AMPA Receptors are Coupled to the Nitric Oxide/Cyclic GMP Pathway in Cerebellar Astroglial Cells

In cultured rat cerebellar astroglia kainate induces cGMP formation with low potency (EC₅₀ 310 μM). In the presence of cyclothiazide, a blocker of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor desensitization, the effect of kainate was potentiated and glutamate and AMPA elicited large increases (>100-fold) in cGMP levels. The response to all three agonists was abolished by the nitric oxide synthase inhibitor N^G-nitro-L-arginine and required extracellular calcium. Uptake of Co²⁺ was induced by AMPA in a limited population of astroglial cells and this effect was potentiated by cyclothiazide. These results indicate that calcium-permeable AMPA receptors mediate stimulation of nitric oxide formation in cerebellar astroglia. This effect may be relevant for glutamate-dependent synaptic plasticity processes in the cerebellum.     

KCC2 activity is critical in limiting the onset and severity of status epilepticus

The K⁺/Cl^– cotransporter (KCC2) allows adult neurons to maintain low intracellular Cl^– levels, which are a prerequisite for efficient synaptic inhibition upon activation of γ-aminobutyric acid receptors. Deficits in KCC2 activity are implicated in epileptogenesis, but how increased neuronal activity leads to transporter inactivation is ill defined. In vitro, the activity of KCC2 is potentiated via phosphorylation of serine 940 (S940). Here we have examined the role this putative regulatory process plays in determining KCC2 activity during status epilepticus (SE) using knockin mice in which S940 is mutated to an alanine (S940A). In wild-type mice, SE induced by kainate resulted in dephosphorylation of S940 and KCC2 internalization. S940A homozygotes were viable and exhibited comparable basal levels of KCC2 expression and activity relative to WT mice. However, exposure of S940A mice to kainate induced lethality within 30 min of kainate injection and subsequent entrance into SE. We assessed the effect of the S940A mutation in cultured hippocampal neurons to explore the mechanisms underlying this phenotype. Under basal conditions, the mutation had no effect on neuronal Cl^– extrusion. However, a selective deficit in KCC2 activity was seen in S940A neurons upon transient exposure to glutamate. Significantly, whereas the effects of glutamate on KCC2 function could be ameliorated in WT neurons with agents that enhance S940 phosphorylation, this positive modulation was lost in S940A neurons. Collectively our results suggest that phosphorylation of S940 plays a critical role in potentiating KCC2 activity to limit the development of SE.Fast synaptic inhibition in the adult brain is largely mediated via the activation of γ-aminobutyric acid receptors (GABA_ARs). The ability of neurons to maintain low intracellular Cl^– is dependent upon the activity of the K⁺/Cl^– cotransporter KCC2. KCC2 expression in the rodent brain is developmentally regulated with low levels evident before birth that then dramatically increase from postnatal day 7 (P7) onwards and correlate with the appearance of hyperpolarizing GABA_AR currents (1). KCC2 null mice die shortly after birth, exhibit high levels of intracellular Cl^– and anomalous excitatory actions of GABA and glycine, highlighting the vital role of KCC2 (2). In addition to regulating Cl^– transport, KCC2 exhibits transporter-independent properties that affect glutamate receptors and dendritic spines (3–6).Status epilepticus (SE) is a state of continuous seizures that is highly lethal and leads to long-term neurological deficits in survivors. A key feature of SE is impaired GABAergic inhibition (7) that manifests clinically as resistance to the GABA_A modulator diazepam (8). The prevailing hypothesis of impaired GABAergic signaling during SE is displacement of GABA_A receptors from the synapse and a reduction in the number of receptors on the cell surface (9). However, an additional component that could contribute to the compromised inhibition during SE is a buildup of intracellular Cl^– due to ionic plasticity and deficits in KCC2 function. KCC2 dysfunction is thought to contribute to traumatic brain injury, neuropathic pain, and acute stress (10). Consistent with this, deficits in the activity and expression levels of KCC2 develop rapidly in animal models of SE and persist after its termination (11–13).Recent evidence indicates that the transport activity and membrane trafficking of KCC2 are modulated by protein kinase C (PKC)-dependent phosphorylation of residue S940 within its cytoplasmic domain. S940 phosphorylation leads to increased KCC2 transport activity as well as increased accumulation on the plasma membrane due to a reduction in endocytosis (14, 15). Using phospho-specific antibodies we demonstrate that SE induced by the chemoconvulsant kainate results in rapid S940 dephosphorylation. Furthermore, genetically ablating S940 phosphorylation accelerates the development and lethality of SE. Thus, our results provide to our knowledge the first direct evidence that deficits in KCC2 activity directly contribute to the pathophysiology of SE.     

