GluR1 mRNA Levels Research Articles

Protein kinase C epsilon delays latency until anoxic depolarization through arc expression and GluR2 internalization

Global cerebral ischemia is a debilitating injury that damages the CA1 region of the hippocampus, an area important for learning and memory. Protein kinase C epsilon (PKCɛ) activation is a critical component of many neuroprotective treatments. The ability of PKCɛ activation to regulate AMPA receptors (AMPARs) remains unexplored despite the role of AMPARs in excitotoxicity after brain ischemia. We determined that PKCɛ activation increased expression of a protein linked to learning and memory, activity-regulated cytoskeleton-associated protein (arc). Also, arc is necessary for neuroprotection and confers protection through decreasing AMPAR currents via GluR2 internalization. In vivo, activation of PKCɛ increased arc expression through a BDNF/TrkB pathway, and decreased GluR2 mRNA levels. In hippocampal cultured slices, PKCɛ activation decreased AMPAR current amplitudes in an arc- and GluR2-dependent manner. Additionally, PKCɛ activation triggered an arc- and GluR2 internalization-dependent delay in latency until anoxic depolarization. Inhibiting arc also blocked PKCɛ-mediated neuroprotection against lethal oxygen and glucose deprivation. These data characterize a novel PKCɛ-dependent mechanism that for the first time defines a role for arc and AMPAR internalization in conferring neuroprotection.

Distinct amygdalar AMPAergic/GABAergic mechanisms promote anxiolitic-like effects in an unpredictable stress model of the hamster.

Studies have pointed to both α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) antagonists and GABA(A) receptor (GABA(A)R) agonists as potent antistress agents. In this work, separate subchronic injections of the AMPAR antagonist, 6-ciano-7-nitroquinoxaline-2,3-dione (CNQX), and α1 GABA(A)R subunit agonist (Zol) within the central amygdala nucleus modified the elevated plus maze performances of hamsters exposed randomly to one of the following stressful conditions: food/water deprivation, forced swimming test, and permanence in cold room. Indeed, stressed hamsters treated with CNQX or Zol displayed a very great (p < 0.001) increase of entrance plus a moderate (p < 0.05) time spent into open arms, respectively. At the cellular level, Zol-treated animals supplied a moderately evident argyrophilic reaction (indicative of neurodegeneration) in the hippocampus while it was absent in the hypothalamus. Interestingly, this reaction was significantly reduced by CNQX supporting its preferential protective role. Furthermore, both agents were responsible for a mixed expression pattern of GluR1 and GluR2 mRNA levels in which Zol overall upregulated GluR1 mRNAs, while they were downregulated by CNQX in the hippocampal oriens-pyramidalis layer and in layer III of the cerebral cortex. These findings support the amygdalar AMPAergic protective response against anxiety states in chronically stressed hamsters, which may constitute useful therapeutic strategies for panic-related mood disorders.

AMPA receptors mediate passive avoidance deficits induced by sleep deprivation

The present study addressed the effects of sleep deprivation (SD) on AMPA receptor (AMPAR) binding in brain regions associated with learning and memory, and investigated whether treatment with drugs acting on AMPAR could prevent passive avoidance deficits in sleep deprived animals. [3H]AMPA binding and GluR1 in situ hybridization signals were quantified in different brain regions of male Wistar rats either immediately after 96h of sleep deprivation or after 24h of sleep recovery following 96h of sleep deprivation. Another group of animals were sleep deprived and then treated with either the AMPAR potentiator, aniracetam (25, 50 and 100mg/kg, acute administration) or the AMPAR antagonist GYKI-52466 (5 and 10mg/kg, acute and chronic administration) before passive avoidance training. Task performance was evaluated 2h and 24h after training. A significant reduction in [3H]AMPA binding was found in the hippocampal formation of SD animals, while no alterations were observed in GluR1 mRNA levels. The highest dose of aniracetam (100mg/kg) reverted SD-induced impairment of passive avoidance performance in both retention tests, whereas GYKI-52466 treatment had no effect. Pharmacological enhancement of AMPAR function may revert hippocampal-dependent learning impairments produced after SD. We argue that such effects might be associated with reduced AMPAR binding in the hippocampus of sleep deprived animals.

Effects of the chinese traditional prescription xiaoyaosan decoction on chronic immobilization stress-induced changes in behavior and ultrastructure in rat hippocampus.

