Changes in splice variant expression and subunit assembly of AMPA receptors during maturation of hippocampal astrocytes

Astrocytes in the hippocampus express glutamate receptors of the AMPA subtype. An increasing body of evidence suggests a contribution of astroglial AMPA receptors to a direct signaling between neurons and glial cells in vivo. Here, we have combined functional analysis with singlecell RT-PCR to investigate whether hippocampal astrocytes express Ca(2+)-permeable AMPA receptors. We show that by postnatal day 5, a mosaic of Ca(2+)-permeable and less Ca(2+)-permeable AMPA receptors coexists in individual astrocytes, while receptors with a more uniform, low divalent permeability dominate in older cells. Moreover, we report an upregulation of the flip form of the GluR2 subunit during maturation, while the splicing status of GluR1 and GluR4 remains unchanged. Due to its specific properties, Ca(2+)-permeable AMPA receptors in astrocytes might strengthen neuron-to-glia signaling and enable proper formation of structural and functional connections between glial cells and glutamatergic synapses in the developing hippocampus.     

Oligodendrocytes but not astrocytes express apolipoprotein E after injury of rat optic nerve

Apolipoprotein E (apoE), a lipid-binding glycoprotein involved in transport and metabolism of phospholipids and cholesterol, is synthesized and secreted at elevated rates following transection of mature rat peripheral and central nerves. In the peripheral nervous system (PNS) infiltrating macrophages express apoE during Wallerian degeneration. Following injury of the optic nerve (ON) apoE synthesis is significantly stimulated but the apoE-expressing cells have thus far not been identified. This study used 1 micron and thin cryosections to identify the cellular source of apoE in transected ON. Serial 1 micron frozen sections were stained by avidin-biotin-peroxidase complex immunocytochemistry by using a specific antiserum to apoE and by antibodies that identify different cell types: glial fibrillary acidic protein (GFAP) for astrocytes, 2',3'-cyclic nucleotide-3' phosphodiesterase (CNP) for oligodendrocytes, and ED1 for macrophages. In normal ON both astrocytes and oligodendrocytes were apoE-positive. One week after ON transection oligodendrocytes accounted for the majority of apoE-positive cells, while apoE immunoreactivity had disappeared from astroglial cell bodies and processes. In contrast to the PNS only a few ED1/apoE-positive macrophages were present in ON 7 days after transection. By using immunogold-labeled ultrathin cryosections apoE could be localized in the Golgi apparatus of oligodendrocytes, indicating synthesis by these cells. Our data suggest that oligodendrocyte-derived apoE protein may participate in the redistribution of myelin lipids after CNS injury.     

Selective and AMPA receptor-dependent astrocyte death following prolonged blockade of glutamate reuptake in rat cerebellar cultures

In this study we examined the effects of prolonged l-trans-pyrrolidine-2,4-dicarboxylate (PDC)-induced glutamate reuptake blockade on the viability of glial cells in cerebellar granule cell cultures. Immunofluorescence staining for the glial-specific intermediate filament protein, GFAP, revealed that the PDC- induced increase of extracellular glutamate concentration was accompanied by increased astrocyte death, while neurons and oligodendrocytes remained intact and viable. Astrocytic cell death was manifested as fragmentation of processes and cell bodies. The selective astrocyte death was completely prevented by the competitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor antagonist, NBQX (10 microM), whereas MK-801 (10 microM), a noncompetitive blocker of N-methyl-D-aspartate receptors, gave only partial protection. Double staining for GFAP and the AMPA receptor subunits GluR2/3 showed that astrocytes had much higher immunoreactivity for GluR2/3 than neurons or oligodendrocytes, suggesting that the number of AMPA receptors is likely to be higher on astrocytes. Furthermore, we employed real-time RT-PCR to measure GluR1-4 subunit mRNA expression in control and PDC-exposed cultures. Following treatment with PDC, GluR1 and GluR4 mRNAs were reduced by 40% and GluR3 was reduced by 70% relative to control levels. In contrast, GluR2 expression was not affected by the PDC treatment, indicating that GluR3 was the dominant type of AMPA receptor subunit expressed on astrocytes. Our results show that astrocytes appear to be more vulnerable than neurons or oligodendrocytes to a gradual increase in the extracellular glutamate concentration, suggesting that astrocytes may be critically involved in the pathophysiology of slowly developing chronic neurodegenerative disorders.     

