Early continuous white noise exposure alters l‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid receptor subunit glutamate receptor 2 and γ‐aminobutyric acid type a receptor subunit β3 protein expression in rat auditory cortex

Auditory experience during the postnatal critical period is essential for the normal maturation of auditory function. Previous studies have shown that rearing infant rat pups under conditions of continuous moderate‐level noise delayed the emergence of adult‐like topographic representational order and the refinement of response selectivity in the primary auditory cortex (A1) beyond normal developmental benchmarks and indefinitely blocked the closure of a brief, critical‐period window. To gain insight into the molecular mechanisms of these physiological changes after noise rearing, we studied expression of the AMPA receptor subunit GluR2 and GABA_A receptor subunit β3 in the auditory cortex after noise rearing. Our results show that continuous moderate‐level noise rearing during the early stages of development decreases the expression levels of GluR2 and GABA_Aβ3. Furthermore, noise rearing also induced a significant decrease in the level of GABA_A receptors relative to AMPA receptors. However, in adult rats, noise rearing did not have significant effects on GluR2 and GABA_Aβ3 expression or the ratio between the two units. These changes could have a role in the cellular mechanisms involved in the delayed maturation of auditory receptive field structure and topographic organization of A1 after noise rearing. © 2009 Wiley‐Liss, Inc.     

Insulin treatment restores glutamate (α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid) receptor function in the hippocampus of diabetic rats

Type 1 diabetes is associated with cognitive dysfunction. Cognitive processing, particularly memory acquisition, depends on the regulated enhancement of expression and function of glutamate receptor subtypes in the hippocampus. Impairment of memory was been detected in rodent models of type 1 diabetes induced by streptozotocin (STZ). This study examines the functional properties of synaptic α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors and the expression of synaptic molecules that regulate glutamatergic synaptic transmission in the hippocampus of STZ‐diabetic rats. The AMPA receptor‐mediated miniature excitatory postsynaptic currents (mEPSCs) and single‐channel properties of synaptosomal AMPA receptors were examined after 4 weeks of diabetes induction. Results show that amplitude and frequency of mEPSCs recorded from CA1 pyramidal neurons were decreased in diabetic rats. In addition, the single‐channel properties of synaptic AMPA receptors from diabetic rat hippocampi were different from those of controls. These impairments in synaptic currents gated by AMPA receptors were accompanied by decreased protein levels of AMPA receptor subunit GluR1, the presynaptic protein synaptophysin, and the postsynaptic anchor protein postsynaptic density protein 95 in the hippocampus of diabetic rats. Neural cell adhesion molecule (NCAM), an extracellular matrix molecule abundantly expressed in the brain, and the polysialic acid (PSA) attached to NCAM were also downregulated in the hippocampus of diabetic rats. Insulin treatment, when initiated at the onset of diabetes induction, reduced these effects. These findings suggest that STZ‐induced diabetes may result in functional deteriorations in glutamatergic synapses in the hippocampus of rats and that these effects may be reduced by insulin treatment. © 2015 Wiley Periodicals, Inc.     

Exercise modifies α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor expression in striatopallidal neurons in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐lesioned mouse

The α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic‐acid‐type glutamate receptor (AMPAR) plays a critical role in modulating experience‐dependent neuroplasticity, and alterations in AMPAR expression may underlie synaptic dysfunction and disease pathophysiology. Using the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mouse model of dopamine (DA) depletion, our previous work showed exercise increases total GluA2 subunit expression and the contribution of GluA2‐containing channels in MPTP mice. The purpose of this study was to determine whether exercise‐dependent changes in AMPAR expression after MPTP are specific to the striatopallidal (D₂R) or striatonigral (D₁R) medium spiny neuron (MSN) striatal projection pathways. Drd₂‐eGFP‐BAC transgenic mice were used to delineate differences in AMPAR expression between striatal D₂R‐MSNs and D₁R‐MSNs. Striatal AMPAR expression was assessed by immunohistochemical (IHC) staining, Western immunoblotting (WB) of preparations enriched for postsynaptic density (PSD), and alterations in the current–voltage relationship of MSNs. We found DA depletion results in the emergence of GluA2‐lacking AMPARs selectively in striatopallidal D₂R‐MSNs and that exercise reverses this effect in MPTP mice. Exercise‐induced changes in AMPAR channels observed after DA depletion were associated with alterations in GluA1 and GluA2 subunit expression in postsynaptic protein, D₂R‐MSN cell surface expression, and restoration of corticostriatal plasticity. Mechanisms regulating experience‐dependent changes in AMPAR expression may provide innovative therapeutic targets to increase the efficacy of treatments for basal ganglia disorders, including Parkinson's disease. © 2013 Wiley Periodicals, Inc.     

