EAAC1 Immunoreactivity Research Articles

Functional integration of human dental pulp stem cell-derived neurons in vivo

Event Abstract Back to Event Functional integration of human dental pulp stem cell-derived neurons in vivo Gábor Gerber1*, Marianna Király2, Kristóf Kádár2, Dénes B. Horváthy3, Péter P. Nardai3, Miklós Weszl3, Attila Cselenyák3, Levente Kiss3, Zsombor Lacza3 and Gábor Varga2 1 Semmelweis University, Department of Anatomy, Histology and Embryology, Hungary 2 Semmelweis University, Department of Oral Biology, Hungary 3 Semmelweis University, Institute of Human Physiology and Clinical Experimental Research, Hungary Pluripotency and the potential for continuous self-renewal make dental pulp stem cells (DPSCs) an attractive donor source for neuronal cell replacement. Despite recent encouraging results in this field, little is known about the functional integration of transplanted DPSC derived neurons Materials and Methods. Human Vybrant DiD labeled neuronally induced DPSCs were transplanted into the cerebrospinal fluid of 2-days-old male rats. Cortical lesion was induced by touching a -60 ˚C metal stamp to the calvaria over the motor cortex. Cell migration was followed by fluorescent microscopy. Neuronal cell markers were detected by immunohistochemistry. Voltage-dependent sodium (NaV) and potassium channels (KDR) were investigated by patch clamp recording. Most transplanted DPSCs migrated into the normal neural progenitor zones and expressed early neuronal marker β-III-tubulin, neuronal IF protein, NeuN, and glial GFAP. The cells displayed TTX sensitive NaV currents and TEA sensitive KDR. Two weeks after the injury, the cells started to migrate towards the lesion, and after four weeks they also expressed the neuron specific EAAC1, GAD65/67, 5-HT and tyrosine hydroxylase immunoreactivity. Their TTX sensitive NaV currents and KDR currents increased. Their input resistance decreased, resting membrane potential increased. Our data demonstrate that at the single-cell level, grafted DPSC-derived neurons undergo morphological and functional integration into the host brain. Immunohistochemical and electrophysiological data both reveal that DPSCs can provide an appropriate model for studying neuro- and gliogenesis in vivo, since they show progression in their neural specific marker expression and electrophysiological properties after brain injury, displaying a behavior very similar to that of the normal progenitor zone cells. However, synaptic integration remains still to be investigated. Supported by NI 69008 OTKA and TÁMOP 4.2.2-08/1/KMR-2008-0004. Conference: IBRO International Workshop 2010, Pécs, Hungary, 21 Jan - 23 Jan, 2010. Presentation Type: Poster Presentation Topic: Development Citation: Gerber G, Király M, Kádár K, Horváthy DB, Nardai PP, Weszl M, Cselenyák A, Kiss L, Lacza Z and Varga G (2010). Functional integration of human dental pulp stem cell-derived neurons in vivo. Front. Neurosci. Conference Abstract: IBRO International Workshop 2010. doi: 10.3389/conf.fnins.2010.10.00010 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 14 Apr 2010; Published Online: 14 Apr 2010. * Correspondence: Gábor Gerber, Semmelweis University, Department of Anatomy, Histology and Embryology, Budapest, Hungary, gerber@ana.sote.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Gábor Gerber Marianna Király Kristóf Kádár Dénes B Horváthy Péter P Nardai Miklós Weszl Attila Cselenyák Levente Kiss Zsombor Lacza Gábor Varga Google Gábor Gerber Marianna Király Kristóf Kádár Dénes B Horváthy Péter P Nardai Miklós Weszl Attila Cselenyák Levente Kiss Zsombor Lacza Gábor Varga Google Scholar Gábor Gerber Marianna Király Kristóf Kádár Dénes B Horváthy Péter P Nardai Miklós Weszl Attila Cselenyák Levente Kiss Zsombor Lacza Gábor Varga PubMed Gábor Gerber Marianna Király Kristóf Kádár Dénes B Horváthy Péter P Nardai Miklós Weszl Attila Cselenyák Levente Kiss Zsombor Lacza Gábor Varga Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Transient ischemia-induced changes of excitatory amino acid carrier 1 in the ventral horn of the lumbar spinal cord in rabbits

