Target engagement and intracellular delivery of mono- and bivalent LDL receptor-binding peptide-cargo conjugates: Implications for the rational design of new targeted drug therapies.

Targeted delivery to specific tissues and subcellular compartments is of paramount importance to optimize therapeutic or diagnostic interventions while minimizing side-effects. Using recently identified LDL receptor (LDLR) -targeting small synthetic peptide-vectors conjugated to model cargos of different nature and size, we investigated in LDLR-expressing cells the impact of vector-cargo molecular engineering and coupling valency, as well as the cellular exposure duration on their target engagement and intracellular trafficking and delivery profiles. All vector-cargo conjugates evaluated were found to be delivered to late compartments together with the natural ligand LDL, although to varying extents and with different kinetics. Partial recycling together with the LDLR was also consistently observed. Under continuous cellular exposure, the extent of intracellular vector-cargo delivery primarily relies on their endosomal unloading potential. In this condition, the highest intracellular delivery potential was observed with a monovalent conjugate displaying a rather high LDLR dissociation rate. On the contrary, under transient cellular exposure followed by chase, low dissociation-rate bivalent conjugates revealed a higher intracellular delivery potential than the monovalent conjugate. This was shown to rely on their ability to undergo multiple endocytosis-recycling rounds, with limited release in the ligand-free medium. The absence of reciprocal competition with the natural ligand LDL on their respective intracellular trafficking was also demonstrated, which is essential in terms of potential safety liabilities. These results demonstrate that not only molecular engineering of new therapeutic conjugates of interest, but also the cellular exposure mode used during in vitro evaluations are critical to anticipate and optimize their delivery potential.

Olfactory stem cells reveal MOCOS as a new player in autism spectrum disorders.

With an onset under the age of 3 years, autism spectrum disorders (ASDs) are now understood as diseases arising from pre- and/or early postnatal brain developmental anomalies and/or early brain insults. To unveil the molecular mechanisms taking place during the misshaping of the developing brain, we chose to study cells that are representative of the very early stages of ontogenesis, namely stem cells. Here we report on MOlybdenum COfactor Sulfurase (MOCOS), an enzyme involved in purine metabolism, as a newly identified player in ASD. We found in adult nasal olfactory stem cells of 11 adults with ASD that MOCOS is downregulated in most of them when compared with 11 age- and gender-matched control adults without any neuropsychiatric disorders. Genetic approaches using in vivo and in vitro engineered models converge to indicate that altered expression of MOCOS results in neurotransmission and synaptic defects. Furthermore, we found that MOCOS misexpression induces increased oxidative-stress sensitivity. Our results demonstrate that altered MOCOS expression is likely to have an impact on neurodevelopment and neurotransmission, and may explain comorbid conditions, including gastrointestinal disorders. We anticipate our discovery to be a fresh starting point for the study on the roles of MOCOS in brain development and its functional implications in ASD clinical symptoms. Moreover, our study suggests the possible development of new diagnostic tests based on MOCOS expression, and paves the way for drug screening targeting MOCOS and/or the purine metabolism to ultimately develop novel treatments in ASD.

Vitamin D3 improves respiratory adjustment to fatigue and H-reflex responses in paraplegic adult rats.

We previously demonstrated that vitamin D2 (ergocalciferol) triggers axon regeneration in a rat model of peripheral nerve transection. In order to confirm the regenerative potential of this neuroactive steroid, we performed a study in which vitamin D3 (cholecalciferol) was delivered at various doses to paralytic rats. After spinal cord compression at the T10 level, rats were given orally either vehicle or vitamin D3 at the dose of 50 IU/kg/day or 200 IU/kg/day. Three months later, M and H-waves were recorded from rat Tibialis anterior muscle in order to quantify the maximal H-reflex (H(max)) amplitude. We also monitored the ventilatory frequency during an electrically induced muscle fatigue known to elicit the muscle metaboreflex and an increase in respiratory rate. Spinal cords were then collected, fixed and immunostained with an anti-neurofilament antibody. We show here that vitamin D-treated animals display an increased number of axons within the lesion site. In addition, rats supplemented with vitamin D3 at the dose of 200 IU/kg/day exhibit (i) an improved breathing when hindlimb was electrically stimulated; (ii) an H-reflex depression similar to control animals and (iii) an increased number of axons within the lesion and in the distal area. Our data confirm that vitamin D is a potent molecule that can be used for improving neuromuscular adaptive mechanisms and H-reflex responses.

Involvement of tissue inhibition of metalloproteinases-1 in learning and memory in mice.

