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 ...
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Univ Paris 06, Paris, France. Hasselt Univ, BIOMED Cell Physiol, Diepenbeek, Belgium. Univ Paris 06, INSERM, U952, Paris, France. Univ Paris 06, CNRS, UMR 7224, Paris, France.
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While the role of cholinergic neurotransmission from motoneurons is well established during neuromuscular development, whether it regulates central nervous system development in the spinal cord is unclear. Zebrafish presents a powerful... more
While the role of cholinergic neurotransmission from motoneurons is well established during neuromuscular development, whether it regulates central nervous system development in the spinal cord is unclear. Zebrafish presents a powerful model to investigate how the cholinergic system is set up and evolves during neural circuit formation. In this study, we carried out a detailed spatiotemporal analysis of the cholinergic system in embryonic and larval zebrafish. In 1-day-old embryos, we show that spinal motoneurons express presynaptic cholinergic genes including choline acetyltransferase (chata), vesicular acetylcholine transporters (vachta, vachtb), high-affinity choline transporter (hacta) and acetylcholinesterase (ache), while nicotinic acetylcholine receptor (nAChR) subunits are mainly expressed in interneurons. However, in 3-day-old embryos, we found an unexpected decrease in presynaptic cholinergic transcript expression in a rostral to caudal gradient in the spinal cord, which c...
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Spontaneous neuronal activity occurs at the onset of the synaptogenesis in the central Nervous System and plays a major role in shaping developing neural networks. How intrinsic properties of neurons evolve during this critical... more
Spontaneous neuronal activity occurs at the onset of the synaptogenesis in the central Nervous System and plays a major role in shaping developing neural networks. How intrinsic properties of neurons evolve during this critical developmental period remains largely unknown. We studied the Renshaw cells because they participate to the early-synchronized neuronal activity in the embryonic spinal cord. We found that these interneurons are subdivided into several functional clusters at the onset of the synaptogenesis and then display a transitory involution process during which they lose their ability to sustain tonic firing. This complex developmental trajectory results from the synergy between a persistent sodium inward current and a delayed rectifier potassium outward current, which are present in most neurons during development and in the adult. Taken together, our results reveal a core mechanism producing functional hetereogeneity in embryonic neurons and likely shaping the ongoing ...
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SUMMARYIn the developing central nervous system, electrical signaling is thought to rely exclusively on differentiating neurons as they acquire the ability to generate action potentials. Accordingly, the neuroepithelial progenitors (NEPs)... more
SUMMARYIn the developing central nervous system, electrical signaling is thought to rely exclusively on differentiating neurons as they acquire the ability to generate action potentials. Accordingly, the neuroepithelial progenitors (NEPs) giving rise to all neurons and glial cells during development have been reported to remain electrically passive. Here, we investigated the physiological properties of NEPs in the mouse spinal cord at the onset of spontaneous neural activity (SNA) initiating motor behavior in embryos. Using patch-clamp recordings, we discovered that spinal NEPs exhibit spontaneous membrane depolarizations during episodes of SNA. These recurrent depolarizations exhibited a ventral-to-dorsal gradient with the highest amplitude located in the floor-plate – the ventral-most part of the neuroepithelium. Paired-recordings revealed that NEPs are extensively coupled via gap-junctions and form a single electrical syncytium. Although other NEPs were electrically passive, we d...
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Zebrafish embryos have small and slow miniature end-plate currents (mEPCs), whereas only a few days later larval mEPCs are an order of magnitude larger and faster, being among the fastest of all neuromuscular synapses. To identify the... more
Zebrafish embryos have small and slow miniature end-plate currents (mEPCs), whereas only a few days later larval mEPCs are an order of magnitude larger and faster, being among the fastest of all neuromuscular synapses. To identify the bases for these changes we compared, in embryos and larvae, the properties and distributions of acetylcholine (ACh) receptors (AChRs) and acetylcholinesterase (AChE) as well as the ultrastructure of the developing neuromuscular junctions (NMJs). To mimic synaptic release, patches of muscle membrane were exposed briefly (for 1 ms) to a saturating concentration (10 mM) of ACh. The AChR deactivation kinetics were twice as slow in embryos compared with larvae. In both embryos and larvae, AChRs demonstrated open channel block by millimolar ACh, and this was detected during mEPCs, indicating that a high concentration of ACh is released at immature and mature NMJs. AChR and AChE distributions were compared using the selective fluorescently conjugated labels α...