Objectives: To examine temporal changes of EAAC1 immunoreactivity and its protein level in the spinal ventral horn after transient ischemia in the rabbit to investigate the correlation between neuronal cell death and EAAC1 in the ventral horn of spinal cord.Methods: White rabbits weighing 2.5–3.0 kg were anesthetized with a mixture of 2.5% isoflurane in 30% oxygen and 70% nitrous oxide, and the abdominal aortic artery below the left renal artery was occluded for 15 minutes. At designated times after reperfusion, the immunohistochemical and Western blot analysis for EAAC1 was conducted using tissues of the seventh lumbar spinal segment.Results: EAAC1 immunoreactivity was detected in the neurons of the normal spinal cord. EAAC1 immunoreactivity and protein level reduced significantly 30 minutes after ischemia/reperfusion, but EAAC1 immunoreactivity and protein level again increased by 80% versus sham 3 hours after ischemia. At this time point, neurological defect in hindlimb was also detected. Thereafter, EAAC1 immunoreactivity and protein levels remained to be attenuated in the ventral horn of spinal cord until 48 hours after ischemia.Conclusion: The significant change in EAAC1 expression and motor defects at early time after transient spinal cord ischemia relates to the acute events following ischemia/reperfusion. These results indicate that EAAC1 has an important role in the modulation of glutamate homeostasis in ischemic neurons in the spinal ventral horn.

Cellular distribution of glutamate transporters in the gastrointestinal tract of mice. An immunohistochemical and in situ hybridization approach

L-Glutamate transport by intestinal epithelial cells is an initial step of the entire glutamate metabolism pathway in the gut mucosa. The present study examined the cellular distribution of glutamate transporters in the digestive tract of adult mice using immunohistochemistry and in situ hybridization technique. Expression of EAAC1 mRNA was more intense in the ileum, where the epithelium in crypts and the basal half of intestinal villi showed high levels of transcripts, suggesting an essential role of EAAC1 in differentiating or premature epithelial cells. Electron-microscopically, EAAC1 immunoreactivity was predominantly localized in the striated border of enterocytes. Immunoreactivity for GLT-1 was found in the lateral membrane of epithelial cells at the bottom of gastric glands and at the intestinal crypts, and also in the lateral membrane of secretory cells at the duodenal gland. GLAST immunoreactivity was restricted to the fundic and pyloric glands, and was especially intense in the neck portion of both glands. However, in situ hybridization analysis failed to confirm the expression of GLT-1 and GLAST at the mRNA level, possibly due to limited sensitivity. The strong and specific luminal localization of EAAC1 in the intestinal epithelium suggests that EAAC1 is a predominant transporter of glutamate, at least in the lower part of the small intestine.

Glutamate transporters alterations in the reorganizing dentate gyrus are associated with progressive seizure activity in chronic epileptic rats.

The expression of glial and neuronal glutamate transporter proteins was investigated in the hippocampal region at different time points after electrically induced status epilepticus (SE) in the rat. This experimental rat model for mesial temporal lobe epilepsy is characterized by cell loss, gliosis, synaptic reorganization, and chronic seizures after a latent period. Despite extensive gliosis, immunocytochemistry revealed only an up-regulation of both glial transporters localized at the outer aspect of the inner molecular layer (iml) in chronic epileptic rats. The neuronal EAAC1 transporter was increased in many somata of individual CA1-3 neurons and granule cells that had survived after SE; this up-regulation was still present in the chronic epileptic phase. In contrast, a permanent decrease of EAAC1 immunoreactivity was observed in the iml of the dentate gyrus. This permanent decrease in EAAC1 expression, which was only observed in rats that experienced progressive spontaneous seizure activity, could lead to abnormal glutamate levels in the iml once new abnormal glutamatergic synaptic contacts are formed by means of sprouted mossy fibers. Considering the steady growth of reorganizing mossy fibers in the iml, the absence of a glutamate reuptake mechanism in this region could contribute to progression of spontaneous seizure activity, which occurs with a similar time course.

Modulation of glutamate transporters (GLAST, GLT-1 and EAAC1) in the rat cerebellum following portocaval anastomosis

