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W N Ross

Publications and source records attributed to W N Ross.

At least 37 records · Page 2Linked to original sources
PubMed1992

Imaging voltage and synaptically activated sodium transients in cerebellar Purkinje cells.

Transient changes in sodium concentration in response to electrical activity were detected in Purkinje cells by using the fluorescent indicator SBFI (Minta & Tsien (J. biol. Chem. 264, 19,449 (1989)). Fast sodium action potentials caused large increases in internal sodium concentration, [Na]i, in the soma and axon, and were generally undetectable in the dendrites. No changes were detected in the dendrites corresponding to calcium action potentials. The spatial distribution of these transients corresponds to that expected if the increase in [Na]i were the result of Na+ entry through voltage-dependent Na channels generating sodium spikes in the axon hillock and soma. The [Na]i transients rapidly recovered (tau less than 1 s) in the axon hillock, probably by Na+ diffusion into the soma. Climbing fibre activation produced distinct [Na]i transients in the dendrites in addition to somatic and axonal signals. As regenerative potentials did not produce transients in this region, these signals may be caused by Na+ entry through ligand-gated channels. These results confirm and extend the description of channel distribution and electrical signalling in Purkinje cells.

Action Potentials↗
PubMed1992

Synaptically activated increases in Ca2+ concentration in hippocampal CA1 pyramidal cells are primarily due to voltage-gated Ca2+ channels.

Changes in intracellular Ca2+ concentration ([Ca2+]i) in the soma and dendrites of hippocampal CA1 pyramidal neurons were measured using intracellularly injected fura-2. A large component of the [Ca2+]i elevation caused by high frequency stimulation of the Schaffer collaterals was correlated with the Na+ spikes triggered by the excitatory postsynaptic potentials (EPSPs). These spikes were generated in the soma and proximal dendrites and stimulated Ca2+ entry through voltage-gated Ca2+ channels. Suppressing spikes by hyperpolarizing the soma or by injecting QX-314 revealed a smaller nonspike component of Ca2+ entry. A substantial fraction of this component was mediated by the action of the EPSPs on voltage-gated Ca2+ channels, because it persisted in 2-amino-5-phosphonovaleric acid and because it was usually reduced when Ca2+ channel activity was suppressed by hyperpolarization. Ca2+ entry through the N-methyl-D-aspartate receptor channel could not be detected with certainty, perhaps because it was highly localized.

2-Amino-5-phosphonovalerate↗
PubMed1992

Calcium transients in cerebellar Purkinje neurons evoked by intracellular stimulation.

1. Purkinje cells in thin slices from the guinea pig cerebellum were injected with fura-2 and high-speed sequences of fluorescence images from the cell body and entire dendritic tree were made while simultaneously recording somatic membrane potential during evoked and spontaneous electrical activity. The changes in fluorescence were interpreted in terms of changes in [Ca2+]i. 2. Individual calcium action potentials were usually accompanied by transient increases in [Ca2+]i all over the dendritic field. During evoked or spontaneous bursts of calcium spikes, [Ca2+]i increased more rapidly and to higher concentrations in fine dendrites than in thicker dendrites. At the end of a burst [Ca2+]i declined faster in thin dendrites than in thicker ones. These variations are most easily understood as deriving from the difference in surface-to-volume ratio of the two kinds of dendrites. 3. During bursts of calcium action potentials [Ca2+]i increases sometimes occurred only in individual dendritic branches, but always including the fine dendrites of that particular branch, showing that calcium action potentials can be regenerative in restrictive parts of the dendritic field without involving the soma or dendritic shaft. 4. Plateau or subthreshold potential changes evoked in the presence of tetrodotoxin (TTX) caused small, widespread increases in [Ca2+]i. The amplitude of these changes was much less than the increase corresponding to spike bursts. The distribution of these plateau Ca signals in thick and thin dendrites was similar to Ca spike-evoked signals, suggesting that the Ca conductances underlying these two potentials are the same or are distributed similarly in the dendrites. 5. Significant increases in [Ca2+]i in the soma were recorded during bursts of sodium-dependent action potentials in normal Ringer. Although much of this increase is due to calcium entry through calcium channels, some of this increase could be due to calcium entry through sodium channels.

