Slow and selective death of spinal motor neurons in vivo by intrathecal infusion of kainic acid: implications for AMPA receptor-mediated excitotoxicity in ALS

Excitotoxicity mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors has been proposed to play a major role in the selective death of motor neurons in sporadic amyotrophic lateral sclerosis (ALS), and motor neurons are more vulnerable to AMPA receptor-mediated excitotoxicity than are other neuronal subclasses. On the basis of the above evidence, we aimed to develop a rat model of ALS by the long-term activation of AMPA receptors through continuous infusion of kainic acid (KA), an AMPA receptor agonist, into the spinal subarachnoid space. These rats displayed a progressive motor-selective behavioral deficit with delayed loss of spinal motor neurons, mimicking the clinicopathological characteristics of ALS. These changes were significantly ameliorated by co-infusion with 6-nitro-7-sulfamobenso(f)quinoxaline-2,3-dione (NBQX), but not with d(-)-2-amino-5-phosphonovaleric acid (APV), and were exacerbated by co-infusion with cyclothiazide, indicative of an AMPA receptor-mediated mechanism. Among the four AMPA receptor subunits, expression of GluR3 mRNA was selectively up-regulated in motor neurons but not in dorsal horn neurons of the KA-infused rats. The up-regulation of GluR3 mRNA in this model may cause a molecular change that induces the selective vulnerability of motor neurons to KA by increasing the proportion of GluR2-lacking (i.e. calcium-permeable) AMPA receptors. This rat model may be useful in investigating ALS etiology.     

Ionotropic glutamate receptor subunits are differentially regulated in the motoneuronal pools of the rat hypoglossal nucleus in response to axotomy

Unilateral hypoglossal nerve axotomy was used as a model to analyse immunohistochemically the expression of the GluR1, GluR2, GluR3, and GluR4 glutamate receptor subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype and the NR1 subunit of the N-methyl-D-aspartate (NMDA) subtype in the different morphofunctional hypoglossal pools from 1 to 45 days postaxotomy. Following hypoglossal nerve axotomy, the percentage of motoneurons that were GluR1-immunopositive and the labeling intensity for this subunit was increased in some hypoglossal pools. Immunolabeling for the GluR2 subunit was undetectable. These results contrast with the unchanged pattern for these two subunits after sciatic nerve axotomy previously described. Image analysis showed a significant decrease in the intensity of immunohistochemical labeling for the GluR2/3 and GluR4 subunits in motoneurons, although most motoneurons were still immunopositive for these 2 subunits after axotomy. The intensity of immunolabeling for the NR1 subunit was slightly decreased postlesion, whereas the percentage of NR1-immunopositive motoneurons increased. Immunoreactivity returned to basal levels 45 days postlesion. These findings show that in axotomized hypoglossal motoneurons, i) AMPA and NMDA receptor subunits are still expressed, ii) the composition of the ionotropic glutamate receptor subunit pool is subjected to continuous changes during the regeneration process, iii) AMPA receptors, if functional, would have physiological properties different to those in intact motoneurons, and iv) the various AMPA receptor subunits are differentially regulated. The present results also suggest a faster recovery of basal levels of immunoreactivity for caudally localised groups of motoneurons which could reflect a caudo-rostral sequential functional revovery in the hypoglossal nucleus.     

Ionotropic glutamate receptor subunit distribution on hypoglossal motoneuronal pools in the rat

