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D K Berg

Publications and source records attributed to D K Berg.

At least 91 records · Page 5Linked to original sources
PubMed1987

Effects of preganglionic denervation and postganglionic axotomy on acetylcholine receptors in the chick ciliary ganglion.

The regulation of nicotinic acetylcholine receptors (AChRs) in chick ciliary ganglia was examined by using a radiolabeled anti-AChR mAb to quantitate the amount of receptor in ganglion detergent extracts after preganglionic denervation or postganglionic axotomy. Surgical transection of the preganglionic input to the ciliary ganglion in newly hatched chicks caused a threefold reduction in the total number of AChRs within 10 d compared with that present in unoperated contralateral control ganglia. Surgical transection of both the choroid and ciliary nerves emerging from the ciliary ganglion in newly hatched chicks to establish postganglionic axotomy led to a nearly 10-fold reduction in AChRs within 5 d compared with unoperated contralateral ganglia. The declines were specific since they could not be accounted for by changes in ganglionic protein or by decreases in neuronal survival or size. Light microscopy revealed no gross morphological differences between neurons in operated and control ganglia. A second membrane component of cholinergic relevance on chick ciliary ganglion neurons is the alpha-bungarotoxin (alpha-Bgt)-binding component. The alpha-Bgt-binding component also declined in number after either postganglionic axotomy or preganglionic denervation, but appeared to do so with a more rapid time course than did ganglionic AChRs. The results imply that cell-cell interactions in vivo specifically regulate both the number of AChRs and the number of alpha-Bgt-binding components in the ganglion. Regulation of these neuronal cholinergic membrane components clearly differs from that previously described for muscle AChRs.

Animals↗
PubMed1987

The properties and regulation of functional acetylcholine receptors on chick ciliary ganglion neurons.

The properties of acetylcholine receptor (AChR) channels on chick ciliary ganglion neurons in culture were examined using patch-clamp recording techniques. Acetylcholine (ACh) was applied by rapid microperfusion. Whole-cell current noise analysis revealed a single class of functional receptors on the neurons. Dose-response studies indicated a Kd of about 36 microM and a Hill coefficient of 1.5-1.7, predicting 2 ACh binding sites per receptor. Both fast and slow components of receptor desensitization were observed. Single-channel recordings from excised outside-out patches of soma membrane exposed to 2-5 microM ACh indicated a single-channel conductance of 40 pS, a reversal potential of -9 mV, a mean open duration of 1 msec, and an opening probability of 0.34. The kinetic behavior of the channels was provisionally described by a 3-closed, 1-open state model for receptor activation. In all of these properties, AChRs of ciliary ganglion neurons resemble those on skeletal muscle fibers. Growing the neurons in an elevated K+ concentration produced a 2-3-fold decrease in peak whole-cell currents induced by ACh under standard test conditions, without altering any of the single-channel properties described above. Neither changes in cholinesterase activity nor receptor distribution accounted for the decrease. Instead, calculations indicated that elevated K+ reduced the ACh response by decreasing the number of functional AChRs on the neurons. No K+-dependent decrease is observed, however, in the number of total receptors on the neurons detected either by a monoclonal antibody specific for the receptor or by an alpha-neurotoxin that binds to the receptor and blocks its function. Moreover, the number of receptors detected by the 2 probes is at least 10-fold greater than the calculated number of functional receptors. The findings suggest that only a small fraction of the AChRs on the neuronal surface is functional and that the cell can alter the ratio of functional and nonfunctional receptors in response to growth conditions.

Acetylcholine↗
PubMed1987

Immunological identification of a nicotinic acetylcholine receptor on bovine chromaffin cells.

