Translocation of Glutamate Transporter Subtype Excitatory Amino Acid Carrier 1 Protein in Kainic Acid-Induced Rat Epilepsy
2003; Elsevier BV; Volume: 163; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)63705-4
ISSN1525-2191
AutoresAkiko Furuta, Mami Noda, Satoshi O. Suzuki, Yoshinobu Goto, Yoshiko Kanahori, Jeffrey D. Rothstein, Toru Iwaki,
Tópico(s)Epilepsy research and treatment
ResumoGlutamate excitotoxicity has been implicated in the pathophysiology of epilepsy. Systemic injection of kainic acid (KA) in the rat produces an animal model of human temporal lobe epilepsy. We examined the temporal expression of the sodium-dependent neuronal glutamate transporter, excitatory amino acid carrier 1 (EAAC1), in KA-induced rat epilepsy. As an early alteration, perinuclear deposits of EAAC1 protein were found mainly in the large pyramidal neurons at the hippocampus, neocortex, piriform cortex, and amygdala with the reduction of neuropil staining 6 hours after KA injection. Immunoelectron microscopic study revealed that the perinuclear EAAC1 immunoreactivity corresponded to the translocation to the Golgi complex. At this time point, EAAC1 mRNA was down-regulated. The intracellular aggregation of EAAC1 primarily disappeared by 24 hours. In vitro studies indicated that internalization of EAAC1 from the plasma membrane to the intracellular compartment by KA treatment was associated with the reduction of electrogenic transporter currents. Our results suggest that the transient EAAC1 internalization participates in the modulation of the transporter function preventing excessive glutamate uptake to pyramidal neurons during the early stage of epilepsy. Glutamate excitotoxicity has been implicated in the pathophysiology of epilepsy. Systemic injection of kainic acid (KA) in the rat produces an animal model of human temporal lobe epilepsy. We examined the temporal expression of the sodium-dependent neuronal glutamate transporter, excitatory amino acid carrier 1 (EAAC1), in KA-induced rat epilepsy. As an early alteration, perinuclear deposits of EAAC1 protein were found mainly in the large pyramidal neurons at the hippocampus, neocortex, piriform cortex, and amygdala with the reduction of neuropil staining 6 hours after KA injection. Immunoelectron microscopic study revealed that the perinuclear EAAC1 immunoreactivity corresponded to the translocation to the Golgi complex. At this time point, EAAC1 mRNA was down-regulated. The intracellular aggregation of EAAC1 primarily disappeared by 24 hours. In vitro studies indicated that internalization of EAAC1 from the plasma membrane to the intracellular compartment by KA treatment was associated with the reduction of electrogenic transporter currents. Our results suggest that the transient EAAC1 internalization participates in the modulation of the transporter function preventing excessive glutamate uptake to pyramidal neurons during the early stage of epilepsy. Glutamate, the principal excitatory neurotransmitter in the mammalian central nervous system,1Fonnum F Glutamate: a neurotransmitter in mammalian brain.J Neurochem. 1984; 42: 1-11Crossref PubMed Scopus (1670) Google Scholar has been implicated in the pathophysiology of epilepsy. Extracellular glutamate released from presynaptic vesicles must be rapidly removed from the synaptic cleft by glutamate transporting proteins to prevent neuronal excitotoxicity.2Choi DW Maulucci-Gedde M Kriegstein AR Glutamate neurotoxicity in cortical cell culture.J Neurosci. 1987; 7: 357-368Crossref PubMed Google Scholar, 3Rosenberg PA Amin S Leitner M Glutamate uptake disguises neurotoxic potency of glutamate agonists in cerebral cortex in dissociated cell culture.J Neurosci. 