Tissue inhibitor of metalloproteinases (TIMP-1) is one of the four-member family (TIMPs-1-4) of multifunctional proteins that inhibit matrix metalloproteinases (MMPs). Its expression in the hippocampus is neuronal-activity-dependent and dramatically induced by stimuli leading to long-term potentiation (LTP), suggesting that TIMP-1 is a candidate plasticity protein potentially involved in learning and memory processes. We tested this hypothesis in a hippocampus-dependent task using the new olfactory tubing maze, with mice carrying a null mutation for TIMP-1 (TIMP-1 KO) and mice overexpressing TIMP-1 (TIMP-1 (tg)). The TIMP-1 KO mice were significantly impaired in making correct odor-reward associations when compared with their respective wild type (WT) littermates, while TIMP-1 overexpressing mice performed better than their WT controls. Both genetically modified mice learned the paradigm and the timing of the task, like their respective WTs, and no olfactory dysfunctioning was observed. These data suggest that TIMP-1 is involved in learning and memory processes related to the hippocampus, and support the hypothesis that the MMP/TIMP ratio, and hence MMP activity, modulates neuronal plasticity in normal learning and memory processes, while altered proteolytic activity could impair cognitive functions.

Tyrosine phosphorylation of ionotropic glutamate receptors by Fyn or Src differentially modulates their susceptibility to calpain and enhances their binding to spectrin and PSD-95.

Both tyrosine phosphorylation and calpain-mediated truncation of ionotropic glutamate receptors are important mechanisms for synaptic plasticity. Previous work from our laboratory has shown that calpain activation results in truncation of the C-terminal domains of several glutamate receptor subunits. To test whether and how tyrosine phosphorylation of glutamate ionotropic receptor subunits modulates calpain susceptibility, synaptic membranes were phosphorylated by Fyn or Src, two members of the Src family tyrosine kinases. Tyrosine phosphorylation of synaptic membranes by Src significantly reduced calpain-mediated truncation of both NR2A and NR2B subunits of NMDA receptors, but not of GluR1 subunits of AMPA receptors. In contrast, phosphorylation with Fyn significantly protected calpain-mediated truncation of GluR1 subunits of AMPA receptors, but enhanced calpain-mediated truncation of NR2A subunits of NMDA receptors. Similar results were observed with NR2A and NR2B C-terminal domain fusion proteins phosphorylated by Fyn or Src before incubation with calpain and calcium. In addition, phosphorylation of NR2A and NR2B C-terminal fusion proteins by Fyn or Src enhanced their binding to spectrin and PSD-95. Thus, tyrosine phosphorylation impairs or facilitates calpain-mediated truncation of glutamate receptor subunits, depending on which tyrosine kinase is activated. Such mechanisms could serve to regulate receptor integrity and location, in addition to modulating channel properties.

Src-mediated tyrosine phosphorylation of NR2 subunits of N-methyl-D-aspartate receptors protects from calpain-mediated truncation of their C-terminal domains.

Src-mediated tyrosine phosphorylation of N-methyl-d-aspartate receptor subunits has been shown to modify the functional properties of N-methyl-d-aspartate receptors. Moreover, calpain-mediated truncation of N-methyl-d-aspartate receptor subunits has been found to alter the structure of the receptors. In the present study, we first used immunoprecipitation with a variety of antibodies against N-methyl-d-aspartate receptor subunits and anti-phosphotyrosine antibodies to show that tyrosine-phosphorylated subunits of N-methyl-d-aspartate receptor are protected against calpain-mediated truncation of their C-terminal domains. A GST fusion protein containing the C-terminal domain of NR2A was used to identify the calpain cutting sites in the C-terminal domain. One site was identified at residues 1278-1279, corresponding to one of the preferred calpain truncation sites. This site is adjacent to a consensus sequence for Src-mediated tyrosine phosphorylation, and Src-mediated tyrosine phosphorylation of the GST-NR2A C-terminal fusion protein also inhibited calpain-mediated truncation of the fusion protein. We propose that phosphorylation of NR2 subunits and the resulting inhibition of calpain-mediated truncation of their C-terminal domains provide for the stabilization of the N-methyl-d-aspartate receptors in postsynaptic structures.

Characterization in cultured cerebellar granule cells and in the developing rat brain of mRNA variants for the NMDA receptor 2C subunit.