Two probes previously shown to distinguish the nicotinic ACh receptor of chick ciliary ganglion neurons also recognize a component on the surface of bovine chromaffin cells in culture that displays the properties expected for the chromaffin nicotinic ACh receptor. The first probe is a monoclonal antibody, mAb 35, raised against ACh receptor from Electrophorus electric organ, and the second is an alpha-neurotoxin, Bgt 3.1, purified from B. multicinctus venom. mAb 35 binds specifically to a single class of high-affinity sites on the chromaffin cells in culture. Scatchard analysis indicates a KD of 2.1 +/- 0.2 nM for the binding and a Bmax of 1.6 +/- 0.1 X 10(4) mAb 35 sites per cell. The number of sites on the cells can be reduced through modulation by exposure of the cells to Bgt 3.1. The modulation can be blocked by the cholinergic ligands d-tubocurarine and carbamylcholine. Long-term exposure to the agonist carbamylcholine alone also reduces the number of mAb 35 binding sites. Bgt 3.1 inhibits nicotine-induced catecholamine release from the cells to the same extent and with the same concentration dependence that it modulates the number of mAb 35 sites on the cells. In addition, mAb 35 treatment of the cells causes a specific and almost complete blockade of nicotine-induced catecholamine release, apparently through a modulation of the receptor. These results indicate that the bovine chromaffin component recognized by mAb 35 and Bgt 3.1 is likely to be the nicotinic ACh receptor on the cells and that it has many similarities to ACh receptors on chick autonomic neurons.

Animals↗
PubMed1987

Neuronal acetylcholine receptors: fate of surface and internal pools in cell culture.

Chick ciliary ganglion neurons have nicotinic ACh receptors that mediate synaptic input to the cells. Ultrastructural studies with a monoclonal antibody that recognizes the neuronal ACh receptor have previously shown that, in addition to a predominantly synaptic location for the receptors on the neuron surface in vivo, substantial amounts of intracellular receptor are present as well. Here we report that intracellular receptor and smaller receptor-related components make up at least two-thirds of the total antibody binding sites associated with the ciliary ganglion neurons in cell culture. The intracellular sites for the most part represent integral membrane components that bind to concanavalin A when solubilized, indicating that the components are glycosylated. Sucrose gradient analysis shows that the intracellular material includes a 10 S component, likely to represent assembled receptor, along with species sedimenting in the 5-9 S range. Blocking the surface sites with unlabeled antibody and measuring the appearance of the new receptor on the cell surface with radiolabeled antibody indicates that the receptors are inserted into the plasma membrane at a rate equivalent to about 4% of the total surface receptor per hour. The transit time for newly synthesized receptor to reach the cell surface appears to be 2-3 hr. These observations suggest that about 5% of the intracellular receptors are transported to the cell surface in culture. The half-life of ACh receptors in the plasma membrane was estimated by 2 different approaches to be about 22 hr. Surface and internal sites respond in a qualitatively similar way to external agents that specifically modulate cholinergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Affinity labeling of neuronal acetylcholine receptor subunits with an alpha-neurotoxin that blocks receptor function.

An alpha-neurotoxin, Bgt 3.1, has previously been shown to recognize the functional acetylcholine receptor (AChR) on chick autonomic neurons, since it specifically blocks receptor function and it binds to a class of sites on the neurons with the pharmacology, kinetics, and affinity expected for the receptor. A monoclonal antibody, mAb 35, to the main immunogenic region of muscle and electric organ AChR alpha subunit cross-reacts with a component on chick autonomic neurons that, from several lines of evidence, also appears to be the functional AChR. The identity of the antibody-binding component has remained in doubt, however, because previous studies indicated that in at least one instance a substantial discrepancy existed between the number of functional AChRs estimated physiologically and the number of putative AChRs detected by mAb 35 on the neurons. The present findings demonstrate that Bgt 3.1 and mAb 35 recognize the same AChRs on the neurons, and provide information about the stoichiometry of binding and the identity of subunits associated with active sites on the receptor. Chick ciliary ganglion neurons examined under a variety of growth and regulatory conditions in culture displayed a constant ratio of about 2:1 for mAb 35 and Bgt 3.1 binding to cell surface sites. Treatment of the cells with mAb 35 induced a substantial decrease in the number of Bgt 3.1 sites and vice versa. AChRs that had been covalently labeled with a photoaffinity derivative of 125I-Bgt 3.1 in situ and then solubilized were specifically immune-precipitated by mAb 35, demonstrating unequivocally that the receptors possessed both Bgt 3.1 and mAb 35 binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Functional blockade of neuronal acetylcholine receptors by antisera to a putative receptor from brain.

