Autoantibodies to the glutamate receptor kill neurons via activation of the receptor ion channel.

Antibodies to the glutamate/AMPA receptor subunit 3 (GluR3), are found in a human epilepsy, Rasmussen's encephalitis [RE], and were hypothesized as the major cause for the neuronal loss, chronic inflammatory changes and epileptic seizures characteristic of the disease. To establish the pathogenic potential and mechanism of action of such antibodies, we raised murine antibodies against specific peptides of the GluR3 protein and studied their ability to bind, activate, and kill neurons. Mice were immunized with two GluR3 specific peptides: GluR3A (amino acids 245-274) and GluR3B (amino acids 372-395), and with a scrambled GluR3B peptide for control. High levels of antibodies to each of these peptides were obtained, with no cross reactivity between them. Antibodies to the GluR3B peptide were found to bind to cultured neurons, evoke GluR ion channel activity, and kill neurons. In contrast, antibodies against GluR3A peptide bound to neurons but failed to activate the receptor or kill neurons. Anti-scrambled-GluR3B antibodies had no effect. Both the activation of the GluRs and the neuronal death induced by anti-GluR3B antibodies were blocked by CNQX, a specific glutamate/AMPA receptor antagonist; killing was independent of complement. This indicates a mechanism of excitotoxicity-neuronal death due to over-activation of the receptor, a phenomenon known to be caused by excess of glutamate. Purified anti-GluR3B IgGs retained the neuronal killing capacity, and killing was completely and specifically blocked by preincubation with the GluR3B peptide. Excitotoxic neuronal death induced by anti-GluR3B antibodies took place primarily via apoptosis. Taken together, these results show that antibodies to a specific peptide of the GluR can kill neurons by an excitotoxic mechanism, thus mimicking the effects of excess of glutamate. This is the first example that antibodies can lead to neuronal death in a non-classical complement-independent manner, via activation of a membranal neurotransmitter receptor.     

Mutation of a glutamate receptor motif reveals its role in gating and delta2 receptor channel properties

Despite its importance in the cerebellum, the functions of the orphan glutamate receptor delta2 are unknown. We examined a mutant delta2 receptor channel in lurcher mice that was constitutively active in the absence of ligand. Because this mutation was within a highly conserved motif (YTANLAAF), we tested its effect on several glutamate receptors. Mutant delta2 receptors showed distinct channel properties, including double rectification of the current-voltage relationship, sensitivity to a polyamine antagonist and moderate Ca 2+ permeability, whereas other constitutively active mutant glutamate channels resembled wild-type channels in these respects. Moreover, the kinetics of ligand-activated currents were strikingly altered. We conclude that the delta2 receptor has a functional ion channel pore similar to that of glutamate receptors. The motif may have a role in the channel gating of glutamate receptors.     

Ion permeation through hyperpolarization-activated membrane channels (Q-channels) in the lobster stretch receptor neurone

In the lobster stretch receptor neurone it is possible to demonstrate a hyperpolarization-activated membrane current,I _Q, which appears to be carried by Na⁺ and K⁺ in combination. The ion permeability of the membrane channel conducting this current (Q-channel) was investigated using conventional electrophysiological techniques including intracellular ion concentration measurements. It was found that none of the ions choline, protonated Tris, Rb⁺, NH ₄ ⁺ , Li⁺, and protonated hydroxylamine was able to pass through theQ-channel which, thus, appears to be permeable to Na⁺ and K⁺ only. With increasing extracellular Na⁺ concentrations,I _Q was increased up to a saturation level. This behaviour could be described by a one-site-two-barriers version of the Eyring rate theory, assuming that the permeant ions are turned over at specific saturable channel sites which sense 70% of the transmembrane potential difference. With increasing extracellular K⁺ concentrations,I _Q was increased in accordance with a simple first-order doseresponse relationship. This finding can be accounted for by assuming that K⁺ increases all rates of turn-over of the permeant ions at their specific sites by similar relative amounts. Changes in extracellular Na⁺ and K⁺ concentrations were found to have no effect on the gating properties of theQ-channel.     

