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N C Spitzer

Publications and source records attributed to N C Spitzer.

At least 73 records · Page 4Linked to original sources
PubMed1984

The absence of calcium blocks impulse-evoked release of acetylcholine but not de novo formation of functional neuromuscular synaptic contacts in culture.

We have examined the role of calcium-dependent, impulse-evoked release of acetylcholine (ACh) in the formation of functional neuromuscular contacts. Dissociated cell cultures prepared from Xenopus laevis embryos were maintained either in standard medium containing calcium or in calcium-free medium. Both the number of neuron-myocyte contacts and the number of terminations of neurites on myocytes were reduced in Ca-free medium. Intracellular recordings from neuron-myocyte pairs during perfusion with a standard saline revealed that functional synaptic contacts were formed in Ca-free medium, although with a reduced frequency (approximately 30% of controls). Postsynaptic potentials elicited by neuronal action potentials were smaller than those observed after growth in standard medium. The frequency of small, spontaneously occurring potentials was reduced by a factor of 10. Ca-free saline prevented the impulse-evoked release of transmitter from neurons in Ca-free and control cultures. Labeling with alpha-bungarotoxin and iontophoretic application of ACh revealed no ACh receptor clusters in the membrane of myocytes grown in Ca-free medium. Our results suggest that evoked, vesicular release is not required for the initial formation of neuromuscular contacts, although it may be involved in further maturation of synapses. Roles for spontaneous quantal or non-quantal release have not been excluded.

Acetylcholine↗
PubMed1983

Enkephalin reduces quantal content at the frog neuromuscular junction.

Opiate peptides, particularly Met-enkephalin, have been shown to block or reduce Ca2+-dependent events in a variety of neurones. Our own observations on the effect of enkephalin on developing Rohon-Beard neurones in Xenopus spinal cord suggested that enkephalin might interact directly with Ca2+ channels in vertebrate neurones. This possibility prompted us to look for an effect of enkephalin on another population of neuronal Ca2+ channels--those in the presynaptic terminals at the frog neuromuscular junction. We were encouraged to try this preparation by an early report that morphine, in rather high concentrations, reduced the amount of acetylcholine (ACh) released from frog muscle by nerve stimulation. Our present results indicate that Met-enkephalin reversibly and specifically reduces the quantal content of transmitter release from nerve terminals in the frog cutaneous pectoris muscle, probably by means of an effect on inward Ca2+ current.

Acetylcholine↗
PubMed1983

Enkephalin reduces calcium action potentials in Rohon-Beard neurons in vivo.

Rohon-Beard neurons of the Xenopus spinal cord develop somatic Ca++-dependent action potentials at early embryonic stages. Because many Ca++-dependent events are blocked or reduced by enkephalin in other neuronal systems, we tested the effect of met-enkephalin on the Ca++ spikes of Rohon-Beard cells. Our results indicate that met-enkephalin, which has no effect on the resting membrane potential or conductance in these cells, reversibly and specifically shortens their Ca++ action potentials, probably by decreasing the Ca++ conductance. Enkephalin has a similar effect on Xenopus dorsal root ganglion cells in vivo. Examination of Rohon-Beard cells at various stages of development shows that enkephalin shortens Ca++ spikes from the earliest times that the spikes appear (approximately 21 hr after fertilization) to the latest times they are detectable (7 to 9 days). This very early response suggests either that enkephalin binds directly to the Ca++ channels or that an "enkephalin receptor" is among the earliest neuronal phenotypes to appear in these cells.

Action Potentials↗
PubMed1982

Voltage- and stage-dependent uncoupling of Rohon-Beard neurones during embryonic development of Xenopus tadpoles.

