The development of the electrical activity of hypothalamic cells was studied using intracellular ... 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.
Glycine is one of the most important inhibitory neurotransmitters in the spinal cord and the brai... 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.
Electrophysiological recordings of outside-out patches to fast-flow applications of glycine were ... 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...
Miniature IPSCs (mIPSCs) recorded in the Mauthner (M)-cell of zebrafish larvae have a broad ampli... 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...
Glycine is one of the most important inhibitory neurotransmitters in the spinal cord and the brai... 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.
Characteristics and functional activity of Angiotensin II (AII) neuronal receptors were studied u... 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)
The whole-cell voltage clamp technique was used to record potassium currents in mouse fetal hypot... 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.
The effects of the anti-ischemic agents ifenprodil and its derivative SL 82.0715 ((+/-)-alpha-(4-... 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.
From morphological characterization and intracellular recordings, monolayer cultures derived from... 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.
The development of the electrical activity of hypothalamic cells was studied using intracellular ... 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.
Glycine is one of the most important inhibitory neurotransmitters in the spinal cord and the brai... 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.
Electrophysiological recordings of outside-out patches to fast-flow applications of glycine were ... 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...
Miniature IPSCs (mIPSCs) recorded in the Mauthner (M)-cell of zebrafish larvae have a broad ampli... 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...
Glycine is one of the most important inhibitory neurotransmitters in the spinal cord and the brai... 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.
Characteristics and functional activity of Angiotensin II (AII) neuronal receptors were studied u... 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)
The whole-cell voltage clamp technique was used to record potassium currents in mouse fetal hypot... 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.
The effects of the anti-ischemic agents ifenprodil and its derivative SL 82.0715 ((+/-)-alpha-(4-... 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.
From morphological characterization and intracellular recordings, monolayer cultures derived from... 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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