Immunohistochemical localization of AMPA-type glutamate receptor subunits in the nucleus of the Edinger-Westphal in embryonic chick

The ischemic damage in the hippocampal CA1 region following transient forebrain ischemia, delayed neuronal death, is a typical apoptotic response, but the underlying mechanisms are not fully understood. We have reported that mild hyperthermia (38 °C) accelerates DNA fragmentation of the gerbil CA1 pyramidal neurons following transient forebrain ischemia. Recently, we reported that galectin-3, a β-galactosidase-binding lectin, is spatio-temporally expressed only by activated microglial cells located within CA1 region following transient forebrain ischemia in gerbils. Furthermore, expression of galectin-3 and Iba-1 (a specific microglial cell marker) are strongly reduced by hypothermia during ischemic insult. To further elucidate the effect of hyperthermia on the expression of galectin-3 by micloglia in delayed neuronal death, we examined immunohistochemical expression of galectin-3 and Iba-1, in situ terminal dUTP-biotin nick end labeling of DNA fragmentation (for determination of cell death) and hematoxylin and eosin staining (for morphological observation). We observed that between 37 °C and 39 °C, there was a temperature-dependent enhancement of galectin-3 expression in microglial cells in the CA1 region following transient ischemia. Apoptotic DNA fragmentation, detected by TUNEL staining, was observed in CA1 region in normothermia. This TUNEL staining was enhanced by hyperthermia at 37.5 °C and 38 °C, but not at 39 °C. Ischemia-induced neuronal degeneration in CA1 region in gerbil hippocampus subjected to hyperthermia (37.5 °C, 38 °C and 39 °C) observed by HE staining is similar to that in normothermic gerbils. These findings imply that galectin-3 expression in microglia may influence the survival of CA1 pyramidal neurons in cases such as hyperthermia-related neuronal injury.     

Immunohistochemical localization of AMPA-type glutamate receptor subunits in the nucleus of the Edinger-Westphal in embryonic chick

The Edinger-Westphal nucleus (EW) in birds is responsible for the control of pupil constriction, accommodation, and choroidal blood flow. The activation of EW neurons is mediated by the neurotransmitter glutamate, in large part through AMPA-type glutamate receptors (GluRs), whose behavior varies according to the subunit composition. We investigated the developmental expression of the GluR subunits in EW of the chick (Gallus gallus) using immunohistochemistry on tissue from embryonic days 10 through 20 (E10–E20). Of the three antibodies used, one recognized the GluR1 subunit, another the GluR4 subunit, and the third recognized a sequence common to GluR2 and GluR3 subunits. No immunolabeling of EW neurons for any GluR subunits was observed prior to E12, although immunolabeling was seen in somatic oculomotor prior to E12. At E12, immunoreactivity for each of the three antibodies was in only approximately 2% of EW neurons. By E14, the abundance of GluR1+ perikarya in EW had increased to 13%, and for GluR2/3 had increased to 48%. The perikaryal abundance of the immunoreactivity for GluR1 and GluR2/3 declined to 3% and 23%, respectively, by E16. At E14, 33% of EW neurons immunolabeled for GluR4, and their frequency increased to 43% by E16, and remained at that approximate percentage through hatching. The increased expression of GluR1 and GluR4 in EW at E14 coincides with the reported onset of the expression of the calcium-binding protein parvalbumin, and the calcium currents associated with AMPA receptors formed by these two subunits may play a role in the occurrence of parvalbumin expression.     

Spontaneous changes in acetylcholine receptor and calcium leakage activity in cell-attached patches from cultured dystrophic myotubes

 Calcium leakage activity (CLA) was recorded in association with acetylcholine receptor (AChR) activity in cell-attached patches from cultured nondystrophic and dystrophic (mdx) myotubes. Cell-attached recordings from dystrophic myotubes exhibited spontaneous transitions in the activity pattern that were characterized by an instability of AChR function and a decrease in the frequency of AChR events. Recordings from nondystrophic myotubes could be maintained for similar time periods without observing any consistent changes in the distribution of CLA and AChR events, thus indicating that the conditions of the experiment were not conducive to developing AChR instability or desensitization in nondystrophic myotubes. In dystrophic myotubes, the decrease in AChR event frequency was associated with an increase in small-conductance events which had the characteristics of CLA, and the subsequent acquisition of an inside-out patch appeared to restore the AChR activity. Examination of baseline current–voltage relationships indicated that dystrophic and nondystrophic patches exhibited the same general pattern of seal maturation with temporal increases in the total-patch circuit resistance. These results are discussed in relation to the AChR aggregation hypothesis, which proposes that the absence of dystrophin leads to abnormal AChR–cytoskeletal interactions and CLA that can be reversed by removing the influence of intracellular signal transduction enzymes that aggregate and stabilize AChR clusters. Received: 9 June 1998 / Received after revision: 2 October 1998 / Accepted: 7 October 1998     

