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.     

Mild hypothermia, but not propofol, is neuroprotective in organotypic hippocampal cultures

The neuroprotective potency of anesthetics such as propofol compared to mild hypothermia remains undefined. Therefore, we determined whether propofol at two clinically relevant concentrations is as effective as mild hypothermia in preventing delayed neuron death in hippocampal slice cultures (HSC). Survival of neurons was assessed 2 and 3 days after 1 h oxygen and glucose deprivation (OGD) either at 37 degrees C (with or without 10 or 100 microM propofol) or at an average temperature of 35 degrees C during OGD (mild hypothermia). Cell death in CA1, CA3, and dentate neurons in each slice was measured with propidium iodide fluorescence. Mild hypothermia eliminated death in CA1, CA3, and dentate neurons but propofol protected dentate neurons only at a concentration of 10 microM; the more ischemia vulnerable CA1 and CA3 neurons were not protected by either 10 microM or 100 microM propofol. In slice cultures, the toxicity of 100 muM N-methyl-D-aspartate (NMDA), 500 microM glutamate, and 20 microM alpha-amino-5-methyl-4-isoxazole propionic acid (AMPA) was not reduced by 100 microM propofol. Because propofol neuroprotection may involve gamma-aminobutyric acid (GABA)-mediated indirect inhibition of glutamate receptors (GluRs), the effects of propofol on GluR activity (calcium influx induced by GluR agonists) were studied in CA1 neurons in HSC, in isolated CA1 neurons, and in cortical brain slices. Propofol (100 and 200 microM, approximate burst suppression concentrations) decreased glutamate-mediated [Ca2+]i increases (Delta[Ca2+]i) responses by 25%-35% in isolated CA1 neurons and reduced glutamate and NMDA Delta[Ca2+]i in acute and cultured hippocampal slices by 35%-50%. In both CA1 neurons and cortical slices, blocking GABAA receptors with picrotoxin reduced the inhibition of GluRs substantially. We conclude that mild hypothermia, but not propofol, protects CA1 and CA3 neurons in hippocampal slice cultures subjected to oxygen and glucose deprivation. Propofol was not neuroprotective at concentrations that reduce glutamate and NMDA receptor responses in cortical and hippocampal neurons.     

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.     

Down-regulation of N-methyl D-aspartate receptor in rat-modeled disuse osteopenia

Lack of mechanical stress may result in osteoporosis; however, the underlying mechanisms of disuse osteoporosis remain unclear. It has been indicated that mechanical loading causes extracellular glutamate accumulation in osteoblasts. We hypothesized that the glutamate receptor mediation on bone cells might also be involved in mechanically stimulated osteogenesis. In this study, we investigated the changes of bone formation and the expressions of osteogenic genes and N-methyl D-aspartate (NMDA) receptors, the major glutamate receptors, in disused bones. Rat modeled disuse osteopenia in hind limbs was induced by a 3-week tail suspension in Sprague-Dawley rats. Bone mineral density and trabecular bone volume of distal femurs were measured to verify the osteopenia of disused bones. The mRNA expressions of cbfa1/Runx2, type I collagen, alkaline phosphatase (ALP) and osteocalcin (OC) in bones were measured as osteogenic markers. The influences of mechanical unloading on the expressions of NMDA receptors (NR₁ and NR₂D) in bones were also examined. The effects of NMDA mediation on osteogenesis were tested by a treatment of MK-801, a non-competitive NMDA receptor antagonist, in cultured osteoblasts and bone marrow stroma cells. Our result showed that mRNA expressions of cbfa1/Runx2, type I collagen, ALP and OC were significantly decreased in disused bones. The mRNA and protein expressions of NR₁ and NR₂D were significantly decreased in disused bones; furthermore, immunolocalization of both receptors showed decreases in osteoblasts, but not in osteoclasts. The results from the in vitro study showed that MK-801 inhibited mRNA expression of cbfa1/Runx2 in bone marrow stroma cells and also inhibited those of collagen type I, ALP and OC of osteoblasts in a dose-dependent manner. These results suggest that NMDA receptor mediation may play an important role in transmitting mechanical loading in bones, and decreases of the expressions of NMDA receptors in disused bones, especially in osteoblasts, may contribute to the decrease of osteogenesis.     

Glutamate does not play a major role in controlling bone growth.

