Biomedical subjects

E Cherubini

Publications and source records attributed to E Cherubini.

At least 55 records · Page 3Linked to original sources

PubMed1996

Postsynaptic induction of mossy fibre long term depression in developing rat hippocampus.

The whole cell configuration of the patch clamp technique was used to study the mechanisms of induction of long term depression (LTD) occurring at the mossy fibre-CA3 synapse between postnatal (P) day 6 and P13. In control conditions, when two pulses were delivered to the mossy fibres with an interval of 50 ms a potentiation of the EPSC evoked by the second pulse associated with a reduction in the number of failures was observed. Tetanization of the mossy fibres induced LTD of the responses to the first and second stimulus without affecting the paired pulse facilitation. Loading the postsynaptic cell with BAPTA prevented the induction of LTD but did not modify the paired pulse facilitation, suggesting that LTD induction occurs at the postsynaptic site.

Animals↗

PubMed1996

Changes in intracellular calcium induced by NMDA in cultured rat hippocampal neurons require exogenous glycine.

Confocal laser scanning microscopy was used to study changes in intracellular free calcium concentration ([Ca2+]i) at the level of the soma of cultured hippocampal neurones following pressure application of glutamate or N-methyl-D-aspartate (NMDA). [Ca2+]i was imaged in the presence of tetrodotoxin after loading cells with the fluorescent dye indicator fluo-3/AM. Responses to glutamate were potently antagonized by 6-cyano-7 nitroquinoxaline-2,3-dione (CNQX: 20 microM). They were also strongly and reversibly depressed by 3-((+/-)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP; 20 microM), leaving a small CNQX-sensitive component. Responses to NMDA were also blocked by CNQX. In the presence of saturating concentrations of glycine (100 microM), the depression of glutamate or NMDA responses by CNQX was greatly reduced. Exogenously applied glycine also potentiated the NMDA response. These data indicate that the glycine binding site of the NMDA receptor channel is not saturated in cultured hippocampal neurones and thus is susceptible to the action of agonists or antagonists.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1996

Zinc modulation of bicuculline-sensitive and -insensitive GABA receptors in the developing rat hippocampus.

Intracellular recordings were used to study the effects of zinc on the bicuculline-sensitive and -insensitive responses evoked by GABA in CA3 rat hippocampal neurons in slices obtained from postnatal day (P) 0 to P8. In the absence of bicuculline, zinc inhibited GABA-induced responses in a concentration-dependent manner. This effect was developmentally regulated, being maximal (50%) between P0 and P5 and then declining to 30% after P5. In the presence of bicuculline, GABA-resistant responses were potentiated in 49% of cases, depressed in 38% and not affected in 13%. The period of maximum potentiation between P0 and P2 coincided with that of maximum expression of the bicuculline-resistant receptors. The effects of zinc were also studied using the whole-cell and outside-out configuration of the patch-clamp technique on bicuculline-sensitive and -insensitive GABA-induced currents elicited in isolated cells acutely dissociated from the same slices as those used for intracellular recordings. At a holding potential of -50 mV in symmetrical chloride solutions, GABA (50 and 100 microM) activated whole-cell inward currents which were reversibly blocked by zinc. The EC50 values for the blocking effect of zinc on currents evoked by 50 and 100 microM GABA were 6.6 nM and 5.8 microM respectively. In the presence of bicuculline (100 microM), zinc potentiated the residual responses to GABA; the response curve was bell-shaped with a peak at 1 microM. When the response to GABA was completely abolished by bicuculline, zinc (1 microM) was often able to restore it. In the presence of bicuculline, however, zinc was not able to restore the response to isoguvacine. In two excised outside-out patches, zinc (1 microM) increased the activity of opening of bicuculline-resistant GABA-evoked single channel currents (Np) from 1 to 1.87 and from 0.25 to 0.42 respectively, without changing single-channel conductance. These data suggest that down- or up-regulation of bicuculline-sensitive or -insensitive GABA receptors may be functionally important in regulating synaptic activity during development.

Animals↗

PubMed1995

Cyclic AMP-dependent modulation of giant depolarizing potentials by metabotropic glutamate receptors in the rat hippocampus.