Glutamate transporters have the important function of removing glutamate released from synapses and keeping extracellular glutamate concentrations below excitotoxic levels. Extracellular glutamate increases in portocaval anastomosis (PCA), so we used a portacaval anastomosis model in rats to analyze the expression of glutamate transporters (GLAST, GLT-1 and EAAC1) in rat cerebellum, 1 and 6 months after PCA, using immunohistochemical methods. In controls, EAAC1 immunoreactivity in Purkinje cells and glial GLAST and GLT-1 immunoreactivities in the molecular layer (ML) increased from young to old rats. One month after PCA, Purkinje cell bodies were not immunostained for neuronal EAAC1 glutamate transporter, whereas glial glutamate transporter expressions (GLAST and GLT-1) were decreased when compared to young controls. In rats with long-term PCA (6 months post-PCA), neuronal and glial glutamate transporter expressions were increased. The expression of the neuronal glutamate transporter EAAC1 was less intense than old controls, whereas glial glutamate transporters (GLAST and GLT-1) increased more than their controls. Since the level of the neuronal glutamate transporter (EAAC1) in long-term PCA did not reach that of the controls, GLAST and GLT-1 glutamate transporters seemed to be required to ensure the glutamate uptake in this type of encephalopathy. EAAC1 immunoreactivity also was expressed by Bergmann glial processes in long-term PCA, but this increase did not suffice to reverse the alterations caused at the early stage. The present findings provide evidence that transitory alteration of glutamate transporter expressions could be a significant factor in the accumulation of excess glutamate in the extracellular space in PCA, which probably makes Purkinje cells more vulnerable to glutamate effect.

Platelet-derived Growth Factor Rapidly Increases Activity and Cell Surface Expression of the EAAC1 Subtype of Glutamate Transporter through Activation of Phosphatidylinositol 3-Kinase

Na(+)-dependent glutamate transporters are the primary mechanism for removal of excitatory amino acids (EAAs) from the extracellular space of the central nervous system and influence both physiologic and pathologic effects of these compounds. Recent evidence suggests that the activity and cell surface expression of a neuronal subtype of glutamate transporter, EAAC1, are rapidly increased by direct activation of protein kinase C and are decreased by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K). We hypothesized that this regulation could be analogous to insulin-induced stimulation of the GLUT4 subtype of glucose transporter, which is dependent upon activation of PI3-K. Using C6 glioma, a cell line that endogenously and selectively expresses EAAC1, we report that platelet-derived growth factor (PDGF) increased Na(+)-dependent L-[(3)H]-glutamate transport activity within 30 min. This effect of PDGF was not due to a change in total cellular EAAC1 immunoreactivity but was instead correlated with an increase cell surface expression of EAAC1, as measured using a membrane impermeant biotinylation reagent combined with Western blotting. A decrease in nonbiotinylated intracellular EAAC1 was also observed. These studies suggest that PDGF causes a redistribution of EAAC1 from an intracellular compartment to the cell surface. These effects of PDGF were accompanied by a 35-fold increase in PI3-K activity and were blocked by the PI3-K inhibitors, wortmannin and LY 294002, but not by an inhibitor of protein kinase C. Other growth factors, including insulin, nerve growth factor, and epidermal growth factor had no effect on glutamate transport nor did they increase PI3-K activity. These studies suggest that, as is observed for insulin-mediated translocation of GLUT4, EAAC1 cell surface expression can be rapidly increased by PDGF through activation of PI3-K. It is possible that this PDGF-mediated increase in EAAC1 activity may contribute to the previously demonstrated neuroprotective effects of PDGF.

Regional and cellular expression of glial (GLT1) and neuronal (EAAC1) glutamate transporter proteins in ovine fetal brain

Glutamate transport is a primary mechanism for regulating extracellular levels of glutamate which can have either neurotrophic or neurotoxic effects in the developing brain, depending on its concentration. Using immunoblotting and immunocytochemistry, we tested the hypotheses that expression of neuronal and glial glutamate transporter proteins was regionally and temporally regulated in the developing ovine brain and that expression of the glial isoform early in development was not cell-type specific. Immunoblots for the neuronal glutamate transporter EAAC1 revealed a major band of immunoreactivity at 69,000 mol. wt, whereas glial glutamate transporter-1 (GLT1) immunoreactivity was observed as 73,000 and 146,000 mol. wt proteins. EAAC1 and GLT1 are regulated differently during development, with EAAC1 immunoreactivity being most abundant at 60 and 71 days completed gestation (term=145 days) and dissipating thereafter, while GLT1 immunoreactivity was most abundant at 136 days gestation. By immunocytochemistry EAAC1 expression is neuronal throughout gestation with intense labelling of dendrites within the telencephalon evident at 60 days. Neuropil, neuronal cell bodies and processes are EAAC1-immunoreactive throughout gestation with no evidence of astrocytic or oligodendroglial immunoreactivity. In contrast, GLT1 is expressed by neuronal and non-neuronal cell types during midgestation with astrocyte selectivity developing by 136 days. During midgestation, GLT1 is transiently expressed in neurons of the subplate, cranial nerve nuclei, basal ganglia, and cerebellar cortex. The major finding of this study, that GLT1 is transiently expressed in various neuronal populations at midgestation demonstrates that the cell-type specificity of the GLT1 phenotype is developmentally regulated and depends on brain maturity.

