The development of the electrical activity of hypothalamic cells was studied using intracellular and patch clamp recording technics on cultured hypothalamic neurones from 14 days mouse embryos. After 24 h of incubation, 15% of recorded... more
The development of the electrical activity of hypothalamic cells was studied using intracellular and patch clamp recording technics on cultured hypothalamic neurones from 14 days mouse embryos. After 24 h of incubation, 15% of recorded cells were spontaneously active. During the first five days of culture this ratio increased exponentially to reach 95% at day 5. Between the 5th and the 9th day the spontaneous activity progressively decreased although the majority of cells remained excitable. Spontaneous activity reappeared after the 9th day and was underlain by a synaptic potential activity. During the first five days of culture, only a TTX sensitive inward current was observed in all cell tested. A calcium inward current appeared after the first week of incubation. It was recorded on 40% of cells at day 11th and in 80% of cells tested after a month of incubation. Our results show that electrical activity of cultured hypothalamic neurones develop in three stages. A first stage characterized by a spontaneous electrical activity without post synaptic potential, an intermediate stage during which a Calcium inward current appeared corresponding to synaptic contact development and a third stage during which post synaptic potential activity was observed, corresponding to synaptic contact maturation.
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Glycine is one of the most important inhibitory neurotransmitters in the spinal cord and the brainstem, and glycinergic synapses have a well-established role in the regulation of locomotor behavior. Research over the last 15 years has... more
Glycine is one of the most important inhibitory neurotransmitters in the spinal cord and the brainstem, and glycinergic synapses have a well-established role in the regulation of locomotor behavior. Research over the last 15 years has yielded new insights on glycine neurotransmission. Glycinergic synapses are now known not to be restricted to the spinal cord and the brainstem. Presynaptic machinery for glycine release and uptake, the structure and function of postsynaptic receptors and the factors (both pre- and postsynaptic) which control the strength of glycinergic inhibition have been extensively studied. It is now established that glycinergic synapses can be excitatory in the immature brain and that some inhibitory synapses can corelease gamma-aminobutyric acid (GABA) and glycine. Moreover, the presence of glycine transporters on glial cells and the capacity of these cells to release glycine suggest that glycine may also act as a neuromodulator. Extensive molecular studies have revealed the presence of distinct subtypes of postsynaptic glycine receptors with different functional properties. Mechanisms of glycine receptors aggregation at postsynaptic sites during development are better understood and functional implications of variation in receptor number between postsynaptic sites are partly elucidated. Mutations of glycine receptor subunits have been shown to underly some human locomotor disorders, including the startle disease. Clearly, recent work on glycine receptor channels and the synapses at which they mediate inhibitory signalling in both young and adult animals necessitates an update of our vision of glycinergic inhibitory transmission.
Research Interests: Neuroscience, Kinetics, Biology, Neurotransmission, Membrane Proteins, and 15 moreMedicine, Brain, Humans, Animals, Glial Cell, Alternative splicing, Neurons, Clinical Sciences, Gephyrin, Glycine receptor, Alternative Splicing, Glycine, Biochemistry and cell biology, Allosteric regulation, and Functional Properties
Refuting the challenges of the developmental shift of
Electrophysiological recordings of outside-out patches to fast-flow applications of glycine were made on patches derived from the Mauthner cells of the 50-h-old zebrafish larva. As for glycinergic miniature inhibitory postsynaptic... more
Electrophysiological recordings of outside-out patches to fast-flow applications of glycine were made on patches derived from the Mauthner cells of the 50-h-old zebrafish larva. As for glycinergic miniature inhibitory postsynaptic currents (mIPSCs), depolarizing the patch produced a broadening of the transient outside-out current evoked by short applications (1 ms) of a saturating concentration of glycine (3 mM). When the outside-out patch was depolarized from −50 to +20 mV, the peak current varied linearly with voltage. A 1-ms application of 3 mM glycine evoked currents that activated rapidly and deactivated biexponentially with time constants of ≈5 and ≈30 ms (holding potential of −50 mV). These two decay time constants were increased by depolarization. The fast deactivation time constant increased e-fold per 95 mV. The relative amplitude of the two decay components did not significantly vary with voltage. The fast component represented 64.2 ± 2.8% of the total current at −50 mV a...
