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D K Berg

Publications and source records attributed to D K Berg.

At least 73 records · Page 4Linked to original sources
PubMed1992

Nicotinic receptors that bind alpha-bungarotoxin on neurons raise intracellular free Ca2+.

Many populations of vertebrate neurons have a membrane component that binds alpha-bungarotoxin and cholinergic ligands. Despite the abundance of this component and its similarities to nicotinic receptors, its function has remained controversial. Using a fluorescence assay, we show here that activation of the component elevates the intracellular concentration of free Ca2+, demonstrating a receptor function for the toxin-binding component. Whole-cell voltage-clamp and intracellular recordings did not detect a significant current resulting from receptor activation, possibly because the currents were small or the receptors rapidly desensitized. The rise in intracellular free Ca2+ caused by the receptor was prevented by Ca2+ channel blockers. This suggests a signaling cascade likely to have important regulatory consequences for the neuron.

Animals↗
PubMed1991

Nicotinic acetylcholine receptor mRNA in dorsal root ganglion neurons.

Nicotinic acetylcholine receptor (AChR) genes are expressed in subpopulations of chick dorsal root ganglion (DRG) neurons. In 18-day embryonic ganglia, 19% of the neurons have material homologous to neuronal AChR alpha 3 gene mRNA, and 8% have material homologous to alpha 4 mRNA as seen with in situ hybridization. RNAase protection experiments confirm that DRG RNA contains alpha 3 and alpha 4 transcripts, and Northern blot analysis establishes the size of the transcripts as being 3.5 and 3.3 kb, respectively. The proportion of DRG neurons containing alpha 3 mRNA does not decline up through 1 year post-hatch, indicating that alpha 3 gene expression is not a developmentally transient event in the ganglion. An antiAChR monoclonal antibody detects cross-reacting material in 16% of the DRG neurons from 18-day embryos, indicating that AChR mRNA is translated into protein. Electrophysiological measurements confirm the presence of functional AChRs on DRG neurons freshly isolated from 18-day embryos: 24% of the neurons have substantial ACh sensitivities, whereas another 23% have small but detectable responses. Staining dorsal root ganglion sections with an anticholine acetyltransferase antiserum reveals cross-reactive material localized in axons in the ganglion; no evidence suggests the presence of cholinergic synaptic structures or AChR clusters on neuronal somata in the ganglion. It is possible that AChRs on DRG neurons participate in a diffuse form of transmission between the cholinergic fibers and a subpopulation of neuronal somata in the ganglion. Alternatively, AChRs on the somata may represent an ectopic distribution of receptors whose primary function is at the terminals of central or peripheral DRG processes.

Acetylcholine↗
PubMed1991

Regulation of acetylcholine receptors on chick ciliary ganglion neurons by components from the synaptic target tissue.

Chick ciliary ganglion neurons have nicotinic acetylcholine receptors (AChRs) that mediate synaptic transmission through the ganglion. A soluble component of about 50 kDa from embryonic eye tissue, the synaptic target of the ganglion, increases the development of ACh sensitivity by the neurons 10-fold over a 1-week period in culture. The increased sensitivity does not arise from a change in agonist affinity or esterase activity. Both the basal ACh response obtained in the absence of the 50-kDa component and the elevated responses obtained with it can be inhibited by neuronal bungarotoxin (nBgt) but not by alpha-bungarotoxin (alpha Bgt). Increases of less than twofold are observed for the binding of anti-AChR monoclonal antibody 35 (mAb 35), nBgt, and alpha Bgt to the neurons under these conditions. Extract fractions containing the 50-kDa component also enable the neurons to enhance their ACh responses through a cAMP-dependent mechanism. Either the 50-kDa fraction induces the appearance of a new type of AChR regulated by cAMP, or it alters the function of existing AChRs. The 50-kDa fraction produces no change in neuronal growth but can increase GABA responses sixfold, indicating that its effects are not confined to AChRs. It is not clear whether a single molecular species is responsible for the diverse regulatory effects or whether several types of active components are present in the fraction. The component which enhances ACh sensitivity is trypsin-sensitive and heat-labile, as expected for a protein. The component may be widely distributed since the 50-kDa fraction from a number of tissues can increase the ACh response. The fraction from eye tissue, however, has a specific activity 5-10 times greater than that of the liver fraction. A wide distribution would suggest multiple targets and roles for the component during development.

