Glutamate Receptors In Hippocampal Neurons Research Articles

In vitro ischemia triggers a transcriptional response to down-regulate synaptic proteins in hippocampal neurons.

Transient global cerebral ischemia induces profound changes in the transcriptome of brain cells, which is partially associated with the induction or repression of genes that influence the ischemic response. However, the mechanisms responsible for the selective vulnerability of hippocampal neurons to global ischemia remain to be clarified. To identify molecular changes elicited by ischemic insults, we subjected hippocampal primary cultures to oxygen-glucose deprivation (OGD), an in vitro model for global ischemia that resulted in delayed neuronal death with an excitotoxic component. To investigate changes in the transcriptome of hippocampal neurons submitted to OGD, total RNA was extracted at early (7 h) and delayed (24 h) time points after OGD and used in a whole-genome RNA microarray. We observed that at 7 h after OGD there was a general repression of genes, whereas at 24 h there was a general induction of gene expression. Genes related with functions such as transcription and RNA biosynthesis were highly regulated at both periods of incubation after OGD, confirming that the response to ischemia is a dynamic and coordinated process. Our analysis showed that genes for synaptic proteins, such as those encoding for PICK1, GRIP1, TARPγ3, calsyntenin-2/3, SAPAP2 and SNAP-25, were down-regulated after OGD. Additionally, OGD decreased the mRNA and protein expression levels of the GluA1 AMPA receptor subunit as well as the GluN2A and GluN2B subunits of NMDA receptors, but increased the mRNA expression of the GluN3A subunit, thus altering the composition of ionotropic glutamate receptors in hippocampal neurons. Together, our results present the expression profile elicited by in vitro ischemia in hippocampal neurons, and indicate that OGD activates a transcriptional program leading to down-regulation in the expression of genes coding for synaptic proteins, suggesting that the synaptic proteome may change after ischemia.

Development of Potent Fluorescent Polyamine Toxins and Application in Labeling of Ionotropic Glutamate Receptors in Hippocampal Neurons

The natural product argiotoxin-636 (ArgTX-636) found in the venom of the Argiope lobata spider is a potent open-channel blocker of ionotropic glutamate (iGlu) receptors, and recently, two analogues, ArgTX-75 and ArgTX-48, were identified with increased potency and selectivity for iGlu receptor subtypes. Here, we have exploited these analogues as templates in the development of fluorescent iGlu receptor ligands to be employed as unique tools for dynamic studies. Eighteen fluorescent analogues were designed and synthesized, and subsequently pharmacologically evaluated at three iGlu receptor subtypes, which resulted in the discovery of highly potent fluorescent iGlu receptor antagonists with IC50 values as low as 11 nM. The most promising ligands were further characterized showing retention of their mechanism of action, as open-channel blockers of iGlu receptors, as well as preservation of the photophysical properties of the incorporated fluorophores. Finally, we demonstrate the applicability of the developed probes for imaging of iGlu receptors in hippocampal neurons.

Variability of calcium responses to agonists of glutamate receptors in hippocampal neurons

The subject of this work was to study the reasons of the variability of the calcium response amplitudes in individual neurons of the hippocampal cell culture to agonists of ionotropic glutamate receptors and the regularities of the calcium response amplitude distribution. Changes of [Ca2+] i in the neurons in response to the NMDA-, AMPA-, and KA-receptor agonists were recorded using fluorescence probe Fura-2. The calcium response amplitudes (expressed as the ratio of fluorescence intensities of Fura-2 upon excitation at wave-lengths 340 and 380 nm) to short-term application of glutamate receptor agonists N-methyl-D-aspartate (NMDA), domoic acid (DA), α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and (S)-(−)-5-fluorowillardiine (FW) were measured. Calcium responses of individual cells differed in shape and amplitude but always reproduced upon the second application of the agonist. To elucidate the nature of calcium response variability, we compared distributions of calcium response amplitudes to the NMDA-, KA-, and AMPA-receptor agonists in cultures of various ages in the presence of receptor desensitization inhibitors and different agonist concentrations. An even increase from 0.05 to 1.6 was characteristic for distributions of calcium response amplitudes. Nevertheless, in 1–3% neurons of the cell culture, calcium response amplitudes reached much higher values. The efficiency of the ligands usually increased in the following order: FW ≈ NMDA > DA. However, this regularity varied with age and depended on the presence of the receptor desensitization inhibitor. In the process of growth and differentiation of neurons in culture from 1 to 14 day in vitro, calcium response amplitude to AMPA- and KA-receptor agonists increased. Desensitization inhibitors transformed the response from pulse-like with a sharp peak into stepwise and increased the amplitude of calcium responses but did not abolish the character of even amplitude distribution. The effect of AMPA- and KA-receptor desensitization inhibitor decreased with calcium response amplitude growth in the control and approached zero in neurons with initially maximal amplitude. KA- and AMPA-receptor agonists at high concentrations possessed a property of desensitization inhibitors and transformed a transient response into a continuous one that lasted throughout the application time. Thus, the amplitude and shape of the calcium response to glutamate receptor agonists is a characteristic parameter of an individual cell.

