Showing papers by "Yehezkel Ben-Ari published in 1996"

Apoptosis and necrosis after reversible focal ischemia: an in situ DNA fragmentation analysis.

Abstract: Apoptosis is one of the two forms of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alterations including membrane blebbing and nuclear and cytoplasmic condensation. Early activation of an endonuclease has been previously demonstrated after a transient focal ischemia in the rat brain (Charriaut-Marlangue C, Margaill I, Plotkine M, Ben-Ari Y (1995) Early endonuclease activation following reversible focal ischemia. J Cereb Blood Flow Metab 15:385–388). We now show that a significant number of striatal and cortical neurons exhibited chromatin condensation, nucleus segmentation, and apoptotic bodies increasing with recirculation time, as demonstrated by in situ labeling of DNA breaks in cryostat sections. Apoptotic nuclei were also detected in the horizontal limb diagonal band, accumbens nucleus and islands of Calleja. Several necrotic neurons, in which random DNA fragmentation occurs, we...

Bidirectional plasticity expressed by GABAergic synapses in the neonatal rat hippocampus.

Abstract: 1. Activity-dependent plasticity of GABAergic synaptic transmission was investigated in neonatal rat hippocampal slices obtained between postnatal day (P) 2-10 using intracellular recording techniques. In all experiments, AMPA receptors were blocked by continual application of CNQX (10 microM). 2. Between P2 and P4, tetanic stimulation (TS) evoked NMDA receptor-dependent long-term depression of monosynaptic GABAA EPSPS (LTDGABAA). In contrast, when NMDA receptors were blocked by D-AP5 (50 microM), the same TS evoke long-term potentiation of GABAA EPSPS (LTPGABAA). 3. Between P6 and P10, TS failed to produce either LTP or LTD or hyperpolarizing monosynaptic GABAA IPSPS under the same recording conditions. However, when GABAergic potentials were rendered depolarizing (KCl-filled electrode) Ts induced either LTPGABAA or LTDGABAA in the presence or absence of D-AP5, respectively. 4. Both LTPGABAA and LTDGABAA were specific to the conditioned pathway and could be sequentially expressed at the same synapses. Potentiation of GABAergic synaptic efficacy was induced more easily following previous induction of LTDGABAA than in naive slices. 5. In conclusion, early in development, bidirectional synaptic plasticity is expressed by GABAA receptors and the activation (or not) of NMDA receptors determines the induction of either LTPGABAA or LTDGABAA.

Spontaneous release of GABA activates GABAB receptors and controls network activity in the neonatal rat hippocampus.

Abstract: 1. We investigated the effects of the selective gamma-aminobutyric acid-B (GABAB) receptor antagonist, P-3 aminopropyl-P-diethoxymethyl phosphoric acid (CGP 35348), on spontaneous and evoked postsynaptic potentials (PSPs) and currents (PSCs) in CA3 pyramidal cells and interneurons of hippocampal slices obtained between postnatal day 3 and 7 with the use of intracellular and whole cell recording techniques. The intracellular pipette solution contained either 2 M CsCl or 50 mM 2(triethylamino)-N-(2,6-dimethylphenyl) acetamine (QX314) dissolved in 2 M KMeSO4. Cesium and QX314 block postsynaptic responses mediated by GABAB receptors. 2. Under control conditions, bath application of CGP 35348 (0.5-1 mM) progressively increased the duration of spontaneous and evoked polysynaptic giant GABAergic PSPs leading to the appearance of ictal-like discharges. The effects of CGP 35348 were dose dependent and voltage independent. 3. In CA3 pyramidal neurons, CGP 35348 (0.5 mM) had no effect on monosynaptic GABAergic inhibitory PSPs (IPSPs) that were isolated in the presence of ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) and D(-)2-amino-5-phosphovaleric acid (D-APV, 50 microM). Similarly, CGP 35348 (0.5 mM) had no effect on monosynaptic glutamatergic excitatory PSPs (EPSPs) that were isolated in the presence of bicuculline (10 microM) and high divalent cation artificial cerebrospinal fluid (ACSF; 6 mM Mg2+/4 mM Ca2+). 4. In CA3 pyramidal neurons exposed to CNQX (20 microM) and D-APV (50 microM), application of the potassium channel blocker 4-aminopyridine (4-AP, 50 microM) generated synchronous giant GABAergic PSPS that were blocked in the presence of high divalent cation ACSF (6 mM Mg2+/4 mM Ca2+) or bicuculline (10 microM). The duration of these synchronous GABAergic PSPs was prolonged in the presence of CGP 35348 (0.5 mM) but did not lead to the appearance of ictal-like discharges. 5. In the presence of bicuculline, interictal giant glutamatergic potentials were observed in simultaneously recorded CA3 pyramidal cells and interneurons. CGP 35348 (0.5 mM) progressively increased the duration of these bicuculline-induced glutamatergic bursts leading to the simultaneous appearance of ictal discharges in both pyramidal cells and interneurons. 6. These results suggest that in the neonatal CA3 hippocampal region, when synchronous giant polysynaptic GABAergic PSPs are present (i.e., under basal, control conditions), spontaneously released GABA reaches a critical level and activates GABAB receptors on both pyramidal cells and interneurons thus regulating the level of glutamatergic and GABAergic activity in the CA3 neuronal network.

