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G D Fischbach

Publications and source records attributed to G D Fischbach.

At least 37 records · Page 2Linked to original sources
PubMed1995

ARIA is concentrated in nerve terminals at neuromuscular junctions and at other synapses.

Skeletal muscle ACh receptors (AChRs) accumulate at neuromuscular junctions (nmjs) at least partly because of the selective induction of AChR subunit genes in subsynaptic myotube nuclei by the motor nerve terminal. Additionally, mammalian AChRs undergo a postnatal change in subunit composition from embryonic (alpha 2 beta gamma delta) to adult (alpha 2 beta epsilon delta) forms, a switch that also depends on innervation. ARIA, a protein purified from chicken brains based on its ability to induce AChR synthesis in primary chick muscle cells, is a strong candidate for being the molecule responsible for these early developmental events. ARIA mRNA has been detected in embryonic motor neurons during synapse formation, and the gene continues to be expressed postnatally. In this report, we provide evidence that ARIA-like immunoreactivity is concentrated in rat motor nerve terminals from early postnatal ages, and that it can be detected in motor neurons in E18 embryos. ARIA is also detectable in axons within colchicine-treated sciatic nerves, suggesting that the protein in the nerve terminal has been transported from the cell body. ARIA mRNA is present in, but not restricted to, cholinergic neurons. Likewise, we report here that ARIA-like immunoreactivity is present in some noncholinergic central synapses. We also present evidence that isoforms of ARIA are differentially distributed among functionally distinct classes of neurons.

Animals↗
PubMed1994

A role for the acetylcholine receptor-inducing protein ARIA in oligodendrocyte development.

ARIA acetylcholine receptor-inducing activity protein, is a member of a family of ligands that includes the Neu differentiation factor, heregulin, and glial growth factor. These ligands all act through one or more receptor tyrosine kinases of approximately 185 kDa. In some conditions these ligands promote proliferation, whereas in others they induce differentiation. ARIA was originally isolated from chick brain on the basis of its ability to induce synthesis of nicotinic acetylcholine receptors in skeletal muscle. In this paper we show that ARIA is expressed in the subventricular zone of the rat brain and that it enhances the development of oligodendrocytes from bipotential (O2A) glial progenitor cells. We have also found that ARIA induces tyrosine phosphorylation of a 185-kDa protein in O2A progenitor cells. ARIA does not increase bromodeoxyuridine incorporation by oligodendrocytes but is mitogenic when added to Schwann cells in vitro. Thus, ARIA accelerates the formation of oligodendrocytes in vitro and is expressed where it could exercise the same influence in vivo.

Animals↗
PubMed1993

ARIA, a protein that stimulates acetylcholine receptor synthesis, is a member of the neu ligand family.

Motor neurons stimulate their postsynaptic muscle targets to synthesize neurotransmitter receptors. Polypeptide signaling molecules may mediate this inductive interaction. Here we report the purification of ARIA, a protein that stimulates the synthesis of muscle acetylcholine receptors, and the isolation of ARIA cDNA. Recombinant ARIA increases acetylcholine receptor synthesis greater than 3-fold, and it induces tyrosine phosphorylation of a 185 kd muscle protein. The ARIA cDNA hybridizes with mRNAs that are expressed in the spinal cord from E4, a time prior to the onset of neuromuscular synapse formation, through adulthood. By E7, hybridizing mRNAs are concentrated in motor neurons. Chicken ARIA is homologous to the rat Neu differentiation factor and human here-gulin, ligands for the receptor tyrosine kinase encoded by the neu (c-erbB2, HER2) proto-oncogene. Our data suggest that members of the ARIA protein family promote the formation and maintenance of chemical synapses and, furthermore, that receptor tyrosine kinases play important roles in this process.

Animals↗
PubMed1993

ARIA, a protein that stimulates acetylcholine receptor synthesis, also induces tyrosine phosphorylation of a 185-kDa muscle transmembrane protein.