1. Intracellular recordings were used to study the role of metabotropic glutamate receptors (mGluRs) in modulating GABA-mediated giant depolarizing potentials (GDPs) in immature rat hippocampal CA3 neurones. 2. The mGluR antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG, 1 mM) reduced the frequency of GDPs. The broad-spectrum ionotropic glutamate receptor antagonist kynurenic acid (1 mM) blocked GDPs. 3. In the presence of kynurenic acid, both tetanic stimulation of the hilus or bath application of quisqualic acid (1 microM) and trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD, 20 microM) induced the appearance of GDPs. These effects were antagonized by MCPG (1 mM) or L(+)-2-amino-3-phosphonopropionic acid (L-AP3) and blocked by bicuculline (10 microM). 4. 8-Bromo-cAMP (8-Br-cAMP, 0.3 mM), 3-isobutyl-1-methylxanthine (IBMX, 200 microM) or forskolin (30 microM) mimicked the effects of mGluR agonists on GDPs. The forskolin analogue 1,9-dideoxyforskolin (30 microM), which does not activate adenylate cyclase, was ineffective. 5. Incubation of slices in the presence of the protein kinase A inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPS) (500 microM) or superfusion of Rp-cAMPS (20 microM) prevented the effects of forskolin or t-ACPD on GDPs. In the presence of kynurenic acid, the protein kinase C activator, phorbol 12,13-diacetate (2 microM) induced the appearance of GDPs. This effect was prevented by staurosporine (1 microM). However, staurosporine (1-3 microM) did not modify the effects of t-ACPD on GDPs. 6. It is suggested that, during development, mGluRs enhance the synchronous release of GABA, responsible for GDPs, through cAMP-dependent protein kinase.

Animals↗

PubMed1995

Functional expression of voltage dependent sodium channels in Xenopus oocytes injected with mRNA from neonatal or adult rat brain.

The two electrode voltage clamp technique was used to study voltage-dependent sodium currents (INa) in Xenopus laevis oocytes previously injected with mRNA extracted from adult (A) or neonatal (N, < 5 days old) rat brains. In the presence of niflumic acid (300 microM) to block endogenous Ca(2+)-activated Cl- currents, depolarizing voltage steps from a holding potential of -100 mV to various voltages elicited in both groups of oocytes fast inward sodium currents which peaked at approximately 0 mV and then slowly declined to approximately 75% of the maximum current at +40 mV. At the peak, A INa was significantly larger than N INa (296 +/- 59 nA vs. 147 +/- 32 nA). Inactivation kinetics of N INa was best fit with one exponential component whereas A INa with two exponential components. A significant difference in the voltage dependence of inactivation was found between A INa or N INa. The values of Vh were -53 +/- 0.9 mV or -59.8 +/- 0.7 mV for A INa or N INa respectively. The recovery from inactivation was fitted in both groups with two exponential functions (tau f and tau s) whose values were not significantly different. However the ratio between tau f and tau s was significantly higher for N INa comparing to A INa (5.7 vs. 2.1). TTX reversibly blocked INa. The IC50 value was 58.2 +/- 6.3 nM for A INa and 20.4 +/- 2.2 nM for N INa. These results suggest that different isoforms of TTX-sensitive, voltage-dependent sodium channel subunits are functionally expressed, may be in different proportions in oocytes injected with A or N mRNA.

Aging↗

PubMed1995

Spontaneous GABA-mediated synaptic currents in cerebellar granule cells in culture.

The whole-cell configuration of the patch clamp technique was used to characterize the electrophysiological properties of spontaneous GABA-mediated synaptic currents in cerebellar granule cells grown in a low (5 mM) potassium medium. In the presence of kynurenic acid (1 mM), to block the excitatory drive, bicuculline-sensitive synaptic events were recorded. Their amplitude distribution could be fitted by several Gaussians having the same interpeak distance. In tetrodotoxin (TTX, 1 microM) spontaneous miniature events occurred at a lower frequency. Spontaneous currents reversed polarity at 8.17 +/- 0.63 mV, a potential close to ECl; the decay phase could be fitted with a single exponential having at -60 mV a time constant of 48.7 +/- 1.5 ms. In low noise recordings, channel closing could be resolved during the decay phase of miniature events. It appeared that a single quantum of GABA opened few channels on the postsynaptic membrane.