Research Interests: Chemistry, Electrophysiology, Kinetics, Neurophysiology, Medicine, and 14 moreIon Channels, Patch-clamp and imaging techniques, Evoked Potentials, Animals, Reaction Time, Constant Time Delay, Glycine receptor, Zebrafish, Glycine, Depolarization, Excitatory Postsynaptic Potentials, Psychology and Cognitive Sciences, Medical and Health Sciences, and In Vitro Techniques
Miniature IPSCs (mIPSCs) recorded in the Mauthner (M)-cell of zebrafish larvae have a broad amplitude distribution that is attributable only partly to the functional heterogeneity of postsynaptic glycine receptors (GlyRs). The role of the... more
Miniature IPSCs (mIPSCs) recorded in the Mauthner (M)-cell of zebrafish larvae have a broad amplitude distribution that is attributable only partly to the functional heterogeneity of postsynaptic glycine receptors (GlyRs). The role of the kinetic properties of GlyRs in amplitude fluctuation was investigated using fast-flow application techniques on outside-out patches. Short applications of a saturating glycine concentration evoked outside-out currents with a biphasic deactivation phase as observed for mIPSCs, and they were consistent with a rapid clearance of glycine from the synaptic cleft. Patch currents declined slowly during continuous applications of 3 mmglycine, but the biphasic deactivation phase of mIPSCs cannot reflect a desensitization process because paired-pulse desensitization was not observed. The maximum open probability (Po) of GlyRs was close to 0.9 with 3 mmglycine. Analyses of the onset of outside-out currents evoked by 0.1 mmglycine are consistent with the prese...
Research Interests: Neuroscience, Biophysics, Chemistry, Kinetics, Medicine, and 15 morePatch-clamp and imaging techniques, Evoked Potentials, Animals, Neurons, Gating, Glycine receptor, Time Factors, SYNAPSES, Zebrafish, Glycine, Binding Site, Time Course, Psychology and Cognitive Sciences, Medical and Health Sciences, and Kinetic model
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Glycine is one of the most important inhibitory neurotransmitters in the spinal cord and the brainstem, and glycinergic synapses have a well-established role in the regulation of locomotor behavior. Research over the last 15 years has... more
Glycine is one of the most important inhibitory neurotransmitters in the spinal cord and the brainstem, and glycinergic synapses have a well-established role in the regulation of locomotor behavior. Research over the last 15 years has yielded new insights on glycine neurotransmission. Glycinergic synapses are now known not to be restricted to the spinal cord and the brainstem. Presynaptic machinery for glycine release and uptake, the structure and function of postsynaptic receptors and the factors (both pre- and postsynaptic) which control the strength of glycinergic inhibition have been extensively studied. It is now established that glycinergic synapses can be excitatory in the immature brain and that some inhibitory synapses can corelease gamma-aminobutyric acid (GABA) and glycine. Moreover, the presence of glycine transporters on glial cells and the capacity of these cells to release glycine suggest that glycine may also act as a neuromodulator. Extensive molecular studies have revealed the presence of distinct subtypes of postsynaptic glycine receptors with different functional properties. Mechanisms of glycine receptors aggregation at postsynaptic sites during development are better understood and functional implications of variation in receptor number between postsynaptic sites are partly elucidated. Mutations of glycine receptor subunits have been shown to underly some human locomotor disorders, including the startle disease. Clearly, recent work on glycine receptor channels and the synapses at which they mediate inhibitory signalling in both young and adult animals necessitates an update of our vision of glycinergic inhibitory transmission.
Research Interests: Neuroscience, Kinetics, Biology, Neurotransmission, Membrane Proteins, and 15 moreMedicine, Brain, Humans, Animals, Glial Cell, Alternative splicing, Neurons, Clinical Sciences, Gephyrin, Glycine receptor, Alternative Splicing, Glycine, Biochemistry and cell biology, Allosteric regulation, and Functional Properties
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Characteristics and functional activity of Angiotensin II (AII) neuronal receptors were studied using binding and intracellular recording methods. Characteristics of AII binding allowed definition of two distinct classes of high and low... more
Characteristics and functional activity of Angiotensin II (AII) neuronal receptors were studied using binding and intracellular recording methods. Characteristics of AII binding allowed definition of two distinct classes of high and low affinity binding sites. The effects of AII on the electrophysiological membrane properties of neurones were investigated using cultured mouse spinal cord (SC). AII induced changes in membrane potential and input resistance which varied according to the applied concentration of peptide. These data agreed with binding results suggesting two classes of AII neuronal receptors. Although angiotensin II-like material is found in the rat brain by RIA and immunochemistry, the presence of authentic angiotensin II (AII) is a point for discussion. Using RIA and radio-receptor assay (RRA), we found AII like peptides. However, chromatographic separation by gel filtration has revealed that this material is not authentic AII but consists of compounds of higher molecular weight. The recognition of the same material, both by AII antibodies (RIA) and AII binding sites (RRA) suggests that precursors containing AII sequences exist in the rat brain. We incubated rat brain with 3H-angiotensin I (AI) at 37 degrees C and analysed the resulting 3H-peptides (HPLC). Authentic 3H-AII was not detected, but two smaller peptides appeared (peak alpha et beta). The same peaks appeared when rat brain was incubated with 3H-AII. We have only been able to reveal 3H-AII formation from 3H-AI by inhibiting AII angiotensinases with excess of AII or low temperature (22 degrees C or 12 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)