1. Electrical coupling of Rohon-Beard neurones in the spinal cord of Xenopus laevis has been studied from the time of closure of the neural tube (stage 20, 22h after fertilization of the egg) to the free swimming tadpole (stage 49, 12d old). Pairs of cells were examined by impaling both simultaneously with single micro-electrodes having current-passing and voltage-recording capabilities.2. At the earliest time studied (stage 20), before Ca(2+) action potentials are detected, cells are electrically coupled. Coupling coefficients as high as 0.6 can be recorded. Tests with small hyperpolarizing and depolarizing current pulses demonstrate that the coupling does not show rectification.3. The coupling is voltage-dependent: depolarization or hyperpolarization of one cell with respect to another, above a threshold, causes relative uncoupling of the cells. The coupling coefficient falls to approximately 10% of its initial value when the difference in potential between the two cell bodies is approximately 75 mV. Cells usually become recoupled at the termination of the current pulse. Other, unidentified cells in the spinal cords of the same embryos show coupling that is not voltage-dependent.4. Voltage-dependent uncoupling and recoupling persist when cells are depolarized by high K(+), and in the presence of 30 mM-Co(2+), suggesting that chemical synapses are not involved. They are also unaffected by addition of Rb(+), Cs(+) or TEA(+) to the extracellular solution, elevated levels of Ca(2+), or replacement of Na(+) with Tris or Cl(-) with isethionate, suggesting that conductance changes in the surface membrane, such as anomalous rectification, are not responsible.5. Lowering the intracellular pH with CO(2)-HCO(3) (-) buffered saline does not abolish electrical coupling but appears to eliminate its voltage dependence.6. Slightly later (e.g. stage 21), cells that do not yet produce Ca(2+) action potentials while coupled will do so when their input resistance is increased by uncoupling them from their neighbours.7. Later still (e.g. stage 23), cells make Ca(2+) action potentials while coupled, and an action potential in one cell can trigger an action potential in other cells to which it is coupled. Ca(2+) action potentials that do not bring other coupled cells to threshold for impulse initiation can transiently reduce the strength of coupling. Repetitive firing of these Ca(2+) action potentials at a low frequency does not cause permanent uncoupling of the cells.8. Rohon-Beard neurones become electrically uncoupled about stage 25 (early tail-bud, 28h old). Coupling disappears around the time of appearance of the Na(+) component of the action potential, although coupling that is voltage-dependent or independent can still be seen between other, unidentified cells. No electrical coupling of Rohon-Beard cells was detected at later stages of development.

Action Potentials↗
PubMed1982

The appearance and development of chemosensitivity in Rohon-Beard neurones of the Xenopus spinal cord.

1. We have examined the onset and subsequent development of chemosensitivity in Rohon-Beard neurones from the Xenopus spinal cord. These cells become sensitive to bath-applied gamma-aminobutyric acid (GABA) around stage 25 (early tailbud, about 1 d old), and remain so at least until stage 49 (9 d old). In contrast, a number of other neurotransmitter candidates tested caused no potential or conductance change during the same period.2. We examined ionophoretic dose-response relations of the cells at stage 26, a couple of hours after the first acquisition of GABA sensitivity. Sensitivities as high as 450 mV/nC were recorded. Comparable sensitivities were recorded between stages 46-49 (5-9 d old).3. Measurements of ionophoretic sensitivities and input resistances during several periods from stage 26 to maturity show that the underlying conductance change for a given GABA dose is likely to increase steadily during this time. A ;sensitivity index' (ionophoretic sensitivity/input resistance) was calculated, which is low at stage 26, higher at intermediate stages (stages 31-42), and highest for mature cells (stages 46-49; 5-9 d of development).4. The reversal potential of the ionophoretic GABA response is the same at stage 26 (-30 mV) as it is in mature cells. Ion substitution experiments show that Na(+) and K(+), but not Cl(-) or Ca(2+), are involved in the response.5. GABA responses at stage 26 are pharmacologically similar to those of mature cells. The responses are blocked by 10 muM-picrotoxin or curare, and muscimol is an agonist in concentrations as low as 1 muM.6. GABA responses at stage 26 desensitize in a manner similar to that seen for mature cells, either with prolonged bath application of GABA or with repetitive ionophoretic application.7. Nearly half of the cells tested at stage 26 respond to glycine, in concentrations as low as 5 muM. This sensitivity is absent by 3(1/2) d of development.8. The responses of Rohon-Beard neurones to GABA are similar to those of other cells in that they involve a conductance increase, are mimicked by muscimol, and are blocked by picrotoxin. These responses are different in that they do not involve Cl(-) and are blocked by low concentrations of curare.9. Many of the characteristics of GABA receptors, i.e. the reversal potential, desensitization, and pharmacology, are constant during development. However, the sensitivity of the cells to GABA and the spectrum of transmitters to which they are sensitive appear to change.