N-Methyl-D-aspartate (NMDA) receptors are heteromeric structures resulting from the association of at least two distantly related subunit types, NR1 and one of the four NR2 subunits (NR2A-NR2D). When associated with NR1, the NR2 subunits impose specific properties to the reconstituted NMDA receptors. Although the NR1 mRNAs are expressed in the majority of central neurons, the NR2 subunits display distinct patterns of expression in the developing and adult rat brain. The NR2C subunit is barely expressed in the rat forebrain, whereas its expression increases substantially in the granule cells in the course of cerebellar development. We have identified novel NR2C splice variants in cultured cerebellar granule cells as well as in the developing cerebellum. When compared with the prototypic NR2C mRNA, these variants carry one (NR2Cb) or two (NR2Cd) insertions or a deletion (NR2Cc) and encode putative NR2C polypeptides that terminate between the third and fourth membrane segments or between the first and second membrane segments. RT-PCR analysis and in situ hybridization show that expression of the splice variants is developmentally regulated, both in the cerebellum and in the hippocampus. Electrophysiological recordings and microfluorimetry emissions in transfected human embryonic kidney 293 cells indicate that the NR2Cb variant, when expressed in combination with NR1, does not contribute to the formation of functional receptor channels. The significance of theses findings is discussed.

Gene expression in developing rat hippocampal pyramidal neurons appears independent of mossy fiber innervation.

In the rat, neonatal gamma-irradiation of the hippocampus induces a selective destruction of dentate granule cells and prevents the development of the mossy fiber-CA3 pyramidal cell connection. In the absence of mossy fiber input, the CA3 pyramidal neurons exhibit morphological alterations and rats deprived of dentate granule cells fail to develop kainate-induced epileptic activity in the CA3 pyramidal neurons. Neonatal elimination of the granule cells also impairs learning and memory tasks in adult rats. In the present work, we assessed by in situ hybridization and semi-quantitative RT-PCR, whether in the pyramidal layers, the absence of mossy fiber input alters the expression of a number of genes involved in activity-dependent signal transduction, in GABAergic neurotransmitter signaling and in neurite development via microtubule organization. Surprisingly, we show that the expression and the developmentally regulated alternative splicing of the genes we examined in the developing hippocampus are not altered in the pyramidal neurons, whether the dentate granule afferents are present or absent. Our results suggest that in the CA3 pyramidal layer, the developmental expression patterns of the mRNAs we studied are independent of extrinsic cues provided by mossy fiber input.

Differential interaction of the tSXV motifs of the NR1 and NR2A NMDA receptor subunits with PSD-95 and SAP97.

The NR1 and NR2 subunits of the N-methyl-D-aspartate (NMDA) receptor are encoded by distinct genes. In the rat brain, four C-terminal variants of the NR1 subunit (NR1-1 to NR1-4) are encoded by a single gene, and are generated by alternative splicing of the C1 and C2 exon cassettes, while four different genes encode the NR2 subunits (NR2 A-D). Functional NMDA receptors result from the heteromultimeric assembly of NR1 variants with distinct NR2 subunits. The NR2B subunit interacts with post-synaptic density protein 95 (PSD-95), SAP97 and members of the membrane-associated guanylate-like kinase (MAGUK) family of proteins. This interaction occurs through the binding of the C-terminal tSXV intracellular motif of the NR2B subunit to the N-terminal PDZ (PSD-95, discs-large, ZO-1) domains of the PSD-95 and SAP97 proteins. Both NR1-3 and NR1-4 also display a consensus C-terminal tSXV motif. Using the two-hybrid genetic system in yeast and site-directed mutagenesis, we compared the binding of the NR2A, NR1-3 and NR1-4 tSXV motifs with the PDZ domains of PSD-95 and SAP97. The main conclusions of the present report are that: (i) while NR2A displays a strong interaction with PSD-95 and SAP97, the NR1-3 and NR1-4 NMDA receptor subunits do not display any interaction despite the presence of tSXV motifs; (ii) the C-terminal tSXV motif of the NR2A subunit is mandatory but not sufficient for efficient interaction with the PSD-95 and SAP97 proteins; (iii) as yet unidentified upstream sequences of the receptor subunits determine whether the tSXV motifs will bind to the PSD-95 and SAP97 PDZ domains; (iv) different tSXV motifs elicit interactions of variable strengths; and (v) residues in positions -3 and -4 modulate the binding affinity of the C-terminal tSXV motifs. Using immunohistochemistry, we also compared the distribution of the PSD-95, NR2A and SAP97 proteins in adult rat brain, and we show that in the cortex, hippocampus and cerebellum, there is evidence for colocalization of these proteins.