Animals↗

PubMed1995

Neurofilament-negative neurones exhibit HVA Ca2+ currents with faster inactivation kinetics.

The whole cell configuration of the patch clamp technique was used to study the biophysical and pharmacological properties of voltage activated Ca2+ channel currents on hippocampal neurones cultured in the presence or absence of foetal calf serum. In the presence of serum cells were intensively immunostained with neurofilament (NF) antibodies. In the absence of serum, cells were non-immunoreactive; they were identified as neurones by their ability to generate action potentials. NF negative cells still expressed both high (HVA) and low (LVA) voltage activated Ca2+ channels. However, in comparison to NF positive neurones, HVA Ca2+ currents present in NF negative neurones had a faster inactivation kinetics.

Action Potentials↗

PubMed1995

Protein kinase A-dependent increase in frequency of miniature GABAergic currents in rat CA3 hippocampal neurons.

The whole-cell configuration of the patch clamp technique was used to study the effect of an intracellular increase in cAMP on the frequency of GABA-mediated miniature post synaptic currents (MPSCs) in neonatal rats from (P6 to P12) CA3 hippocampal neurons in slices. In the presence of tetrodotoxin (1 microM), and kynurenic acid (1 mM) to block ionotropic glutamatergic currents, forskolin, an activator of adenylate cyclase, markedly increased the frequency of MPSCs without affecting their amplitude or kinetics. The inactive forskolin analog, 1,9-dideoxyforskolin (30 microM), had no effect on the frequency of MPSCs. The effect of forskolin was prevented by the specific protein kinase A (PKA) antagonist Rp-cAMP (30 microM). It is concluded that stimulation of PKA potentiates spontaneous GABA release in immature hippocampal neurons.

Animals↗

PubMed1995

Whole cell and single channel properties of a new GABA receptor transiently expressed in the Hippocampus.

1. The patch-clamp technique was used to characterize, in acutely dissociated CA3 rat hippocampal neurons, the whole cell and single channel properties of a novel response to gamma-aminobutyric acid (GABA) present only during a restricted period of postnatal development. 2. At postnatal days 0-10 (P0-P10), both GABA (100 microM) and isoguvacine (50 microM) evoked at a holding potential of -50 mV, in symmetrical chloride solution, whole cell inward currents. Bicuculline blocked the response to isoguvacine but only reduced the response to GABA (from 512 +/- 137 pA to 60 +/- 13 pA, mean +/- SE). After P12, bicuculline abolished the response to GABA. 3. The bicuculline-insensitive GABA currents were Cl- mediated and antagonized by picrotoxin. The desensitization rate was slower than the conventional bicuculline-sensitive response. The peak to plateau ratio induced by 0.1 or 1 mM of GABA shifted from 4.6 +/- 0.4 and 17.7 +/- 2.6 to 1.5 +/- 0.1 and 3.1 +/- 0.5 in the absence or in the presence of bicuculline, respectively. The recovery from desensitization was significantly faster for the bicuculline-insensitive responses. 4. In excised outside-out patches, GABA (20 microM) activated, in the presence of bicuculline (100 microM), single channel currents having conductances of 14, 22, and 31 pS. These values were similar to those obtained in the same preparation, in the absence of bicuculline. 5. These findings suggest that this new receptor type, which mediates bicuculline-insensitive responses with slow kinetics, may potentiate the depolarizing action of GABA during a critical period of postnatal development and therefore play a crucial role in synaptogenesis.

Animals↗

PubMed1995

Persistent current oscillations produced by activation of metabotropic glutamate receptors in immature rat CA3 hippocampal neurons.

