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C R Bader

Publications and source records attributed to C R Bader.

At least 37 records · Page 2Linked to original sources
PubMed1993

Purification of human muscle satellite cells by flow cytometry.

To purify satellite cells directly from human muscle biopsies, we have developed a method based on size separation of dissociated cells by flow cytometry. Immediately after tryptic dissociation of human muscle biopsies and elimination of erythrocytes, microscopic observation and flow cytometry analysis of cell suspensions revealed two populations of cells differing in size and nucleocytoplasmic ratio. Clonal cultures of these two cell types with a manual procedure demonstrated that only the small cells were myogenic satellite cells. Flow cytometry-sorting and analysis of the small cell population showed that (1) all sorted cells contained desmin immediately after dissociation and plating; (2) more than 98% of the cells expressed the 5.1.H11 epitope after 2 weeks of proliferation in culture; and (3) 90% of the sorted cells were able to form myotubes when cultivated at low density or in clonal cultures. Thus, human muscle satellite cells can be directly purified from human muscle samples using flow cytometry.

Adolescent↗
PubMed1993

A voltage-dependent proton current in cultured human skeletal muscle myotubes.

1. A voltage-dependent proton current, IH, was studied in cultured myotubes obtained from biopsies of human muscle, using whole-cell recording with the patch-clamp technique. 2. With a pHo of 8.0 and a calculated pHi of 6.3, IH was activated at voltages more depolarized than -50 mV and its conductance reached its maximum value at voltages more depolarized than +10 mV. 3. Studies of the reversal potential of IH during substitution of K+, Na+, Ca2+, Cl-, Cs+ and H+ in the extracellular solution indicated that protons were the major charge carriers of IH. 4. IH was also activated during a voltage step to +22 mV with a pHo of 7.3 and a calculated pHi of 7.3. 5. Acidification of the extracellular solution led to a shift towards depolarized voltages of the conductance-voltage relationship. 6. Stationary noise analysis of IH suggested that the elementary event underlying IH was very small with a conductance of less than 0.09 pS. 7. Extracellular application of various divalent cations blocked IH. The block by divalent cations was voltage dependent, being more efficient at hyperpolarized than at depolarized voltages. For Cd2+, the Michaelis-Menten constant (Km) for the block was 0.6 microM at -28 mV and 10.4 microM at +12 mV. 8. Ca2+ was a less efficient blocker than Cd2+ but could block IH at physiological concentrations (the Km values for the block were 0.9 mM at -38 mV and 7.3 mM at -8 mV). 9. The voltage-dependent properties of IH and its ability to be affected by pH and Ca2+ suggest that IH might be used by skeletal muscle cells to extrude protons during action potentials. 10. A model of IH activation suggests that under extreme conditions, the conductance of IH can reach 40% of its maximum value after less than ten action potentials.

Action Potentials↗
PubMed1991

Development of anomalous rectification (Ih) and of a tetrodotoxin-resistant sodium current in embryonic quail neurones.

1. The developmental expression of an inwardly rectifying current activated by membrane hyperpolarization (Ih) and of a tetrodotoxin (TTX)-resistant Na+ current (INa(TR)) was studied using freshly dissociated ganglionic quail neurones of various embryonic ages. This work was carried out on parasympathetic (ciliary) and sensory (trigeminal and dorsal root) ganglion neurones with the whole-cell configuration of the patch-clamp technique. 2. In sensory and parasympathetic neurones, Ih was activated at potentials more negative than -60 mV and displayed strong inward rectification. No sign of time- or voltage-dependent inactivation was apparent. Ih was carried by both Na+ and K+ ions and was selectively and reversibly blocked by extracellular Cs+. 3. During the development of sensory neurones, Ih was observed for the first time between embryonic day 10 (E10) and E11 and the percentage of neurones expressing the current increased subsequently, reaching a plateau level of about 80% at E14. In the parasympathetic neurones of the ciliary ganglion, Ih was already detected at E10 and the percentage of neurones possessing the current increased until E16, a stage at which all neurones were found to express Ih. 4. In the presence of TTX (1 microM), an inward Na+ current, INa(TR), was recorded in sensory neurones after E12. This current was activated at potentials more depolarized than -30 mV and its amplitude was maximal at +5 mV. INa(TR) showed time- and voltage-dependent inactivation. Half-maximal steady-state inactivation was observed at -40 mV. 5. INa(TR) was observed for the first time after E12 in sensory neurones and the percentage of neurones with INa(TR) increased until E14. Thereafter, 80% of the neurones had the current. In contrast, INa(TR) was never observed in the parasympathetic neurones of the ciliary ganglion during embryonic development. 6. Our results with parasympathetic and sensory neurones suggest that the expression of INa(TR) is linked to the phenotype and not to the embryonic origin of a neurone.