Antisera to a putative acetylcholine receptor purified from chick brain specifically inhibit the acetylcholine response of chick ciliary ganglion neurons in cell culture. The putative brain receptor and a similar membrane component previously identified on ciliary ganglion neurons appear to be functional nicotinic acetylcholine receptors in the nervous system and are clearly distinct from membrane components in the tissues that bind alpha-bungarotoxin.

Acetylcholine↗
PubMed1986

Preganglionic neurons from the Edinger-Westphal nucleus: growth and histochemical characterization in cell culture.

The Edinger-Westphal (EW) nucleus, also known as the accessory oculomotor nucleus in the chick, provides the cholinergic preganglionic input to the parasympathetic ciliary ganglion. In addition to acetylcholine, many EW neurons have been shown to contain enkephalin-like and/or substance P-like immunoreactivity. Establishment of EW neurons in culture would make possible study of their interactions with ciliary ganglion neurons in vitro and in addition would provide a valuable system for studying cholinergic/peptidergic neurons of the vertebrate central nervous system. We describe here dissociated cell cultures established from midbrain tissue containing the EW nucleus. In these cultures, 86% of the cells with neuronal morphology were positive for intracellular acetylcholinesterase activity, 54% were positive for enkephalin-like immunoreactivity, and 4% were positive for substance P-like immunoreactivity. The proportions of neurons that scored as labeled were even higher if the number of positive cells was compared to the number of cells in sister cultures immunoreactive for the large neurofilament protein polypeptide. When the cultures were stained simultaneously for acetylcholinesterase activity and enkephalin-like immunoreactivity, 34% of the cells with neuronal morphology were positive for both. In cultures derived from adjacent tissue regions very few cells expressed both activities. These results suggest that the cells expressing both acetylcholinesterase activity and enkephalin-like immunoreactivity in culture are EW neurons. The putative EW neurons survive for weeks in vitro in the absence of their normal target, the ciliary ganglion.

Acetylcholinesterase↗
PubMed1986

Enkephalin and substance P modulate synaptic properties of chick ciliary ganglion neurons in cell culture.

Since enkephalin- and substance P-like immunoreactive materials have been identified in preganglionic terminals of the avian ciliary ganglion, we tested the effects of enkephalin and substance P directly on chick ciliary ganglion neurons in dissociated cell culture. Under these conditions the neurons form cholinergic synapses with each other that are spontaneously active. Both peptides modulate properties of membrane components associated with synaptic transmission between the neurons. Enkephalin causes a 60% reduction in the mean amplitude of the excitatory synaptic potentials, and the effect appears to be presynaptic in origin: enkephalin does not alter acetylcholine sensitivity on the neurons, but does inhibit Ca2+ influx as reflected by a 38% shortening of the Ca2+ component of the action potential. Both the reduction in synaptic potential amplitude and the shortening of the Ca2+ action potential produced by enkephalin are blocked by naloxone. Substance P, on the other hand, has no effect on Ca2+ action potentials but does reduce the time course of acetylcholine responses in the neurons by a mechanism consistent with enhanced receptor desensitization. Decay of the acetylcholine voltage response in the absence of substance P is described by a single exponential process with a time constant of 4-5 s. Coapplication of acetylcholine and substance P results in a second exponential decay process with a time constant of about 1 s that appears after a 200-400 ms lag period. Preincubation with substance P alone does not decrease the peak voltage response or shorten the lag, suggesting that either agonist or activated receptor is necessary for the substance P effect. These findings suggest modulatory roles for the peptides in ganglionic transmission.