1992; 12: 56-61Crossref PubMed Google Scholar EAAC1, neuronal/epithelial glutamate transporter subtype, is widely distributed in the neurons of the central nervous system.4Rothstein JD Martin L Levey AI Dykes HM Jin L Wu D Nash N Kuncl RW Localization of neuronal and glial glutamate transporters.Neuron. 1994; 13: 713-725Abstract Full Text PDF PubMed Scopus (1449) Google Scholar Besides in neurons, EAAC1 is also localized in immature oligodendrocytes,5Gottlieb M Domercq M Matute C Altered expression of the glutamate transporter EAAC1 in neurons and immature oligodendrocytes after transient forebrain ischemia.J Cerebr Blood Flow Metab. 2000; 20: 678-687Crossref PubMed Scopus (47) Google Scholar a subset of normal astrocytes6Conti F DeBiasi S Minelli A Rothstein JD Melone M EAAC1, a high-affinity glutamate transporter, is localized to astrocytes and GABAergic neurons besides pyramidal cells in the rat cerebral cortex.Cereb Cortex. 1998; 8: 108-116Crossref PubMed Scopus (186) Google Scholar and neoplastic astrocytes,7Palos TP Ramachandran B Boado R Howard BD Rat C6 and human astrocytic tumor cells express a neuronal type of glutamate transporter.Mol Brain Res. 1996; 37: 297-303Crossref PubMed Scopus (68) Google Scholar as well as in peripheral tissue including the kidney,8Shayakul C Kanai Y Lee WS Brown D Rothstein JD Hediger MA Localization of the high-affinity glutamate transporter EAAC1 in rat kidney.Am J Physiol. 1997; 273: F1023-F1029PubMed Google Scholar heart,9King N Williams H McGivan JD Suleiman MS Characteristics of L-aspartate transport and expression of EAAC-1 in sarcolemmal vesicles and isolated cells from rat heart.Cardiovasc Res. 2001; 52: 84-94Crossref PubMed Scopus (15) Google Scholar and small intestine.10Erickson RH Gum Jr, JR Lindstrom MM McKean D Kim YS Regional expression and dietary regulation of rat small intestinal peptide and amino acid transporter mRNAs.Biochem Biophys Res Commun. 1995; 216: 249-257Crossref PubMed Scopus (120) Google Scholar Among the five subtypes of glutamate transporter, the critical role of the astrocytic glutamate transporter, GLT-1 has been emphasized in the epileptic brain because GLT-1-deficient mice develop lethal seizures.11Tanaka K Watase K Manabe T Yamada K Watanabe M Takahashi K Iwama H Nishikawa T Ichihara N Kikuchi T Okuyama S Kawashima N Hori S Takimoto M Wada K Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1.Science. 1997; 276: 1699-1702Crossref PubMed Scopus (1457) Google Scholar However, the regulation of EAAC1 during epilepsy has not been understood. Although EAAC1-deficient mice display only a reduced spontaneous locomotor activity without neurodegeneration,12Peghini P Janzen J Stoffel W Glutamate transporter EAAC-1-deficient mice develop dicarboxylic aminoaciduria and behavioral abnormalities but no neurodegeneration.EMBO J. 1997; 16: 3822-3832Crossref PubMed Scopus (274) Google Scholar administration of the anti-sense oligonucleotide of EAAC1 into a rat brain produces epilepsy.13Rothstein JD Dykes HM Pardo CA Bristol LA Jin L Kuncl RW Kanai Y Hediger MA Wang Y Schielke JP Welty DF Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate.Neuron. 1996; 16: 675-686Abstract Full Text Full Text PDF PubMed Scopus (2110) Google Scholar The regulation of EAAC1 in epileptic brains is rather inconsistent: up-regulation of EAAC1 in amygdala-kindled rats,14Miller HP Levey AI Rothstein JD Tzingounis AV Conn PJ Alterations in glutamate transporter protein levels in kindling-induced epilepsy.J Neurochem. 