AMPA receptor protein expression and function in astrocytes cultured from hippocampus.

Glutamate receptors guide the proliferation, migration, and differentiation of glial cells. Here, we characterize AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) and NMDA receptor protein expression and function and mRNA expression in hippocampal glial cultures. By immunocytochemistry, GluR2 (the subunit that limits the Ca(2+) permeability of AMPA receptors) exhibited prominent labeling in hippocampal glial cultures. Double-labeling of GluR2 with GFAP and with A2B5 revealed GluR2 subunit expression on type-1 and type-2 astrocyte lineage cells. GluR1 subunit expression was more prominent in type-1 than in type-2 astrocytes. To characterize functional properties of glutamate receptors expressed in cultured hippocampal astrocytes, we performed whole-cell patch clamp recording. Application of L-glutamate, AMPA, and kainate, but not NMDA, to small, rounded cells (morphologically identified as type-2 astrocytes) elicited inward currents which were blocked by the AMPA/kainate antagonist 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX). Cyclothiazide potentiated AMPA- and kainate-elicited currents, indicative of AMPA-preferring receptors. Current voltage analysis indicated that type-2 astrocyte AMPA receptors were electrically linear, indicative of GluR2-containing, Ca(2+)-impermeable AMPA receptors. By Northern blot analysis, GluR1 mRNA was highest in astrocyte cultures from cerebellum and hippocampus and moderate in astrocyte cultures from neocortex and striatum. GluR3 mRNA was detectable in astrocyte cultures from cerebellum and neocortex. GluR2 and NR1 mRNA expression were not detected in astrocytes cultured from any brain region examined. In situ hybridization studies showed wide expression of GluR1 mRNA in cultured astrocytes; GluR2 and GluR3 mRNAs were near background levels. Thus, cultured type-2 astrocytes express functional AMPA receptors in a cell-specific and region-specific manner, consistent with their role in neuronal-glial communication.     

Transient Ca2+-permeable AMPA receptors in postnatal rat primary auditory neurons

Fast excitatory transmission in the nervous system is mostly mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors whose subunit composition governs physiological characteristics such as ligand affinity and ion conductance properties. Here, we report that AMPA receptors at inner hair cell (IHC) synapses lack the GluR2 subunit and are transiently Ca2+-permeable before hearing onset as evidenced using agonist-induced Co2+ accumulation, Western blots and GluR2 confocal microscopy in the rat cochlea. AMPA (100 microM) induced Co2+ accumulation in primary auditory neurons until postnatal day (PND) 10. This accumulation was concentration-dependent, strengthened by cyclothiazide (50 microM) and blocked by GYKI 52466 (80 microM) and Joro spider toxin (1 microM). It was unaffected by D-AP5 (50 microM), and it could not be elicited by 56 mM K+ or 1 mM NMDA + 10 microM glycine. Western blots showed that GluR1 immunoreactivity, present in homogenates of immature cochleas, had disappeared by PND12. GluR2 immunoreactivity was not detected until PND10 and GluR3 and GluR4 immunoreactivities were detected at all the ages examined. Confocal microscopy confirmed that the GluR2 immunofluorescence was not located postsynaptically to IHCs before PND10. In conclusion, AMPA receptors on maturing primary auditory neurons differ from those on adult neurons. They are probably composed of GluR1, GluR3 and GluR4 subunits and have a high Ca2+ permeability. The postsynaptic expression of GluR2 subunits may be continuously regulated by the presynaptic activity allowing for variations in the Ca2+ permeability and physiological properties of the receptor.     