Action Potentials↗
PubMed1981

The mature electrical properties of identified neurones in grasshopper embryos.

1. We have examined the mature electrical properties of five identified neurones in embryos of the grasshopper Schistocerca nitens. The five cells arise from two different precursor cells: the median neuroblast, whose first three progency are called DUM 3,4,5; DUM 4,5; and DUM 5; and mid-line precursor 3, which divides once to produce the H cell and the H cell sibling.2. Electrical coupling was investigated by dual intracellular penetrations. Action potentials were elicited by intracellular stimulation of cell bodies and by extracellular stimulation of axons. The ionic basis of action potentials was investigated by changing the ionic environment and by applying various blocking agents.3. Most of the mature electrical properties of all five cells appear by day 13 of embryonic development. They change little through hatching on day 20.4. The recorded resting potential for all five cells varies from -55 to -60 mV and the recorded input resistance varies from 200 to 450 MOmega. All five cells show delayed rectification, much of which is blocked by tetraethylammonium (TEA). Their resistance increases as they are hyperpolarized.5. The five cells do not appear to be electrically coupled between days 13 and 20.6. All five neurones generate mature action potentials and in several cases show cell-specific electrical properties by day 13. The ionic dependence and depolarizing phase of the action potential change little between days 13 and 20; some changes occur in the after-hyperpolarization.7. One cell-specific difference is observed in normal saline. Four of the cells have axons, median neurites, and somata which generate action potentials in normal saline; but one of the cells (the H cell sibling) has an inexcitable soma and generates action potentials only in its axon.8. Another cell-specific difference in the soma action potentials of DUM 3,4,5,; DUM 4,5; DUM 5; and the H cell is observed when outward current is blocked by TEA. In three of the cells, TEA causes the short-duration action potential (2-4 msec) to be converted into a long-duration action potential (100-1000 msec) in which there is an initial spike (Na(+)-dependent) followed by a long plateau (Ca(2+)-dependent). In the other cell, DUM 5, at resting potential the addition of TEA only causes a shoulder (Ca(2+)-dependent) on the falling phase of the action potential. DUM 5 and DUM 4,5 thus have different electrical properties, even though they differ only by a single cell division from the precursor cell.9. In all four neurones which normally generate soma action potentials (DUM 3,4,5; DUM 4,5; DUM 5; and the H cell), the inward current is carried by both Na(+) and Ca(2+). On day 13, either inward current alone can generate the overshooting action potential; in contrast, by day 18, neither inward current alone can generate an overshooting response. The inward current of the axon action potential in all five cells is carried predominantly by Na(+).10. Thus, the progeny of two different embryonic precursor cells (median neuroblast and mid-line precursor 3) show a broad spectrum of electrical properties. The mature phenotype of electrical excitability is not a property shared in common by all the progeny of a single embryonic precursor cell in the grasshopper. Conversely, progeny from different precursor cells can share the same mature phenotype of electrical excitability.

Action Potentials↗
PubMed1981

The development of electrical properties of identified neurones in grasshopper embryos.

