Biomedical subjects

M E Spira

Publications and source records attributed to M E Spira.

At least 19 recordsLinked to original sources

PubMed1997

Low mobility of the Ca2+ buffers in axons of cultured Aplysia neurons.

Cellular Ca2+ buffers determine amplitude and diffusional spread of neuronal Ca2+ signals. Fixed Ca2+ buffers tend to retard the signal and to lower the apparent diffusion coefficient (D(app)) of Ca2+, whereas mobile buffers contribute to Ca2+ redistribution. To estimate the impact of the expression of specific Ca2+-binding proteins or the errors in Ca2+ measurement introduced by indicator dyes, the diffusion coefficient De and the Ca2+-binding ratio kappa(e) of endogenous Ca2+ buffers must be known. In this study, we obtain upper bounds to these quantities (De < 16 microm2/s; kappa(e) < 60) for axoplasm of metacerebral cells of Aplysia california. Due to these very low values, even minute concentrations of indicator dyes will interfere with the spatiotemporal pattern of Ca2+ signals and will conceal changes in the expression of specific Ca2+-binding proteins, which in the native neuron are expected to have significant effects on Ca2+ signals.

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PubMed1996

Use of Aplysia neurons for the study of cellular alterations and the resealing of transected axons in vitro.

The present report describes the experimental advantages offered by the combined use of Aplysia neurons and contemporary techniques to analyze the cellular events associated with nerve injury in the form of axotomy. The experiments were performed by transecting, under visual control, the main axon of identified Aplysia neurons in primary culture while monitoring several related parameters. We found that in cultured Aplysia neurons axotomy leads to the elevation of the [Ca2+]i in both the proximal and distal axonal segments from a resting level of 100 nM up to the millimolar range for a duration of 3-5 min. This increase in [Ca2+]i led to identical alterations in the cytoarchitecture of the proximal and distal segments. The formation of a membrane seal over the transected ends by their constriction and the subsequent fusion of the membrane is a [Ca2+]i-dependent process and is triggered by the elevation of [Ca2+]i to the microM level. Seal formation was followed by down-regulation of the [Ca2+]i to control levels. Following the formation of the membrane seal an increase in membrane retrieval was observed. We hypothesize that the retrieved membrane serves as an immediately available membrane reservoir for growth cone extension.

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PubMed1996

Acceleration of membrane recycling by axotomy of cultured aplysia neurons.

The rapid transition of a stationary axon into a motile growth cone requires the recruitment of membrane and its strategic insertion into the neurolemma. The source of membrane to support the initial rapid growth postaxotomy is not known. Using membrane capacitance measurements, we examined quantitative aspects of membrane dynamics following axotomy of cultured Aplysia neurons. Axotomy activates two processes in parallel: membrane retrieval and exocytosis. Unexpectedly, membrane retrieval is the dominant process in the majority of the experiments. Thus, while a growth cone is vigorously extending, the total neuronal surface area decreases. We suggest that the initial rapid extension phase of the newly formed growth cone postaxotomy is supported by a pool of intracellular membrane that is rapidly retrieved from the neurolemma.

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PubMed1995

A new family of conotoxins that blocks voltage-gated sodium channels.

Conus peptides, including omega-conotoxins and alpha-conotoxins (targeting calcium channels and nicotinic acetylcholine receptors, respectively) have been useful ligands in neuroscience. In this report, we describe a new family of sodium channel ligands, the mu-O-conotoxins. The two peptides characterized, mu-O-conotoxins MrVIA and MrVIB from Conus marmoreus potently block the sodium conductance in Aplysia neurons. This is in marked contrast to standard sodium channel blockers that are relatively ineffective in this system. The sequences of the peptides are as follows. mu-O-conotoxin MrVIA: ACRKKWEYCIVPIIGFIYCCPGLICGPFVCV mu-O-conotoxin MrVIB: ACSKKWEYCIVPILGFVYCCPGLICGPFVCV mu-O-conotoxin MrVIA was chemically synthesized and proved indistinguishable from the natural product. Surprisingly, the mu-O-conotoxins show no sequence similarity to the mu-O-conotoxins. However, ananalysis of cDNA clones encoding the mu-O-conotoxin MrVIB demonstrated striking sequence similarity to omega- and delta-conotoxin precursors. Together, the omega-, delta-, and mu-O-conotoxins define the O-superfamily of Conus peptides. The probable biological role and evolutionary affinities of these peptides are discussed.