The expression of ionotropic glutamate receptor subunits in the motoneuronal pools of the hypoglossal nucleus was studied using specific antibodies against subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) subtypes. The highest numbers of intensely immunolabelled motoneurons were found in the dorsal tier and caudoventromedial part of the hypoglossal nucleus with all antibodies except that against the GluR1 AMPA subunit. Labelling for the GluR1 subunit was weak except for caudally located groups of motoneurons which innervate tongue muscles related to respiratory activity. By contrast, most motoneurons were intensely immunostained with antibodies against GluR2/3 and GluR4 subunits of the AMPA subtype. The low staining observed using an antibody specific for the GluR2 subunit (which prevents Ca²⁺-entry through AMPA channels) strongly suggests that AMPA receptors in hypoglossal motoneurons are Ca²⁺-permeable. Immunolabelling for the GluR5/6/7 kainate receptor subunits was found in many motoneuronal somata as well as in thin axon-like profiles and puncta that resembled synaptic boutons. Most motoneurons were intensely immunostained for the NMDA receptor subunit NR1. These results show that the hypoglossal nucleus contains five heterogeneous pools of motoneurons which innervate functionally defined groups of tongue muscles. The uneven expression of the different receptor subunits analysed here could reflect diverse phenotypic properties of hypoglossal motoneurons which might be expected to generate different patterns of motor responses under different physiological or pathological conditions.     

Developmental characteristics of AMPA receptors in chick lumbar motoneurons

Ca2+ fluxes through ionotropic glutamate receptors regulate a variety of developmental processes, including neurite outgrowth and naturally occurring cell death. In the CNS, NMDA receptors were originally thought to be the sole source of Ca2+ influx through glutamate receptors; however, AMPA receptors also allow a significant influx of Ca2+ ions. The Ca2+ permeability of AMPA receptors is regulated by the insertion of one or more edited GluR2 subunits. In this study, we tested the possibility that changes in GluR2 expression regulate the Ca2+ permeability of AMPA receptors during a critical period of neuronal development in chick lumbar motoneurons. GluR2 expression is absent between embryonic day (E) 5 and E7, but increases significantly by E8 in the chick ventral spinal cord. Increased GluR2 protein expression is correlated with parallel changes in GluR2 mRNA in the motoneuron pool. Electrophysiological recordings of kainate-evoked currents indicate a significant reduction in the Ca2(+)-permeability of AMPA receptors between E6 and E11. Kainate-evoked currents were sensitive to the AMPA receptor blocker GYKI 52466. Application of AMPA or kainate generates a significant increase in the intracellular Ca2+ concentration in E6 spinal motoneurons, but generates a small response in older neurons. Changes in the Ca(2+)-permeability of AMPA receptors are not mediated by age-dependent changes in the editing pattern of GluR2 subunits. These findings raise the possibility that Ca2+ influx through Ca(2+)-permeable AMPA receptors plays an important role during early embryonic development in chick spinal motoneurons.     

GluR2 knockdown reveals a dissociation between [Ca2+]i surge and neurotoxicity

Reduction in GluR2 subunit expression and subsequent increases in AMPA receptor mediated Ca(2+) currents were postulated to exacerbate glutamate neurotoxicity following seizures or global ischemia. To directly test the effects of shifting the GluR1/GluR2 subunit ratio on excitotoxicity, GluR2 antisense deoxyoligonucleotides (AS-ODNs) were applied to dissociated hippocampal cultures for 1-8 days. The GluR1/GluR2 protein ratio was examined immunohistochemically and by Western blotting. [Ca(2+)](i) concentrations were determined by ratiometric imaging of Fura 2-loaded cells. The cultures were exposed to glutamate, AMPA, NMDA or kainic acid (KA) 3 days after GluR2 knockdown and cell viability was determined 1 day later by MTT reduction assay or Trypan blue exclusion. Although GluR2 AS-ODNs increased the GluR1/GluR2 protein ratio in a time dependent manner, neurons and glia appeared healthy and MTT reduction values were similar to untreated and sense controls. Basal [Ca(2+)](i) levels were unchanged but [Ca(2+)](i) was selectively increased by agonist stimulation of AMPA receptors. Unexpectedly, delayed neurotoxicity was attenuated at saturating doses of glutamate while little difference in cell viability was observed at lower doses or with the other excitotoxins at any concentration. Therefore, there was a dissociation between rises in AMPA receptor-mediated Ca(2+) influx and neurotoxicity despite marked decreases in GluR2 but not GluR1 immunoreactivity. It is proposed that a modification of AMPA receptor stochiometry that raises agonist-stimulated Ca(2+) influx during an excitotoxic insult may have eventual neuroprotective effects.     