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The whole-cell voltage clamp technique was used to record potassium currents in mouse fetal hypothalamic neurons developing in culture medium from days 1 to 17. The neurons were derived from fetuses of IOPS/OF1 mice on the 14th day of... more
The whole-cell voltage clamp technique was used to record potassium currents in mouse fetal hypothalamic neurons developing in culture medium from days 1 to 17. The neurons were derived from fetuses of IOPS/OF1 mice on the 14th day of gestation. The mature neurons (greater than six days in culture) showed both a transient potassium current and a non-inactivating delayed rectifier potassium current. These were identified pharmacologically by using the potassium channel blockers tetraethyl ammonium chloride and 4-aminopyridine, and on the basis of their kinetics and voltage sensitivities. The delayed rectifier potassium current had a threshold of-20 mV, a slow time-course of activation, and was sustained during the voltage pulse. The 4-aminopyridine-sensitive current was transient, and was activated from a holding potential more negative (-80 mV) than that required for evoking the delayed rectifier potassium current (-40 mV). The delayed rectifier potassium current was detectable from day 1 onwards, while the transient potassium current showed a distinct developmental trend. The time-constant of inactivation became faster with age in culture. The half steady-state inactivation potential showed a shift towards less negative membrane potentials with age, and the relationship was best described by a logarithmic regression equation. The developmental trend of the transient potassium current may relate functionally to the progressive morphological changes, and the appearance of synaptic connections during ontogenesis.
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The effects of the anti-ischemic agents ifenprodil and its derivative SL 82.0715 ((+/-)-alpha-(4-chlorophenyl)-4-[(4-fluorophenyl) methyl]-1-piperidineethanol] have been analyzed in a number of models indicative of N-methyl-D-aspartate... more
The effects of the anti-ischemic agents ifenprodil and its derivative SL 82.0715 ((+/-)-alpha-(4-chlorophenyl)-4-[(4-fluorophenyl) methyl]-1-piperidineethanol] have been analyzed in a number of models indicative of N-methyl-D-aspartate (NMDA) antagonistic potential in vitro and in vivo. Ifenprodil and SL 82.0715 potently and noncompetitively antagonize the stimulatory effects of NMDA on cyclic GMP production in immature rat cerebellar slices (IC50 values, 0.4 and 10 microM, respectively), as well as the NMDA-evoked [3H]acetylcholine release in adult rat striatal slices (IC50 values, 1.6 and 6.6 microM, respectively). Ifenprodil is 10 times more potent than (+/-)3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) but less active than the reference noncompetitive NMDA channel blockers [MK 801, ((+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclohepten-5,10-imine ], phencyclidine and 1-[1-(2-thienyl)cyclohexyl]piperidine (TCP)] in these models. Ifenprodil and SL 82.0715 partially displace (maximal displacement 40-50% at 10 microM) the NMDA receptor ligand [3H]CPP from its binding site to rat brain membranes (IC50 values, 0.1 and 0.3 microM, respectively) in a noncompetitive manner; in the micromolar range the two agents also partially displace the NMDA channel ligand [3H]TCP from its binding site to rat brain membranes, and noncompetitively antagonize the L-glutamate-induced increase in [3H]TCP binding. Ifenprodil (0.01-1 microM) partially antagonizes the depolarizing effects of NMDA on the immature rat hemisected spinal cord in vitro. In mouse cultured spinal cord neurons, ifenprodil dose-dependently antagonizes the depolarizing effects of micropressure applied NMDA. Inhibition of the effects of NMDA in this model by ifenprodil and SL 82.0715 is noncompetitive. In vivo and after systemic i.p. administration, ifenprodil and SL 82.0715 antagonize the stimulatory effects of intrastriatally dialyzed NMDA on striatal dopamine release in rats (ID50 values, 0.9 and 0.3 mg/kg, respectively), and block the harmaline-evoked increase in cerebellar cyclic GMP production in mice (ID50 values, 3 and 4 mg/kg, respectively). These results indicate that ifenprodil is a noncompetitive NMDA antagonist which has a mechanism of action distinct from either the reference competitive NMDA receptor antagonists (CPP and 2-amino-5-phosphonovalerate) or the noncompetitive NMDA channel blockers (phencyclidine, TCP and MK 801). The potent NMDA antagonistic effects of the ifenprodil class of compounds are likely to be related to the demonstrated anti-ischemic potential of these compounds.