Xiaoyaosan (XYS) decoction has been widely used as a traditional medicine for treating stress and depression-related disorders in China for thousands of years. Aim of the Study. To observe the potential mechanism of XYS decoction's antidepressant-like effect in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors related to synaptic plasticity in the hippocampus rats induced by chronic immobilization stress (CIS). Materials and Methods. Animals were randomly divided into five groups: (1) control group; (2) sham-operated group; (3) CIS group, in which rats were conducted CIS for 21 days; (4) XYS decoction treatment group; (5) 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) positive group, in which the amygdala of CIS rats was unilaterally microinjected with a competitive glutamate receptor antagonist, CNQX. After CIS for 21 days, the open field test (OPT) and elevated plus-maze test (EPM) were measured, the ultrastructure of hippocampus CA1 subregion was observed by the electron microscopy; both the GluR1 and GluR2 mRNA level of AMPA receptor subunits in hippocampus CA1 subregion were detected by real-time qPCR. Results. Rats subjected to CIS exhibited increases in time in central zone and decreases in total distance traveled in the OPT. In the EPM, they also showed decreases in center zone time and entries, open arm time and entries, and an increase in close arm time. Ultrastructural damage in the hippocampus CA1 was also observed. XYS decoction and CNQX showed significant improvement behavioral changes and the ultrastructural damage of the hippocampus CA1; XYS decoction also reversed CIS-induced decreases in GluR2 mRNA and increases in GluR1 mRNA in the hippocampus CA1 as well as CNQX. Conclusions. XYS decoction may effectively produce an antidepressant-like effect, which appears to be involved AMPA receptors related synaptic plasticity of hippocampus.

Chromatin structure and expression of the AMPA receptor subunit Glur2 in human glioma cells: Major regulatory role of REST and Sp1

It has been suggested that reduced glutamate receptor expression protects glioma cells from glutamate toxicity. GluR2 is the critical subunit of the GluR2 subtype of AMPA glutamate receptors as this subunit determines the Ca(2+) permeability of the receptor. The gene encoding the GluR2 subtype of AMPA receptors has been described as a target gene for the transcription repressor REST. However, we recently showed that the GluR2 gene is not regulated by REST in several neuronal and neuroendocrine cell lines, due to a repressive chromatin environment. Here, we show that the GluR2 gene has an open chromatin configuration in human glioma cells. Overexpression of REST reduced GluR2 mRNA levels while shRNA-mediated depletion of REST or expression of a REST mutant, that contained a transcriptional activation domain, enhanced GluR2 expression. Incubation with trichostatin A (TSA), a histone deacetylase inhibitor, induced acetylation of histone 4 of the GluR2 locus in glioma cells, leading to an upregulation of GluR2 expression. Together, these data suggest that REST is responsible for the reduced expression of GluR2 in glioma cells. The transcription factor Sp1 additionally binds under physiological conditions to the GluR2 gene in human glioma cells and expression of a dominant-negative mutant of Sp1 reduced expression of GluR2. Thus, the regulation via Sp1 represents a further control point for GluR2 expression in glioma cells. Together, we show that the GluR2 gene is embedded into an open chromatin configuration in glioma cells and expression of GluR2 is controlled by REST and Sp1.

Exposure of neurons to excitotoxic levels of glutamate induces cleavage of the RNA editing enzyme, adenosine deaminase acting on RNA 2, and loss of GLUR2 editing

AMPA receptors are glutamate receptors that are tetramers of various combinations of GluR1-4 subunits. AMPA receptors containing GluR1, 3 and 4 are Ca2+ permeable, however, AMPA receptors containing even a single subunit of GluR2 are Ca2+ impermeable. Most AMPA receptors are Ca2+ impermeable due to the presence of GluR2. GluR2 confers special properties on AMPA receptors through the presence of arginine at the pore apex; other subunits (GluR1, 3, 4) contain glutamine at the pore apex and allow Ca2+ influx. Normally, an RNA editing step changes DNA-encoded glutamine to arginine, introduces arginine in the GluR2 pore apex. GluR2 RNA editing is carried out by an RNA-dependent adenosine deaminase (ADAR2). Loss of GluR2 editing leads to the formation of highly excitotoxic AMPA channels [Mahajan and Ziff (2007) Mol Cell Neurosci 35:470-481] and is shown to contribute to loss of motor neurons in amyotrophic lateral sclerosis (ALS). Relatively higher levels of Ca2+-permeable AMPA receptors are found in motor neurons and this has been correlated with lower GluR2 mRNA levels. However, the reason for loss of GluR2 editing is not known. Here we show that exposure of neurons to excitotoxic levels of glutamate leads to specific cleavage of ADAR2 that leads to generation of unedited GluR2. We demonstrate that cleaved ADAR2 leads to a decrease or loss of GluR2 editing, which will further result in high Ca2+ influx and excitotoxic neuronal death.