Animals↗
PubMed1990

Potassium current activated by intracellular sodium in quail trigeminal ganglion neurons.

Whole-cell voltage clamp and single-channel recordings were performed on cultured trigeminal ganglion neurons from quail embryos in order to study a sodium-activated potassium current (KNa). When KNa was activated by a step depolarization in voltage clamp, there was a proportionality between KNa and INa at all voltages between the threshold of INa and ENa. Single-channel recordings indicated that KNa could be activated already by 12 mM intracellular sodium and was almost fully activated at 50 mM sodium. 100 mM lithium, 100 mM choline, or 5 microM calcium did not activate KNa. The relationship between the probability for the channel to be open (Po) vs. the sodium concentration and the relationship of KNa open time-distributions vs. the sodium concentration suggest that two to three sodium ions bind cooperatively before KNa channels open. KNa channels were sensitive to depolarization; at 12 mM sodium, a 42-mV depolarization caused an e-fold increase in Po. Under physiological conditions, the conductance of the KNa channel was 50 pS. This conductance increased to 174 pS when the intra- and extracellular potassium concentrations were 75 and 150 mM, respectively.

Animals↗
PubMed1989

Transient expression of a Ca2+-activated Cl- current during development of quail sensory neurons.

The expression of a calcium-activated chloride current (ICl(Ca)) was studied during the development of the sensory neurons of quail trigeminal ganglia. This current is expressed in 20% of the neurons by the 5th day of embryonic development; it can be found in nearly all neurons by the 7th day and subsequently disappears in half of them. Similar results were obtained with dorsal root ganglion neurons. The disappearance of ICl(Ca) in part of the sensory neurons during development is not due to a selective death of the neurons possessing this current and our results suggest that it is mediated by an interaction of the sensory neurons with their target tissue.

Animals↗
PubMed1989

Single-channel current simulation and recording using a photodiode as current generator.

A device which can generate rectangular currents in the picoampere range is described. The current generator is a photodiode connected to the head stage of a single-channel recording amplifier. The photodiode is activated by a light-emitting diode controlled by a computer or any other current source. The device can transmit signals corresponding to simulated single-channel behaviour. Since the kinetic parameters of the simulation are known, the user can test the data acquisition and analysis system under conditions similar to those prevailing during recording from a biological membrane. This current generator can also be used for the tuning of patch-clamp amplifiers; rectangular currents generated by the photodiode allow the frequency response of the amplifier to be properly adjusted.

Electric Stimulation↗
PubMed1989

Expression of substance P and of a Ca2+-activated Cl- current in quail sensory trigeminal neurons.

A chloride current activated by an increase in intracellular calcium concentration is not present in all neurons of the trigeminal ganglion. It is not known whether the trigeminal neurons expressing calcium-activated chloride current belong to a defined class of neurons or whether they could belong to any class of sensory neurons. An answer to this question would be of importance because the physiological role of calcium-activated chloride current in neurons has not yet been completely established, nonetheless it is clear that this current, when activated, would act to modulate neuronal excitability. The goal of this study was to determine whether there was a difference in the expression of calcium-activated chloride current between neurons with and without substance P. The rationale was that the use of this morphological marker, which is present in a substantial fraction of embryonic trigeminal neurons, may give a first estimate of a possible inhomogeneity in the expression of calcium-activated chloride current among different classes of sensory neurons. The study was done on freshly dissociated neurons in order to minimize the influence of the culture conditions on the expression of the current or of substance P. By recording from large samples of neurons in cultures either enriched or depleted in substance P-containing neurons, we found that neurons with substance P expressed calcium-activated chloride current three times less frequently than neurons without substance P. This observation was confirmed by performing the immunocytochemical labelling for substance P immediately after the electrophysiological assessment of the presence or absence of calcium-activated chloride current. This result indicates that calcium-activated chloride current may not be randomly distributed in neurons of a sensory ganglion. It raises the possibility that neurons belonging to certain sensory modalities may need calcium-activated chloride current for their physiological functioning.

Animals↗
PubMed1989

Characterization of dissociated monolayer cultures of human spinal cord.

