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N C Spitzer

Publications and source records attributed to N C Spitzer.

At least 37 records · Page 2Linked to original sources
PubMed1997

Purposeful patterns of spontaneous calcium transients in embryonic spinal neurons.

Embryonic cultured Xenopus spinal neurons generate two types of spontaneous elevation of intracellular calcium that encode developmental information in the frequency with which they are produced. Calcium spikes regulate the appearance of GABA and maturation of potassium current. Calcium waves in growth cones regulate neurite extension. Spikes and waves are also observed in neurons differentiating in situ. Because differentiation is dependent on the frequency of calcium transients, neurons that are coactive and fire spikes in concert would be expected to differentiate together. Consistent with this prediction, segmentally arrayed clusters of putative motoneurons on the ventral aspect of the neural tube fire together during development.

Journal Article↗
PubMed1997

Breaking the code: regulation of neuronal differentiation by spontaneous calcium transients.

Calcium ions play critical roles in neuronal development. Stimulation of transient elevations of intracellular calcium (Ca2+i) activates protein kinases, regulates transcription and influences motility and morphology. Embryonic Xenopus spinal neurons exhibit a Ca(2+)-sensitive period in culture; removing extracellular Ca2+ during this period affects several aspects of neuronal differentiation. However, both the mechanisms that generate natural fluctuations in Ca2+i and the signals they transduce are not well understood. Spontaneous, transient and repeated elevations of Ca2+i in embryonic Xenopus spinal neurons have been observed over periods up to 5 h in vitro and in vivo, confocally imaging fluo 3-loaded cells. Developing neurons generate two distinctive types of spontaneous Ca2+i transients, calcium spikes and calcium waves. We have investigated the mechanisms by which they are generated and their roles in directing neuronal differentiation. Spikes are generated by spontaneous action potentials, and thus are rapidly propagated throughout entire neurons. Ca2+ entry triggers Ca2+ release from intracellular stores, and spikes have a characteristic double exponential decay. In contrast, the generation of waves does not involve conventional voltage-dependent Ca2+ channels, but an unknown Ca2+ entry pathway that can be blocked by Ni2+ at a higher concentration than required to block classical voltage-dependent Ca2+ channels. Waves rise and decay slowly, and unlike spikes, are local events. However both spikes and waves are abolished by removal of extracellular Ca2+. Developmentally, the incidence and frequency of spikes decrease while the incidence and frequency of waves are constant. To study the roles of Ca2+ transients, we have imaged Ca2+ in spinal neurons throughout an extended period of early development, and find that spikes and waves are expressed at distinct frequencies. Neuronal differentiation is altered when they are eliminated by preventing Ca2+ influx. By reimposing different frequency patterns of Ca2+ transients, we demonstrate that natural spike activity is sufficient to promote normal neurotransmitter expression and channel maturation, while wave activity at growth cones is sufficient to regulate neurite extension. On the other hand, suppression of spontaneous Ca2+ elevations with BAPTA, a rapid Ca2+ chelator, indicates that they are also necessary to direct differentiation. Ca2+ transients appear to encode information in their frequency. Thus, they act like action potentials, although they are 10(4) times longer in duration and less frequent and implement an intrinsic development program.

Animals↗
PubMed1996

Mitochondrial dysfunction is a primary event in glutamate neurotoxicity.

Excitotoxic neuronal death, associated with neurodegenerative disorders and hypoxic insults, results from excessive exposure to excitatory neurotransmitters. Glutamate neurotoxicity is triggered primarily by massive Ca2+ influx arising from overstimulation of the NMDA subtype of glutamate receptors. The underlying mechanisms, however, remain elusive. We have tested the hypothesis that mitochondria are primary targets in excitotoxicity by confocal imaging of intracellular Ca2+ ([Ca2+]i) and mitochondrial membrane potential (delta psi) on cultured rat hippocampal neurons. Sustained activation of NMDA receptors (20 min) elicits reversible elevation of [Ca2+]i. Longer activation (50 min) renders elevation of [Ca2+]i irreversible (Ca2+ overload). Susceptibility to NMDA-induced Ca2+ overload is increased when the 20 min stimuli are applied to neurons pretreated with electron transport chain inhibitors, thereby implicating mitochondria in [Ca2+]i homeostasis during excitotoxic challenges. Remarkably, delta psi exhibits prominent and persistent depolarization in response to NMDA, which closely parallels the incidence of neuronal death. Blockade of the mitochondrial permeability transition pore by cyclosporin A allows complete recovery of delta psi and prevents cell death. These results suggest that early mitochondrial damage plays a key role in induction of glutamate neurotoxicity.