Amino Acid Sequence↗

PubMed1995

Alterations of voltage-activated sodium current by a novel conotoxin from the venom of Conus gloriamaris.

1. The novel peptide toxin delta-conotoxin-GmVIA, recently purified by us from the mollusk-hunting snail Conus gloriamaris, induces convulsive-like contractions when injected into land snails but has no detectable effects in mammals. 2. At concentrations of 0.5-0.75 microM, the toxin induces action potential broadening and increased excitability of cultured Aplysia neurons. 3. Whole cell patch-clamp experiments on cultured Aplysia neurons revealed that the toxin does not alter potassium or calcium currents, but induces action potential broadening by slowing the inactivation kinetics of the sodium current. Under control conditions, the inactivation kinetics of the sodium current follows a single exponential with tau = 0.47 +/- 0.14 (SE) ms. After toxin application the sodium current inactivation is composed of two phases: an early phase with tau = 0.86 +/- 0.12 ms and a late phase of slowly inactivating sodium current with tau = 488 +/- 120 ms. In addition, the toxin shifts the voltage-dependent steady-state inactivation curve to more positive values and the steady-state activation curve to more negative values. These alterations are not associated with changes in the rise time or the peak value of the sodium current. 4. The novel delta-conotoxin-GmVIA, and the previously described "King Kong peptide," purified from another mollusk-hunting cone (Conus textile), share a similar cystein framework also found in the calcium channel blocking peptide omega-conotoxin but represent a new class of conotoxins with unusual specificity for molluscan sodium channels.

Action Potentials↗

PubMed1995

Axotomy induces a transient and localized elevation of the free intracellular calcium concentration to the millimolar range.

1. Axonal transection triggers a cascade of pathological processes that frequently lead to the degeneration of the injured neuron. It is generally believed that the degenerative process is triggered by an overwhelming influx of calcium through the cut end of the axon. 2. Theoretical considerations and indirect observations suggest that axotomy is followed by an increase in the free intracellular calcium concentration ([Ca2+]i) to the millimolar level. In contrast, only relatively modest and transient elevation in [Ca2+]i to the micromolar level was revealed by recent fura-2 studies. 3. In the current study we used the low-affinity Ca2+ indicator mag-fura-2 to reexamine the spatiotemporal distribution pattern of Ca2+ after axotomy and to map the free intracellular Mg2+ concentration gradients. 4. We report that axotomy elevates [Ca2+]i well beyond the "physiological" range of calcium concentrations, to levels > 1 mM near the tip of the cut axon and to hundreds of micromolars along the axon further away from the cut end. Nevertheless, [Ca2+]i recovers to the control levels within 2-3 min after the resealing of the cut end. 5. A comparison of the behavior of fura-2 and mag-fura-2 in the cytosol of the axotomized neurons reveals that the determination of [Ca2+]i by fura-2 largely underestimates the actual intracellular Ca2+ concentrations. 6. Experiments in which one branch of a bifurcated axon was transected revealed that the elevation in [Ca2+]i is confined to the transected axonal branch and does not spread beyond the bifurcation point. 7. After axotomy, the intracellular Mg2+ concentration equilibrates rapidly with the external concentration and then recovers at a rate somewhat slower than that of [Ca2+]i. 8. To the best of our knowledge, this study is the first direct demonstration that axotomy elevates [Ca2+]i to the millimolar range and that neurons are able to recover from these extreme calcium concentrations.

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PubMed1994

The survival of transected axonal segments of cultured Aplysia neurons is prolonged by contact with intact nerve cells.

