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G D Fischbach

Publications and source records attributed to G D Fischbach.

At least 19 recordsLinked to original sources
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↗
PubMed1978

A transition temperature for acetylcholine channel conductance in chick myoballs.

1. The temperature dependence of ACh channel conductance (gamma) and channel open time (tau) was determined by analysing ACh induced membrane current fluctuations in voltage clamped chick myoballs. 2. gamma decreased from 25-30 pmho at 37 degrees C to less than 5 phmo at 10 degrees C. An Arrhenius plot of gamma vs. temperature exhibited a clear break or 'transition temperature' at 20 degrees C. 3. tau increased from 2 msec at 37 degrees C to 16 msec at 10 degrees C. The Arrhenius plot of tau vs. temperature was linear. No transition temperature was detected. 4. Submicellar concentrations of the non-ionic detergent, Triton X-100 reversibly blocked ACh respnses. The effect was all-or-none at the molecular level. 5. These results are consistent with the possibility that the fluidity of membrane lipids in the ACh receptor micro-environment may influence the degree to which the channel can open.

Acetylcholine↗
PubMed1978

Accumulation of acetylcholine receptors and acetylcholinesterase at newly formed nerve-muscle synapses.

Nerve-muscle synapses form and the postsynaptic membrane begins to differentiate rapidly in vitro. New clusters of AChRs have been located at transmitting synapses within 3 hours after a competent neurite contacted a receptive myotube. Receptors within new subneural clusters are not identical with receptors at adult end-plates: they are metabolized rapidly and their mean channel open time is prolonged. In this sense synapse formation must be considered a drawn out affair. AChE also accumulates at synapses soon after functional contacts are established. The cleft enzyme is apparently regulated differently than receptors in the postsynaptic membrane. Muscle activity is necessary for the early accumulation of AChE, but not for the clustering of receptors. Further studies in vitro should be useful for analysis of the role of muscle activity, of the local neural influence on the postsynaptic membrane, and of the mechanism of the muscle response.

Acetylcholine↗
PubMed1977

The action potential of chick dorsal root ganglion neurones maintained in cell culture.

1. The directly evoked action potential of dissociated, embryonic, chick, dorsal root ganglion (DRG) neurones maintained in cell culture is prolonged compared to spinal cord cell spikes and the re-polarization phase is marked by a plateau. 2. Evidence was obtained that both Ca2+ and Na+ carry inward current across the active soma membrane. Ca2+ because: overshooting spikes persist in tetrodotoxin (TTX) or Na+-free media; in the presence of TTX (or absence of Na+) spike size varies directly with extracellular Ca2+ and spikes are eliminated by Co2+. Na+ because: spikes persist in the presence of Co2+ or Ca2+-free media; in the presence of Co2+ (or absence of Ca2+) spike varies directly with extracellular Na+ and spikes are blocked by TTX. 3. On the other hand, Ca2+ plays less if any role in action potentials conducted along sensory nerve cell processes. Conducted spikes could not be evoked in TTX containing or Na+-free media. 4. A long-lasting depolarization follows the action potential in some neurones. This depolarization is associated with an increase in membrane conductance and appears to drive the membrane potential to ca. -30mV. It persists when conducted impulses are blocked so it is probably not a recurrent synaptic potential. 5. It is suggested that combined Ca2+-Na+ spikes observed in isolated sensory neurones in vitro reflect the action potential of adult sensory cells but the possibility that they represent an early stage in development is also discussed.

Action Potentials↗