1997; 68: 1564-1570Crossref PubMed Scopus (124) Google Scholar no change of EAAC1 protein level in genetically epilepsy-prone rats,15Akbar MT Rattray M Williams RJ Chong NW Meldrum BS Reduction of GABA and glutamate transporter messenger RNAs in the severe-seizure genetically epilepsy-prone rat.Neuroscience. 1998; 85: 1235-1251Crossref PubMed Scopus (59) Google Scholar and down-regulation in kainic acid (KA) seizures.16Simantov R Crispino M Hoe W Broutman G Tocco G Rothstein JD Baudry M Changes in expression of neuronal and glial glutamate transporters in rat hippocampus following kainate-induced seizure activity.Mol Brain Res. 1999; 65: 112-123Crossref PubMed Scopus (88) Google ScholarSystemic or intracerebral injection of KA, a rigid analogue of glutamate, in the rat is frequently used to produce an animal model of human temporal lobe epilepsy. Since the description by Olney and colleagues,17Olney JW Rhee V Ho OL Kainic acid: a powerful neurotoxic analogue of glutamate.Brain Res. 1974; 77: 507-512Crossref PubMed Scopus (621) Google Scholar the neurotoxicity of KA has been extensively studied neuropathologically and electrophysiologically.18Sperk G Kainic acid seizures in the rat.Prog Neurobiol. 1994; 42: 1-32Crossref PubMed Scopus (629) Google Scholar In the present study, we found transient translocation of EAAC1 protein to the Golgi complex with the reduction of neuropil staining in the pyramidal neurons 6 hours after KA injection. To test the hypothesis that the EAAC1 internalization by KA treatment is associated with functional impairment, in vitro studies were also performed with C6 glioma cells. We used the C6 glioma cell line because it endogenously expresses only EAAC1 among sodium-dependent glutamate transporter subtypes7Palos TP Ramachandran B Boado R Howard BD Rat C6 and human astrocytic tumor cells express a neuronal type of glutamate transporter.Mol Brain Res. 1996; 37: 297-303Crossref PubMed Scopus (68) Google Scholar and it has been used to investigate the membrane trafficking of the EAAC1 through protein kinase C and phosphatidylinositol 3-kinase.19Davis KE Straff DJ Weinstein EA Bannerman PG Correale DM Rothstein JD Robinson MB Multiple signaling pathways regulate cell surface expression and activity of the excitatory amino acid carrier 1 subtype of Glu transporter in C6 glioma.J Neurosci. 1998; 18: 2475-2485Crossref PubMed Google Scholar, 20Dowd LA Coyle AJ Rothstein JD Pritchett DB Robinson MB Comparison of Na+-dependent glutamate transport activity in synaptosomes, C6 glioma, and Xenopus oocytes expressing excitatory amino acid carrier 1 (EAAC1).Mol Pharmacol. 1996; 49: 465-473PubMed Google Scholar, 21Sims KD Straff DJ Robinson MB Platelet-derived growth factor rapidly increases activity and cell surface expression of the EAAC1 subtype of glutamate transporter through activation of phosphatidylinositol 3-kinase.J Biol Chem. 2000; 275: 5228-5237Crossref PubMed Scopus (115) Google Scholar Moreover, cultured hippocampal neurons express not only EAAC1 but also GLT-1.22Brooks-Kayal AR Munir M Jin H Robinson MB The glutamate transporter, GLT-1, is expressed in cultured hippocampal neurons.Neurochem Int. 1998; 33: 95-100Crossref PubMed Scopus (51) Google Scholar In our result, the reduction of the reversed transporter current with the change of subcellular localization was observed by KA treatment. The intracellular trafficking and functional role of EAAC1 in epilepsy have been discussed.Materials and MethodsAnimal Procedures and Tissue PreparationThis study was approved by the Animal Committee of Kyushu University. Male Wistar rats weighing 180 to 200 g (8-weeks-old; Kyudo, Japan) were divided into seven groups of five rats each. Rats were injected intraperitoneally with 10 mg/kg KA (Sigma, St. Louis, MO or Ocean Produce Int., Nova