Expression of estrogen receptor α and β in reactive astrocytes at the male rat hippocampus after status epilepticus

Estrogen is neuroprotective against status epilepticus (SE)‐induced hippocampal damage in female animals. In male animals, estrogen is converted from testosterone via aromatization the activity of which is upregulated by brain damage. However, it is controversial whether estrogen is neuroprotective or neuroinvasive against male hippocampal damage after SE. In order to understand the role of estrogen, it is important to elucidate the distribution manner of estrogen receptor (ER)α and β as the targets of estrogen. In this study, we examined the time course changes of ERs in adult male rat hippocampus after SE using anti‐ERα antibodies (MC‐20 and PA1‐309) and anti‐ERβ antibodies (PA1‐310B and PA1‐311). In control rats, both ERα and β were expressed in the pyramidal cells predominantly at CA1 and CA3. ERα was expressed in the cytoplasm and the nucleus, whereas ERβ was expressed in the cytoplasm of the pyramidal cells. After SE, according to the pyramidal cell loss at CA1, the number of ERα‐ and β‐immunoreactive pyramidal cells decreased up to day 21. On the other hand, reactive astrocytes, which newly appeared after SE and formed gliosis at CA1, were confirmed to express both ERs in the nucleus, cytoplasm, and process. There were no differences in immunoreactivity between antibodies. Our results indicate that endogenous estrogen affects the pyramidal cells through ERα and β under normal circumstances in adult male rats, whereas the targets of estrogen shift to the reactive astrocytes through ERα and β after SE.     

Non‐classical mechanism of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor channel block by fluoxetine

Antidepressants have many targets in the central nervous system. A growing body of data demonstrates the influence of antidepressants on glutamatergic neurotransmission. In the present work, we studied the inhibition of native Ca²⁺‐permeable and Ca²⁺‐impermeable α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors in rat brain neurons by fluoxetine. The Ca²⁺‐impermeable AMPA receptors in CA1 hippocampal pyramidal neurons were weakly affected. The IC₅₀ value for the inhibition of Ca²⁺‐permeable AMPA receptors in giant striatal interneurons was 43 ± 7 μm . The inhibition of Ca²⁺‐permeable AMPA receptors was voltage dependent, suggesting deep binding in the pore. However, the use dependence of fluoxetine action differed markedly from that of classical AMPA receptor open‐channel blockers. Moreover, fluoxetine did not compete with other channel blockers. In contrast to fluoxetine, its membrane‐impermeant quaternary analog demonstrated all of the features of channel inhibition typical for open‐channel blockers. It is suggested that fluoxetine reaches the binding site through a hydrophobic access pathway. Such a mechanism of block is described for ligands of sodium and calcium channels, but was never found in AMPA receptors. Molecular modeling suggests binding of fluoxetine in the subunit interface; analogous binding was proposed for local anesthetics in closed sodium channels and for benzothiazepines in calcium channels.     

α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazoleproprionic acid receptor activation protects against phencyclidine‐induced caspase‐3 activity by activating voltage‐gated calcium channels

Phencyclidine (PCP) is a noncompetitive, open channel blocker of the N‐methyl‐D‐aspartate (NMDA) receptor–ion channel complex. When administered to immature animals, it is known to cause apoptotic neurodegeneration in several regions, and this is followed by olanzapine‐sensitive, schizophrenia‐like behaviors in late adolescence and adulthood. Clarification of its mechanism of action could yield data that would help to inform the treatment of schizophrenia. In our initial experiments, we found that α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazoleproprionic acid (AMPA) inhibited PCP‐induced apoptosis in organotypic neonatal rat brain slices in a concentration‐dependent and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione‐sensitive manner. Calcium signaling pathways are widely implicated in apoptosis, and PCP prevents calcium influx through NMDA receptor channels. We therefore hypothesized that AMPA could protect against this effect by activation of voltage‐dependent calcium channels (VDCCs). In support of this hypothesis, pretreatment with the calcium channel blocker cadmium chloride eliminated AMPA‐mediated protection against PCP. Furthermore, the L‐type VDCC inhibitor nifedipine (10 µM) fully abrogated the effects of AMPA, suggesting that L‐type VDCCs are required for AMPA‐mediated protection against PCP‐induced neurotoxicity. Whereas the P/Q‐type inhibitor ω‐agatoxin TK (200 nM) reduced AMPA protection by 51.7%, the N‐type VDCC inhibitor ω‐conotoxin (2 µM) had no effect. Decreased AMPA‐mediated protection following cotreatment with K252a, a TrkB inhibitor, suggests that brain‐derived neurotrophic factor signaling plays an important role. By analogy, these results suggest that activation of L‐type, and to a lesser extent P/Q‐type, VDCCs might be advantageous in treating conditions associated with diminished NMDAergic activity during early development. © 2014 Wiley Periodicals, Inc.     