Acetylcholine↗
PubMed1990

Cyclic AMP-dependent phosphorylation of a neuronal acetylcholine receptor alpha-type subunit.

Chick ciliary ganglion neurons have nicotinic acetylcholine receptors (AChRs) that mediate synaptic transmission through the ganglion. A cAMP-dependent process has previously been shown to enhance the ACh response of the neurons 2- to 3-fold without requiring the synthesis of new receptors. We show here that the receptors can be phosphorylated in situ by a cAMP-dependent process. The phosphorylation occurs predominantly on components of 50 and 58 kDa. Both derive from putative ligand-binding alpha 3 subunits, with the smaller phosphorylated species probably representing a degradation product of the larger. The increase in receptor phosphorylation caused by incubating the neurons with a cAMP analog parallels the increase observed in the ACh response, with respect to both time course and relative extent. The phosphorylation of ciliary ganglion AChRs differs from that reported for electric organ AChRs, which occurs primarily on the non-ligand-binding gamma and delta subunits and increases the rate of agonist-induced receptor desensitization.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Subunit composition of nicotinic acetylcholine receptors from chick ciliary ganglia.

Nicotinic ACh receptors were immunoaffinity-purified from chick ciliary ganglia, radioiodinated, and examined by SDS-PAGE. Components with Mr's of 49, 52, and 60 kDa were obtained. Limited proteolysis produced different peptide maps from the components, confirming the 3 as distinct species. All are glycoproteins since treatment with glycopeptidase F altered their migration during electrophoresis. The 60 kDa component appears to be encoded by the AChR alpha 3 gene since it was selectively immunoprecipitated by an antiserum to a fusion protein containing a putative cytoplasmic region of the predicted alpha 3 gene product. The 49 kDa component selectively cross-reacted on immunoblots with 4 monoclonal antibodies that recognize a component of similar size in AChR preparations from chicken brain. The 52 kDa component is a novel species not previously identified in preparations of brain AChRs. If all 3 components represent integral AChR subunits, they may compose 2 receptor subtypes in the ganglion, e.g., one containing 49 and 60 kDa subunits and another containing 52 and 60 kDa subunits. This is supported by the finding that a receptor preparation can be obtained containing only the 49 and 60 kDa components and is consistent with reports of brain AChRs having only 2 types of subunits. Alternatively, ganglionic AChRs may contain 3 or more types of subunits, with at least one being selectively lost under certain conditions.

Animals↗
PubMed1989

Regulating the number and function of neuronal acetylcholine receptors.

Nicotinic acetylcholine receptors (AChRs) on vertebrate neurons represent a family of receptors distinct from the well-characterized AChR of skeletal muscle. New probes for neuronal AChRs are now being used to examine the regulation of receptor number and function. The results suggest that neuronal AChRs differ from muscle AChRs in regulation by presynaptic input and by at least one second messenger system, and that neuronal AChRs are additionally regulated by retrograde signals from the synaptic target tissue. The forms of regulation provide potential mechanisms by which cell-cell interactions could stabilize synaptic contacts on neurons and modulate synaptic function.

Animals↗
PubMed1989

Differential effects of nerve transection on the ACh and GABA receptors of chick ciliary ganglion neurons.