Rapid Communication The glutamate transporter, GLT-1, is expressed in cultured hippocampal neurons

There are multiple subtypes of Na + -dependent glutamate transporters. Several studies suggest that EAAC1 and EAAT4 are expressed in neurons, while GLT-1 and GLAST expression is thought to be restricted to glia. In the present study, expression of GLT-1 and EAAC1 was examined in cultured rat hippocampal neurons using single cell mRNA amplification and immu- nocytochemistry with subtype specific antibodies. GLT-1 and EAAC1 mRNAs were observed in all neurons examined. Neuronal phenotype was confirmed in these cells by expression of neurofilament (NF-L) mRNA and absence of glial fibrillary acidic protein (GFAP) mRNA. EAAC1 immunoreactivity was observed in essentially all cells which expressed neuron specific enolase (NSE) and GLT-1 immunoreactivity was detected in the majority (approximately 90%) of NSE-positive cells. Consistent with the glial expression of GLT-1, GLT-1 immunoreactivity was also observed in NSE-negative cells. These studies provide evidence that GLT-1 expression is not intrinsically restricted to glial cells, but can occur in neurons under certain circumstances.

Spinal cord GLT-1 glutamate transporter and blood glutamic acid alterations in motor neuron degeneration (Mnd) mice

This study characterizes for the first time neurochemical mechanisms in Mnd mice, initially described as a model of motor neuron disease and more recently proposed as a model for neuronal ceroid lipofuscinosis. A selective decrease (−30%) of [ 3H]glutamate uptake was found in spinal cord but not cortical synaptosomes of Mnd mice aged 28 weeks, when they show histopathological alterations, complete blindness and moderate neurological deficits. In spite of the widespread presence of stored material in neurons in many brain regions and spinal cord, the active transport of [ 3H]serotonin, [ 3H]dopamine and depolarization-induced [ 3H]serotonin release were not affected. Spinal EAAC1 glutamate transporter protein was significantly decreased in some but not all aged mice by 36% on average, possibly due to the loss of motor neurons. GLT-1 immunoreactivity was reduced by 34% in 28-week-old Mnd mice, while GLAST immunoreactivity was not affected. In Mnd mice aged 14 weeks, when there was no apparent alteration of motor function, the defect in the glial transporter protein GLT-1 was similar to that in 28-week-old mice (25%). Blood glutamic acid concentration was increased in Mnd mice aged 14–22 weeks. We suggest that the early decrease of GLT-1 protein might raise the extrasynaptic glutamic acid concentration, and contribute to the loss of motor neurons in affected mice, resulting in low [ 3H]glutamate uptake, low EAAC1 immunoreactivity and neurological deficits.

EAAC1, a high-affinity glutamate tranporter, is localized to astrocytes and gabaergic neurons besides pyramidal cells in the rat cerebral cortex.

High-affinity uptake of glutamate from the synaptic cleft plays a crucial role in regulating neuronal activity in physiological and pathological conditions. We have used affinity-purified specific polyclonal antibodies raised against a synthetic peptide corresponding to the C-terminal region of rabbit and rat EAAC1, a glutamate (Glu) transporter believed to be exclusively neuronal, to investigate its cellular and subcellular localization and whether it is expressed exclusively in glutamatergic cells of infragranular layers, as suggested by previous studies. Light microscopic studies revealed that EAAC1 immunoreactivity (ir) is localized to neurons and punctate elements in the neuropil. EAAC1-positive neurons were more numerous in layers II-III and V-VI, i.e. throughout all projection layers. Most EAAC1-positive neurons were pyramidal, although nonpyramidal cells were also observed. Some EAAC1-positive non-pyramidal neurons stained positively with an antiserum to GAD, thus demonstrating that EAAC1 is not confined to glutamatergic neurons. Non-neuronal EAAC1-positive cells were also observed in the white matter, and some of them stained positively with an antiserum to GFAP. Ultrastructural studies showed that EAAC1-ir was in neuronal cell bodies, dendrites and dendritic spines, but not in axon terminals, i.e. exclusively postsynaptic. Analysis of the type of axon terminals synapsing on EAAC1-ir profiles showed that 97% of them formed asymmetric contacts, thus indicating that EAAC1 is located at the very sites of excitatory amino acid release. Unexpectedly, EAAC1-ir was also found in a few astrocytic processes located in both the gray and the white matter. The localization of EAAC1 may explain the pathological symptoms that follow EAAC knockout (seizures and mild toxicity), as seizures could be due to the loss of EAAC1-mediated fine regulation of neuronal excitability at axodendritic and axospinous synapses, whereas the mild toxicity may be related to the functional inactivation of astrocytic EAAC1.