Scotia, Canada). After being deeply anesthetized with ether, rats were intra-aortically perfused with 4% paraformaldehyde and brain tissue samples were obtained at 2 hours, 6 hours, 24 hours, 1 week, and 4 weeks after the KA injection. In the two control groups, the rats were injected intraperitoneally with 0.1 mol/L of phosphate-buffered saline (200 μl) and killed at 6 hours and 4 weeks after the injection. After the KA injection, behavioral changes were observed to determine the seizure stages according to the criteria of Racine.23Racine RJ Modification of seizure activity by electrical stimulation. II. Motor seizure.Electroencephalogr Clin Neurophysiol. 1972; 32: 281-294Abstract Full Text PDF PubMed Scopus (5677) Google Scholar Intraperitoneal KA injection (10 mg/kg) produced the following acute-stage behavioral changes in each Wistar rat: within 2 hours, the rats developed masticatory movements, head nodding, and wet dog shakes (class 1 and 2 by Racine23Racine RJ Modification of seizure activity by electrical stimulation. II. Motor seizure.Electroencephalogr Clin Neurophysiol. 1972; 32: 281-294Abstract Full Text PDF PubMed Scopus (5677) Google Scholar), followed by forelimb clonus and rearing (class 3 and 4); between 2 and 4 hours after KA injection, the rats exhibited severe, repeated status epilepticus with rearing and falling and reached class 5 motor seizures; the seizures declined and rats appeared exhausted between 4 and 6 hours; spontaneous motor seizures were occasionally observed in the late stage 1 week after KA injection.Western BlottingFresh-frozen samples from the hippocampus of the control and KA-treated rats killed at 6 hours and 4 weeks were used. Western blotting was performed as described previously.24Fukamachi S Furuta A Ikeda T Ikenoue T Kaneoka T Rothstein JD Iwaki T Altered expressions of glutamate transporter subtypes in rat model of neonatal cerebral hypoxia-ischemia.Dev Brain Res. 2001; 132: 131-139Crossref PubMed Scopus (58) Google Scholar Samples were homogenized in protein lysis buffer. Each sample (10 μg/lane) was separated by 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membranes were incubated overnight with EAAC1 antibody (1:1000)4Rothstein JD Martin L Levey AI Dykes HM Jin L Wu D Nash N Kuncl RW Localization of neuronal and glial glutamate transporters.Neuron. 1994; 13: 713-725Abstract Full Text PDF PubMed Scopus (1449) Google Scholar in blocking buffer at 4°C, then washed and incubated with peroxidase-conjugated secondary antibody (1:20,000; Chemicon, Temecula, CA) for 1 hour. The immunoreactive proteins were visualized with enhanced chemiluminescence (Amersham, Buckinghamshire, UK). Then, the membrane was washed and incubated with actin antibody (1:5000, Amersham) and developed with enhanced chemiluminescence as a loading control.Northern BlottingTotal RNA was isolated by the guanidine isothiocyanate method25Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62983) Google Scholar from each hippocampus from which a contralateral sample was used for Western blotting. RNA (10 μg/lane) was diluted in loading buffer (0.5× 3-(N-morpholino)propanesulfonic acid (MOPS), 50% formamide, 6.7% formaldehyde, 10 μg/ml ethidium bromide), electrophoresed in 1% denaturing agarose gel containing 1× MOPS and 6.7% formaldehyde, and transferred onto nylon membrane (Roche, Mannheim, Germany). The membrane was hybridized with 32P-labeled EAAC1 cDNA probes overnight at 65°C. After washing twice in 6× standard saline citrate (SSC) at room temperature for 10 minutes, once in 2× SSC/0.1% SDS and 0.2× SSC/0.1% SDS at 65°C for 20 minutes, the membrane was exposed to X-ray film (Eastman-Kodak, Rochester, NY) overnight