AMPA/kainate receptors in mouse spinal cord cell-specific display of receptor subunits by oligodendrocytes and astrocytes and at the nodes of Ranvier

Spinal cord white matter is susceptible to AMPA/kainate (KA)-type glutamate receptor-mediated excitotoxicity. To understand this vulnerability, it is important to characterize the distribution of AMPA/KA receptor subunits in this tissue. Using immunohistochemistry and laser confocal microscopy, we studied the expression sites of AMPA/KA receptor subunits in mouse spinal cord. The white matter showed consistent immunoreactivity for AMPA receptor subunit GluR2/3 and KA receptor subunits GluR6/7 and KA2. In contrast, antibodies against GluR1, GluR2, GluR4 (AMPA), and GluR5 (KA) subunits showed only weak and occasional labeling of white matter. However, gray matter neurons did express GluR1 and GluR2, as well as GluR2/3. The white matter astrocytes were GluR2/3 and GluR6/7 immunopositive, while the gray matter astrocytes displayed primarily GluR6/7. Both exclusively and abundantly, KA2 labeled oligodendrocytes and myelin, identified by CNPase expression. Interestingly, myelin basic protein, another myelin marker, showed less correlation with KA2 expression, placing KA2 at specific CNPase-containing subdomains. Focal points of dense KA2 labeling showed colocalization with limited, but distinct, axonal regions. These regions were identified as nodes of Ranvier by coexpressing the nodal marker, ankyrin G. Overall, axonal tracts showed little, if any, AMPA/KA receptor expression. The proximity of oligodendrocytic KA2 to the axonal node and the paucity of axonal AMPA/kainate receptor expression suggest that excitotoxic axonal damage may be secondary and, possibly, mediated by oligodendrocytes. Our data demonstrate differential expression of glutamate AMPA and KA receptor subunits in mouse spinal cord white matter and point to astrocytes and oligodendrocytes as potential targets for pharmacological intervention in white matter glutamate excitotoxicity.     

Factors regulating AMPA-type glutamate receptor subunit changes induced by sciatic nerve injury in rats

Excitatory glutamatergic neurotransmission at Ia afferent-motoneuron synapses is enhanced shortly after physically severing or blocking impulse propagation of the afferent and/or motoneuron axons. We considered the possibility that these synaptic changes occur because of alterations in the number or properties of motoneuron alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors. Therefore, we quantitatively analyzed glutamate receptor (GluR)1, GluR2/3, and GluR4 AMPA subunit immunoreactivity (ir) in motoneurons 3, 7, or 14 days after axotomy or continuous tetrodotoxin (TTX) block of the sciatic nerve. GluR1-ir remained low in experimental and control motoneurons with either treatment and at any date. However, there was a large reduction of GluR2/3-ir (peak at 7 days >60% reduced) and a smaller, but statistically significant, reduction of GluR4-ir (around 10% reduction at days 3, 7, and 14) in axotomized motoneurons. TTX sciatic blockade did not affect AMPA subunit immunostainings. Axonal injury or interruption of the trophic interaction between muscle and spinal cord, but not activity disruption, appears therefore more likely responsible for altering AMPA subunit immunoreactivity in motoneurons. These findings also suggest that synaptic plasticity induced by axotomy or TTX block, although similar in the first week, could be related to different mechanisms. The effects of axotomy or TTX block on motoneuron expression of the metabotropic glutamate receptor mGluR1a were also studied. mGluR1a-ir was also strongly decreased after axotomy but not after TTX treatment. The time course of the known stripping of synapses from the cell somas of axotomized motoneurons was studied by using synaptophysin antibodies and compared with AMPA and mGluR1a receptor changes. Coverage by synaptophysin-ir boutons was only clearly decreased 14 days post axotomy and not at shorter intervals or after TTX block.     