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From morphological characterization and intracellular recordings, monolayer cultures derived from fetal mouse hypothalami were found to include functionally differentiated peptide neurons, a number of which appear to contain vasopressin.... more
From morphological characterization and intracellular recordings, monolayer cultures derived from fetal mouse hypothalami were found to include functionally differentiated peptide neurons, a number of which appear to contain vasopressin. These cells exhibited particular patterns of slow, calcium-dependent membrane depolarizations, resembling in their periodicity and duration the phasic activity of vasopressin neurons recorded extracellularly in vivo.
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... Dopamine affects two voltage-dependent K+ currents of identified rat lactotroph cells PM Lledo, P. Legendre, J. Zhang, JM Israel and JD Vincent INSERM U.176, rue Camille Saint-Saans, 33077 Bordeaux Cedex, France Introduction ...
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The effects of dopamine (DA) on voltage-dependent potassium currents were investigated in rat lactotrophs maintained in primary culture. Lactotroph cells were identified using the reverse hemolytic plaque assay. Membrane currents and... more
The effects of dopamine (DA) on voltage-dependent potassium currents were investigated in rat lactotrophs maintained in primary culture. Lactotroph cells were identified using the reverse hemolytic plaque assay. Membrane currents and potentials of lactotroph cells were recorded using the patch-clamp recording technique in the 'whole-cell' configuration. In the presence of cobalt (2 mM), two types of voltage-dependent K+ currents were recorded, a voltage-activated delayed K+ current (IK) and a voltage-activated transient K+ current (IA). The current IK was activated at membrane potentials varying from -20 to +40 mV and did not inactivate during prolonged voltage steps (up to 25 s); it was blocked by tetraethylammonium (10 mM). The current IA was activated at membrane potentials higher than -45 mV and showed a voltage-dependent inactivation between -110 and -40 mV; it was slightly inhibited by 4-aminopyridine (5 mM). Under current-clamp conditions, the majority of the cells (60%) showed spontaneous Ca2(+)-dependent action potentials (APs) while silent cells (40%) were excitable by depolarizing current pulses. Bath application of 10 nM DA evoked a hyperpolarizing response, blocked spontaneous APs and decrease the amplitude of evoked APs. Only the hyperpolarizing response faded during the course of the whole cell recording experiments. Under voltage-clamp conditions, DA induced a reversible increase in both voltage-dependent outward K+ currents, without modifying their thresholds. Steady-state inactivation of IA was not affected by DA. These DA-induced responses were dose-dependent and they involved D2 receptor activation. They were mimicked by the specific D2 receptor agonist bromocriptine (10 nM) and blocked by the specific D2 receptor antagonist sulpiride (100 nM), the D1 antagonist SCH 23390 being ineffective. The ability of DA to increase voltage-dependent K+ currents cannot be observed without GTP in the recording pipette. It was pertussis-toxin-sensitive but was affected neither by bath application of 1 mM forskolin nor by the presence of 500 microM cyclic AMP with 500 microM 3-isobutyl-1-methylxanthine in the pipette solutions. We conclude that in lactotroph cells DA specifically increases two voltage-dependent K+ currents via a pertussis-toxin-sensitive guanine nucleotide regulatory protein and appears to be independent of intracellular cyclic AMP. This effect leads to a decrease in the excitability of the cell, explaining in part the inhibitory effect of DA on prolactin release.
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The effects of dopamine (DA) on voltage-dependent Ca2+ currents were investigated in cultured rat lactotroph cells using the patch clamp recording technique. Each recorded cell was identified by the reverse hemolytic plaque assay. In the... more
The effects of dopamine (DA) on voltage-dependent Ca2+ currents were investigated in cultured rat lactotroph cells using the patch clamp recording technique. Each recorded cell was identified by the reverse hemolytic plaque assay. In the whole-cell configuration, two types of Ca2+ currents, L and T, were characterized on the basis of their kinetics, voltage sensitivity, and pharmacology. The L component had a threshold of -25 mV, showed little inactivation during a 150-msec voltage step, and was maximal at +10 mV. Cadmium ions (100 microM) significantly reduced its amplitude (75%). The T component was activated at a membrane potential close to -50 mV, was maximal at -10 mV, and showed a voltage-dependent inactivation between -90 and -30 mV. It was quickly inactivated during a maintained depolarization (time constant, 27 ms at -30 mV) and was strongly reduced (80%) by nickel ions (100 microM). Bath application of DA (10 nM) caused a markedly general depression of inward Ca2+ currents, acting differently on the T- and L-type currents. DA application shifted the voltage-dependence of the L-type current activation toward depolarization values (8 mV) without modifying its time- and voltage-dependent inactivation. In contrast, DA enhanced the inactivation of the T-type current by accelerating its time-dependent inactivation (25% decrease in the time constant of inactivation) and by shifting the voltage-dependence of the T-type current inactivation toward hyperpolarizing values (-63 mV in control vs. -77 mV in the presence of DA). These effects of DA were dose-dependent and involved the activation of a D2 receptor type. They were mimicked by bromocriptine application (10 nM), whereas sulpiride (100 nM) blocked the DA-evoked response. The D1 antagonist SCH 23390 was ineffective up to 100 microM. All of these DA-induced modifications in Ca2+ currents were abolished using a GTP-free pipette solution or after pretreatment of cells with pertussis toxin, suggesting that DA can regulate the function of Ca2+ channels through GTP-binding proteins (G-proteins). Our results show that DA acts simultaneously by reducing both voltage-dependent Ca2+ currents on lactotroph cells. Thus, DA reduces the entry of Ca2+ ions across the surface membrane and thereby influences electrical activity and the cytosolic free Ca2+ concentration involved in both basal and evoked PRL release.
