Biomedical subjects

E Cherubini

Publications and source records attributed to E Cherubini.

At least 37 records · Page 2Linked to original sources

PubMed1999

Long-term synaptic changes induced by intracellular tetanization of CA3 pyramidal neurons in hippocampal slices from juvenile rats.

Minimal excitatory postsynaptic potentials were evoked in CA3 pyramidal neurons by activation of the mossy fibres in hippocampal slices from seven- to 16-day-old rats. Conditioning intracellular depolarizing pulses were delivered as 50- or 100-Hz bursts. A statistically significant depression and potentiation was induced in four and five of 13 cases, respectively. The initial state of the synapses influenced the effect: the amplitude changes correlated with the pretetanic paired-pulse facilitation ratio. Afferent (mossy fibre) tetanization produced a significant depression in four of six inputs, and no significant changes in two inputs. Quantal content decreased or increased following induction of the depression or potentiation, respectively, whereas no significant changes in quantal size were observed. Compatible with presynaptic maintenance mechanisms of both depression and potentiation, changes in the mean quantal content were associated with modifications in the paired-pulse facilitation ratios, coefficient of variation of response amplitudes and number of response failures. Cases were encountered when apparently "presynaptically silent" synapses were converted into functional synapses during potentiation or when effective synapses became "presynaptically silent" when depression was induced, suggesting respective changes in the probability of transmitter release. It is concluded that, in juvenile rats, it is possible to induce lasting potentiation at the mossy fibre-CA3 synapses by purely postsynaptic stimulation, while afferent tetanization is accompanied by long-lasting depression. The data support the existence not only of a presynaptically induced, but also a postsynaptically induced form of long-term potentiation in the mossy fibre-CA3 synapse. Despite a postsynaptic induction mechanism, maintenance of both potentiation and depression is likely to occur presynaptically.

Animals↗

PubMed1999

Glutamate controls the induction of GABA-mediated giant depolarizing potentials through AMPA receptors in neonatal rat hippocampal slices.

Glutamate controls the induction of GABA-mediated giant depolarizing potentials through AMPA receptors in neonatal rat hippocampal slices. Giant depolarizing potentials (GDPs) are generated by the interplay of the depolarizing action of GABA and glutamate. In this study, single and dual whole cell recordings (in current-clamp configuration) were performed from CA3 pyramidal cells in hippocampal slices obtained from postnatal (P) days P1- to P6-old rats to evaluate the role of ionotropic glutamate receptors in GDP generation. Superfusion of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10-40 microM) completely blocked GDPs. However, in the presence of CNQX, it was still possible to re-induce the appearance of GDPs with GABA (20 microM) or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxadepropionate (AMPA) (5 microM). This effect was prevented by the more potent and selective AMPA receptor antagonist GYKI 53655 (50-100 microM). In the presence of GYKI 53655, both kainic or domoic acid (0.1-1 microM) were unable to induce GDPs. In contrast, bath application of D-(-)-2-amino-5-phosphonopentanoic acid (50 microM) or (+)-3-(2carboxy-piperazin-4-yl)-propyl-L-phosphonic acid (20 microM) produced only a 37 +/- 9% (SE) and 36 +/- 11% reduction in GDPs frequency, respectively. Cyclothiazide, a selective blocker of AMPA receptor desensitization, increased GDP frequency by 76 +/- 14%. Experiments were also performed with an intracellular solution containing KF to block GABAA receptor-mediated responses. In these conditions, a glutamatergic component of GDP was revealed. GDPs could still be recorded synchronous with those detected simultaneously with KCl-filled electrodes, although their amplitude was smaller. Similar results were found in pair recordings obtained from minislices containing only a small portion of the CA3 area. These data suggest that GDP generation requires activation of AMPA receptors by local release of glutamate from recurrent collaterals.

Animals↗

PubMed1998

Cholinergic function in the hippocampus of juvenile rats chronically deprived of NGF.