Global ischemia-induced modifications in the expression of AMPA receptors and inflammation in rat brain

Alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPAR) and inflammatory processes have been related to ischemia-induced damage, but there are few studies addressing their response in different brain areas. Here we compare AMPAR expression after ischemia in several brain areas (hippocampus, cerebral cortex and caudate-putamen) in an attempt to correlate it with their different vulnerabilities. We found outstanding decreases in GluR1 and GluR2 mRNA levels after global ischemia and 48 h reperfusion (I/R) in all the areas studied, however, protein levels maintained in some areas such as CA3, suggesting different post-transcriptional control in different areas of the brain. To characterize the inflammatory response in these areas, we measured the mRNA levels of CD11b/CD18 membrane integrin (a reactive microglia marker), which showed an important but similar up-regulation in all brain areas studied, which was confirmed by immunohistochemistry. We conclude that the down-regulation of AMPAR gene expression following I/R does not explain differences in the vulnerability of different areas. Additionally, our data indicate that the level of inflammation is independent of the vulnerability of the different brain areas and does not explain differences in the AMPAR expression observed in the brain areas studied.

Chromatin structure and expression of the AMPA receptor subunit GluR2 in human glioma cells and the role of REST

The AMPA receptors are postsynaptic ion channels that open following stimulation with glutamate. The regulatory region of the gene encoding the GluR2 subtype of AMPA receptors contains a binding site for the transcriptional repressor REST. The cell-type specific microenvironment, in particular the cell type-specific structure of the chromatin, is crucial for the ability of REST to control target gene transcription. Using antibodies directed against methylated lysine residues 4 or 9 (H3K4 or H3K9) of histone H3, we show that the GluR2 gene has an open chromatin configuration in human U87MG glioma cells, with nucleosomes carrying di-and trimethylated H3K4. In contrast, the GluR2 gene is embedded into a repressed chromatin environment in non-neuronal hepatoma cells and keratinocytes. Chromatin immunoprecipitation experiments revealed binding of REST and histone deacetylase-1 to the GluR2 gene under physiological conditions. While overexpression of REST reduced GluR2 mRNA levels, expression of a mutant of REST that contained a transcriptional activation domain enhanced GluR2 gene transcription in U87MG glioma cells. Treatment of the cells with the histone deacetylase inhibitor trichostatin A (TSA) induced an upregulation of GluR2 expression, indicating that the transcription of the GluR2 gene is dependent of the balance between histone acetylation and deactylation. Transcription of the GluR2 gene was also inducible by TSA following inhibition of Sp1 target gene transcription, indicating that the inhibition of Sp1 is not required for the upregulation of GluR2 expression following histone deacetylase inhibition.

TNFα siRNA reduces brain TNF and EEG delta wave activity in rats

Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine with several CNS physiological and pathophysiological actions including sleep, memory, thermal and appetite regulation. Short interfering RNAs (siRNA) targeting TNFα were incubated with cortical cell cultures and microinjected into the primary somatosensory cortex (SSctx) of rats. The TNFα siRNA treatment specifically reduced TNFα mRNA by 45% in vitro without affecting interleukin-6 or gluR1–4 mRNA levels. In vivo the TNFα siRNAα reduced TNFα mRNA, interleukin-6 mRNA and gluR1 mRNA levels compared to treatment with a scrambled control siRNA. After in vivo microinjection, the density of TNFα-immunoreactive cells in layer V of the SSctx was also reduced. Electroencephalogram (EEG) delta wave power was decreased on days 2 and 3 on the side of the brain that received the TNFα siRNA microinjection relative to the side receiving the control siRNA. These findings support the hypothesis that TNFα siRNA attenuates TNFα mRNA and TNFα protein in the rat cortex and that those reductions reduce cortical EEG delta power. Results also are consistent with the notion that TNFα is involved in CNS physiology including sleep regulation.

