol. 2000; 417: 88-102Crossref PubMed Scopus (71) Google Scholar). Excitatory postsynaptic currents (EPSCs) were recorded using whole-cell voltage clamp techniques (14.Zhou H.Y. Chen S.R. Chen H. Pan H.L. Opioid-induced long-term potentiation in the spinal cord is a presynaptic event.J. Neurosci. 2010; 30: 4460-4466Crossref PubMed Scopus (109) Google Scholar, 23.Chen S.R. Chen H. Yuan W.X. Pan H.L. Increased presynaptic and postsynaptic α2-adrenoceptor activity in the spinal dorsal horn in painful diabetic neuropathy.J. Pharmacol. Exp. Ther. 2011; 337: 285-292Crossref PubMed Scopus (39) Google Scholar). The impedance of the glass electrode was 4–7 MΩ when the pipette was filled with the internal solution containing (in mm) 135 potassium gluconate, 5 KCl, 2.0 MgCl2, 0.5 CaCl2, 5.0 HEPES, 5.0 EGTA, 5.0 ATP-Mg, 0.5 Na-GTP, and 10 QX314 (adjusted to pH 7.25 with 1.0 m CsOH, 280–300 mosm). The slice was continuously perfused with Krebs solution at 5.0 ml/min at 34 °C. Spontaneous EPSCs (sEPSCs) were recorded in the presence of 2 μm strychnine and 10 μm bicuculline at a holding potential of −60 mV. In addition, EPSCs were evoked from the dorsal root using a bipolar tungsten electrode connected to a stimulator (0.2 ms, 0.3–0.5 mA, 0.1 Hz; Grass Instruments, Quincy, MA). NMDAR-mediated EPSCs (NMDAR-EPSCs) were recorded in the presence of 2 μm strychnine, 10 μm bicuculline, and 20 μm 6-cyano-7-nitroquinoxaline-2,3-dione at a holding potential of +40 mV (24.Li D.P. Yang Q. Pan H.M. Pan H.L. Pre- and postsynaptic plasticity underlying augmented glutamatergic inputs to hypothalamic presympathetic neurons in spontaneously hypertensive rats.J. Physiol. 2008; 586: 1637-1647Crossref PubMed Scopus (77) Google Scholar). To determine the paired-pulse ratio, two EPSCs were evoked by a pair of stimuli given at 50-ms intervals. The paired-pulse ratio was expressed as the ratio of the amplitudes of the second and first synaptic responses (14.Zhou H.Y. Chen S.R. Chen H. Pan H.L. Opioid-induced long-term potentiation in the spinal cord is a presynaptic event.J. Neurosci. 2010; 30: 4460-4466Crossref PubMed Scopus (109) Google Scholar). The input resistance was monitored, and the recording was abandoned if it changed by more than 15%. EPSCs were recorded using an amplifier (MultiClamp 700A, Axon Instruments, Foster City, CA), filtered at 1–2 kHz, and digitized at 10 kHz. To directly determine postsynaptic NMDAR activity, currents were elicited by puff application of 100 μm NMDA to the recorded neuron in the extracellular solution containing a low concentration of Mg2+ (0.1 mm), 10 μm glycine, and 1 μm tetrodotoxin at a holding potential of −60 mV and using the pipette internal solution containing (in mm) 110.0 Cs2SO4, 2.0 MgCl2, 0.1 CaCl2, 1.1 EGTA, 10.0 HEPES, 2.0 MgATP, and 0.3 Na2GTP (pH was adjusted to 7.25 with 1.0 m CsOH (280–300 mosm)) (24.Li D.P. Yang Q. Pan H.M. Pan H.L. Pre- and postsynaptic plasticity underlying augmented glutamatergic inputs to hypothalamic presympathetic neurons in spontaneously hypertensive rats.J. Physiol. 2008; 586: 1637-1647Crossref PubMed Scopus (77) Google Scholar). Similarly, to examine postsynaptic AMPAR function, currents were elicited similarly but with 100 μm AMPA (25.Li D.P. Byan H.S. Pan H.L. Switch to glutamate receptor 2-lacking AMPA receptors increases neuronal excitability in hypothalamus and sympathetic drive in hypertension.J. Neurosci. 2012; 32: 372-380Crossref PubMed Scopus (44) Google Scholar). The puff electrode was placed about 150 μm away from the neuron being recorded. Puff application of NMDA or AMPA was performed using a Pressure System IIe (4 p.s.i., 15 ms; Toohey Co., Fairfield, NJ). All drugs were freshly prepared in artificial cerebrospinal fluid before the experiments and delivered via syringe pumps to reach their final concentrations. Chelerythrine, NMDA, and AMPA were purchased from Sigma-Aldrich. (Rp)-cAMPS was obtained from Tocris. Ro 25-6981, AP5, 6-cyano-7-nitroquinoxaline-2,3-dione, bicuculline, and tetrodotoxin were purchased from Ascent Scientific. All data are expressed as means ± S.E. To determine the amplitude of the evoked EPSCs, at least 10 consecutive EPSCs were averaged and analyzed off-line with Clampfit 10.0 