MK-801 upregulates NR2A protein levels and induces functional recovery of the ipsilateral hemidiaphragm following acute C2 hemisection in adult rats

BACKGROUND: C2 hemisection results in paralysis of the ipsilateral hemidiaphragm. Recent data indicate that an upregulation of the N-methyl-D-aspartate (NMDA) receptor 2A subunit following chronic C2 hemisection is associated with spontaneous hemidiaphragmatic recovery following injury. MK-801, an antagonist of the NMDA receptor, upregulates the NR2A subunit in neonatal rats. HYPOTHESIS: We hypothesized that administration of MK-801 to adult, acute C2-hemisected rats would result in an increase of NR2A in the spinal cord. Furthermore, we hypothesized that upregulation of NR2A would be associated with recovery of the ipsilateral hemidiaphragm as in the chronic studies. DESIGN: To develop a dose-response curve, adult rats were treated with varying doses of MK-801 and their spinal cords harvested and assessed for NR2A as well as AMPA GluR1 and GluR2 subunit protein levels. In the second part of this study, C2-hemisected animals received MK-801. Following treatment, the animals were assessed for recovery of the hemidiaphragm through electromyographic recordings and their spinal cords assessed for NR2A, GluR1, and GluR2. RESULTS: Treatment with MK-801 leads to an increase of the NR2A subunit in the spinal cords of adult noninjured rats. There were no changes in the expression of GluR1 and GluR2 in these animals. Administration of MK-801 to C2-hemisected rats resulted in recovery of the ipsilateral hemidiaphragm, an increase of NR2A, and a decrease of GluR2. CONCLUSION: Our findings strengthen the evidence that the NR2A subunit plays a substantial role in mediating recovery of the paralyzed hemidiaphragm following C2 spinal cord hemisection.     

MK-801 Upregulates NR2A Protein Levels and Induces Functional Recovery of the Ipsilateral Hemidiaphragm Following Acute C2 Hemisection in Adult Rats

Abstract

Background: C2 hemisection results in paralysis of the ipsilateral hemidiaphragm. Recent data indicate that an upregulation of the N-methyl-D-aspartate (NMDA) receptor 2A subunit following chronic C2 hemisection is associated with spontaneous hemidiaphragmatic recovery following injury. MK-801, an antagonist of the NMDA receptor, upregulates the NR2A subunit in neonatal rats.

Hypothesis: We hypothesized that administration of MK-801 to adult, acute C2-hemisected rats would result in an increase of NR2A in the spinal cord. Furthermore, we hypothesized that upregulation of NR2A would be associated with recovery of the ipsilateral hemidiaphragm as in the chronic studies.

Design: To develop a dose-response curve, adult rats were treated with varying doses of MK-801 and their spinal cords harvested and assessed for NR2A as well as AMPA GluRI and GluR2 subunit protein levels. In the second part of this study, C2-hemisected animals received MK-801. Following treatment, the animals were assessed for recovery of the hemidiaphragm through electromyographic recordings and their spinal cords assessed for NR2A, GluRI, and GluR2.

Results: Treatment with MK-801 leads to an increase of the NR2A subunit in the spinal cords of adult noninjured rats. There were no changes in the expression of GluRI and GluR2 in these animals. Administration of MK-801 to C2-hemisected rats resulted in recovery of the ipsilateral hemidiaphragm, an increase of NR2A, and a decrease of GluR2.

Conclusion: Our findings strengthen the evidence that the NR2A subunit plays a substantial role in mediating recovery of the paralyzed hemidiaphragm following C2 spinal cord hemisection.     

Effects of synaptic activity on dendritic spine motility of developing cortical layer v pyramidal neurons

It is increasingly clear that dendritic spines play an important role in compartmentalizing post-synaptic signals and that their dynamic morphological properties have functional consequences. Here, we examine this issue using two-photon microscopy to characterize spine motility on layer V pyramidal neurons in acute slices of the developing mouse cortex. In this system, all spine classes except filopodia become less dynamic as development proceeds. General manipulations of activity (TTX or KCl treatment) do not alter spine dynamics, although increased glutamatergic transmission (AMPA or NMDA treatment) stabilizes developing cortical spines. These effects on spine dynamics do not appear to be related to AMPA or NMDA receptor expression as assessed with immunolabeling, as there is no correlation between spine motility and AMPA (GluR1/2) or NMDA (NR1/NR2B) receptor subunit expression on a spine by spine basis. These results indicate that activity through glutamatergic synapses is important for regulating spine motility in the developing mouse cortex, and that the relative complement of receptors, while different across morphological classifications, cannot account for differences in dynamic structural changes in dendritic spines.     

