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Mutations in doublecortin (DCX) are associated with intractable epilepsy in humans, due to a severe disorganization of the neocortex and hippocampus known as classical lissencephaly. However, the basis of the epilepsy in lissencephaly remains unclear. To address potential functional redundancy with murin Dcx, we targeted one of the closest homologues, doublecortin-like kinase 2 (Dclk2). Here, we report that Dcx; Dclk2-null mice display frequent spontaneous seizures that originate in the hippocampus, with most animals dying in the first few months of life. Elevated hippocampal expression of c-fos and loss of somatostatin-positive interneurons were identified, both known to correlate with epilepsy. Dcx and Dclk2 are coexpressed in developing hippocampus, and, in their absence, there is dosage-dependent disrupted hippocampal lamination associated with a cell-autonomous simplification of pyramidal dendritic arborizations leading to reduced inhibitory synaptic tone. These data suggest that hippocampal dysmaturation and insufficient receptive field for inhibitory input may underlie the epilepsy in lissencephaly, and suggest potential therapeutic strategies for controlling epilepsy in these patients. epilepsy | receptive field | pyramidal neuron | dendrites | delamination

Presynaptic ionotropic glutamate receptors are emerging as key players in the regulation of synaptic transmission. Here we identify GluR7, a kainate receptor (KAR) subunit with no known function in the brain, as an essential subunit of presynaptic autoreceptors that facilitate hippocampal mossy fiber synaptic transmission. [GluR7.sup.-/-] mice display markedly reduced short- and long-term synaptic potentiation. Our data suggest that presynaptic KARs are GluR6/ GluR7 heteromers that coassemble and are localized within synapses. We show that recombinant GluR6/GluR7 KARs exhibit low sensitivity to glutamate, and we provide evidence that presynaptic KARs at mossy fiber synapses are likely activated by high concentrations of glutamate, Overall, from our data, we propose a model whereby presynaptic KARs are localized in the presynaptic active zone close to release sites, display low affinity for glutamate, are likely [Ca.sup.2+]-permeable, are activated by single release events, and operate within a short time window to facilitate the subsequent release of glutamate. kainate receptors | presynaptic glutamate receptors | short-term plasticity | synaptic plasticity

We report the crystal structure of the glycosylated ligand-binding ($1S2) domain of the kainate receptor subunit GluR6, in complex with the agonist domoate. The structure shows the expected overall homology with AMPA and NMDA receptor subunit structures but reveals an unexpected binding mode for the side chain of domoate, in which contact is made to the larger lobe only (lobe I). In common with the AMPA receptor subunit GluR2, the GluR6 S1S2 domain associates as a dimer, with many of the interdimer contacts being conserved. Subtle differences in these contacts provide a structural explanation for why GluR2 L483Y and GluR3 L507Y are nondesensitizing, but GluR6, which has a tyrosine at that site, is not. The structure incorporates native glycosylation, which has not previously been described for ionotropic glutamate receptors. The position of the sugars near the subunit interface rules out their direct involvement in subunit association but leaves open the possibility of indirect modulation. Finally, we observed several tetrameric assemblies that satisfy topological constraints with respect to connection to the receptor pore, and which are therefore candidates for the native quaternary structure. crystal structure | domoate | glutamate receptor

Although it is widely agreed that expression of LTP at mossy fiber synapses occurs in the presynaptic terminal (1, 2), the site of induction of this N-methyl-D-aspartate (NMDA) receptor-independent form [...], The site of induction of long-term potentiation (LTP) at mossy fiber-CA3 synapses in the hippocampus is unresolved, with data supporting both pre- and postsynaptic mechanisms. Here we report that mossy fiber LTP was reduced by perfusion of postsynaptic neurons with peptides and antibodies that interfere with binding of EphB receptor tyrosine kinases (EphRs) to the PDZ protein GRIP. Mossy fiber LTP was also reduced by extracellular application of soluble forms of B-ephrins, which are normally membrane-anchored presynaptic ligands for the EphB receptors. The application of soluble ligands for presynaptic ephrins increased basal excitatory transmission and occluded both tetanus and forskolin-induced synaptic potentiation. These findings suggest that PDZ interactions in the postsynaptic neuron and trans-synaptic interactions between postsynaptic EphB receptors and presynaptic B-ephrins are necessary for the induction of mossy fiber LTP.

