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Y Ben-Ari

Publications and source records attributed to Y Ben-Ari.

At least 163 records · Page 9Linked to original sources
PubMed1994

Glutamate-induced neuronal death in cerebellar culture is mediated by two distinct components: a sodium-chloride component and a calcium component.

The relative contribution of sodium, chloride and calcium ions in the neuronal death induced by glutamate is controversial. We have therefore reassessed the effects of extracellular ion substitution on glutamate-induced neuronal death in cerebellar granule cell culture. Sodium or chloride substitution by impermeant ions prevented the initial swelling observed after glutamate exposure (100 microM, 15 min) in balanced salt solution but did not prevent the progressive degeneration of cerebellar neurons over the next few hours. In low calcium medium, glutamate exposure also led to degeneration of granule neurons. In contrast, sodium or chloride substitution and calcium omission prevented both the initial swelling and the delayed neuronal death after glutamate exposure. These morphological observations were confirmed both by measurement of the intracellular water space with [3H]methylglucose and by quantification of cell viability by 3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyl tetrazolium bromide (MTT) staining. We conclude that glutamate-induced neuronal death is mediated by two distinct components: a calcium-independent sodium-chloride dependent component and a calcium-dependent component. Each one of these components leads to the death of cerebellar neurons after glutamate exposure.

Animals↗
PubMed1994

A fraction enriched in rat hippocampal mossy fibre synaptosomes contains trophic activities.

Subcellular fractions prepared from the rat hippocampus, were assessed for the presence of trophic activities. The cytosol of synaptosomal fractions induced mitotic reinitiation of confluent 3T3 fibroblasts. The synaptosomal fraction, enriched in mossy fibre terminals, contained the highest mitotic activity. The mitogenic activity was heat and trypsin sensitive, suggesting that polypeptides are involved. The cytosol of the mossy fibre synaptosomal fraction promoted neuritic outgrowth of PC 12 cells and embryonic hippocampal neurones in primary cultures. These results suggest that mossy fibres contain both mitogenic and neurotrophic activities. These factors could participate in mossy fibre sprouting that occur following brief seizures or experimental lesions.

Animals↗
PubMed1994

Inhibition of protein synthesis by the NMDA channel blocker MK-801.

MK-801 is a selective non-competitive N-methyl-D-aspartate (NMDA) channel blocker which has been extensively used in order to determine the properties of NMDA receptors and their role in epilepsy and ischaemic cell death. We now report that MK-801 (10 microM) inhibits [35S]methionine incorporation into polypeptides by 45 +/- 7.5% in adult rat hippocampal slices and by 35 +/- 10% in cultured glial cells. This effect is NMDA-receptor independent since (i) only high doses of MK-801 induced such an effect and (ii) APV (50 microM), a competitive NMDA receptor antagonist, or Mg2+ (10 mM), a NMDA channel blocker, did not reduce protein synthesis.

Animals↗
PubMed1994

Subcellular fractionation on Percoll gradient of mossy fiber synaptosomes: evoked release of glutamate, GABA, aspartate and glutamate decarboxylase activity in control and degranulated rat hippocampus.

Using discontinuous density gradient centrifugation in isotonic Percoll sucrose, we have characterized two subcellular fractions (PII and PIII) enriched in mossy fiber synaptosomes and two others (SII and SIII) enriched in small synaptosomes. These synaptosomal fractions were compared with those obtained from adult hippocampus irradiated at neonatal stage to destroy granule cells and their mossy fibers. Synaptosomes were viable as judged by their ability to release aspartate, glutamate and GABA upon K+ depolarization. After irradiation, compared to the control values, the release of glutamate and GABA was decreased by 57 and 74% in the PIII fraction, but not in the other fractions and the content of glutamate, aspartate and GABA was also decreased in PIII fraction by 62, 44 and 52% respectively. These results suggest that mossy fiber (MF) synaptosomes contain and release glutamate and GABA. Measurement of the GABA synthesizing enzyme, glutamate decarboxylase, exhibited no significant difference after irradiation, suggesting that GABA is not synthesized by this enzyme in mossy fibers.