Expression of AMPA/kainate receptors during development of chick embryo retina cells: in vitro versus in vivo studies

The activity and the subunit expression of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate ionotropic glutamate receptors were studied in retina cells developing in chick embryos and in retina cells cultured as retinospheroids, at the same stages of development. In the retinospheroids, the activity of the AMPA/kainate receptors was monitored by following the changes in the intracellular free calcium concentration ([Ca(2+)](i)), in response to AMPA, kainate or to L-glutamate, and the expression of the receptor subunits GluR1, GluR2/3, GluR4 and GluR6/7 was determined in the retinospheroids and in chick retinas by immunodetection using polyclonal antibodies. The changes in [Ca(2+)](i) in response to 400 microM kainate increased from 5h in vitro to 3 days, and remained constant until day 14, whereas the [Ca(2+)](i) in response to 500 microM L-glutamate or 400 microM AMPA increased from 5h in vitro to 3 days, and thereafter decreased slightly until day 14. The [Ca(2+)](i) responses to kainate are mainly due to AMPA receptor stimulation, since the signals were abolished by LY303070, the AMPA receptor antagonist, and were not affected by MK-801, the NMDA receptor antagonist. In retinospheroids, the levels of expression of GluR1 subunit increased from 5h in vitro until day 7, then decreased until day 14. The levels of expression of GluR2/3 and GluR4 subunits increased from 5h in vitro until day 10, and remained constant until day 14. The levels of kainate receptor subunits GluR6/7 increased from 5h in vitro until day 3, and thereafter decreased slightly until day 14. In the retinas, the expression of GluR1 and GluR6/7 subunits increased from day 8 until day 15, and then decreased until day 22 (post-natal 1). The subunits GluR2/3 and GluR4 increased from day 8 until day 18, and remained constant until day 22. The results suggest that AMPA/kainate receptors are expressed at early embryonic stages, although at low levels and before synapse formation (E12). However, the AMPA receptors are not completely functional at the first stage studied since they do not respond to the agonist AMPA. Also, the patterns of AMPA/kainate receptor subunit expression in retinospheroids of chick embryo retina cells cultured in vitro and in retina cells developing in the embryo (in vivo) were similar, indicating that the AMPA/kainate receptor subunits expression in these primary cultures mimics their expression in the developing chick retina.     

GluR2 deficiency accelerates motor neuron degeneration in a mouse model of amyotrophic lateral sclerosis

AMPA receptor-mediated excitotoxicity has been implicated in the selective degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). Motor neurons in vitro are particularly vulnerable to excessive AMPA receptor stimulation and one of the factors underlying this selective vulnerability is the presence of a large proportion of Ca2+ -permeable (i.e. GluR2-lacking) AMPA receptors. However, the precise role of GluR2-lacking AMPA receptors in motor neuron degeneration remains to be defined. We therefore studied the impact of GluR2 deficiency on motor neuron death in vitro and in vivo. Cultured motor neurons from GluR2-deficient embryos displayed an increased Ca2+ influx through AMPA receptors and an increased vulnerability to AMPA receptor-mediated excitotoxicity. We deleted the GluR2 gene in mutant SOD1G93A mice by crossbreeding them with GluR2 knockout mice. GluR2 deficiency clearly accelerated the motor neuron degeneration and shortened the life span of mutant SOD1G93A mice. These findings indicate that GluR2 plays a pivotal role in the vulnerability of motor neurons in vitro and in vivo, and that therapies that limit Ca2+ entry through AMPA receptors might be beneficial in ALS patients.     

大鼠实验性脑创伤挫伤灶内皮屏障抗原和Ⅳ型水通道蛋白免疫反应性的动态改变

目的研究实验性脑创伤中内皮细胞屏障的损伤修复以及与位于星形细胞足突的Ⅳ型水通道的关系。方法成年雄性SD大鼠32只,随机分入对照组,伤后1h组,4h组,1d组,3d组,6d组和11d组。在大鼠重度冲击加速性伤模型中,应用免疫组化法观察脑创伤灶中内皮屏障抗原(EBA)和Ⅳ型水通道蛋白(AQP4)免疫反应性在不同时点的动态改变。采用图像分析技术对挫伤病灶免疫反应性进行定量分析。结果在血脑屏障损伤的皮质挫伤灶,伤后1dAQP4和EBA免疫反应性明显消失,AQP4阴性反应区面积明显大于EBA阴性反应区(P<0.05)。伤后3d,EBA表达重新出现。伤后6d,EBA表达延伸至挫伤中心区,AQP4表达始见于边缘。伤后11d,二者免疫反应性基本恢复。结论挫伤灶血管源性水肿形成同时伴有内皮屏障功能和星形细胞足突水通道的改变。同一创伤强度下,星形细胞足突AQP4损伤范围较内皮EBA大,恢复较慢。     

