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.     

The human temporal cortex: characterization of neurons expressing nitric oxide synthase, neuropeptides and calcium-binding proteins, and their glutamate receptor subunit profiles.

Immunocytochemical techniques were used to examine the distribution of neurons immunoreactive (-ir) for nitric oxide synthase (nNOS), somatostatin (SOM), neuropeptide Y (NPY), parvalbumin (PV), calbindin (CB) and calretinin (CR), in the inferotemporal gyrus (Brodmann's area 21) of the human neocortex. Neurons that colocalized either nNOS or SOM with PV, CB or CR were also identified by double-labeling techniques. Furthermore, glutamate receptor subunit profiles (GluR1, GluR2/3, GluR2/4, GluR5/6/7 and NMDAR1) were also determined for these cells. The number and distribution of cells containing nNOS, SOM, NPY, PV, CB or CR differed for each antigen. In addition, distinct subpopulations of neurons displayed different degrees of colocalization of these antigens depending on which antigens were compared. Moreover, cells that contained nNOS, SOM, NPY, PV, CB or CR expressed different receptor subunit profiles. These results show that specific subpopulations of neurochemically identified nonpyramidal cells may be activated via different receptor subtypes. As these different subpopulations of cells project to specific regions of pyramidal cells, facilitation of subsets of these cells via different receptor subunits may activate different inhibitory circuits. Thus, various distinct, but overlapping, inhibitory circuits may act in concert in the modulation of normal cortical function, plasticity and disease.     

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.     

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.     

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.     

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 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.     

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.     

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.     

NMDA receptors in cortical development are essential for the generation of coordinated increases in [Ca2+](i) in "neuronal domains"

Spontaneous correlated activity regulates the precision of developing neural circuits. A synchronized elevation of intracellular calcium ion concentration, [Ca(2+)](i), occurred in 5-50 adjacent neurons--known as a "neuronal domain"--in developing neocortex. This coordinated response of neuronal cells is mediated by the diffusion of inositol trisphosphate (IP(3)) via gap-junction channels. In this study, we utilized the N-methyl-D-aspartate (NMDA)-type glutamate receptor epsilon 2 (GluR epsilon 2/NR2B)(-/-) mouse, which does not possess any functional NMDA receptors in the developing neocortex, and showed that NMDA receptors are essential for the generation of "neuronal domains". First, the frequency of spontaneously occurring neuronal domains in brain slices from GluR epsilon 2(-/-) mice was significantly reduced compared to that seen in brain slices from wild-type mice. Secondly, IP(3) injection into a single neuron in a cortical slice from a GluR epsilon 2(-/-) brain resulted in very few neuronal domains being observed, but an injection similarly made into a neuron in a wild-type slice promptly resulted in neuronal domains. Even in the GluR epsilon 2(-/-) brain, the elevation of intracellular [Ca2+](i) was observed frequently in single neurons and microinjection of IP(3) produced an elevation of [Ca2+](i) in the injected cells. These results suggest that the diffusion of IP(3) into the surrounding neurons via gap junctions is almost completely absent in the GluR epsilon 2(-/-) brain. Our results may reflect the critical role of NMDA receptors in the formation of cortical circuitry, probably via the regulation of gap-junction channels between immature cortical neurons.     

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.     

Neuronal and glial localization of NR1 and NR2A/B subunits of the NMDA receptor in the human cerebral cortex

N-Methyl-D-aspartate (NMDA) receptors play a critical role in many cortical functions and are implicated in several neuropsychiatric diseases. In this study, the cellular expression of the NMDAR1 (NR1) and NMDAR2A and B (NR2A and B) subunits was investigated in the human cerebral cortex by immunocytochemistry with antibodies that recognize the NR1 or the NR2A and B subunits of the NMDA receptor. In frontal (areas 10 and 46) and temporal (area 21) association cortices and the cingulofrontal transition cortex (area 32), NR1 and NR2A/B immunoreactivity (ir) were similar and were localized to numerous neurons in all cortical layers. NR1- and NR2A/B-positive neurons were mostly pyramidal cells, but some nonpyramidal neurons were also labeled. Electron-microscopic observations showed that NR1 and NR2A/B ir were similar. In all cases, labeling of dendrites and dendritic spines was intense. In addition, both NR1 and NR2A/B were consistently found in the axoplasm of some axon terminals and in distal astrocytic processes. This investigation revealed that numerous NMDA receptors are localized to dendritic spines, and that they are also localized to axon terminals and astrocytic processes. These findings suggest that the effects of cortical NMDA activation in the human cortex do not depend exclusively on the opening of NMDA channels located at postsynaptic sites, and that the localization of NMDA receptors is similar in a variety of mammalian species.     

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.     

