Enkephalin (ENK)‐containing neurons in the spinal superficial dorsal horn (SDH) play an important role in regulating the transmission of nociceptive information. Using BAC transgenic mice, in which ENK‐containing neurons can be identified by green fluorescent protein expressed under the control of preproenkephalin gene promoter, we have observed that ENK‐containing neurons in the SDH express the NMDA receptor 2B (NR2B) subunit and large conductance calcium‐activated potassium (BK) channels. In experiments, we investigated the effects on nociceptive behavior of intrathecally administrating NR2B subunit antagonist and BK channel antagonist in peripheral nerve‐injured mice.The experiments were performed in 6‐ to 8‐week‐old male ICR mice. Partial sciatic nerve ligation was performed as described previously (PSL, Seltzer model). To assess the development of mechanical allodynia, we measured the frequency of withdrawal responses to 10 repetitive stimuli with von Frey filaments. Behavioral assessments were performed beginning 3 days prior to the ligation surgery. On post‐surgical day 7, effects on nociceptive behavior of the BK channel antagonist, charybdotoxin (CTX), and the NR2B antagonist, ifenprodil (IFN), were analyzed. Additionally, in order to clarify whether the actions of CTX and IFN are mediated by an alteration in the release of enkephalin, we investigated the effect of the selective δ‐opioid receptor antagonist naltrindole (NTI).CTX (1 pmol/10 μL per mouse), IFN (50 nmol/10 μL per mouse) and saline as a control (10 μL per mouse) was intrathecally injected according to the modified method originally described by Hylden. All behavioral experiments were performed in double‐blind fashion.PSL significantly increased the occurrence of withdrawal reflex to von Frey stimuli being indicative of the development of mechanical allodynia. Intrathecal CTX significantly reduced the occurrence of withdrawal reflex as compared to saline group. Additionally, intraperitoneal administration of NTI at the dose of 5 mg/kg body weight attenuated the effect on nociceptive behavior of CTX. Intrathecal IFN significantly reduced the occurrence of withdrawal reflex. This effect was also blocked by intraperitoneal NTI.These behavioral observations might suggest that NMDA receptors and BK channels inhibit the activity of enkephalin‐containing neurons in the spinal dorsal horn, which is speculated to be involved in peripheral nerve injury‐induced chronic pain state.Support or Funding InformationThis work was supported by Dokkyo Medical University Endowment Fund.

Destabilization refers to a memory that becomes unstable when reactivated and is susceptible to disruption by amnestic agents. Here we delineated the cellular mechanism underlying the destabilization of drug memory. Mice were conditioned with methamphetamine (MeAM) for 3 d, and drug memory was assessed with a conditioned place preference (CPP) protocol. Anisomycin (ANI) was administered 60 min after the CPP retrieval to disrupt reconsolidation. We found that destabilization of MeAM CPP after the application of ANI was blocked by the N-methyl-d-aspartate receptor (NMDAR) antagonist MK-801 and the NR2B antagonist ifenprodil (IFN) but not by the NR2A antagonist NVP-AAM077 (NVP). In addition, decrease in the phosphorylation of GluR1 at Serine845 (p-GluR1-Ser845), decrease in spine density, and a reduction in the AMPAR/NMDAR ratio in the basolateral amygdala (BLA) were reversed after the MK-801 treatment. The effect of ANI on destabilization was prevented by the protein phosphatase 2B (calcineurin, CaN) inhibitors cyclosporine A (CsA) and FK-506 and the protein phosphatase 1 (PP1) inhibitors calyculin A (CA) and okadaic acid (OA). These results suggest that memory destabilization involves the activation of NR2B-containing NMDARs, which in turn allows the influx of Ca2+. Increased intracellular Ca2+ stimulates CaN, leading to the dephosphorylation and inactivation of inhibitor 1 and the activation of PP1. PP1 then dephosphorylates p-GluR1-Ser845 to elicit AMPA receptor (AMPAR) endocytosis and destabilization of the drug memory.