Research Interests: Endocrinology, Biophysics, Chemistry, Kinetics, Biology, and 15 moreMedicine, Biological Sciences, Dopamine, Female, Animals, Calcium channels, Dopamine Receptors, Nickel, Cadmium, Guanosine Triphosphate, Membrane Potential, Electric Conductivity, Bromocriptine, Depolarization, and Medical and Health Sciences
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Vasopressin neurons in hypothalamic cell culture display regenerative calcium-dependent plateau potentials. The present study was undertaken to investigate the mechanisms underlying their time course and periodicity. Intracellular... more
Vasopressin neurons in hypothalamic cell culture display regenerative calcium-dependent plateau potentials. The present study was undertaken to investigate the mechanisms underlying their time course and periodicity. Intracellular recordings showed that the duration of the plateaux was controlled by a progressive activation of a voltage- and calcium-dependent potassium conductance, but not by a progressive inactivation of calcium conductances. The refractory period appeared to be due to a calcium-dependent potassium conductance activated by membrane potential depolarization.
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Recent work at the zebrafish neuromuscular junction (NMJ) has shown that positively charged acetylcholine (ACh), at the high concentrations reached in the cleft during neuromuscular transmission, blocks acetylcholine receptors (AChRs) as... more
Recent work at the zebrafish neuromuscular junction (NMJ) has shown that positively charged acetylcholine (ACh), at the high concentrations reached in the cleft during neuromuscular transmission, blocks acetylcholine receptors (AChRs) as soon as they open. Thus after two ACh molecules bind and open the channel, a third molecule enters and blocks the pore at a site resembling that for block by local anesthetics, suggesting that ACh is the endogenous anesthetic of the NMJ. Recovery from open channel block results in a rebound synaptic current only after ACh is cleared from the cleft. Kinetic modeling of other AChRs suggests that a rebound current is generated at all vertebrate NMJs, from fish to frogs to mammals. Open channel block prolongs the current at fast zebrafish NMJs in order to more effectively spread charge along the fibers, akin to multiple central synapses spread over dendrites. Together these findings indicate the need for a fundamental revision of current thinking about neuromuscular transmission at many levels, including channel structure, function and pharmacology.
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The action of zinc on chloride currents evoked by gamma-aminobutyric acid (GABA) was examined on cultured hippocampal neurons using whole cell voltage clamp and outside-out patch recording. Zn (5-30 microM) noncompetitively blocked... more
The action of zinc on chloride currents evoked by gamma-aminobutyric acid (GABA) was examined on cultured hippocampal neurons using whole cell voltage clamp and outside-out patch recording. Zn (5-30 microM) noncompetitively blocked responses evoked by GABA (0.5-100 microM), but did not affect either the time-to-peak or desensitization of the macroscopic current. In outside-out patches, Zn had no effect on the mean conductance or lifetime of the 19 or 30 pS openings of the GABA channel; however, the frequency of channel opening was markedly decreased in a voltage-independent manner. Zn inhibition of GABA responses appeared to be independent of the benzodiazepine binding site as Zn was effective in the presence of either diazepam or Ro15-1788, a competitive antagonist of benzodiazepine agonists and inverse agonists. In contrast to prior reports, Zn also inhibited GABA currents in a similar manner on cultured superior cervical ganglion neurons. These results suggest that Zn acts at an extracellular site on the GABAA receptor complex, which is distinct from either the GABA or benzodiazepine binding sites. The structural similarity of the Cys-Cys loop of the alpha and gamma GABAA receptor subunits to some Zn-binding proteins suggests one possible region for a Zn binding site.
