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Biomedical subjects

Y Ben-Ari

Publications and source records attributed to Y Ben-Ari.

At least 127 records · Page 7Linked to original sources
PubMed1996

Enhanced NMDAR-dependent epileptiform activity is controlled by oxidizing agents in a chronic model of temporal lobe epilepsy.

1. Graded N-methyl-D-aspartate receptor (NMDAR)-dependent epileptiform discharges were recorded from ex vivo hippocampal slices obtained from rats injected a week earlier with an intracerebroventricular dose of kainic acid. Intracellular recordings from pyramidal cells of the CA1 area showed that glutamate NMDAR actively participated in synaptic transmission, even at resting membrane potential. When NMDAR were pharmacologically isolated, graded burst discharges could still be evoked. 2. The oxidizing reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, 200 microM, 15 min) suppressed the late part of the epileptiform burst that did not recover after wash but could be reinstated by the reducing agent tris (2-carboxyethyl) phosphine (TCEP, 200 microM, 15 min) and again abolished with the NMDA antagonist D-2-amino-5-phosphonovaleric acid (D-APV). 3. Pharmacologically isolated NMDAR-mediated responses were decreased by DTNB (56 +/- 10%, mean +/- SD, n = 6), an effect reversed by TCEP. 4. When only the fast glutamateric synaptic component was blocked, NMDA-dependent excitatory postsynaptic potentials (EPSPs) could be evoked despite the presence of underlying fast and slow inhibitory postsynaptic potentials (IPSPs). DTNB decreased EPSPs to 48 +/- 12% (n = 5) of control. 5. Since a decrease of the NMDAR-mediated response by +/- 50% is sufficient to suppress the late part of the burst, we suggest that epileptiform activity can be controlled by manipulation of the redox sites of NMDAR. Our observations raise the possibility of developing new anticonvulsant drugs that would spare alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-R (AMPAR)-mediated synaptic responses and decrease NMDAR-mediated synaptic transmission without blocking it completely.

Animals↗
PubMed1996

MAP2d promotes bundling and stabilization of both microtubules and microfilaments.

Two low molecular weight MAP2 variants have been described, MAP2c and MAP2d. These variants are produced from a single gene by alternative splicing, and in their C-terminal regions contain, respectively, 3 and 4 tandem repeats, some of which are known to be involved in binding to microtubules. Substantial differences in the developmental expression pattern of MAP2c and MAP2d suggest they have different functions in neural cells. In order to investigate the respective roles of these MAP2 variants, we have analyzed the effects of MAP2c and MAP2d expression on microtubule and microfilament organization in transiently transfected cells. Our results show that both variants stabilize microtubules, but only MAP2d stabilizes microfilaments.

Actin Cytoskeleton↗
PubMed1996

Tenascin-C mRNA and tenascin-C protein immunoreactivity increase in astrocytes after activation by bFGF.

Tenascin-C is an extracellular matrix glycoprotein with trophic and repulsive properties, involved in migratory processes in CNS. Previous reports demonstrated that this molecule is produced and secreted by astrocytes. Preliminary data on fibroblasts and astrocytes have suggested that bFGF may modulate tenascin-C expression. bFGF is a mitogenic growth factor, involved in cell differentiation and neovascularization. In the present study, we examined whether bFGF modulates the expression of tenascin-C in hippocampal astrocytes from newborn rats. Our results suggest that bFGF increases the production of tenascin-C by cultured hippocampal astrocytes. We found that both tenascin-C mRNA and protein immunoreactivity were increased after bFGF treatment. Our results also demonstrated that tenascin-C polypeptides were secreted into the extracellular medium. In agreement with previous studies, we suggest that secreted tenascin-C is mainly the high molecular weight form. In addition, our results suggest that a cleavage of the high molecular weight form. In addition, our results suggest that a cleavage of the high molecular weight form may occur in the extracellular medium causing production of proteolytic fragments, that may modify the biological properties of tenascin-C. The present results may be relevant to the understanding of lesion scarring and regeneration process.

Animals↗
PubMed1995

A cautionary note on the use of the TUNEL stain to determine apoptosis.

Detection of DNA fragments in situ using the terminal deoxyribonucleotidyl transferse (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL assay) is now commonly used to investigate apoptosis. Previous reports suggested that physiologically appropriate death is due to apoptosis and that pathological mechanisms involve necrosis. Strong evidence of apoptosis, following ischaemia and epilepsy, has been recently provided by combining genomic DNA gel electrophoresis, light and electron microscopy and in situ, DNA-break labelling. However, only an observation in light microscopy with high magnification permits the detection of chromatin condensation and apoptotic bodies. We report that a positive TUNEL assay reaction should not be considered as a specific marker of apoptosis but can also indicate necrotic cell death.