Objective To investigate the preventive effect of intrathecal KN93 or Ifenprodil administration on remifentanil-induced incisional hyperalgesiaafter surgery iincisional pain in rats.Methods Thirty SD rats were randomly divided into 5 groups (n=6):control group (C group),incision pain group (Ⅰ group),Remifentanil group (R group),KN93 group (K group) and Ifenprodil group(F group).A hind paw incisional model was used to induce incisional painexcept for control group.At the same time,0.04 mg/kg remifentanil was infused subcutaneously 30 min in R group,K group and F group,30 min before incisional model,20 μl dimethyl sulfoxide (DMSO,10％),20 μl KN93 (50 μg/10 μl) and 20 μl Ifenprodil (10 μg/20 μl) were intrathecally injected to rats in R group,K group or F group respectively.Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were tested on 24 h before and 2,6,24,48 h after operation.Results The PWTL and PMWT on the ipsilateral side significantly decreased after operation in Ⅰ group compared with C group; and evenshortenin R group than that in Ⅰ group.Compared with R group,the PWTL(15.32±0.73),(15.79±0.32),(14.86±0.98),(14.76±0.82) s and PMWT (31.8±1.4),(29.3±1.2),(30.8±1.0),(30.5±1.2) g significantly increased in K group(P＜0.05) at each time point.Meanwhile,the PWTL(14.24±0.79),(15.33±0.29),(15.29±0.74),(15.21±0.45) s and PMWT(29.3±1.8),(30.2±2.1),(29.5±1.5),(30.2±1.0) g obviously increased in F group(P＜0.05) at each time point.Conclusions Intrathecal injection of KN93 or Ifenprodil coulde effectively alleviate remifentanil-induced hyperalgesia. Key words: Ca2+/calmodulin dependent protein kinase Ⅱ ; N-methyl-D-aspartate receptor 2B; Remifentanil; Hyperalgesia; Incision pain

Objective To explore the possible mechanism for the neuroprotective effect of ifenprodil by investigating its effects on inducible nitric oxide synthase (iNOS) expression and activity and apoptosis in the ischemic penumbra following focal cerebral ischemia-reperfusion (I/R) in rats.Methods Fifty-four adult male SD rats weighing 280-320 g were randomly divided into 3 groups ( n = 18 each) : I sham operation group (group S) ; II focal cerebral I/R group (group I/R) and Ⅲ ifenpradil preconditioning group (group IF) received intraperitoneal ifenprodil 10 mg/kg before focal cerebral I/R. Focal cerebral I/R was induced by middle cerebral artery occlusion (MCAO) . A 3-0 nylon thread with rounded tip was inserted into right internal jugular vein and threaded cranially until resistance was met. MCAO was maintained for 2 h. At 48 h after reperfusion, the animals were assessed for neurological function which was scored (0 = no functional deficit, 4 = unable to crawl, unconscious) and then decapitated. The brains were immediately removed for microscopic examination and determination of iNOS protein expression and activity, NO content and apoptosis in the ischemic core (IC) and penumbra (IP). Results Ifenprodil pretreatment significantly decreased the cerebral infarct size and neurological scores in group IF as compared with group I/R. In group I/R the iNOS activity was increased compared with group S.The iNOS activity and NO content were significantly lower in IP than in IC in group IR and IF. The TUNEL-positive cells were also mainly confined to IP. Compared with group I/R, in group IF the iNOS protein expression was significantly down-regulated in IC and IP and the iNOS activity and NO content in IC and IP were suppressed and TUNEL-positive cells were significantly reduced in IP. Conclusion Ifenprodil pretreatment has protective effect against cerebral I/R injury by inhibiting iNOS protein expression in IP, suppressing iNOS activity and NO content and reducing apoptosis. Key words: Piperidines; Reperfusion injury; Nitric oxide synthase; Apoptosis