Research Interests: Biophysics, Electrophysiology, Electron Microscopy, Kinetics, Biology, and 15 moreNeurotransmission, Cell Biology, Neurophysiology, Medicine, Animals, Acetylcholinesterase, Acetylcholine, Synaptic Transmission, SYNAPSES, Nervous System, Neuromuscular Junction, Psychology and Cognitive Sciences, Cholinergic antagonists, Medical and Health Sciences, and Neuromuscular transmission
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Zinc has been reported to potentiate glycine receptors (GlyR), but the physiological significance of this observation has been put in doubt by the relatively high values of the EC50, 0.5–1 μM, since such concentrations may not be attained... more
Zinc has been reported to potentiate glycine receptors (GlyR), but the physiological significance of this observation has been put in doubt by the relatively high values of the EC50, 0.5–1 μM, since such concentrations may not be attained in the synaptic cleft of glycinergic synapses. We have re-evaluated this observation in the frame of the hypothesis that contaminant heavy metals present in usual solutions may have lead to underestimate the affinity of the zinc binding site, and therefore to underestimate the potential physiological role of zinc. Using chelators either to complex heavy metals or to apply zinc at controlled concentrations, we have examined the action of zinc on GlyR kinetics in outside-out patches from 50-h-old zebrafish Mauthner cells. Chelating contaminating heavy metals with tricine or N,N,N′,N′-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) decreased the duration of the currents evoked by glycine, confirming that traces of heavy metals alter the GlyR respons...
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We used whole cell and outside-out patch-clamp techniques with reticulospinal Mauthner neurons of zebrafish embryos to investigate the developmental changes in the properties of glycinergic synaptic currents in vivo from the onset of... more
We used whole cell and outside-out patch-clamp techniques with reticulospinal Mauthner neurons of zebrafish embryos to investigate the developmental changes in the properties of glycinergic synaptic currents in vivo from the onset of synaptogenesis. Miniature inhibitory postsynaptic currents (mIPSCs) were isolated and recorded in the presence of TTX (1 μM), kynurenic acid (1 mM), and bicuculline (10 μM) and were found to be sensitive to strychnine (1 μM). The mIPSCs were first observed in 26–29 h postfertilization (hpf) embryos at a very low frequency of ∼0.04 Hz, which increased to ∼0.5 Hz by 30–40 hpf, and was ∼10 Hz in newly hatched (>50 hpf) larvae, indicating an accelerated increase in synaptic activity. At all embryonic stages, the amplitudes of the mIPSCs were variable but their means were similar (∼100 pA), suggesting rapid formation of the postsynaptic matrix. The 20–80% rise times of mIPSCs in embryos were longer (0.6–1.2 ms) than in larvae (∼0.3 ms), likely due to slow...
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Microglia are known to regulate several aspects of the development of the central nervous system. When microglia colonize the spinal cord, from E11.5 in the mouse embryo, they interact with growing central axons of dorsal root ganglion... more
Microglia are known to regulate several aspects of the development of the central nervous system. When microglia colonize the spinal cord, from E11.5 in the mouse embryo, they interact with growing central axons of dorsal root ganglion sensory neurons (SNs), which suggests that they may have some functions in SN development. To address this issue, we analyzed the effects of embryonic macrophage ablation on the early development of SNs using mouse embryo lacking embryonic macrophages (PU.1 knock‐out mice) and immune cell ablation. We discovered that, in addition to microglia, embryonic macrophages contact tropomyosin receptor kinase (Trk) C+ SN, TrkB+ SN, and TrkA+ SN peripheral neurites from E11.5. Deprivation of immune cells resulted in an initial reduction of TrkC+ SN and TrkB+ SN populations at E11.5 that was unlikely to be related to an alteration in their developmental cell death (DCD), followed by a transitory increase in their number at E12.5. It also resulted in a reduction ...
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At larval zebrafish neuromuscular junctions (NMJs), miniature end plate currents (mEPCs) recordedin vivohave an unusually fast time course. We used fast-flow application of acetylcholine (ACh) onto outside-out patches to mimic the effect... more
At larval zebrafish neuromuscular junctions (NMJs), miniature end plate currents (mEPCs) recordedin vivohave an unusually fast time course. We used fast-flow application of acetylcholine (ACh) onto outside-out patches to mimic the effect of synaptic release onto small numbers of ACh receptor channels (AChRs). Positively charged ACh acted at hyperpolarized potentials and at millimolar concentrations as a fast (“flickering”) open channel blocker of AChRs. Because of filtering, the open channel block resulted in reduced amplitude of single channel currents. Immediately after brief (1 msec) application (without significant desensitization) of millimolar ACh at hyperpolarized potentials, a slower, transient current appeared because of delayed reversal of the block. This rebound current depended on the ACh concentration and resembled in time course the mEPC. A simple kinetic model of the AChR that includes an open channel-blocking step accounted for our single channel results, as well as ...