Selective estrogen receptor‐α but not ‐β agonist treatment modulates brain α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptors

Estradiol was previously reported to decrease brain alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-receptor-specific binding. The contributions of estrogen receptor subtypes in the estradiol modulation of AMPA receptors and its predominant subunit GluR2 are unknown. These experiments investigated whether an estrogenic receptor subtype is involved in the estradiol effect on AMPA-receptor-specific binding and GluR2 mRNA levels. Ovariectomized Sprague-Dawley rats were treated 2 days after ovariectomy for 2 weeks with 17beta-estradiol, an agonist for ERalpha 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), or an agonist for ERbeta 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) and compared with intact control rats. Uterus weights, used as aperipheral measure of estrogenic activity, were decreased after ovariectomy and increased by estradiol and PPT but not DPN treatments. In prefrontal and cingulate cortices, the striatum, and the nucleus accumbens, ovariectomy increased [3H]AMPA-specific binding compared with intact controls, which was corrected by estradiol treatment. In all these brain regions, PPT, but not DPN, mimicked the estradiol decrease of AMPA-receptor-specific binding; in the cingulate cortex, the effect of PPT did not reach statistical significance. GluR2 mRNA levels of vehicle-treated ovariectomized rats remained unchanged compared with intact rats in the brain regions investigated. Estradiol and PPT treatment but not DPN decreased GluR2 subunit mRNA levels in the prefrontal cortex and the striatum of ovariectomized rats, whereas no significant change was observed in the cingulate cortex or the nucleus accumbens. The present results suggest that an ERalpha is involved in the estradiol modulation of AMPA receptors in the cortex, striatum, and nucleus accumbens.

Transforming growth factor alpha attenuates the functional expression of AMPA receptors in cortical GABAergic neurons

In the developing neocortex, brain-derived neurotrophic factor (BDNF) exerts a trophic activity to increase the expression and channel activity of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor subunits. Here, we demonstrate that the epidermal growth factor (EGF) receptor (ErbB1) ligands exert the opposite biological activity in cultured neocortical neurons. Subchronic stimulation of ErbB1 with transforming growth factor α (TGFα), EGF, or heparin-binding EGF (HB-EGF) down-regulated protein expression of the GluR1 AMPA receptor subunit in cultured neocortical neurons. In agreement, TGFα treatment decreased the B max of [ 3H] AMPA binding and GluR1 mRNA levels. Immunocytochemistry revealed that the decrease in GluR1 was most pronounced in multipolar GABAergic neurons. To examine the physiological consequences, we recorded AMPA-evoked currents as well as miniature excitatory postsynaptic currents in morphologically identified putative GABAergic neurons in culture. Subchronic TGFα treatment decreased AMPA-triggered currents as well as the amplitude and frequency of miniature excitatory postsynaptic currents. An ErbB1 tyrosine kinase inhibitor, PD153035, inhibited the TGFα effect. Moreover, TGFα counteracted the neurotrophic activity of BDNF on AMPA receptor expression. Co-application of TGFα with BDNF blocked the BDNF-triggered up-regulation of AMPA receptor expression and currents. These observations reveal a negative regulatory activity of the ErbB1 ligand, TGFα, which reduces the input sensitivity of cortical GABAergic neurons to attenuate their inhibitory function.

Ischemia-induced changes of AMPA-type glutamate receptor subunit expression pattern in the rat retina: a real-time quantitative PCR study.