Amino Acids↗
PubMed1994

Induction of c-fos mRNA expression in an in vitro hippocampal slice model of adult rats after kainate but not gamma-aminobutyric acid or bicuculline treatment.

Levels of gene expression following in vitro treatment of rat hippocampal slices with kainate, gamma-aminobutyric acid (GABA), or bicuculline were measured by the reverse transcription-coupled polymerase chain reaction method. Following a short-term exposure to kainate, c-fos gene expression was induced by 12-fold in the adult, but not the newborn, hippocampus. Under the same experimental conditions, zifl268 and brain-derived neurotrophic factor (BDNF) gene expression were unchanged. Our results also demonstrate a lack of induction of c-fos, zifl268 and BDNF after short-time treatment of either adult or newborn hippocampal slices with GABA or bicuculline. The relevance of the differential induction of gene expression in the adult and newborn in an in vitro hippocampal slice model as compared to previously described in vivo models is discussed.

Animals↗
PubMed1994

aFGF, bFGF and flg mRNAs show distinct patterns of induction in the hippocampus following kainate-induced seizures.

We report that kainic acid-induced seizures lead to marked increases in mRNAs encoding basic and acidic fibroblast growth factors (bFGF and aFGF, respectively) and flg, one of their receptors, in the rat hippocampus. Anticonvulsant pretreatment inhibits the up-regulation of these mRNAs. The observed increase in flg mRNA levels involves the pyramidal cells of all hippocampal subfields and the granular cells of the dentate gyrus. The increased expression of aFGF and bFGF mRNAs is limited to neuron populations that are resistant to seizure-induced injury, the granular cells of dentate gyrus and pyramidal cells of CA1 region, respectively. The results suggest that the increase in the FGFs and flg may play pivotal roles in neuron survival and in long-term changes occurring in the hippocampus following seizure activity.

Animals↗
PubMed1994

Glial reaction after seizure induced hippocampal lesion: immunohistochemical characterization of proliferating glial cells.

Kainic acid treatment (a model of temporal lobe epilepsy) induces Ammon's horn sclerosis, which is characterized by degeneration of CA3 pyramidal neurons and reactive gliosis. In the present study we have combined autoradiographic analysis of 3H-thymidine incorporation and immunocytochemistry to investigate this glial scarring phenomenon. The present results demonstrate that in the fields showing neuronal degeneration (i.e. CA3-CA4 fields of Ammon's horn and dentate hilus) the glial reaction consists of a proliferation and hypertrophy of astrocytes and microglia-macrophages. In the regions showing exclusively terminal axonal degeneration (i.e. the molecular layer of kainate-treated rats), glial cells do not proliferate but astrocytes show a transient hypertrophy. These results also demonstrate that oligodendrocytes do not proliferate in the hippocampus of kainate-treated rats. In agreement with our previous report we find that hippocampal astrocytes from kainate-treated rats express A2B5 immunoreactivity, a marker of type-2 astrocytes. A2B5 immunoreactivity was expressed by astrocytes not only in areas showing glial proliferation such as CA3-CA4 fields, but also in the molecular layer, where astrocytes do not proliferate. This suggests that in the CNS, normal resident astrocytes acquire the phenotypic properties of type-2 astrocytes.

Animals↗
PubMed1994

Anoxic LTP sheds light on the multiple facets of NMDA receptors.

Hippocampal neurones in the CA1 region have become a model system to study the mechanisms of long-term potentiation (LTP) and memory processes. The CA1 region is also highly vulnerable to ischaemic or anoxic episodes which induce a selective and delayed degeneration of pyramidal neurones. In CA1 neurones, anoxic episodes generate a novel form of LTP to which we refer as anoxic LTP. In common with tetanic LTP, the induction of anoxic LTP is voltage- and NMDA receptor-dependent. However, in contrast with tetanic LTP, the expression of anoxic LTP is mediated exclusively by NMDA receptors. These observations suggest that anoxic-ischaemic episodes trigger a switch in favour of NMDA receptor-operated synaptic transmission. We suggest that the multiple forms of NMDA receptor-dependent LTPs are determined by extracellular and intracellular modulatory sites of this receptor.