Research Interests: Neuroscience, Biophysics, Chemistry, Kinetics, Neurotransmission, and 15 moreMedicine, Animals, Acetylcholine, Synaptic Transmission, Larva, Lumping Kinetic Model, Neuromuscular Junction, Time Course, Excitatory Postsynaptic Potentials, Motor Neurons, Single Channel, Psychology and Cognitive Sciences, Medical and Health Sciences, Kinetic model, and Electric stimulation
Calcium-dependent inactivation of NMDA channels was examined on cultured rat hippocampal neurons using whole-cell voltage-clamp and cell-attached single-channel recording. An ATP regeneration solution was included in the patch pipette to... more
Calcium-dependent inactivation of NMDA channels was examined on cultured rat hippocampal neurons using whole-cell voltage-clamp and cell-attached single-channel recording. An ATP regeneration solution was included in the patch pipette to retard current “rundown.” In normal [Ca2+]o (1-2 mM) and 10 microM glycine, macroscopic currents evoked by 15 sec applications of NMDA (10 microM) inactivated slowly following an initial peak. At -50 mV in cells buffered to [Ca2+]i < 10(- 8) M with 10 mM EGTA, the inactivation time constant (tau inact) was approximately 5 sec. Inactivation did not occur at membrane potentials of +40 mV and was absent at [Ca2+]o < or = 0.2 mM, suggesting that inactivation resulted from transmembrane calcium influx. The percentage inactivation and tau inact were dependent on [Ca2+]o. The tau inact was also longer with BAPTA in the whole-cell pipette compared to EGTA, suggesting that tau inact reflects primarily the rate of accumulation of intracellular calcium. ...
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Virtually all oligodendrocyte precursors cells (OPCs) receive glutamatergic and/or GABAergic synapses that are lost upon their differentiation into oligodendrocytes in the postnatal and adult brain. Although OPCs are generated at... more
Virtually all oligodendrocyte precursors cells (OPCs) receive glutamatergic and/or GABAergic synapses that are lost upon their differentiation into oligodendrocytes in the postnatal and adult brain. Although OPCs are generated at mid-embryonic stages, several weeks before the onset of myelination, it remains unknown when and where OPCs receive their first synapses and become susceptible to the influence of neuronal activity. In the embryonic spinal cord, neuro-epithelial precursors in the pMN domain cease generating cholinergic motor neurons (MNs) to produce OPCs when the first synapses are formed in the ventral-lateral marginal zone. We discovered that when the first synapses form onto MNs, axoglial synapses also form onto the processes of neuro-epithelial precursors located in the marginal zone as they differentiate into OPCs. After leaving the neuro-epithelium, these pioneer OPCs preferentially accumulate in the marginal zone where they are contacted by functional glutamatergic a...
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The postnatal maturation pattern of glycine receptor channels (GlyRs) expressed by dopaminergic (DA) neurones of the rat substantia nigra pars compacta (SNc) was investigated using single‐channel and whole‐cell patch‐clamp recordings in... more
The postnatal maturation pattern of glycine receptor channels (GlyRs) expressed by dopaminergic (DA) neurones of the rat substantia nigra pars compacta (SNc) was investigated using single‐channel and whole‐cell patch‐clamp recordings in brain slices from rats aged 7–21 postnatal days (P). In neonatal rats (P7‐P10), GlyRs exhibited a main conductance state of 100–110 pS with a mean open time of 16 ms. In juvenile rats (P19‐P22), both the GlyR main conductance state (46‐55 pS) and the mean open time (6.8 ms) were decreased. In neonatal rats, application of 30 μm picrotoxin, which is known to block homomeric GlyRs, strongly reduced glycine‐evoked responses, while it was much less effective in juvenile rats. These results suggest that these GlyRs correspond functionally to α2 homomeric GlyRs in neonatal rats and α1/β heteromeric GlyRs in juvenile rats. A drastic but transient decrease in the glycine responsiveness of DA neurones occurred around P17 concomitant to the functional switch f...