Immunohistochemical localization of the neuron-specific glutamate transporter EAAC1 (EAAT3) in rat brain and spinal cord revealed by a novel monoclonal antibody

Neuronal regulation of glutamate homeostasis is mediated by high-affinity sodium-dependent and highly hydrophobic plasma membrane glycoproteins which maintain low levels of glutamate at central synapses. To further elucidate the molecular mechanisms that regulate glutamate metabolism and glutamate flux at central synapses, a monoclonal antibody was produced to a synthetic peptide corresponding to amino acid residues 161–177 of the deduced sequence of the human neuron-specific glutamate transporter III (EAAC1). Immunoblot analysis of human and rat brain total homogenates and isolated synaptosomes from frontal cortex revealed that the antibody immunoreacted with a protein band of apparent M r ∼70 kDa. Deglycosylation of immunoprecipitates obtained using the monoclonal antibody yielded a protein with a lower apparent M r (∼65 kDa). These results are consistent with the molecular size of the human EAAC1 predicted from the cloned cDNA. Analysis of the transfected COS-1 cells by immunocytochemistry confirmed that the monoclonal antibody is specific for the neuron-specific glutamate transporter. Immunocytochemical studies of rat cerebral cortex, hippocampus, cerebellum, substantia nigra and spinal cord revealed intense labeling of neuronal somata, dendrites, fine-caliber fibers and puncta. Double-label immunofluorescence using antibody to glial fibrillary acidic protein as a marker for astrocytes demonstrated that astrocytes were not co-labeled for EAAC1. The localization of EAAC1 immunoreactivity in dendrites and particularly in cell somata suggests that this transporter may function in the regulation of other aspects of glutamate metabolism in addition to terminating the action of synaptically released glutamate at central synapses.

Non-synaptic localization of the glutamate transporter EAAC1 in cultured hippocampal neurons.

It has been postulated for several years that the high affinity neuronal glutamate uptake system plays a role in clearing glutamate from the synaptic cleft. Four different glutamate transporter subtypes are now identified, the major neuronal one being EAAC1. To be a good candidate for the reuptake of glutamate at the synaptic cleft, EAAC1 should be properly located at synapses, either at pre- or postsynaptic sites. We have investigated the distribution of EAAC1 in primary cultures of hippocampal neurons, which represent an advantageous model for the study of synaptogenesis and synaptic specializations. We have demonstrated that EAAC1 immunoreactivity is segregated in the somatodendritic compartment of fully differentiated hippocampal neurons, where it is localized in the dendritic shaft and in the spine neck, outside the area facing the active zone. No co-localization of EAAC1 immunoreactivity with the stainings produced by typical presynaptic and postsynaptic markers was ever observed, indicating that EAAC1 is not to be considered a synaptic protein. Accordingly, the developmental pattern of expression of EAAC1 was found to be different from that of typical synaptic markers. Moreover, EAAC1 was expressed in the somatodendritic compartment of hippocampal neurons already at stages preceding the formation of synaptic contacts, and was also expressed in GABAergic interneurons with identical subcellular distribution. Taken together, these data rule against a possible role for EAAC1 in the clearance of glutamate from within the cleft and in the regulation of its time in the synapse. They suggest an unconventional non-synaptic function of this high-affinity glutamate carrier, not restricted to glutamatergic fibres.

