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D K Berg

Publications and source records attributed to D K Berg.

108 records · Page 6Linked to original sources
PubMed1981

Two components from eye tissue that differentially stimulate the growth and development of ciliary ganglion neurons in cell culture.

Survival and development of chick ciliary ganglion neurons in vivo appear to depend on information from the embryonic eye structure that contains the postsynaptic targets of the neurons. We have tested embryonic eye extracts on ciliary ganglion neurons in dissociated cell culture for stimulation of growth and development. Control conditions were chosen that permitted the long term maintenance of the neurons in the absence of tissue extracts of conditioned medium. The conditions included coating the culture substratum with fibroblast material and increasing the K+ concentration in the culture medium to 25 mM. Neurons survived for at least 3 weeks in control conditions. Two major components were resolved in eye extracts that stimulated growth and development of the neurons above the basal levels obtained with control conditions. One component, with an apparent molecular weight of about 2 X 10(4) by gel filtration analysis, stimulated neuronal growth without increasing the levels of choline acetyltransferase activity per neuron. The second component, with an apparent molecular weight of about 5 X 10(4), increased development of choline acetyltransferase levels per neuron but had no effect on neuronal growth. Both components were effective in normal K+ as well as 25 mM K+. These components may represent mechanisms by which the postsynaptic target tissue acts in vivo to direct the growth and development of ciliary ganglion neurons.

Animals↗
PubMed1981

Internalization of alpha-bungarotoxin on neurons induced by a neurotoxin that blocks neuronal acetylcholine sensitivity.

A protein neurotoxin (Bgt 3.1) present as a minor component in the venom of Bungarus multicinctus has been shown previously to block acetylcholine (ACh) sensitivity on chick ciliary ganglion (CG) neurons in cell culture. Alpha-bungarotoxin (Bgt. 2.2) binds to the neurons but does not block ACh sensitivity; the function of the Bgt. 2.2 binding site is unknown. The present studies demonstrate that Bgt 3.1 can induce the rapid internalization of Bgt 2.2 bound on the surface of CG and sympathetic neurons. The rapid internalization of bound Bgt 2.2 caused by Bgt. 3.1 can be seen with fluoresence microscopy using rhodamine-labeled Bgt 2.2 as the probe and by immunological techniques using anti-Bgt 2.2 antiserum to locate the bound 125I-Bgt 2.2. The rapid internalization is blocked by low temperature or by high concentrations of Bgt 2.2 and is not induced by Bgt 2.2 itself or by small cholinergic ligands. Bound 125I-Bgt 2.2 is released into the medium as degraded material after internalization is induced. Bgt 3.1 does not induce internalization of Bgt 2.2 bound to skeletal myotubes in culture nor does it induce the internalizaton of rhodamine-labeled nerve growth factor bound to sympathetic neurons, suggesting that its effect on neuronally bound Bgt 2.2 might be a specific one. Competition binding studies suggest that Bgt 3.1 may trigger the internalization of bound Bgt 2.2 by direct interaction with a Bgt 2.2 binding site. The effect of Bgt 3.1 on neuronal ACh sensitivity, however, does not depend on internalization of Bgt 2.2 binding sites since full inhibition of ACh sensitivity is still achieved by Bgt 3.1 under conditions where internalization is blocked. Neurons may have more than one class of Bgt 2.2 on the neurons. The internalization of Bgt 2.2 binding sites induced by Bgt 3.1 provides an unusual opportunity to study cellular mechanisms by which neurons can regulate the number and distribution of their surface components.

Acetylcholine↗
PubMed1979

Inhibition of neuronal acetylcholine sensitivity by alpha-toxins from Bungarus multicinctus venom.

Bungarus multicinctus venom contains several alpha-toxins in addition to the widely used alpha-bungarotoxin (Bgt 2.2). We have found that two of the alpha-toxins (Bgt 3.1 and 3.3) inhibit neuronal acetylcholine (AcCho) sensitivity when tested on ciliary ganglion neurons in cell culture. Over 90% of the AcCho sensitivity recorded in response to iontophoretic application of AcCho was blocked when the neurons were incubated with either of the toxins at 10(-7) M for 1 hr at 37 degrees C. The blockade could be partially reversed by incubating the neurons for 1-2 hr in medium lacking the toxins. The neurons also had a high-affinity binding site for Bgt 2.2, as indicated by binding studies with rhodamine-labeled Bgt 2.2. Concentrations of Bgt 2.2(10(-7) M) that should be nearly adequate to saturate the high-affinity site, however, had no detectable effect on AcCho sensitivity of the neurons. Higher concentrations of Bgt 2.2(10(-5) M) produced a partial inhibition of AcCho sensitivity, suggesting either that the neurons had two classes of binding sites for Bgt 2.2 (with the low-affinity site affecting AcCho sensitivity) or that the preparation of Bgt 2.2 contained minor components (e.g., Bgt 3.1 or 3.3) that were responsible for the blockade. The mechanisms by which Bgt 3.1 and 3.3 inhibit neuronal AcCho sensitivity remain unknown. If they bind specifically to the AcCho receptor, they will be useful agents for studying the distribution and regulation of this membrane component.

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↗
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↗
PubMed1977

Dissociated ciliary ganglion neurons in vitro: survival and synapse formation.