Modulation of dendritic release of dopamine by metabotropic glutamate receptors in rat substantia nigra

A superfusion system was used to study the effects of metabotropic glutamate receptor (mGluR) ligands upon the release of [(3)H]dopamine ([(3)H]DA) previously taken up by rat substantia nigra (SN) slices. trans-(+/-)-1-Amino-(1S,3R)-cyclopentane dicarboxylic acid (trans-ACPD; 100 and 600 microM), a group I and II mGluR agonist, evoked the release of [(3)H]DA from nigral slices. This last effect was reduced significantly by (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)-glycine (MCCG; 300 microM), an antagonist of group II mGluR, or by the addition of tetrodotoxin (D-APV; 1 microM) to the superfusion medium. D-(-)-2-Amino-5-phosphono-valeric acid (100 microM), an N-methyl-D-aspartate receptor antagonist, or the presence of Mg(2+) (1.2mM) in the superfusion medium did not modify trans-ACPD-induced [(3)H]DA release. In addition, a group II mGluR agonist such as (2S,1'R,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)-glycine (DCG-IV; 100 microM) significantly induced the release of [(3)H]DA from nigral slices, whereas a group I mGluR agonist such as (RS)-3,5-dihydroxyphenylglycine (DHPG; 50 and 100 microM) did not modify the release of the [(3)H]-amine. Further experiments showed that the NMDA (100 microM)-evoked release of [(3)H]DA was decreased significantly by prior exposure of SN slices to trans-ACPD. Finally, partial denervation of the DA nigro-striatal pathway with 6-hydroxydopamine (6-OH-DA) increased trans-ACPD-induced release of [(3)H]DA, whereas it decreased trans-ACPD inhibitory effects on NMDA-evoked release of [(3)H]DA from nigral slices. The present results suggest that the dendritic release of DA in the SN is regulated by mGluR activation. Such nigral mGluR activation may produce opposite effects upon basal and NMDA-evoked release of DA in the SN. In addition, such mGluR-induced effects in the SN are modified in response to partial denervation of the DA nigro-striatal pathway.     

Adult rat optic nerve oligodendrocyte progenitor cells express a distinct repertoire of voltage- and ligand-gated ion channels

Cultured oligodendrocyte progenitor cells derived from the developing central nervous system (CNS) express a pattern of ion channels that is distinct from mature oligodendrocytes and other cell types of the CNS. In the present study, we used the whole-cell patch-clamp technique and the fura-2-based Ca⁺⁺ imaging system to study the ion channel expression of oligodendrocyte progenitor cells derived from the optic nerves of adult rats. We found that the adult oligodendrocyte progenitor cell membrane is dominated by K⁺ currents, both delayed outward and inward rectifying. The inwardly rectifying K⁺ currents were often as large as the outward delayed rectifying K⁺ currents. The delayed rectifying outward currents were partially blocked by 50 mM tetraethylammonium or 1 mM 4-aminopyridine, but not by 2 or 5 mM BaCl₂. This suggests that the delayed rectifier channels expressed by adult progenitor cells are different from those expressed by perinatal cells. Most adult oligodendrocyte progenitor cells showed no or only small A-type K⁺ currents. Both Ca⁺⁺ and Na⁺ channels were also detected in these cells. Furthermore, adult progenitor cells responded to the neurotransmitters GABA and kainate and the pharmacology of these responses indicated that these cells express GABA_A receptors and kainate receptors that are Ca⁺⁺ -permeable. Our study suggests that adult oligodendrocyte progenitor cells are electrophysiologically distinct and that these cells share electrophysiological characteristics with both perinatal progenitor cells and immature oligodendrocytes. © 1995 Wiley-Liss, Inc.     

Selective excitotoxic pathology in the rat hippocampus

The pattern of cell loss and neuronal degeneration resulting from multiple microinjections of N -methyl-D-aspartate (NMDA), ibotenate (IBO), quisqualate (QUIS), and kainate (KA) into hippocampus was studied, together with the protection provided by the NMDA antagonist 3-(±)-2-carboxypiperazin-4-yl-propyl-l-phosphonate (CPP). Histological evaluation was carried out after 7 days of survival. NMDA and IBO resulted in an extensive loss of all cells in the hippocampus including dentate gyrus, hilar cells, and CA3-CA1 pyramidal cells, but there was an absence of damage to areas and structures outside hippocampus. After QUIS and KA injections the hippocampal damage was limited to hilar cells in the dentate gyrus, CA3 pyramidal cells, and partial loss of CA1 cells; there was extensive extrahippocampal damage including entorhinal cortex, amygdala, layers III, V, and VI of ventral neocortex, olfactory areas, and various thalamic nuclei. CPP provided almost complete protection from the effects of intrahippocampal injections of NMDA and IBO, but did not affect the hippocampal cell loss found after QUIS and KA (with the exception of minor protection of some CA1 cells). CPP protected most extrahippocampal sites from the damage resulting from QUIS and KA, indicating that such excitotoxic cell death is indirect and involves NMDA receptor activation by an endogenous agent. The use of multiple microinjections as opposed to single injections allows a clearer interpretation of selective excitotoxic vulnerability.     