Intracellular and extracellular recordings were used to assess the cholinergic function in hippocampal slices from juvenile rats chronically deprived of NGF. NGF was neutralised by implanting into the lateral ventricle of postnatal (P) day 2 rats, alphaD11 hybridoma cells (secreting monoclonal antibodies specific for NGF). Parental myeloma cells (P3U) were used as controls. At P15-P18, slow cholinergic EPSPs could be elicited in cells from both alphaD11- and P3U-treated rats. However, slices from alphaD11-implanted rats exhibited a 50% reduction in acetylcholine release following stimulation of cholinergic fibres. This effect was associated to a significant increase in the sensitivity of pyramidal cells to carbachol, as suggested by the shift to the left of the dose/response curve. This may reflect a compensatory mechanism for the reduced efficacy of cholinergic innervation in NGF-deprived rats. In both alphaD11- and P3U-treated rats, carbachol was able to induce a similar concentration-dependent depression of the field EPSPs, evoked by Schaffer collateral stimulation, suggesting that presynaptic muscarinic receptors were not altered. In rats implanted with alphaD11 cells at P15 and sacrificed at P21-P24, no changes in the sensitivity to carbachol were found. At this developmental stage, no differences in acetylcholine release were observed between P3U- and alphaD11-treated animals. These results provide physiological evidence for a regulatory role of NGF in the cholinergic function of the hippocampus during postnatal development.

Acetylcholine↗

PubMed1998

Two distinct forms of long-term depression coexist at the mossy fiber-CA3 synapse in the hippocampus during development.

During a critical period of postnatal development, between postnatal days 6 and 14, a high-frequency stimulation train (100 Hz for 1 s) to the mossy fibers induces a long-term depression (LTD) of synaptic efficacy of 29 +/- 5.2%. This form of LTD is homosynaptic. It is independent of the activation of N-methyl-D-aspartate or metabotropic glutamate receptors but needs an increase in calcium into the postsynaptic cell for its induction. At the same synapse LTD also could be induced by low-frequency stimulation of the mossy fibers (1 Hz for 15 min). In this case the magnitude of the depression is 37 +/- 4.2%. This form of LTD is N-methyl-D-aspartate independent but requires the activation of metabotropic glutamate receptors because it is prevented by (S)-alpha-methyl-4-carboxyphenylglycine (1 mM). Moreover its induction appears to be presynaptic, because, in contrast with the high-frequency one, it is not blocked by loading the postsynaptic cell with the calcium chelator EGTA or bis-(-o-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid (BAPTA). Saturation of one form of LTD does not occlude the other, suggesting that high and low frequency-induced LTD depend on distinct mechanisms of induction and expression. Quantal (noise deconvolution) analysis of minimal excitatory postsynaptic potentials shows, similarly to high-frequency LTD, a decrease in quantal content without any change in quantal size after low-frequency LTD, suggesting that in both forms of LTD the site where maintenance mechanisms are located is presynaptic.

Animals↗

PubMed1998

Nitric oxide sensitive depolarization-induced hyperpolarization: a possible role for gap junctions during development.

Electrical coupling is a widespread feature of developing neuronal circuits and it contributes to the generation of patterned activity. In the developing rat hippocampus, release of GABA by coactive hilar interneurones generates widespread synchronized activity. Here it is shown that hilar interneurones strongly rectify in the outward direction when depolarized. This depolarization-induced hyperpolarization, abolished by gap junction uncouplers, is modulated by nitric oxide. This phenomenon might represent a current-shunting mechanism of the excess current by providing functional inhibition at a developmental stage when GABA is excitatory. Spatial buffering of the current might represent an osmotic mechanism for growth and differentiation.

Anesthetics, Inhalation↗

PubMed1998

A novel form of long-term depression in the CA1 area of the adult rat hippocampus independent of glutamate receptors activation.