To investigate whether the previously observed decrease in immunoreactivity of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor subunits GluR1, -2, -3, and -4 after ischemia-reperfusion in the rat retina is associated with changes at the mRNA expression level. Furthermore, to study possible changes in the ratios of alternative splice variants of GluR2 and -4 and possible changes in the subunit composition of the receptor complex after ischemia-reperfusion. The ischemia-induced changes were related to expression levels of immediate early genes, c-fos and c-jun, and to expression levels of different cell-type-specific transcripts. A 60-minute ischemic event was induced unilaterally in the rat eye by cannulating the anterior chamber and raising the intraocular pressure. Reperfusion was allowed to occur for 2 hours up to 28 days. Total RNA was isolated from the retinas and transcript levels were assessed by real-time quantitative PCR (qPCR). A differential decrease was observed in the expression levels of all AMPA-type GluR subunits 2 hours after ischemia-reperfusion, with a significant downregulation of GluR2 and -3 transcript levels. At the long-term (72 hours-4 weeks), expression levels for all four subunits were decreased by approximately 64%. No changes were observed, either in the expression ratio of GluR2 and -4 splice variants, or in the relative expression of the different subunits. Immediate early genes c-fos and c-jun were transiently upregulated. Expression levels of the ganglion-cell-specific transcripts Thy-1 and neurofilament and of the AII-amacrine-specific transcript parvalbumin decreased after ischemia-reperfusion, whereas the ON bipolar cell transcripts mGluR6 and PKCalpha did not show ischemia-induced changes. Shortly after ischemia-reperfusion immunolabeling of GluR1, -2/3, and -4 is strongly decreased, whereas the corresponding mRNA levels are not affected, indicating degradation at the protein level. In contrast, the GluR2 mRNA level is reduced, whereas immunostaining is not yet affected, suggesting that the GluR2 protein is relatively stable under postischemia conditions. The long-term decrease in mRNA levels of all AMPA-type GluR subunits suggests that ischemia affects a main component of the excitatory retinal neurotransmission. It remains to be investigated whether these changes contribute to the subsequent neurodegeneration.

Neuronal activity regulates protein and gene expressions of GluR2 in postnatal rat visual cortical neurons in culture.

Ionotropic glutamate receptors, the principal excitatory neurotransmitter receptors in the CNS, are classified into NMDA and non-NMDA subtypes. Previously, we found a direct relationship between neuronal activity and NMDA receptor subunit 1 in rat primary neuronal cultures and monkey visual cortex. The present study focused on the relationship between neuronal activity and subunit 2 of AMPA glutamate receptor, GluR2. GluR2 controls Ca(2+) permeability of AMPA receptors, and the transcription of its gene is activated by nuclear respiratory factor 1, which also activates the transcription of a few subunit genes of cytochrome oxidase (CO). Primary neuronal cultures of postnatal rat visual cortex were subjected to impulse blockade with tetrodotoxin (TTX) for 6 days, or 20 mM KCl depolarizing treatment for 1, 2, 5, 10, 20, 30, and 40 hrs. After 20 hrs of KCl treatment, GluR2 immunoreactivity and CO activity were significantly increased above controls (P < 0.01), and both remained high at 30 and 40 hrs of treatment. However, GluR2 mRNA level as shown by in situ hybridization was already up-regulated above controls after 10 hrs of KCl treatment (P < 0.01) and remained elevated with longer periods of depolarization. TTX blockade, on the other hand, induced a significant down-regulation of GluR2 immunoreactivity, GluR2 gene expression as well as CO activity (P < 0.01 for all). Our results indicate that both protein and mRNA expressions of GluR2 in cultured visual cortical neurons are tightly controlled by neuronal activity.

Acute and long-term changes in the mesolimbic dopamine pathway after systemic or local single nicotine injections.

We have examined several neurochemical and behavioural parameters related to the function of the mesolimbic dopamine (DA) pathway in animals treated with nicotine following three modes of drug administration, i.e. systemic intraperitoneal injection, intra-accumbens (Acb) infusion or intraventral tegmental area (intra-VTA) microinjection. The present modes of systemic, intra-Acb and intra-VTA nicotine administration elicited comparable acute increases in dialysate DA levels from the Acb. The increase in extracellular DA levels was paralleled by a significant enhancement of locomotion in a habituated environment in the case of systemic or intra-VTA nicotine administration, whereas unilateral or bilateral intra-Acb nicotine infusion was ineffective, showing that accumbal DA increase is not sufficient to elicit locomotion in this experimental paradigm. Intra-VTA, but not systemic or intra-Acb, nicotine administration caused a long-term (at least 24-h) increase in basal dialysate DA levels from the Acb. In addition, significant increases in tyrosine hydroxylase (TH) and GluR1 (but not dopamine transporter or NR1) mRNA levels in the VTA were detected 24 h after intra-VTA nicotine administration. Systemic nicotine injection caused only an increase in TH mRNA levels while intra-Acb infusion did not modify any of the mRNAs tested. The long-term increase in basal DA levels in the Acb and TH, and GluR1 mRNA levels in the VTA upon intra-VTA nicotine microinjection indicates that even a single nicotine injection can induce plastic changes of the mesolimbic DA pathway.