Axonal segments transected from their cell body in vivo commonly undergo degeneration within 3-4 days (Wallerian degeneration). In lower vertebrates and invertebrates, however, some transected axonal segments survive for long periods ranging between 30 and 200 days. To circumvent the technical complications of studying the mechanisms underlying long-term survival of transected axons in vivo, we developed an in vitro system. We found previously that isolated axonal segments of cultured Aplysia neurons preserved their morphological integrity for an average duration of 7.6 days (range 2-14 days) and maintain their passive and excitable membrane properties. This survival occurred in the absence of de novo protein synthesis. In the present study we examined the influence of homologous neurons on the survival of transected axonal segments. We found that the average survival time of transected axons was doubled when co-cultured in physical contact with intact homologous neurons (average 15.3 days, range 2-27 days). During this period, the transected axons extended neurites, maintained normal passive and excitable membrane properties, formed electrotonic junctions with the intact neurons and maintained normal free intracellular Ca2+ levels. Consistent with these observations, electron micrographs of the transected axon revealed that the cytoskeletal elements of the axon appeared normal even 20 days after transection. In contrast, the mitochondria and smooth endoplasmic reticulum appeared damaged. As the prolonged survival was conditional on physical contact between the transected axon and the surrounding intact neurons, we suggest that the prolongation of survival time is promoted by the direct transfer of material from the intact neurons to the transected axon. However, co-culture of transected axons with homologous neurons did not fully mimic in vivo conditions, in which transected axons can survive for several months.

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PubMed1994

Delta-conotoxin GmVIA, a novel peptide from the venom of Conus gloriamaris.

A novel peptide toxin, delta-conotoxin GmVIA, was purified from the venom of Conus gloriamaris, a mollusc-hunting snail. It consists of 29 amino acids, including six Cys residues: [sequence: see text] The pattern of disulfide connectivity (4-19, 12-24, and 18-29) is the same as for the omega-conotoxins, which are Ca2+ channel ligands. However, the peptide does not compete with omega-conotoxin for binding to membrane preparations from frog, rat, and chick brain. Instead, initial electrophysiological results suggest that the peptide induces action potential broadening in molluscan neurons by slowing down Na+ current inactivation. Synthetic delta-conotoxin GmVIA was prepared by solid-phase methods and appeared identical in all respects to the natural material. The chromatographic behavior of native and reduced delta-conotoxins is quite remarkable, suggesting that the disulfides form a core which forces hydrophobic residues to point out toward the solvent.

Amino Acid Sequence↗

PubMed1994

New mollusc-specific alpha-conotoxins block Aplysia neuronal acetylcholine receptors.

Two mollusc-specific neurotoxic peptides from the venom of the molluscivorous snail Conus pennaceus are described. These new toxins block acetylcholine receptors (AChR) of cultured Aplysia neurons. Bath application of 0.5-1 microM toxin induces 5-10-mV membrane depolarization, which recovers to the control level within 1-3 min in the presence of the toxin. This response is blocked by 1 mM hexamethonium. Concomitantly with the transient depolarization, the toxins block approximately 90% of the depolarizing responses evoked by brief iontophoretic application of acetylcholine. The pharmacology and amino acid sequences of the toxins (alpha PnIA, GCCSLPPCAANNPDYC-NH2; alpha PnIB, GCCSLPPCALSNPDYC-NH2) enable their classification as novel alpha-conotoxins. The sequences differ from those of previously described alpha-conotoxins in a number of features, the most striking of which is the presence of a single negatively charged residue in the C-terminal loop. This loop contains a positively charged residue in piscivorous venom alpha-conotoxins. In contrast to other alpha-conotoxins, which are selective for vertebrate skeletal muscle nicotinic ACh receptors, these Conus pennaceus toxins block neuronal ACh receptors in molluscs. As such they are new probes which can be used to define subtypes of ACh receptors, and they should be useful tools in the study of structure-function relationships in ACh receptors.

Amino Acid Sequence↗

PubMed1993

Spatiotemporal distribution of Ca2+ following axotomy and throughout the recovery process of cultured Aplysia neurons.