In young rats, low frequency (1-2 Hz) stimulation of the Schaffer collaterals for 15 min induces in the CA1 area of the hippocampus a homosynaptic and N-methyl-D-aspartate receptor-dependent form of long-term depression (LTD) of synaptic efficacy. In the adults, while a similar stimulation paradigm is able to depress previously potentiated synapses, it leads to conflicting results when applied to naive synapses. In the present experiments, different stimulation paradigms have been used to induce LTD in the CA1 area of the adult rat hippocampus in vitro. Thus, stimulation of the afferent pathway at frequencies higher than those used to produce LTD in young animals (5-10 Hz, for 15 min) reliably induced a homosynaptic form of LTD. This form of LTD was associated with a significant increase in paired-pulse facilitation ratio and was insensitive to ionotropic (CNQX, 10 microM and CPP, 20 microM) and metabotropic (S-MCPG, 1 mM) glutamate receptors antagonists, suggesting a presynaptic mechanism for both LTD induction and expression. In conclusion, our experiments clearly show that LTD is not a purely developmental phenomenon but is present also in mature rats, which possess the whole machinery for LTD induction and this will greatly enhance the flexibility and the storing capacity of neuronal circuits.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1998

Changes in intracellular calcium concentration affect desensitization of GABAA receptors in acutely dissociated P2-P6 rat hippocampal neurons.

The whole cell configuration of the patch-clamp technique was used to study the effects of different cytosolic calcium concentrations [Ca2+]i on desensitization kinetics of gamma-aminobutyric acid (GABA)-activated receptors in acutely dissociated rat hippocampal neurons. Two different intrapipette concentrations of the calcium chelator 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; 11 and 0.9 mM, respectively) were used to yield a low (1.2 x 10(-8) M) or a high (2.2 x 10(-6) M) [Ca2+]i. In low [Ca2+]i, peak values of GABA-evoked currents (20 microM) evoked at -30 mV, were significantly larger than those recorded in high calcium [2,970 +/- 280 (SE) pA vs. 1,870 +/- 150 pA]. The extent of desensitization, assessed from steady-state to peak ratio was significantly higher in high calcium conditions (0.14 +/- 0.007 vs. 0.11 +/- 0.008). Similar effects of -Ca2+-i on desensitization were observed with GABA (100 microM). Recovery from desensitization, measured at 30 s interval with double pulse protocol was significantly slower in high [Ca2+]i than in low [Ca2+]i (54 +/- 3% vs. 68 +/- 2%). The current-voltage relationship of GABA-evoked currents was linear in the potential range between -50 and 50 mV. The kinetics of desensitization process including the rate of onset, extent of desensitization, and recovery were voltage independent. The run down of GABA-evoked currents was faster with the higher intracellular calcium concentration. The run down process was accompanied by changes in desensitization kinetics: in both high and low [Ca2+]i desensitization rate was progressively increasing with time as the slow component of the desensitization onset was converted into the fast one. In excised patches, the desensitization kinetics was much faster and more profound than in the whole cell configuration, indicating the involvement of intracellular factors in regulation of this process. In conclusion, [Ca2+]i affects the desensitization of GABAA receptors possibly by activating calcium-dependent enzymes that regulate their phosphorylation state. This may lead to modifications in cell excitability because of changes in GABA-mediated synaptic currents.

Animals↗

PubMed1998

Dynamics of excitatory transmitter release: analysis of synaptic responses in CA3 hippocampal neurons after repetitive stimulation of afferent fibers.

The patch-clamp technique (whole cell configuration) was used to record excitatory postsynaptic currents (EPSCs) evoked by repetitive stimulation (4 pulses at 50-ms intervals) of afferent fibers in the stratum lucidum-radiatum. Different synaptic behaviors (EPSC patterns) were classified in terms of facilitation or depression of the mean amplitude of the second, third, and fourth EPSC with respect to the previous one. A large variety of EPSC patterns was observed by stimulating different afferent fibers. Experiments with the mGluR2/mGluR3 agonist 2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) (1 microM), a compound that reduces release at mossy but not at associative commissural fibers and therefore allows to identify the origin of synaptic responses, showed that particular EPSC patterns could not be associated to the activation of a specific type of synaptic input. To investigate the role of the probability of release in the dynamics of synaptic activity, the extracellular calcium concentration was varied from 0.8 to 4 mM in several experiments. EPSC patterns dominated by depression, characteristics of high release probability conditions, could be observed in the majority of the cases in the presence of higher calcium concentrations. A quantitative model for dynamics of transmitter release has been developed. Experimental results were compared with data computed with the model taking into account the probability of release and the time course of reavailability. This work indicates that short-term changes of presynaptic conditions occurring during a train of action potentials can account for the high variability of EPSC responses. The model that is proposed also suggests a general method of experimental data analysis to investigate the possible presynaptic mechanisms underlying long-lasting changes in synaptic efficacy.