Motoneurons promote the accumulation of acetylcholine receptors (AChRs) at developing neuromuscular synapses. The AChR-inducing activity protein ARIA, which is purified from chicken brain and increases the synthesis of AChRs in chicken myotubes, may play a crucial role in this process. Here we show that ARIA induces the rapid tyrosine phosphorylation of a M(r) 185,000 protein (p185) in muscle cells. Phosphorylation of p185 correlates with AChR induction at each stage of ARIA purification. Moreover, medium conditioned by spinal cord motoneurons stimulates AChR synthesis and p185 phosphorylation. Studies with membrane-impermeant reagents and 125I-labeled ARIA indicate that p185 is a transmembrane ARIA-receptor tyrosine kinase. Our data suggests that muscle AChR synthesis can be regulated through tyrosine phosphorylation.

Animals↗
PubMed1993

The number of Na+ channels in cultured chick muscle is increased by ARIA, an acetylcholine receptor-inducing activity.

ARIA is a glycoprotein purified from chick brain on the basis of its ACh receptor-inducing activity (ARIA). In this study we present evidence that ARIA increases the number of voltage-gated sodium channels in chick muscle as well as the number of ACh receptors (AChRs). Exposure of chick myotubes to ARIA increased by twofold the number of 3H-saxitoxin binding, an effect that is comparable to the increase of AChRs assayed by 125I-alpha-bungarotoxin (125I-alpha-BTX) binding. We also documented effects of ARIA on myoblasts: the number of 125I-alpha-BTX binding sites in the mononucleated muscle cells was increased by 1.5-fold, and the peak TTX-sensitive inward currents increased by the same amount. No change was detected in the voltage dependence of channel activation, in mean channel current, or in mean channel open time. Thus, the Na+ channel is the first molecule, other than AChR subunits, whose expression has been shown to be induced by ARIA. Since sodium channels are concentrated at motor end plates, our results provide circumstantial evidence that ARIA may regulate several genes expressed at developing neuromuscular junctions. Moreover, the finding that ARIA's effects extend to mononucleated myoblasts suggests that this protein may be important during the earliest stages of muscle formation and innervation.

Animals↗
PubMed1992

Mind and brain.

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Animals↗
PubMed1992

The development of ACh- and GABA-activated currents in embryonic chick ciliary ganglion neurons in the absence of innervation in vivo.

Chemical synaptic transmission in the chick ciliary ganglion is mediated by nicotinic ACh receptors. Ciliary ganglion neurons also express GABAA receptors, although there is no known source of GABAergic innervation of the ganglion, and the function of GABA receptors on these neurons is not known. We examined whether ACh and GABA receptors on embryonic chick ciliary ganglion neurons are regulated by presynaptic inputs. Whole-cell currents evoked by ACh or GABA in neurons soon after dissociation were taken to estimate the level of functional receptors in intact ganglia. We destroyed the accessory oculomotor nucleus (AON), the only source of synaptic input to the ganglion, on embryonic day (E) 4. We determined that 80% of the operations resulted in the virtual elimination of synaptic contacts in the ganglion, using P65 immunohistochemistry (a synaptic vesicle antigen) and direct ultrastructural examination. Previous experiments have shown that during normal development, ACh-activated currents increase over sevenfold between E6 and E18; GABA-activated currents increase only twofold, in proportion to cell size. We found that ACh-activated currents of uninnervated neurons at E14 and E18 were as large as control responses. Furthermore, ACh receptor-like molecules, visualized with monoclonal antibody 35 immunofluorescence, were concentrated in high density clusters on the surface of E18 neurons from AON-ablated embryos. GABA-activated currents were also not affected by AON destruction. We conclude that ACh and GABA receptors are not induced in embryonic chick ciliary ganglion neurons during development by contact with or soluble factors released from AON synaptic terminals.

Acetylcholine↗
PubMed1991

A prion-like protein from chicken brain copurifies with an acetylcholine receptor-inducing activity.

The mammalian prion protein (PrPC) is a cellular protein of unknown function, an altered isoform of which (PrPSc) is a component of the infectious particle (prion) thought to be responsible for spongiform encephalopathies in humans and animals. We report here the isolation of a cDNA that encodes a chicken protein that is homologous to PrPC. This chicken prion-like protein (ch-PrLP) is identical to the mouse PrP at 33% of its amino acid positions, including an uninterrupted stretch of 24 identical residues, and it displays the same structural domains. In addition, ch-PrLP, like its mammalian counterpart, is attached to the cell surface by a glycosyl-phosphatidylinositol anchor. We find that ch-PrLP is the major protein in preparations of an acetylcholine receptor-inducing activity that has been purified greater than 10(6)-fold from brain on the basis of its ability to stimulate synthesis of nicotinic receptors by cultured myotubes. The ch-PrLP gene is expressed in the spinal cord and brain as early as embryonic day 6; and in the spinal cord, the protein appears to be concentrated in motor neurons. Our results therefore raise the possibility that prion proteins serve normally to regulate the chemoreceptor number at the neuromuscular junction and perhaps in the central nervous system as well.