Transient increase of tenascin-C in immature hippocampus: astroglial and neuronal expression.

Abstract: In the present report we describe the anatomical localization of cells expressing tenascin-C, an extracellular matrix glycoprotein, in the hippocampal complex of developing rats. We report a development-dependent down regulation of both tenascin-C protein and mRNA. The highest levels of expression of tenascin-C was observed in rat pups from embryonic day 18 to postnatal day 7. Double labelling experiments performed with a tenascin-C antibody or tenascin-C probes combined with specific markers of astrocytes (GFAP) or neurons (MAP2 and Tau) allowed us to demonstrate that tenascin-C is expressed by both immature astrocytes and neurons in immature hippocampus. The temporal and topographic distribution of cells expressing of post-mitotic cells. In view of these data we discuss the hypothesis that tenascin-C, as a mediator of neuron-glia interaction, may contribute to the development of hippocampal cells.

Seizures induce tenascin-C mRNA expression in neurons

Abstract: Tenascin-C, an extracellular matrix glycoprotein that exhibits both growth-promoting and growth-inhibiting properties, is produced in the CNS mainly by astrocytes. In the present study we show that kainate-induced seizures result in an increased expression of tenascin-C in rat brain. Tenascin-C mRNA was increased mainly in the granule cell layer of the hippocampal complex, but tenascin-C mRNA expression was also observed in the pyriform cortex and amygdalo-cortical nucleus. Double labelling experiments using tenascin-C probes and MAP2 (a neuronal microtubule associated protein) antibodies revealed many neurons in these layers that express tenascin-C mRNA. These results support our previous findings of an increased tenascin-C immunoreactivity associated with the axons of granule cells. Tenascin-C expression is rapidly induced by seizures (6h), preceding any lesion and glial reaction. In this pathological condition tenascin-C appears to be produced by both glia and neurons. The functional repercussions on the scarring and remodelling processes are also discussed.

Intracellular injection of a Ca2+ chelator prevents generation of anoxic LTP.

