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D K Berg

Publications and source records attributed to D K Berg.

At least 37 records · Page 2Linked to original sources
PubMed1999

Neuronal acetylcholine receptors with alpha7 subunits are concentrated on somatic spines for synaptic signaling in embryonic chick ciliary ganglia.

Nicotinic acetylcholine receptors containing alpha7 subunits are widely distributed in the vertebrate nervous system. In the chick ciliary ganglion such receptors generate large synaptic currents but appear to be excluded from postsynaptic densities on the cells. We show here that alpha7-containing receptors are concentrated on somatic spines in close proximity to putative sites of presynaptic transmitter release. Intermediate voltage electron microscopy on thick sections, together with tomographic reconstruction, permitted three-dimensional analysis of finger-like projections emanating from cell bodies. The projections were identified as spines based on their morphology, cytoskeletal content, and proximity to presynaptic elements. Both in situ and after ganglionic dissociation, the spines were grouped on the cell surface and tightly folded into mats. Immunogold labeling of receptors containing alpha7 subunits showed them to be preferentially concentrated on the somatic spines. Postsynaptic densities were present in vivo both on the soma near spines and occasionally on the spines themselves. Synaptic vesicle-filled projections from the presynaptic calyx were interdigitated among the spines. Moreover, the synaptic vesicles often abutted the membrane and sometimes included Omega profiles as if caught in an exocytotic event, even when no postsynaptic densities were juxtaposed on the spine. The results suggest several mechanisms for delivering transmitter to alpha7-containing receptors, and they support new ideas about synaptic signaling via spines. They also indicate that neurons must have specific mechanisms for targeting alpha7-containing receptors to desired locations.

Animals↗
PubMed1999

Rapsyn variants in ciliary ganglia and their possible effects on clustering of nicotinic receptors.

Nicotinic acetylcholine receptors (nAChRs) containing the alpha7 gene product can influence a range of cellular events in neurons, depending on receptor location. On chick ciliary neurons, the receptors are concentrated on somatic spines, but little is known about mechanisms responsible for sequestering them there. Rapsyn is a 43-kDa protein essential for clustering nicotinic receptors at the vertebrate neuromuscular junction. RT-PCR confirmed previous studies showing that the chick ciliary ganglion expresses rapsyn transcripts, including several splice variants lacking part or all of exon 2. Heterologous expression of rapsyn constructs, together with nicotinic receptor constructs, shows that chicken full-length rapsyn can induce clustering of both muscle and neuronal nicotinic receptors. Splice variants lacking one or both leucine zipper motifs of exon 2 are unable to cluster the receptors, though, like full-length rapsyn, they cluster themselves. Immunological analysis demonstrates the presence of full-length rapsyn in chick muscle extracts but fails to detect either full-length or splice-variant versions of rapsyn at significant levels in ganglion extracts. The results suggest that rapsyn does not cluster alpha7-nAChRs on ciliary neurons in any way similar to that of receptors at the neuromuscular junction where rapsyn and the receptors are present in approximately equimolar amounts.

Alternative Splicing↗
PubMed1999

Developmental changes in the nicotinic responses of ciliary ganglion neurons.

The accumulation of functional neurotransmitter receptors by neurons during development is an essential part of synapse formation. Chick ciliary ganglion neurons express two kinds of nicotinic receptors. One is abundant, contains the alpha7 gene product, rapidly desensitizes, and binds alpha-bungarotoxin. The other is less abundant, contains multiple gene products (alpha3, beta4, alpha5, and beta2 subunits), slowly desensitizes, and binds the monoclonal antibody mAb 35. Rapid application of agonist to freshly dissociated neurons elicits responses from both classes of receptors. Between embryonic days 8 and 15, the whole cell response of alpha3-containing receptors increases fivefold in peak amplitude and, normalized for cell growth, 1.7-fold in current density. In addition, the response decays more slowly in older neurons, suggesting a developmental decrease in the rate of desensitization. The whole cell response of alpha7-containing receptors increases 10-fold in peak amplitude over the same period and 3-fold in current density. No change in the rate of desensitization was apparent for alpha7-containing receptors with developmental age, but analysis was limited by overlap in responses from the two kinds of receptors. Indirect immunofluorescence measurements on dissociated neurons showed that the relative levels of alpha7-containing receptors on the soma increased during development to the same extent as the whole cell response attributed to them. In contrast, the relative levels of alpha3-containing receptors increased more during the same time period than did the whole cell response they generated. The immunofluorescence analysis also showed that both classes of receptors become distributed in prominent clusters on the cell surface as a function of developmental age. The results indicate that during this period of synaptic consolidation on the neurons, the two major classes of functional nicotinic receptors undergo substantial upregulation; alpha3-containing receptors as a class may undergo changes in receptor properties as well.