Animals↗
PubMed1996

Spontaneous calcium transients regulate myofibrillogenesis in embryonic Xenopus myocytes.

Spontaneous transient elevations of intracellular calcium (Ca2+i) have functional roles in the development of Xenopus spinal neurons. However, less is known about the roles of elevations of Ca2+i in the differentiation of other cell types. We have examined Xenopus myocytes as a first step in determining if Ca2+i transients are a more general feature of differentiation in excitable cells. We find that cultured myocytes, like neurons, exhibit spontaneous Ca2+i transients during an early developmental period. These transients average 1.4 min in duration and occur at an average frequency of 6/hr in cultures containing myocytes and neurons. Culture conditions can influence transient production; for example, myocyte-enriched cultures have a lower incidence of transient-producing cells. Transients persist in 0-Ca2+ medium, indicating that they arise from intracellular stores. Caffeine-sensitive Ca2+ stores are present in these cells, and depletion or block of these stores eliminates transient production. To determine if transients play a functional role during development, we blocked their production with intracellular BAPTA, a rapid Ca2+ chelator. Cellular differentiation is significantly inhibited only when BAPTA is applied early in development, during the period of transient production, while later BAPTA treatments have no effect. Blocking transient production severely perturbed myofibril organization and sarcomere assembly. However, other aspects of myocyte differentiation were not affected by transient blockade, indicating that not all myogenic differentiation programs are regulated in this manner. Our results suggest that spontaneous Cai2+ transients play a role in cytoskeletal organization during myofibrillogenesis.

Animals↗
PubMed1996

Temporal regulation of Shaker- and Shab-like potassium channel gene expression in single embryonic spinal neurons during K+ current development.

A developmental increase in density of delayed rectifier potassium current (IKv) in embryonic Xenopus spinal neurons shortens action potential durations and limits calcium influx governing neuronal differentiation. Although previous work demonstrates that maturation of IKv depends on general mRNA synthesis, it is not known whether increases in K+ channel gene transcripts direct maturation of the current. Accordingly, the developmental appearance of specific Kv potassium channel genes was determined using single-cell reverse transcription-PCR techniques after whole-cell recording of IKv during the period of its development. Detection of a coexpressed housekeeping gene along with the potassium channel gene controlled for successful aspiration of cellular mRNA and allowed scoring of cells in which Kv gene transcripts were not detected. Diverse types of Xenopus spinal neurons exhibit homogeneous development of IKv both in vivo and in culture. In contrast, transcripts of two genes encoding delayed rectifier current, Kv1.1 (Shaker) and Kv2.2 (Shab), are expressed heterogeneously during the period in which the current develops. Kv1.1 mRNA achieves maximal appearance in approximately 30% of cells, while IKv is immature; Kv2.2 mRNA appears later in approximately 60% of mature neurons. Kv1.1 and 2.2 are thus candidates for generation of IKv, and spinal neurons are a heterogeneous population with respect to potassium channel gene expression. Moreover, correlation of gene expression with current properties shows that neurons lacking Kv2.2 have a characteristic voltage dependence of activation of IKv.

Animals↗
PubMed1996

Regulation of intracellular Cl- levels by Na(+)-dependent Cl- cotransport distinguishes depolarizing from hyperpolarizing GABAA receptor-mediated responses in spinal neurons.