Action Potentials↗
PubMed1992

Calcium transients evoked by climbing fiber and parallel fiber synaptic inputs in guinea pig cerebellar Purkinje neurons.

1. Calcium transients related to climbing fiber (CF) and parallel fiber (PF) synaptic potentials were recorded from Purkinje cells in guinea pig cerebellar slices. Transients were measured using either absorbance changes of arsenazo III or fluorescence changes of fura-2, which were injected into individual cells in the slice. 2. All-or-none somatically recorded CF potentials elicited by white matter stimulation had all-or-none Ca transients. These signals began with a delay of > or = 2 ms from the start of the electrically recorded synaptic potential. The recovery time of CF-induced arsenazo III absorbance transients was < 50 ms in the fine dendrites in conditions that minimized the effects of dye buffering. 3. Ca2+ entry through voltage-gated Ca channels opened by Ca action potentials was the dominant source of the rise in [Ca2+]i after CF activation. There was no significant change in [Ca2+]i corresponding to the plateau potential that followed the large CF response. 4. The appearance and amplitude of distal CF-evoked Ca signals was more variable than proximal signals, suggesting that CF potentials do not reliably spread to the fine distal dendrites. The distal transient could be enhanced by intrasomatic depolarizing pulses, suggesting that it was a property of the postsynaptic membrane and not the presynaptic side of the CF synapse that was responsible for this variability. 5. Parallel fiber responses were evoked by electrical stimulation near the pial surface. Graded synaptic potentials and related Ca transients were reversibly blocked by 2 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Small synaptic potentials induced small, localized Ca transients. With increasing stimulus intensity, the PF electrical response developed a regenerative component. Larger dendritic Ca transients were detected corresponding to this component. Ca transients evoked by the regenerative responses had the same rapid rise times and fall times as those related to somatically stimulated Ca action potentials, suggesting that they also were due to Ca2+ entry through voltage-sensitive channels. 6. During trains of PF responses, we observed an increase in the spatial extent of related Ca transients. This effect could be modulated by changes in the resting potential, suggesting that the same intrinsic mechanism was affecting the spread of both CF and PF signals.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1991

High time resolution fluorescence imaging with a CCD camera.

We have built a high speed, sensitive camera system capable of capturing sequences of low-light level images synchronized with recordings of membrane potential. The camera system is based on a cooled, scientific grade CCD camera controlled by a PC/AT computer. It can take 100 frames/sec of 18 X 18 element images and 40 frames/sec of 50 X 50 element images with no lag in response to step changes in light intensity. High accuracy and dynamic range of the measurements result from the fact that light levels of the picture elements are digitized with 12 bit accuracy with intrinsic camera noise levels typically less than 1/10,000 of the maximum detectable light level. We have used this system to record calcium dependent fura-2 fluorescence transients in the dendrites of cerebellar Purkinje cells and from different regions of leech neurons in segmental ganglia or isolated in culture.

Animals↗
PubMed1990

Spatial and temporal analysis of calcium-dependent electrical activity in guinea pig Purkinje cell dendrites.

We have used the calcium indicator dye arsenazo III, together with a photodiode array, to record intracellular calcium changes simultaneously from all regions of individual guinea pig cerebellar Purkinje cells in slices. The optical signals, recorded with millisecond time resolution, are good indicators of calcium-dependent electrical events. For many cells the sensitivity of the recordings was high enough to detect signals from each array element without averaging. Consequently, it was possible to use these signals to follow the complex spatial and temporal patterns of plateau and spike potentials. Calcium entry corresponding to action potentials was detected from all parts of the dendritic field including the fine spiny branchlets, demonstrating that calcium action potentials spread over the entire arbor. Usually, the entire dendritic tree fired at once. But sometimes only restricted areas had signals at any one moment with transients detected in different regions at other times. In one cell, six separate zones were distinguished. These results show that calcium action potentials could be regenerative in some dendrites and could fail to propagate into others. Signals from plateau potentials were also detected from extensive areas in the dendritic field but were always smaller than those caused by a burst of action potentials.

