Biomedical subjects
G D Fischbach
Publications and source records attributed to G D Fischbach.
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.
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.
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.
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.
Variation in the ratio of acetylcholine receptors and the Mr 43,000 receptor-associated protein in embryonic chick myotubes and myoblasts.
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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.
Mr 42,000 ARIA: a protein that may regulate the accumulation of acetylcholine receptors at developing chick neuromuscular junctions.
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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.
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.
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.
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.
A novel 87,000-Mr protein associated with acetylcholine receptors in Torpedo electric organ and vertebrate skeletal muscle.
To identify proteins associated with nicotinic postsynaptic membranes, mAbs have been prepared to proteins extracted by alkaline pH or lithium diiodosalicylate from acetylcholine receptor-rich (AChR) membranes of Torpedo electric organ. Antibodies were obtained that recognized two novel proteins of 87,000 Mr and a 210,000:220,000 doublet as well as previously described proteins of 43,000 Mr, 58,000 (51,000 in our gel system), 270,000, and 37,000 (calelectrin). The 87-kD protein copurified with acetylcholine receptors and with 43- and 51-kD proteins during equilibrium centrifugation on continuous sucrose gradients, whereas a large fraction of the 210/220-kD protein was separated from AChRs. The 87-kD protein remained associated with receptors and 43-kD protein during velocity sedimentation through shallow sucrose gradients, a procedure that separated a significant amount of 51-kD protein from AChRs. The 87- and 270-kD proteins were cleaved by Ca++-activated proteases present in crude preparations and also in highly purified postsynaptic membranes. With the exception of anti-37-kD antibodies, some of the monoclonals raised against Torpedo proteins also recognized determinants in frozen sections of chick and/or rat skeletal muscle fibers and in permeabilized chick myotubes grown in vitro. Anti-87-kD sites were concentrated at chick and rat endplates, but the antibodies also recognized determinants present at lower site density in the extrasynaptic membrane. Anti-210:220-kD labeled chick endplates, but studies of neuron-myotube cocultures showed that this antigen was located on neurites rather than the postsynaptic membrane. As reported in other species, 43-kD determinants were restricted to chick endplates and anti-51-kD and anti-270-kD labeled extrasynaptic as well as synaptic membranes. None of the cross reacting antibodies recognized determinants on intact (unpermeabilized) myotubes, so the antigens must be located on the cytoplasmic aspect of the surface membrane. The role that each intracellular determinant plays in AChR immobilization at developing and mature endplates remains to be investigated.
Variation among acetylcholine receptor clusters induced by ciliary ganglion neurons in vitro.
We have examined the variation in receptor density and area among neurite-associated acetylcholine receptor patches (NARPs) induced by chick ciliary ganglion neurons on nearby myotubes in vitro. Quantitative analysis of rhodamine-alpha-bungarotoxin (RBTX) NARPs revealed that about 15% of the NARPs were "outstanding" in terms of size (greater than 60 micron 2) and fluorescence intensity (greater than 100 units on a 0-255 scale). The total number of receptors at different NARPs ranged over 3 orders of magnitude. It is likely that variation in NARP size and intensity reflects regional variation in the ability of myotubes to respond to the neuronal influence because (1) no gradient in NARP size or intensity with distance from the soma was evident; (2) the intensities and areas of uninnervated receptor clusters (hot spots) were similar to those of NARPs; (3) acetylcholinesterase was present at the same proportion of hot spots and NARPs at all times examined. We found no physiological or morphological evidence that outstanding NARPs were more effective sites of transmitter release. Outstanding NARPs were restricted to the longest neurite of individual neurons, so they may signal trophic interactions of the sort that promote neurite outgrowth and survival.
Rapid desensitization of glutamate receptors in vertebrate central neurons.