Action Potentials↗

PubMed1998

Facilitation of miniature GABAergic currents by ruthenium red in neonatal rat hippocampal neurons.

The whole cell configuration of the patch-clamp technique was used to study the modulation gamma-aminobutyric acid (GABA)-mediated postsynaptic currents by ruthenium red in CA3 hippocampal neurons in slices obtained from postnatal (P) days P6-P10 old rats. In the presence of kynurenic acid (1 mM), ruthenium red (100 microM) completely blocked stimulus-elicited GABA-mediated postsynaptic currents and reduced by 50% the amplitude of the spontaneous ones. Ruthenium red (100 microM) increased the frequency but not the amplitude of miniature GABAergic currents recorded in the presence of tetrodotoxin (1 microM) and kynurenic acid (1 mM), an effect that was prevented by heparin (100 microM). Ruthenium red did not modify the kinetics of miniature postsynaptic currents and the currents induced by exogenous application of GABA (10 microM) in the presence of tetrodotoxin, suggesting that its action was presynaptic in origin. The effects of ruthenium red on quantal GABA release was independent of external calcium. In a nominally Ca2+-free solution the potentiating effect induced by this polyvalent cation on miniature postsynaptic currents was still present. Intracellular calcium stores were not involved in ruthenium red action, because this polyvalent cation was able to facilitate miniature currents also in the presence of thapsigargin (10-20 microM). These results indicate that ruthenium red has a dual action on GABA release from GABAergic interneurons: it reduces the amplitude of spontaneous events and increases the frequency of miniature currents. The former effect is calcium-dependent, whereas the latter is calcium independent.

Animals↗

PubMed1998

GABA excites immature CA3 pyramidal cells through bicuculline-sensitive and -insensitive chloride-dependent receptors.

Intracellular and patch clamp recording techniques were used to investigate the role of GABA in immature CA3 hippocampal neurons. During the first postnatal week spontaneous GABA release was detected as spontaneous ongoing synaptic potentials (SPSPs) or giant depolarizing potentials (GDPs). GDPs were generated at regular intervals and regulated by ionotropic glutamate receptors (GluRs), whereas SPSPs occurred randomly and were unaffected by ionotropic GluRs. Both GDPs and SPSPs were positively modulated by metabotropic GluRs through cyclic AMP-dependent protein kinase. Moreover GABA controlled its own release through GABAA and GABAB receptors, probably localized on GABAergic nerve terminals. At this developmental stage, GABA depolarized CA3 pyramidal cells through two distinct classes of chloride-permeable receptors: bicuculline sensitive and insensitive, respectively. The bicuculline-insensitive responses were blocked by picrotoxin in a noncompetitive way. Whole-cell GABA currents, recorded in the presence of bicuculline, had a slower desensitization rate and faster recovery from desensitization. In excised outside-out patches, in the presence of bicuculline, GABA activated single-channel currents with conductances of 14, 22, and 31 pS. These values were similar to those obtained when GABA was applied in the absence of bicuculline. Interestingly, GABA responses obtained in the absence of bicuculline, were sensitive to the blocking effect of zinc, whereas bicuculline-resistant responses were almost unaffected by this divalent cation. Expression of different subunits in native receptors (particularly of the alpha and rho type) may account for the functional differences observed in the present experiments. Activation of bicuculline-insensitive receptors would strengthen and prolong the depolarizing action of GABA, thus favoring the entry of calcium through voltage-dependent calcium channels. This calcium signal may be essential in promoting stabilization of synaptic contacts during a critical period of postnatal development.

Animals↗

PubMed1997

Immunoglobulins from motoneurone disease patients enhance glutamate release from rat hippocampal neurones in culture.