Animals↗
PubMed1994

Structure, regional and developmental expression of rat MAP2d, a MAP2 splice variant encoding four microtubule-binding domains.

MAP2, a major component of microtubule polymers in neurons consists of high molecular weight (HMW) proteins MAP2a, MAP2b and a low molecular weight (LMW) MAP2c, expressed in the developing brain. These isoforms are produced from a single gene by alternative splicing and share identical C-termini encompassing 3 tandem repeats, critical in microtubule binding. We describe the structure, regional and developmental expression of a novel MAP2 splice variant, MAP2d, containing an insertion whose sequence is homologous to the three and four repeats of MAP2 and Tau respectively. This insertion is absent from the mRNAs encoding HMW MAP2. MAP2d mRNAs are expressed at higher levels than MAP2c in all adult nervous tissues of the rat, and are found at low levels in glial cell cultures when compared to primary cultures of cerebellar neurons. Splicing of the fourth repeat in mature Tau precedes that in MAP2d during rat brain development. The tardive expression of a four microtubule-binding domain LMW MAP2 suggests it could play in extended neurites a similar role as mature Tau in axons.

Alternative Splicing↗
PubMed1994

Kainate-induced apoptotic cell death in hippocampal neurons.

We have examined the role apoptosis plays in epileptic brain damage using intra-amygdaloid injection of kainate. With the silver staining technique of Gallyas, argyrophylic (dying) neurons were observed, a few hours after the injection, in the amygdala and in the vulnerable pyramidal neurons of the hippocampal CA3 region. In both areas, cell death has apoptotic features, including: (i) nuclear chromatin condensation and marginalization with light and electron microscopy; (ii) DNA fragmentation with a typical ladder pattern on agarose gel electrophoresis; (iii) positive nuclear labelling with a selective in situ DNA fragmentation staining method. Combined in situ DNA labelling and silver staining showed that the DNA fragmentation occurred in dying neurons. CA1 or granule cells which do not degenerate following intra-amygdaloid injection of kainate were not stained with the in situ DNA labelling or the argyrophylic technique. Administration of diazepam blocked the kainate-induced seizures and prevented DNA fragmentation in CA3 but not in the amygdala. Therefore, apoptosis contributes to the local and distant damage induced by kainate.

Animals↗
PubMed1994

Correlation between reactive sprouting and microtubule protein expression in epileptic hippocampus.

Temporal lobe epilepsy in both human and rats is associated with a collateral sprouting of hippocampal mossy fibers (i.e. the axons of granule cells). This sprouting generates abnormal recurrent synaptic connections. We previously showed that in the experimental model of temporal lobe epilepsy induced by an intra-amygdaloid injection of kainate, the synaptic remodeling of mossy fibers was preceded by a transient increased expression of alpha-tubulin in granule cells. This suggests that an overproduction of tubulin polymers may be responsible, at least in part, for the elongation and side-branching of mossy fibers, which occurs 12-30 days after seizures. In the present study we show that this increased expression of alpha-tubulin is accompanied by an increased expression of the microtubule-associated proteins MAP2 and TAU. Thus, using in situ hybridization, we observe that MAP2 messenger RNA levels increased in granule cell bodies and dendrites from day 3 to two weeks after kainate treatment. This rise is associated with a concomitant transient increase of MAP2 immunoreactivity in the granule cell dendrites. TAU messenger RNA also increases in granule cell bodies, while TAU immunoreactivity increases in their axons, the mossy fibers. The time course of these changes parallels that of alpha-tubulin, and develops before and during the axonal mossy fiber sprouting. Since MAP2 and TAU are important for the initiation, elongation and stabilization of neurites, we suggest that the overexpression of these proteins via the formation of microtubules may play an important role in the sprouting of mossy fibers in epileptic rats.

Amygdala↗
PubMed1994

gamma-Aminobutyric acid (GABA): a fast excitatory transmitter which may regulate the development of hippocampal neurones in early postnatal life.