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Research Interests: Biology, Cell Biology, Macrophages, Apoptosis, Medicine, and 15 moreGene expression, Signal Transduction, Adenosine, Mice, Animals, Amino Acids, In Vivo, Phagocytosis, Retinoic Acid, Cell communication, Gtp Binding Proteins, Thymocyte, Adenosine Triphosphate, coculture techniques, and Medical biochemistry and metabolomics
[Brone, Bert; Smolders, Silke; Smolders, Sophie; Swinnen, Nina; Rigo, Jean-Michel] Hasselt Univ, BIOMED, Agoralaan Gebouw C, B-3590 Diepenbeek, Belgium. [Swinnen, Nina; Legendre, Pascal] Univ Paris 06, INSERM, U952, Paris, Ile De France,... more
[Brone, Bert; Smolders, Silke; Smolders, Sophie; Swinnen, Nina; Rigo, Jean-Michel] Hasselt Univ, BIOMED, Agoralaan Gebouw C, B-3590 Diepenbeek, Belgium. [Swinnen, Nina; Legendre, Pascal] Univ Paris 06, INSERM, U952, Paris, Ile De France, France. [Swinnen, Nina; Legendre, Pascal] Univ Paris 06, CNRS, UMR 7224, Paris, Ile De France, France. [Swinnen, Nina; Legendre, Pascal] Univ Paris 06, Paris, Ile De France, France.
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Research Interests: Genetics, Functional Analysis, Electrophysiology, Long Term Potentiation, Biology, and 15 moreImmunohistochemistry, Intellectual Disability, Medicine, Interneuron, Biological Sciences, Learning and Memory, Autism Spectrum Disorder, Deletion, Glycine receptor, Locomotor Activity, Language Delay, De Novo, Biotinylation, Knock Out Mice, and Medical and Health Sciences
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A remarkable feature of early neuronal networks is their endogenous ability to generate spontaneous rhythmic electrical activity independently of any external stimuli. In the mouse embryonic SC, this activity starts at an embryonic age of... more
A remarkable feature of early neuronal networks is their endogenous ability to generate spontaneous rhythmic electrical activity independently of any external stimuli. In the mouse embryonic SC, this activity starts at an embryonic age of ∼ 12 d and is characterized by bursts of action potentials recurring every 2-3 min. Although these bursts have been extensively studied using extracellular recordings and are known to play an important role in motoneuron (MN) maturation, the mechanisms driving MN activity at the onset of synaptogenesis are still poorly understood. Because only cholinergic antagonists are known to abolish early spontaneous activity, it has long been assumed that spinal cord (SC) activity relies on a core network of MNs synchronized via direct cholinergic collaterals. Using a combination of whole-cell patch-clamp recordings and extracellular recordings in E12.5 isolated mouse SC preparations, we found that spontaneous MN activity is driven by recurrent giant depolari...
Research Interests: Neuroscience, Gap Junctions, Biology, Medicine, Pregnancy, and 15 moreMice, Female, Animals, Acetylcholine, Glycine, Action Potentials, Glutamic Acid, Glutamate Receptor, Cholinergic agents, Depolarization, Motor Neurons, Gabaergic, Psychology and Cognitive Sciences, Medical and Health Sciences, and In Vitro Techniques
Rhythmic electrical activity is a hallmark of the developing embryonic CNS and is required for proper development in addition to genetic programs. Neurotransmitter release contributes to the genesis of this activity. In the mouse spinal... more
Rhythmic electrical activity is a hallmark of the developing embryonic CNS and is required for proper development in addition to genetic programs. Neurotransmitter release contributes to the genesis of this activity. In the mouse spinal cord, this rhythmic activity occurs after embryonic day 11.5 (E11.5) as waves spreading along the entire cord. At E12.5, blocking glycine receptors alters the propagation of the rhythmic activity, but the cellular source of the glycine receptor agonist, the release mechanisms, and its function remain obscure. At this early stage, the presence of synaptic activity even remains unexplored. Using isolated embryonic spinal cord preparations and whole-cell patch-clamp recordings of identified motoneurons, we find that the first synaptic activity develops at E12.5 and is mainly GABAergic. Using a multiple approach including direct measurement of neurotransmitter release (i.e., outside-out sniffer technique), we also show that, between E12.5 and E14.5, the ...
Research Interests: Neuroscience, Biology, Medicine, Pregnancy, Mice, and 15 moreFemale, Animals, Spinal Cord, Embryonic Stem Cell, Periodicity, Glycine receptor, Glycine, Neurotransmitter, Patch Clamp, Spontaneous Activity, Motor Neurons, Psychology and Cognitive Sciences, CHO cells, Cricetinae, and Medical and Health Sciences
Glycine and GABA mediate inhibitory neurotransmission in the spinal cord and central nervous system. The general concept of neurotransmission is now challenged by the contribution of both phasic activation of postsynaptic glycine and... more
Glycine and GABA mediate inhibitory neurotransmission in the spinal cord and central nervous system. The general concept of neurotransmission is now challenged by the contribution of both phasic activation of postsynaptic glycine and GABA(A) receptors (GlyRs and GABA(A)Rs, respectively) and tonic activity of these receptors located at extrasynaptic sites. GlyR and GABA(A)R kinetics depend on several parameters, including subunit composition, subsynaptic localization and activation mode. Postsynaptic and extrasynaptic receptors display different subunit compositions and are activated by fast presynaptic and slow paracrine release of neurotransmitters, respectively. GlyR and GABA(A)R functional properties also rely on their aggregation level, which is higher at postsynaptic densities than at extrasynaptic loci. Finally, these receptors can co-aggregate at mixed inhibitory postsynaptic densities where they cross-modulate their activity, providing another parameter of functional complex...