software. sEPSCs were analyzed off-line using the Mini Analysis peak detection program (Synaptosoft, Leonia, NJ). The cumulative probability of the amplitude and the inter-event interval of the sEPSCs were compared using the Kolmogorov-Smirnov test, which estimates the probability that two distributions are similar. We used Student's t test to compare two groups and one-way analysis of variance followed by Dunnett's post hoc test to compare more than two groups. Two-way analysis of variance followed by Bonferroni's post hoc test was used to determine the difference in the development of hyperalgesia and opioid tolerance between the vehicle-treated and AP5-treated groups. A p value of < 0.05 was considered to be statistically significant. We previously showed that brief exposure to the μ-opioid receptor agonist (D-Ala2, N-Me-Phe4, Gly-ol5)-enkephalin increases glutamatergic input to dorsal horn neurons (14.Zhou H.Y. Chen S.R. Chen H. Pan H.L. Opioid-induced long-term potentiation in the spinal cord is a presynaptic event.J. Neurosci. 2010; 30: 4460-4466Crossref PubMed Scopus (109) Google Scholar). To determine the effect of chronic morphine treatment on glutamatergic input to dorsal horn neurons, we recorded sEPSCs in the spinal cord slices from vehicle- and morphine-treated rats. The frequency of sEPSCs of lamina II neurons was significantly higher in the morphine-treated group (n = 11 neurons) than in the vehicle-treated group (n = 13 neurons), but the difference in the amplitude of sEPSCs was not significantly different (Fig. 1, A–C). To determine the role of NMDARs in the control of glutamatergic input to dorsal horn neurons, we tested the effect of AP5, a selective NMDAR antagonist (26.Olverman H.J. Jones A.W. Watkins J.C. l-Glutamate has higher affinity than other amino acids for [3H]-d-AP5 binding sites in rat brain membranes.Nature. 1984; 307: 460-462Crossref PubMed Scopus (224) Google Scholar), on sEPSCs. We have shown that 50 μm AP5 completely blocks NMDAR-mediated EPSCs (24.Li D.P. Yang Q. Pan H.M. Pan H.L. Pre- and postsynaptic plasticity underlying augmented glutamatergic inputs to hypothalamic presympathetic neurons in spontaneously hypertensive rats.J. Physiol. 2008; 586: 1637-1647Crossref PubMed Scopus (77) Google Scholar). Bath application of 50 μm AP5 significantly decreased the frequency, but not the amplitude, of sEPSCs of lamina II neurons in the same morphine-treated rats (n = 11 neurons, Fig. 1, B and C). However, AP5 application had no significant effects on the frequency and amplitude of sEPSCs of lamina II neurons in vehicle-treated rats (n = 13 neurons, Fig. 1, A and C). To determine the role of presynaptic NMDARs in glutamatergic input increased by morphine treatment, we recorded sEPSCs in separate lamina II neurons by including the NMDAR channel blocker MK-801 in the pipette solution to block the postsynaptic NMDARs. We have shown that intracellular dialysis of 1 mm MK-801 completely blocked the postsynaptic NMDARs within 15 min (14.Zhou H.Y. Chen S.R. Chen H. Pan H.L. Opioid-induced long-term potentiation in the spinal cord is a presynaptic event.J. Neurosci. 2010; 30: 4460-4466Crossref PubMed Scopus (109) Google Scholar). In all 11 neurons recorded with MK-801, AP5 still significantly reduced the frequency of sEPSCs in morphine-treated rats (p < 0.05, Fig. 1D). These results suggest that chronic morphine increases glutamatergic input to spinal dorsal horn neurons by enhancing presynaptic NMDAR activity. To determine whether NMDARs at primary afferent terminals play a role in increased glutamate release to spinal dorsal horn neurons, monosynaptic EPSCs were evoked from the dorsal root in vehicle- and morphine-treated rats. These EPSCs were identified on the basis of the constant latency and absence of conduction failure of evoked EPSCs in response to a 20-Hz electrical stimulation, as we described previously (14.Zhou H.Y. Chen S.R. Chen H. Pan H.L. Opioid-induced long-term potentiation in the spinal cord is a presynaptic event.J. Neurosci. 