1. The whole-cell configuration of the patch-clamp technique was used to study the effects of immunoglobulins (IgGs) from patients affected by amyotrophic lateral sclerosis (ALS) on spontaneous glutamatergic currents in rat hippocampal cells in culture. 2. Focal application of ALS IgGs (100 micrograms ml-1) to hippocampal cells induced a rise in frequency but not in amplitude of spontaneous excitatory postsynaptic currents (SEPSC) which outlasted the period of IgG application. The mean frequency ratio (ALS over control) was 3.2 +/- 0.6 (n = 19). No changes in frequency or amplitude of SEPSCs were observed after treatment with IgGs obtained from healthy donors (n = 5) or from patients with Alzheimer's disease (n = 4). 3. ALS IgGs also increased the frequency (by a factor of 2.0 +/- 0.3) but not the amplitude of miniature excitatory postsynaptic currents (mEPSC) recorded in the presence of TTX (n = 19). A rise in frequency of mEPSC was also seen in cells superfused with a calcium-free solution (n = 4). 4. In the presence of TTX, ALS IgGs did not modify the amplitude or the shape of currents evoked by AMPA (100 microM), recorded at a holding potential of -50 mV. 5. It is concluded that ALS IgGs enhance both SEPSCs and mEPSCs through a presynaptic type of action. The excessive release of glutamate from nerve endings may be the cause of motoneurone death in ALS patients.

Adult↗

PubMed1997

NGF antibodies impair long-term depression at the mossy fibre-CA3 synapse in the developing hippocampus.

Nerve growth factor (NGF) and other neurotrophins are proteins involved in neuronal survival and differentiation. Much experimental evidence is now drawing attention into a role of neurotrophins in activity-dependent synaptic plasticity processes. We now show that slices from rats chronically deprived of NGF, by intraventricular injection of alpha D11 hybridoma cells, which produce monoclonal antibodies against NGF, display a reduced probability of induction of long-term depression at the mossy fibre-CA3 synapse.

Animals↗

PubMed1997

A pacemaker current in dye-coupled hilar interneurons contributes to the generation of giant GABAergic potentials in developing hippocampus.

The establishment of synaptic connections and their refinement during development require neural activity. Increasing evidence suggests that spontaneous bursts of neural activity within an immature network are mediated by gamma-aminobutyric acid via a paradoxical excitatory action. Our data show that in the developing hippocampus such synchronous burst activity is generated in the hilar region by transiently coupled cells. These cells have been identified as neuronal elements because they fire action potentials and they are not positive for the glial fibrillary acidic protein staining. Oscillations in hilar cells are "paced" by a hyperpolarization-activated current, with properties of Ih. Coactivated interneurons synchronously release GABA, which via its excitatory action may serve a neurotrophic function during the refinement of hippocampal circuitry.

Aging↗

PubMed1997

Neurotrophin-3 promotes the survival of oligodendrocyte precursors in embryonic hippocampal cultures under chemically defined conditions.

embryonic rat hippocampal cells were cultured in basal medium with or without addition of the neurotrophin NT-3. After culturing in these extreme conditions, the effects of NT-3 on the neuronal and on the glial components were assessed. Neurons survived even in the absence of NT-3 but failed to reach terminal differentiation. On the other hand, NT-3 promoted the survival but not the proliferation and/or the differentiation of oligodendrocytes precursors present in the same culture, an effect that was reversed by the addition of neutralizing antibodies against NT-3. Type I or II astrocytes were not affected by NT-3. These results reinforce the role for NT-3 in oligodendrocyte lineage development and allow to dissect the roles of this neurotrophin in survival and in proliferation/differentiation of oligodendrocytes.

Animals↗

PubMed1997

Glycine-activated whole cell and single channel currents in rat cerebellar granule cells in culture.

The patch clamp technique was used to study whole cell and single channel currents evoked by glycine in cerebellar granule cells in culture. Whole cell concentration response curve gave a Kd value for glycine of 73 microM and a Hill slope of 1.58. Glycine-activated currents reversed close to the predicted Cl- equilibrium potential. The responses to glycine were antagonized by strychnine and picrotoxin with an IC50 of 58 nM and 172 microM, respectively. Furthermore, glycine-evoked currents were potentiated by zinc in a dose-dependent way. In outside-out membrane patches, glycine opened channels with conductances of 32, 52, 84 and 96 pS. The most frequently occurring was the 52 pS channel. The single channel current/voltage relationship was linear in the potential range between -60 and 60 mV. The 52, 84 and 96 pS channels exhibited prolonged openings whereas the 32 pS was characterized by fast (< 10 ms) openings. Open and closed time histograms of the 52 pS channel could be fitted with the sum of two or three exponentials, respectively, whereas burst duration histograms could be fitted with the sum of two exponentials. Glycine current density change drastically during days in culture, the maximal expression being between day 4 and 7, suggesting that the expression of glycine receptor channels is developmentally regulated.