The Cre/IoxP system is increasingly showing promise for investigating genes involved in neural function. Here, we demonstrate that in vivo modification of genes in the mouse brain can be accomplished in a spatial- and temporal-specific manner by targeted delivery of an adeno-associated virus (AAV) encoding a green fluorescent protein/Cre recombinase (GFP/Cre) fusion protein. By using a reporter mouse, in which Cre recombinase activates [beta]-galactosidase expression, we demonstrate long-term recombination of neurons in the hippocampus, striatum, and septum as early as 7 days after stereotaxic injection of virus. Recombined cells were observed for at least 6 months postinjection without evidence of cell loss or neural damage. AAV-mediated delivery of GFP/Cre provides a valuable approach to alter the mouse genome, as AAV delivers genes efficiently to neurons with low toxicity. This approach will greatly facilitate the study of genetic modifications in the mouse brain. gene transfer | Cre recombinase | transgenic mouse | IoxP | ROSA26

The actions of glutamate in the central nervous system are mediated through interaction with fast activating ionotropic receptors and G protein-coupled metabotropic glutamate receptors (mGluRs). Studies of these receptors have relied on the availability of agonists and antagonists selective for each receptor class. Compounds that were thought to be selective for mGluRs have been extensively used to study the role of these receptors in the brain. Their use has implicated mGluRs in a wide range of physiological and pathological processes including the modulation of N-methyl-D-aspartate (NMDA) receptors and NMDA receptor-dependent processes. We report that some of the most commonly used mGluR compounds act as antagonists on NMDA receptors at concentrations commonly used to activate or block mGluRs. In addition, several of the drugs also act as agonists at higher concentrations due at least in part to high levels of contaminant amino acids. Our results indicate that caution should be used when using these drugs to study the roles of mGluRs in various NMDA-dependent processes. The antagonist effects were dependent on the concentration of the NMDA receptor coagonists, preventing reappraisal of previously published work.

Kainate receptors assembled with the GluR6 subunit are significant for both the sensitivity of CA3 neurons to kainate and for synaptic transmission at the mossy fibre-CA3 synapse, according to research using mutant mice lacking the kainate-receptor subunit GluR6. It was established that GluR6-deficient mice are less susceptible to systemic administration of kainate. This research indicates a vital role for kainate receptors, rather than alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, in kainate-induced seizures and excitoxic neuronal death in the hippocampus.

NMDA receptors serve many functions in the developing and adult central nervous system [1]. However, activation of these receptors also can contribute to the pathophysiology of epilepsy [2] and stroke [...], The function of the N-methyl-D-aspartate (NMDA)-preferring glutamate receptor can be regulated by extracellular pH, a process that may be important during ischemia in the brain or during seizures. Protons inhibit NMDA receptor function by 50 percent at pH 7.3 through interactions with the NR1 subunit, and both polyamines and NR1 exon 5 potentiate receptor function through relief of the tonic proton inhibition present at physiological pH. A single amino acid (lysine 211) was identified that mediates the effects of exon 5 in the rat brain. Electroneutral Substitutions at this position restored pH sensitivity and, consequently, polyamine relief of tonic inhibition. This effect, together with the structural similarities between polyamines and the surface loop encoded by exon 5, suggest that exon 5 may act as a tethered pH-sensitive constitutive modulator of NMDA receptor function.

A comparative study of RNA editing of the glutamate receptor subunit GluR1-GluR4 sequences, which have a high degree of sequence identity, reveals that editing process is specific only to the GluR2 subunit RNA and an intron sequence in the GluR2 gene is responsible for the selective RNA editing. Alterations in these helix regions by mutations lead to significant changes in the editing efficiency. Deletion studies suggest that both base pairing in the exon sequence and integrity of the whole inverted repeat are essential for editing.

Rasmussen's encephalitis is a rare, progressive, catastrophic disease of unknown pathogenesis that begins in the first decade of life, affecting previously normal children[1]. The disease affects the cortex of a [...], Rasmussen's encephalitis is a progressive childhood disease of unknown cause characterized by severe epilepsy, hemiplegia, dementia, and inflammation of the brain. During efforts to raise antibodies to recombinant glutamate receptors (GluRs), behaviors typical of seizures and histopathologic features mimicking Rasmussen's encephalitis were found in two rabbits immunized with GluR3 protein. A correlation was found between the presence of Rasmussen's encephalitis and serum antibodies to GluR3 detected by protein immunoblot analysis and by immunoreactivity to transfected cells expressing GluR3. Repeated plasma exchanges in one seriously ill child transiently reduced serum titers of GluR3 antibodies, decreased seizure frequency, and improved neurologic function. Thus, GluR3 is an autoantigen in Rasmussen's encephalitis, and an autoimmune process may underlie this disease.