Colocalization of glutamate ionotropic receptor subunits in the human temporal neocortex

alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) receptors represent major classes of glutamate receptors (GluR) which play fundamental roles in normal excitatory synaptic activity and, probably, in the etiology of several brain diseases. These receptors are composed of multiple receptor subunit proteins, and the differential expression of these subunits in cortical neurons is considered to be one of the substrates for the functional diversity of cortical excitatory circuitry. In the monkey neocortex, different subpopulations of neurons have been identified on the basis of immunocytochemical colocalization studies using subunit-specific antibodies, but no comparable investigations have been made in the human neocortex. The aim of the present study was to determine quantitatively GluR subunit combinations in the human temporal neocortex by double-labeling immunocyto- chemical experiments. We quantified the neuronal populations expressing different receptor subtypes with fluorescent tags visualizing them with confocal laser microscopy. We studied AMPA, kainate- and NMDA-receptor subunits, using antibodies against GluR1, GluR2, GluR2/3, GluR2/4, GluR5/6/7 and NMDAR1 subunits. A high degree of colocalization (93-100%) using combinations of antibodies against GluR2 with GluR2/3, GluR2/3 with GluR2/4, and GluR2 or GluR2/4 with NMDAR1 was found, whereas for other combinations the degree of colocalization varied between 38% and 88%. Some of the percentages reported here are similar to those found in the monkey cortex, whereas others differ considerably. These results emphasize the diversity of excitatory circuits in the human neocortex, and suggest species differences with regard to some of these GluR-mediated circuits.     

Mechanical loading modulates glutamate receptor subunit expression in bone

The cellular mechanisms coupling mechanical loading with bone remodeling remain unclear. In the CNS, the excitatory amino acid glutamate (Glu) serves as a potent neurotransmitter exerting its effects via various membrane Glu receptors (GluR). Nerves containing Glu exist close to bone cells expressing functional GluRs. Demonstration of a mechanically sensitive glutamate/aspartate transporter protein and the ability of glutamate to stimulate bone resorption in vitro suggest a role for glutamate linking mechanical load and bone remodeling. We used immunohistochemical techniques to identify the expression of N-methyl-d-aspartate acid (NMDA) and non-NMDA (AMPA or kainate) ionotropic GluR subunits on bone cells in vivo. In bone sections from young adult rats, osteoclasts expressed numerous GluR subunits including AMPA (GluR2/3 and GluR4), kainic acid (GluR567) and NMDA (NMDAR2A, NMDAR2B and NMDAR2C) receptor subtypes. Bone lining cells demonstrated immunoexpression for NMDAR2A, NMDAR2B, NMDAR2C, GluR567, GluR23, GluR2 and GluR4 subunits. Immunoexpression was not evident on osteocytes, chondrocytes or vascular channels. To investigate the effects of mechanical loading on GluR expression, we used a Materials Testing System (MTS) to apply 10 N sinusoidal axial compressive loads percutaneously to the right limbs (radius/ulna, tibia/fibula) of rats. Each limb underwent 300-load cycles/day (cycle rate, 1 Hz) for 4 consecutive days. Contralateral, non-loaded limbs served as controls. Mechanically loaded limbs revealed a load-induced loss of immunoexpression for GluR2/3, GluR4, GluR567 and NMDAR2A on osteoclasts and NMDAR2A, NMDAR2B, GluR2/3 and GluR4 on bone lining cells. Both neonatal rabbit and rat osteoclasts were cultured on bone slices to investigate the effect of the NMDA receptor antagonist, MK801, and the AMPA/kainic acid receptor antagonist, NBQX, on osteoclast resorptive activity in vitro. The inhibition of resorptive function seen suggested that both NMDAR and kainic acid receptor function are required for normal osteoclast function. While the exact role of ionotropic GluRs in skeletal tissue remains unclear, the modulation of GluR subunit expression by mechanical loading lends further support for participation of Glu as a mechanical loading effector. These ionotropic receptors appear to be functionally relevant to normal osteoclast resorptive activity.     