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Research Interests: Cognitive Science, Biology, Motor neuron, Medicine, Gene expression, and 15 moreMolecular and cellular biology, Mice, Animals, Gene, Degeneration, Cellular and Molecular Neuroscience, Growth Factor, Neurotrophic Factors, Neurosciences, Gene Expression Regulation, Transforming Growth Factor, Motor Neurons, reverse transcriptase polymerase chain reaction, Nerve degeneration, and In Vitro Techniques
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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... more
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 and its GABAAR in immature hypoglossal motoneurons of the spastic (SPA) mouse, a model of human hyperekplexic syndrome.
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The amplitude of glycinergic miniature inhibitory postsynaptic currents (mIPSCs) varies considerably in neurons recorded in the isolated hindbrain of 50‐h‐old zebrafish larvae. At this age, glycinergic synapses are functionally mature. In... more
The amplitude of glycinergic miniature inhibitory postsynaptic currents (mIPSCs) varies considerably in neurons recorded in the isolated hindbrain of 50‐h‐old zebrafish larvae. At this age, glycinergic synapses are functionally mature. In order to measure the occupancy level of postsynaptic glycine receptors (GlyRs) and to determine the pre‐ and/or postsynaptic origin of its variability, we analysed mIPSCs within bursts evoked by α‐latrotoxin (0.1–1 nm). Two types of burst were observed according to their mIPSC frequencies: ‘slow’ bursts with clearly spaced mIPSCs and ‘fast’ bursts characterised by superimposed events. Non‐stationary noise analysis of mIPSCs in some ‘slow’ bursts recorded in the presence or in the absence of Ca2+ denoted that mIPSC amplitude variance did not depend on the quantity of neurotransmitters released (presynaptic origin), but rather on intrinsic stochastic behaviour of the same group of GlyRs (postsynaptic origin). In these bursts, the open probability mea...
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Microglia are known to invade the mammalian spinal cord (SC) at an early embryonic stage. While the mechanisms underlying this early colonization of the nervous system are still unknown, we recently found that it is associated, at least... more
Microglia are known to invade the mammalian spinal cord (SC) at an early embryonic stage. While the mechanisms underlying this early colonization of the nervous system are still unknown, we recently found that it is associated, at least partially, with the ability of microglia to proliferate at the onset of motoneuron developmental cell death and of synaptogenesis in mouse embryo (E13.5).In vitrostudies have shown that the proliferation and activation of adult microglia can be influenced by the purinergic ionotropic receptor P2X7 via a coupling with Pannexin-1. By performing patch-clamp recordingsin situusing a whole-mouse embryonic SC preparation, we show here that embryonic microglia already express functional P2X7R. P2X7R activation evoked a biphasic current in embryonic microglia, which is supposed to reflect large plasma membrane pore opening. However, although embryonic microglia express pannexin-1, this biphasic current was still recorded in microglia of pannexin-1 knock-out ...