Determinants of the Sensitivity of AMPA Receptors to Xenon

Background: There is substantial and growing literature on the actions of general anesthetics on a variety of neurotransmitter-gated ion channels, with the greatest attention being focused on inhibitory [gamma]-amino butyric acid type A receptors. In contrast, glutamate receptors, the most important class of fast excitatory neurotransmitter-gated receptor channels, have received much less attention, and their role in the production of the anesthetic state remains controversial.

Methods: [alpha]-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors formed from a variety of different subunits were expressed in Xenopus oocytes and HEK-293 cells, and their sensitivities to the inhalational general anesthetics xenon, isoflurane, and halothane were determined using two-electrode voltage clamp and patch clamp techniques. The effects of desensitization on anesthetic sensitivity were investigated using cyclothiazide and site-directed mutagenesis. An ultrarapid application system was also used to mimic rapid high-concentration glutamate release at synapses.

Results: The authors show that xenon can potently inhibit AMPA receptors when assayed using bath application of kainate. However, when the natural neurotransmitter l-glutamate is used under conditions in which the receptor desensitization is blocked and the peak of the glutamate-activated response can be accurately measured, the pattern of inhibition changes markedly. When desensitization is abolished by a single-point mutation (L497Y in GluR1 and the equivalent mutation L505Y in GluR4), the xenon inhibition is eliminated. When AMPA receptors are activated by glutamate using an ultrarapid application system that mimics synaptic conditions, sensitivity to xenon, halothane, and isoflurane is negligible.     

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.     

Selective postsynaptic co-localization of MCT2 with AMPA receptor GluR2/3 subunits at excitatory synapses exhibiting AMPA receptor trafficking

MCT2 is the main neuronal monocarboxylate transporter needed by neurons if they are to use lactate as an additional energy substrate. Previous evidence suggested that some MCT2 could be located in postsynaptic elements of glutamatergic synapses. Using post-embedding electron microscopic immunocytochemistry, it is demonstrated that MCT2 is present at postsynaptic density of asymmetric synapses, in the stratum radiatum of both rat hippocampal CA1 and CA3 regions, as well as at parallel fibre-Purkinje cell synapses in mouse cerebellum. MCT2 levels were significantly lower at mossy fibre synapses on CA3 neurons, and MCT2 was almost absent from symmetric synapses on CA1 pyramidal cells. It could also be demonstrated using quantitative double-labeling immunogold cytochemistry that MCT2 and AMPA receptor GluR2/3 subunits have a similar postsynaptic distribution at asymmetric synapses with high levels expressed within the postsynaptic density. In addition, as for AMPA receptors, a significant proportion of MCT2 is located on vesicular membranes within the postsynaptic spine, forming an intracellular pool available for a putative postsynaptic endo/exocytotic trafficking at these excitatory synapses. Altogether, the data presented provide evidence for MCT2 expression in the postsynaptic density area at specific subsets of glutamatergic synapses, and also suggest that MCT2, like AMPA receptors, could undergo membrane trafficking.     

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.     

Determinants of the sensitivity of AMPA receptors to xenon

BACKGROUND: There is substantial and growing literature on the actions of general anesthetics on a variety of neurotransmitter-gated ion channels, with the greatest attention being focused on inhibitory gamma-amino butyric acid type A receptors. In contrast, glutamate receptors, the most important class of fast excitatory neurotransmitter-gated receptor channels, have received much less attention, and their role in the production of the anesthetic state remains controversial. METHODS: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors formed from a variety of different subunits were expressed in Xenopus oocytes and HEK-293 cells, and their sensitivities to the inhalational general anesthetics xenon, isoflurane, and halothane were determined using two-electrode voltage clamp and patch clamp techniques. The effects of desensitization on anesthetic sensitivity were investigated using cyclothiazide and site-directed mutagenesis. An ultrarapid application system was also used to mimic rapid high-concentration glutamate release at synapses. RESULTS: The authors show that xenon can potently inhibit AMPA receptors when assayed using bath application of kainate. However, when the natural neurotransmitter l-glutamate is used under conditions in which the receptor desensitization is blocked and the peak of the glutamate-activated response can be accurately measured, the pattern of inhibition changes markedly. When desensitization is abolished by a single-point mutation (L497Y in GluR1 and the equivalent mutation L505Y in GluR4), the xenon inhibition is eliminated. When AMPA receptors are activated by glutamate using an ultrarapid application system that mimics synaptic conditions, sensitivity to xenon, halothane, and isoflurane is negligible. CONCLUSIONS: AMPA receptors, when assayed in heterologous expression systems, showed a sensitivity to inhalational anesthetics that was minimal when glutamate was applied rapidly at high concentrations. Because these are the conditions that are most relevant to synaptic transmission, the authors conclude that AMPA receptors are unlikely to play a major role in the production of the anesthetic state by inhalational agents.     

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.