Bone cells express glutamate-gated Ca2+-permeable N-methyl-D-aspartate (NMDA) receptors and GLAST glutamate transporters. Blocking NMDA receptors has been reported to reduce the number of bone resorption pits produced by osteoclasts, and mechanical loading alters GLAST transporter expression, which should change the extracellular glutamate concentration and NMDA receptor activation. Thus, by analogy with the brain, glutamate is postulated to be an important intercellular messenger in bone, controlling bone formation and resorption. We found that activating or blocking NMDA receptors had no effect on bone formation by rat osteoblasts in culture. The number of resorption pits produced by osteoclasts was reduced by the NMDA receptor blocker MK-801 but not by another blocker AP-5, implying that this effect of MK-801 is unrelated to its glutamate-blocking action. By contrast, MK-801, AP-5, and NMDA had no consistent effect on the volume of pits. In mice with GLAST glutamate transporters knocked out, no differences were detected in mandible and long bone size, morphology, trabeculation, regions of muscle attachment, resorption lacunae, or areas of formation versus resorption of bone, compared with wild-type siblings. These data suggest that glutamate does not play a major role in controlling bone growth.     

Expression of an N-Methyl-D-Aspartate-Type Receptor by Human and Rat Osteoblasts and Osteoclasts Suggests a Novel Glutamate Signaling Pathway in Bone

Signaling between the various types of cells found in bone is responsible for controlling the activity of osteoblasts and osteoclasts, and therefore the regulation of bone mass. Our identification of a neuronal glutamate transporter in osteoblasts and osteocytes suggests the possibility that bone cells may use the excitatory amino acid glutamate as a signaling molecule. In these studies we report the expression of different subtypes of glutamate receptors in osteoblasts and osteoclasts in vitro and in vivo. We have identified expression in human and rat bone cells of N-methyl-D-aspartate receptor-1 (NMDAR-1) and 2D subunits and PSD-95, the NMDA receptor clustering protein associated with signaling in the central nervous system. In situ hybridization and immunohistochemistry localized NMDAR-1 expression to osteoblasts and osteoclasts in human tissue sections. These findings strengthen the suggestion that glutamate is involved in signaling between bone cells.     

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.     

Propofol inhibits phosphorylation of N-methyl-D-aspartate receptor NR1 subunits in neurons

BACKGROUND: Anesthetics may interact with ionotropic glutamate receptors to produce some of their biologic actions. Cellular studies reveal that the ionotropic glutamate receptors, N-methyl-D-aspartate receptors (NMDARs), can be phosphorylated on their NR1 subunits at the C-terminal serine residues, which is a major mechanism for the regulation of NMDAR functions. It is currently unknown whether anesthetics have any modulatory effects on NMDAR NR1 subunit phosphorylation. METHODS: The possible effect of a general anesthetic propofol on phosphorylation of NR1 subunits at serine 897 (pNR1S897) and 896 (pNR1S896) was detected in cultured rat cortical neurons. RESULTS: Propofol consistently reduced basal levels of pNR1S897 and pNR1S896 in a concentration-dependent manner. This reduction was rapid as the reliable reduction of pNR1S896 developed 1 min after propofol administration. Pretreatment of cultures with the protein phosphatase 2A inhibitors okadaic acid or calyculin A blocked the effect of propofol on the NR1 phosphorylation, whereas okadaic acid or calyculin A alone did not alter basal pNR1S897 and pNR1S896 levels. In addition, propofol decreased tyrosine phosphorylation of protein phosphatase 2A at tyrosine 307, resulting in an increase in protein phosphatase 2A activity. In the presence of propofol, the NMDAR agonist-induced intracellular Ca2+ increase was impaired in neurons with dephosphorylated NR1 subunits. CONCLUSIONS: Together, these data indicate an inhibitory effect of a general anesthetic propofol on NMDAR NR1 subunit phosphorylation in neurons. This inhibition was mediated through a signaling mechanism involving activation of protein phosphatase 2A.     

Propofol Inhibits Phosphorylation of N-methyl-d-aspartate Receptor NR1 Subunits in Neurons

Background: Anesthetics may interact with ionotropic glutamate receptors to produce some of their biologic actions. Cellular studies reveal that the ionotropic glutamate receptors, N-methyl-d-aspartate receptors (NMDARs), can be phosphorylated on their NR1 subunits at the C-terminal serine residues, which is a major mechanism for the regulation of NMDAR functions. It is currently unknown whether anesthetics have any modulatory effects on NMDAR NR1 subunit phosphorylation.