at −80°C. After stripping the EAAC1 probe in 2× SSC/50% formamide at 65°C overnight, the membrane was reprobed with glyceraldehyde phosphate dehydrogenase (GAPDH) cDNA as a loading control as described above.Immunohistochemistry and Immunoelectron MicroscopyImmunohistochemistry and immunoelectron microscopy were performed as described previously.24Fukamachi S Furuta A Ikeda T Ikenoue T Kaneoka T Rothstein JD Iwaki T Altered expressions of glutamate transporter subtypes in rat model of neonatal cerebral hypoxia-ischemia.Dev Brain Res. 2001; 132: 131-139Crossref PubMed Scopus (58) Google Scholar For histopathological examination, the coronal sections at the level of the dorsal hippocampi and the sagittal sections of the cerebellum were stained with hematoxylin and eosin (H&E). For immunohistochemistry, paraffin-embedded 5-μm-thick sections were deparaffinized and incubated with 0.3% hydrogen peroxide in absolute methanol for 30 minutes. Sections were autoclaved in 0.01 mol/L of citrate buffer, pH 6.0, to enhance the immunoreactivity. After washing with Tris-HCl buffer (50 mmol/L Tris-HCl, pH 7.6), the sections were incubated at 4°C overnight with primary antibodies diluted 1:100 for EAAC14Rothstein JD Martin L Levey AI Dykes HM Jin L Wu D Nash N Kuncl RW Localization of neuronal and glial glutamate transporters.Neuron. 1994; 13: 713-725Abstract Full Text PDF PubMed Scopus (1449) Google Scholar (rabbit, polyclonal), N-methyl-d-aspartate receptor (NR) 1 (rabbit, polyclonal; Chemicon) and glutamate receptor (GluR) 2/3 (rabbit, polyclonal; Chemicon). The sections were then washed and incubated with biotinylated rabbit antibodies diluted 1:200 and peroxidase-conjugated streptavidin-biotin complex or fluorescein isothiocyanate-conjugated streptavidin (Amersham) diluted 1:100 sequentially. The colored reaction product was developed with 3,3′-diaminobenzidine tetrahydrochloride solution. The sections for immunofluorescence were counterstained with propidium iodide and were observed under a confocal laser microscope (LSM-GB200; Olympus, Tokyo, Japan). For immunoelectron microscopy, rats were perfused with 2% paraformaldehyde/2% glutaraldehyde in 0.1 mol/L of phosphate buffer. Vibratome sections (40 μm) were pretreated by 1% sodium borohydride for 10 minutes, then immunohistochemistry was performed using the labeled biotin-streptavidin method with 3,3′-diaminobenzidine tetrahydrochloride reaction the same as with paraffin sections. Ultrathin sections without lead staining were observed under an electron microscope (JEM-100CX; JEOL, Japan).In Situ HybridizationNonradioisotopic in situ hybridization was performed as described previously.26Suzuki SO Iwaki T Non-isotopic in situ hybridization of CD44 transcript in formalin-fixed paraffin-embedded sections.Brain Res Protoc. 1999; 4: 29-35Crossref PubMed Scopus (8) Google Scholar To detect the EAAC1 transcripts, a 149-bp cDNA fragment of rat EAAC1 (GenBank accession number U21107; position 504 to 652) was amplified by polymerase chain reaction from rat cerebral cDNA and was cloned in TA-cloning vector (Invitrogen Corp., Carlsbad, CA). Digoxigenin-labeled anti-sense and sense cRNA probes were generated using a DIG RNA Labeling Kit (SP6/T7) (Roche). Paraffin-embedded sections were deparaffinized, digested with 20 μg/ml of proteinase K for 15 minutes at room temperature, acetylated with 0.25% acetic anhydride/0.1 mol/L triethanolamine (TEA), pH 8.0, for 10 minutes, and treated with 0.2 N HCl for 10 minutes. Then, sections were hybridized with the probes overnight at 50°C. After being rinsed in 2× SSC/50% formamide at 65°C for 30 minutes, sections were treated with 50 