Rohon-Beard (RB) spinal neurons of Xenopus larvae are depolarized by GABA. To study the mechanisms underlying this distinctive response, intracellular and patch-clamp recordings were made from RB neurons in situ. The intracellularly recorded GABA reversal potential (EREV) was near -30 mV in normal saline and was approximately 25 mV more negative in Na(+)-free saline. Whole-cell recordings from RB neurons and from neighboring dorsolateral interneurons (DLi) revealed that GABA responses of both cells were mediated by GABAA receptors. Currents elicited by GABA were mimicked by muscimol and reversibly blocked by bicuculline, and EREV shifted with changes in Cl- concentration ([Cl]) in agreement with Cl- selectivity. In perforated patch recordings, EREV for RB cells was significantly more positive than for DLi cells (-38 vs -63 mV), indicating that intact RB cells maintain higher levels of intracellular Cl-. Replacement of external Na+ or exposure to the Cl- transport inhibitor bumetanide (100 microM) shifted RB cell EREV to move negative values, consistent with Na+(-)dependent Cl cotransport contributing to higher internal [Cl]. In contrast, these treatments did not change DLi cell EREV. The results indicate that a Na+(-)dependent Cl- transport mechanism underlies GABAA receptor-mediated depolarizing Cl- conductances in RB neurons. Thus, both inhibitory and excitatory GABA responses appear to be present during the same developmental period in vivo. GABA may stimulate Ca2+ influx in RB neurons because the intracellular GABA EREV is above the threshold for low voltage-activated Ca2+ channels.

Animals↗
PubMed1995

Distinct aspects of neuronal differentiation encoded by frequency of spontaneous Ca2+ transients.

Stimulation of transient increases in intracellular calcium (Cai2+) activates protein kinases, regulates transcription and influences motility and morphology. Developing neurons generate spontaneous Cai2+ transients, but their role in directing neuronal differentiation and the way in which they encode information are unknown. Here we image Ca2+ in spinal neurons throughout an extended period of early development, and find that two types of spontaneous events, spikes and waves, are expressed at distinct frequencies. Neuronal differentiation is altered when they are eliminated by preventing Ca2+ influx. Reimposing different frequency patterns of Ca2+ elevation demonstrates that natural spike activity is sufficient to promote normal neurotransmitter expression and channel maturation, whereas wave activity is sufficient to regulate neurite extension. Suppression of spontaneous Ca2+ elevations by BAPTA loaded intracellularly indicates that they are also necessary for differentiation. Ca2+ transients appear to encode information in their frequency, like action potentials, although they are 10(4) times longer in duration and less frequent, and implement an intrinsic development programme.

Action Potentials↗
PubMed1995

Convertible modes of inactivation of potassium channels in Xenopus myocytes differentiating in vitro.

1. Voltage-dependent inactivating single-channel potassium currents were recorded in cell-attached and inside-out patches from embryonic Xenopus myocytes differentiating in culture. 2. Channels with rapid inactivation (time constants < 25 ms) and with slow inactivation (time constants > 80 ms) recorded after one day in vitro appear to belong to two functionally different classes. Rapidly and slowly inactivating channels show steady-state inactivation with potentials of half-inactivation of -74 +/- 7 and -44 +/- 9 mV. They exhibit voltage-dependent activation, with times to half-maximal activation of 0.79 +/- 0.09 and 1.17 +/- 0.22 ms when stepped from -120 to +40 mV. Rapidly inactivating channels also have a lower open probability than slowly inactivating ones. The channels have similar conductances of 23 +/- 6 and 17 +/- 4 pS and extrapolated reversal potentials close to the potassium equilibrium potential. 3. In cell-attached patches, inactivation behaviours of channels with rapid or slow inactivation do not change during recording. After patch excision, rapidly inactivating channels usually switch to a slow inactivation mode. Slowly inactivating channels derived from rapidly inactivating channels after patch excision retain their conductance and extrapolated reversal potential, but are not distinguishable from native slowly inactivating channels with respect to steady-state inactivation, activation and inactivation times, as well as open probabilities. 4. The change in inactivation behaviour of rapidly inactivating channels after patch excision is reversed by application of reduced dithiothreitol (DTT). In contrast, channels with slow inactivation in the cell-attached mode do not change in to rapidly inactivating channels after application of DTT in the excised configuration, suggesting that these channels belong to a structurally different class. 5. Frequent observation of superposing channel openings indicates clustering of inactivating potassium channels in the myocyte membrane, since many patches lack channel activity. Clustering does not depend on the presence of differentiating neurones. 6. Channels with rapid inactivation increase 6-fold in density during the first day in culture in the presence of neurones; channel density decreases in their absence. Channels with slow inactivation increase 2-fold in density in the presence or absence of differentiating neurones during this period. 7. Channels with rapid or slow inactivation in cell-attached membrane belong to functionally distinct classes that are developmentally regulated differently. Reversible changes from rapid to slow inactivation mode after patch excision suggest that the channels may be structurally related.