This study investigates the alterations in the spatiotemporal distribution pattern of the free intracellular Ca2+ concentration ([Ca2+]i) during axotomy and throughout the recovery process of cultured Aplysia neurons, and correlates these alterations with changes in the neurons input resistance and trans-membrane potential. For the experiments, the axons were transected while imaging the changes in [Ca2+]i with fura-2, and monitoring the neurons' resting potential and input resistance (Ri) with an intracellular microelectrode inserted into the cell body. The alterations in the spatiotemporal distribution pattern of [Ca2+]i were essentially the same in the proximal and the distal segments, and occurred in two distinct steps: concomitantly with the rupturing of the axolemma, as evidenced by membrane depolarization and a decrease in the input resistance, [Ca2+]i increased from resting levels of 0.05-0.1 microM to 1-1.5 microM along the entire axon. This is followed by a slower process in which a [Ca2+]i front propagates at a rate of 11-16 microns/s from the point of transection towards the intact ends, elevating [Ca2+]i to 3-18 microM. Following the resealing of the cut end 0.5-2 min post-axotomy, [Ca2+]i recovers in a typical pattern of a retreating front, travelling from the intact ends towards the cut regions. The [Ca2+]i recovers to the control level 7-10 min post-axotomy. In Ca(2+)-free artificial sea water (2.5 mM EGTA) axotomy does not lead to increased [Ca2+]i and a membrane seal is not formed over the cut end. Upon reperfusion with normal artificial sea water, [Ca2+]i is elevated at the tip of the cut axon and a membrane seal is formed. This experiment, together with the observations that injections of Ca2+, Mg2+ and Na+ into intact axons do not induce the release of Ca2+ from intracellular stores, indicates that Ca2+ influx through voltage gated Ca2+ channels and through the cut end are the primary sources of [Ca2+]i following axotomy. However, examination of the spatiotemporal distribution pattern of [Ca2+]i following axotomy and during the recovery process indicates that diffusion is not the dominating process in shaping the [Ca2+]i gradients. Other Ca2+ regulatory mechanisms seem to be very effective in limiting these gradients, thus enabling the neuron to survive the injury.

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PubMed1993

Alteration of sodium currents by new peptide toxins from the venom of a molluscivorous Conus snail.

TxIA and TxIB, peptides with 27-amino acid residues recently isolated from the molluscivorous marine snail Conus textile neovicarius, exhibit strong paralytic activity in molluscs, with no paralytic effects on athropods and vertebrates. At concentrations of 0.25-0.5 microM the toxins cause spontaneous repetitive firing and dramatic broadening of the action potential of cultured Aplysia neurons. The action potential duration partially recovers within 30 min in the presence of the toxins. Under these conditions a second toxin application does not change the spike duration. TxI-induced spike broadening occurs when potassium and calcium conductances are blocked. Voltage-clamp experiments revealed that the toxins alter the kinetics of the sodium current either by slowing down the rate of sodium current inactivation or by recruiting silent sodium channels with slower activation and inactivation kinetics. The toxins shift the voltage-dependent steady-state Na+ current inactivation curve to more positive values by 6 mV. These changes are not associated with alteration in the rate of sodium current activation, in the peak sodium current, or the sodium current reversal potential. TxI apparently represents a new class of conotoxins with an unusual phylogenic specificity and may therefore be useful as a probe for the study of molluscan neuronal sodium channels.

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PubMed1993

Resealing of the proximal and distal cut ends of transected axons: electrophysiological and ultrastructural analysis.