Methods: The possible effect of a general anesthetic propofol on phosphorylation of NR1 subunits at serine 897 (pNR1S897) and 896 (pNR1S896) was detected in cultured rat cortical neurons.

Results: Propofol consistently reduced basal levels of pNR1S897 and pNR1S896 in a concentration-dependent manner. This reduction was rapid as the reliable reduction of pNR1S896 developed 1 min after propofol administration. Pretreatment of cultures with the protein phosphatase 2A inhibitors okadaic acid or calyculin A blocked the effect of propofol on the NR1 phosphorylation, whereas okadaic acid or calyculin A alone did not alter basal pNR1S897 and pNR1S896 levels. In addition, propofol decreased tyrosine phosphorylation of protein phosphatase 2A at tyrosine 307, resulting in an increase in protein phosphatase 2A activity. In the presence of propofol, the NMDAR agonist-induced intracellular Ca2+ increase was impaired in neurons with dephosphorylated NR1 subunits.     

Vocalization responses after intrathecal administration of ionotropic glutamate receptor agonists in rats

Inotropic glutamate receptors in the spinal cord (N-methyl-D-aspartic acid [NMDA], alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA], and kainate receptors) seem to play a key role in acute pain transmission and the neuronal plasticity in chronic pain states. Vocalization responses produced by activation of these receptors on the pain pathways can be quantified semiautomatically and thus could be used as a research tool. We studied vocalization responses induced by intrathecal administration of various agonists acting at the glutamate receptors in normal rats and in the presence of peripheral inflammation and a chronic constriction injury model of neuropathic pain. The nonselective endogenous agonist, glutamate, and the NMDA receptor glycine site agonist D-serine did not produce vocalization, whereas selective agonists acting at AMPA, NMDA, and kainate receptors produced dose-related vocalization responses. The vocalization response evoked by the administration of AMPA was significantly increased in the neuropathic pain model. In conclusion, spinal administration of ionotropic glutamate receptor agonists produce short-lasting, dose-related vocalization responses that can be used as a basic research and screening tool for analgesic studies. However, peripheral inflammation or nerve injury did not substantially alter vocalization responses overall, possibly indicating that the vocalization test is not a good tool for studying the role of excitatory amino acids in these pathological pain conditions. IMPLICATIONS: Vocalization responses evoked by spinal administration of ionotropic glutamate receptor agonists can be used for experimental analgesic studies. However, pathological pain models did not substantially alter vocalization responses, possibly indicating that this test is not suitable for studying the role of spinal excitatory amino acids in central sensitization.     

Boutons containing vesicular zinc define a subpopulation of synapses with low AMPAR content in rat hippocampus

Cortical regions of the brain stand out for their high content in synaptic zinc, which may thus be involved in synaptic function. The relative number, chemical nature and transmitter receptor profile of synapses that sequester vesicular zinc are largely unknown. To address this, we combined pre-embedding zinc histochemistry and post-embedding immunogold electron microscopy in rat hippocampus. All giant mossy fibre (MF) terminals in the CA3 region and approximately 45% of boutons making axospinous synapses in stratum radiatum in CA1 contained synaptic vesicles that stained for zinc. Both types of zinc-positive boutons selectively expressed the vesicular zinc transporter ZnT-3. Zinc-positive boutons further immunoreacted to the vesicular glutamate transporter VGLUT-1, but not to the transmitter gamma-aminobutyric acid. Most dendritic spines in CA1 immunoreacted to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) subunits GluR1-3 (approximately 80%) and to N-methyl-D-aspartate receptor (NMDAR) subunits NR1 + NR2A/B (approximately 90%). Synapses made by zinc-positive boutons contained 40% less AMPAR particles than those made by zinc-negative boutons, whereas NMDAR counts were similar. Further analysis indicated that this was due to the reduced synaptic expression of both GluR1 and GluR2 subunits. Hence, the levels of postsynaptic AMPARs may vary according to the presence of vesicular zinc in excitatory afferents to CA1. Zinc-positive and zinc-negative synapses may represent two glutamatergic subpopulations with distinct synaptic signalling.     