Animals↗
PubMed1999

Extracellular calcium regulates responses of both alpha3- and alpha7-containing nicotinic receptors on chick ciliary ganglion neurons.

Neuronal nicotinic receptors are generally both permeable to calcium and potentiated by it. We have examined acute calcium regulation of both native alpha7-containing and the less abundant alpha3-containing nicotinic receptors on chick ciliary ganglion neurons. Most of the receptors are concentrated on somatic spines tightly overlaid in situ by a large presynaptic calyx. Whole cell patch-clamp recording from dissociated neurons using perforated patch-clamp techniques indicates that the rapidly desensitizing nicotinic response of alpha7-containing receptors achieves maximum amplitude in 2 mM calcium; both lower and higher concentrations of calcium are less effective. Barium and strontium but not magnesium can substitute for calcium in potentiating the response. Neither calcium current through the receptors nor calcium action at intracellular sites is necessary. These latter conclusions are supported by current-voltage analysis of the nicotine-induced response, ion substitution experiments, and internal perfusion of the cells with 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) via a conventional patch pipette. Varying the agonist concentration indicates that some of the calcium-dependent enhancement may involve a shift in the dose-response curve for agonist binding, but much of the effect is also likely to involve increased receptor responsiveness. Blockade of alpha7-containing receptors with alpha-bungarotoxin showed that the heteromeric alpha3-containing nicotinic receptors also undergo calcium-dependent potentiation. Calcium did not have a major effect on the desensitization rate of either receptor class but did have a selective effect on the rise time of alpha7-containing receptors. Analysis of stably transfected cells expressing an alpha7 gene construct showed that the calcium potentiation observed for native receptors did not require neuron-specific modifications or components and that it could be seen with the natural agonist acetylcholine. Receptor dependence on extracellular calcium may provide a regulatory mechanism for constraining synaptic signaling, avoiding local depletion of external calcium, and limiting calcium buildup in postsynaptic compartments.

Animals↗
PubMed1998

Mammalian nicotinic receptors with alpha7 subunits that slowly desensitize and rapidly recover from alpha-bungarotoxin blockade.

One of the most abundant nicotinic receptors in the nervous system is a species that contains the alpha7 gene product, rapidly desensitizes, and binds alpha-bungarotoxin with great affinity. The receptor has a high relative permeability to calcium and performs a variety of functions including presynaptic modulation of transmitter release and postsynaptic generation of synaptic currents. Fast excitatory transmission in mammalian intracardiac ganglia is mediated primarily by nicotinic receptors, and although intracardiac ganglion neurons express the alpha7 gene, no toxin-sensitive response has been detected previously in them. We report here that whole-cell patch-clamp recordings from freshly dissociated intracardiac ganglion neurons reveal a nicotinic response that desensitizes slowly and is blocked by alpha-bungarotoxin in a rapidly reversible manner. The only rat gene previously thought capable of forming such receptors was alpha9, but no evidence suggests that the alpha9 gene is expressed in neurons. We find that reverse transcription (RT)-PCR detects alpha7 but not alpha9 mRNA in the ganglia. In addition, the pharmacology of the nicotinic response is typical of alpha7-containing receptors but differs in several respects from that expected for alpha9. Binding experiments with immunotethered receptors identifies a ganglionic species that contains the alpha7 gene product. Moreover, intracellular perfusion of the cells with an anti-alpha7 monoclonal antibody specifically reduces the amplitude of the toxin-sensitive response. The results indicate that alpha7-containing receptors are responsible for the slowly desensitizing, toxin-reversible response and suggest that the receptors are modified in cell-specific ways to influence their functional properties.