AMPA receptor subunit GluR2 gates injurious signals in ischemic stroke

Ischemic stroke, or a brain attack, is the third leading cause of death in developed countries. A critical feature of the disease is a highly selective pattern of neuronal loss; certain identifiable subsets of neurons--particularly CA1 pyramidal neurons in the hippocampus are severely damaged, whereas others remain intact. A key step in this selective neuronal injury is Ca2+/Zn2+ entry into vulnerable neurons through alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor channels, a principle subtype of glutamate receptors. AMPA receptor channels are assembled from glutamate receptor (GluR)1, -2, -3, and -4 subunits. Circumstance data have indicated that the GluR2 subunits dictate Ca2+/Zn2+ permeability of AMPA receptor channels and gate injurious Ca2+/Zn2+ signals in vulnerable neurons. Therefore, targeting to the AMPA receptor subunit GluR2 can be considered a practical strategy for stroke therapy.     

Down-Regulation of AMPA Receptor Subunit GluR2 in Amygdaloid Kindling

Abstract: Alterations in glutamatergic transmission are postulated to be important in kindling and epilepsy. The levels of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunits (GluR1, 2, and 4) were compared in amygdalakindled and sham-operated animals using subunit-specific antibodies and quantitative western blotting. Four limbic regions were examined: limbic forebrain, piriform cortex/amygdala, hippocampus, and entorhinal cortex. When subunit levels were examined 24 h after the last stage 5 seizure, levels of GluR2 were found to be selectively reduced in limbic forebrain (30%) and piriform cortex/amygdala (25%), with no changes in other regions examined. In addition, no changes in the other subunits were observed in any region. The decrease in GluR2 that was observed in kindled animals at 24 h was no longer present at 1 week and 1 month after the last stage 5 seizure. Because the GluR2 subunit uniquely determines the calcium permeability of these receptors and because the piriform cortex has been implicated as a source of excitatory drive for limbic seizures, reduced GluR2 expression may be important in increasing neuronal excitability in kindling-induced epilepsy, or may reflect a compensatory mechanism resulting from kindling.     

Expression of 15 glutamate receptor subunits and various splice variants in tissue slices and single neurons of brainstem nuclei and potential functional implications

Abstract: Brainstem nuclei serve a diverse array of functions in many of which ionotropic glutamate receptors are known to be involved. However, little detailed information is available on the expression of different glutamate receptor subunits in specific nuclei. We used RT‐PCR in mice to analyze the glutamate receptor subunit composition of the pre‐Bötzinger complex, the hypoglossal nucleus, the nucleus of the solitary tract, and the inferior olive. Analyzing 15 receptor subunits and five variants, we found all four α‐amino‐3‐hydroxy‐5‐methyl‐4‐propionic acid (AMPA) and six NMDA receptor (NR) subunits as well as three of five kainate (KA) receptors (GluR5, GluR6, and KA1) to be expressed in all nuclei. However, some distinct differences were observed: The inferior olive preferentially expresses flop variants of AMPA receptors, GluR7 is more abundant in the pre‐Bötzinger complex than in the other nuclei, and NR2C is most prominent in the nucleus of the solitary tract. In single hypoglossal motoneurons and interneurons of the pre‐Bötzinger complex investigation of GluR2 editing revealed strong expression of the GluR2‐R editing variant, suggesting low Ca²⁺ permeability of AMPA receptors. Thus, Ca²⁺ ‐permeable AMPA receptors are unlikely to be the cause for the reported selective vulnerability of hypoglossal motoneurons during excitotoxic events.     