μg/ml of RNase A in 10 mmol/L of Tris-HCl, pH 7.6/500 mmol/L NaCl/1 mmol/L ethylenediaminetetraacetic acid at 37°C for 30 minutes, and washed once in 2× SSC/0.1% SDS at 65°C for 20 minutes and twice in 0.2× SSC/0.1% SDS at 65°C for 20 minutes. Sections were then incubated with alkaline phosphatase-conjugated anti-digoxigenin monoclonal antibody (1:500) overnight at 4°C (DIG Nucleic Acid Detection kit, Roche). Sections were developed in 350 μg/ml nitro blue tetrazolium, 175 μg/ml 5-bromo-4-chloro-3-indolyl phosphate/100 mmol/L Tris-HCl, pH 9.5, 100 mmol/L NaCl, and 50 mmol/L MgCl for 16 to 24 hours in the dark. To determine the specificity of the digoxigenin-labeled EAAC1 anti-sense probe, hybridization with an EAAC1 sense probe as well as competitive hybridization with digoxigenin-labeled and nonlabeled probes was performed at the same time.Densitometric AnalysisImage analysis of immunohistochemistry and in situ hybridization for EAAC1 was performed on a Macintosh computer using the public domain NIH Image program 1.62 (National Technical Information Service, Springfield, VA) as described previously.27Furuta A Iida T Nakabeppu Y Iwaki T Expression of hMTHI in the hippocampi of control and Alzheimer's disease.NeuroReport. 2001; 12: 2895-2899Crossref PubMed Scopus (44) Google Scholar The optical densities at large neurons were obtained from five fields (0.025 inches) of each section of CA3 at the hippocampus, layer 5 at the neocortex, and mediodorsal nuclei at the thalamus, and then averaged for three rats. Statistical analysis for the differences of regional changes between control and KA-treated rats was performed with a Kruskal-Wallis test as well as a Mann-Whitney U-test using a StatView J4.5 program (Abacus Concepts, Berkeley, CA).Cell CultureC6 rat glioma cells (CL 107, American Type Culture Collection, Rockville, MD) that endogenously express EAAC17Palos TP Ramachandran B Boado R Howard BD Rat C6 and human astrocytic tumor cells express a neuronal type of glutamate transporter.Mol Brain Res. 1996; 37: 297-303Crossref PubMed Scopus (68) Google Scholar were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated calf serum. For electrophysiology, cells were plated in 35-mm dishes and incubated with 500 nmol/L of phorbol 12-myristate 13-acetate (PMA) for 3 hours. Then, KA (10 μmol/L) was applied for 1.5 hours before use. For immunocytochemistry, culture cells were plated in Lab-Tek chamber slides (Nunc Inc., Naperville, IL), and fixed with 4% paraformaldehyde, pretreated with 1% Nonidet P-40 for 10 minutes, and incubated overnight with EAAC1 antibody diluted 1:10. Then cells were incubated with fluorescein isothiocyanate-conjugated anti-rabbit IgG (Amersham) and propidium iodide (Sigma). The slides were observed under a laser scan confocal microscopy (LSM-GB200; Olympus, Japan).Electrophysiological MeasurementsPatch-clamp recordings were made as reported previously.28Noda M Nakanishi H Akaike N Glutamate release from microglia via glutamate transporter is enhanced by amyloid-beta peptide.Neuroscience. 1999; 92: 1465-1474Crossref PubMed Scopus (137) Google Scholar, 29Noda M Nakanishi H Nabekura J Akaike N AMPA-kainate subtypes of glutamate receptor in rat cerebral microglia.J Neurosci. 