Perampanel,a selective,noncompetitive α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor antagonist,as adjunctive therapy for refractory partial‐onset seizures: Interim results from phase III,extension study 307

Purpose: To evaluate safety, tolerability, and seizure outcome data during long‐term treatment with once‐daily adjunctive perampanel (up to 12 mg/day) in patients with refractory partial‐onset seizures. Methods: Study 307 was an extension study for patients completing the double‐blind phase of three pivotal phase III trials (studies 304, 305, and 306). The study consisted of two phases: an open‐label treatment phase (including a 16‐week blinded conversion period and a planned 256‐week maintenance period) and a 4‐week follow‐up phase. Patients were blindly titrated during the conversion period to their individual maximum tolerated dose (maximum 12 mg/day). Adverse events (AEs) were monitored throughout the study and seizure frequency recorded. The interim data cutoff date for analyses was December 1, 2010. Key Findings: In total, 1,218 patients were enrolled in the study. At the interim cutoff date, 1,186 patients were in the safety analysis set; 1,089 (91.8%) patients had >16 weeks of exposure to perampanel, 580 (48.9%) patients had >1 year of exposure, and 19 (1.6%) patients had >2 years of exposure. At the interim analysis, 840 (70.8%) patients remained on perampanel treatment. The large majority of patients (n = 1,084 [91%]) were titrated to 10 mg or 12 mg/day. Median (range) duration of exposure was 51.4 (1.1–128.1) weeks. Treatment‐emergent AEs were reported in 87.4% of patients. The most frequent were dizziness (43.9%), somnolence (20.2%), headache (16.7%), and fatigue (12.1%). Serious AEs were reported in 13.2% of patients. In the intent‐to‐treat analysis set (n = 1,207), the frequency of all seizures decreased over the first 26 weeks of perampanel treatment in patients with at least 26 weeks of exposure to perampanel (n = 1,006 [83.3%]); this reduction was maintained in patients with at least 1 year of exposure (n = 588 [48.7%]). The overall median percent changes in seizure frequency in patients included in each 13‐week interval of perampanel treatment were ?39.2% for weeks 14–26 (n = 1,114), ?46.5% for weeks 40–52 (n = 731), and ?58.1% for weeks 92–104 (n = 59). Overall responder rates in patients included in each 13‐week interval of perampanel treatment were 41.4% for weeks 14–26 (n = 1,114), 46.9% for weeks 40–52 (n = 731), and 62.7% for weeks 92–104 (n = 59). During the blinded conversion period, the reduction in seizure frequency in patients previously randomized to placebo (?42.4%, n = 369) was similar to that in patients previously randomized to perampanel (?41.5%, n = 817). Significance: Consistent with pivotal phase III trials, these interim results demonstrated that perampanel had a favorable tolerability profile in patients with refractory partial‐onset seizures over the longer term. The decrease in seizure frequency was consistent and maintained in those patients over at least 1 year of perampanel exposure.     

Glutamate receptor 1-immunopositive neurons in the gliotic CA1 area of the mouse hippocampus after pilocarpine-induced status epilepticus

Significant reduction in glutamate receptor 1 (GluR1)- and GluR2/3-immunopositive neurons was demonstrated in the hilus of the dentate gyrus in mice killed on days 1, 7 and 60 after pilocarpine-induced status epilepticus (PISE). In addition, GluR1 and GluR2/3 immunostaining in the strata oriens, radiatum and lacunosum moleculare of areas CA1-3 decreased drastically on days 7 and 60 after PISE. Neuronal loss observed in the above regions may account, at least in part, for a decrease in GluR immunoreactivity. By contrast, many GluR1-immunopositive neurons were observed in the gliotic area of CA1. Of these, about 42.8% were immunopositive for markers for hippocampal interneurons, namely calretinin (7.6%), calbindin (12.8%) and parvalbumin (22.4%). GluR1 or GluR2/3 and BrdU double-labelling showed that the GluR1- and GluR2/3-immunopositive neurons at 60 days after PISE were neurons that had survived rather than newly generated neurons. Furthermore, anterograde tracer and double-labelling studies performed on animals at 60 days after PISE indicated a projection from the hilus of the dentate gyrus to gliotic areas in both CA3 and CA1, where the projecting fibres apparently established connections with GluR1-immunopositive neurons. The projection to CA1 was unexpected. These novel findings suggest that the intrinsic hippocampal neuronal network is altered after PISE. We speculate that GluR1-immunopositive neurons in gliotic CA1 act as a bridge between dentate gyrus and subiculum contributing towards epileptogenesis.     