Effects of electrostimulation on glycogenolysis in cultured rat myotubes

A model for electrostimulation causing contractions of primary cultures of rat myotubes was established. The kinetics of glycogen degradation was investigated for a 2-h period to elucidate the coupling between contraction and glycogenolytic flux. Electrostimulation caused contraction and increased glycogenolytic flux, but had no effect on glycogen phosphorylase-a activity. Forskolin increased glycogenolytic flux more than electrostimulation, and caused a fast activation of glycogen phosphorylase, while it did not elicit contraction. The effects of electrostimulation and forskolin on glycogenolytic flux were partly additive. The metabolism of glucose and glycogen was almost equally anaerobic and aerobic. The ATP content remained constant during glycogenolysis, but phosphocreatine decreased with the largest decrease in electrostimulated cells. The calculated ATP turnover rate increased about 3 times by electrostimulation. For all conditions, pH_i decreased from about 7.0 to about 6.6 at 2 h. It is concluded that in the present in vitro system glycogenolytic flux may be enhanced without eliciting contraction, a condition normally not observed in vivo. The system also shows much less dynamic range of energy metabolism than in vivo, primarily because of a high resting ATP turnover.     

Involvement of metabotropic glutamate receptor 5 in cardiomyocyte differentiation from mouse embryonic stem cells

Metabotropic glutamate receptors (mGluRs) are G-protein coupled receptors (GPCRs) activated by glutamate. The function of mGluRs is not restricted to the regulation of synaptic transmission. Although some roles of mGluR5 in mouse embryonic stem cells (ESCs) have been proposed, little is known about the significance of mGluR5 in cardiomyocyte differentiation from ESCs. We demonstrated that mGluR5 expression increased during cardiomyocyte differentiation. Activation of mGluR5 with (RS)-3, 5-dihydroxy phenylglycine (DHPG) promoted cardiomyocyte differentiation in a dose-dependent manner. DHPG significantly enhanced PI 3-kinase enhancer (PIKE) and PI3K p110α expression, but had no significant effect on Homer1b/c. The coexpression of PIKE or PI3K p110α together with Troponin T in embryoid bodies (EBs) treated with DHPG was elevated to 9.51% and 12.05%, respectively. Inhibition of mGluR5 with 2-methyl-6-(phenylethynyl)pyridine (MPEP) treating the ESCs, did hold back the cardiogenesis from the ESCs at the early differentiation stage. However, EBs applied by MPEP could not inhibit cardiomyocyte differentiation. Small interfering RNA (siRNA) of mGluR5 blocked cardiomyocyte differentiation by repressing PIKE and PI3K p110α expression, but had no notable influence on Homer1b/c. mGluR5 siRNA also decreased the DHPG-induced Ca2? transient peak amplitude in the isolated ESC-derived cardiomyocytes. The amplitude of Ca2? oscillation was reduced by ～90% with si-mGluR5-3 compared with si-control. The protein expression of T-type Ca2? channel and L-type Ca2? channel was decreased in si-mGluR5-3-treated EBs. Taken together, these results revealed that mGluR5/PIKE/PI3K signaling pathway was involved in cardiomyocyte differentiation from ESCs. The key function of mGluR5 is probably associated with cardiogenesis and Ca2? signal in ESC-derived cardiomyocytes.     