Chick ciliary ganglion neurons have nicotinic acetylcholine receptors (AChRs) that mediate chemical transmission through the ganglion, and GABAA receptors of unknown significance. Previous experiments examining the role of cell-cell interactions in regulating neuronal AChRs have shown that postganglionic axotomy of ciliary ganglia in newly hatched chicks causes a 10-fold decline in total AChRs within 5 d compared with unoperated contralateral ganglia and that preganglionic denervation causes a 3-fold decline within 10 d. Many of the AChRs are known to be intracellular; of those present on the cell surface, only a small fraction appears to be functionally available normally. In the present experiments, the effects of the operations on functional AChRs and GABAA receptors in the plasma membrane of the neurons were examined by removing the ganglia 5 d after axotomy or 10 d after denervation, dissociating them into single cells, and immediately measuring their ACh and GABA sensitivities with intracellular recording techniques. The ACh sensitivity of axotomized ciliary ganglion neurons was reduced 10-fold compared with neurons from unoperated contralateral ganglia of the same chicks. The reduction could be largely accounted for by a decrease in the maximum response and did not arise from a change either in the dose-response curve or the acetylcholinesterase activity of the neurons. Autoradiographic studies using a radiolabeled anti-AChR monoclonal antibody also demonstrated a substantial decrease in the total number of surface AChRs associated with axotomized neurons. In contrast, axotomy had no unilateral effect on the GABA response.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1989

Specific down-regulation of the alpha-bungarotoxin binding component on chick autonomic neurons by ciliary neuronotrophic factor.

Chick ciliary ganglion neurons have a cholinergic membrane component that binds alpha-bungarotoxin with high affinity but has no known function. The component is different from the nicotinic ACh receptor on the neurons that mediates cholinergic transmission through the ganglion. Ciliary neuronotrophic factor (CNTF) has been shown to enhance the survival of ciliary ganglion neurons in cell culture and has been postulated to act as a target-derived trophic factor for the neurons in vivo. We show here that a factor indistinguishable from CNTF specifically down-regulates alpha-bungarotoxin binding sites on the neurons while increasing cell growth and the number of ACh receptors on the cells. Similar effects, though reduced in magnitude, are seen with chick sympathetic neurons. CNTF has no effect on the number of ACh receptors found on chick myotubes in culture. The down-regulation of alpha-bungarotoxin binding sites on neurons caused by CNTF occurs with a half-time of about 19 hr and is largely reversed within a 4 d period following CNTF removal. It is distinct from the down-regulation caused by cholinergic agonists. Nerve growth factor and fibroblast growth factor have no apparent effect on the number of alpha-bungarotoxin binding sites on the neurons, though fibroblast growth factor does stimulate neuronal growth. The results indicate that the effects of CNTF on the alpha-bungarotoxin binding component are both novel for a growth factor and specific, and they suggest a relationship between the component and the regulation of growth by the target tissue.

Animals↗
PubMed1988

Expression and regulation of neuronal acetylcholine receptor mRNA in chick ciliary ganglia.

A chicken genomic clone encoding a portion of the neuronal acetylcholine receptor (AChR) alpha 3 subunit was used to identify homologous mRNA in embryonic chick ciliary ganglia. In situ hybridization indicated that the mRNA was neuronal. Northern blot analysis revealed a major hybridizing species of 3.5 kb. Protection experiments confirmed that ganglionic RNA contained material indistinguishable by RNAase digestion from the 300 nucleotide probe used. No transcripts were detected by in situ hybridization or Northern blot analysis for chick neuronal AChR alpha 2 or alpha 4 genes. alpha 3 transcripts were present at all times examined (E6 to 1 year posthatch). Both postganglionic axotomy and preganglionic denervation of ciliary ganglia in newly hatched chicks produced declines in alpha 3 mRNA levels, implying regulation of neuronal AChR mRNA by cell-cell interactions.

Animals↗
PubMed1988

Metabolic stability and antigenic modulation of nicotinic acetylcholine receptors on bovine adrenal chromaffin cells.