Repeated brief episodes of hypoxia modulate the calcium responses of ionotropic glutamate receptors in hippocampal neurons

The aim of this study was to evaluate the intracellular cytosolic calcium concentration ([Ca 2+] i) changes induced by activation of ionotropic glutamate receptors in cultured hippocampal neurons after repeated brief episodes of hypoxia. To investigate what kinds of ionotropic glutamate receptors are involved we used specific agonists for AMPA- and NMDA-type glutamate receptors. Measurements of [Ca 2+] i in cultured hippocampal neurons were made by imaging Fura-2AM loaded hippocampal cells. In the rat hippocampal slice method, field potential measurements in CA1 pyramidal neurons were used. The main result of our study is that brief hypoxic episodes progressively depress the [Ca 2+] i increases induced by agonists of AMPA and NMDA glutamate receptors in cultured hippocampal neurons. An effectiveness of this depression is increased from the first hypoxic episode to the third one. Hypoxic preconditioning effect is observed during 10–20 min after termination of hypoxic episode and depends on [Ca 2+] i response amplitudes to agonists before hypoxia. In contrast to AMPA receptor activation, NMDA receptor activation before hypoxia induce the spontaneous [Ca 2+] i increase about 3 min after each hypoxic episode. These spontaneous [Ca 2+] i increases may be an indicator of the development of posthypoxic hyperexcitability in hippocampal neurons. Our results suggest that brief hypoxia-induced depression of the glutamate receptor-mediated [Ca 2+] i responses contributes to the development of rapid hypoxic preconditioning in hippocampal CA1 neurons.

Activity-dependent endocytic sorting of kainate receptors to recycling or degradation pathways.

Kainate receptors (KARs) play important roles in the modulation of neurotransmission and plasticity, but the mechanisms that regulate their surface expression and endocytic sorting remain largely unknown. Here, we show that in cultured hippocampal neurons the surface expression of GluR6-containing KARs is dynamically regulated. Furthermore, internalized KARs are sorted into recycling or degradative pathways depending on the endocytotic stimulus. Kainate activation causes a Ca2+- and PKA-independent but PKC-dependent internalization of KARs that are targeted to lysosomes for degradation. In contrast, NMDAR activation evokes a Ca2+-, PKA- and PKC-dependent endocytosis of KARs to early endosomes with subsequent reinsertion back into the plasma membrane. These results demonstrate that GluR6-containing KARs are subject to activity-dependent endocytic sorting, a process that provides a mechanism for both rapid and chronic changes in the number of functional receptors.

Reversible Translocation and Activity-Dependent Localization of the Calcium–Myristoyl Switch Protein VILIP-1 to Different Membrane Compartments in Living Hippocampal Neurons

Visinin-like protein-1 (VILIP-1) belongs to the family of neuronal calcium sensor (NCS) proteins, a neuronal subfamily of EF-hand [corrected] calcium-binding proteins that are myristoylated at their N termini. NCS proteins are discussed to play roles in calcium-dependent signal transduction of physiological and pathological processes in the CNS. The calcium-dependent membrane association, the so-called calcium-myristoyl switch, localizes NCS proteins to a distinct cellular signaling compartment and thus may be a critical mechanism for the coordinated regulation of signaling cascades. To study whether the biochemically defined calcium-myristoyl switch of NCS proteins can occur in living neuronal cells, the reversible and stimulus-dependent translocation of green fluorescent protein (GFP)-tagged VILIP-1 to subcellular targets was examined by fluorescence microscopy in transfected cell lines and hippocampal primary neurons. In transiently transfected NG108-15 and COS-7 cells, a translocation of diffusely distributed VILIP-1-GFP but not of myristoylation-deficient VILIP-1-GFP to the plasma membrane and to intracellular targets, such as Golgi membranes, occurred after raising the intracellular calcium concentration with a calcium ionophore. The observed calcium-dependent localization was completely reversed after depletion of intracellular calcium by EGTA. Interestingly, a fast and reversible translocation of VILIP-1-GFP and translocation of endogenous VILIP-1 to specialized membrane structures was also observed after a depolarizing stimulus or activation of glutamate receptors in hippocampal neurons. These results show for the first time the reversibility and stimulus-dependent occurrence of the calcium-myristoyl switch in living neurons, suggesting a physiological role as a signaling mechanism of NCS proteins, enabling them to activate specific targets localized in distinct membrane compartments.