Amino Acid Sequence↗
PubMed1991

Acetylcholine receptor-inducing activity stimulates expression of the epsilon-subunit gene of the muscle acetylcholine receptor.

Motor neurons regulate the transcription of acetylcholine receptor subunit genes in postsynaptic muscle fibers both through muscle electrical activity produced by motor neuron acetylcholine release and by mechanisms independent of such transmitter release. Factors secreted by the motor neuron may mediate activity-independent regulation, including the postnatal switch from alpha 2 beta gamma delta (embryonic type) to alpha 2 beta epsilon delta (adult type) receptors. We have investigated the effect of putative trophic factors, agents affecting second-messenger systems, and muscle activity on the levels of acetylcholine receptor subunit mRNAs in primary mouse muscle cultures. We found that ARIA (acetylcholine receptor-inducing activity), a 42-kDa glycoprotein purified on the basis of its ability to increase the synthesis of acetylcholine receptors in chick myotubes, increases epsilon-subunit mRNA levels up to 10-fold. In addition, ARIA stimulated alpha-, gamma-, and delta-subunit mRNA levels 2-fold but had no effect on the expression of the beta-subunit gene. These effects of ARIA were independent of muscle activity, and they were not mimicked by calcitonin gene-related peptide nor by thyroxine, forskolin, phorbol 12-myristate 13-acetate, the calcium ionophore A23187, basic fibroblast growth factor, or transforming growth factor beta. Based on these data, we suggest that ARIA may act at the mammalian neuromuscular junction to induce adult-type acetylcholine receptors.

Acetylcholine↗
PubMed1990

A role for cAMP in the development of functional neuromuscular transmission.

We have found that the incidence of functionally connected neuron-myotube pairs in chick ciliary-myotube cultures increases from 58% to more than 90% when the cells are treated for several hours with 8-bromo-cyclic adenosine monophosphate (8-br-cAMP) or with agents known to increase intracellular cAMP. The increase in connectivity was not accompanied by a change in neuron survival, or in the length of neurite-myotube contact. Moreover, there was no change in the shape of the presynaptic action potential, in mean end plate potential (epp) amplitude or in the sensitivity of postsynaptic acetylcholine receptors (AChRs). One interpretation of these results in that a cAMP-dependent phosphorylation acts as a trigger to activate a previously "silent" synapse.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

The development of ACH- and GABA-activated currents in normal and target-deprived embryonic chick ciliary ganglia.

We have examined the expression of functional ACh and GABA receptors on embryonic chick ciliary ganglion neurons between Stages (St) 29 and 44 (Embryonic Day 6 to Embryonic Day 18). Whole-cell currents activated by ACh or GABA were measured in neurons 3-6 hr after dissociation to estimate the level of functional receptors in vivo. The mean peak IACh increased sevenfold between St 29 (321 pA) and St 44 (2345 pA) in two steps, separated by a plateau between St 35 and St 38 (E9 to E12). Cell size, estimated from measurements of membrane capacitance, increased only threefold over the same interval. Moreover, IACh and cell size were not well correlated at any stage examined. IGABA increased twofold between St 29 and St 38; the change was gradual and without any indication of two phases. The increase in IACh during development was not dependent on innervation of target cells within the eye. We removed the primordial eye between St 11 and St 13 (E2) and allowed the embryos to mature to various stages. Despite a small (20-50%) reduction in IACh at every stage examined, IACh still increased dramatically (about 10-fold) between St 29 and St 44 in target-deprived neurons. IACh was not uniquely affected by early target removal; IGABA and capacitance were also slightly reduced in target-deprived neurons.

Acetylcholine↗
PubMed1989

Differential activation of myotube nuclei following exposure to an acetylcholine receptor-inducing factor.