Bovine adrenal chromaffin cells have nicotinic acetylcholine receptors (AChRs) that are activated by the splanchnic nerve, resulting in release of catecholamines from the cells. Examination of the AChRs can provide information about the regulation and turnover of synaptic components on neurons and endocrine cells. Previous studies have shown that mAb 35 recognizes the AChR on the cells. Here we show that mAb 35 can remove AChRs from the surface of the cells by antigenic modulation, and that the modulation can be used together with other methods to examine the stability and turnover of the receptors in the plasma membrane. Unexpectedly, the results indicate a disparity between the rate at which AChRs reappear on the cells and the rate at which the ACh response recovers after preexisting AChRs have been removed. Exposure of bovine adrenal chromaffin cultures to mAb 35 results in a parallel decrease in the magnitude of the nicotinic response and the number of AChRs on the cells. The decrease depends on the concentration and divalence of mAb 35, and on the time and temperature of the incubation. The antibody induces receptor aggregation in the plasma membrane under conditions where receptor loss subsequently occurs. After binding to receptor, mAb 35 appears to be internalized, degraded, and released from the cells through a temperature sensitive pathway that requires lysosomal function. These features are characteristic of antigenic modulation. Appearance of new AChRs on the cells either after antigenic modulation or after blockade of existing AChRs with monovalent antibody fragments occurs at a rate equivalent to 3% of the receptors present on control cells per hour. The rate of receptor loss from the cells was measured in the presence of either tunicamycin or puromycin to block appearance of new receptors. Both conditions indicated a receptor half-life of approximately 24 h and a rate of loss of approximately 3%/h. The finding that the rate of receptor loss equaled the rate of receptor appearance was consistent with the observation that the total number of AChRs on untreated cells did not increase with time. In the presence of tunicamycin, loss of receptor-mediated response to nicotine also occurred with a half-time of 24 h. Paradoxically, the rate of recovery of the nicotinic response, determined using two procedures, was more than twice as great as the rate at which new AChRs appeared on the cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Glands↗
PubMed1988

Cyclic AMP-dependent mechanism regulates acetylcholine receptor function on bovine adrenal chromaffin cells and discriminates between new and old receptors.

Bovine adrenal chromaffin cells have nicotinic acetylcholine receptors (AChRs) that mediate release of catecholamines from the cells in response to synaptic input, and resemble neuronal AChRs in pharmacology and antigenic profile. Results presented here show that a cAMP-dependent process enhances the function of adrenal chromaffin AChRs as a population in the plasma membrane. This was demonstrated by showing that cAMP analogues cause specific increases both in the level of nicotine-induced catecholamine release from the cells and in the level of the nicotine-induced conductance change occurring in the cells. Neither de novo synthesis of receptors nor transport of preexisting intracellular receptors to the plasma membrane is necessary for the enhancement. The responsiveness of AChRs to regulation by the cAMP-dependent process appears to depend on the length of time the receptors have been on the cell surface. AChRs newly inserted into the plasma membrane generate a greater nicotinic response than do older AChRs and, unlike older AChRs, their response to agonist is not enhanced after treatment of the cells with cAMP analogues. The findings indicate that the AChRs and/or associated components undergo a maturation in the plasma membrane that alters their function and their regulation by secondary messenger systems.

Adrenal Glands↗
PubMed1988

Benzodiazepine interactions with GABAA receptors on chick ciliary ganglion neurons.