Animals↗
PubMed1995

Ruthenium red protects against glutamate-induced neuronal death in cerebellar culture.

Intracytosplasmic concentration of calcium dramatically increases after glutamate exposure in cultured neurons. Here, we investigated the effects of glutamate (100 microM, 15 min) on the intramitochondrial calcium homeostasis using 45Ca as a radioactive tracer. Calcium content increased in both cytoplasmic and mitochondrial fractions during the first 15 min following glutamate exposure. Whereas calcium content declined in the cytosolic fraction during the next 45 min, it remained elevated in the mitochondrial fraction. We next investigated the effects of ruthenium red, a mitochondrial calcium uptake blocker, on calcium homeostasis and neuronal viability after glutamate. Ruthenium red (100 microM) fully protected granule cells from glutamate exposure when performed in low sodium-chloride solution. Furthermore, ruthenium red substantially lessened calcium accumulation in mitochondrial fraction after glutamate exposure.

Animals↗
PubMed1995

Synaptic GABAA activation induces Ca2+ rise in pyramidal cells and interneurons from rat neonatal hippocampal slices.

1. Changes in intracellular Ca2+ concentration ([Ca2+]i) induced by activation of GABAA receptors (synaptic stimulation or application of the GABAA agonist isoguvacine) were studied on pyramidal cells and interneurons from hippocampal slices of rats from two age groups (postnatal days (P) 2-5 and P12-13) using the fluorescent dye fluo-3 and a confocal laser scanning microscope. Cells were loaded with the dye either intracellularly, using patch pipettes containing fluo-3 in the internal solution, or extracellularly, using pressure pulses applied to an extracellular pipette containing the permeant dye fluo-3 AM. 2. Interneurons and pyramidal cells from P2-5 slices loaded with fluo-3 AM responded by an increase in [Ca2+]i to isoguvacine and to glutamate, in contrast to cells from P12-13 slices which responded to glutamate but not to isoguvacine. 3. The isoguvacine-induced rise in [Ca2+]i was reversibly blocked by bath application of the GABAA receptor antagonist bicuculline (20 microM), suggesting the specific involvement of GABAA receptors. The sodium channel blocker tetrodotoxin (TTX, 1 microM in the bath) did not prevent the isoguvacine-induced rise in [Ca2+]i. 4. The isoguvacine-induced rise in [Ca2+]i was reversibly blocked by bath application of the calcium channel blocker D600 (50 microM) suggesting the involvement of voltage-dependent Ca2+ channels. 5. Electrical stimulation of afferent fibres induced a transient increase in [Ca2+]i in neonatal pyramidal cells and interneurons (P5) loaded non-invasively with fluo-3 AM. This elevation of [Ca2+]i was reversibly blocked by bicuculline (20 microM) but not by APV (50 microM) and CNQX (10 microM). 6. During simultaneous electrophysiological recording in the current-clamp mode and [Ca2+]i monitoring from P5 pyramidal cells, electrical stimulation of afferent fibres, in the presence of APV (50 microM) and CNQX (10 microM), caused synaptic depolarization accompanied by a few action potentials and a transient increase in [Ca2+]i. In voltage clamp (-70 mV) however, there was no increase in [Ca2+]i following synaptic stimulation, showing that it is depolarization dependent. 7. Using a non-invasive method of [Ca2+]i monitoring, we demonstrate here that in neonatal (P2-5) hippocampus, GABA is an excitatory neurotransmitter which can cause an elevation of [Ca2+]i in interneurons and pyramidal cells via activation of voltage-dependent Ca2+ channels. This action may underlie the trophic role of GABA in hippocampal development.

Animals↗
PubMed1995

Non-involvement of the redox site of NMDA receptors in bidirectional synaptic plasticity in the CA1 area of the rat hippocampus in vitro.

We have examined the effects of the redox reagent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) on synaptic potentials recorded extracellularly from the CA1 area in hippocampal slices following low frequency stimulation (LFS) and tetanic stimulation (TS). Application of DTNB (200 microM) neither changed synaptic responses, nor prevented the expression of TS-induced long-term potentiation of synaptic responses and their depotentiation by LFS. Conversely, in naive slices, LFS still induced long-term depression of synaptic responses following application of DTNB. This depression could be subsequently reversed with a TS. It is concluded that the redox state of N-methyl-D-aspartate receptors does not affect the expression of long-term potentiation and depression of synaptic responses.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1995

NMDA-dependent GABAA-mediated polysynaptic potentials in the neonatal rat hippocampal CA3 region.