A glycoprotein purified from chick brain, of relative molecular mass 42,000, increases the rate of appearance of acetylcholine receptors (AChRs) on the surface of chick myotubes. RNase protection assays have shown that this AChR-inducing activity (ARIA) increases the amount of mRNA encoding the alpha-subunit of the AChR, with little or no effect on the amounts of gamma- and delta-mRNAs2. Here, we report that the mRNAs encoding the alpha- and gamma-subunits of the receptor detected by in situ hybridization are concentrated around nuclei in cultured myotubes. Consistent with previous results, ARIA selectively increased the amount of alpha-subunit mRNA, but we now find that all nuclei were not activated to the same extent, with a substantial number not responding at all. Assuming that ARIA is released by motor nerve terminals, our results indicate that only a subset of muscle nuclei are capable of contributing to the accumulation of AChRs at developing neuromuscular junctions.

Actins↗
PubMed1989

Calcium-dependent, slow desensitization distinguishes different types of glutamate receptors.

1. L-Glutamate, the most likely transmitter of rapid excitatory synaptic interactions in the brain and spinal cord, is a potent neurotoxin. Mechanisms that terminate the action of glutamate are, therefore, likely to be important for maintaining the integrity of glutaminoceptive neurons. In this study, we show that glutamate currents evoked in voltage-clamped chick motoneurons fade during prolonged or repeated application of glutamate by pressure ejection from nearby pipettes. 2. The magnitude of the decline depends on the Ca2+/Mg2+ ratio in the extracellular medium. With Ca2+ = 10.0 mM and no added Mg, the steady-state glutamate current amounted to 50% of the initial value. 3. Single-channel measurements indicate that the fade is due to receptor desensitization rather than to agonist-induced channel blockade, as the mean channel open time within bursts is independent of the agonist concentration. 4. Application of more selective agonists showed that Ca2+-dependent slow desensitization involved only G1 (NMDA) receptors. G2 responses (activated by kainate and quisqualate) did not exhibit this slow phase of desensitization under the same conditions.

Amino Acids↗
PubMed1989

Glutamate receptor desensitization and its role in synaptic transmission.

Responses of excitatory amino acid receptors to rapidly applied glutamate were measured in outside-out membrane patches from chick spinal neurons. The peak current varied with glutamate concentration, with a half-maximal response at 510 microM and a Hill coefficient near 2. Currents activated by 1 mM glutamate desensitized and recovered in two phases. The faster time constant was identical to the time constant of decay of synaptic currents, suggesting that glutamatergic synaptic currents are terminated, in part, by receptor desensitization. Steady-state desensitization was evident following application of only 2-3 microM glutamate, concentrations comparable to levels in the extracellular space in the intact brain. Thus, glutamate receptor desensitization can affect synaptic efficacy in two ways: at high concentrations, rapid desensitization of receptors may curtail synaptic currents; at low concentrations, there is a significant reduction in the number of activatable receptors.

Animals↗
PubMed1989

Formation of acetylcholine receptor clusters in chick myotubes: migration or new insertion?

Experiments were performed to study the feasibility of two mechanisms of acetylcholine receptor (ACHR) accumulation in chick myotubes: diffusion and trapping of previously dispersed surface receptors and localized insertion of new receptors at accumulation sites. Fluorescence photobleaching recovery (FPR) measurements indicated that the majority of diffusely distributed ACHRs in chick myotube membranes were mobile whereas nearly all receptors within high density clusters were effectively immobile. Unlike previous reports, two rates of ACHR movement characterized the mobile population. Moreover, we found that the estimated diffusion coefficient depended critically on the objective (spot size) used to assay recovery from bleaching. Implications of this finding for mechanisms of receptor immobilization are discussed. Extracts of chick brain, known to increase the number of surface receptors, did not alter receptor mobility. Extracts of Torpedo electric organ that increase the number of receptor aggregates, decreased the mobile fraction of ACHRs. Simulations of the diffusion and trapping mechanism indicated that captured receptors should congregate around the periphery of a receptor patch during the first hour after they were inserted into the membrane. However, newly inserted ACHRs were found to be located centrally within receptor patches under neurites, and this was not consistent with an exclusive diffusion-trapping mechanism. We also studied the mobility of ACHRs near points of contact made by cholinergic growth cones. The rate of receptor movement was increased in the vicinity of growth cones, but the magnitude of this effect was small.

Animals↗