Effects of ionotropic glutamate receptor channel blockers on the development of pentylenetetrazol kindling in mice

Experiments on mice were performed to study the ability of monocationic and dicationic adamantane and phenylcyclohexyl derivatives to prevent the development of kindling induced by i.p. administration of pentylenetetrazol (Corasol, 35 mg/kg). The monocationic phenylcyclohexyl derivative IEM-1921 effectively slowed the development of kindling, this being seen over a wide range of doses (0.0001–0.1 μmol/kg). A monocationic adamantane derivative (memantine), also a selective non-competitive blocker of NMDA receptors, produced a similar effect at doses 100 times higher. The anticonvulsive activity of the dicationic phenylcyclohexyl derivative IEM-1925, which could block both types of glutamate receptors, differed from the activity of the monocationic derivative by having a more complex dose-response relationship. Thus, the development of kindling was suppressed by essentially the same doses as needed for the monocation IEM-1921 (0.001 μmol/kg). However, on reducing the dose by a factor of 10 (0.0001 μmol/kg), IEM-1925 facilitated the development of kindling. This difference in the prophylactic activities of selective NMDA receptor blockers and substances able to block both NMDA and AMPA receptors provides evidence that the mechanism of kindling involves both types of ionotropic glutamate receptor and the effects of compounds depend not only on the ratio of the contributions of these receptors, but also on the kinetic characteristics of the blocking action. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 91, No. 11, pp. 1241–1251, November, 2005.     

Ion channel properties underlying axonal action potential initiation in pyramidal neurons

A high density of Na+ channels in the axon hillock, or initial segment, is believed to determine the threshold for action potential initiation in neurons. Here we report evidence for an alternative mechanism that lowers the threshold in the axon. We investigated properties and distributions of ion channels in outside-out patches from axons and somata of layer 5 pyramidal neurons in rat neocortical slices. Na+ channels in axonal patches (<30 microm from the soma) were activated by 7 mV less depolarization than were somatic Na+ channels. A-type K+ channels, which were prominent in somatic and dendritic patches, were rarely seen in axonal patches. We incorporated these findings into numerical simulations which indicate that biophysical properties of axonal channels, rather than a high density of channels in the initial segment, are most likely to determine the lowest threshold for action potential initiation.     

Some peculiarities in the antigenic properties of human embryonic tissues when cultured on various media

Summary The human antigenic properties are retained by the human embryonic muscular and connetive tissues in culturing for the period of 3 months in homo- or heterogenic medium. The antigenic properties of these tissues are changed during the process of culturing: There is a gradual weakening of the properties inherent to the embryonic tissues with the intensification of these characteristic of the muscular and connective tissues of adult.(Presented by Active member of the AMN SSSR N. N. Zhukov-Verezhnikov) Translated from Byulleten Èksperimental' noi Biologii i Meditsiny Vol. 49, No. 4, pp. 105–109, April, 1960.     

Functions and mechanisms of receptor tyrosine kinase Torso signaling: lessons from Drosophila embryonic terminal development.

The Torso receptor tyrosine kinase (RTK) is required for cell fate specification in the terminal regions (head and tail) of the early Drosophila embryo. Torso contains a split tyrosine kinase domain and belongs to the type III subgroup of the RTK superfamily that also includes the platelet-derived growth factor receptors, stem cell or steel factor receptor c-Kit proto-oncoprotein, colony-stimulating factor-1 receptor, and vascular endothelial growth factor receptor. The Torso pathway has been a model system for studying RTK signal transduction. Genetic and biochemical studies of Torso signaling have provided valuable insights into the biological functions and mechanisms of RTK signaling during early Drosophila embryogenesis.     

Group I and II metabotropic glutamate receptor expression in cultured rat spinal cord astrocytes

We have examined the development of expression of group I and II metabotropic glutamate receptors (mGluRs) in pure rat spinal cord astrocyte cultures, using immunocytological and calcium imaging techniques. mGluR1alpha and mGluR2/3 antibodies were found to label roughly 10% of the total astrocyte population at all time points examined, whereas mGluR5 was poorly expressed in our culture system. Results from intracellular Ca2+ imaging experiments, measured using fura-2 ratio imaging, suggest that 20% of these cultured astrocytes express functional group I mGluRs (mGluR1 and/or 5). Our results contrast with previously published work in cultured cortical astrocytes where mGluR5 and not mGluR1 is expressed, suggesting that cultured astrocytes from different parts of the CNS exhibit different patterns of mGluR expression.