Treatment responsive GABA(B)‐receptor limbic encephalitis presenting as new‐onset super‐refractory status epilepticus (NORSE) in a deployed U.S. soldier

A 23‐year‐old, previously healthy, deployed U.S. soldier presented with bilateral temporal lobe seizures recalcitrant to multiple antiepileptic drugs and anti‐seizure anaesthetic agents. He received methylprednisolone, intravenous immunoglobulins, plasma exchange, and rituximab for presumed autoimmune encephalitis before achieving seizure freedom. Six weeks after presentation, the aetiology of his refractory seizures was found to be due to autoantibodies targeting the anti‐GABA(B)‐receptor. This case is noteworthy for being the first reported case of anti‐GABA(B)‐receptor limbic encephalitis presenting with new‐onset refractory status epilepticus (NORSE), a clinical syndrome that often carries a grave prognosis and in which a treatable aetiology is often never discovered. Our case also supports testing for GABA‐receptor autoantibodies and the upfront use of multi‐modal immunotherapy in patients presenting with limbic encephalitis and new refractory seizures.     

Distinct properties of murine α5 γ‐aminobutyric acid type a receptors revealed by biochemical fractionation and mass spectroscopy

γ‐Aminobutyric acid type A receptors (GABA_ARs) that contain the α5 subunit are expressed predominantly in the hippocampus, where they regulate learning and memory processes. Unlike conventional postsynaptic receptors, GABA_ARs containing the α5 subunit (α5 GABA_ARs) are localized primarily to extrasynaptic regions of neurons, where they generate a tonic inhibitory conductance. The unique characteristics of α5 GABA_ARs have been examined with pharmacological, immunostaining, and electrophysiological techniques; however, little is known about their biochemical properties. The aim of this study was to modify existing purification and enrichment techniques to isolate α5 GABA_ARs preferentially from the mouse hippocampus and to identify the α5 subunit by using tandem mass spectroscopy (MS/MS). The results showed that the detergent solubility of the α5 subunits was distinct from that of α1 and α2 subunits, and the relative distribution of the α5 subunits in Triton X‐100‐soluble fractions was correlated with that of the extracellular protein radixin but not with that of the postsynaptic protein gephyrin. Mass spectrometry identified the α5 subunit and showed that this subunit associates with multiple α, β, and γ subunits, but most frequently the β3 subunit. Thus, the α5 subunits coassemble with similar subunits as their synaptic counterparts yet have a distinct detergent solubility profile. Mass spectroscopy now offers a method for detecting and characterizing factors that confer the unique detergent solubility and possibly cellular location of α5 GABA_ARs in hippocampal neurons. © 2009 Wiley‐Liss, Inc.     

Propofol effectively inhibits lithium-pilocarpine-induced status epilepticus in rats via downregulation of N-methyl-D-aspartate receptor 2B subunit expression

Status epilepticus was induced via intraperitoneal injection of lithium-pilocarpine.The inhibitory effects of propofol on status epilepticus in rats were judged based on observation of behavior,electroencephalography and 24-hour survival rate.Propofol(12.5-100 mg/kg) improved status epilepticus in a dose-dependent manner,and significantly reduced the number of deaths within 24 hours of lithium-pilocarpine injection.Western blot results showed that,24 hours after induction of status epilepticus,the levels of N-methyl-D-aspartate receptor 2A and 2B subunits were significantly increased in rat cerebral cortex and hippocampus.Propofol at 50 mg/kg significantly suppressed the increase in N-methyl-D-aspartate receptor 2B subunit levels,but not the increase in N-methyl-D-aspartate receptor 2A subunit levels.The results suggest that propofol can effectively inhibit status epilepticus induced by lithium-pilocarpine.This effect may be associated with downregulation of N-methyl-D-aspartate receptor 2B subunit expression after seizures.     