Nimodipine improves kainic acid induced neurotoxicity in cerebellar granular cell culture: a double-blind dose-response study

Summary— The neuroprotective role of nimodipine was tested in kainic acid (50 and 100 μM) induced neurotoxicity in cerebellar granular cell cultures of 4 to 7 day-old rat pups. Nimodipine was applied in 50, 100 and 200 μM concentrations. Kainate, in either dose, induced cerebellar granular cell death in respect to controls and the results were statistically significant ( P = 0.000 for both doses). However, kainic acid in 100 μM concentration led to higher rates of cell death than 50 μM ( P = 0.017). The neuroprotective role of nimodipine in kainate induced neurotoxicity was dose dependent. Kainate toxicity in 50 μM concentration was blocked by 50 and 100 μM nimodipine concentrations ( P = 0.006 and P = 0.002, respectively) while 200 μM nimodipine was found ineffective. The most effective nimodipine dose for 100 μM kainic acid neurotoxicity was 200 μM ( P = 0.000) while 50 and 100 μM concentrations of nimodipine were found ineffective. In this study, we have proven the dose-dependent neuroprotective role of nimodipine in kainate induced neurotoxicity in cerebellar granular cell cultures of rat pups.     

Activation of Class II or III Metabotropic Glutamate Receptors Protects Cultured Cortical Neurons Against Excitotoxic Degeneration

Trans-1-aminocyclopentane-1,3-dicarboxylic acid, a mixed agonist of all metabotropic glutamate receptor (mGluR) subtypes, is known to produce either neurotoxic or neuroprotective effects. We have therefore hypothesized that individual mGluR subtypes differentially affect neurodegenerative processes. Selective agonists of subtypes which belong to mGluR class II or III, such as (2_s, 1′_R,2′_R,3′_R)-2-(2,3-dicarboxycyclopropyl)-glycine (DCG-IV) (specific for subtypes mGluR2 or 3) or L-2-amino-4-phosphonobutanoate and L-serine-O-phosphate (specific for subtypes mGluR4, 6 or 7), were highly potent and efficacious in protecting cultured cortical neurons against toxicity induced by either a transient exposure to N-methyl-D-aspartate (NMDA) or a prolonged exposure to kainate. In contrast, agonists that preferentially activate class I mGluR subtypes (mGluR1 or 5), such as quisqualate or trans-azetidine-2,3-dicarboxylic acid, were inactive. DCG-IV was still neuroprotective when applied to cultures after the toxic pulse with NMDA. This delayed rescue effect was associated with a reduction in the release of endogenous glutamate, a process that contributes to the maturation of neuronal damage. We conclude that agonists of class II or III mGluRs are of potential interest in the experimental therapy of acute or chronic neurodegenerative disorders.     

Do kainate-lesioned hippocampi become epileptogenic?

Kainic acid lesions of hippocampal subfields CA3–CA4 produced dramatic synchronous afterdischarge activity in subfield CA1 when studied 2–4 weeks post-lesion in the in vitro slice preparation. This epileptiform discharge was correlated with a loss of intrinsic firing-induced afterhyperpolarizations and synaptic IPSPs. Two to 4 months post-lesion, intrinsic afterpotentials and synaptic inhibition appeared normal in most cells studied.     

l-[H]Glutamate binds to kainate-, NMDA- and AMPA-sensitive binding sites: an autoradiographic analysis

The anatomical distribution ofl-[³H]glutamate binding sites was determined in the presence of various glutamate analogues using quantitative autoradiography. The binding ofl-[³H]glutamate is accounted for the presence of 3 distinct binding sites when measured in the absence of Ca²⁺, Cl⁻ and Na⁺ ions. The anatomical distribution and pharmacological specificity of these binding sites correspond to that reported for the 3 excitatory amino acid binding sites selectively labeled byd-[³H]2-amino-5-phosphonopentanoate (d-[³H]AP5), [³H]kainate ([³H]KA) and [³H]α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([³H]AMPA) which are thought to be selective ligands for the N-methyl-d-aspartate (NMDA), KA and quisqualate (QA) receptors, respectively.