Acetylcholine↗
PubMed1986

Surface and intracellular distribution of a putative neuronal nicotinic acetylcholine receptor.

Chick ciliary ganglion neurons have a membrane component that shares an antigenic determinant with the main immunogenic region (MIR) of nicotinic acetylcholine receptors from skeletal muscle and electric organ. Previous studies have shown that the component has many of the properties expected for a ganglionic nicotinic acetylcholine receptor, and that its distribution on the neuron surface in vivo is restricted predominantly to synaptic membrane. Here we report the presence of a large intracellular pool of the putative receptor in embryonic neurons and demonstrate that it is associated with organelles known to comprise the biosynthetic and regulatory pathways of integral plasma membrane proteins. Embryonic chick ciliary ganglia were lightly fixed, saponin-permeabilized, incubated with an anti-MIR monoclonal antibody (mAb) followed by horseradish peroxidase-conjugated secondary antibody, reacted for peroxidase activity, and examined by electron microscopy. Deposits of reaction product were associated with synaptic membrane, small portions of the pseudodendrite surface membrane, most of the rough endoplasmic reticulum, small portions of the nuclear envelope, some Golgi complexes, and a few coated pits, coated vesicles, multivesicular bodies, and smooth-membraned vacuoles. No other labeling was present in the neurons. The labeling was specific in that it was not present when the anti-MIR mAb was replaced with either nonimmune serum or mAbs of different specificity. Chick dorsal root ganglion neurons thought to lack nicotinic acetylcholine receptors were not labeled by the anti-MIR mAb. Substantial intracellular populations have also been reported for the muscle acetylcholine receptor and brain voltage-dependent sodium channel alpha-subunit. This may represent a general pattern for multisubunit membrane proteins during development.

Animals↗
PubMed1986

Cholinergic modulation of an acetylcholine receptor-like antigen on the surface of chick ciliary ganglion neurons in cell culture.

Chick ciliary ganglion neurons have a membrane component that shares an antigenic determinant with the "main immunogenic region" of the alpha subunits in nicotinic ACh receptors from skeletal muscle and electric organ. Ultrastructural studies on antibody binding in the ganglion have shown that the cross-reacting antigen on the neuron surface is located predominantly in synaptic membrane. Biochemical studies have shown that the cross-reacting component has a number of other properties expected for the ganglionic nicotonic ACh receptor and that it is distinct from the alpha-bungarotoxin binding component in the tissue. Here we show that ciliary ganglion neurons grown in dissociated cell culture express a similar component that cross-reacts with monoclonal antibodies to ACh receptors, and that the number of antibody-binding sites on the neurons can be modulated by exposure to cholinergic agonists and a protein neurotoxin that reversibly inhibits ACh receptors on the neurons. In most, though not all, cases, levels of ACh sensitivity associated with the neurons are specifically comodulated in parallel with the changes in number of antibody binding sites. The results suggest that at least a portion of the cross-reacting sites on the surface of ciliary ganglion neurons is likely to represent nicotinic ACh receptors. The fact that in some instances levels of ACh sensitivity can be altered without changing the number of cross-reacting sites, however, leaves open the possibility that not all of the sites are associated with receptors or that the neurons can alter the proportion of receptors that is functional.

Animals↗
PubMed1986

Identification of a nicotinic acetylcholine receptor on neurons using an alpha-neurotoxin that blocks receptor function.