The kainate selective glutamate receptor GluR6 undergoes editing at a level of 70% of its mRNA transcripts. The edited versioncontains a glutamine residue at position 621, while the unedited version contains an arginine at the same postion. The Ca2+ permeability of the edited and unedited versions of the GluR6 receptor was investigated. The results showed that the edited version of the receptor exhibits lower Ca2+ permeability. The level of permeability was dependent on the level of editing, which in turn, may regulate synaptic activity.

The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate (AMPA/KA) receptortype of glutamete receptors are the primary mediators of excitatory synaptic transmission in the mammalian central nervous system. A pharmacological distinction between AMPA/KA receptor subtypes was determined using the biphenylderivative of 1,3-naphthalene disulfonic acid, Evans blue, which acted as an antagonist for some receptor subtypes, but did not affect other subtypes. The structure of Evans blue could be used as a starting point for the development of substances to reduce excitatory activity of specific brain regions.

The possibility of the involvement of the glutamate receptor (GluR) in the pathogenesis of amyotrophic lateral sclerosis (ALS) was investigated. Previous mappings of the genes coding for subunits 1 to4 of GluR had eliminated the possibility of their involvement in ALS. However, the gene coding for the GluR5 subunit was mapped to human chromosome 21q21.1-22.1. This map coordinate is concordant with the previously determined ALS locus. This finding provides evidence for the involvement of a mutated GluR5 in the pathogenesis of ALS.

The metabotropic glutamate receptors (mGluRs) comprise a second class of glutamate receptors which show variation by alternative splicing events. A splice variant of mGluR1a, termed mGluR1c, was isolated and characterized. The results showed that the the spliceevent replaced 312 C-terminal amino acids by a stretch of only 10 residues. Theparent and the variant receptors elicited different patterns of calcium releasein Xenopus oocytes. In addition, in situ hybridizations showed that mGluR1c is expressed at lower levels than other splice variants. These results show that the mGluR1 receptor can modify its function and distribution by alternative splicing.

The expression of a G-protein-coupled receptor (OR3) involved in murine olfaction was studied. Polymerase chain reaction techniques were used to isolate and amplify the OR3 sequence which was subsequently used for Southern and Northern blot analyses. The results showed that OR3 is expressed solely by a subset of olfactory cells that have a characteristic bilateral symmetry in the adult olfactory epithelium. These receptors appear to originate from the central nervous system, suggesting that they are also involved in the elaboration of specific pathways between the olfactory bulb and olfactory epithelium during development.

INFLUX OF CA THROUGH GLUTAMATE receptors (GlurR) is thought to play a key role in long-term potentiation, excitotoxic neuron damage, and epilepsy [1]. In many neurons, non-NMDA glutamate receptors have [...]

We report the cloning and characterization of rat [Alpha]10, a previously unidentified member of the nicotinic acetylcholine receptor (nAChR) subunit gene family. The protein encoded by the [Alpha]10 nAChR subunit gene is most similar to the rat [Alpha]9 nAChR, and both [Alpha]9 and [Alpha]10 subunit genes are transcribed in adult rat mechanosensory hair cells. Injection of Xenopus laevis oocytes with [Alpha]10 cRNA alone or in pairwise combinations with either [Alpha]2-[Alpha]6 or [Beta]2-[Beta]4 subunit cRNAs yielded no detectable ACh-gated currents. However, coinjection of [Alpha]9 and [Alpha]10 cRNAs resulted in the appearance of an unusual nAChR subtype. Compared with homomeric [Alpha]9 channels, the [Alpha]9[Alpha]10 nAChR subtype displays faster and more extensive agonist-mediated desensitization, a distinct currentvoltage relationship, and a biphasic response to changes in extracellular [Ca.sup.2+] ions. The pharmacological profiles of homomeric [Alpha]9 and heteromeric [Alpha]9[Alpha]10 nAChRs are essentially indistinguishable and closely resemble those reported for endogenous cholinergic eceptors found in vertebrate hair cells. Our data suggest that efferent modulation of hair cell function occurs, at least in part, through heteromeric nAChRs assembled from both [Alpha]9 and [Alpha]10 subunits.

Programmed cell death regulates a number of biological phenomena, and the apoptotic signal must itself be tightly controlled to avoid inappropriate cell death. We established a genetic screen to search for molecules that inhibit the apoptotic signal from the Fas receptor. Here we report the isolation of a gene, LFG, that protects cells uniquely from Fas but not from the mechanistically related tumor necrosis factor [Alpha] death signal. LFG is widely distributed, but remarkably is highly expressed in the hippocampus. LFG can bind to the Fas receptor, but does not regulate Fas expression or interfere with binding of an agonist antibody. Furthermore LFG does not inhibit binding of FADD to Fas.