2010; 30: 4460-4466Crossref PubMed Scopus (109) Google Scholar, 27.Zhou H.Y. Chen S.R. Chen H. Pan H.L. Sustained inhibition of neurotransmitter release from nontransient receptor potential vanilloid type 1-expressing primary afferents by μ-opioid receptor activation-enkephalin in the spinal cord.J. Pharmacol. Exp. Ther. 2008; 327: 375-382Crossref PubMed Scopus (20) Google Scholar). In this protocol, we used a fixed stimulation intensity to evoke AMPAR-EPSCs in vehicle-treated and morphine-treated rats. The amplitude of AMPAR-EPSCs of lamina II neurons was significantly higher in morphine-treated rats than in vehicle-treated rats (n = 9 neurons, Fig. 2, A and B). Bath application of 50 μm AP5 significantly reduced the amplitude of monosynaptic EPSCs of lamina II neurons in morphine-treated rats (n = 11 neurons, Fig. 2, A and B). Also, the effect of AP5 on evoked monosynaptic EPSCs of morphine-treated rats was associated with a decrease in the paired-pulse ratio (Fig. 2C). However, AP5 had no significant effect on the amplitude of monosynaptic EPSCs of lamina II neurons recorded from vehicle-treated rats (n = 14 neurons, Fig. 2, A and B). In another nine lamina II neurons from morphine-treated rats, 1 mm MK-801 was included in the internal pipette solution and dialyzed for 15 min to block postsynaptic NMDARs before EPSCs were recorded. In all nine neurons, bath application of 50 μm AP5 still significantly reduced the amplitude of monosynaptic AMPAR-EPSCs evoked from the dorsal root (Fig. 2, A and B). Taken together, these results indicate that chronic morphine increases presynaptic NMDAR activity at primary afferent terminals in the spinal cord. Chronic opioid treatment results in significant increases in adenylyl cyclase activity and in basal levels of cAMP in the central nervous system (28.Avidor-Reiss T. Nevo I. Saya D. Bayewitch M. Vogel Z. Opiate-induced adenylyl cyclase superactivation is isozyme-specific.J. Biol. Chem. 1997; 272: 5040-5047Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 29.Jolas T. Nestler E.J. Aghajanian G.K. Chronic morphine increases GABA tone on serotonergic neurons of the dorsal raphe nucleus: association with an up-regulation of the cyclic AMP pathway.Neuroscience. 2000; 95: 433-443Crossref PubMed Scopus (99) Google Scholar). To determine whether cAMP-dependent PKA is involved in increased presynaptic NMDAR activity by chronic morphine, we used (Rp)-cAMPS, a highly selective and membrane-permeable PKA inhibitor. (Rp)-cAMPS competes with cAMP for the binding sites on the regulatory subunit of PKA and, when bound, prevents release of the free catalytic subunit (30.Connelly P.A. Botelho L.H. Sisk R.B. Garrison J.C. A study of the mechanism of glucagon-induced protein phosphorylation in isolated rat hepatocytes using (Sp)-cAMPS and (Rp)-cAMPS, the stimulatory and inhibitory diastereomers of adenosine cyclic 3′,5′-phosphorothioate.J. Biol. Chem. 1987; 262: 4324-4332Abstract Full Text PDF PubMed Google Scholar). It has been shown that 100 μm (Rp)-cAMPS inhibits PKA activity in brain slices (31.Xie C.W. Lewis D.V. Involvement of cAMP-dependent protein kinase in μ-opioid modulation of NMDA-mediated synaptic currents.J. Neurophysiol. 1997; 78: 759-766Crossref PubMed Scopus (30) Google Scholar). Incubation with 100 μm (Rp)-cAMPS in the spinal cord slices from morphine-treated rats for 1–2 h had no significant effect on the base-line frequency of sEPSCs (n = 11 neurons) or the amplitude of monosynaptic EPSCs (n = 12 neurons) of lamina II neurons evoked from the dorsal root (Fig. 3, A and C). Furthermore, bath application of 50 μm AP5 still significantly inhibited the frequency of sEPSCs and the amplitude of evoked EPSCs in all lamina II neurons tested in the spinal cord slices treated with (Rp)-cAMPS (Fig. 3, B and D). Intrathecal administration of PKC inhibitors can reverse or attenuate opioid tolerance induced by chronic morphine administration (32.Granados-Soto V. Kalcheva I. Hua X. Newton A. Yaksh T.L. Spinal PKC activity and expression: role in tolerance produced by continuous spinal morphine infusion.Pain. 2000; 85: 395-404Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Increased PKC activity can promote NMDAR surface expression and regulate the channel gating (33.Lan J.Y. Skeberdis V.A. Jover T. Grooms S.Y. Lin Y. Araneda R.C. Zheng X. Bennett M.V. Zukin R.S. Protein kinase C modulates NMDA receptor trafficking and gating.Nat. Neurosci.