Research Interests: Neuroscience, Biophysics, Biology, Enzyme Inhibitors, Cell Biology, and 15 moreMedicine, Chemokines and chemokine receptors, Cell Differentiation, Mice, Animals, Microglia, Embryonic Stem Cell, Caspase, HUMAN MEDICINE, Adenosine Triphosphate, Cell Adhesion Molecules, Pannexin, Connexins, Medical and Health Sciences, and Electric stimulation
Research Interests: Neuroscience, Monte Carlo Simulation, Kinetics, Science, Biology, and 14 moreMedicine, Multidisciplinary, Ion Channels, Stochastic processes, Diffusion, Animals, Synaptic Transmission, Goldfish, Transmitter release, SYNAPSES, Zebrafish, Statistical models, Neuromuscular Junction, and Monte Carlo Method
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In a previous study, miniature inhibitory synaptic events recorded in the Mauthner cell of the 52-hour-old zebrafish larvae (Brachydanio rerio) were found to be mainly glycinergic. Their amplitude distribution was not Gaussian and it was... more
In a previous study, miniature inhibitory synaptic events recorded in the Mauthner cell of the 52-hour-old zebrafish larvae (Brachydanio rerio) were found to be mainly glycinergic. Their amplitude distribution was not Gaussian and it was proposed that their large amplitude variation might reflect the activation of immature synapses. However, ultrastructural studies of the synaptic contacts over the M-cell soma of 52 h larvae described here, revealed that numerous synaptic contacts on this neuron are already mature at this developmental stage and that most of them already contain a single active zone. As in the adult goldfish, immunohistochemistry indicates the presence of both glycine- and GABA-immunoreactive boutons which establish synaptic contacts. We also found that, in addition to the predominant glycinergic postsynaptic inhibitory currents, some postsynaptic currents are also GABAergic since they are specifically inhibited by bicuculline (20 microM). GABAergic miniature events (time to peak close to 0.8 ms and decay time-constant close to 45 ms) were only detected in the presence of 11.5 mM [KCl]o. Their amplitude distributions were well fitted by one, or at most two, Gaussian curves. Outside-out recordings showed one class of GABA receptors with a main conductance state of 23 pS. This indicates that the smallest GABAergic miniature inhibitory synaptic events correspond to the opening of 14-20 chloride channels Pre- and postsynaptic factors which contribute to the predominance of glycinergic synaptic currents over GABAergic ones in untreated preparations and to the striking differences between their frequencies and their respective amplitude distribution histograms are discussed with reference to the morphological characteristics of the mature synaptic endings impinging on this still developing neuron.
Research Interests: Neuroscience, Psychology, Biology, Neurotransmission, Immunohistochemistry, and 15 moreTransmission Electron Microscopy, Medicine, Brain, Animals, Constant Time Delay, Glycine receptor, Time Factors, SYNAPSES, Axons, Neurosciences, Patch Clamp, GABA receptor, Phosphate Buffer Saline, Gabaergic, and Active Zone
Glycine acts as a neuromodulator to regions rich in glutamatergic synapses, such as the forebrain. However, recent evidences for synaptic release of glycine in hippocampal cultured neurons and synaptosomes argue for the existence of... more
Glycine acts as a neuromodulator to regions rich in glutamatergic synapses, such as the forebrain. However, recent evidences for synaptic release of glycine in hippocampal cultured neurons and synaptosomes argue for the existence of functional glycinergic synapses in the hippocampus. It is well established that GABA and glycine act in concert at inhibitory synapses, while the existence of synapses which utilize both glutamate and glycine is less common. The purpose of the present study was to investigate the distribution of glycine and its role in hippocampal neurotransmission. Using immunohistochemistry, we demonstrate that vesicular glycine is preferentially stored in glutamatergic, rather than GABAergic presynaptic terminals. Using the sniffer patch technique, we found that glycine could be released upon presynaptic activity. Furthermore, using whole-cell patch-clamp recordings, we show for the first time the presence of a postsynaptic strychnine-sensitive chloride current in response to presynaptic stimulation. The small amplitude of this current is likely due to the paucity of postsynaptic glycine receptors rather than a low level of glycine release. Taken together, our results suggest that glycine is stored in glutamatergic presynaptic terminals. It is likely that the major role of glycine that is released from presynaptic terminals is to modulate N-methyl-d-aspartate receptor function but may also play a role in decreasing neuronal excitability by opposing glutamatergic neurotransmission in pathological states such as epilepsy or ischemia.
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Research Interests: Neuroscience, Psychology, Biophysics, Kinetics, Biology, and 15 moreMedicine, Low Frequency, Markov chains, Animals, Neurons, Frequency Dependence, Constant Time Delay, Glycine receptor, Receptor, Glycine, Neurosciences, GABA receptor, Markov model, Neurotransmitter Release, and Electric stimulation
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A fully automatized software package for detection and measurements of randomly occurring synaptic transients embedded in background noise is described. It is based on waveform recognition protocols, allows analysis of long data segments,... more
A fully automatized software package for detection and measurements of randomly occurring synaptic transients embedded in background noise is described. It is based on waveform recognition protocols, allows analysis of long data segments, and provides quantitative information about event amplitudes and kinetics. Simulated postsynaptic recordings have been used to assess its performance over a wide range of conditions mimicking those seen in physiological experiments.