An alpha-neurotoxin, Bgt 3.1, that reversibly blocks the ACh response of chick ciliary ganglion neurons has been used to identify 2 classes of high-affinity binding sites on the cells in culture. The first class appears to be the alpha-bungarotoxin binding site on the neurons. The second class of Bgt 3.1 sites is distinct from the alpha-bungarotoxin binding sites and has the properties expected for the functional nicotinic ACh receptor on the cells. Equilibrium binding and kinetic studies indicate a Kd value of 5-6 nM for Bgt 3.1 at the second class of sites. The kinetics and affinity of binding are consistent with those inferred from previous physiological studies for Bgt 3.1 inhibition of receptor function. Bgt 3.1 binding to the sites is completely inhibited by each of the cholinergic ligands ACh, carbachol, nicotine, d-tubocurarine, and trimethaphan, but not by alpha-bungarotoxin. Highest site densities are found in cultures of ciliary and sympathetic ganglion neurons, cell types known to have ganglionic nicotinic ACh receptors. Low levels of sites may be present in cultures of spinal cord and dorsal root ganglion neurons; no binding is found in cultures of skeletal myotubes or cardiac cells when alpha-bungarotoxin is used to block Bgt 3.1 binding to alpha-bungarotoxin sites. These results demonstrate that Bgt 3.1 can be used as a specific probe for the nicotinic ACh receptor on chick autonomic neurons.

Animals↗
PubMed1985

Characterization of a component in chick ciliary ganglia that cross-reacts with monoclonal antibodies to muscle and electric organ acetylcholine receptor.

Chick ciliary ganglion neurons have previously been shown to contain a component that shares an antigenic determinant with the "main immunogenic region" of the alpha-subunit in nicotinic acetylcholine receptor from skeletal muscle and electric organ. Ultrastructural studies of antibody binding in the ganglion have shown that the cross-reacting antigen exposed on the surface of the neurons is located predominantly in synaptic membrane. Here we show that the neuronal antigen can be identified in detergent extracts of ciliary and sympathetic ganglia, but not in extracts of heart, liver, spinal cord, retina, or dorsal root ganglia. In the ciliary ganglion the component is present as an integral membrane constituent, and, when detergent solubilized, it sediments as a 10 S species and binds to concanavalin A. The component is distinct from the alpha-bungarotoxin-binding site on the neurons since toxin-binding sites and antibody-binding sites can be precipitated separately in ganglion extracts. The component reaches peak levels per ganglionic protein between embryonic days 8 and 12. These are some of the properties expected for the nicotinic acetylcholine receptor on ciliary ganglion neurons.

Animals↗
PubMed1985

gamma-Aminobutyric acid receptors on chick ciliary ganglion neurons in vivo and in cell culture.

In the chick ciliary ganglion, preganglionic terminals maintain cholinergic synapses on the choroid neurons and both cholinergic and electrical synapses on the ciliary neurons. The preganglionic terminals also contain enkephalin- and substance P-like immunoreactivity, suggesting that transmission through the ganglion is more complicated than is indicated by the known synaptic connections. We report here that embryonic chick ciliary ganglion neurons also have gamma-aminobutyric acid (GABA) receptors and that GABA applied to the ganglion can block transmission elicited by preganglionic stimulation. Studies on the neurons in cell culture indicate that the GABA response is mediated by GABAA receptors: GABA activates a Cl- conductance, and the response can be mimicked by muscimol and blocked by bicuculline or picrotoxin. The GABA receptors are regulated independently from acetylcholine (ACh) receptors on the neurons since the levels of ACh and GABA sensitivity are influenced differently by culture age and by chronic exposure to GABA or elevated K+ concentrations. Application of GABA to intact ciliary ganglia increases the membrane conductance of ganglionic neurons (as in culture), reduces to subthreshold the amplitude of excitatory postsynaptic potentials in the neurons elicited by preganglionic stimulation and completely blocks transmission through the ganglion. A native source of ligand for the receptors in vivo has yet to be identified.

Acetylcholine↗
PubMed1984

Shared antigenic determinant between the Electrophorus acetylcholine receptor and a synaptic component on chicken ciliary ganglion neurons.