1. The single-electrode voltage-clamp technique was used to study the effects of the metabotropic glutamate receptors (mGluRs) agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD, ACPD, 3-10 microM) on CA3 hippocampal neurons during the 1st 10 days of postnatal (P) life and in adulthood. 2. Repeated applications of 1S,3R-ACPD, in the presence of tetrodotoxin (TTX, 1 microM), tetraethylammonium chloride (TEACl 10 mM), and CsCl (2 mM), induced in immature but not in adult neurons periodic inward currents (PICs) that persisted for several hours after the last application of the agonist. 3. PICs, which were generated by nonspecific cationic currents, reversed polarity at 2.8 +/- 3 (SD) mV. They were reversibly blocked by kynurenic acid (1 mM), suggesting that they were mediated by glutamate acting on ionotropic receptors. They were also abolished in a nominally Ca(2+)-free medium. 4. PICs were irreversibly abolished by thapsigargin (10 microM) but were unaffected by ryanodine (10-40 microM). Caffeine (2 mM) also reversibly blocked PICs; this effect was independent from adenosine 3',5'-cyclic monophosphate (cAMP) accumulation, inhibition of voltage-dependent Ca2+ current, or blockade of adenosine receptors. 5. We suggest that, in neonatal slices, mGluRs-induced PICs are triggered by elevation of [Ca2+]i, after mobilization of Ca2+ from inositol 1,4,5-trisphosphate (InsP3)-sensitive stores. This will lead to a persistent, pulsatile release of glutamate from presynaptic nerve terminals, a phenomenon that is probably maintained via a calcium-induced-calcium release process.

Animals↗

PubMed1995

Activation of metabotropic glutamate receptors increase the frequency of spontaneous GABAergic currents through protein kinase A in neonatal rat hippocampal neurons.

1. The tight-seal whole cell recording technique was used to study the effects of the metabotropic glutamate receptor (mGluR) agonist, trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) on spontaneous gamma-aminobutyric acid (GABA)-mediated synaptic currents in neonatal rat CA1 hippocampal neurons in slices obtained from postnatal (P) days P6-P12. 2. Bath application of t-ACPD (3-30 microM), in the presence of kynurenic acid, induced a concentration-dependent increase in frequency but not in amplitude of spontaneous GABAergic currents. The mean frequency ratio (t-ACPD 10 microM over control) was 2.6 +/- 1 (mean +/- SD), whereas the mean amplitude ratio was 1.1 +/- 0.3. 3. The effect of t-ACPD was partially antagonized by the mGluR antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG, 1 mM). 4. t-ACPD (10-30 microM) did not modify the frequency of miniature GABAergic synaptic currents recorded in tetrodotoxin (the mean frequency ratio of t-ACPD over control was 0.7 +/- 0.3). 5. Forskolin (30 microM), but not its analogue 1,9 dideoxyforskolin (30 microM), mimicked the effect of t-ACPD. Similar effects were obtained with 3-isobutyl-1-methylxanthine (IBMX, 200 microM). 6. The potentiating effect of t-ACPD on spontaneous GABAergic currents was prevented by Rp-cAMPS (30 microM), a specific antagonist of protein kinase A. This suggests that mGluRs localized at the soma-dendritic level of GABAergic interneurons and positively coupled to cyclic AMP may modulate GABA release during a critical period of postnatal development.

Animals↗

PubMed1994

Developmental shift from long-term depression to long-term potentiation at the mossy fibre synapses in the rat hippocampus.

During development, in the CA1 hippocampal region, long-term potentiation (LTP) starts appearing at postnatal (P) day 7 and reaches its maximal expression towards the end of the second postnatal week. However, LTP is often preceded by long-term depression (LTD), an activity-dependent and long-lasting reduction of synaptic strength. LTD can be induced by sustained, low-frequency stimulation of the afferent pathway and is dependent on activation of N-methyl-D-aspartate (NMDA) receptors. We report here that, in the CA3 hippocampal region, during a critical period of postnatal development, between P6 and P14, a high-frequency stimulation train (100 Hz, 1 s) to the mossy fibres in the presence of the NMDA receptor antagonist (+)-3-(2-carboxy-piperazin-4-yl)-propyl-1-phosphonic acid (CPP; 20 microM) induced LTD. The depression of the amplitude of the field excitatory postsynaptic potential (EPSP) was 28 +/- 7% (n = 21). This form of LTD was NMDA-independent and synapse-specific. When a tetanus was applied in the presence of CPP and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 microM), which blocked the field EPSP, it failed to induce LTD upon washout of CNQX. LTD was probably postsynaptic in origin since it did not affect paired-pulse facilitation. A rise in extracellular calcium concentration (from 2 to 4 mM) produced LTP instead of LTD. At the end of the second postnatal week, the same high-frequency stimulation train to the mossy fibres induced LTP as in adult neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Transient expression of a novel type of GABA response in rat CA3 hippocampal neurones during development.