Animals↗

PubMed1997

Functionally distinct chloride-mediated GABA responses in rat cerebellar granule cells cultured in a low-potassium medium.

The patch-clamp technique was used to study whole cell currents evoked by gamma-aminobutyric acid (GABA) in rat cerebellar granule cells cultured in 5 mM potassium, a condition that favors the development of functional GABAergic synapses. GABA activated both high- and low-sensitivity receptors. The high-sensitivity receptor had an effective concentration producing half the maximum response (EC50) of 13 microM, whereas the low-sensitivity one had an EC50 of 255 microM. The GABAA receptor agonist isoguvacine activated only the high-sensitivity receptor with an EC50 of 16 microM. When GABA was applied during the desensitized phase of the response elicited by a saturating concentration of isoguvacine, it was still able to induce a small response, whereas when isoguvacine was applied during the desensitizing phase of GABA-evoked current no response was detected. GABA responses were highly heterogeneous regarding their sensitivity to bicuculline. In a small number of cells (3 of 25), bicuculline (10 microM) completely abolished GABA-evoked currents. In the majority of the neurons (22 of 25) the blocking effect of bicuculline (100 microM) was 64 +/- 4% (mean +/- SE). The bicuculline-resistant component was abolished by picrotoxin (100 microM). In bicuculline, the dose-response curve for GABA was fitted with a sigmoidal curve with an EC50 value of 209 microM. These data indicate that functional new GABA receptor types with unusual pharmacology could be switched on by conditions that maintain cells in their undifferentiated state.

Animals↗

PubMed1996

The calcineurin inhibitor cyclosporin A-cyclophilin A complex reduces desensitization of GABAA-mediated responses in acutely dissociated rat hippocampal neurons.

The effects of the selective inhibitor of calcineurin, cyclosporin A-cyclophilin A (CC) complex on the desensitization kinetics of GABAA receptors was studied in acutely dissociated hippocampal neurons, using the patch clamp technique in the whole cell configuration. In control conditions, the decay of GABA-evoked current could be fitted by a biexponential function having time constants of 0.65 +/- 0.24 s and 3.75 +/- 2 s. The plateau to peak ratio was 0.087 +/- 0.034. Recovery from desensitization was obtained in more than 2 min. In cells dialyzed with the CC complex, the decay of the currents could be fitted with the sum of two exponentials having time constants similar to controls (0.81 +/- 0.47 s and 3.62 +/- 2.1 s), but the percentage of the fast component was smaller. The plateau to peak ratio was significantly larger than control (0.185 +/- 0.07). With CC complex, recovery from desensitization was completed in almost 30 s. The cyclosporin A derivative PSC 833, which does not inhibit calcineurin, did not affect desensitization kinetics. These results suggest that phosphatase 2B regulates desensitization of GABAA receptors.

Animals↗

PubMed1996

ALS IgGs suppress [Ca2+]i rise through P/Q-type calcium channels in central neurones in culture.

Confocal laser scanning microscopy (with the fluorescent calcium dye fluo-3) was used to test the effect of IgG obtained from patients with amyotrophic lateral sclerosis (ALS) on the KCl-induced [Ca2+] rise in rat hippocampal neurones in culture. In the presence of tetrodotoxin and ionotropic glutamate receptor antagonists, ALS IgGs depressed (by 30-40%) Ca2+ transients evoked by influx of Ca2+ through voltage-activated channels; such an effect did not occur with IgG obtained from healthy donors. The depressant action of ALS IgG was selectively prevented by the inhibitor of P/Q-type Ca2+ channels, omega-agatoxin IVA (which alone reduced Ca2+ transients by 40%). The reduced Ca2+ transients might impair Ca(2+)-dependent glutamate receptor desensitization and thus facilitate excitotoxic damage.

Amyotrophic Lateral Sclerosis↗