Evoked inhibitory postsynaptic potentials (IPSPs) were studied in CA3 hippocampal neurons from brain slice preparations of rats ranging from 5 to 18 days of age (P5-18) using intracellular recording techniques. With KMeSO4-filled electrodes the evoked inhibitory response consisted of fast and slow IPSPs mediated by GABAA and GABAB receptors respectively. In recordings obtained with electrodes filled with 2-(triethylamino)-N-(2,6-dimethylphenyl) acetamide and KMeSO4, electrical stimulation evoked monophasic IPSPs in mature slices (P10-18) and biphasic IPSPs with an early and a late phase in neonatal slices (P4-7). In neonates both the early and late phases of the IPSP were mediated by GABAA receptors. Pharmacological investigation revealed that the early phase arose from both direct and feedforward activation of GABAergic interneurons involving non-NMDA receptors, while the late phase resulted from polysynaptic activation of GABAergic interneurons mediated by NMDA receptors.

Animals↗
PubMed1995

Expression of an acidic isoform of calponin in rat brain: western blots on one- or two-dimensional gels and immunolocalization in cultured cells.

Calponin, an actin- and Ca(2+)-calmodulin-binding protein characterized as an inhibitory factor of the smooth-muscle actomyosin activity, has also been shown to be present in some non-muscle cells. However, there is a controversy as to whether calponin is present or not in brain. Several laboratories indicate that this protein is absent in chicken or bovine brains, while Applegate et al. [Applegate, Feng, Green and Taubman (1994) J. Biol. Chem. 269, 10683-10690] have recently reported the identification of an mRNA specific for a 36 kDa non-muscle calponin analogue in homogenates of rat brains. For the first time we demonstrate, by Western blots and in situ immunofluorescence localization using monoclonal as well as affinity-purified polyclonal antibody to gizzard calponin, that a 36-37 kDa and a 35-36 kDa calponin-like proteins are expressed respectively in pig and rat brains and in rat cerebellar cultured cells. The acidic pI (5.2-5.4) of the rat brain protein revealed by isoelectric focusing is in good agreement with that of the protein coded for by the calponin isoform mRNA described by Applegate et al. and is different from that of the protein from chicken gizzard (pI 9.9). Brain calponin-like protein is different from two other Ca(2+)-calmodulin-binding proteins previously identified in brain, namely caldesmon and adducin, and from tropomyosin.

Animals↗
PubMed1995

Calcium-dependent inactivation of heteromeric NMDA receptor-channels expressed in human embryonic kidney cells.

1. Whole-cell current through heteromeric NR1-NR2A and NR1-NR2B subunit combinations of NMDA channels transiently expressed in human embryonic kidney cells (HEK 293) were studied using the patch-clamp technique. 2. With 4 mM Mg-ATP in the internal pipette solution, the responses of cells expressing NR1-NR2A channels to glutamate application gradually decreased, reaching 50% of control during the first 20 min of recording. This process was accompanied by acceleration of desensitization. 3. Conditioning (5-15 s) applications of glutamate (100 microM) induced a transient inactivation of NR1-NR2A and NR1-NR2B channels (20-40%) with a slow time course of recovery (tau r = 10-60 s). Both the degree of inactivation and the time constant of recovery increased with the duration of conditioning applications of glutamate, and with an elevation of Ca2+ in the external solution. 4. These results show that both NR1-NR2A and NR1-NR2B recombinant NMDA receptor-channels expressed in HEK 293 cells can be transiently inhibited by Ca2+ ions in a similar way to that described for hippocampal neurones.

Calcium↗
PubMed1995

Postnatal maturation of gamma-aminobutyric acidA and B-mediated inhibition in the CA3 hippocampal region of the rat.

In the adult central nervous system, GABAergic synaptic inhibition is known to play a crucial role in preventing the spread of excitatory glutamatergic activity. This inhibition is achieved by a membrane hyperpolarization through the activation of postsynaptic gamma-aminobutyric acidA (GABAA) and GABAB receptors. In addition, GABA also depress transmitter release acting through presynaptic GABAB receptors. Despite the wealth of data regarding the role of GABA in regulating the degree of synchronous activity in the adult, little is known about GABA transmission during early stages of development. In the following we report that GABA mediates most of the excitatory drive at early stages of development in the hippocampal CA3 region. Activation of GABAA receptors induces a depolarization and excitation of immature CA3 pyramidal neurons and increases intracellular Ca2+ ([Ca2+]i)] during the first postnatal week of life. During the same developmental period, the postsynaptic GABAB-mediated inhibition is poorly developed. In contrast, the presynaptic GABAB-mediated inhibition is well developed at birth and plays a crucial role in modulating the postsynaptic activity by depressing transmitter release at early postnatal stages. We have also shown that GABA plays a trophic role in the neuritic outgrowth of cultured hippocampal neurons.