1. Intracellular recordings were used to study the effects of gamma-aminobutyric acid (GABA) on rat CA3 hippocampal neurones during the first two weeks of postnatal life. 2. In the presence of tetrodotoxin (TTX, 1 microM), from postnatal day 0 (P0) to P12 both associated with an increase in input conductance whereas baclofen (30-100 microM) produced a membrane hyperpolarization. 3. Bicuculline (50 microM) reduced the effects of GABA and abolished the response to isoguvacine without affecting the response to baclofen. 4. This novel bicuculline-insensitive GABA response was chloride dependent and was blocked by picrotoxin (10-100 microM) in an uncompetitive way. In bicuculline and picrotoxin, a GABAB-mediated hyperpolarization appeared. 5. Towards the end of the second postnatal week, bicuculline blocked the GABA-induced depolarization and revealed a small hyperpolarizing response which was blocked by the GABAB antagonist CGP 35348 (0.5-1 mM). 6. It is suggested that, during development, the GABA response was mediated through the conventional GABAA and GABAB receptors as well as a new bicuculline-baclofen-insensitive type of receptor.

Aging↗

PubMed1994

Serum fractions from amyotrophic lateral sclerosis patients depress voltage-activated Ca2+ currents of rat cerebellar granule cells in culture.

Whole-cell patch clamp recording from rat cerebellar granule cells in culture was used to study the effect of immune protein fractions extracted from the serum of amyotrophic lateral sclerosis (ALS) patients on voltage-activated Ca2+ currents. The inward currents, carried by Ba2+, were induced by depolarizing step commands positive to -50 mV and showed typical voltage-dependent inactivation. Application of immunoprotein fractions obtained from the serum of ALS patients produced a strong depression of the inward current amplitude without changing its threshold potential at which the maximum was attained, or its time course. These data support the hypothesis that the serum of ALS patients contains an immunoprotein capable of interacting with high threshold Ca2+ channels of central neurones.

Adult↗

PubMed1994

Developmental changes in spontaneous GABAA-mediated synaptic events in rat hippocampal CA3 neurons.

Ongoing spontaneous postsynaptic potentials (SPSPs) were intracellularly recorded at 34-36 degrees C from hippocampal CA3 neurons in slices obtained from postnatal days (P) 0-6 and 7-31. SPSPs occurred randomly, and their frequency distribution was fitted by a single exponential function. They were little affected by kynurenic acid, but were reversibly blocked by bicuculline, implying that they were mediated by GABAA receptors. The mean amplitude was 4.53 +/- 0.89 mV in control conditions and 4.07 +/- 0.79 mV in kynurenic acid. In kynurenic acid (with CsCl-filled microelectrodes), SPSPs reversed polarity at 2.4 +/- 2 mV. When tetrodotoxin (1 microM) was added to kynurenic acid solution, GABAA-mediated miniature postsynaptic potentials (MPSPs) were recorded. Under these conditions large events disappeared. The mean amplitude of MPSPs was 2.51 +/- 0.43 mV. The mean frequency decreased from 2.96 +/- 1.04 Hz in kynurenic acid to 0.4 +/- 0.15 Hz in kynurenic acid plus tetrodotoxin. In contrast with P0-P6, at P7-P31 SPSPs were significantly affected by kynurenic acid. The mean amplitude of SPSPs shifted from 4.71 +/- 0.82 mV in control conditions to 3.79 +/- 0.76 mV in kynurenic acid. At this developmental stage, the reversal potential of GABAA-mediated SPSPs shifted towards more negative values (-23.7 +/- 1.3 mV). Addition of tetrodotoxin to kynurenic acid solution abolished larger events and revealed GABAergic MPSPs. The mean amplitude of MPSPs was 2.72 +/- 0.5 mV, a value very close to that observed at P0-P6. Synaptic currents were recorded at 22-24 degrees C from voltage-clamped CA3 pyramidal neurons (at P6) using the tight-seal whole-cell recording technique.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗

PubMed1994

Characterization of a voltage-dependent anionic channel in fused synaptosomes isolated from rat hippocampi.

