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G D Fischbach

Publications and source records attributed to G D Fischbach.

At least 73 records · Page 4Linked to original sources
PubMed1981

Neurotransmitters decrease the calcium conductance activated by depolarization of embryonic chick sensory neurones.

Several neurotransmitters including noradrenaline (NA), gamma-aminobutyric acid (GABA) and serotonin (5-HT), and also certain peptides, decrease the duration of the Na+-Ca2+ action potential recorded in cell bodies of embryonic chick dorsal root ganglion neurones maintained in cell culture. To determine if these agents decreased action potential duration by affecting Ca2+ channels (inward current) or K+ channels (outward current) membrane currents were recorded in voltage-clamped sensory neurone somata. 1. Depolarization produced a prominent inward Na+ current and a smaller and slower inward Ca2+ current (ICa). The inactivation of ICa was not simply dependent on membrane potential but apparently required prior entry of Ca2+. Two components of outward current, voltage-activated and Ca2+-activated, were evident in most cells. 2. The effect of NA, and also of GABA and 5-HT, was shown to result from a direct effect on ICa because: NA decreased the TTX-resistant tail current recorded at EK and also the inward current recorded in the presence of 125 mM-TEA and TTX (in which Na+ and K+ currents were blocked). 3. The decrease in ICa is most likely due to an effect on the number of available Ca2+ channels and/or the single Ca2+ channel conductance rather than to a shift in either the kinetics of channel activation or the Ca2+ equilibrium potential. 4. No effect of the several transmitters on the voltage-dependent Na+ and K+ currents was observed. 5. Implications of ICa modulation for the phenomenon of presynaptic inhibition are discussed.

Action Potentials↗
PubMed1981

Peptide and amine transmitter effect on embryonic chick sensory neurons in vitro.

It has been known for some time that NE and ACh can affect voltage-sensitive channels in the heart but it has only recently been appreciated that neurotransmitters (and certain peptides) can modulate voltage-sensitive channels in neurons. In addition to the effect on the action potential of embryonic chick sensory neurons described here, NE decreases the duration of spikes in rat superior cervical ganglion neurons (7). Serotonin prolongs action potentials recorded in Aplysia sensory neurons (9) and an as yet unidentified transmitter decreases an inward Ca++ current in the same cells (20). In the heart, one important consequence of the effect of NE and ACh on the action potential is a change in the strength and/or duration of contraction. In neurons, attention has been focused on the possibility that modulation of voltage-sensitive channels might result in a change in transmitter release. In Aplysia, the 5-HT induced prolongation of sensory neuron some spikes is associated with a dramatic augmentation of transmitter release at sensory nerve-motorneurone synapses (9) and the decrease in some inward Ca++ current is associated with presynaptic inhibition (20). Enkephalin, NE, GABA, and 5-HT can inhibit the evoked release of Substance P from cultured embryonic chick sensory neurons. These same drugs decrease ICa, apparently by decreasing the number of the conductance of voltage-sensitive Ca++ channels. The two phenomena may be related. It is significant in this regard that the same variability (between platings) in the ability of enkephalin to reduce Substance P release was also observed in the effect of enkephalin on action potential duration. Cells that released normal amounts of Substance P in the presence of enkephalin also exhibit spikes of normal duration in the presence of the peptide.

Acetylcholine↗
PubMed1980

Regulation of acetylcholinesterase appearance at neuromuscular junctions in vitro.

The appearance of acetylcholinesterase (AChE) at newly formed nerve-muscle synapses depends on synaptic transmission. Synapses form when cultures are grown in the presence of acetylcholine receptor antagonists, but AChE does not accumulate at these synapses. The important component of transmission seems to be muscle activity. Treatment with dibutyryl cyclic GMP mimics muscle activity, directly inducing synaptic AChE appearance.

Acetylcholinesterase↗
PubMed1980

A post-natal decrease in acetylcholine channel open time at rat end-plates.

1. The mean channel open time (tau) of ACh receptors in adult developing rat skeletal muscle was estimated by analysis of ACh current fluctuations. 2. At adult end-plates, tau was 1.4 msec at 21 degrees C. Time constants of miniature end-plate current decays (taus) were equally brief. 3. On the other hand, tau and taus were long (4.5 msec, 21 degrees C) at neonatal rat end-plates. These estimates are comparable to values obtained at extrajunctional sites on embryonic rat myotubes grown in culture and on adult denervated muscle fibres. 4. Both tau and taus decreased to values characteristic of adult end-plates during the first 2 weeks after birth. 5. Complex ACh power spectra were obtained at most end-plates during the transition period and at some, miniature synaptic current decays were doubly exponential. End-plates in transition might contain a mixture of slow, neonatal channels and fast, adult channels.