Spinal axonal injury induces brief downregulation of ionotropic glutamate receptors and no stripping of synapses in cord-projection central neurons

Spinal cord injury often damages the axons of cord-projecting central neurons. To determine whether their excitatory inputs are altered following axonal injury, we used rat rubrospinal neurons as a model and examined their excitatory input following upper cervical axotomy. Anterograde tracing showed that the primary afferents from the cerebellum terminated in a pattern similar to that of control animals. Ultrastructurally, neurons in the injured nucleus were contacted by excitatory synapses of normal appearance, with no sign of glial stripping. Since cerebellar fibers are glutamatergic, we examined the expression of ionotropic receptor subunits GluR1-4 and NR1 for AMPA and NMDA receptors, respectively, in control and injured neurons using immunolabeling methods. In control neurons, GluR2 appeared to be low as compared to GluR1, GluR3, and GluR4, while NR1 labeling was intense. Following unilateral tractotomy, the levels of expression of each subunit in axotomized neurons appeared to be normal, with the exception that they were lower than those of control neurons of the nonlesioned side at 2-6 days postinjury. These findings suggest that axotomized neurons are only temporarily protected from excitotoxicity. This is in sharp contrast to the responses of central neurons that innervate peripheral targets, in which both synaptic stripping and reduction of their ionotropic glutamate receptor subunits persist following axotomy. The absence of an injury-induced trimming of afferents and stripping of synapses and the lack of a persistent downregulation of postsynaptic receptors might enable injured cord-projection neurons to continue to control their supraspinal targets during most of their postinjury survival. Although this may support neurons by providing trophic influences, it nevertheless may subject them to excitotoxicity and ultimately lead to their degenerative fate.     

The role of N-methyl-D-aspartate (NMDA) receptors in pain and morphine tolerance

To determine the importance of the N-methyl-D-aspartate (NMDA) receptor in pain hypersensitivity following injury, the NMDAR1 subunit was selectively deleted in the lumbar spinal cord of adult mice by the localized injection of an adeno-associated virus expressing the Cre recombinase into floxed NR1 mice. This procedure resulted in more than an 80% reduction in the expression of both NR1 mRNA and protein and a corresponding loss of NMDA, but not AMPA currents, in the lamina II neurons in the injected area. This spatially and temporally restricted knockout dramatically reduced the response to pain hypersensitivity resulting from the intraplantar injection of formalin but did not alter heat or cold paw withdrawal latencies, mechanical threshold, or motor function. Thus, the NMDA receptor in the spinal cord dorsal horn is essential for central sensitization, the central facilitation of pain transmission produced by peripheral injury. Agents that act on these spinal receptors may provide a therapeutic approach to ameliorate injury-induced pain.     

The effect of three inhaled anesthetics in mice harboring mutations in the GluR6 (kainate) receptor gene

Combinations of GluR5-GluR7, KA1, and KA2 subunits form kainate receptors, a subtype of excitatory ionotropic glutamate receptors. Isoflurane enhances the action of kainate receptors comprising GluR6 subunits expressed in oocytes. To test whether alterations of the GluR6 subunit gene affect the actions of inhaled anesthetics in vivo, we measured the minimum alveolar concentration of desflurane, isoflurane, and halothane in mice lacking the kainate receptor subunit GluR6 (GluR6 knockout mice) and mice with a dominant negative glutamine/arginine (Q/R) editing mutation in membrane domain 2 of the GluR6 receptor (GluR6 editing mutants), which increases the calcium permeability of kainate receptors containing GluR6Q. We also measured the capacity of isoflurane to interfere with Pavlovian fear conditioning to a tone and to context. Absence of the GluR6 subunit did not change the minimum alveolar concentration of isoflurane, desflurane, or halothane. Possibly, kainate receptors assembled from the remaining kainate receptor subunits compensate for the absent subunits and thereby produce a normal minimum alveolar concentration. A Q/R mutation that dominantly affects kainate receptors containing the GluR6 subunit in mice increased isoflurane minimum alveolar concentration (by 12%; P < 0.01), decreased desflurane minimum alveolar concentration (by 18%; P < 0.001), and did not change halothane minimum alveolar concentration (P = 0.25). These data may indicate that kainate receptors containing GluR6Q subunits differently modulate, directly or indirectly, the mechanism by which inhaled anesthetics cause immobility. The mutations of GluR6 that were studied did not affect the capacity of isoflurane to interfere with fear conditioning.     