Research Interests: Biophysics, Chemistry, Biology, Membrane Proteins, Molecular Pharmacology, and 15 moreMedicine, Ion Channels, Hippocampus, Pubmed, Animals, Rats, Sympathetic Nervous System, Electric Conductivity, Neurosciences, Binding Site, Extracellular, Chlorides, GABAA receptor, binding sites, and Pharmacology and pharmaceutical sciences
The inhibition of N-methyl-D-aspartate (NMDA) receptor channels by the vasodilatory and anti-ischemic agent ifenprodil was examined on cultured rat hippocampal neurons. Whole-cell and single-channel patch recordings were used. Ifenprodil... more
The inhibition of N-methyl-D-aspartate (NMDA) receptor channels by the vasodilatory and anti-ischemic agent ifenprodil was examined on cultured rat hippocampal neurons. Whole-cell and single-channel patch recordings were used. Ifenprodil inhibition of NMDA currents could be separated into two components, with IC50 values of 0.75 and 161 microM. The high and low affinity components were both voltage independent but could be separated by their kinetics and dependence on extracellular calcium and glycine. The maximal inhibition of inward current by ifenprodil (approximately 90%) was equally divided between the two components in 0.3 mM extracellular calcium and 500 nM glycine. The low affinity action of ifenprodil had rapid kinetics and appeared to result from allosteric inhibition of the glycine modulatory site on the NMDA receptor. The macroscopic kinetics of the high affinity component were slow. The rate of onset was concentration dependent, and complete recovery required 1-2 min. Unlike open-channel blockers, ifenprodil block was not use dependent, and pre-exposure to ifenprodil also reduced subsequent NMDA responses. Low concentrations of ifenprodil were less effective after calcium-dependent inactivation of whole-cell currents, but the IC50 was unaffected, suggesting that calcium and ifenprodil act on a common set of channels. On outside-out membrane patches, ifenprodil reduced the frequency of channel opening without altering the single-channel conductance. Open time histograms of the large conductance events revealed two mean open times of approximately 2 and 8 msec, but only the duration of the long openings was decreased by ifenprodil. This effect was concentration dependent and revealed a blocking rate constant of 6 x 10(7) M-1sec-1. However, the proportion of current blocked by low concentrations of ifenprodil was larger in outside-out patches than in whole-cell recordings, suggesting that intracellular factors may influence ifenprodil efficacy. These results indicate that high affinity ifenprodil binding is extracellular and does not require agonist binding or channel opening. Because low concentrations of ifenprodil only partially inhibited the current and affected only the long openings, ifenprodil may promote a modal shift in channel gating.
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1. Whole-cell, voltage-clamp recordings were obtained from neurones of the supraoptic area of neonatal rats in dissociated cell culture. Recordings were made from neurones having the same morphology as those which were vasopressin or... more
1. Whole-cell, voltage-clamp recordings were obtained from neurones of the supraoptic area of neonatal rats in dissociated cell culture. Recordings were made from neurones having the same morphology as those which were vasopressin or oxytocin immunoreactive. 2. Three types of voltage-activated K+ current were identified on the basis of their kinetics, voltage sensitivities, Ca2+ dependence and pharmacology. The currents corresponded to the delayed rectifier current (IK), the A-current (IA), and the Ca2+-dependent current (IK(Ca] described in other neurones. 3. IK had a threshold of -40 mV, a sigmoidal time course of activation, and was sustained during voltage steps lasting less than 300 ms. The underlying conductance was voltage dependent reaching a maximum at +30 mV (mean maximum conductance 4.09 nS). The activation time constant was also voltage dependent declining exponentially from 4.5 ms at -30 mV to 1.8 ms at +50 mV. 4. IA was transient, and was activated from holding potentials negative to -70 mV; the maximum conductance (mean 5.9 nS) underlying the current was obtained at +10 mV. The activation and inactivation time constants were voltage dependent: the activation time constant declined exponentially between -40 mV (2.2 ms) and +40 mV (0.65 ms). 5. IK and IA were attenuated by the K+ channel blockers tetraethylammonium (TEA) and 4-aminopyridine (4-AP). TEA blocked the conductance underlying IK but appeared to alter the kinetics of IA. In contrast, 4-AP blocked the conductance underlying IA and, to a lesser extent, IK. 6. IK and IA were activated independently of external Ca2+ and the voltage activation of Ca2+ channels since these currents were recorded in the presence of Co2+, a Ca2+ channel blocker. 7. IK(Ca) was recorded only when Ca2+ (2 mM) was present in the external medium. From a holding potential of -30 mV, IK(Ca) had a threshold of -20 mV, was maximal at about +20 mV and declined at more positive potentials. This current was sustained during voltage steps lasting 100 ms and was abolished by addition of Co2+ (2 mM) to the medium. 8. The possible roles of the three K+ currents in regulating the characteristic firing behaviour of supraoptic neurones previously recorded in vivo and in vitro are discussed.