Animals↗
PubMed1995

Multiple forms of long-term potentiation and multiple regulatory sites of N-methyl-D-aspartate receptors: role of the redox site.

Long-term potentiation (LTP) is a form of synaptic plasticity thought to be involved in learning and memory. Although extensively studied, mainly in the CA1 region of the hippocampus, the mechanisms underlying the induction and expression of LTP are poorly elucidated. This is probably due to the fact that LTP is not a unique process and indeed recent studies have shown that several forms of LTP could be generated depending on the experimental conditions. Furthermore, LTP is generally associated with a long-lasting increase of the synaptic efficacy of AMPA receptors but an increasing number of data also suggested that NMDA receptors could be potentiated as well. NMDA receptor responses are modulated by a large number of extracellular and intracellular events, providing additional possibilities for the generation of LTP. The role of these different modulatory sites of the NMDA receptor and their relation with LTP are reviewed with a particular attention to the redox site which seems to be a selective target to distinguish between AMPA and NMDA-LTP.

Animals↗
PubMed1995

Cell death, gliosis, and synaptic remodeling in the hippocampus of epileptic rats.

Seizures set in motion complex molecular and morphological changes in vulnerable structures, such as the hippocampal complex. A number of these changes are responsible for neuronal death of CA3 and hilar cells, which involves necrotic and apoptotic mechanisms. In surviving dentate granule cells seizures induce an increased expression of tubulin subunits and microtubule-associated proteins, suggesting that an overproduction of tubulin polymers would lead to a remodeling of mossy fibers (the axons of granule cells). In fact, these fibers sprout in the dentate gyrus to innervate granule cell dendrites, creating recurrent excitatory circuits. In contrast, terminal mossy fibers do not sprout in the CA3 field. Navigation of mossy fiber's growth cones may be facilitated by astrocytes, which would exert differential effects by producing and excreting cell adhesion and substrate molecules. In the light of the results discussed here, we suggest that in adult brain activated-resident astrocytes (nonproliferating, tenascin-negative, neuronal cell-adhesion molecule-positive astrocytes) could contribute to the process of axonal outgrowth and synaptogenesis in the dentate gyrus, while proliferating astrocytes, tenascin-positive, could impede any axonal rearrangement in CA3.

Animals↗
PubMed1995

Molecular correlates between reactive and developmental plasticity in the rat hippocampus.

Area CA3 of the hippocampus is the most epileptogenic structure of the brain. Various studies have shown that kainate-induced experimental epilepsy in rats and human cases of epilepsy are associated with sprouting of the mossy fibers of the dentate granule neurons and selective loss of pyramidal neurons, notably in the CA3-CA4 areas of Ammon's horn. In experimental models of epilepsy, brief seizure activity initiates a cascade of molecular alterations that will contribute to changes in the expression of numerous genes, which can last several weeks. The products of some of these genes will contribute to the permanent state of enhanced synaptic efficiency, to the sprouting and formation of novel excitatory synapses, and possibly to neuronal cell loss. The expression of genes encoding transcription factors and numerous growth factors is rapidly altered following seizure episodes. Based on observations in vivo and in vitro in cultured hippocampal neurons, it is hypothesized that an interplay between transcription and growth factors, because of their pleiotropic effects on the regulation of effector genes, may be instrumental in coupling transient extracellular stimuli to irreversible cellular alterations.

Animals↗
PubMed1995

Distribution of caldesmon and of the acidic isoform of calponin in cultured cerebellar neurons and in different regions of the rat brain: an immunofluorescence and confocal microscopy study.

Caldesmon and calponin are two F-actin-binding and calcium-calmodulin-dependent proteins. In smooth muscle and nonmuscle cells both proteins are localized on actin filaments. Using one- or two-dimensional gel electrophoresis followed by the Western blot technique, and by immunofluorescence studies, we have given evidence that calponin is also present in rat and pig brain. In the present study, for the first time, we demonstrate caldesmon- and calponin-specific immunoreactivities in cerebellar cultured neurons. In the rat central nervous system these antibodies mainly stain neuronal cell bodies and dendrites. By confocal analysis we observed that calponin and caldesmon are located in the actomyosin domain although the total actin and myosin were not saturated. In many cases it is clear that these two proteins are adjacent rather than superimposed in the same domain of the cell. These results are compatible with the functional role of caldesmon and calponin in the regulation of the actomyosin activity as described by others and suggest that they are part of the contractile apparatus of neural cells.

Actins↗