The properties of neonatal GABAergic synapses were investigated in neurones of the hippocampal CA3 region. GABA, acting on GABAA receptors, provides most of the excitatory drive on immature CA3 pyramidal neurones at an early stage of development, whereas glutamatergic synapses (in particular, those mediated by AMPA receptors) are mostly quiescent. Thus, during the first postnatal week of life, bicuculline fully blocked spontaneous and evoked depolarising potentials, and GABAA receptor agonists depolarised CA3 pyramidal neurones. GABAA mediated currents also had a reduced sensitivity to benzodiazepines. In the presence of bicuculline, between P0 and P4, increasing the stimulus strength reveals an excitatory postsynaptic potential which is mostly mediated by NMDA receptors. During the same developmental period, pre- (but not post) synaptic GABAB inhibition is present. Intracellular injections of biocytin showed that the axonal network of the GABAergic interneurones is well developed at birth, whereas the pyramidal recurrent collaterals are only beginning to develop. Finally, chronic bicuculline treatment of hippocampal neurones in culture reduced the extent of neuritic arborisation, suggesting that GABA acts as a trophic factor in that period. In conclusion, it is suggested that during the first postnatal week of life, when excitatory inputs are still poorly developed, GABAA receptors provide the excitatory drive necessary for pyramidal cell outgrowth. Starting from the end of the first postnatal week of life, when excitatory inputs are well developed, GABA (acting on both GABAA and GABAB receptors) will hyperpolarise the CA3 pyramidal neurones and, as in the adult, will prevent excessive neuronal discharges. Our electrophysiological and morphological studies have shown that hippocampal GABAergic interneurones are in a unique position to modulate the development of CA3 pyramidal neurones. Developing neurones require a certain degree of membrane depolarisation, and a consequent rise in intracellular calcium, for stimulating neurite outgrowth; the GABAergic network, which develops prior to the glutamatergic one, appears to provide this depolarisation. Starting from the end of the first postnatal week of life, at a time when excitatory pathways are developing, GABA (acting on both GABAA and GABAB receptors) would reverse its action, and start to play its well-known role as an inhibitory neurotransmitter.

Animals↗
PubMed1994

Subcellular fractionation on Percoll gradient of mossy fiber synaptosomes: morphological and biochemical characterization in control and degranulated rat hippocampus.

A method for preparation of hippocampal mossy fiber synaptosomes directly from the postnuclear pellet is presented. This method represents an adaptation of that previously described for the isolation of synaptosomes by centrifugation through Percoll gradients directly from the supernatant fraction. We have characterized by electron microscopy two fractions, PII and PIII, enriched in mossy fiber synaptosomes; fraction PIII had 75% mossy fiber synaptosomes with well-preserved morphology (large size 3 microns, complex morphology, high synaptic vesicle density, multisynapses), whereas fraction PII contained 12%. These fractions were enriched in lactate dehydrogenase activity indicating that the integrity of synaptosomes was preserved. Compared with the other synaptosomal fractions, these fractions showed greater levels of dynorphin A (1-8) immunoreactivity and endogenous zinc, which are particularly concentrated in hippocampal mossy fiber terminals. Furthermore, we prepared synaptosomes from adult hippocampus after neonatal irradiation, which destroys the majority of granule cells and associated mossy fibers. The levels of dynorphin and zinc decreased by 88 and 70% in fraction PII and by 95 and 90%, respectively, in PIII. These results suggest that the rapid Percoll procedure is convenient for the purification of mossy fiber synaptosomes.

Animals↗
PubMed1994

Neonatal irradiation prevents the formation of hippocampal mossy fibers and the epileptic action of kainate on rat CA3 pyramidal neurons.