Glutamate receptor subunit 3 (GluR3) immunoreactivity delineates a subpopulation of parvalbumin-containing interneurons in the rat hippocampus

Rasmussen's encephalitis is a childhood disease resulting in intractable seizures associated with hippocampal and neocortical inflammation. An autoantibody against the GluR3 subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors is implicated in the pathophysiology of Rasmussen's encephalitis. AMPA receptors mediate excitatory neurotransmission in the brain and contain combinations of four subunits (GluR1-4). Although the distributions of GluR1, GluR2, and GluR4 are known in some detail, the cellular distribution of GluR3 in the mammalian brain remains to be described. We developed and characterized a GluR3-specific monoclonal antibody and quantified the cellular distribution of GluR3 in CA1 of the rat hippocampus. GluR3 immunoreactivity was detected in all pyramidal neurons and astrocytes and in most interneurons. We quantified the intensity of GluR3 immunoreactivity in interneuron subtypes defined by their calcium-binding protein content. GluR3 immunofluorescence, but not GluR1 or GluR2 immunofluorescence, was significantly elevated in somata of parvalbumin-containing interneurons compared to pyramidal somata. Strikingly, increased GluR3 immunofluorescence was not observed in calbindin- and calretinin-containing interneurons. Furthermore, 24% of parvalbumin-containing interneurons could be distinguished from surrounding neurons based on their intense GluR3 immunoreactivity. This subpopulation had significantly elevated GluR3 immunoreactivity compared to the rest of parvalbumin-containing interneurons. Electron microscopy revealed enriched GluR3 immunoreactivity in parvalbumin-containing perikarya at cytoplasmic and postsynaptic sites. Parvalbumin-containing interneurons, potent inhibitors of cortical pyramidal neurons, are vulnerable in the brains of epileptic patients. Our findings suggest that the somata of these interneurons are enriched in GluR3, which may render them vulnerable to pathological states such as epilepsy and Rasmussen's encephalitis.     

Changes in glial cell white matter AMPA receptor expression after spinal cord injury and relationship to apoptotic cell death

Increasing evidence suggests that AMPA receptors (AMPARs) play a key role in mediating excitotoxic cell damage after acute spinal cord injury (SCI). However, the role of glial AMPARs in posttraumatic white matter injury requires further clarification. In the present study we examined the changes in AMPAR expression after SCI, the cellular distribution of these changes, and their association with apoptosis. Western blots revealed expression of GluR1, 3, and 4, but not GluR2, in spinal cord white matter. Immunohistochemistry was used to examine the distribution of AMPARs in spinal cord white matter. Quantification of AMPAR-expressing cells in spinal cord white matter indicated predominantly GluR3 expression in oligodendrocytes and predominantly GluR4 expression in astrocytes. A clip compression model of SCI was used to examine the changes in AMPAR expression in dorsal column white matter after injury. Quantitative analysis of GluR3 levels of expression indicated a significant decrease at 3 days postinjury compared to uninjured animals, followed by a recovery of expression by 2 weeks. GluR4 subunits followed a similar expression pattern. Gene message expression of GluR3 and GluR4 flip/flop mRNA splice variants exhibited a pattern of expression that correlated with protein expression. GluR3-expressing glia appeared to be more susceptible to apoptosis than GluR4-expressing cells. A large decline in GluR3-expressing oligodendrocytes suggests that this subunit may be associated with the induction of apoptosis in white matter glia, thus contributing to secondary injury mechanisms.     

The trophic factors S-100beta and basic fibroblast growth factor are increased in the forebrain reactive astrocytes of adult callosotomized rat.