gamma-Aminobutyric acidA (GABAA) receptors on chick ciliary ganglion neurons can be modulated by benzodiazepines and identified by radiolabeled benzodiazepine binding. Enhancement of submaximal GABA responses by benzodiazepines was demonstrated using a multibarrel pipette to construct complete benzodiazepine dose-response curves for single cells in culture. EC50 values of 22 +/- 5 nM, 1.1 +/- 0.3 microM, and 4.6 +/- 0.5 microM were obtained for flunitrazepam, clonazepam, and chlordiazepoxide, respectively. Chlordiazepoxide shifted the GABA dose-response curve to lower GABA concentrations without increasing the maximal response to GABA, demonstrating that benzodiazepines enhance the GABA response by increasing the receptor affinity for GABA. The imidazodiazepine Ro15-1788 potentiated the GABA response with an EC50 of 250 +/- 70 nM, and Ro5-4864 (chlorodiazepam) partially blocked the GABA response both in the presence and absence of chlordiazepoxide. Scatchard analysis of data from binding studies with [3H]flunitrazepam to ganglion membrane homogenates was consistent with the presence of a single class of high affinity sites with a KD of 34 +/- 6 nM and a Bmax of 145 +/- 26 fmol/mg of protein. Several lines of evidence indicated that the sites were associated with GABAA receptors. The KD of [3H]flunitrazepam binding was similar to the EC50 for flunitrazepam modulation of the GABA response. The level of [3H]flunitrazepam binding was enhanced approximately 50% over control levels by GABA. The binding was decreased both by clonazepam and by Ro5-4864 at concentrations similar to those required for the compounds to modulate the GABA response. These studies demonstrate that ciliary ganglion GABAA receptors are similar in major respects to GABAA receptors in the central nervous system but may differ in minor pharmacological properties.

Animals↗
PubMed1988

The distribution of acetylcholine receptors in chick ciliary ganglion neurons following disruption of ganglionic connections.

Chick ciliary ganglion neurons have nicotinic acetylcholine receptors (AChRs) that mediate primary chemical synaptic transmission through the ganglion. Previous studies have shown that preganglionic denervation reduces the total number of AChRs in the ganglion about 3-fold in 10 d, while postganglionic axotomy reduces AChR levels about 10-fold in 5 d. Since the neurons contain large numbers of intracellular AChRs in addition to the surface AChRs, the present studies were undertaken to determine whether either surface or internal AChR pools are changed selectively by the operations. An anti-AChR monoclonal antibody followed by an HRP-conjugated secondary antibody was used to visualize AChR distributions on neurons in ciliary ganglia 5 d after postganglionic axotomy and 10 d after preganglionic denervation. Ganglia were permeabilized by treatment with saponin to obtain access to intracellular receptors. The results show that the operations do not qualitatively change the subcellular localization of AChRs, but they do alter the levels relative to control ganglia. Axotomy produces substantial declines both in the number of synaptic AChRs and in the number of intracellular AChRs. Denervation produces a significant, though less extensive decline in the number of intracellular receptors while having no detectable effect on the number of synaptic AChRs. Small alterations in receptor distribution would have gone undetected by the present techniques. Regulation of neuronal AChRs differs in several respects from that described for muscle AChRs: presynaptic input appears to be less important for controlling neuronal AChRs, while signals from the postsynaptic target tissue may be essential for maintaining synaptic receptors on the neurons.

Animals↗
PubMed1988

A desensitized form of neuronal acetylcholine receptor detected by 3H-nicotine binding on bovine adrenal chromaffin cells.