1. The effects of unilateral gamma-ray irradiation at birth on the properties of adult CA3 pyramidal neurons have been studied in hippocampal slices. 2. Neonatal gamma-ray irradiation reduced by 80% the number of granule cells and prevented the formation of mossy fiber synapses without reducing the number of CA3 pyramidal cells. The destruction of the mossy fibers was also confirmed with extracellular recordings. 3. Excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) evoked by stimulation of the stratum radiatum had similar properties in nonirradiated and irradiated hippocampi: the EPSP reversed polarity near 0 mV, was reduced in amplitude by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) and D(-)-2-amino-5-phosphonovalerate (APV, 50 microM); the fast and slow IPSPs reversed at -75 and -100 mV, were blocked by bicuculline (10 microM), and reduced by phaclofen (0.5 mM), respectively. 4. Bath application of kainate (300-500 nM) evoked epileptiform activity in 81.5% of nonirradiated hippocampal CA3 regions and only in 29% of the irradiated CA3 regions. In contrast, bath application of high potassium (7 mM) and bicuculline (10 microM) generated spontaneous and evoked epileptiform activity in both nonirradiated and irradiated CA3 regions. 5. In nonirradiated and irradiated CA3 regions, kainate (200-300 nM) reduced the amplitude of the fast and slow IPSPs, reduced spike accommodation, and increased the duration of the action potential generated by a depolarizing pulse. 6. The postsynaptic responses of CA3 neurons to bath application of glutamatergic agonists were similar in nonirradiated and irradiated hippocampi in terms of amplitude, reversal potential, and pharmacology. 7. It is concluded that the most conspicuous effect of neonatal gamma-ray irradiation is to prevent the epileptic action of kainate. We propose that kainate generates epileptiform activity in the intact CA3 region by activating high-affinity binding sites located on the mossy fiber terminals.

2-Amino-5-phosphonovalerate↗
PubMed1994

Kainate-induced inactivation of NMDA currents via an elevation of intracellular Ca2+ in hippocampal neurons.

1. Ionic currents and the cytosolic free calcium concentration ([Ca2+]i) were recorded in rat hippocampal neurons in culture using the whole-cell configuration of the patch-clamp technique and confocal laser scanning microscopy with the fluorescent Ca2+ indicator Fluo-3 or dual-emission microspectrofluorimetry with the fluorescent Ca2+ indicator Indo-1. The excitatory amino acids, kainate and N-methyl-D-aspartate (NMDA), were repeatedly applied to the neurons using either a fast perfusion system or pressure-ejection from micropipettes. 2. Conditioning (1-10 s) applications of NMDA induced desensitization of NMDA currents. Recovery from desensitization, estimated from analysis of the amplitudes of short (20-50 ms) test NMDA currents, was double exponential. The time constant of the first phase was < 2 s and for the second phase it was in the range 10-50 s. 3. Conditioning applications of kainate decreased the amplitude of NMDA currents. Recovery of NMDA currents from kainate-induced inactivation was slow and could be fitted with a single exponential. The time constant of recovery was in the range 10-50 s and increased with prolongation of the conditioning pulse of kainate. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 microM) prevented kainate-induced inactivation of NMDA currents. 4. Depolarizing voltage pulses (1-10 s) also induced an inactivation of NMDA currents with a slow recovery. The time course of the recovery increased with prolongation of depolarizing pulses and with an elevation of external calcium. Cadmium, a blocker of voltage-gated channels, prevented development of the depolarization-induced inactivation of NMDA currents. 5. Simultaneous recording of ionic currents and fluorescence of Ca(2+)-sensitive dyes showed that application of kainate, NMDA, or depolarizing pulses resulted in a rise of [Ca2+]i. Cadmium (100 microM) reversibly blocked [Ca2+]i transients induced by depolarizing pulses without modification of kainate-induced rise in fluorescence intensity. 6. For equal inward currents the elevation of [Ca2+]i was approximately 3.5-fold higher for applications of NMDA than for kainate. 7. Strong buffering of [Ca2+]i prevented the inactivation of NMDA currents induced by kainate or by depolarization. 8. Our results suggest that in the hippocampal neurons kainate produces inactivation of NMDA currents via an elevation of [Ca2+]i.

Animals↗
PubMed1994

Glutamate metabotropic receptors increase a Ca(2+)-activated nonspecific cationic current in CA1 hippocampal neurons.