Q/R editing of the rat GluR5 and GluR6 kainate receptors in vivo and in vitro: evidence for independent developmental, pathological and cellular regulation.

Kainate (KA) is a potent neuroexcitatory agent in several areas of the adult brain, with convulsant and excitotoxic properties that increase as ontogeny proceeds. Besides its depolarizing actions, KA may enhance intracellular accumulation of Ca2+ to promote selective neuronal damage. The effects of KA are mediated by specific receptors recently considered to be involved in fast neurotransmission and that can be activated synaptically. KA receptors, e.g. GluR5 and GluR6 have been characterized by molecular cloning. Structure-function relationships indicate that in the MII domain of these KA receptors, a glutamine (Q) or arginine (R) residue determines ion selectivity. The arginine stems from post-transcriptional editing of the GluR5 and GluR6 pre-RNAs, and the unedited and edited versions of GluR6 elicit distinct Ca2+ permeability. Using a PCR-based approach, we show that in vivo, Q/R editing in the GluR5 and GluR6 mRNAs is modulated during ontogeny and differs substantially in a variety of nervous tissues. GluR5 editing is highest in peripheral nervous tissue, e.g. the dorsal root ganglia, where GluR6 expression is barely detectable. In contrast, GluR6 editing is maximal in forebrain and cerebellar structures where GluR5 editing is lower. Intra-amygdaloid injections of KA provide a model of temporal lobe epilepsy, and we show that following seizures, the extent of GluR5 and GluR6 editing is altered in the hippocampus. However, in vitro, high levels of glutamate and potassium-induced depolarizations have no effect on GluR5 and GluR6 Q/R editing. GluR6 editing is rapidly enhanced to maximal levels in primary cultures of cerebellar granule neurons but not in cultured hippocampal pyramidal neurons. Finally, we show that cultured glial cells express partially edited GluR6 mRNAs. Our results indicate that Q/R editing of GluR5 and GluR6 mRNAs is structure-, cell type- and time-dependent, and suggest that editing of these mRNAs is not co-regulated.

Increased cyclin D1 in vulnerable neurons in the hippocampus after ischaemia and epilepsy: a modulator of in vivo programmed cell death?

Several observations suggest that delayed neuronal death in ischaemia, epilepsy and other brain disorders includes an apoptotic component, involving programmed cell death (PCD). PCD is hypothesized to result, in part, from aberrant control of the cell cycle. Because they are instrumental in mitosis, cyclins D are key markers to evaluate whether neurons indeed progress into the cell cycle in situations of pathology. Therefore, we investigated in rat brains, the expression of cyclins D in the delayed neuronal death that occurs following transient global ischaemia and kainate-induced seizures. Following a four-vessel occlusion insult, quantitative in situ hybridization revealed a highly significant and persistent 100% increase of cyclin D1 mRNA in the vulnerable pyramidal neurons of the CA1 hippocampal region. Ischaemia also induced a smaller and transient cyclin D1 mRNA increase in the resistant CA3 area and dentate gyrus. In contrast, the cyclin D2 and D3 mRNAs, expressed constitutively in the adult rat hippocampus, were not upregulated. Following kainate-induced seizures, cyclin D1 mRNA was induced in the vulnerable CA3 region, and to a lesser extent, in non-vulnerable regions. Cyclin D1 immunohistochemistry revealed increased protein levels in the cytoplasm and nucleus of neurons commited to die after ischaemia. Double labelling experiments indicate that cyclin D1 is also expressed in reactive astrocytes but not in microglial cells. Finally, we report that in neurons, cyclin D1 expression peaks before nuclear condensation and the appearance of DNA fragmentation. We propose that cyclin D1, when expressed at high levels in lesioned neurons, may act as a modulator of apoptosis.

GABA modulates cytotoxicity of immunocompetent cells expressing GABAA receptor subunits.

C57 black mouse splenic T lymphocytes effector cells were co-cultivated with Balb/c mouse splenic cells for sensitization; P815 DBA mouse mastocytoma target cells were then added and specific T cell-dependent cytotoxicity determined. This cytotoxicity increased after gamma-aminobutyric acid (GABA) treatment of the sensitized effectors, but decreased after GABA treatment of the targets. These GABA effects seemed to be specific since they were partially mimicked by linear but not ramified GABA analogues. Furthermore, they were likely mediated by GABAA receptor since GABAA receptor subunit mRNAs and protein could be demonstrated in effector or target immune specific cells, suggesting that under yet to be defined circumstances, GABA may affect T cell functions.