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Research Interests: Biology, Immunohistochemistry, Enzyme Inhibitors, Flavonoids, Medicine, and 15 moreCell Differentiation, Glutamate, Nitriles, Animals, Cell Death, Mitogen Activated Protein Kinase, Rats, Chromones, MAP Kinase, Optimality Condition, Neurosciences, Ly, Glutamate decarboxylase, Dorsal Horn, and Chemical Neuroanatomy
Research Interests: Epilepsy, Biology, Hippocampus, Cellular, Humans, and 15 moreGlutamate receptors, Female, Animals, Calcium Signaling, Dendrites, Clinical Sciences, Adult, Glycine, Cellular and Molecular Medicine, Action Potentials, Biochemistry and cell biology, Hippocampal formation, Chlorides, Excitatory Postsynaptic Potentials, and Gabaergic
Research Interests: Neuroscience, Chemistry, Electrophysiology, Kinetics, Biology, and 15 moreCalcium, Biological Chemistry, Medicine, Biological Sciences, French horn, Animals, Phosphorylation, CHEMICAL SCIENCES, Glycine receptor, Calcineurin, Glycine, Protein Binding, Bicuculline, GABAA receptor, and Medical and Health Sciences
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Research Interests: Chemistry, Biological Chemistry, Medicine, Patch-clamp and imaging techniques, Biological Sciences, and 13 moreEvoked Potentials, Humans, Animals, CHEMICAL SCIENCES, Glycine receptor, Receptor, Transfection, Protein Conformation, Glycine, Protein Binding, CHO cells, Cricetinae, and Medical and Health Sciences
Microglia are the immune cells of the central nervous system. They are suspected to play important roles in adult synaptogenesis and in the development of the neuronal network. Microglial cells originate from progenitors in the yolk sac.... more
Microglia are the immune cells of the central nervous system. They are suspected to play important roles in adult synaptogenesis and in the development of the neuronal network. Microglial cells originate from progenitors in the yolk sac. Although it was suggested that they invade the cortex at early developmental stages in the embryo, their invasion pattern remains largely unknown. To address this issue we analyzed the pattern of cortical invasion by microglial cells in mouse embryos at the onset of neuronal cell migration using in vivo immunohistochemistry and ex vivo time‐lapse analysis of microglial cells. Microglial cells begin to invade the cortex at 11.5 days of embryonic age (E11.5). They first accumulate at the pial surface and within the lateral ventricles, after which they spread throughout the cortical wall, avoiding the cortical plate region in later embryonic ages. The invasion of the cortical parenchyma occurs in different phases. First, there is a gradual increase of ...
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Glycine receptor (GlyR) alpha3 is involved in vision, and processing of acoustic and nociceptive signals, and RNA editing of GLRA3 transcripts was associated with hippocampal pathophysiology of mesial temporal lobe epilepsy (TLE).... more
Glycine receptor (GlyR) alpha3 is involved in vision, and processing of acoustic and nociceptive signals, and RNA editing of GLRA3 transcripts was associated with hippocampal pathophysiology of mesial temporal lobe epilepsy (TLE). However, neither the role of GlyR alpha3 splicing in hippocampal neurons nor the expression of splice variants have yet been elucidated. We report here that the long (L) splice variant of GlyR alpha3 predominates in the brain of rodents. Cellular analysis using primary hippocampal neurons and hippocampus cryosections revealed preferential association of synaptic alpha3L clusters with glutamatergic nerve endings in strata granulare and pyramidale. In primary hippocampal neurons GlyR alpha3L clusters also preferred glutamatergic nerve endings while alpha3K was mainly in a diffuse state. Co-expression of GlyR beta subunit with alpha3L or alpha3K produced heteromeric receptor clusters and favoured their association with GABAergic terminals. However, heteromeric alpha3L was still more efficient than heteromeric alpha3K in associating with glutamatergic nerve endings. To give physiological relevance to these results we have finally analysed GlyR alpha3 splicing in human hippocampus obtained from patients with intractable TLE. As up-regulation of alpha3K occurred at the expense of alpha3L in TLE patients with a severe course of disease and a high degree of hippocampal damage, our results again involve post-transcriptional processing of GLRA3 transcripts in the pathophysiology of TLE.