Animals↗
PubMed1995

Spontaneous calcium transients regulate neuronal plasticity in developing neurons.

Calcium ions play critical roles in neuronal differentiation. We have recorded transient, repeated elevations of calcium in embryonic Xenopus spinal neurons over periods of 1 h in vitro and in vivo, confocally imaging fluo 3-loaded cells at 5 s intervals. Calcium spikes and calcium waves are found both in neurons in culture and in the intact spinal cord. Spikes rise rapidly to approximately 400% of baseline fluorescence and have a double exponential decay, whereas waves rise slowly to approximately 200% of baseline fluorescence and decay slowly as well. Imaging of fura 2-loaded neurons indicates that intracellular calcium increases from 50 to 500 nM during spikes. Both spikes and waves are abolished by removal of extracellular calcium. Developmentally, the incidence and frequency of spikes decrease, whereas the incidence and frequency of waves are constant. Spikes are generated by spontaneous calcium-dependent action potentials and also utilize intracellular calcium stores. Waves are produced by a mechanism that does not involve classic voltage-dependent calcium channels. Spikes are required for expression of the transmitter GABA and for potassium channel modulation. Waves in growth cones are likely to regulate neurite extension. The results demonstrate the roles of a novel signaling system in regulating neuronal plasticity, that operates on a time scale 10(4) times slower than that of action potentials.

Animals↗
PubMed1995

Spontaneous activity: functions of calcium transients in neuronal differentiation.

Calcium ions play critical roles in neuronal development. Many excitable cells promote calcium influx across their surface membrane during early stages of differentiation, which can trigger further elevation of intracellular calcium by release from stores. Several distinct types of spontaneous elevations of intracellular calcium occur during development of amphibian spinal neurons, both in culture and in the intact spinal cord. Rapid spikes and slow waves originate by different mechanisms and have separate functions. Spikes are required for neurotransmitter expression and channel modulation. Waves occurring in growth cones appear to regulate neurite extension.

Action Potentials↗
PubMed1994

Action potentials, calcium transients and the control of differentiation of excitable cells.

Calcium influx via action potentials in differentiating nerve and muscle is regulated principally by the expression of potassium currents. Transient elevations of intracellular calcium in spontaneously active cells are necessary for normal neuronal development. The mechanisms that connect calcium elevations to long term developmental change are likely to be utilized in the mature nervous system.

Action Potentials↗
PubMed1994

Spontaneous neuronal calcium spikes and waves during early differentiation.

Calcium ions play critical roles in neuronal development, but the factors that govern spontaneous fluctuations in intracellular calcium are not well understood. Transient, repeated elevations of calcium in embryonic Xenopus spinal neurons have been recorded over periods of 1 hr in vitro and in vivo, confocally imaging fluo-3-loaded cells at 5 sec intervals. Calcium spikes and calcium waves are found both in neurons in culture and in the intact spinal cord. Spikes rise rapidly to approximately 400% of baseline fluorescence and have a characteristic double exponential decay, while waves rise slowly to approximately 200% of baseline fluorescence and decay slowly as well. Imaging of fura-2-loaded neurons indicates that intracellular calcium increases from 50 to 500 nM during spikes. Both spikes and waves are abolished by removal of extracellular calcium. Developmentally, the incidence and frequency of spikes decrease while the incidence and frequency of waves are constant. Spikes are generated by spontaneous calcium-dependent action potentials that can be triggered by low-threshold, T-type calcium current and are eliminated by agents that block voltage-dependent calcium channels. They can be elicited by depolarization, are generated in an all-or-none manner, and are rapidly and bidirectionally propagated. Spikes also utilize intracellular calcium stores, since blocking release from stores substantially reduces their amplitude. Waves are not elicited by depolarization nor by activation of glutamate receptors, and are propagated at a rate consistent with diffusion of calcium. Waves are blocked by Ni2+ at a higher concentration than required to block classical voltage-dependent calcium channels. Previous work now suggests that spikes are required for expression of the transmitter GABA and for potassium channel modulation. The present study indicates that waves in growth cones are likely to regulate neurite extension.

Action Potentials↗
PubMed1993

Calcium dependence of differentiation of GABA immunoreactivity in spinal neurons.