S-100 is a calcium-binding protein that is predominantly found in astrocytes of the central nervous system. In the present study, we investigated the temporal and spatial changes of S-100beta immunoreactivity after a stereotaxic mechanical lesion of the adult rat corpus callosum performed with an adjustable wire knife. Rats were killed 7, 14 and 28 days after surgery. S-100beta immunoreactivity was found within the cytoplasm and processes of quiescent putative astrocytes that were observed throughout the gray and white matters of the forebrain of sham-operated rats. Following callosotomy, the S-100beta immunoreactive profiles showed increased size and thick processes, as well as increased amount of S-100beta immunoreactivity. Unbiased stereologic analysis revealed a sustained and widespread increase of the Areal Fraction of S-100beta immunoreactive profiles in the medial and lateral regions of the white matter of callosotomized rats at the studied time-intervals. In the cerebral cortex of callosotomized rats, the estimated total number of S-100beta immunoreactive profiles was also increased 7 and 14 days after the lesion. Since the cellular and temporal changes in S-100beta immunoreactivity were closely similar to those described for basic fibroblast growth factor (bFGF) following brain lesions, we co-localized the S-100beta and bFGF immunoreactivities after callosotomy. bFGF immunoreactivity was found in the nuclei of S-100beta immunoreactive glial profiles throughout the forebrain regions of the sham-operated rats. bFGF immunoreactivity was increased in the nuclei of reactive S-100beta immunoreactive putative astrocytes in the forebrain white matter and in the cerebral cortex of callosotomized rats. These results indicate that after transection of the corpus callosum of adult rats, the reactive astrocytes may exert paracrine trophic actions through S-100beta and bFGF. Interactions between S-100beta and bFGF may be relevant to the events related to neuronal maintenance and repair following brain injury.     

Delayed treatment of secondary degeneration following acute optic nerve transection using a combination of ion channel inhibitors

Studies have shown that a combined application of several ion channel inhibitors immediately atfer cen-tral nervous system injury can inhibit secondary degeneration. However, for clinical use, it is necessary to determine how long atfer injury the combined treatment of several ion channel inhibitors can be delayed and effcacy maintained. In this study, we delivered Ca2+ entry-inhibiting P2X7 receptor antagonist oxi-dized-ATP and AMPA receptor antagonist YM872 to the optic nerve injury sitevia an iPRECIO@ pump immediately, 6 hours, 24 hours and 7 days atfer partial optic nerve transection surgery. In addition, all of the ion channel inhibitor treated rats were administered with calcium channel antagonist lomerizine hy-drochloride. It is important to note that as a result of implantation of the particular pumps required for programmable delivery of therapeutics directly to the injury site, seromas occurred in a signiifcant propor-tion of animals, indicating infection around the pumps in these animals. Improvements in visual function were observed only when treatment was delayed by 6 hours; phosphorylated Tau was reduced when treat-ment was delayed by 24 hours or 7 days. Improvements in structure of node/paranode of Ranvier and reductions in oxidative stress indicators were also only observed when treatment was delayed for 6 hours, 24 hours, or 7 days. Beneifts of ion channel inhibitors were only observed with time-delayed treatment, suggesting that delayed therapy of Ca2+ ion channel inhibitors produces better neuroprotective effects on secondary degeneration, at least in the presence of seromas.     

Secondary degeneration of the optic nerve following partial transection: The benefits of lomerizine