Objective To investigate the analgesic effect of intrathecal injection of NR2B antagonist ifenprodil in mouse of bone caner pain. Methods The mouse model of bone cancer pain was developed by intra-femur inoculations of α-minimal essence media (α-MEM) with osteolytic NCTC 2472 cells in C3H/HeJ mice(group T). The sham group (group S,n =10) were inoculated by α-MEM without any cells. Pain ethology indexes such as the spontaneous lifting behaviors, the paw withdrawal mechanical threshold(PWMT) and the paw withdrawal thermal latency (PWTL) were observed on 1 d before inoculation and on 3 d,5 d,7 d,10 d,14 d after inoculation to evaluate the mouse model of bone cancer. On the 14th d after inoculation, the succeed mouse model of bone cancer pain was divided randomly into 2.5 μg, 5 μg, 10 μg ifenprodil group(group I1, I2, I3,n =10) and solventia group (20%DMSO group, group C,n =10). Group I1～I3 were treated by intrathecal injection of corresponding dose of ifenprodil. Group C and group S were treated by intrathecal injection of solventia. All the volume of physic liquor of intrathecal injection was 5 μl. Pain ethology indexes mentioned above was observed at 2 h, 12 h, 24 h after administration. Results (1) The significant increasing of spontaneous lifting behaviors(13.16±1.78), the obvious decreasing of PWMT (0.47±0.19)g and PWTL (10.01±1.42)s were observed on 14th d after inoculation in group T compared with group S and preoperative base level( P ＜0.05). (2) The spontaneous lifting behaviors [I2:(9.00±1.80),(10.90±1.17);I3:(6.80± 2.51),(8.80±1.64)] was decreased, the PWMT [I2:(1.12±0.27)g,(0.82±0.30)g;I3:(1.44±0.34)g,(1.16±0.37)g] and PWTL [I2:(13.86±1.51)s,(12.12±1.27)s;I3:(15.95±1.70)s,(13.74±1.98)s] was increased in group I2 and I3 compared with the basal level on 14th d before administration and group C at 2 h and 12 h after administration ( P ＜0.05) The analgesic effect of group I3 was more conspicuous compared with group I2( P ＜0.05). No obvious change of pain ethology indexes was observed in group I1 ( P ＞0.05). Conclusion Intrathecal injection of ifenprodil can efficiently relieve mechanical hyperalgia and thermal hyperalgia in the mouse model of bone cancer pain. Key words: Ifenprodil; NR2B; Bone cancer pain; Spinal cord

Objective To investigate the effect of intrathecal injection of ifenprodil on the expression of 2B subunit mRNA of NMDA receptor (NR2B) in spinal cord in a mouse model of bone cancer pain. Methods One hundred and forty male C3H/HeJ mice weighing 20-25 g were randomly divided into 5 groups (n=28 each):group S sham operation; group B bone cancer pain; group I1- ifenprodil 2.5, 5 and 10 μg. Bone cancer pain was performed by intra-right-femur inoculations of osteolytic NCTC 2472 cells in group I1- and B. On the 14 d after inoculations, group I1-3 received intrathecal injection of ifenprodil 2.5, 5 and 10 μg dissolved in 5 μl solvent respectively, while group B and S received intrathecal injection of solvent. Seven mice was selected randomly from each group at 1 d before inoculations, at 1 h before, intrathecal injection of ifenprodil or solvent and at 2, 12 and 24 h after the injection (T1-5) tor determination of mechanical and thermal pain threshold. The mice were decapitated at T2.5 after determination of pain threshold and the L3-5 part of spinal cord was removed for determination of the expression of NR2B mRNA by RT-PCR. Results The mechanical and thermal pain threshold were significantly lower in group B and I1-3(P 0.05), and the expression of NR2B mRNA was significantly higher in group B and I3 while lower in group I2.3 than in group S (P 0.05). The mechanical and thermal pain threshold were significantly higher and the expression of NR2B mRNA was lower in group I3 than in group I2 (P <0.05). Conclusion lntratheeal injection of ifenprodil can attenuate bone cancer pain through blocking NR2B and inhibit hyperalgesia through down-regulating the NR2B mRNA expression in spinal cord. Key words: Piperidines; Receptors, N-methyl-D-aspartate; Spinal cord; Bone neoplasms; Injections, spinal