Embryonic human spinal cord cells have been grown in dissociated monolayer cultures for 1 to 7 weeks. Using cell type specific markers, it was possible to show that the cultures contain neurons, astrocytes and fibroblasts. Electrical membrane properties were studied with patch electrodes using the whole cell recording technique. Neurons had short duration action potentials that could be blocked by tetrodotoxin. The membrane currents in these neurons were studied in voltage clamp experiments. Three types of voltage-dependent currents were observed: a sodium current; a potassium current made up of two components, IA and IK; and a calcium current. Both cholinergic and GABAergic neurons are present in the cultures. There is more choline acetyltransferase activity in cultures prepared from the anterior as compared to the posterior part of the spinal cord, suggesting that the cultures contain motoneurons. This tissue culture preparation was developed for the study of amyotrophic lateral sclerosis; we have been unable to detect the presence of any toxic agent from the serum of these patients on the cultured cells. Experiments are in progress to purify the motoneurons using Percoll gradients.

Acetylcholine↗
PubMed1988

Electrophysiology of a chick neuronal nicotinic acetylcholine receptor expressed in Xenopus oocytes after cDNA injection.

Brain nicotinic acetylcholine receptors (nAChRs) are made up of protein subunits that differ from those constituting muscle nAChRs. To characterize the physiological properties of one class of avian brain nicotinic receptor, we injected the nuclei of Xenopus oocytes with full-length cDNAs for the ligand binding (alpha 4) and structural (n alpha) subunits. Injected oocytes had large ACh-induced currents in the microampere range that were insensitive to alpha-bungarotoxin, as expected for neuronal nAChRs. We found that these brain nAChRs incorporate at least two alpha 4 subunits and that their functional properties differ from muscle nAChRs in at least two respects: the elementary conductance is considerably smaller (20 pS), and channels in outside out patches stop functioning within a few minutes.

Animals↗
PubMed1988

Membrane currents of rat satellite cells attached to intact skeletal muscle fibers.

Muscle satellite cells play an important role in the postnatal growth of skeletal muscle and in the regeneration of damaged muscle during adult life. Little is known about the physiological properties of satellite cells in their dormant state as they lie adjacent to the intact muscle fibers, underneath the basement membrane. Our recent experiments, using patch clamp techniques, indicate that no tight electrical coupling is present between satellite cells and the muscle fiber dissociated from rat flexor digitorum brevis. Satellite cells possess sodium channels with low sensitivity to tetrodotoxin and at a much lower density than muscle. In addition, satellite cells are insensitive to acetylcholine (ACh) for at least 24 hr after having been removed from the animal, even when detached from their muscle fiber. However, we could measure ACh-evoked currents from satellite cells 48-72 hr in culture, indicating that ACh sensitivity develops with time.

Acetylcholine↗
PubMed1987

Calcium-activated chloride current in cultured sensory and parasympathetic quail neurones.

1. Sensory (trigeminal and dorsal root) and autonomic (ciliary) ganglia from embryonic quail were dissociated and the neurones were grown in tissue culture. 2. Intracellular recordings were made in voltage clamp using patch electrodes and the whole-cell recording technique. In order to investigate a calcium-activated chloride current, the sodium and potassium currents were blocked. 3. Depolarizing voltage steps from a holding potential of -100 mV to a test potential of +20 mV triggered an early inward and a delayed outward current. The latter persisted as a long-lasting inward tail current when the membrane was depolarized to -100 mV. 4. These currents were all blocked by extracellular cobalt suggesting that they were calcium dependent. During a test depolarization to +20 mV, in the presence of intracellular EGTA (20 mM), the inward current persisted but the outward current was suppressed. EGTA (20 mM) also suppressed the long-lasting inward tail current at -100 mV. This suggested the presence of a calcium-activated current. 5. The reversal potential of the calcium-activated current was near the equilibrium potential for chloride ions and was shifted as predicted by the Nernst equation when the extracellular chloride concentration was changed. 6. The calcium-activated current was partially blocked by adding 4-acetamido-4'-isothiocyanatostilbene-disulphonic acid (SITS) at a concentration of 1 mM to the external superfusion medium. This effect of a compound known to interfere with chloride channels together with the results of point (5) suggested the existence of a calcium-activated chloride current (ICl(Ca)). 7. ICl(Ca) could be activated by transient and sustained components of the calcium current present in the cultured neurones. 8. ICl(Ca) was present in 80% of the sensory neurones but only in 10% of the parasympathetic neurones.

Animals↗
PubMed1986

DATAC: a multipurpose biological data analysis program based on a mathematical interpreter.