Acetylcholine↗
PubMed1979

Enkephalin inhibits release of substance P from sensory neurons in culture and decreases action potential duration.

Sensory neurons grown in dispersed cell culture in the absence of non-neuronal cell types contain immunoreactive substance P that is chemically similar to synthetic substance P. When depolarized in high-K+ media (30-120 mM), the neurons release this peptide by a Ca2+-dependent mechanism. An enkephalin analogue, [D-Ala2]enkephalin amide, at 10 micron inhibits the K+-evoked release of substance P. At the same or lower concentrations, [D-Ala2]enkephalin amide and enkephalin decrease the duration of the Ca2+ action potential evoked and recorded in dorsal root ganglion cell bodies without affecting the resting membrane potential or resting membrane conductance. This modulation of voltage-sensitive channels may account for the inhibition of substance P release.

Action Potentials↗
PubMed1979

Induction of acetylcholine receptors on cultured skeletal muscle by a factor extracted from brain and spinal cord.

Extracts of chicken brain and spinal cord increase the total number of acetylcholine receptors and the number of acetylcholine receptors and the number of receptor clusters on uninnervated skeletal myotubes in culture. The active component in these extracts may be unique to neural tissue. Spinal cord cells grown in culture contain the active factor and they secrete it into the medium. Most of the activity is associated with a small molecule possibly a peptide. Such a factor may be responsible for the clustering of receptors at newly formed nerve-muscle synapses.

Acetylcholine↗
PubMed1979

Uptake and release of [3H]gamma-aminobutyric acid by embryonic spinal cord neurons in dissociated cell culture.

We have investigated the uptake and release of [3H]gamma-aminobutyric acid (GABA) by embryonic chick spinal cord cells maintained in culture. Cells dissociated from 4- or 7-d-old embryos were studied between 1 and 3 wk after plating. At 3 degrees C, [3H]GABA was accumulated by a high affinity (Km approximately equal to 4 microM) and a low affinity (Km approximately equal to 100 microM) mechanism. The high affinity transport was markedly inhibited in low Na+ media, by ouabain, at 0 degrees C, and by 2,4-diaminobutyric acid. Autoradiography, after incubation in 0.1 microM [3H]GABA, showed that approximately 50% (range = 30-70%) of the multipolar cells were labeled. These cells were neurons rather than glia; action potentials and/or synaptic potentials were recorded in cells subsequently found to be labeled. Non-neuronal, fibroblast-like cells and co-cultured myotubes were not labeled under the same conditions. The fact that not all of the neurons were labeled is consistent with the suggestion, based on studies of intact adult tissue, that high affinity transport of [3H]GABA may be unique to neurons that use GABA as a neurotransmitter. Our finding that none of fifteen physiologically identified cholinergic neurons, i.e., cells that innervated nearby myotubes, were heavily labeled after incubation in 0.1 microM [3H]GABA is significant in this regard. The newly taken up [3H]GABA was not metabolized in the short run. It was stored in a form that could be released when the neurons were depolarized in a high K+ (100 mM) medium. As expected for a neurotransmitter, the K+-evoked release was reversibly inhibited by reducing the extracellular Ca++/Mg++ ratio.

Aminobutyrates↗
PubMed1979

Early events in neuromuscular junction formation in vitro: induction of acetylcholine receptor clusters in the postsynaptic membrane and morphology of newly formed synapses.

The development of clusters of acetylcholine (ACh) receptors at newly formed synapses between embryonic chick spinal cord and muscle cells grown in vitro has been studied by iontophoretic mapping with ACh. A semi-automated technique using on-line computer analysis of ACh responses and a photographic system to record the position of each ACh application permit the rapid construction of extensive and detailed maps of ACh sensitivity. Clusters of receptors, evident as peaks of ACh sensitivity, are present on many uninnervated myotubes. The distribution of ACh sensitivity closely parallels the distribution of 125I-alpha-bungarotoxin binding sites on the same muscle cell. In all cases where individual myotubes were adequately mapped before and after synapse formation, ingrowing axons induced new clusters of receptors rather than seeking out preexisting clusters. Synapses can form at active growth cones within 3 h of nerve-muscle contact. New receptor clusters can appear beneath neurites within a few hours. Many of the uninnervated clusters on innervated myotubes disappear with time. In contrast, receptor clusters on uninnervated myotubes remain in the same location for many hours. Synaptic clusters and clusters on uninervated myotubes are stable even though individual receptors are metabolized rapidly. The morphology of several identified sites of transmitter release was examined. At the scanning EM level, synapses appeared as small, rough-surfaced varicosities with filopodia that radiated outwards over the muscle surface. One synapse was studied by transmission EM. Acetylcholinesterase and a basement lamina were present within the synaptic cleft.