Secondary degeneration is a form of ‘bystander’ damage that can affect neural tissue both nearby and remote from an initial injury. Partial optic nerve transection is an excellent model in which to unequivocally differentiate events occurring during secondary degeneration from those resulting from primary CNS injury. We analysed the primary injury site within the optic nerve (ON) and intact areas vulnerable to secondary degeneration. Areas affected by the primary injury showed morphological disruption, loss of β-III tubulin axonal staining, reduced myelinated axon density, greater proteoglycan expression (phosphacan), increased microglia and macrophage numbers and increased oxidative stress. Similar, but less extreme, changes were seen in areas of the optic nerve undergoing secondary degeneration. The CNS-specific L- and T-type calcium channel blocker lomerizine alleviated some of the changes in areas vulnerable to secondary degeneration. Lomerizine reduced morphological disruption, oxidative stress and phosphacan expression, and limited early increases in macrophage numbers. However, lomerizine failed to prevent progressive de-myelination of ON axons. Within the retina, secondary retinal ganglion cell (RGC) death was significant in areas vulnerable to secondary degeneration. Lomerizine protected RGCs from secondary death at 4 weeks but did not fully restore behavioural function (optokinetic nystagmus). We conclude that blockade of calcium channels is neuroprotective and limits secondary degenerative changes following CNS injury. However such an approach may need to be combined with other treatments to ensure long-term maintenance of full visual function.     

Effect of ionotropic glutamate receptor antagonists on Fos-like immunoreactivity in the dorsal horn following transection of the rat sciatic nerve

Fos-like immunoreactivity (FLI) was investigated in the lumbar dorsal horn 2 h after transection of the rat sciatic nerve and sham operation. FLI following nerve transection was distributed through the medio-lateral extension of the superficial layer of the dorsal horn, while FLI after sham operation, tissue injury, was restricted to the lateral one-third of this layer. The number of FLI neurons in the lateral one-third was similar in the two operations, indicating that neurons expressing FLI in the medial two-thirds and in the lateral one-third of the superficial layer after nerve transection are derived from nerve injury and tissue injury, respectively. FLI in the lateral one-third, but not the medial two-thirds, after nerve transection was significantly reduced by pretreatment with NMDA and AMPA/KA receptor antagonists, indicating that there is a considerable difference in the contributions of ionotropic glutamate receptors to FLI in this layer induced by nerve injury and tissue injury.     

Co-localization of AMPA-type glutamate receptor immunoreactivity in neurons of the rat subthalamic nucleus

In order to determine the precise cellular localization of the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type glutamate receptor subunit immunoreactivity in the rat subthalamic nucleus, single and double immunofluorescence was performed. Intense level of GluR1, GluR2, GluR2/3 and GluR4 immunoreactivity was found in almost all neurons of the subthalamic nucleus. By double immunofluorescence, the subthalamic neurons in the same sections that displayed a strong immunoreactivity for GluR1 were found to display a robust GluR2 immunoreactivity and the subthalamic neurons that displayed GluR2 immunoreactivity were also found to express GluR4 immunoreactivity. The present results thus demonstrate that individual neurons of the subthalamic nucleus are likely to co-express GluR1 and GluR2, and GluR2 and GluR4 immunoreactivity. The native AMPA channels in the subthalamic neurons may, therefore, be composed of heteromeric subunits. The present results provide information of the neuroanatomical localization of AMPA receptor subunits in neurons of the subthalamic nucleus. The localization of AMPA receptor subunits may be related to functional characteristics of AMPA channels in the subthalamic neurons.     