Acetylcholine↗
PubMed1998

Organizing effects of rapsyn on neuronal nicotinic acetylcholine receptors.

Targeting receptors to appropriate locations on the cell surface is a critical task for neurons. We have examined the possibility that rapsyn controls the distribution of nicotinic receptors on neurons as it does nicotinic receptors on muscle fibers. Cotransfection of QT6 cells with rapsyn and neuronal nicotinic receptor cDNA constructs produced receptor aggregates or clusters that codistributed in part with rapsyn protein. Though all nicotinic receptor subtypes tested were affected by rapsyn, receptors containing the alpha 7 gene product were among the most responsive. In addition, rapsyn caused a portion of the nicotinic receptors containing alpha 7 subunits to become resistant to solubilization with nonionic detergent and to display a marked increase in metabolic stability. The results are consistent with rapsyn linking the receptors to cytoskeletal elements and suggest that it may play an organizing role determining the fate and location of nicotinic receptors on neurons.

Animals↗
PubMed1998

Nicotinic receptor subtypes in the developing chick brain: appearance of a species containing the alpha4, beta2, and alpha5 gene products.

Increasing evidence suggests nicotinic receptors regulate developmental events in the nervous system. We used [3H]epibatidine and 125I-alpha-bungarotoxin, together with subunit-specific monoclonal antibodies, to distinguish and quantify nicotinic receptor subtypes in developing chick brain. The results show that more than three fourths of the epibatidine-binding receptors at both early and late embryonic stages contain alpha4 and beta2 subunits, representing receptors previously distinguished by high affinity nicotine binding. A fraction of these also contain the alpha5 gene product, which is consistent with studies on transfected cells showing that the alpha4, beta2, and alpha5 gene products coassemble to produce epibatidine-binding receptors. A small portion of the receptors contain alpha3 and beta4 subunits, assembled in part with either alpha4 or beta2 subunits. The most abundant nicotinic receptors, however, at both early and late embryonic stages are those having high affinity for alpha-bungarotoxin rather than epibatidine. Most contain alpha7 subunits, whereas about half contain alpha8 subunits as well. The sharpest developmental increase between embryonic days 8 and 17/18 occurs with receptors containing alpha5 subunits, whereas receptors containing alpha3 or beta4 subunits undergo no specific increase. The three major receptor species (containing alpha4 and beta2 but not alpha5 subunits; alpha7 subunits; or alpha7 and alpha8 subunits) each increase approximately 3-fold during the same period. The results indicate greater receptor complexity than appreciated previously; they provide information about the rules governing subunit assembly in neuronal nicotinic receptors and draw attention to the role of alpha5 subunits in late development.

Animals↗
PubMed1997

Detection of functional nicotinic receptors blocked by alpha-bungarotoxin on PC12 cells and dependence of their expression on post-translational events.

A major class of nicotinic receptors in the nervous system is one that binds alpha-bungarotoxin and contains the alpha7 gene product. PC12 cells, frequently used to study nicotinic receptors, express the alpha7 gene and have binding sites for the toxin, but previous attempts to elicit currents from the putative receptors have failed. Using whole-cell patch-clamp recording techniques and rapid application of agonist, we find a rapidly desensitizing acetylcholine-induced current in the cells that can be blocked by alpha-bungarotoxin. The current amplitude varies dramatically among three populations of PC12 cells but correlates well with the number of toxin-binding receptors. In contrast, the current shows no correlation with alpha7 transcript; cells with high levels of alpha7 mRNA can be negative for toxin binding and yet have other functional nicotinic receptors. Northern blot analysis and reverse transcription-PCR reveal no defects in alpha7 RNA from the negative cells, and immunoblot analysis demonstrates that they contain full-length alpha7 protein, although at reduced levels. Affinity purification of toxin-binding receptors from cells expressing them confirms that the receptors contain alpha7 protein. Transfection experiments demonstrate that PC12 cells lacking native toxin-binding receptors are deficient at producing receptors from alpha7 gene constructs, although the same cells can produce receptors from other transfected gene constructs. The results indicate that nicotinic receptors that bind alpha-bungarotoxin and contain alpha7 subunits require additional gene products to facilitate assembly and stabilization of the receptors. PC12 cells offer a model system for identifying those gene products.