Inhibition of NR2B phosphorylation restores alterations in NMDA receptor expression and improves functional recovery following traumatic brain injury in mice

Traumatic brain injury (TBI) triggers a massive glutamate efflux, hyperactivation of N-methyl-D-aspartate receptors (NMDARs) and neuronal cell death. Previously it was demonstrated that, 15 min following experimentally induced closed head injury (CHI), the density of activated NMDARs increases in the hippocampus, and decreases in the cortex at the impact site. Here we show that CHI-induced alterations in activated NMDARs correlate with changes in the expression levels of the major NMDARs subunits. In the hippocampus, the expression of NR1, NR2A, and NR2B subunits as well as the GluR1 subunit of the AMPA receptor (AMPAR) were increased, while in the cortex at the impact site, we found a decrease in the expression of these subunits. We demonstrate that CHI-induced increase in the expression of NMDAR subunits and GluR1 in the hippocampus, but not in the cortex, is associated with an increase in NR2B tyrosine phosphorylation. Furthermore, inhibition of NR2B-phosphorylation by the tyrosine kinase inhibitor PP2 restores the expression of this subunit to its normal levels. Finally, a single injection of PP2, prior to the induction of CHI, resulted in a significant improvement in long-term recovery of motor functions observed in CHI mice. These results provide a new mechanism by which acute trauma contributes to the development of secondary damage and functional deficits in the brain, and suggests a possible role for Src tyrosine kinase inhibitors as preoperative therapy for planned neurosurgical procedures.     

Remifentanil directly activates human N-methyl-D-aspartate receptors expressed in Xenopus laevis oocytes

BACKGROUND: Clinical studies suggest that intraoperative administration of the clinical remifentanil formulation Ultiva (GlaxoWellcome GmbH & Co, Bad Oldesloe, Germany) increases postoperative pain and postoperative analgesic requirements, but mechanisms remain unclear. N-methyl-D-aspartate (NMDA) receptors are thought to play a major role in development of postoperative pain and opiate tolerance. The authors hypothesized that Ultiva directly stimulates human NMDA receptors. METHODS: To test this hypothesis, the authors expressed human NR1A/NR2A and NR1A/NR2B NMDA receptors in Xenopus laevis oocytes by injection of messenger RNA prepared in vitro. After protein expression, they used a two-electrode voltage clamp to measure currents induced by NMDA receptor agonists and opioids. RESULTS: Noninjected cells were unresponsive to all compounds tested. Glutamate/glycine (1 nM-1 mM each) or Ultiva (0.01 pM-0.1 mM) stimulated NMDA receptors concentration dependently. NR1A/2A EC50 values were 8.0 microM/12 microM for glutamate/glycine and 3.5 nM for Ultiva, and NR1A/2B EC50 values were 3.9 microM/1.9 microM for glutamate/glycine and 0.82 microM for Ultiva. Glycine in combination with Ultiva showed no additive effect compared with Ultiva alone. Ultiva-induced currents were inhibited by MK-801 (pore blocker) but not by 7-CK (glycine antagonist), D-AP5 (glutamate antagonist), or naloxone. Fentanyl (10 microM) did not stimulate NMDA receptors. CONCLUSION: These data indicate that Ultiva but not fentanyl stimulates NMDA receptors of different subunit combinations (NR1A/2A, NR1A/2B). The mechanism seems to be allosteric activation of the NMDA receptor.     

Modulation of AMPA receptor GluR1 subunit phosphorylation in neurons by the intravenous anaesthetic propofol

Background: The ionotropic glutamate receptor is a potential molecular sitein the central nervous system that general anaesthetics mayinteract with to produce some of their biological actions. Proteinphosphorylation has been well documented to occur in the intracellularC-terminal domain of -amino-3-hydroxy-5-methylisoxazole-4-propionicacid (AMPA) subtype of glutamate receptors, which representsa pivotal mechanism for the post-translational modulation ofAMPA receptor functions. In this study, we investigated a possibleinfluence of an i.v. anaesthetic agent propofol on the phosphorylationof AMPA receptor GluR1 subunits in cultured neurons. Methods: The effect of propofol on phosphorylation of GluR1 subunitsat serine 831 and 845 was assayed in cultured rat striatal andcortical neurons by western blot with phospho- and site-specificantibodies. Results: Propofol consistently elevated phosphorylation of GluR1 subunitsat the C-terminal serine 845 site in both striatal and corticalneurons. The elevation in phosphorylation was concentration-dependentand started at a low concentration (3 µM). This increasein serine 845 phosphorylation was rapid and sustained duringthe entire course of propofol exposure. In contrast to serine845, phosphorylation of GluR1 at serine 831 was not alteredby propofol in striatal and cortical neurons. Total GluR1 abundanceremained unchanged in response to propofol incubation. Conclusions: These data indicate that propofol possesses the ability to upregulateAMPA receptor GluR1 subunit phosphorylation at a specific serine845 site in neurons and provide evidence supporting the AMPAreceptor as a molecular target for general anaesthetics.     