1. We have examined the development of the electrical properties of five identified neurones in grasshopper embryos between days 10 and 13 of embryogenesis (hatching occurs on day 20). DUM 3,4,5; DUM 4,5; DUM 5; the H cell; and the H cell sibling are the progeny of two different precursor cells. Electrical coupling and electrical excitability were assayed by intracellular recordings. 2. Midway through embryogenesis, on day 10, the five cells are highly electrically coupled to each other and are electrically inexcitable. The temporal sequence of the development of electrical excitability and electrical coupling is described for DUM 3,4,5; 4,5; and 5. The H cell and H cell sib undergo the same sequence one day later. 3. The first non-linear membrane property to appear is delayed rectification which appears on day 11 and can be blocked by tetraethylammonium (TEA). In some cells at about day 11, the addition of TEA to normal saline unmasks a Na+-dependent action potential in the axon. 4. The first action potential in normal saline is a Na+-dependent response that appears in the axon at day 11-11.5. 5. The next stage of excitability in normal saline is the appearance about day 11.5 of a Na+-dependent action potential in the median neurite between the soma and the two axons. In some cells at about day 11.5, the addition of TEA unmasks an excitable response in the soma. 6. Overshooting action potentials appear in the soma about day 12; the inward current is carried by both Na+ and Ca2+; TEA causes a prolonged shoulder on the falling phase of the action potential. A short time later, TEA causes a long-duration CA2+ plateau. 7. A progressive decrease in the degree of electrical coupling among the cells occurs between days 10 and 12.5. Complete uncoupling is never observed before day 11, but has always occurred by day 12.5. 8. Two methods were used to demonstrate that electrical coupling does not mask the presence of excitable inward current channels and thus make the cells appear inexcitable. First, we exposed the cells to veratridine. The cells which normally generate excitable Na+ response are depolarized by it; the younger inexcitable cells are not. Secondly, we electrically isolated the cells by killing the somata of their neighbours. The input resistance increased, yet the extent of excitability remained unchanged. 9. There is variability in the precise temporal relationship of excitability and uncoupling. Pairs of cells from different embryos of the same ate can generate the same type of action potentials and yet be coupled in one embryo and uncoupled in another. Electrical excitability and uncoupling appear to be causally unrelated and independent events, occurring at about the same developmental stage.

Action Potentials↗
PubMed1981

Embryonic development of identified neurons: segment-specific differences in the H cell homologues.

Although all of the segmental ganglia of the grasshopper come from identical sets of embryonic precursor cells, there are striking differences in the number and properties of the neurons in the mature ganglia. In every segment, midline precursor 3 (MP3) divides once and gives rise to two progeny. We describe the fate of the two MP3 progeny in different segments. The morphology, physiology, and survival of the MP3 progeny depend upon their segmental position in the embryo. In the meso- and metathoracic segments (T2 and T3), one of the two progeny of MP3 transforms into the H cell. In many of the abdominal segments, beginning with the fourth (A4), both MP3 progeny die. A gradient of transformation and cell death is seen in the intervening first three abdominal segments (A1 to A3). From A1 to A3, the MP3 progeny acquire fewer of the morphological phenotypes of the H cell. This is paralleled by the partial acquisition of the physiological phenotypes of the H cell. Thus, cells that are the progeny of the same precursor cell in different segments can either live or die and, if they survive, can develop different morphological and physiological properties.

Animals↗
PubMed1981

Embryonic development of identified neurons: origin and transformation of the H cell.

We describe the origin and the transformation of a single neuron, the H cell, which assumes two different roles during grasshopper embryogenesis. The H cell originates from the single cell division of midline precursor 3 (MP3). In the metathoracic (T3) segment, the H cell first appears as one of a pair of central pioneer fibers and later transforms into an unpaired identified neuron. During the course of its transformation, the H cell loses its original morphology and acquires new morphological and physiological properties. The H cell acquires many of the same phenotypes as the first progeny of the median neuroblast (MNB); the processes of the first MNB progeny contact the soma of the H cell and these cells are electrically coupled prior to the H cell transformation.