A novel role for involvement of N-methyl-D-aspartate (NMDA) glutamate receptors in sperm acrosome reaction

Ionotropic glutamate receptors are expressed in mouse and human spermatozoa. However, the possible role of these receptors has not been reported in the sperm acrosome reaction. This study was conducted to demonstrate the function of N-methyl-D-aspartate (NMDA) glutamate receptors in the acrosome reaction of mouse spermatozoa. Epididymal spermatozoa from adult mice were release in a culture medium. The sperm suspension was then divided into six groups: (1) spermatozoa at 0 min, (2) spermatozoa at 60 min (control), (3) spermatozoa treated with NMDA glutamate receptor agonist (L-glutamate, LG), (4) spermatozoa treated with α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainite glutamate receptor agonist (kainic acid), (5) spermatozoa treated with NMDA glutamate receptor antagonist (MK-801)+LG and (6) spermatozoa treated with ethylene glycol tetraacetic acid (EGTA, as a calcium chelator)₊LG. The sperm samples were examined for the acrosome reaction and intracellular calcium concentration. After 60 min, LG but not kainic acid significantly increased both the acrosome reaction and intracellular calcium levels in the spermatozoa compared with the control group. Co-administration of MK-801 or EGTA+LG could significantly reverse the effect of LG in the acrosome reaction and the level of intracellular calcium as compared to the LG group. The possibility that LG induced the acrosome reaction and elevated inter-cellular calcium concentration in mouse spermatozoa and that MK-801 could reverse the effects of LG, may suggest the involvement of NMDA glutamate receptors, at least in the initiation of the acrosome reaction in vitro.     

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.     

A novel variant of ionotropic glutamate receptor regulates somatostatin secretion from delta-cells of islets of Langerhans

Many metabolic factors affect the secretion of insulin from beta-cells and glucagon from alpha-cells of the islets of Langerhans to regulate blood glucose. Somatostatin from delta-cells, considered a local inhibitor of islet function, reduces insulin and glucagon secretion by activating somatostatin receptors in islet cells. Somatostatin secretion from delta-cells is increased by high glucose via glucose metabolism in a similar way to insulin secretion from beta-cells. However, it is unknown how low glucose triggers somatostatin secretion. Because L-glutamate is cosecreted with glucagon from alpha-cells under low-glucose conditions and acts as a primary intercellular messenger, we hypothesized that glutamate signaling triggers the secretion of somatostatin. In this study, we showed that delta-cells express GluR4c-flip, a newly identified splicing variant of GluR4, an (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptor of rat. After treatment with L-glutamate, AMPA, or kainate, secretion of somatostatin from isolated islets was significantly stimulated under low-glucose conditions. The glutamate-dependent somatostatin secretion was Ca(2+) dependent and blocked by 6-cyano-7-nitroquinoxaline-2,3-dione. Somatostatin in turn inhibited the secretion of L-glutamate and glucagon from alpha-cells. These results indicate that L-glutamate triggers somatostatin secretion from delta-cells by way of the GluR4c-flip receptor under low-glucose conditions. The released somatostatin may complete the feedback inhibition of alpha-cells. Thus, alpha- and delta-cells may communicate with each other through L-glutamate and somatostatin signaling.     

Inflammation-induced up-regulation of ionotropic glutamate receptor expression in human skin

Background: N-methyl-D-aspartate (NMDA), -amino-3-hydroxy-5-methylisoxazolone-4-propionicacid (AMPA), and kainate (KA) receptors are members of the ionotropicglutamate receptor (iGluR) family and are increased in inflamedrat skin. These receptors contribute to inflammatory pain. Inthis study, we have examined whether there is a similar increasein iGluRs in inflamed human skin in the presence of inflammatorypain. Methods: Normal and inflamed-skin biopsies were obtained from eight patientsundergoing elective wound-debridement surgery. Real-time-polymerasechain reaction (PCR) and western blot analysis were used forquantitation of iGluR mRNA and protein in normal and inflamedhuman skin. Results: A significant increase in mRNA and protein for NMDA, AMPA, andKA receptor subunits was detected in inflamed compared withnormal skin. The amounts of NMDA (NR1 subunit), AMPA (GluR2subunit), and KA (GluR6 subunit) mRNA in inflamed skin weremean 6 (SD 1.6-fold), 2.5 (0.6-fold), and 3.8 (0.9-fold) (P<0.05),respectively, greater than that measured in normal skin. Theratio of NR1, GluR2, and GluR6 protein in inflamed comparedwith normal skin was 5.7 (1.2), 2.4 (0.5), and 3.6 (0.9) (P<0.05),respectively. Conclusions: These results, in human tissue, demonstrate that iGluR mRNAand protein expression are increased during persistent inflammationand that this increased activity may be involved in mediatingclinical inflammatory pain in human skin.     

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.     

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.