不同剂量氯胺酮对老年大鼠认知功能的影响

目的 探讨腹腔注射不同剂量氯胺酮对老年大鼠认知功能的影响.方法 老年SD大鼠40只,15月龄,体重470～570 g,雌雄各半,随机分为4组(n=10),对照组(C组)腹腔注射生理盐水2 ml,K1组、K2组和K3组分别腹腔注射氯胺酮10、20和100 mg/kg(溶于2 ml生理盐水),连续3 d.于停药后1 d(TI)、2 d(T2)、3 d(T3)行水迷宫实验,记录潜伏期及游泳路程.末次水迷宫测试结束后1 h处死大鼠,采用RT-PCR法测定海马N-甲基-D-天门冬氨酸(NMDA)受体亚基NR1 mRNA和NR2BmRNA的表达,免疫组织化学法测定海马NR1和NB2B蛋白的表达.结果 与T1时比较,C组和K2组T2,3时、K1,3组T3时潜伏期缩短,C组T2,3时、K1,2组T3时游泳路程缩短(P＜0.05);与C组比较,K3组T2,3时潜伏期和游泳路程延长,K1组NR2B蛋白表达上调,K3组NR2B mRNA及蛋白表达下调(P＜0.05);各组NR1 mRNA及蛋白表达差异无统计学意义(P＞0.05).结论 亚麻醉剂量氯胺酮对老年大鼠认知功能无明显影响,而麻醉剂量氯胺酮可致老年大鼠认知功能减退,其机制可能与含NR2B亚基的NMDA受体表达下调有关.     

Blockade of glutamate receptors and barbiturate anesthesia: increased sensitivity to pentobarbital-induced anesthesia despite reduced inhibition of AMPA receptors in GluR2 null mutant mice.

BACKGROUND: Barbiturates enhance gamma-aminobutyric acid type A (GABA(A)) receptor function and also inhibit the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptor. The relative contribution of these actions to the behavioral properties of barbiturates is not certain. Because AMPA receptor complexes that lack the GluR2 subunit are relatively insensitive to pentobarbital inhibition, GluR2 null mutant mice provide a novel tool to investigate the importance of AMPA receptor inhibition to the anesthetic effects of barbiturates. METHODS: GluR2 null allele (-/-), heterozygous (+/-), and wild-type (+/+) mice were injected with pentobarbital (30 and 35 mg/kg intraperitoneally). Sensitivity to anesthetics was assessed by measuring the latency to loss of righting reflex, sleep time, and the loss of corneal, pineal, and toe-pinch withdrawal reflexes. In addition, patch-clamp recordings of acutely dissociated CA1 hippocampal pyramidal neurons from (-/-) and (+/+) mice were undertaken to investigate the effects of barbiturates on kainate-activated AMPA receptors and GABA-activated GABA(A) receptors. RESULTS: Behavioral tests indicate that sensitivity to pentobarbital was increased in (-/-) mice. In contrast, AMPA receptors from (-/-) neurons were less sensitive to inhibition by pentobarbital (concentrations that produced 50% of the maximal inhibition [IC50], 301 vs. 51 microM), thiopental (IC50, 153 vs. 34 microM), and phenobarbital (IC50, 930 vs. 205 microM) compared with wild-type controls, respectively. In addition, the potency of kainate was greater in (-/-) neurons, whereas no differences were observed for the potentiation of GABA(A) receptors by pentobarbital. CONCLUSIONS: The GluR2 null mutant mice were more sensitive to pentobarbital anesthesia despite a reduced sensitivity of GluR2-deficient AMPA receptors to barbiturate blockade. Our results indicate that the inhibition of AMPA receptors does not correlate with the anesthetic effects of barbiturates in this animal model. We postulate that the increase in the sensitivity to anesthetics results from a global suppression of excitatory neurotransmission in GluR2-deficient mice.     