The fates of the proximal and distal segments of transected axons differ. Whereas the proximal segment usually recovers from injury and regenerates, the distal segment degenerates. In the present report we studied the kinetics of the recovery processes of both proximal and distal axonal segments following axotomy and its temporal relations to the alterations in the cytoarchitecture of the injured neuron. The experiments were performed on primary cultured metacerebral neurons (MCn) isolated from Aplysia. We transected axons while monitoring the changes in transmembrane potential and input resistance (Rn) by inserting intracellular microelectrodes into the soma and axon. Correlation between the electrophysiological status of the injured axon and its ultrastructure was provided by rapid fixation of the neuron at selected times postaxotomy. Axotomy leads to membrane depolarization from a mean of -55.7 S.D. 12.8 mV to -12.7 S.D. 3.3 mV and decreased Rn from tens of M omega to 1-3 M omega. The transected axons remained depolarized for a period of 10-260 s for as long as the axoplasm was in direct contact with the bathing solution. Rapid repolarization and partial recovery of Rn was associated with the formation of a membrane seal over the cut ends by the constriction and subsequent fusion of the axolema. Prior to the formation of a membraneous barrier, electron-dense deposits aggregate at the tip of the cut axon and appear to form an axoplasmic "plug." Electrophysiological analysis revealed that this "plug" does not provide resistance for current flow and that the axoplasmic resistance is homogenously distributed. The kinetics of injury and recovery processes as well as the ultrastructural changes of the proximal and distal segments are identical suggesting that the different fates of the segments cannot be attributed to differences in the immediate response of the segments to axotomy.

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PubMed1993

Survival of isolated axonal segments in culture: morphological, ultrastructural, and physiological analysis.

Some transected distal axons survive for months in vivo, generate new neurites, and reconnect to proximal segments before degenerating. To determine the factors regulating these phenomena, we studied the behavior of transected axons in cultured metacerebral neurons (MCn) of Aplysia. The neurons were isolated from the ganglia and cultured at 18 degrees C. The morphology, ultrastructure, and electrophysiological properties of the transected axons, as well as their ability to synthesize protein, were examined at different times postaxotomy. Follow-up studies revealed that cultured isolated axonal segments can preserve their morphological integrity for up to 14 days, maintain their passive and active membrane properties for at least 10 days, and extend new neurites and form electrotonic junctions with their proximal segments and intact MCns. De novo protein synthesis is an unlikely mechanism to account for the survival of the isolated axons since they did not incorporate [35S]methionine. We conclude that the viability of transected axons in culture devoid of other cells depends on pools of proteins synthesized prior to the transection and energy stores sufficiently large to maintain neuronal homeostasis.

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PubMed1993

Membrane depolarization combined with release of calcium from internal stores does not trigger secretion from PC 12 cells.

The mechanisms underlying catecholamine release from pheochromocytoma (PC 12) cells were examined. Whereas application of 1 microM bradykinin (BK) induced an increase in intracellular calcium ([Ca2+])i), either in medium containing 1.8 mM Ca2+ or in medium prepared without the addition of CaCl2 ("Ca(2+)-free medium"), norepinephrine ([3H]NE) release was induced only in Ca(2+)-containing medium. Similarly depolarization by 50 mM potassium induced [3H]NE release only in 1.8 mM calcium-containing medium. The combination of membrane depolarization (50 mM KCl) with increased [Ca2+]i secondary to BK application in "Ca(2+)-free medium" did not induce catecholamine secretion. It was concluded that Ca2+ entry through calcium channels at the plasma membrane is essential for the activation of catecholamine release. A rise in [Ca2+]i (4-5 times x basal) released from internal stores is not sufficient to trigger secretion from PC 12 cells, either by itself or in combination with membrane depolarization.

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PubMed1993

Chemical and electrophysiological characterization of new peptide neurotoxins from the venom of the molluscivorous snail Conus textile neovicarius: a review.