Animals↗
PubMed1980

Reassembly of protein-lipid complexes into large bilayer vesicles: perspectives for membrane reconstitution.

Protein-lipid complexes in apolar solvents reassemble into large bilayer protein-lipid vesicles (PLVs) with diameters of several micrometers. PLVs form spontaneously upon hydration of the protein-lipid complex residue after solvent removal. This procedure has been applied to the following membrane proteins: bovine and squid rhodopsin, reaction centers from Rhodopseudomonas sphaeroides, beef heart cytochrome c oxidase, and acetylcholine receptors from Torpedo californica. PLVs have a large internal aqueous space (e.g., 790 mul/mg of lipid for cattle rhodopsin vesicles). Freeze-fracture replicas of PLVs revealed that both internal and external leaflets contained numerous intramembranous particles with diameters between 80 and 120 A, depending on the specific protein incorporated in the membrane. The optical spectral properties of rhodopsin and reaction centers in PLVs were similar to those recorded in the respective natural membrane. Furthermore, bovine rhodopsin in PLVs was chemically regenerable with 9-cis-retinal. Actinic illumination induced proton efflux from reaction center vesicles that was abolished by proton ionophores. Therefore, this method is suitable for the incorporation of some membrane proteins in their functional state. PLVs were penetrated with microelectrodes and visualized by the injection of a fluorescent dye. Preliminary electrical recordings were obtained by sealing PLVs to a hole in a septum separating two aqueous compartments. These studies suggest that PLVs assembled by this procedure permit the simultaneous analysis of reconstituted membranes by chemical, optical, and electrical techniques.

Animals↗
PubMed1980

Electrical excitability: a spectrum of properties in the progeny of a single embryonic neuroblast.

We have examined the range of some properties of the progeny of a single embryonic precursor cell in the grasshopper. The approximately 100 progeny of this single neuroblast share certain features such as their transmitter and some aspects of their morphology; at the same time, however, they demonstrate a broad spectrum of electrical properties, from spiking to non-spiking neurons. The first-born progeny are spiking neurons with peripheral axons. Many of the progeny, including all of the last-born, do not generate action potentials. The nonspiking progeny are local intraganglionic neurons and appear to compose a major proportion of the progeny of this neuroblast. All of the nonspiking neurons have calcium inward current channels and can make action potentials when outward current channels are blocked. We propose a model for grasshopper neurogenesis based on cell lineage such that (i) certain features (e.g., transmitter) are shared by the progeny of all cell divisions from a single neuroblast, and (ii) other features (e.g., electrical properties) are shared by the progeny of a given birth position (e.g., first versus last born) from all of the neuroblasts. According to this model, the first-born progeny from all neuroblasts are spiking neurons, whereas the last-born are nonspiking.

Action Potentials↗
PubMed1979

Embryonic development of identified neurones: differentiation from neuroblast to neurone.

Individually identified neurones are sufficiently large and accessible in grasshopper embryos to permit visualisation and impalement with intracellular microelectrodes from the time of their birth to their maturation. In this article part of the temporal pattern of differentiation from an identified neuroblast (precursor cell) to a group of identified neurones is described.

Acetylcholine↗
PubMed1979

Embryonic development of identified neurons: temporal pattern of morphological and biochemical differentiation.

Individually identified neurons can be recognized in grasshopper embryos, and are accessible to examination by morphological, physiological, and biochemical techniques from their birth to their maturation. Only after the axon of an identified neuron reaches its postsynaptic target does the neurotransmitter accumulate, the soma rapidly enlarge, and the central arborizations greatly expand.

Animals↗
PubMed1979

Ultrastructural development of Rohon-Beard neurons: loss of intramitochondrial granules parallels loss of calcium action potentials.