Monoclonal antibodies raised against purified acetylcholine receptor from muscle and electric organ were tested for cross-reaction with surface components on chicken ciliary ganglion neurons. Indirect immunofluorescence indicated that antibodies to a determinant in the "main immunogenic region" of the receptor bind to the neurons in culture. Ultrastructural studies on 16-day embryonic ganglia, using horseradish peroxidase-conjugated monoclonal antibody, revealed that most of the conjugate labeling was associated with synaptic membrane on the neurons. A lesser amount of labeling was associated with the short processes extending from the neuronal somata in the region of preganglionic innervation. The labeling was blocked by coincubation with unlabeled antibodies of appropriate specificity and not by nonimmune serum. The pattern of labeling was clearly different from that previously found for a horseradish peroxidase conjugate of alpha-bungarotoxin: the toxin conjugate bound extensively to the short processes but not to synaptic membrane on the neurons. The synaptic antigen identified here by the cross-reacting antibodies is a candidate for the synaptic acetylcholine receptor on chicken ciliary ganglion neurons.

Animals↗
PubMed1983

Differential regulation of acetylcholine sensitivity and alpha-bungarotoxin-binding sites on ciliary ganglion neurons in cell culture.

Levels of acetylcholine (ACh) sensitivity and numbers of alpha-bungarotoxin (alpha-Bgt)-binding sites have been measured for chick ciliary ganglion neurons grown in cell culture under various conditions. The two properties were found not to change in parallel. Neurons maintained in culture medium supplemented with embryonic eye extract developed high levels of ACh sensitivity and low numbers of alpha-Bgt-binding sites, whereas neurons grown in medium containing elevated K+ concentrations displayed the reverse. Neurons from media containing both eye extract and elevated K+ concentrations had both low levels of sensitivity and low numbers of toxin sites. The growth conditions do not alter the basic binding properties of the ACh receptors and alpha-Bgt-binding sites. Both the ACh receptor dose-response characteristics and the pharmacological properties of the toxin-binding sites were similar for neurons grown in media containing eye extract or elevated K+ concentrations. The inhibitory effects of eye extract on development of alpha-Bgt-binding sites appeared to be specific: eye extract had previously been shown to stimulate neuronal growth and cholinergic development, and in the present study eye extract enhanced development of ACh sensitivity and had no effect on mechanisms responsible for binding and accumulation of tetanus toxin. Eye extract did not block alpha-Bgt binding in competition binding experiments and did not cause redistribution of toxin sites away from the neuronal soma. These results demonstrate that ACh sensitivity and alpha-Bgt-binding sites can be independently regulated on the neurons and suggest that the two membrane properties are associated with separate membrane components.

Acetylcholine↗
PubMed1983

The ultrastructural localization of alpha-bungarotoxin binding sites in relation to synapses on chick ciliary ganglion neurons.

The distribution of alpha-bungarotoxin binding sites on chick ciliary ganglion neurons was examined at the ultrastructural level by incubating ganglia with horseradish peroxidase-conjugated toxin and examining the peroxidase-stained and thin-sectioned ganglia with the electron microscope. Both in embryonic and in adult ganglia heavy labeling was restricted to the surface membrane of short processes emerging from the ciliary and choroid cell somata in the region of preganglionic innervation. Less dense labeling occasionally was present on the smooth surface membrane of the soma in the same region. In contrast, the pre- and postsynaptic membranes of most synapses were clearly not labeled even in the immediate vicinity of heavily labeled processes. The labeling represented specific binding of the toxin conjugate since it could be prevented by d-tubocurarine and hexamethonium or by unconjugated toxin. The conjugated toxin was not excluded from the synaptic cleft on the basis of size because a substantially larger protein conjugate, a horseradish peroxidase-labeled monoclonal antibody, was able to enter the cleft and heavily label synaptic membranes as well as soma membranes. Even neurons in adult ganglia had very little synaptic labeling after exposure to the conjugated toxin. These results strongly suggest that the high affinity alpha-bungarotoxin binding sites on chick ciliary ganglion neurons are different from the synaptic ACh receptors which would be expected to be concentrated in the postsynaptic membrane. Clustering of the alpha-bungarotoxin binding sites in the vicinity of synapses, however, may reflect a related synaptic function.

Animals↗