2000; 20: 251-258PubMed Google Scholar C6 glioma cells were whole cell-clamped using a patch pipette containing 90 mmol/L NaCl, 10 mmol/L l-glutamate, 3 mmol/L MgATP, 5 mmol/L HEPES, 1 mmol/L CaCl2, 4 mmol/L MgCl2, and 5 mmol/L EGTA. The pH of the solution was adjusted to 7.3 with N-methyl-d-glucamine. The pipette resistance was 6 to 9 MΩ. The external solution contained 0 or 10 mmol/L KCl, 110 or 100 mmol/L choline chloride, 0.5 mmol/L MgCl2, 3 mmol/L CaCl2, 5 mmol/L HEPES, 15 mmol/L glucose, 6 mmol/L BaCl2, and 0.1 mmol/L ouabain. The pH of the solution was adjusted to 7.4 with N-methyl-d-glucamine. The external potassium solution was applied rapidly using the Y-tube technique,30Min BI Kim CJ Rhee JS Akaike N Modulation of glycine-induced chloride current in acutely dissociated rat periaqueductal gray neurons by mu-opioid agonist, DAGO.Brain Res. 1996; 734: 72-78Crossref PubMed Scopus (39) Google Scholar which allows the complete exchange of the external solution surrounding a cell within 20 ms. The temperature monitored in the recording dishes was 33 to 34°C.ResultsWestern and Northern Blot Analysis of EAAC1Western blotting of the hippocampal homogenates from control and KA-treated rats at 6 hours and 4 weeks demonstrated a single band that corresponded to EAAC1 protein using C-terminal EAAC1 antibody. Total expression level of EAAC1 protein was not altered 6 hours after KA treatment. Degradation products were not observed in the homogenates from KA-treated rats (Figure 1A). Northern blotting of the hippocampal homogenates revealed a single band at 3.8 kb that corresponds to EAAC1 transcripts using 32P-labeled EAAC1 cDNA probes (Figure 1B). Neither the truncated nor aberrant form of EAAC1 mRNA was detected in KA-treated rats.Early Transient Changes of EAAC1 Expression in the Large Pyramidal NeuronsIn the control rat brains, EAAC1 was enriched in the large pyramidal neurons and neuropil of the hippocampus, caudate putamen, amygdala, piriform cortex, olfactory bulb, and cerebellar cortex (Figure 2; A, C, and E). Early histopathological changes 2 hours after KA injection were characterized by edema in the neuropil throughout the forebrain. Despite the marked neuropil changes in H&E sections, EAAC1 protein expressions were generally retained even in the shrunken neurons until 2 hours after KA injection during status epilepticus. Expression of EAAC1 increased slightly at the piriform cortex at 2 hours. The most dramatic change was disclosed at 6 hours; ie, perinuclear deposition of EAAC1 immunoreactivity was found together with the reduction of neuropil staining mainly in the large pyramidal neurons of the neocortex (Figure 2B), hippocampus (Figure 2D), amygdala, and piriform cortex. These changes were partly seen in the putaminal neurons and small pyramidal neurons of the neocortex, but not observed in the cerebellar Purkinje cells (Figure 2F), spinal motor neurons, or astrocytes. In the immunoelectron microscopy, EAAC1-immunoreactivity at 6 hours was associated mainly with the outer membrane of the Golgi complex, which was consistent with the perinuclear staining of the large pyramidal neurons at 6 hours (Figure 2G). Less immunoreactivity for EAAC1 was found in the dendrites, axons, plasma membranes, and nucleus. This intracellular relocation of EAAC1 is specific, because other membrane proteins such as one of the N-methyl D-aspartate (NMDA) receptors, NR1, and one of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, GluR 2/3, did not undergo the same spatial reorganization in the immediate postseizure period (Figure 3). The intracellular aggregation of EAAC1 at 6 hours (Figure 4, B and F) was not observed at 24 hours (Figure 4, C and G), 1 week, and 4 weeks (Figure 4, D and H) after KA injection. EAAC1 expression partly decreased at the piriform cortex, the lateral part of amygdala, and the medial part of the thalamic nuclei at 1 week and 4 weeks.Figure 2Immunohistochemistry (A–F) and immunoelectron microscopy (G) in layer 5 of neocortex (A, B, and G), CA3 pyramidal neurons at hippocampus (C, D), and cerebellar Purkinje cells (E, F). A–D: In