Repeated Administration of Amphetamine or Cocaine Does Not Alter AMPA Receptor Subunit Expression in the Rat Midbrain

We previously reported that ventral tegmental area (VTA) dopamine neurons are supersensitive to AMPA when recorded three days after discontinuing repeated amphetamine or cocaine administration. By increasing dopamine cell activity, this may contribute to the induction of behavioral sensitization. The goal of this study was to determine if increased sensitivity to AMPA reflects increased AMPA receptor expression in the midbrain. Immunolabeling for GluR1, GluR2, GluR2/3, and GluR4 was quantified by immunohistochemistry with 35S-labeled secondary antibodies in VTA, substantia nigra, and a transitional area. First, rats were treated for five days with saline or amphetamine (5 mg/kg) and killed three or 14 days after the last injection. No significant changes in immunolabeling were observed for any subunit at either withdrawal time. GluR1 immunolabeling was further examined in rats killed 16–18 hrs or 24 hrs after a single injection of amphetamine or repeated injections of saline, amphetamine (5 mg/kg × 5 days) or cocaine (20 mg/kg × 7 days). No significant differences were observed in any region. Finally, neither repeated amphetamine or cocaine administration significantly altered GluR1 mRNA levels as quantified by reverse transcriptase-polymerase chain reaction. Our results suggest that enhanced responsiveness of VTA dopamine neurons to AMPA after withdrawal from repeated stimulant administration involves mechanisms more complex than increased expression of AMPA receptor subunits.

Differential expression of glutamate and GABA-A receptor subunit mRNA in cortical dysplasia

Focal cortical dysplasia is characterized by disorganized cortical lamination, dysplastic and heterotopic neurons, and an association with epilepsy. The contribution that dysplastic and heterotopic neurons make to epileptogenesis in focal cortical dysplasia is unknown and the phenotype of these cells may be distinct. The authors hypothesized that the expression of genes encoding glutamatergic (glutamate [GluR] and N-methyl-D-aspartate NMDA receptors [NR]) and gamma-aminobutyric acid A receptor (GABA(A)R) subunits is distinct in dysplastic and heterotopic neurons and that changes in receptor gene expression could be defined in a cell-specific pattern. Single immunohistochemically labeled dysplastic and heterotopic neurons were microdissected from human focal cortical dysplasia specimens obtained during epilepsy surgery. Pyramidal neurons were microdissected from postmortem control cortex and from temporal cortex without dysplasia resected during temporal lobectomy. Poly (A) messenger RNA (mRNA) from single neurons was amplified, radiolabeled, and used to probe complementary DNA (cDNA) arrays containing GluR(1-6), NR(1A,1B), NR(2A-D), and GABA(A)Ralpha(1-6), and -Rbeta(1-3) subunit cDNAS: The relative hybridization intensities of each mRNA-cDNA hybrid were quantified by phosphorimaging. GluR, NR, and GABA(A)R subunit mRNA expression did not differ between control neurons and nondysplastic epilepsy specimens. Expression of GluR(4), NR(2B), and NR(2C) subunit mRNA was increased, and NR(2A) and GABA(A)Rbeta(1) subunit mRNA was decreased in dysplastic compared with pyramidal and heterotopic neurons. In contrast, GABA(A)Ralpha(1), -Ralpha(2), and -Rbeta(2) as well as GluR(1) mRNA levels were reduced in both dysplastic and heterotopic neurons. Differential expression of GluR, NR, and GABA(A)R mRNA in dysplastic and heterotopic neurons demonstrates cell specific gene transcription changes in focal cortical dysplasia. These results suggest that dysplastic and heterotopic neurons may be pharmacologically distinct and make differential contributions epileptogenesis in focal cortical dysplasia.

Gene expression of two glutamate receptor subunits in response to repeated stress exposure in rat hippocampus.

1. Glutamatergic mechanisms are thought to be involved in stress-induced alterations of brain function, especially in the hippocampus. We have hypothesized that repeated stress exposure may evoke changes of hippocampal glutamate receptors at the level of gene expression. 2. The study was designed to analyze the levels of mRNA coding for NMDAR1, the essential subunit of the N-methyl-D-aspartate (NMDA) receptor subtype, and for GluR1, an AMPA glutamate receptor subunit, after repeated immobilization stress in rat hippocampus. Toward this aim, we applied a competitive RT-PCR technique which allowed precise and reliable quantification of the transcripts. 3. We found that repeated immobilization stress for 7 days significantly increased GluR1 mRNA levels, by 27% (P<0.01), as measured 24 hr after the last stress exposure. Levels of mRNA coding for NMDAR1 were slightly elevated, but the difference failed to be significant. 4. These results demonstrate selective changes in the gene expression of glutamate receptor subunits, which are likely to take part in the mechanisms leading to enhanced excitability and vulnerability of hippocampal neurons and to potential damage during repeated or chronic stress exposure.