Expression and localization of ionotropic glutamate receptor subunits in the goldfish retina—an in situ hybridization and immunocytochemical study

The expression and distribution of AMPA, kainate and NMDA glutamate receptor subunits was studied in the goldfish retina. For the immunocytochemical localization of the AMPA receptor antisera against GluR2, GluR2/3 and GluR4 were used, and for in situ hybridization rat specific probes for GluR1 and GluR2 and goldfish specific probes for GluR3 and GluR4 were used. The localization of the low affinity kainate receptor and NMDA receptor was studied using antisera against GluR5-7 and NR1. All AMPA receptor subtypes were demonstrated to be present in the goldfish retina both by immunocytochemistry and in situ hybridization. In situ hybridization revealed expression of all AMPA receptors subunit at the inner border of the INL. Only GluR3 was also strongly expressed in the outer border of the INL. Some of the ganglion cells displayed a strong signal for GluR1, GluR3 and GluR4. GluR1-immunoreactivity was present in subsets of bipolar, amacrine, and ganglion cells. GluR2 and GluR2/3-immunoreactivity was mainly localized in the outer plexiform layer. GluR2 and GluR2/3-immunoreactivity are associated with the photoreceptor synaptic terminals. GluR4-immunoreactivity is present on Müller cells in the inner retina and on dendrites of bipolar cells in the OPL, whereas GluR5-7-immunoreactivity was prominently present on horizontal cell axon terminals. Finally, NR1-immunoreactivity was confined to amacrine cells, the inner plexiform layer and ganglion cells. This study shows that there is a strong heterogeneity of glutamate receptor subunit expression in the various layers of the retina. Of the AMPA receptor subunits GluR3 seems to be expressed the most widely in all layers with strong glutamatergic synaptic interactions whereas all the other subunits seem to have a more restricted expressed pattern.     

谷氨酸下调培养海马神经元AMPA受体G1uR2亚单位的表达

目的 研究在癫痫发病过程中,谷氨酸对AMPA受体G1uR2亚单位表达变化的影响。方法 用RT-PCR和Western Blot方法观察谷氨酸诱导培养大鼠海马神经元AMPA受体G1uR2亚单位mRNA和蛋白的表达变化。结果 在谷氨酸刺激后2h,8h,12h,培养海马神经元G1uR2 mRNA和蛋白表达明显下降,与对照组相比,差异有显著性（P〈0．05）,而非NMDA受体拮抗剂CNQX能阻断此变化。结论 在癫痫等疾病中,谷氨酸能通过激活AMAP／KA受体下调AMPA受体G1uR2亚单位的表达,参与发病过程。     

谷氨酸下调培养海马神经元AMPA 受体GluR2亚单位的表达

目的研究在癫痫发病过程中,谷氨酸对AMPA受体Glu R2亚单位表达变化的影响。方法用RT-PCR和Western Blot方法观察谷氨酸诱导培养大鼠海马神经元AMPA受体Glu R2亚单位mRNA和蛋白的表达变化。结果在谷氨酸刺激后2h,8h,12h,培养海马神经元Glu R2mRNA和蛋白表达明显下降,与对照组相比,差异有显著性(P<0.05),而非NMDA受体拮抗剂CNQX能阻断此变化。结论在癫痫等疾病中,谷氨酸能通过激活AMAP/KA受体下调AMPA受体GluR2亚单位的表达,参与发病过程。     

Roles of ionotropic glutamate receptors in early developing neurons derived from the P19 mouse cell line