1. We studied the currents evoked in CA1 pyramidal neurons by the selective metabotropic glutamate receptor (mGluR) agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD; 100 microM, 2.30-5 min) with the single-electrode voltage-clamp technique in the continuous presence of tetrodotoxin (1 microM), bicuculline (10 microM), 6-cyano-7-nitroquinoxaline-2,3-dione (15 microM), and D-2-amino-5-phosphonovaleric acid (50 microM) to depress action potentials and synaptic activity. Microelectrodes were filled with 3M CsCl or 2 M Cs2SO4. 2. With CsCl-filled microelectrodes, bath application of 1S,3R-ACPD induced an inward current of -308 +/p 50 (SE) pA amplitude [holding potential (VH -60 mV, n = 12)] associated with a conductance decrease (26.5 +/- 5.6%, P < or = 0.0022, n = 12). The current-voltage (I-V) relation of the 1S,3R-ACPD-induced (difference) current investigated using ramp voltage commands from -130 to +10 mV had a V shape with two reversal potentials: -99.6 +/- 3.4 and -17.5 +/- 3.0 mV (n = 12). 3. In contrast, in the presence of external K+ channel blockers (2 mM Ba2+ and 6 mM Cs+ or 25 mM tetraethylammonium, 6 mM Cs+, and 3 mM 4-aminopyridine), 1S,3R-ACPD also generated an inward current, albeit of smaller amplitude (-114.2 +/- 27.5 pA, P < or = 0.003, VH -60 mV, n = 8). This current was associated with a conductance increase (20.7 +/- 3.1%, P < or = 0.0117, n = 8), decreased linearly with depolarization (from -130 to -60 mV), and reversed polarity at an estimated potential of -20.7 +/- 3.6 mV (n = 8). We refer to this current recorded in the presence of K+ channel blockers as IACPD. 4. In the presence of Cd2+ (200 microM, to block voltage-dependent Ca2+ channels that are readily activated in the presence of K+ channel blockers) and a low Ca2+ concentration (100 microM), IACPD decreased linearly from -130 to +10 mV and reversed polarity at -15.8 +/- 8.5 mV (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Anoxic LTP is mediated by the redox modulatory site of the NMDA receptor.

1. The effects of redox reagents, 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and tris(carboxyethyl)phosphine (TCEP), on anoxia-induced long-term potentiation (LTP) were investigated in CA1 hippocampal neurons using extracellular recording techniques. Experiments were performed in the presence of 0.1 mM MgCl2 and 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to pharmacologically isolate N-methyl-D-aspartate (NMDA) receptor-mediated responses. 2. DTNB (200 microM), a thiol oxidizing reagent, reduces by 52 +/- 9% (mean +/- SE) (n = 9/9) NMDA-receptor field potentials evoked by electrical stimulation of Schaffer collaterals and this effect could not be reversed by extensive washing. Nearly the same reduction of the initial response was obtained with different concentrations of DTNB (100 and 500 microM), but the time required to reach the maximal inhibition was concentration-dependent. 3. In keeping with an earlier study oxygen and glucose deprivation for 2-3 min induced a long-term potentiation (LTP) of the NMDA receptor response (+65 +/- 16%, n = 4/6). This potentiation was reversed by DTNB (100-500 microM) (-47 +/- 18%; n = 4/4) and the initial LTP could not be restored upon extensive washing of the drug. 4. TCEP (200 microM), a reagent which reduces S-S bond, amplified the electrically evoked NMDA-receptor EPSP (+27 +/- 12%; n = 3). In addition, TCEP (200 microM), nearly completely reversed the effect of DTNB (200 microM) on anoxia-induced LTP (+56 +/- 19%; n = 3/3). Preliminary results also indicate that TCEP occlude anoxic-LTP (n = 3/4). 5. Following DTNB (200 microM) treatment, oxygen and glucose deprivation did not generate anoxic LTP and extensive washing did not restore a potentiated NMDA field potential. 6. These observations strongly suggest that the redox site of the NMDA receptor is involved in the induction and the maintenance of the anoxic LTP of the NMDA receptor-mediated response in CA1.

6-Cyano-7-nitroquinoxaline-2,3-dione↗