The inside out configuration of the patch-clamp technique was used to study single-channel anionic currents from purified hippocampal synaptosomes fused into liposomes to form giant proteoliposomes. At least six different anionic channels with unitary conductances of 22-150 pS were found. The most frequently observed was the 32-pS conductance channel. This was voltage-dependent; the open probability increased from 0.20 at -40 mV to 0.46 at 40 mV. This channel may be involved in the repolarization of nerve terminal membranes after an action potential, thus, limiting the duration of the spike and the transmitter release.

Animals↗

PubMed1994

The effect of intracellular Ca2+ on GABA-activated currents in cerebellar granule cells in culture.

The patch clamp technique was used to study the effects of intracellular free calcium ([Ca2+]i) on GABAA-evoked whole-cell and single channel currents of cultured cerebellar granule cells. Changes in [Ca2+]i were obtained by adding to the extracellular solution the calcium ionophore A23187 (2 microM). The relationship between [Ca2+]i and [Ca2+]o in the presence or absence of A23187 was assessed using fluorimetric measurements from Fura-2 loaded cells. In 2 mM [Ca2+]o and A23187, [Ca2+]i was about 1.5 microM, whereas in the absence of A23187 it was about 250 nM. In whole-cell experiments (symmetrical chloride concentrations) at -50 mV, GABA (0.5 microM) evoked inward currents that did not desensitize. Bath application of A23187 significantly reduced the steady-state amplitude of GABA currents by 37 +/- 6%. Single channel currents activated by GABA (0.5 microM) were also recorded in the outside-out configuration of the patch clamp technique. Kinetic analysis of single channel events revealed that A23187 significantly increased the long closed time constant (tau c3) without affecting the open time constants (tau o1 and tau o2) or the short and medium closed time constants (tau c1 and tau c2). Moreover, application of A23187 induced a significant reduction of burst duration (tau b). We conclude that a rise in [Ca2+]i by A23187 may decrease the binding affinity of GABA for the GABAA receptor.

Animals↗

PubMed1993

Currents activated by GABA and their modulation by Zn2+ in cerebellar granule cells in culture.

Whole-cell and single-channel currents evoked by gamma-aminobutyric acid (GABA) were recorded from rat cerebellar granule cells in culture. The electrophysiological properties of these currents were studied in control condition and in the presence of external Zn2+ (10-30 microM). GABA (10 microM) induced bicuculline-sensitive whole-cell currents which desensitized. The desensitization was more rapid for higher concentrations of GABA (30-300 microM). The current-voltage relation of GABA currents was linear from -70 to +50 mV. Two different types of cells were found with respect to the stoichiometry for agonist binding, one with Hill coefficient 1.5 and another one with coefficient 1. The half-maximum concentration displayed more variability, with values varying from 10 to 50 microM. The time constant of recovery from desensitization (tau r) was estimated to be 36 s. Zn2+ (30 microM) blocked GABA-activated whole-cell currents in a non-competitive and voltage-independent way without a significant change in the current kinetics. In excised outside-out patches, GABA (0.5 microM) activated single-channel events of 19 and 31 pS. Kinetic analysis yielded two mean shut times (tau c1 = 2.70 ms, tau c2 = 205 ms) and one mean open time (tau o = 3.64 ms). Zn2+ (10 microM) did not affect single-channel conductances and mean open and shut times, but significantly reduced the probability of opening from 0.17 to 0.06. It is probable that Zn2+ binds to a site located on the extracellular part of the GABAA receptor channel complex.

Animals↗