The developmental regulation of neurotransmitter synthesis has been extensively studied and appears in many cases to depend on electrical activity. The central nervous system of the Xenopus embryo and young larva is an attractive subject for such studies, since action potentials first elicited from Xenopus spinal neurons at the time of closure of the neural tube are long in duration and calcium-dependent. Moreover, cells exhibit spontaneous elevations of intracellular calcium during this early period as a consequence of calcium influx through voltage-dependent channels, which induces calcium release from intracellular stores. Since the early differentiation of Xenopus spinal neurons in dissociated cell culture parallels development in vivo, we have examined the maturation of gamma-aminobutyric acid (GABA) immunoreactivity in cultured neurons and explored its dependence on spontaneous calcium influx at early stages of development. We find that specific GABA immunoreactivity develops in spinal neurons in dissociated cell culture with the same time course previously defined in vivo. Additionally, this process requires calcium influx that occurs spontaneously through voltage-dependent channels. The appearance of GABA immunoreactivity is blocked by transcriptional inhibitors. The early appearance of GABA raises the possibility that it may play additional roles at early stages of development.

Animals↗
PubMed1993

Calcium regulates neuronal differentiation both directly and via co-cultured myocytes.

Control of neuronal development by cellular interactions can be regulated by both extracellular and intracellular calcium. Removal of extracellular calcium affects the differentiation of amphibian spinal neurons in vitro by preventing neuronal calcium influx during the production of calcium-dependent action potentials (Holliday and Spitzer, Dev. Biol. 141:13-23, 1990). However, this culture condition affects differentiation through other mechanisms as well. We have investigated the interaction between neurons and myocytes to distinguish direct effects of low extracellular calcium on neuronal differentiation and indirect effects due to interference with neuron-myocyte interactions. We have examined the initiation of neurite outgrowth and the subsequent extension and orientation of processes. We find that (1) the number of neurons that initiate process outgrowth is reduced by the presence of myocytes in a standard medium containing calcium. Experiments with muscle-conditioned medium indicate that the production and/or secretion of inhibitory cues is calcium dependent. (2) When neurite initiation occurs, neuronal architecture in the absence of myocytes is similar to that in their presence, either in standard or in calcium-free medium, although neurite extension is enhanced by the absence of calcium. (3) Conditioned medium (CM) experiments additionally demonstrate that the orientation of neurite outgrowth to myocyte-derived cues is calcium dependent, although the production of directional cues by myocytes is calcium independent.

Animals↗
PubMed1993

Low-threshold Ca2+ current and its role in spontaneous elevations of intracellular Ca2+ in developing Xenopus neurons.

Amphibian spinal neurons exhibit spontaneous elevations of intracellular calcium at early stages of development. The underlying calcium influx involves high-voltage-activated (HVA) currents. To begin to understand how they are triggered, we have studied the biophysical properties and developmental function of low-voltage-activated (LVA) T-type calcium current of neurons cultured from the embryonic neural plate. T current was recorded from young neurons (6-9 hr in vitro) and from mature neurons (18-48 hr in vitro) using whole-cell voltage clamp. For both young and mature neurons, T current has a low threshold and is activated at membrane potentials positive to -60 mV in 2 mM extracellular calcium. The current is maximal at -35 mV with a mean peak amplitude of approximately 50 pA. Nickel blocks both LVA and HVA currents, but the former are 20-fold more sensitive. Amiloride also blocks T current selectively. T current is recorded in 87% of young neurons. This percentage drops to 67% in mature neurons after 1 d in culture and to 35% in mature neurons after 2 d in culture. There are no significant developmental changes in T current threshold, peak density, time course of activation and inactivation, and pharmacological sensitivity to blockers from 6 to 48 hr in culture. Spontaneous transient calcium elevations in young neurons assayed by fluo-3 fluorescence are blocked by nickel or amiloride at concentrations that specifically block T current. T current has the lowest threshold among other inward currents in young neurons. Moreover, mathematical simulations show that T current lowers the threshold of the action potential by 15 mV. We conclude that T current can depolarize cells and trigger action potentials, and constitutes part of the cascade of events leading to spontaneous elevations of intracellular calcium in cultured neurons at early stages of differentiation.

Amiloride↗