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

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

Hippocampal AMPA and NMDA mRNA levels correlate with aberrant fascia dentata mossy fiber sprouting in the pilocarpine model of spontaneous limbic epilepsy.

There is considerable controversy whether aberrant fascia dentata (FD) mossy fiber sprouting is an epiphenomena related to neuronal loss or a pathologic abnormality responsible for spontaneous limbic seizures. If mossy fiber sprouting contributes to seizures, then reorganized axon circuits should alter postsynaptic glutamate receptor properties. In the pilocarpine-status rat model, this study determined if changes in alpha amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and n-methyl-D-aspartic acid (NMDA) receptor subunit mRNA levels correlated with mossy fiber sprouting. Sprague-Dawley rats were injected with pilocarpine (320 mg/kg; i.p.) and maintained in status epilepticus for 6 to 8 hours (pilocarpine-status). Rats were killed during the: (1) latent phase after neuronal loss but before spontaneous limbic seizures (day 11 poststatus; n = 7); (2) early seizure phase after their first seizures (day 25; n = 7); and (3) chronic seizure phase after many seizures (day 85; n = 9). Hippocampi were studied for neuron counts, inner molecular layer (IML) neo-Timm's staining, and GluR1-3 and NMDAR1-2b mRNA levels. Compared with controls, pilocarpine-status rats in the: (1) latent phase showed increased FD GluR3, NMDAR1, and NMDAR2b; greater CA4 and CA1 NMDAR1; and decreased subiculum GluR1 hybridization densities; (2) early seizure phase showed increased FD GluR3, increased CA1 NMDAR1, and decreased subiculum NMDAR2b densities; and (3) chronic seizure phase showed increased FD GluR2; increased FD and CA4 GluR3; decreased CA1 GluR2; and decreased subiculum GluR1, GluR2, NMDAR1, and NMDAR2b levels. In multivariate analyses, greater IML neo-Timm's staining: (1) positively correlated with FD GluR3 and NMDAR1 and (2) negatively correlated with CA1 and subiculum GluR1 and GluR2 mRNA levels. These results indicate that: (1) hippocampal AMPA and NMDA receptor subunit mRNA levels changed as rats progressed from the latent to chronic seizure phase and (2) certain subunit alterations correlated with mossy fiber sprouting. Our findings support the hypothesis that aberrant axon circuitry alters postsynaptic hippocampal glutamate receptor subunit stoichiometry; this may contribute to limbic epileptogenesis.

Excitatory Amino Acid Receptors and Puberty

Glutamate is an important excitatory signal in the hypothalamus for the steroid-mediated preovulatory gonadotropin surge. Steroids may exert this action by regulating glutamate receptor levels or glutamate release, or both. Work in our laboratory found no changes in NMDA and kainate receptor binding in the hypothalamus of castrated or castrated plus steroid-replaced male and female rats. Likewise, we found that NMDA and kainate binding did not change over the onset of puberty in the female rat. A competitive quantitative RT-PCR assay using exogenous internal standards was used to measure NMDAR1, GluR1, and β-actin mRNAs levels. NMDAR1 and GluR1 expression was examined in the preoptic hypothalamic area and in the medial basal hypothalamus at Postnatal Days 10, 15, 20, 25, 30, 32, 34, 36, 40, and 63. A transient increase in GluR1 mRNA levels in the preoptic hypothalamic area was observed on Day 20, with all other time points showing comparable levels. NMDAR1 levels in the POA and medial basal hypothalamus did not change significantly at any of the time points; in contrast, however, AMPA receptor binding levels were increased in the hypothalamus at the time of puberty in the female rat. Thus, in addition to the previously reported elevation of glutamate release rates in the hypothalamus at the time of puberty, AMPA receptors may also be elevated and play a role in mediating glutamate regulatory effects on the timing of puberty in the female rat.