Circuit mechanisms of GluA1‐dependent spatial working memory

Spatial working memory (SWM), the ability to process and manipulate spatial information over a relatively short period of time, requires an intact hippocampus, but also involves other forebrain nuclei in both in rodents and humans. Previous studies in mice showed that the molecular mechanism of SWM includes activation of AMPA receptors containing the GluA1 subunit (encoded by gria1) as GluA1 deletion in the whole brain (gria1^–/–) results in strong SWM deficit. However, since these mice globally lack GluA1, the circuit mechanisms of GluA1 contribution to SWM remain unknown. In this study, by targeted expression of GluA1 containing AMPA receptors in the forebrain of gria1^–/– mice or by removing GluA1 selectively from hippocampus of mice with “floxed” GluA1 alleles (gria1^fl/fl), we show that SWM requires GluA1 action in cortical circuits but is only partially dependent on GluA1‐containing AMPA receptors in hippocampus. We further show that hippocampal GluA1 contribution to SWM is temporally restricted and becomes prominent at longer retention intervals (≥30 s). These findings provide a novel insight into the neural circuits required for SWM processing and argue that AMPA mediated signaling across forebrain and hippocampus differentially contribute to encoding of SWM. © 2013 Wiley Periodicals, Inc.     

Acute down-regulation of adenosine A(1) receptor activity in status epilepticus

Purpose: Status epilepticus (SE) remains a potentially devastating condition that quickly becomes refractory to antiepileptic drug treatment and arises as a result of a failure of the brain’s endogenous antiepileptic mechanisms. Understanding these mechanisms and how they are disrupted in SE is necessary in order to identify novel therapeutic approaches. Adenosine is considered an endogenous anticonvulsant. Extracellular concentrations increase coinciding with seizure termination; activation of A₁ receptors (A₁Rs) reduces seizure‐induced damage and epileptiform activity. The present study examines the effectiveness of focal drug delivery in a model of limbic SE that closely resembles the human condition and describes, for the first time, alterations in A₁R signaling during prolonged seizures that may contribute to the progression from self‐terminating seizures to self‐sustaining SE (SSSE). Methods: We developed a rat perforant path stimulation model in which 50% of rats develop SSSE and tested whether modulation of A₁Rs influenced SSSE development when drugs were infused to the dentate gyrus. We further determined the ability of A₁Rs to modulate perforant path to granule cell transmission in hippocampal slices taken from sham‐operated control and post‐SE animals. Key Findings: Adenosine (3 μm ) and the A₁R‐selective agonist 2‐chloro‐N⁶‐cyclopentyladenosine (CCPA; 10 μm ) reduced the severity of SSSE as measured by spike count, electroencephalography power and behavioral seizure score. In addition, CCPA suppressed the progression to SSSE. Surprisingly, the A₁R‐selective antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX; 1 μm ) had no effect on the severity of or progression to SSSE, suggesting a lack of intrinsic A₁R activation. Immunohistochemistry revealed no alterations in total A₁R expression. However, we observed a marked down‐regulation of A₁R modulation of neurotransmission in vitro, indicating acute A₁R desensitization. Significance: These findings indicate that A₁R activation can prevent the progression to SE and suggest that reduced A₁R signaling promotes the transition of seizures to SSSE.     

Antisense oligonucleotide to GABAA receptor γ2 subunit induces limbic status epilepticus

γ-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the brain. A deficiency of GABAergic inhibition mediated via the GABA_A receptor complex has for a long time been suspected to be a central factor in epileptogenesis. Status epilepticus is a condition of sustained and prolonged excitation of neuronal circuits, as detected by epileptiform discharges in the electroencephalogram (EEG). Reduction of GABA_A receptor-mediated hippocampal inhibition has been implicated in the development of status epilepticus. The present study provides direct evidence of a link between the GABA_A receptor and epilepsy. We show that selective inhibition of the expression of the GABA_A receptor γ2 subunit in the rat hippocampus by means of antisense oligonucleotides leads to spontaneous electrographic seizures that evolve into profound limbic status epilepticus, ultimately resulting in severe neurodegenerative changes. Concurrent treatment with diazepam prevents the development of status epilepticus and markedly reduces neuronal cell loss. These findings strongly support the hypothesis that the GABA_A receptor is critically involved in the pathogenesis of seizures and status epilepticus. J. Neurosci. Res. 54:863–869, 1998. © 1998 Wiley-Liss, Inc.