AMPA RECEPTOR REGULATION MECHANISMS: FUTURE TARGET FOR SAFER NEUROPROTECTIVE DRUGS

The post-synaptic AMPA receptors play an important role in mediating fast excitatory transmission in the mammalian brain. Over-activated AMPA receptors induce excitotoxicity, implicated in a number of chronic neuro-de-gen-era-tive disorders such as Parkinson's disease, Huntington's disease, and AIDS encephalitis. AMPA receptor antagonists offer protection against neurodegeneration in the experimental models even if they are given 24 h after the injury. Because AMPA receptors seem to be involved in the neurodegenerative diseases, modulating the activity of the AMPA receptors could be an attractive approach to reduce or prevent excitotoxicity. Studies conducted recently have exhibited a number of new mechanisms for AMPA receptor regulation. Modulations of these were found to have protective implications. AMPA receptor depolarization and desensitization are protective to the neurons. Receptor desensitization depends on the receptor subunit composition. The R/G editing site and the flip/flop cassettes in AMPA receptor subunits contribute to a great extent in receptor desensitization and recovery rates. Molecules that could quicken receptor desensitization or delay recovery could be of use. AMPA receptors limit neuronal entry of Ca2+ ions by regulating Ca2+-permeability. Ca2+-permeable receptor channels are made up of GluR1, GluR3, or GluR4 subunits, whereas presence of the GluR2 subunit restricts Ca2+ entry and renders the receptor Ca2+-impermeable. GluR2 levels, however, experience a fall after neuronal insult rendering the AMPA receptors Ca2+-permeable, thus factors that could interfere with this event might prove to be very beneficial- against excitotoxicity. AMPA receptor clusters are stabilized by PSD-95, which requires palmitoylation at two sites. Targeting palmitoylation of the PSD-95 can also be a useful approach to disperse AMPA clusters at the synapse. In the perisynaptic region, mGluRs are present a little away from the synapse and are among the glutamate transporters, which require high-frequency firing for activation. On activation they might enhance the activity of NMDA receptors at the synapse to increase the levels of AMPA receptors. AMPA receptors surfaced at this juncture can contribute to heavy Ca2+ influx. Thus, blocking this pathway could be of considerable importance in preventing the excitotoxicity. A number of proteins such as the GRIP, PICK, and NSF also modulate the functions of AMPA receptors. Polyamines also block Ca2+ permeable AMPA receptors and thus are pro--tec-tive. NO and cGMP also play an important role in negatively regulating AMPA receptors and thus could offer protection. Modulation of AMPA receptor by different mechanisms has been discussed in the present review to implicate importance of these targets/pathways for safer and future neuro-protective drugs.     

AMPA receptor regulation mechanisms: future target for safer neuroprotective drugs

The post-synaptic AMPA receptors play an important role in mediating fast excitatory transmission in the mammalian brain. Over-activated AMPA receptors induce excitotoxicity, implicated in a number of Chronic neurodegenerative disorders such as Parkinson's disease, Huntington's disease, and AIDS encephalitis. AMPA receptor antagonists offer protection against neurodegeneration in the experimental models even if they are given 24 h after the injury. Because AMPA receptors seem to be involved in the neurodegenerative diseases, modulating the activity of the AMPA receptors could be an attractive approach to reduce or prevent excitotoxicity. Studies conducted recently have exhibited a number of new mechanisms for AMPA receptor regulation. Modulations of these were found to have protective implications. AMPA receptor depolarization and desensitization are protective to the neurons. Receptor desensitization depends on the receptor subunit composition. The R/G editing site and the flip/flop cassettes in AMPA receptor subunits contribute to a great extent in receptor desensitization and recovery rates. Molecules that could quicken receptor desensitization or delay recovery could be of use. AMPA receptors limit neuronal entry of Ca2+ ions by regulating Ca2+-permeability. Ca2+-permeable receptor channels are made up of GluR1, GluR3, or GluR4 subunits, whereas presence of the GluR2 subunit restricts Ca2+ entry and renders the receptor Ca2+-impermeable. GluR2 levels, however, experience a fall after neuronal insult rendering the AMPA receptors Ca2+-permeable, thus factors that could interfere with this event might prove to be very beneficial against excitotoxicity. AMPA receptor clusters are stabilized by PSD-95, which requires palmitoylation at two sites. Targeting palmitoylation of the PSD-95 can also be a useful approach to disperse AMPA clusters at the synapse. In the perisynaptic region, mGluRs are present a little away from the synapse and are among the glutamate transporters, which require high-frequency firing for activation. On activation they might enhance the activity of NMDA receptors at the synapse to increase the levels of AMPA receptors. AMPA receptors surfaced at this juncture can contribute to heavy Ca2+ influx. Thus, blocking this pathway could be of considerable importance in preventing the excitotoxicity. A number of proteins such as the GRIP, PICK, and NSF also modulate the functions of AMPA receptors. Polyamines also block Ca2+ permeable AMPA receptors and thus are protective. NO and cGMP also play an important role in negatively regulating AMPA receptors and thus could offer protection. Modulation of AMPA receptor by different mechanisms has been discussed in the present review to implicate importance of these targets/pathways for safer and future neuroprotective drugs.