Long-lasting enhanced expression in the rat hippocampus of NMDAR1 splice variants in a kainate model of epilepsy.

Chronic epilepsy is associated with increased excitability which may result from abnormal glutamatergic synaptic transmission involving altered properties of N-methyl-D-aspartate (NMDA) receptors. To date two gene families encoding NMDA receptor subunits have been cloned, NR1 and NR2. Eight NR1 mRNAs are generated by alternative splicing of exons 5, 21 and 22; the NR1-1 to NR1-4 C-terminal variants exist in the a or b version depending on the presence or absence of the domain encoded by exon 5. Epilepsy was induced in rats by unilateral intra-amygdalar injection of kainate and animals were killed from 6 h to 4 months following the injection. Increased NR1 mRNA levels were observed during status epilepticus (6-24 h after the injection), both psilateral and contralateral, while a second wave of NMDAR1 mRNA increase occurred in chronic epileptic animals, between 21 days and 4 months following kainate injection. Our data show: (i) a permanent increase of the NR1-2a and NR1-2b mRNA species (containing exon 22) in all hippocampal fields, both ipsilateral and contralateral, and (ii) an increase of the NR1-3 (a and b) mRNAs (containing exon 21) in the ipsilateral CA1, and NR1-3a mRNA in the ipsilateral dentate gyrus. No long-term changes were observed for the NR1-1 and NR14 splice variants. In the ipsilateral CA3 area a globally decreased mRNA expression was associated with neuronal loss. A possible contribution to the maintenance of the epileptic state by an increased expression of NMDA receptors is discussed.

MAP2d mRNA is expressed in identified neuronal populations in the developing and adult rat brain and its subcellular distribution differs from that of MAP2b in hippocampal neurones.

The brain microtubule-associated protein MAP2 family is composed of high-molecular-weight (MAP2a and MAP2b) and low-molecular-weight (MAP2c and MAP2d) isoforms. The common C-terminal region of HMW MAP2 and MAP2c contains three repeated microtubule-binding domains while MAP2d comprises four repeats. MAP2c mRNA is known to be expressed at high levels in the immature brain. We show that in the brains of rat pups, MAP2c mRNAs are indeed expressed at high levels compared with MAP2d. However, in adult rat brains, MAP2d mRNA levels are higher than MAP2c. In order to identify the neural cells expressing MAP2d, we used in situ hybridization. In vivo, we show that MAP2d mRNA is expressed in well-identified neuronal populations of the brain. In primary cultures of hippocampal neurones, double-labelling experiments confirm that MAP2d is clearly expressed in neurones. We also evaluated in this study the subcellular distribution of the MAP2d mRNAs in cultured hippocampal neurones and we report that in contrast with MAP2b mRNAs, mostly localized in dendrites, MAP2d mRNAs are essentially located in neuronal cell bodies.

Characterization of rat porin isoforms: cloning of a cardiac type-3 variant encoding an additional methionine at its putative N-terminal region.

In vivo, the outer mitochondrial membrane presents a restriction of diffusion for ADP in heart and slow twitch skeletal muscles, but not in fast twitch skeletal muscle. Mitochondrial porins constitute the main pathway for the transit of metabolites across the outer mitochondrial membrane. We decided, therefore, to characterize, by cloning, rat heart VDAC and to follow their expression in different striated muscles. We cloned three isoforms, one being HVDAC1-like porin (RVDAC1) whereas the other two are MVDAC3-like porins (RVDAC3 and RVDAC3v). These three isoforms are ubiquitously expressed among striated muscles. RVDAC3v differs from RVDAC3 by one additional amino acid, a Met, located between Val39 and Glu40 in RVDAC3 sequence. This study constitutes a first step in order to further characterize striated muscle porin isoforms.

Specific interaction between the XNP/ATR-X gene product and the SET domain of the human EZH2 protein.

Mutations in the XNP gene result in different inherited disorders, including the ATR-X syndrome which is characterized by mental retardation (MR) associated with alpha-thalaessemia. Amino acid sequence analysis revealed that the XNP protein is a new member of the SNF2-like family, which comprises numerous members involved in a broad range of biological functions: transcriptional regulation, DNA repair and chromosome segregation. Since experiments on fibroblasts from ATR-X patients have provided no evidence for either a DNA repair defect or abnormal chromosome breakage or segregation, it seems more likely that the XNP protein is somehow involved in regulation of gene expression. Recent genetic and biochemical studies have led to the emerging concept that SNF2-like proteins are components of a large protein complex which may exert its functions by modulating chromatin structure. To investigate whether XNP could mediate the activity of gene-specific activators through chromatin remodelling, we performed a yeast two-hybrid analysis using XNP and several human heterochromatin-associated proteins. We found a specific interaction between the XNP and the EZH2 proteins. In light of these observations, we discuss how the XNP protein may regulate gene transcription at the chromatin level.