Amino Acid Isomerases↗
PubMed1997

Direct recording of nicotinic responses in presynaptic nerve terminals.

Nicotinic acetylcholine receptors are widely expressed in the nervous system, but their functions remain poorly understood. One attractive hypothesis is that the receptors act presynaptically to modulate synaptic transmission. We provide a direct demonstration of presynaptic nicotinic receptors in situ by using whole-cell patch-clamp techniques to record currents in large presynaptic calyces that midbrain neurons form on ciliary neurons. Bath application of nicotine induced inward currents in the calyces capable of generating action potentials that overrode the limited space clamp achievable. The inward currents reversed near 0 mV and showed inward rectification common for neuronal nicotinic receptors. Tetrodotoxin (TTX) blocked the action potentials but not the inward currents. alpha-Bungarotoxin blocked both, consistent with the presynaptic receptors containing alpha7 subunits. Recording from the postsynaptic ciliary neurons during nicotine exposure revealed EPSCs that TTX blocked, presumably by blocking presynaptic action potentials. The postsynaptic cells also displayed bimodal inward currents caused by their own nicotinic receptors; the bimodal currents were not blocked by TTX but were blocked partially by alpha-bungarotoxin and completely by D-tubocurarine. Dye-filling with Lucifer yellow from the recording pipette confirmed the identity of patched structures and showed no dye transfer between calyx and ciliary neuron. When calyces or ciliary neurons were labeled en mass with neurobiotin and biocytin through nerve roots, dye transfer was rarely observed. Thus, electrical synapses were infrequent and unlikely to influence calyx responses. Immunochemical analysis of preganglionic nerve extracts identified receptors that bind alpha-bungarotoxin and contain alpha7 subunits. The results unambiguously document the existence of functional presynaptic nicotinic receptors.

Animals↗
PubMed1997

Differences in the fate of neuronal acetylcholine receptor protein expressed in neurons and stably transfected cells.

Ligand-gated ion channels are structurally complex transmembrane proteins that all neurons must synthesize for rapid chemical synaptic transmission. The most abundant nicotinic acetylcholine receptor serving as a ligand-gated ion channel in the nervous system is a species that contains alpha7 subunits, binds alpha-bungarotoxin, and has a high relative permeability to calcium. The ability of neurons to make such receptors was compared with that of non-neuronal cells stably transfected with an alpha7 cDNA to determine whether neuron-specific machinery is likely to aid in their assembly or stabilization. Transfected cells expressed alpha7 protein and assembled it into a species that was indistinguishable in size and pharmacology from native receptors, but much of the alpha7 protein they synthesized was rapidly degraded without becoming receptor. Neurons were not only more efficient than the best transfectants at assembling the receptors but also produced a subpopulation of receptors on the cell surface that was relatively stable and resistant to solubilization. This subpopulation, which was absent from transfected cells, may be tethered to cytoskeletal elements in the neurons. The results support the contention that neurons contain components that facilitate the production and stabilization of ligand-gated ion channels.

Animals↗
PubMed1997

Expression of the nicotinic receptor alpha 7 gene in tendon and periosteum during early development.

One of the most abundant nicotinic acetylcholine receptors expressed in the central and peripheral nervous systems is a species that contains the alpha 7 gene product, binds alpha-bungarotoxin with high affinity, and has a high relative permeability to calcium. The alpha 7 gene is also expressed at low levels in embryonic muscle tissue. We show here that the alpha 7 gene is expressed in tendon fibroblasts and periosteal cells during development. In situ hybridizations identify alpha 7 transcripts in tissue sections containing embryonic tendon and periosteum. RNase protection experiments demonstrate alpha 7 mRNA in primary tendon cells grown in culture. Immunofluorescence with subunit-specific monoclonal antibodies reveals alpha 7 protein in embryonic tendon. Immunoprecipitation assays with the antibodies indicate that the alpha 7-containing species in tendon is capable of binding alpha-bungarotoxin and that a similar species can be identified at low levels on the surface of fibroblasts in culture. The results show that the alpha 7 gene product is expressed in a range of tissues, including cells thought to be nonexcitable. The distribution of alpha 7 expression early in development and the ability of alpha 7-containing receptors to elevate intracellular calcium suggest that the gene may influence a variety of calcium-dependent events during embryogenesis.