A nicotinic acetylcholine receptor (AChR) on bovine adrenal chromaffin cells in culture has previously been identified using the alpha-neurotoxin n-Bgt and the monoclonal antibody mAb 35. Here, we report that the cells have 2 classes of high-affinity binding sites for 3H-nicotine, one being associated with the AChR and the other being associated with the alpha-bungarotoxin binding component that is distinct from the AChR. Scatchard analysis of 3H-nicotine binding to the AChR site yields a KD of 20 +/- 3 nM and a Bmax of 104 +/- 12 fmol/mg protein. Nicotinic antagonists block 3H-nicotine binding to the AChR site with the same rank order of potency and affinity with which they block nicotine-induced catecholamine release from the cells. About 80% of the AChRs are on the cell surface, as judged by the distribution of both 3H-nicotine binding and 125I-mAb 35 binding to the receptor, and the ratio of nicotine/mAb 35 binding to the AChR on the cell surface is approximately 1:1. Chronic treatment of the cells with mAb 35 results in receptor modulation such that all of AChR-related nicotine binding is lost from the cell surface, and all of the functional response to nicotine is lost as well. The results confirm that 3H-nicotine binding is associated with AChRs on the cells. The 3H-nicotine binding observed to the AChR represents binding to a desensitized form of the receptor having increased affinity for agonists and unchanged affinity for antagonists. This conclusion derives from the following observations. The KiS for agonist competition of 3H-nicotine binding indicate agonist affinities several orders of magnitude greater than do the KDS measured for receptor activation. Exposing cultures to low concentrations of nicotine and substance P causes receptor densensitization, and the concentration dependence of the nicotine-induced desensitization displays an EC50 of 20 nM, in good agreement with the KD obtained from equilibrium binding studies with 3H-nicotine. In addition, the rate of 3H-nicotine binding is increased both by substance P, which enhances the rate of agonist-induced desensitization on adrenal chromaffin cells, and by preincubation with nicotine. The increased rate of association, together with the dissociation rate, yields a kinetically derived KD of 19 nM, again in good agreement with the KD obtained from equilibrium binding studies. These results demonstrate that the bovine adrenal chromaffin AChR is similar to AChRs from muscle and electric organ in undergoing an agonist-induced conversion to a desensitized state having increased affinity for agonists.

Adrenal Glands↗
PubMed1987

Formation of calyx-like contacts preferentially on appropriate target neurons in culture.

The availability of culture systems for both Edinger Westphal and ciliary ganglion neurons has made it possible to examine the interactions in culture between two populations of vertebrate neurons that synapse in vivo. In the chick, Edinger Westphal neurons provide the sole presynaptic input to the ciliary ganglion and, through this projection, are responsible for the control of lens curvature (accommodation), iris constriction, and possibly smooth muscle function in the choroid layer of the eye. When embryonic chick Edinger Westphal and ciliary ganglion neurons were combined in culture and stained for enkephalin-like immunoreactivity to visualize Edinger Westphal terminals, stained calyx-like contacts were observed that resemble the calyciform terminals formed between Edinger Westphal processes and ciliary neurons in the ciliary ganglion in vivo. Although stained calyx-like contacts could also be found in Edinger Westphal-alone and ciliary ganglion-alone cultures, many more were observed when the two cell types were cultured together. The increase depended specifically on the ciliary ganglion neurons since substitution of either dorsal root ganglion or sympathetic ganglion neurons for them in the cocultures did not increase the number of calyx-like contacts staining positive for enkephalin over those present in cultures of Edinger Westphal neurons alone. When Edinger Westphal neurons were grown simultaneously with dorsal root and ciliary ganglion neurons, calyx-like contacts with enkephalin-like immunoreactivity were found to terminate preferentially on the latter. These findings suggest that vertebrate neurons can form morphologically specific contacts preferentially on appropriate target cells in culture in the absence of many of the potential cues present in the intact tissue.

Animals↗
PubMed1987

Cyclic AMP regulates the proportion of functional acetylcholine receptors on chicken ciliary ganglion neurons.

Previous studies have shown that the number of functional acetylcholine receptors (AcChoRs) on chicken ciliary ganglion neurons in culture is considerably smaller than the total number of AcChoRs detected on the neurons by labeled receptor probes. Here we use patch-clamp recording to show that a cAMP-dependent process enhances the AcCho response of the neurons by a mechanism likely to involve an increase in the number of functional AcChoRs. The increase occurs without requiring protein synthesis and without involving a detectable increase in the total number of AcChoRs on the cell surface measured with a labeled receptor probe. The results imply that the neurons have functional and nonfunctional pools of AcChoRs and that functional receptors can be recruited from intracellular receptors or from nonfunctional receptors on the cell surface by a cAMP-dependent process. A cAMP-dependent regulation of the number of functional neurotransmitter receptors would provide a reversible mechanism by which cell-cell interactions could modulate synaptic transmission in the nervous system.

1-Methyl-3-isobutylxanthine↗