Abstract: 1. The effects of intracellular injection of Ca2+ chelator 1,2-bis (2-aminophenoxy) ethane N,N,N',N'-tetra-acetic acid (BAPTA, 50 mM) on anoxia-aglycemia-induced long-term potentiation (LTP) were investigated in the CA1 region of hippocampal slices with the use of extra- and intracellular recording techniques. Experiments were performed in artificial cerebrospinal fluid (ACSF) containing 10 microM bicuculline and 10 microM 6-cyano-7-nitroquinoxaline- 2,3-dione (CNQX) to pharmacologically isolate N-methyl-D-aspartate (NMDA)-receptor-mediated responses. NMDA-receptor-mediated excitatory postsynaptic potentials (EPSPs) and field potentials were evoked by stimulation of the Schaffer collateral/commissural pathway in the presence of 0.3 mM MgCl2 and 10 microM glycine to promote NMDA-receptor-mediated responses. Under these conditions, application of 50 microM D-2-amino-phosphono-valerate (D-APV) abolished EPSPs and field potentials. 2. Anoxic-aglycemic (AA) episodes (duration 2-2.5 min) potentiated the initial slope (measured within 3 ms from the onset of the synaptic responses) of EPSPs by 108 +/- 14.3% (mean +/- SE, P = 0.0012, n = 7). We refer to this LTP of NMDA-receptor-mediated synaptic responses as anoxic LTP. 3. Intracellular injection of the Ca2+ chelator BAPTA (with the intracellular recording electrode filled with 50 mM BAPTA in 3 M KCl) prevented anoxic LTP. Thirty to 40 min after the AA episode, in BAPTA-loaded cells, the initial slope of the EPSPs was not significantly changed (+7.12 +/- 5%, P = 0.35, n = 5). In contrast, the initial slope of the field potentials, measured at the same time in the same slices, was persistently increased (+49 +/- 2.8%, P = 0.0022, n = 5). 4. High-frequency tetanic stimulation (100 Hz for 500 ms, 2 times, 30 s apart) of the Schaffer collateral/commissural pathway, applied > 0.5 h after the AA episode, induced an additional significant and persistent increase in the initial slope of the field potential (tetanic LTP, +35.4 +/- 9.8%, P = 0.012, n = 5). In BAPTA-loaded cells, there was no further change in the initial slope of the EPSP (+3.9 +/- 3.4%, P = 0.205, n = 5) after the tetanic stimulation. 5. We also report that AA episodes or tetanic stimulation induced a persistent increase in a late synaptic component that was blocked by 50 microM D-APV. This late component was mediated polysynaptically, because its time to peak decreased with increasing stimulation intensities and it was strongly reduced by high-divalent-cation superfusate (ACSF containing 7 mM Ca2+). This component, which had a delay of approximately 8-30 ms, contaminated mainly the peak amplitude and the decay of the monosynaptic response without affecting its initial slope. Thus the measure of the initial slope takes into account only the early phase of the monosynaptic response. 6. We conclude that 1) a rise in intracellular Ca2+ is necessary to generate anoxic LTP of NMDA-receptor-mediated responses, as is the case for tetanic LTP; and 2) in the presence of bicuculline and low extracellular Mg2+, AA episodes and tetanic stimulations induced a long-lasting enhancement of a polysynaptic component mediated or controlled by NMDA receptors.

The HIV-1 envelope protein gp120 induces neuronal apoptosis in hippocampal slices.

Abstract: The HIV-1 envelope protein gp120 produces neuronal cell damage in primary cultures of a variety of cell types including hippocampal and retinal ganglion cell neurons. The properties of primary cell cultures are, however, often markedly different from those of cells living in their normal environment. We now report that gp120 induces widespread chromatin condensation and lesions in pyramidal granular neurones and in interneurones of rat hippocampal organotypic slice cultures. This damage is clearly of an apoptotic (programmed cell death) type. The use of an in vitro organized structure will enable the molecular and cellular mechanism of action of gp120 to be examined in conditions which are particularly suitable and relevant to the in vivo situation.

MAP2d promotes bundling and stabilization of both microtubules and microfilaments

Abstract: 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.

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

Abstract: 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.

Reactive glial cells express a vitronectin-like protein in the hippocampus of epileptic rats.

Abstract: Injection of kainic acid into the amygdala induces in addition to a local cell loss a seizure related distal damage of the hippocampal complex, in particular in the CA3 field and hilus. This neuronal lesion is associated with hypertrophy and proliferation of astroglial cells which start around 3 days after kainate and peaks within 20 days of kainate. We now report that reactive astrocytes are labelled with antibodies against vitronectin in the CA3 field and hilus. In the present study we also exclude that the presence of vitronectin into the brain is due to an extravasation from serum throughout a blood brain barrier leakage. The present results constitute the first demonstration for a glial expression of vitronectin in vivo. Vitronectin is an extracellular matrix glycoprotein involved in axonal growth. The glial expression of vitronectin may therefore contribute to the synaptic remodeling of mossy fibers induced in the hippocampus by such treatment.