Normally, about half of the ciliary ganglion neurons in 8-day-old chick embryos die before day 14 in ovo. However, when dissociated ciliary ganglion neurons were prepared from either 8- or 14-day-old embryos and grown in cell culture with skeletal myotubes, essentially all of the neurons survived for at least 3 weeks. Many of the neurons formed functional synapses on myotubes under these conditions; some neuromuscular synapses could be detected as early as 20 hr after addition of the ganglion cells to muscle cultures. In contrast, most neurons from 8-day embryos survived for only a few days when grown alone on either polyornithine- or collagen-coated dishes. These results suggest that neurons destined to die in ovo can be rescued when grown in cell culture with myotubes and that under these conditions the neurons develop and express differentiated properties.

Animals↗
PubMed1975

Increased extrajunctional acetylcholine sensitivity produced by chronic acetylcholine sensitivity produced by chronic post-synaptic neuromuscular blockade.

1. Anaesthetized rats were paralysed for periods of up to 3 days by chronic administration of D-tubocurarine (DTC), succinylcholine or alpha-bungarotoxin. 2. After 3 days of treatment with DTC, the phrenic nerve remained active. Neuromuscular transmission and spontaneous miniature end-plate potentials (m.e.p.p.s) were restored after removal of the DTC. Resting potentials and input resistances of muscle fibres that had been paralysed for 3 days were similar to those in denervated fibers. 3. Chronic neuromuscular blockade increased the binding of [125-I]-alpha-bungarotoxin by extrajunctional regions of muscle. The time course of the increase was similar to that seen after denervation. Binding to muscles from animals that were anaesthetized and respirated, but not paralysed, was not increased. 4. Three days of paralysis increased the sensitivity of the extrajunctional muscle membrane to acetylcholine (ACh) applied by iontophoresis. 5. Approximately the same proportion of muscle fibres from muscles paralysed for 3 days gave overshooting action potentials in the presence of tetrodotoxin 10-minus 6 g/ml. as did fibres form muscles denervated for 3 days. 6. Chronic paralysis did not change the accumulation of acetylcholinesterase above a ligation in the sciatic nerve. 7. These results are consistent with the idea that extrajunctional ACh sensitivity is normally controlled by muscle activity.

Acetylcholine↗
PubMed1975

Loss of alpha-bungarotoxin from junctional and extrajunctional acetylcholine receptors in rat diaphragm muscle in vivo and in organ culture.

Acetylcholine (ACh) receptors in rat diaphragm muscle were blocked by intrathoracic injection of alpha-bungarotoxin (alpha-BuTx) or [125I]alpha-bungarotoxin ([125I]alpha-BuTx). The stability in vivo of the toxin-receptor complex formed by receptors in normal muscles and receptors in extrajunctional regions of denervated muscles was compared. Toxin was lost from junctional regions of normal muscles with a half-time of approximately 6 days. The loss of toxin was accompanied by a corresponding increase in the number of free toxin-binding sites. In contrast, 65% of the toxin bound to extrajunctional regions of denervated muscle was lost in 24 hr. 2. In a second series of experiments, animals were injected with [125I]alpha-BuTx and the muscle subsequently cultured for 24 hr. Loss of toxin again occurred more rapidly from extrajunctional receptors than from junctional receptors. The loss from extrajunctional receptors was described by a single first-order rate constant whose corresponding half-time was 8-11 hr. Loss was almost completely blocked by sodium cyanide and dinitrophenol and was inhibited by puromycin and cycloheximide. The radioactivity recovered in the medium was largely monoiodotyrosine. These results are consistent with the hypothesis that toxin loss reflects intracellular degradation of toxin-receptor complex. 3. Neonatal rats were injected with [125I]alpha-BuTx and the diaphragms cultured. Radioactive toxin was lost rapidly from extrajunctional regions of muscle and more slowly from regions containing end-plates. 4. These results could be explained by a difference in turnover rates for junctional and extrajunctional receptors.

Animals↗
PubMed1974

Fate of alpha-bungarotoxin bound to acetylcholine receptors of normal and denervated muscle.

In organ culture, alpha-[(125)I]bungarotoxin bound to extrajunctional receptors of denervated muscle is lost from the tissue at a more rapid rate than the toxin bound to the junctional receptors of normal muscle. The rapid loss of toxin from denervated muscle can be blocked by inhibitors of energy production and protein synthesis, and may reflect turnover of the toxin-receptor complex in the membrane.

Animals↗
PubMed1972

Binding of -bungarotoxin to acetylcholine receptors in mammalian muscle (snake venom-denervated muscle-neonatal muscle-rat diaphragm-SDS-polyacrylamide gel electrophoresis).

Experiments were performed to determine the specificity of [(125)I]alpha-bungarotoxin binding to skeletal muscle. In adult rat diaphragm, [(125)I]alpha-bungarotoxin was found to bind almost exclusively to those regions of the muscle that contain endplates and are known to be sensitive to acetylcholine. In contrast, chronically denervated adult muscle and muscle from neonatal rats, both of which are sensitive along their entire lengths, bound substantial amounts of toxin in all regions. Toxin binding to all muscles was inhibited by d-tubocurarine and by carbamylcholine, but not by atropine. The bound [(125)I]toxin was solubilized by homogenization of the tissue in 1% Triton X-100 and was recovered as a single band, distinct from free toxin, after zone sedimentation. Treatment of the solubilized, toxin-bound complex with 2-mercaptoethanol and sodium dodecyl sulfate resulted in the recovery of free toxin. A toxin-bound complex was also obtained when toxin was incubated directly with extracts of muscle endplate regions prepared by homogenization in Triton X-100. No such complex was observed with extracts prepared from muscle lacking endplates. These results are consistent with the interpretation that alpha-bungarotoxin binds specifically to the acetylcholine receptor of mammalian skeletal muscle.

Acrylamides↗