Background: Studies indicate that the NR2B subunits of N-methyl-D-aspartate (NMDA) receptors are involved in the rewarding effects of morphine. In the present study we further investigated the role and action sites of the NR2B subunit of NMDA receptors in morphine rewarding and other behaviors related to drug addiction, such as drug seeking and behavioral sensitization. Methods: A selective antagonist of the NR2B subunit of NMDA receptors, ifenprodil, was locally injected into the nucleus accumbens (NAc) or ventral tegmental area (VTA) in male Sprague Dawley (S.D.) rats to block the NMDA receptors that contain NR2B subunits. A conditioned place preference (CPP) test was used to examine the rewarding and drugseeking effects. Locomotor activity tests were used to determine the behavioral sensitization induced by chronic morphine treatment. Results: Morphine-induced rewarding and drug-seeking behavior were abolished when the NR2B subunits of NMDA receptors at NAc were blocked by ifenprodil that was either coadministered with morphine during CPP conditioning or posttreated during the morphine-withdrawal period. In contrast, morphine still induced rewarding, drug-seeking behavior, and behavioral sensitization when ifenprodil was injected at the VTA. On the other hand, only when ifenprodil was coadministered with morphine did it partially inhibit the behavioral sensitization induced by morphine. Conclusions: These data imply that at least the NR2B subunits of NMDA receptors in the NAc, but not VTA, are involved in morphine-induced rewarding and drug-seeking effects.

Ifenprodil and SL 82.0715 antagonize N-methyl-D-aspartate (NMDA)-coupled glycine receptor responses in vivo

Pharmacological effects of ifenprodil (IP), 4-benzyl-alpha-(p-hydroxyphenyl)-beta-methyl-1-piperidineethanol-L-(+)-tartrate monohydrate (Funai Pharmaceutical), a potential vasodilator, were studied in the dog, rabbit, guinea-pig and mouse. 1) Intravenous administration of IP (0.1 approximately 1 mg/kg) exhibited a continuous fall in blood pressure, an increase in heart rate and an increase in cardiac contractile force in the dog. The depressor and chronotropic effects of IP appeared likewise in the pithed dog, and the depressor responses were slightly more evident in the rabbit. The cardiovascular responses to IP were not affected by pretreatment with atropine or diphenhydramine, however, the chronotropic and inotropic effects were completely removed and the depressor response was depressed to some extent by propranolol. On the contrary, a pressor response to adrenaline was reversed to a depressor one after IP in the dog. 2) In heart-lung preparation of dogs, IP brought about slight cardiac stimulating effects, such as an increase in contractile force and a rise in aortic pressure. 3) IP induced a transient slight excitation in in situ movements of the rabbit intestine, and a similar excitation followed with a inhibition in movements of isolated intestine (10(-6), 2.5 X 10(-5) g/ml. This slight excitation was not affected by pretreatment with atropine, whereas the inhibition was depressed to a certain degree by simultaneous pretreatment with phenoxybenzamine and propranolol. IP produced a slight tonic increase in the guinea-pig isolated intestine and inhibited markedly the excitatory effect of histamine in the intestine. 4) IP did not affect the bronchial muscle but inhibited prominently a contractile respose of the muscle to histamine in in situ experiments on guinea-pig. 5) Locomotor activity was reduced dose-dependently and thiopental hypnosis was potentiated similarly after subcutaneous administration of IP (5 approximately 40 mg/kg), while skeletal muscle relaxant effects were not observed. 6) Survival time under a condition of acute anoxia was not changed with a dose of 0.5 and 1 mg/kg, but shortened with a dose of 5 mg/kg. The results suggest that IP has a alpha-blocking, a beta-stimulating and a moderate antihistaminic effects, and shows some cardiovascular effects, such as continuous depressor and chronotropic effects, and a moderate central inhibition.