不同浓度七氟烷对老年大鼠认知功能的影响

目的 探讨不同浓度七氟烷对老年大鼠认知功能的影响.方法 18月龄雄性SD大鼠40只,体重500～650 g,随机分为3组,对照组(C组,n=8)吸入空气,1.5%七氟烷组(S1组,n=16)吸入1.5%七氟烷2 h,3.0%七氟烷组(S2组,n=16)吸入3.0%七氟烷2 h.于吸入七氟烷后1、7 d(T1.2)S1组和S2组随机取8只大鼠,采用Y型迷宫实验行认知功能测试,认知功能测试后12 h时处死大鼠,断头取脑,采用RT-PCR方法测定左侧海马N-甲基-D-天冬氨酸受体2B亚基(NR2B)mRNA表达,采用免疫组化法检测右侧海马NR2B蛋白表达.结果 与C组比较,S2组吸入七氟烷后1 d大鼠认知功能减退,右侧海马NR2B蛋白、左侧海马NR2B mRNA表达上调(P<0.05),S1.组上述指标差异无统计学意义(P0.05).结论 吸入1.5%七氟烷后老年大鼠认知功能无明显变化;吸入3%七氟烷后1 d老年大鼠认知功能减退,可能与其上调海马含2B亚基的NMDA受体表达有关.     

Blockade of AMPA receptors and volatile anesthetics: reduced anesthetic requirements in GluR2 null mutant mice for loss of the righting reflex and antinociception but not minimum alveolar concentration

BACKGROUND: The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptor mediates fast excitatory neurotransmission in the central nervous system. Many general anesthetics inhibit AMPA receptors in vitro; however, it is not certain if this inhibition contributes to the behavioral properties of these drugs. AMPA receptors lacking the GluR2 subunit are resistant to blockade by barbiturates in vitro. Paradoxically, GluR2 null mutant (-/-) mice are more sensitive to barbiturate-induced loss of the righting reflex (LORR) compared with wild-type (+/+) littermates. To determine if interactions between anesthetics and AMPA receptors account for the increased sensitivity of (-/-) mice, the effects of volatile anesthetics that do not directly inhibit AMPA receptors were examined. METHODS: Isoflurane, halothane, desflurane, or sevoflurane were administered to (-/-) and (+/+) littermate controls. Anesthetic requirements for LORR, movement to tail clamp (minimum alveolar concentration [MAC]), and hind-paw withdrawal latency (HPWL) were determined. Electrophysiologic methods examined the inhibition of AMPA receptors by isoflurane and halothane. RESULTS: Anesthetic requirements for LORR and HPWL were decreased, whereas MAC values were unchanged in (-/-) mice. Isoflurane and halothane caused minimal inhibition of AMPA receptors at clinically relevant concentrations. CONCLUSIONS: Direct blockade of AMPA receptors did not account for the increased sensitivity to volatile anesthetics in GluR2 null mutant mice for HPWL or LORR. Thus, the deficiency of GluR2-containing AMPA receptors increases the sensitivity of neuronal circuitry mediating these end points, but not MAC. GluR2-containing receptors do not contribute appreciably to MAC in this mouse model. These results illustrate the difficulties in attributing behavioral responses to drug-receptor interactions in genetically engineered animals.     

鞘内注射艾芬地尔对骨癌痛小鼠脊髓NR2B mRNA表达的影响

目的 探讨鞘内注射艾芬地尔对骨癌痛小鼠脊髓N-甲基-D-天冬氨酸(NMDA)受体2B亚基(NR2B)mRNA表达的影响.方法 雄性C3H/HeJ小鼠140只,体重20～25 g,4～6周龄,随机分为5组(n=28):假手术组(S组)、骨癌病组(B组)和艾芬地尔2.5μg、5μg、10μg组(I1-3组).I1-3组和B组于小鼠右侧股骨远端骨髓腔接种NCTC 2472溶骨性纤维肉瘤细胞,建立骨癌痛模型;S组不接种肿瘤细胞.I1～3组于接种肿瘤细胞后14 d分别鞘内注射艾芬地尔2.5、5、10 μg,B组和S组鞘内注射艾芬地尔溶媒.各组于接种肿瘤细胞前1 d、鞘内注射艾芬地尔或溶媒前1 h、注射后2、12和24 h(T1～5)时随机取7只小鼠测定机械痛阈和热痛阈,并于T2～5,时测定后断头处死,取L3～5脊髓,采用RT-PCR法测定脊髓组织NR2B mRNA的表达水平.结果 与S组比较,除I3,组T3时热痛阈差异无统汁学意义(P0.05)外,B组和I1～3组机械痛阈和热痛阈均降低(P<0.05),B组和I1组脊髓组织NR2B mRNA表达上调,I2组该指标表达下调(P<0.05);与B组比较,I2.3组机械痈阈和热痛阈升高,脊髓组织NR2B mRNA表达下调(P<0.05),I1组各时点以上指标差异均无统计学意义(P0.05);与I2组比较,I3组机械痛阈和热痛阈升高,脊髓组织NR2B mRNA表达下调(P<0.05).结论 鞘内注射艾芬地尔可通过阻断含2B亚基的NMDA受体缓解小鼠骨癌痛,并下凋脊髓组织NR2B mRNA的表达抑制痛敏反应.     