We have examined glutamate receptor desensitization in voltage-clamped embryonic chicken spinal cord neurons and postnatal rat hippocampal neurons maintained in culture. Rapid currents that rose in 0.8-3.6 msec were evoked when glutamate was ionophoresed with 0.5- to 1.0-msec pulses. With prolonged pulses or brief, repetitive pulses, glutamate-evoked currents decayed rapidly in a manner that was independent of holding potential. A similar desensitization occurred following close-range pressure ejection of glutamate. The rapid, desensitizing glutamate current exhibited a linear current-voltage relation and it was not blocked by 2-amino-5-phosphonovalerate, suggesting that it was mediated by N-methyl-D-aspartate-insensitive (G2) receptors. Desensitization of G2 receptors may be agonist-dependent: currents evoked by kainate, a selective G2 agonist, did not decay, whereas prior application of glutamate did reduce the size of kainate responses. The appearance of the rapid current depended critically on the position of the ionophoretic pipette. Such glutamate-receptor "hot spots" often corresponded to points of contact with neighboring neurites, which raises the possibility that they are located at synapses.
Acetylcholine receptor-inducing factor from chicken brain increases the level of mRNA encoding the receptor alpha subunit.
A 42-kDa glycoprotein isolated from chicken brain, referred to as acetylcholine receptor-inducing activity (ARIA), that stimulates the rate of incorporation of acetylcholine receptors into the surface of chicken myotubes may play a role in the nerve-induced accumulation of receptors at developing neuromuscular synapses. Using nuclease-protection assays, we have found that ARIA causes a 2- to 16-fold increase in the level of mRNA encoding the alpha subunit of the receptor, with little or no change in the levels of gamma- and delta-subunit messengers. ARIA also increases the amount of a putative nuclear precursor of alpha-subunit mRNA, consistent with an activation of gene transcription. These results suggest that the concentration of alpha subunit may limit the rate of biosynthesis of the acetylcholine receptors in chicken myotubes. They also indicate that neuronal factors can regulate the expression of receptor subunit genes in a selective manner. Tetrodotoxin, 8-bromo-cAMP, and forskolin also increase the amount of alpha-subunit mRNA, with little change in the amount of gamma- and delta-subunit mRNAs. Unlike, ARIA, however, these agents have little effect on the concentration of the alpha-subunit nuclear precursor.
Rapid desensitization of glutamate receptors in vertebrate central neurons.
We have examined glutamate receptor desensitization in voltage-clamped embryonic chicken spinal cord neurons and postnatal rat hippocampal neurons maintained in culture. Rapid currents that rose in 0.8-3.6 msec were evoked when glutamate was ionophoresed with 0.5- to 1.0-msec pulses. With prolonged pulses or brief, repetitive pulses, glutamate-evoked currents decayed rapidly in a manner that was independent of holding potential. A similar desensitization occurred following close-range pressure ejection of glutamate. The rapid, desensitizing glutamate current exhibited a linear current-voltage relation and it was not blocked by 2-amino-5-phosphonovalerate, suggesting that it was mediated by N-methyl-D-aspartate-insensitive (G2) receptors. Desensitization of G2 receptors may be agonist-dependent: currents evoked by kainate, a selective G2 agonist, did not decay, whereas prior application of glutamate did reduce the size of kainate responses. The appearance of the rapid current depended critically on the position of the ionophoretic pipette. Such glutamate-receptor "hot spots" often corresponded to points of contact with neighboring neurites, which raises the possibility that they are located at synapses.
Changes in the number of chick ciliary ganglion neuron processes with time in cell culture.
The purpose of this study was to describe the shape of chick ciliary ganglion neurons dissociated from embryonic day 8 or 9 ganglia and maintained in vitro. Most of the neurons were multipolar during the first three days after plating, with an average of 6.0 processes extending directly from the cell body. The neurons became unipolar with time. The remaining primary process accounted for greater than 90% of the total neuritic arbor. This striking change in morphology was not due to the selective loss of multipolar cells, or to an obvious decline in the health of apparently intact cells. The retraction of processes was neither prevented nor promoted by the presence of embryonic muscle cells. Process pruning occurred to the same extent and over the same time course whether the cells were plated on a monolayer of embryonic myotubes or on a layer of lysed fibroblasts. Process retraction is not an inevitable consequence of our culture conditions. Motoneurons dissociated from embryonic spinal cords remained multipolar over the same period of time. We conclude that ciliary ganglion neurons breed true in dissociated cell culture in that the multipolar-unipolar transition reflects their normal, in vivo, developmental program.