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1. Conflicting evidence exists concerning the expression and properties of N-methyl-D-aspartate (NMDA) receptors on cerebellar Purkinje cells during development. We used whole-cell and single-channel recording to examine NMDA receptors on... more
1. Conflicting evidence exists concerning the expression and properties of N-methyl-D-aspartate (NMDA) receptors on cerebellar Purkinje cells during development. We used whole-cell and single-channel recording to examine NMDA receptors on acutely dissociated Purkinje cells from newborn rats (postnatal day 0-4). 2. NMDA channels were present on > 80% of identified Purkinje cells and had pharmacological and single-channel properties that were indistinguishable from NMDA receptors on other neurons. In particular, responses were glycine-dependent and Mg2+ produced flickery open-channel block. 3. Our results demonstrate the transient expression of NMDA receptor/channels on Purkinje cells early in development. As NMDA receptors have been implicated in developmental plasticity in other regions of the CNS, a similar role is feasible during climbing fiber innervation of Purkinje cells.
Research Interests: Neuroscience, Chemistry, Electrophysiology, Neurophysiology, Medicine, and 15 moreGene expression, Ion Channels, Cell Culture, Cerebellum, Animals, Long Term Depression, Purkinje Cells, Climbing Fiber, Purkinje cell, Glutamate Receptor, Kainic acid, NMDA receptor, Psychology and Cognitive Sciences, Medical and Health Sciences, and In Vitro Techniques
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Research Interests: Physiology, Algorithms, Biophysics, Chemistry, Kinetics, and 15 moreNeurotransmission, Medicine, Biological Sciences, Computer Simulation, Animals, Synaptic Transmission, Glycine receptor, SYNAPSES, Transfection, Glycine, Dimerization, Automatic data processing, CHO cells, Cricetinae, and Medical and Health Sciences
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The magnocellular neurones of the hypothalamo-neurohypophysial system have in recent years proved a valuable model in which to examine the complex control of peptidergic secretion, in particular oxytocin and vasopressin. The unique... more
The magnocellular neurones of the hypothalamo-neurohypophysial system have in recent years proved a valuable model in which to examine the complex control of peptidergic secretion, in particular oxytocin and vasopressin. The unique properties of the nuclei containing these neurones — principally homogeneity and high density of the neuronal population — have meant that a variety of in vivo and in vitro studies have been possible.
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1. The kinetics and mechanisms underlying the voltage dependence of inhibitory postsynaptic currents (IPSCs) recorded in the Mauthner cell (M cell) were investigated in the isolated medulla of 52-h-old zebrafish larvae, with the use of... more
1. The kinetics and mechanisms underlying the voltage dependence of inhibitory postsynaptic currents (IPSCs) recorded in the Mauthner cell (M cell) were investigated in the isolated medulla of 52-h-old zebrafish larvae, with the use of whole cell and outside-out patch-clamp recordings. 2. Spontaneous miniature IPSCs (mIPSCs) were recorded in the presence of 10(-6) M tetrodotoxin (TTX), 10 mM MgCl2, and 0.1 mM [CaCl2]o. Depolarizing the cell from -50 to +50 mV did not evoke any significant change in the distribution of mIPSC amplitudes, whereas synaptic currents were prolonged at positive voltages. The average decay time constant was increased twofold at +50 mV. 3. The voltage dependence of the kinetics of glycine-activated channels was first investigated during whole cell recording experiments. Currents evoked by voltage steps in the presence of glycine (50 microM) were compared with those obtained without glycine. The increase in chloride conductance (gCl-) evoked by glycine was time and voltage dependent. Inactivation and reactivation of the chloride current were observed during voltage pulses from 0 to -50 mV and from -50 to 0 mV, respectively, and they occurred with similar time constants (2-3 s). During glycine application, voltage-ramp analysis revealed a shift in the reversal potential (ECl-) occurring at all [Cl-]i tested. 4. The basis of the voltage sensitivity of glycine-evoked gCl- was first analyzed by measuring the relative changes in the total open probability (NPo) of glycine-activated channels with voltage.(ABSTRACT TRUNCATED AT 250 WORDS)
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Microglial cells invade the central nervous system during embryonic development, but their developmental functional roles in vivo remain largely unknown. Accordingly, their invasion pattern during early embryonic development is still... more
Microglial cells invade the central nervous system during embryonic development, but their developmental functional roles in vivo remain largely unknown. Accordingly, their invasion pattern during early embryonic development is still poorly understood. To address this issue, we analyzed the initial developmental pattern of microglial cell invasion in the spinal cord of CX3CR1‐eGFP mouse embryos using immunohistochemistry. Microglial cells began to invade the mouse embryonic spinal cord at a developmental period corresponding to the onset of spontaneous electrical activity and of synaptogenesis. Microglial cells reached the spinal cord through the peripheral vasculature and began to invade the parenchyma at 11.5 days of embryonic age (E11.5). Remarkably, at E12.5, activated microglial cells aggregated in the dorsolateral region close to terminals of dying dorsal root ganglia neurons. At E13.5, microglial cells in the ventral marginal zone interacted with radial glial cells, whereas ramified microglial cells within the parenchyma interacted with growing capillaries. At this age, activated microglial cells (Mac‐2 staining) also accumulated within the lateral motor columns at the onset of the developmental cell death of motoneurons. This cell aggregation was still observed at E14.5, but microglial cells no longer expressed Mac‐2. At E15.5, microglial cells were randomly distributed within the parenchyma. Our results provide the essential basis for further studies on the role of microglial cells in the early development of spinal cord neuronal networks in vivo. © 2011 Wiley‐Liss, Inc.