Glutamate Receptor Activation Regulates mRNA at Both Transcriptional and Posttranscriptional Levels

Previous studies from this laboratory have demonstrated that extracellular calcium entry through the NMDA subtype of glutamate receptors in hippocampal neurons selectively down-regulated ligatin gene expression in a rapid and long-lasting manner. Here we investigated the molecular mechanism that underlies this phenomenon. We demonstrate that glutamate receptor activation transiently increased the transcriptional activity of the ligatin gene and simultaneously shortened the half-life of its message. Using nuclear run-on assays and northern analyses of total RNA from alpha-amanitin-treated cells, we measured the effects of glutamate on the transcriptional activity and mRNA stability of the ligatin gene. The transcriptional activity of ligatin was found to be transiently increased (1.4-fold) 20 min after the addition of glutamate, with a return to basal levels by 60 min. Thus, the glutamate-dependent decrease in ligatin message could not be explained by a decline in its synthesis. Instead, concurrent with transcriptional up-regulation, glutamate shortened the half-life of the ligatin message from 10 h to 58 min, leading to a net decrease (0.7-fold) in its steady-state levels by 60 min. This posttranscriptional destablization of ligatin mRNA was mimicked by the translation inhibitor, cycloheximide, but not by puromycin. This finding indicated that the stability of ligatin mRNA was translation independent and distinguished this posttranscriptional regulatory mechanism from those previously described. Moreover, using in situ hybridization and confocal microscopy, we showed that control of message stability occurred both in the cell body and in the dendritic regions distant from the nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of Mitogen‐Activated Protein Kinase in Cultured Rat Hippocampal Neurons by Stimulation of Glutamate Receptors

Mitogen-activated protein kinase (MAP kinase) was activated by stimulation of glutamate receptors in cultured rat hippocampal neurons. Ten micromolar glutamate maximally stimulated MAP kinase activity, which peaked during 10 min and decreased to the basal level within 30 min. Experiments using glutamate receptor agonists and antagonists revealed that glutamate stimulated MAP kinase through NMDA and metabotropic glutamate receptors but not through non-NMDA receptors. Glutamate and its receptor agonists had no apparent effect on MAP kinase activation in cultured cortical astrocytes. Addition of calphostin C, a protein kinase C (PKC) inhibitor, or down-regulation of PKC activity partly abolished the stimulatory effect by glutamate, but the MAP kinase activation by treatment with ionomycin, a Ca2+ ionophore, remained intact. Lavendustin A, a tryrosine kinase inhibitor, was without effect. In experiments with 32P-labeled hippocampal neurons, MAP kinase activation by glutamate was associated with phosphorylation of the tyrosine residue located on MAP kinase. However, phosphorylation of Raf-1, the c-raf protooncogene product, was not stimulated by treatment with glutamate. Our observations suggest that MAP kinase activation through glutamate receptors in hippocampal neurons is mediated by both the PKC-dependent and the Ca(2+)-dependent pathways and that the activation of Raf-1 is not involved.

Seizure-like activity and glutamate receptors in hippocampal neurons in culture

Hippocampal neurons that were grown for prolonged periods in the continuous presence of agents that interfere with synaptic transmission, especially excitatory synaptic transmission, appeared to become seizure-prone. Washout of the synaptic blocking agents, that had been continuously present for several weeks to several months, caused the population of neurons to produce an abnormal and intense electrical activity. This consisted of two major components: spontaneously arising phasic responses that closely resembled paroxysmal depolarization shifts and, less frequently, slowly rising depolarizations similar to the sustained depolarizations observed during ictus-like episodes in intact cortex or cortical slices. We describe here observations on the role of the N-methyl-D-aspartate (NMDA) and non-NMDA types of glutamate receptors in the generation of these activities.