Animals↗
PubMed1997

Neuronal-type acetylcholine receptors and regulation of alpha 7 gene expression in vertebrate skeletal muscle.

Several neuronal nicotinic acetylcholine receptor (AChR) genes are expressed in chick skeletal muscle during development. One of the most abundantly expressed is alpha 7, which produces a protein capable of binding alpha-bungarotoxin and is physically distinct from muscle AChRs containing the alpha 1 gene product. We show here that the alpha 7-containing species in muscle is indistinguishable pharmacologically from alpha 7-containing AChRs in neurons. In addition, immunologic analysis with subunit-specific muscle antibodies shows that the alpha 7-containing species in muscle lacks the beta 1 and delta muscle AChR gene products as it does the alpha 1. RNase protection experiments measuring alpha 7 mRNA levels indicate that the alpha 1 and alpha 7 genes may, in part, be subject to similar kinds of regulation in the tissue. Surgical denervation of leg muscle in newly hatched chicks caused a small and transient increase in alpha 7 mRNA after 8 days, while alpha 1 transcripts underwent a large and sustained increase in number. Similarly, treating myotube cultures with tetrodotoxin caused a modest increase in alpha 7 transcript levels and a large increase in alpha 1. Calcitonin gene-related peptide (CGRP) increased both kinds of transcripts in myotube cultures equally as did treatment with 8-bromo-cyclic AMP; CGRP is thought to work via a cyclic AMP-dependent pathway in muscle. In at least one respect, however, alpha 7 expression in muscle differs qualitatively from that of alpha 1: AChR-inducing activity (ARIA) increased alpha 1 mRNA levels in culture while slightly depressing alpha 7 mRNA levels. The regulatory pattern of alpha 7 expression in muscle may combine features of both alpha 7 expression in neurons and alpha 1 expression in muscle.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Comparison of seasonal changes in reproductive parameters of adult male European fallow deer (Dama dama dama) and hybrid Mesopotamian x European fallow deer (D. d. mesopotamica x D. d. dama).

In a study, aimed at comparing seasonal reproductive development of European fallow deer (Dama dama dama) with Mesopotamian (D. d. mesopotamico) x European F1 hybrids, five adult males of each genotype, which had been raised together since birth, were maintained as a bachelor group. Morphometric (body weight, neck circumference and testis diameter), endocrine (plasma testosterone concentrations) and seminal (ejaculate volume, spermatozoa per ejaculate and spermatozoa motility) parameters were recorded at fortnightly or monthly intervals for a 15-month period, and antler status was noted daily during the general periods of casting and velvet stripping. In addition, two bucks of each genotype were blood sampled via indwelling jugular catheters every 30 min for 24-h periods on five occasions (2-3 months intervals) during the year, and plasma was analysed for concentrations of testosterone and LH. Parameter profiles of the two genotypes were compared by global and time series ante-dependence covariance analysis to investigate overall profile similarity and the seasonal nature of any observed differences. Plasma hormone profiles from high-frequency blood sampling were subjected to PULSAR analysis to determine pulse frequency and amplitude. Throughout the study hybrid males were approximately 30% heavier than European males. However, both genotypes exhibited dramatic but parallel patterns of body weight change (global P = 0.054). Neck circumference was correlated with body weight throughout (P < 0.05), with similar regression slopes between the genotypes at any sampling time (P > 0.10). Covariance adjustment to a common initial body weight was performed to eliminate the effects of large body weight differences on muscle hypertrophy and regression. While profiles of corrected neck circumference were significantly different at the global level (P < 0.01), analysis by time revealed differences occurring only during the latter period of muscular regression in spring. However, profiles of other parameters, including testis diameter, plasma testosterone concentrations, spermatozoa per ejaculate and percentage motile spermatozoa, exhibited significant displacement between genotypes (global P < 0.05) evident as 2-4 weeks advancement in the sexual development (late summer/autumn) and quiescence (spring) phases for hybrid males relative to European males. Furthermore, mean dates of antler casting and velvet stripping were significantly earlier by 2-3 weeks for hybrid males than European males (P < 0.05). High frequency blood sampling revealed markedly seasonal patterns of secretion of testosterone and LH, with hybrid males exhibiting an apparent earlier onset of high-amplitude testosterone 'surges' in February (late summer) compared to those occurring in April (autumn) for European males. When viewed collectively, the data indicate strongly that the Mesopotamian influence is evident in the earlier attainment of sexual development and fertility in late summer and autumn, and earlier onset of sexual quiescence in spring. This is in accord with anecdotal information on earlier reproductive patterns in purebred Mesopotamian fallow deer.