We cultured a P19 mouse teratocarcinoma cell line and induced its neuronal differentiation to study the function of ionotropic glutamate receptors (GluRs) in early neuronal development. Immunocytochemical studies showed 85% neuronal population at 5 days in vitro (DIV) with microtubule-associated protein 2-positive staining. Thirty percent and 50% of the cells expressed the alpha-amino-3-hydroxy-5-methyl-4-isopropinonate (AMPA) receptor subunit, GluR2/3, and the kainate (kainic acid; KA) receptor subunit, GluR5/6/7, respectively. In Western blot analysis, the temporal expression of GluR2/3 began to appear at 3 DIV, whereas GluR5/6/7 was already expressed in the undifferentiated cells. P19-derived neurons began to respond to glutamate, AMPA and KA, but not to the metabotropic GluR agonist trans-1-aminocyclopentane-1,3-decarboxylic acid, by 5 DIV in terms of increases in intracellular calcium and phospholipase C-mediated poly-phosphoinositide turnover. Furthermore, KA reduced cell death of P19-derived neurons in both atmospheric and hypobaric conditions in a phospholipase C-dependent manner. The common AMPA/KA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, but not the AMPA receptor antagonist, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium, profoundly increased hypobaric insult-induced neurotoxicity. In a flow cytometry study, the nerve growth factor-mediated antiapoptotic effect was facilitated by AMPA, with an induction of TrkA, but not p75(NTR) expression. Therefore, AMPA and KA receptors might mediate neurotrophic functions to facilitate neurotrophic factor signaling to protect neurons against hypoxic insult in early neuronal development.     

Orexin decreases mRNA expressions of NMDA and AMPA receptor subunits in rat primary neuron cultures

Orexin is one of the orexigenic neuropeptides in the hypothalamus. Orexin neurons in the lateral hypothalamus (LH) project into the cerebral cortex and hippocampus in which the receptors are distributed in high concentrations. Therefore, to elucidate the actions of orexin in the cerebral cortex, we examined its effects on the mRNA expressions of N-methyl-d-aspartate (NMDA) receptor subunits (NR1, NR2A, NR2B) and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits (GluR1, GluR2) following 6-day application of orexin-A or orexin-B to rat primary cortical neuron cultures. The mRNAs of NR1 and NR2A subunits were significantly decreased by orexin-A and orexin-B at concentrations over 0.1 μM and 0.01 μM, respectively. The mRNA expression of NR2B subunit was also significantly decreased by orexin-A and orexin-B only at the concentration of 1 μM. Moreover, orexin-A and orexin-B at concentrations over 0.01 μM significantly decreased the mRNA expressions of AMPA receptor subunits, GluR1 and GluR2. The present study demonstrated that orexins significantly suppressed RNA expressions of NMDA and AMPA receptor subunits in cortical neuron cultures, suggesting that orexin may regulate the higher functions of the cerebral cortex as well as be involved in energy regulation in the hypothalamus.     

Heteromeric AMPA receptors assemble with a preferred subunit stoichiometry and spatial arrangement.

AMPA receptors are thought to be a tetrameric assembly of the subunits GluR1-4. We have examined whether two coexpressed subunits (GluR1/2) combine at random to form channels, or preferentially assemble with a specific stoichiometry and spatial configuration. The subunits carried markers controlling ion permeation and desensitization, and these properties were monitored as a function of relative expression level and subunit composition. Homomeric receptors assembled stochastically while heteromeric receptors preferentially formed with a stoichiometry of two GluR1 and two GluR2 subunits, and with identical subunits positioned on opposite sides of the channel pore. This structure will predominate if GluR1 binds to GluR2 more rapidly during receptor assembly than other subunit combinations. The practical outcome of selective heteromeric assembly is a more homogenous receptor population in vivo.     

Neuroadaptations in Ionotropic and Metabotropic Glutamate Receptor mRNA Produced by Cocaine Treatment