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Renshaw cells (V1R) are excitable as soon as they reach their final location next to the spinal motoneurons and are functionally heterogeneous. Using multiple experimental approaches, in combination with biophysical modeling and dynamical... more
Renshaw cells (V1R) are excitable as soon as they reach their final location next to the spinal motoneurons and are functionally heterogeneous. Using multiple experimental approaches, in combination with biophysical modeling and dynamical systems theory, we analyzed, for the first time, the mechanisms underlying the electrophysiological properties of V1R during early embryonic development of the mouse spinal cord locomotor networks (E11.5–E16.5). We found that these interneurons are subdivided into several functional clusters from E11.5 and then display an unexpected transitory involution process during which they lose their ability to sustain tonic firing. We demonstrated that the essential factor controlling the diversity of the discharge pattern of embryonic V1R is the ratio of a persistent sodium conductance to a delayed rectifier potassium conductance. Taken together, our results reveal how a simple mechanism, based on the synergy of two voltage-dependent conductances that are ...
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The Author(s) 2013. This article is published with open access at Springerlink.com Abstract The axon initial segment (AIS) is responsible for both the modulation of action potentials and the maintenance of neuronal polarity. Yet, the... more
The Author(s) 2013. This article is published with open access at Springerlink.com Abstract The axon initial segment (AIS) is responsible for both the modulation of action potentials and the maintenance of neuronal polarity. Yet, the molecular mechanisms controlling its assembly are incompletely understood. Our study in single electroporated motor neu-rons in mouse embryos revealed that AnkyrinG (AnkG), the AIS master organizer, is undetectable in bipolar migrating motor neurons, but is already expressed at the beginning of axonogenesis at E9.5 and initially distributed homogeneously along the entire growing axon. Then, from E11.5, a stage when AnkG is already apposed to the membrane, as observed by electron microscopy, the protein progressively becomes restricted to the proximal axon. Analysis on the global motor neurons population indicated that Neurofascin follows an identical spatio-temporal dis-tribution, whereas sodium channels and b4-spectrin only appear along AnkG? segments ...
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Copyright © 2011 Anne-Emilie Allain et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work... more
Copyright © 2011 Anne-Emilie Allain et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. γ-aminobutyric acid (GABA) acting on Cl −-permeable ionotropic type A (GABAA) receptors (GABAAR) is the major inhibitory neurotransmitter in the adult central nervous system of vertebrates. In immature brain structures, GABA exerts depolarizing effects mostly contributing to the expression of spontaneous activities that are instructive for the construction of neural networks but GABA also acts as a potent trophic factor. In the present paper, we concentrate on brainstem and spinal motoneurons that are largely targeted by GABAergic interneurons, and we bring together data on the switch from excitatory to inhibitory effects of GABA, on the maturation of the GABAergic system and GABAAR subunits. We finally discuss the role of GABA a...
Super-resolution imaging of synapses has revealed that key synaptic proteins are dynamically organized within sub-synaptic domains (SSDs). At mixed inhibitory synapses in spinal cord neurons, both GlyRs and GABAARs reside at the same... more
Super-resolution imaging of synapses has revealed that key synaptic proteins are dynamically organized within sub-synaptic domains (SSDs). At mixed inhibitory synapses in spinal cord neurons, both GlyRs and GABAARs reside at the same post-synaptic density (PSD). To examine how the different inhibitory receptors are organized and regulated, we carried out dual-color direct stochastic optical reconstruction microscopy (dSTORM). We found that endogenous GlyRs and GABAARs as well as their common scaffold protein gephyrin form SSDs that align with pre-synaptic RIM1/2, thus forming trans-synaptic nanocolumns. Strikingly, GlyRs and GABAARs occupy different sub-synaptic spaces, exhibiting only a partial overlap at mixed inhibitory synapses. When network activity was increased by pharmacological treatment using the K+ channel blocker 4-aminopyridine (4-AP), the GABAAR copy numbers of as well as the number of GABAAR SSDs were reduced, while GlyRs remained largely unchanged. This differential ...