Animals↗
PubMed1996

Post-translational regulation of neuronal acetylcholine receptors stably expressed in a mouse fibroblast cell line.

Second messenger regulation of neuronal acetylcholine receptors (AChRs) was investigated in a mouse fibroblast cell line, M10, stably transfected with chicken alpha 4 and beta 2 cDNAs. Both forskolin and 8-bromo-cyclic adenosine 3',5'-monophosphate (cAMP) induced large increases in the numbers of AChRs. The increases were due in part to increased transcription and translation of the alpha 4 and beta 2 genes. Blockade of protein synthesis with cycloheximide, however, revealed that forskolin also exerts a post-translational effect, increasing the number of surface receptors by twofold. Immunoblot analysis of sucrose gradient fractions confirmed that the cells had a large fraction of unassembled subunits potentially available for receptor assembly. The post-translational effect of forskolin was blocked by H-89, an inhibitor of cAMP-dependent protein kinase, and by okadaic acid, an inhibitor of phosphatases 1 and 2A. Nicotine also acted post-translationally to induce a twofold increase in the number of surface receptors, but the mechanism differed from that utilized by forskolin, since the effects of the two agents were additive and were differentially affected by okadaic acid. The results suggest that protein phosphorylation-dephosphorylation mechanisms act post-translationally to increase the number of neuronal AChRs maintained on the cell surface. This could be achieved by increasing the efficiency of receptor assembly, transport, or stabilization on the cell surface.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Tyrosine kinase inhibitors alter composition of nicotinic receptors on neurons.

Protein tyrosine kinase (PTK) inhibitors were used to examine the roles of tyrosine phosphorylation in synaptic function. We show here that two different PTK inhibitors, herbimycin A and lavendustin A, both selectively downregulate a subpopulation of nicotinic acetylcholine receptors (AChRs) on chick ciliary ganglion neurons in culture. The downregulation requires a number of hours to occur and involves only those receptors containing the alpha 3, alpha 5, and beta 4 gene products. Not affected are AchRs that additionally contain the beta 2 gene product or AchRs that are made up of the alpha 7 gene product. The downregulation preferentially targets receptors destined for the cell surface and has little effect on the large pool of intracellular receptors. The receptor loss is not additive with that seen in the presence of either cycloheximide or tunicamycin, two compounds that the block appearance of new receptors. The downregulation induced by herbimycin A in surface receptors is accompanied by a specific decrement in the amount of alpha 3 protein in the cells. The results indicate that PTKs, either by phosphorylating AChR gene products directly or by acting through intermediary proteins, regulate the size and composition of the AChR pool maintained on the cell surface. Receptor regulation by PTKs may provide a mechanism for long-term control of synaptic signaling between neurons.

Animals↗
PubMed1996

Elevation of intracellular calcium levels in neurons by nicotinic acetylcholine receptors.

The recognition that intracellular free calcium serves as a ubiquitous intracellular signal has motivated efforts to elucidate mechanisms by which cells regulate calcium influx. One route of entry that may offer both spatial and temporal fine resolution for altering calcium levels is that provided by cation-permeable, ligand-gated ion channels. Biophysical measurements as well as calcium imaging techniques demonstrate that neuronal nicotinic acetylcholine receptors as a class have a high relative permeability to calcium; some subtypes equal or exceed all other known receptors in this respect. Activation of nicotinic receptors on neurons can produce substantial increases in intracellular calcium levels by direct passage of calcium through the receptor channel. When multiple classes of nicotinic receptors are expressed by the same neuron, each appears capable of increasing calcium in the cell but may differ with respect to location, temporal response, agonist sensitivity, or regulation in achieving it. As a result, nicotinic receptors must be considered strong candidates for signaling molecules through which neurons regulate a diverse array of cellular events.

Acetylcholine↗