Animals↗
PubMed1978

Channel open time and metabolic stability of synaptic and extrasynaptic acetylcholine receptors on cultured chick myotubes.

The mean channel open time and metabolic stability of acetylcholine receptors were studied in developing chick muscle fibers in vitro. Analysis of acetylcholine noise recorded from small patches of surface membrane on uninnervated myotubes indicates that the mean ionic channel open time is independent of receptor density. On myotubes innervated in vitro by spinal cord neurons, the mean open time of synaptic receptors was identical to that of extrasynaptic receptors on the same fibers. Receptor stability was estimated by autoradiography of cultures labeled with 125I-labeled alpha-bungarotoxin. Synaptic and extrasynaptic toxin--receptor complexes disappear at the same, relatively rapid rate. Both the mean channel open time and the apparent rate of receptor degradation are comparable to values obtained at extrasynaptic sites on denervated adult muscles in other species.

Acetylcholine↗
PubMed1978

Clusters of intramembranous particles on cultured myotubes at sites that are highly sensitive to acetylcholine.

Electrophysiological and autoradiographic studies have shown that the distribution of acetylcholine (AcCho) receptors on uninnervated cultured chicken muscle cells is not uniform. Regions of high receptor density (hot spots 10--40 times more sensitive than surrounding areas are localized as discrete patches or clusters about 10 micrometer in diameter o myotube muscle membranes. Hot spots were also found on fusion-arrested mononucleated myoblasts. We have developed a method for freeze-fracturing monolayer cultures that allows the unambiguous reidentification of membrane patches previously assayed for ACCho sensitivity. The freeze-fractured membranes at physiologically defined hot spots contain aggregates of many (10--20) small clusters of large (10--19 nm in diameter) intramembranous particles. Clusters are found on both fracture faces, but the particle density is much greater on the protoplasmic (P) face than on the extracellular (E) face (about 2000/micrometer2 vs. 700/micrometer2). Some of the particles appear to be composed of five or six "subunits" arranged cylindrically around a central dark dot. Because the aggregates are present at sites of high AcCho sensitivity, it is likely that the intramembranous particles are in some way related to the AcCho receptor molecule.

Acetylcholine↗
PubMed1978

Enrichment of spinal cord cell cultures with motoneurons.

Spinal cord cell cultures contain several types of neurons. Two methods are described for enriching such cultures with motoneurons (defined here simply as cholinergic cells that are capable of innervating muscle). In the first method, 7-day embryonic chick spinal cord neurons were separated according to size by 1 g velocity sedimentation. It is assumed that cholinergic motoneurons are among the largest cells present at this stage. The spinal cords were dissociated vigorously so that 95-98% of the cells in the initial suspension were isolated from one another. Cells in leading fractions (large cell fractions: LCFs) contain about seven times as much choline acetyltransferase (CAT) activity per unit cytoplasm as do cells in trailing fractions (small cell fractions: SCFs). Muscle cultures seeded with LCFs develop 10-70 times as much CAT as cultures seeded with SCFs and six times as much CAT as cultures seeded with control (unfractionated) spinal cord cells. More than 20% of the large neurons in LCF-muscle cultures innervate nearby myotubes. In the second method, neurons were gently dissociated from 4-day embryonic spinal cords and maintained in vitro. This approach is based on earlier observations that cholinergic neurons are among the first cells to withdraw form the mitotic cycle in the developing chick embryo (Hamburger, V. 1948. J. Comp. Neurol. 88:221-283; and Levi-Montalcini, R. 1950. J. Morphol. 86:253-283). 4-Day spinal cord-muscle cultures develop three times as much CAT as do 7-day spinal cord-muscle plates, prepared in the same (gentle) manner. More than 50% of the relatively large 4-day neurons innervate nearby myotubes. Thus, both methods are useful first steps toward the complete isolation of motoneurons. Both methods should facilitate study of the development of cholinergic neurons and of nerve-muscle synapse formation.

Animals↗
PubMed1978

Acetylcholine noise in cultured chick myoballs: a voltage clamp analysis.

1. Large spherical muscle cells were grown in vitro by allowing dissociated chick myoblasts to fuse in suspension culture. 2. Myoball membranes could be adequately voltage clamped even in the face of large inward currents (ca. 100 nA) induced by ACh. 3. The single channel conductance, gamma, estimated from the ratio of the mean to the variance of ACh currents was 25-40 pmho at temperatures between 25 degrees and 37 degrees C. 4. Spectra of ACh current fluctuations declined with 1/f2. The mean channel open time (tau) estimated from the half-power frequency was 2 msec at 37 degrees C and -90 mV holding potential. At lower membrane potentials tau was decreased and on hyperpolarization tau was prolonged.

Acetylcholine↗