Blockade of Glutamate Receptors and Barbiturate Anesthesia: Increased Sensitivity to Pentobarbital-induced Anesthesia Despite Reduced Inhibition of AMPA Receptors in GluR2 Null Mutant Mice

Background: Barbiturates enhance [gamma]-aminobutyric acid type A (GABAA) receptor function and also inhibit the [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptor. The relative contribution of these actions to the behavioral properties of barbiturates is not certain. Because AMPA receptor complexes that lack the GluR2 subunit are relatively insensitive to pentobarbital inhibition, GluR2 null mutant mice provide a novel tool to investigate the importance of AMPA receptor inhibition to the anesthetic effects of barbiturates.

Methods: GluR2 null allele (-/-), heterozygous (+/-), and wild-type (+/+) mice were injected with pentobarbital (30 and 35 mg/kg intraperitoneally). Sensitivity to anesthetics was assessed by measuring the latency to loss of righting reflex, sleep time, and the loss of corneal, pineal, and toe-pinch withdrawal reflexes. In addition, patch-clamp recordings of acutely dissociated CA1 hippocampal pyramidal neurons from (-/-) and (+/+) mice were undertaken to investigate the effects of barbiturates on kainate-activated AMPA receptors and GABA-activated GABAA receptors.

Results: Behavioral tests indicate that sensitivity to pentobarbital was increased in (-/-) mice. In contrast, AMPA receptors from (-/-) neurons were less sensitive to inhibition by pentobarbital (concentrations that produced 50% of the maximal inhibition [IC50], 301 vs. 51 [mu]M), thiopental (IC50, 153 vs. 34 [mu]M), and phenobarbital (IC50, 930 vs. 205 [mu]M) compared with wild-type controls, respectively. In addition, the potency of kainate was greater in (-/-) neurons, whereas no differences were observed for the potentiation of GABAA receptors by pentobarbital.     

Remifentanil Directly Activates Human N-methyl-d-aspartate Receptors Expressed in Xenopus laevis Oocytes

Background: Clinical studies suggest that intraoperative administration of the clinical remifentanil formulation Ultiva(R) (GlaxoWellcome GmbH & Co, Bad Oldesloe, Germany) increases postoperative pain and postoperative analgesic requirements, but mechanisms remain unclear. N-methyl-d-aspartate (NMDA) receptors are thought to play a major role in development of postoperative pain and opiate tolerance. The authors hypothesized that Ultiva(R) directly stimulates human NMDA receptors.

Methods: To test this hypothesis, the authors expressed human NR1A/NR2A and NR1A/NR2B NMDA receptors in Xenopus laevis oocytes by injection of messenger RNA prepared in vitro. After protein expression, they used a two-electrode voltage clamp to measure currents induced by NMDA receptor agonists and opioids.

Results: Noninjected cells were unresponsive to all compounds tested. Glutamate/glycine (1 nm-1 mm each) or Ultiva(R) (0.01 pm-0.1 mm) stimulated NMDA receptors concentration dependently. NR1A/2A EC50 values were 8.0 [mu]M/12 [mu]M for glutamate/glycine and 3.5 nM for Ultiva(R), and NR1A/2B EC50 values were 3.9 [mu]M/1.9 [mu]M for glutamate/glycine and 0.82 [mu]M for Ultiva(R). Glycine in combination with Ultiva(R) showed no additive effect compared with Ultiva(R) alone. Ultiva(R)-induced currents were inhibited by MK-801 (pore blocker) but not by 7-CK (glycine antagonist), D-AP5 (glutamate antagonist), or naloxone. Fentanyl (10 [mu]M) did not stimulate NMDA receptors.