Glutamate receptors in dysplasic cortex: an in situ hybridization and immunohistochemistry study in rats with prenatal treatment with methylazoxymethanol

Injection of the antimitotic drug methylazoxymethanol (MAM) in the pregnant rat at E14 leads in the offsprings to a severe malformation with microcephaly and cortical heterotopiae in the white matter and in the CA1 field of the hippocampus. These animals suffer cognitive and epileptic disorders. Since these pathologies have been associated with glutamatergic transmission abnormalities, we have examined by in situ hybridization and immunohistochemistry the distribution and expression levels of several glutamate receptors subunits in these rats. Examination of the GluR2 flip and flop, NR1, NR2A and NR2B subunit gene transcripts showed a qualitatively similar distribution in both the neocortex and hippocampus of MAM and control rats. Quantitative analysis revealed an altered proportion of the GluR2 flip and flop subunits in the CA1 region of MAM animals as compared to controls. Moreover, a 26% reduction in the expression of the NR1 subunit and a 40% increase in the expression of the GluR2 flip subunit were noted in cortical heterotopiae, as compared to the adjacent neocortex. Immunostaining for GluR2/3, NR1 or NR2 showed, in both MAM and control animals, that glutamate receptors were mainly concentrated in the soma and dendrites of neocortical and hippocampal pyramidal cells, including in heterotopiae, and in the apical dendrites of hippocampal granule cells. Abnormalities in the expression of glutamate receptor subtypes in cortical heterotopiae and in the hippocampal CA1 region could contribute to functional disorders previously reported in MAM animals such as memory impairments and epilepsy. Copyright 1997 Elsevier Science B.V.

Glutamate receptors in dysplasic cortex: an in situ hybridization and immunohistochemistry study in rats with prenatal treatment with methylazoxymethanol.

Injection of the antimitotic drug methylazoxymethanol (MAM) in the pregnant rat at E14 leads in the offsprings to a severe malformation with microcephaly and cortical heterotopiae in the white matter and in the CA1 field of the hippocampus. These animals suffer cognitive and epileptic disorders. Since these pathologies have been associated with glutamatergic transmission abnormalities, we have examined by in situ hybridization and immunohistochemistry the distribution and expression levels of several glutamate receptors subunits in these rats. Examination of the GluR2 flip and flop, NR1, NR2A and NR2B subunit gene transcripts showed a qualitatively similar distribution in both the neocortex and hippocampus of MAM and control rats. Quantitative analysis revealed an altered proportion of the GluR2 flip and flop subunits in the CA1 region of MAM animals as compared to controls. Moreover, a 26% reduction in the expression of the NR1 subunit and a 40% increase in the expression of the GluR2 flip subunit were noted in cortical heterotopiae, as compared to the adjacent neocortex. Immunostaining for GluR2/3, NR1 or NR2 showed, in both MAM and control animals, that glutamate receptors were mainly concentrated in the soma and dendrites of neocortical and hippocampal pyramidal cells, including in heterotopiae, and in the apical dendrites of hippocampal granule cells. Abnormalities in the expression of glutamate receptor subtypes in cortical heterotopiae and in the hippocampal CA1 region could contribute to functional disorders previously reported in MAM animals such as memory impairments and epilepsy.

Serum deprivation-induced apoptosis in cultured hippocampi is prevented by kainate.

Serum deprivation of hippocampal organotypic cultures induced cell death within 6 h in dentate gyrus granule cells and hilar interneurons whereas neurons from other hippocampal regions were spared. Dying neurons exhibited condensed chromatin in the nuclei, as revealed by cresyl violet, Hoescht staining, and electron microscopy. Cell death was abolished by cycloheximide. KA, an agonist of AMPA/KA receptors that induces depolarization, also prevented neuronal death. This effect was antagonized by the AMPA/KA receptor antagonist DNQX, but not by APV, an antagonist of NMDA receptors. PTX, a GABA(A) receptor antagonist, reduced neuronal death by 50% after serum withdrawal. These data indicate that protein synthesis-dependent programmed cell death (PCD) occurs in the dentate gyrus upon trophic support withdrawal and suggest that neuronal activity contributes to cellular homeostasis.