Abstract : The expression of glutamate receptor/subunit mRNAs was examined 3 weeks after discontinuing 1 week of daily injections of saline or cocaine. The level of mRNA for GluR1-4, NMDAR1, and mGluR5 receptors was measured with in situ hybridization and RT-PCR. In nucleus accumbens, acute cocaine treatment significantly reduced the mRNA level for GluR3, GluR4, and NMDAR1 subunits, whereas repeated cocaine reduced the level for GluR3 mRNA. Acute cocaine treatment also reduced the NMDAR1 mRNA level in dorsolateral striatum and ventral tegmental area. In prefrontal cortex, repeated cocaine treatment significantly increased the level of GluR2 mRNA. The GluR2 mRNA level was not changed by acute or repeated cocaine in any other brain regions examined. Repeated cocaine treatment also significantly increased mGluR5 mRNA levels in nucleus accumbens shell and dorsolateral striatum. Functional properties of the ionotropic glutamate receptors are determined by subunit composition. In addition, metabotropic glutamate receptors can modulate synaptic transmission and the response to stimulation of ionotropic receptors. Thus, the observed changes in levels of AMPA and NMDA receptor subunits and the mGluR5 metabotropic receptor may alter excitatory neurotransmission in the mesocorticolimbic dopamine system, which could play a significant role in the enduring biochemical and behavioral effects of cocaine.     

Dramatic Increase of the RNA Editing for Glutamate Receptor Subunits During Terminal Differentiation of Clonal Human Neurons

Abstract: RNA editing plays an important role in determining physiological characteristics of certain glutamate-gated receptor (GluR) channels such as Ca²⁺ permeability and desensitization kinetics. In one case, the editing changes a gene-encoded glutamine (Q) to an arginine (R) codon located in the channel-forming domain of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit GluR-B and also the kainate receptor subunits GluR5 and GluR6. Another case of RNA editing alters an arginine (R) to a glycine (G) codon at a position termed the "R/G" site of AMPA subunits GluR-B, C, and D. Double-stranded RNA-specific adenosine deaminases (DRADA) have been implicated as agents involved in the editing. By using a human teratocarcinoma cell line, NT2, we investigated the change of the RNA editing of GluR subunits in conjunction with the expression of two DRADA members, DRADA1 and DRADA2 genes, during neuronal differentiation. Whereas Q/R and R/G site RNA editing both become progressively activated in differentiating NT2 cells, the expression of the two DRADA genes can already be detected even in the undifferentiated NT2 cells. Development of the editing machinery appears to require, in addition to DRADA enzymes, a currently unidentified mechanism(s) that may become activated during neuronal differentiation.     

GABAA and alpha-amino-3-hydroxy-5-methylsoxazole-4-propionate receptors are differentially affected by aging in the rat hippocampus

We have investigated the age-dependent modifications in the expression of eight different subunits of the gamma-aminobutyric acid, type A (GABA(A)) receptor (alpha1, alpha2, alpha3, alpha5, beta2, beta3, gamma2S, and gamma2L) and all four subunits of the alpha-amino-3-hydroxy-5-methylsoxazole-4-propionate (AMPA) receptor (GluR1-4) in the hippocampus of 24-month-old rats. All aged hippocampi displayed a remarkable increase (aged/adult ratio, 3.53 +/- 0.54) in the mRNA levels of the short version of the gamma2 subunit in parallel with a similar increase in the gamma2 subunit protein (aged/adult ratio, 2.90 +/- 0.62). However, this increase was not observed in the mature receptor. On the other hand, the expression of the different alpha subunit mRNAs increased moderately with aging, displaying a heterogeneous pattern. The most frequent modification consisted in an increase in the expression of the alpha1 subunit mRNA (aged/adult ratio, 1.26 +/- 0.18), in parallel with a similar increase on the alpha1 protein (aged/adult ratio, 1. 27 +/- 0.12) and in the alpha1 incorporated to the assembled GABA(A) receptor (tested by immunoprecipitation; aged/adult ratio, = 1.20 +/- 0.10). However, in the same hippocampal samples, no major modifications were observed on the expression of the AMPA receptor subunits. As a whole, these results indicated the existence of an increased expression of the GABA(A) receptor subunits and a preservation of the AMPA receptor at the hippocampal formation. These modifications could reflect the existence of specific deficiencies (neuronal loss and/or deafferentiation) on the GABAergic system in the aged rats.