The use of a mathematical command interpreter combined with the structural facility of the C-language allowed us to design a data treatment program having considerable flexibility and being able to handle any types of data (electrophysiological, biochemical and theoretical data). Ensembles of data are treated by the interpreter as if they were simple variables so that an elaborate computation can be performed on the spot by simply writing the appropriate equation on the terminal. These facilities combined with the ability of editing macrocommands at run time provide the user with data treatment possibilities that extend far beyond the possibilities actually implemented in the program. The originality of this program is that the user can easily implement the commands he most often needs, writing them in a language that most scientists will know, algebra.

Biometry↗
PubMed1985

Evoked mechanical responses of isolated cochlear outer hair cells.

Intracellular current administration evokes rapid, graded, and bidirectional mechanical responses of isolated outer hair cells from the mammalian inner ear. The cells become shorter in response to depolarizing and longer in response to hyperpolarizing currents in the synaptic end of the cell. The cells respond with either an increase or decrease in length to transcellular alternating current stimulation. The direction of the movement with transcellular stimuli appears to be frequency dependent. Iontophoretic application of acetylcholine to the synaptic end of the cell decreases its length. The microarchitecture of the organ of Corti permits length changes of outer hair cells in a manner that could significantly influence the mechanics of the cochlear partition and thereby contribute to the exquisite sensitivity of mammalian hearing.

Acetylcholine↗
PubMed1985

Voltage-dependent potassium currents in developing neurones from quail mesencephalic neural crest.

Neurones in explants cultured from quail mesencephalic neural crest were studied at different stages of their development using the voltage-clamp technique. A voltage-dependent outward current activated by membrane depolarization was identified as a potassium current by the sensitivity of its reversal potential to extracellular potassium. The voltage-dependent potassium current is made up of two components which differ in their sensitivity to 4-aminopyridine (4-AP) and tetraethylammonium (TEA). The component most sensitive to 4-AP has fast activation kinetics and inactivates quickly at sustained depolarized voltages. By analogy with a current described in other preparations, this current was called IA. The component most sensitive to TEA has slower activation kinetics and inactivates more slowly at sustained depolarized voltages. This current was called IK. IA and IK were already present in neurones cultured for 24 h. The ratio between the peak of IK and that of IA increased significantly between 24 h and 4 days in culture. This means that the two components of the voltage-dependent potassium current follow a different time course during development.

4-Aminopyridine↗
PubMed1985

Human spinal cord neurons in dissociated monolayer cultures: morphological, biochemical, and electrophysiological properties.

The preparation of dissociated monolayer cultures from embryonic human spinal cord is described. Optimal survival was achieved with embryonic tissue between the eighth and ninth week. The neurons survive for as long as 7 weeks in culture and they grow in a standard tissue culture medium which contains 13% decomplemented human serum. The neurons have been identified by indirect immunofluorescence techniques using antibodies to tetanus toxin and neurofilament protein. Our biochemical studies demonstrate the presence of cholinergic and GABAergic neurons. Cholinergic neurons develop in culture and are more numerous in the cultures prepared from the anterior part of the spinal cord as compared to those from the posterior part. Therefore, it is possible that a large part of the cholinergic neurons derive from the motoneuron pool. Electrical membrane properties were studied with patch electrodes using the whole cell recording technique. Neurons had short duration action potentials that could be blocked by tetrodotoxin (TTX). Voltage clamp experiments combined with the use of pharmacological blocking agents revealed the presence of several voltage- and time-dependent currents: a sodium current sensitive to TTX, a potassium current made up of two components, sensitive to tetraethylammonium and 4-aminopyridine, and a calcium current sensitive to cobalt. From a biochemical and electrophysiological point of view the properties of human spinal cord neurons in culture closely resemble the properties of spinal cord neurons from other species.

Acetylcholine↗
PubMed1985

Study of a neuronal potassium current in different culture conditions.

The potassium current of neurons in explants cultured from quail mesencephalic neural crest were studied in voltage clamp, using the whole cell recording technique. Two voltage-dependent potassium currents were identified; they differed in their sensitivity to blocking agents and to sustained depolarizing voltages. The potassium current component most sensitive to 4-aminopyridine had fast activation kinetics and inactivated quickly at sustained depolarized voltages. By analogy with a current described in other preparations, this current was called IA. The current component most sensitive to tetraethylammonium had slower activation kinetics and inactivated more slowly than IA at sustained depolarized voltages. This current was called IK. The properties of IA and IK were examined in neurons cultured in a defined medium and in neurons co-cultured with striated muscle. The rate of inactivation of IA appeared to be increased when neural crest neurons were cultured in the presence of striated muscle. The change in the properties of IA could be due to a direct effect of the co-culture with muscle on the membrane current; another possibility could be that co-culture favors the survival of a neuronal population that does not survive well when cultured in a defined medium.

4-Aminopyridine↗