Three peptide toxins exhibiting strong paralytic activity to molluscs, but with no paralytic effects on arthropods or vertebrates, were purified from the venom of the molluscivorous snail Conus textile neovicarius from the Red Sea. The amino acid sequences of these mollusc specific toxins are: TxIA, WCKQSGEMCNLLDQNCCDGYCIVLVCT (identical to the so-called 'King Kong peptide'); TxIB, WCKQSGEMCNVLDQNCCDGYCIVFVCT; TxIIA, WGGYSTYC gamma VDS gamma CCSDNCVRSYCT (gamma = gamma-carboxyglutamate). There is a similarity of the Cys framework of these toxins to that of the omega-conotoxins; however, their net negative charges, high content of hydrophobic residues, and uneven number of Cys residues in TxIIA are highly unusual for conotoxins. When assayed on isolated cultured Aplysia neurons, all three toxins induced spontaneous repetitive firing. The TxI toxins also induced a marked prolongation of the action potential duration. Voltage clamp experiments revealed that the TxI toxins alter the kinetics of the sodium current either by slowing down the rate of sodium current inactivation, or by recruiting silent sodium channels with slower activation and inactivation kinetics. The toxins shift the voltage-dependent steady-state Na+ current inactivation curve to more positive values by 6 mV. These changes are not associated with alteration in the rate of INa+ activation, in the peak INa+, or the sodium current reversal potential. TxI represents a new class of conotoxins with an unusual phylogenic specificity and may therefore be useful as a probe for the study of voltage gated sodium channels. (This review summarizes previously published papers).

Amino Acid Sequence↗

PubMed1991

Mollusc-specific toxins from the venom of Conus textile neovicarius.

Three peptide toxins exhibiting strong paralytic activity to molluscs, but with no paralytic effects on arthropods or vertebrates, were purified from the venom of the molluscivorous snail Conus textile neovicarius from the Red Sea. The amino acid sequences of these mollusc specific toxins are: TxIA, WCKQSGEMCNLLDQNCCDGYCI-VLVCT (identical to the so called 'King Kong peptide'); TxIB, WCKQSGEMCNVLDQNCCDGYCIVFVCT; TxIIA, WGGYSTYC gamma VDS gamma CCSDNCVRSYCT (gamma = gamma-carboxyglutamate). There is a similarity of the Cys framework of these toxins to that of the omega-conotoxins; however, their net negative charges, high content of hydrophobic residues and uneven number of Cys residues in TxIIA, are highly unusual for conotoxins. When assayed on isolated cultured Aplysia neurons, all three toxins induced membrane depolarization and spontaneous repetitive firing. The TxI toxins also induce a marked prolongation of the action potential duration, which is sodium dependent. These effects differ significantly from the blocking activities of piscivorous venom conotoxins. These mollusc specific conotoxins may therefore serve as new and selective probes for ion-channel functions in molluscan neuronal systems.

Amino Acid Sequence↗

PubMed1990

Facilitatory and inhibitory transmitters modulate calcium influx during action potentials in aplysia sensory neurons.

Serotonin (5-HT) produces presynaptic facilitation and FMRFamide produces presynaptic inhibition in Aplysia sensory neurons. These effects may involve the modulation of Ca2+ influx into sensory neuron terminals during action potentials. Here, we have used the Ca2+ indicator dye fura-2 to monitor directly the effects of 5-HT and FMRFamide on internal Ca2+ concentration ([Ca2+]i). 5-HT caused a 50% increase in the transient rise in [Ca2+]i in response to action potentials, whereas FMRFamide decreased the [Ca2+]i transient by 40%. Neither transmitter altered the resting [Ca2+]i, the time course of recovery of the [Ca2+]i transient, or the [Ca2+]i transients produced by intracellular injection of CaCl2 or inositol 1,4,5-trisphosphate. We conclude that the effects of the transmitters on the action potential-induced [Ca2+]i transient are due to changes in Ca2+ influx and not in intracellular Ca2+ homeostasis.

Action Potentials↗

PubMed1989

Sequential model to describe the nicotinic synaptic current.

An analytical formula is derived to describe the synaptic end plate current (epc) at the nicotinic receptor. Various concurrently occurring underlying processes, including (a) diffusion, (b) hydrolysis of acetylcholine, and (c) its binding to the dimeric receptor, were considered in order to develop the equation. Numeric solution of the equations that describe the events underlying the epc showed that these events occur in sequence, rather than concurrently. This sequential occurrence of the processes allowed for simplifications, which were used as the basis for the new description of the epc. The resulting formula serves as a tool for evaluating the relative contribution of the various processes in formation of the natural occurring transient epc.

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