We have examined the ultrastructure of the cell body of a vertebrate spinal neuron, the Rohon-Beard cell of Xenopus laevis, at four stages during its development (Nieuwkoop and Faber stages: 22, 29/30, 37/38 and 42). At this time it has attained its electrical excitability and the action potential mechanism in the cell body is maturing through a sequence of stages in which the inward current is carried by Ca++ (stages 20-25), later by Ca++ and Na+ (stages 25-40), and finally by Na+ (stages 40-51) (Spitzer and Baccaglini, '76; Baccaglini and Spitzer, '77). There is a change in the abundance and distribution of the organelles in the perikaryon during this period, characteristic of other developing neurons. Mitochondria and Golgi apparatus become localized progressively more in the interior of the cells, and rough endoplasmic reticulum progressively more to the periphery where it often appears in orderly tiers parallel to the plasma membrane. The mitochondria contain dense intramitochondrial granules which are known in other cells to contain concentrations of divalent cations. The number of granules declines over the course of the developmental period studied. The presence of the intramitochondrial granules was examined quantitatively because electrophysiological data indicate that the amount of Ca++ entering the cells in early stages should raise the internal Ca++ concentration by several orders of magnitude, and that Ca++ is rapidly sequestered (Baccaglini and Spitzer, '77). A minimum of 200 mitochondrial profiles from at least four Rohon-Beard cells were scored for the presence of dense intramitochondrial granules at each stage studied. In stage 22 Rohon-Beard cells 75 +/- 5% (mean +/- SD, n = 4) of the mitochondrial profiles scored contained granules; in stage 29/30, 56 +/- 10% (n = 7); in stage 37/38, 3 +/- 3% (n = 5); and in stage 42, 0.5 +/- 0.25% (n = 4). Therefore, dense intramitochondrial granules, an indication of calcium accumulation in mitochondria, decrease in parallel with the loss of the Ca++ component of the inward current of the action potential in Rohon-Beard neurons.

Action Potentials↗
PubMed1977

Developmental changes in the inward current of the action potential of Rohon-Beard neurones.

1. Rohon-Beard cells in the spinal cord of Xenopus tadpoles have been studied in animals from early neural tube to free-swimming larval stages. The onset and further development of electrical excitability of these neurones has been investigated in different ionic environments, to determine the ionic species carrying the inward current of the action potential.2. The cells appear inexcitable at early stages (Nieuwkoop & Faber stages 18-20) and do not give action potentials to depolarizing current pulses.3. The action potential is first recorded at stage 20. (A) The inward current is carried by Ca(2+) at stages 20-25, since it is blocked by mm quantitites of La(3+), Co(2+) or Mn(2+) and is unaffected by removal of Na(+) or the addition of tetrodotoxin (TTX). (B) The action potential is an elevated plateau of long duration (mean 190 msec at stages 20-22). The duration decreases exponentially with repetitive stimulation. (C) The specific Ca(2+) conductance (g(Ca)) at the onset of the plateau of the action potential is 2.6 x 10(-4) mho/cm(2). Calculations show that a single action potential raises [Ca(2+)](1) by more than 100-fold.4. At later times (stages 25-40), the inward current of the action potential is carried by both Na(+) and Ca(2+): the action potential has two components, an initial spike which is blocked by removal of Na(+) or addition of TTX, followed by a plateau which is blocked by La(3+), Co(2+) or Mn(2+).5. Finally (stages 40-51), the inward current is primarily carried by Na(+), since the action potential is blocked only by removal of Na(+) or addition of TTX, and the overshoot agrees with the prediction of the Nernst equation for a Na-selective membrane. When the outward current channel is blocked and cells exposed to Na-free solutions, 67% of cells at the latest stages studied were incapable of producing action potentials in which the inward current is carried by divalent cations.6. The duration of the action potential decreases from a maximum of about 1000 msec to about 1 msec during development. The maximum input resistance (R(in)) decreases from ca. 1000 to 100 MOmega.7. The calcium action potential may play a role in the development of excitability and the growth of the neurones.

Action Potentials↗