Research Interests: Neuroscience, Psychology, Cognitive Science, Biology, Confocal Microscopy, and 15 moreMedicine, Hippocampus, Glutamate, Humans, Animals, Neurons, European, Fluorescent Antibody Technique, Rats, Glycine receptor, Protein isoforms, Neurosciences, Glutamic Acid, Hippocampal formation, and reverse transcriptase polymerase chain reaction
Research Interests: Neuroscience, Psychology, Cognitive Science, Biophysics, Chemistry, and 15 moreElectrophysiology, Neurotransmission, Medicine, Ion Channels, Brain, Animals, Synaptic Transmission, Conductance, Glycine receptor, Tetrodotoxin, Zebrafish, Glycine, Electric Conductivity, Neurosciences, and In Vitro Techniques
In the hypoglossal nucleus of wild‐type mice, early mixed glycinergic‐GABAergic inhibitory transmission becomes mainly glycinergic during postnatal maturation. In spastic mice (SPA), a model of human hyperekplexic syndrome, an insertion... more
In the hypoglossal nucleus of wild‐type mice, early mixed glycinergic‐GABAergic inhibitory transmission becomes mainly glycinergic during postnatal maturation. In spastic mice (SPA), a model of human hyperekplexic syndrome, an insertion into the gene of the glycine receptor (GlyR) β subunit results in a decreased accumulation of GlyRs at postsynaptic sites and an impaired glycinergic neurotransmission. In SPA mice displaying a mild phenotype (B6C3Fe strain), a compensatory process involving an increased aggregation of GABAA receptors (GABAARs) at postsynaptic sites was proposed to explain survival of mutant animals until adulthood. However, C57BL/6J strain SPA mice which express a lower amount of GlyR β subunit die 2–3 weeks after birth, suggesting that GABAergic compensation does not necessarily take place. We performed a morphofunctional study of inhibitory synapses in the developing hypoglossal nucleus of C57BL/6J SPA mice. In this mutant, the inhibitory synaptic activity was mai...
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In the hypoglossal nucleus, GABA and glycine mediate inhibition at separate or mixed synapses containing glycine receptors (GlyRs) and/or GABA(A) receptors (GABA(A)Rs). The functional development of mixed inhibitory synapses depends on... more
In the hypoglossal nucleus, GABA and glycine mediate inhibition at separate or mixed synapses containing glycine receptors (GlyRs) and/or GABA(A) receptors (GABA(A)Rs). The functional development of mixed inhibitory synapses depends on the brain area studied, but their relative proportion to total synapses generally decreases with time. We have determined the sequential process of inhibitory synapse maturation in the hypoglossal nucleus in vivo. Immunocytochemistry and confocal microscopy were used for codetection of VIAAT, the common presynaptic vesicular transporter of glycine and GABA, GlyRs, GABA(A)R alpha1 and gamma2 subunits, and gephyrin, the scaffold protein implicated in the synaptic localization of inhibitory receptors. In E17 embryos, GlyRs were already clustered while GABA(A)R alpha1 and gamma2 subunit immunoreactivity (IR) displayed both diffuse and clustered patterns. Quantitative analysis at this stage revealed that the majority of GlyR clusters were apposed to VIAAT-IR accumulation and that 30% of them colocalized with gamma2GABA(A)R clusters. This proportion increased with age to 50% at P30. GlyR clusters that did not colocalize with gamma2GABA(A)R clusters were associated with GABA(A)R gamma2 diffuse IR. Interestingly, the percentage of GlyR clusters surrounded by GABA(A)R gamma2 diffuse IR decreased with age, while GlyR clusters colocalized with gamma2GABA(A)R clusters increased. The developmental coclustered pattern of gephyrin and GABA(A)R alpha1 and gamma2 subunits paralleled the coclustered pattern of GlyRs and GABA(A)R alpha1 and gamma2 subunits. Our results indicate that the proportion of GlyR-GABA(A)R coclusters increases until adulthood. A developmental sequence of the postsynaptic events is proposed in which diffuse extrasynaptic GABA(A)Rs accumulate at inhibitory synapses to form postsynaptic clusters, most of them being colocalized with GlyR clusters in the adult.
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The hypothalamo-neurohypophysial system has proved an excellent model for peptidergic neurons in the central nervous system. Electrophysiological studies using in vivo and in vitro preparations with extracellular and intracellular... more
The hypothalamo-neurohypophysial system has proved an excellent model for peptidergic neurons in the central nervous system. Electrophysiological studies using in vivo and in vitro preparations with extracellular and intracellular recording techniques have determined some of the intrinsic and extrinsic mechanisms that generate the striking firing patterns that the neurons exhibit. We have developed a dissociated cell preparation of these neurons and used patch clamp recording techniques to enable detailed studies of membrane properties underlying such activities. Cultured neonatal supraoptic neurons fired spontaneous action potentials which in some cells were distinctively patterned. Under voltage clamp, voltage-activated Na+, K+, and Ca2+ currents were recorded. K+ and Ca2+ currents were modulated by application of alpha-adrenergic agonists, and Ca2+ currents were also modulated by kappa-opioid agonists. The neurons were also sensitive to gamma-aminobutyric acid which acted directly on Cl- channels. Spontaneous, patterned activity, the presence of functional receptors for neurotransmitters and the ability to study the neurons under voltage clamp suggest that this is an excellent model system for studying these peptidergic neurons.