Differential Abundance of Glutamate Transporter Subtypes in Amyotrophic Lateral Sclerosis (ALS)-Vulnerable versus ALS-Resistant Brain Stem Motor Cell Groups

Previous studies have suggested that defective high-affinity glutamate uptake, due mainly to a major loss of the astroglial-specific GLT-1 glutamate transporter, underlies the selective motoneuron degeneration observed in sporadic ALS (24, 28). If a defect in glutamate transport underlies the pathogenesis of sporadic ALS, the glutamate transporter subtype found to be lost in sporadic ALS should be present in abundance in the affected motor nuclei under normal conditions. To investigate this, we used immunohistochemical methods to analyze the localization of two subtypes of high-affinity glutamate transporters in the cranial motor nuclei of normal monkey brain stem: GLT-1, localized to astroglia; and EAAC1, localized to neurons. Our results indicated that all motor cell groups of monkey brain stem are rich in the GLT-1 glutamate transporter, which is localized to astroglial cells and processes that surround and envelop motoneuron cell bodies and dendrites. Image analysis indicated that the abundance of GLT-1 immunoreactive astroglial elements in ALS-vulnerable motor cell groups (i.e., the trigeminal, facial, and hypoglossal motor cell groups) is higher than in ALS-resistant motor cell groups (i.e., the oculomotor, trochlear, and abducens motor cell groups), and statistical analysis showed that this difference is significant. Our results also indicated that both ALS-vulnerable and ALS-resistant motor cell groups of monkey brain stem are relatively poor in EAAC1 immunoreactivity. Therefore, in the case of a loss in the GLT-1 glutamate transporter in sporadic ALS, glutamate may increase in the vicinity of motoneurons in all brain-stem motor cell groups, but especially in the ALS-vulnerable motor cell groups, which are normally richer in GLT-1. Increased extracellular glutamate could lead to excess entry of Ca2+into motoneurons via glutamate-gated or voltage-activated Ca2+channels and produce degeneration of those motoneurons unable to resist the insult. Since motoneurons in the ALS-resistant motor cell groups of the brain stem are enriched in the Ca2+buffering protein parvalbumin, they should be better able to resist the damage than the majority of motoneurons in the ALS-vulnerable motor cell groups, which lack parvalbumin (20).

Fimbria-fornix transections selectively down-regulate subtypes of glutamate transporter and glutamate receptor proteins in septum and hippocampus.

The effects of CNS axotomy on glutamate transporter and glutamate receptor expression were evaluated in adult rats following unilateral fimbria-fornix transections. The septum and hippocampus were collected at 3, 7, 14, and 30 days postlesion. Homogenates were immunoblotted by using antibodies directed against glutamate transporters (GLT-1, GLAST, and EAAC1) and glutamate receptors (GluR1, GluR2/3, GluR6/7, and NMDAR1), and they were assayed for glutamate transport by D-[3H]aspartate binding. GLT-1 was decreased at 7 and 14 days postlesion within the ipsilateral septum and at 7 days postlesion in the hippocampus. GLAST was decreased within the ipsilateral septum and hippocampus at 7 and 14 days postlesion. No postlesion alterations in EAAC1 immunoreactivity were observed. D-[3H]Aspartate binding was decreased at 7, 14, and 30 days postlesion within the ipsilateral septum and 14 days postlesion in the hippocampus. GluR2/3 expression was down-regulated at 30 days postlesion within the ipsilateral septum, whereas GluR1, GluR6/7, and NMDAR1 immunoreactivity was unchanged. In addition, no alterations in glutamate receptor expression were detected within hippocampal homogenates. This study demonstrates a selective down-regulation of primarily glial, and not neuronal, glutamate transporters and a delayed, subtype-specific down-regulation of septal GluR2/3 receptor expression after regional deafferentation within the CNS.

Regional deafferentation down-regulates subtypes of glutamate transporter proteins.

Low extracellular glutamate content is maintained primarily by high-affinity sodium-dependent glutamate transport. Three glutamate transporter proteins have been cloned: GLT-1 and GLAST are astroglial, whereas EAAC1 is neuronal. The effects of axotomy on glutamate transporter expression was evaluated in adult rats following unilateral fimbria-fornix and corticostriatal lesions. The hippocampus and striatum were collected at 3, 7, 14, and 30 days postlesion. Homogenates were immunoblotted using antibodies directed against GLT-1, GLAST, EAAC1, and glial fibrillary acidic protein and assayed for glutamate transport by D-[3H]aspartate binding. GLT-1 immunoreactivity was decreased within the ipsilateral hippocampus and striatum at 14 days postlesion. GLAST immunoreactivity was decreased within the ipsilateral hippocampus and striatum at 7 and 14 days postlesion. No alterations in EAAC1 immunoreactivity were observed. D-[3H]Aspartate binding was decreased at 14 days postlesion within the ipsilateral hippocampus and at 7 and 14 days postlesion within the ipsilateral striatum. By 30 days postlesion, glutamate transporters and D-[3H]aspartate binding returned to control levels. This study demonstrates the down-regulation of primarily glial, and not neuronal, glutamate transporters following regional disconnection.