the control rats, pyramidal neurons and neuropil are immunoreactive for EAAC1 (A, C). EAAC1 immunoreactivity is found as perinuclear deposits at pyramidal neurons with reduced neuropil staining 6 hours after the KA injection (B, D), whereas immunoreactivities for EAAC1 are not altered with KA treatment in the Purkinje cells (E, F). G: Ultrastructural localization of EAAC1 in cortical pyramidal neurons 6 hours after the KA injection. Electron micrographs show enrichment of EAAC1 immunoreactivity around the Golgi apparatus (arrows) compared to that at the plasma membranes (arrowheads). Scale bars: 20 μm (A–F); 1 μm (G).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Immunohistochemistry for EAAC1 (A and B, green), NR1 (C and D, green), and GluR2/3 (E and F, green) in the hippocampal CA3 in the control (A, C, E) and 6 hours after KA injection (B, D, F). Although perinuclear immunoreactivity for EAAC1 was found in the 6 hours after KA injection (B), immunoreactivities for the glutamate receptors NR1 and GluR2/3 were not altered in the large pyramidal neurons. Nuclear staining with propidium iodide (A–F, red). Scale bar, 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Immunohistochemistry (A–H, EAAC1) and in situ hybridization with digoxigenin-labeled anti-sense probes (I–P, EAAC1-ISH) in the hippocampus (A–D, I–L) and the magnification of CA3 (E–H, M–P) at control (A, E, I, M), KA 6 hours (6h; B, F, J, N), KA 24 hours (24h; C, G, K, O), and KA 4 weeks (4w; D, H, L, P). A and E: Hippocampal pyramidal neurons and neuropil are immunoreactive for EAAC1 in the control. B and F: EAAC1 immunoreactivity is found as perinuclear deposits at pyramidal neurons (inset in F) 6 hours after the KA injection. C, D, G, and H: Distribution and immunoreactivity for EAAC1 at the hippocampus have almost recovered to the control level 24 hours as well as 4 weeks after the KA injection. I and M: EAAC1 transcripts are enriched in the pyramidal neurons of the control hippocampus. J and N: Expression of EAAC1 mRNA decreases 6 hours after the KA injection. K, L, O, and P: Expression of EAAC1 mRNA has recovered 24 hours as well as 4 weeks after the KA injection. Scale bars: 500 μm (A–D and I–L); 50 μm (E–H); 40 μm (M–P).View Large Image Figure ViewerDownload Hi-res image Download (PPT)In the in situ hybridization, the EAAC1 mRNA expression was ubiquitously found in the pyramidal neurons of the forebrain in the control cases. In the control hippocampus, pyramidal neurons were preferentially labeled compared to the granule cells of dentate gyrus (Figure 4I). Expression of EAAC1 mRNA decreased in the forebrain 6 hours after KA injection (Figure 4, J and N). The expression of EAAC1 mRNA at 24 hours had recovered to the normal level (Figure 4, K and O). The positive signal was not detected in the hybridization with the EAAC1 sense probe as well as competitive hybridization with 50-fold nonlabeled probes.Densitometric Analysis of EAAC1 ExpressionsDensitometric analysis of EAAC1 immunohistochemistry and in situ hybridization was performed at early phase (Figure 5A, KA 6 hours and KA 24 hours) and late phase (Figure 5B, KA 4 weeks). Optical densities for EAAC1 immunohistochemistry demonstrated that perikaryal staining (P in Figure 5A) was increased in hippocampus and neocortex, whereas increased perikaryal staining was not seen in the mediodorsal nuclei of thalamus. Optical densities for neuropil staining in the immunohistochemistry (N in Figure 5A) and the in situ hybridization became reduced in the hippocampus, neocortex, and thalamus at 6 hours. Such changes recovered to the control level at 24 hours. Significant difference is show
Referência(s)