Action Potentials↗
PubMed1989

Spatially and temporally resolved calcium concentration changes in oscillating neurons of crab stomatogastric ganglion.

Calcium concentration changes during oscillations of the membrane potential of crab (Cancer irroratus or Cancer borealis) stomatogastric neurons were monitored at many positions by using the calcium indicator dye arsenazo III and a photodiode array. Data analysis algorithms using signal averaging techniques were developed to improve the time resolution of the measured calcium changes. As previously reported, calcium oscillations were detected from all regions of the neuropil but not from the soma or axon. In some cells step increases in intracellular neuropil calcium were correlated with each of the action potentials in the burst (on the peak of the voltage oscillation). In other cells we observed calcium oscillations phase-locked to the membrane potential with no spike-related component. A few cells had both spike-evoked and graded potential components to the calcium oscillations. In those cells, the spatial distribution of the spike-correlated calcium influx differed from that of the voltage-oscillation-correlated calcium influx, suggesting that different neurites might interact with their postsynaptic targets with different mixtures of graded and spike-correlated transmitter release.

Action Potentials↗
PubMed1988

Influence of substrate on the distribution of calcium channels in identified leech neurons in culture.

The Retzius neuron from the leech, growing in culture on the plant lectin concanavalin A as substrate, produces broad flat growth cones and thick bundles of processes. The same cell extends fine straight processes with numerous branches when grown on a laminin-like substrate extracted from leech central nervous system extracellular matrix, referred to as "leech laminin extract." Cells growing on these two different substrates also show marked differences in the pattern of Ca2+ entry following evoked impulses, as detected optically by local changes in absorbance of the Ca2+-sensitive dye arsenazo III. Ca2+ enters the soma and initial segment of Retzius cells grown on both substrates. However, detectable Ca2+ entry only occurs into the processes of cells growing on leech laminin but not of those growing on concanavalin A. Optical recordings of changes in membrane potential made with the voltage-sensitive dye RH 155 taken from cells growing on either substrate indicate that a depolarization initiated in the soma spreads to the most distant processes with little or no distortion in amplitude or time course. This implies that all voltage-sensitive Ca2+ channels in the cell membrane are equally activated by depolarizing stimuli. Therefore, the fact that impulses evoke Ca2+ entry into processes of Retzius cells grown on leech laminin extract but not of cells grown on concanavalin A shows that the nature of the growth substrate can affect the number and distribution of their functional Ca2+ channels.

Animals↗
PubMed1987

Mapping calcium transients in the dendrites of Purkinje cells from the guinea-pig cerebellum in vitro.

1. A 10 X 10 photodiode array was used to detect stimulation-dependent absorbance changes simultaneously from many positions in the dendrite field of guinea-pig Purkinje cells which had been injected with the calcium indicator Arsenazo III in thin cerebellar slices. Signals from each element of the array were matched to positions on the cells by mapping them onto fluorescence photographs of Lucifer Yellow which had been co-injected into the cells with the Arsenazo III. 2. In response to intrasomatic stimulation the rising phase of the absorbance signals corresponded in time with the calcium spikes recorded with an intracellular electrode. There was no increase in absorbance during bursts of fast sodium spikes. Absorbance signals persisted after the sodium spikes were blocked by tetrodotoxin (TTX). In addition, the signals were largest at 660 nm and small signals of opposite polarity were found at 540 nm. These results indicate that the absorbance signals came from calcium entry into the cell resulting from the turning on of voltage-dependent calcium conductances. 3. In these experiments signals were usually seen all over the dendritic field and were weak or totally absent over the soma. In some cases signals were seen over a more restricted area. With a spatial resolution of 25 microns we were not able to see any evidence for highly localized sites of calcium entry. 4. Sometimes the rising phase of the calcium signals was separated by almost 13 ms in different parts of the dendritic field, too long to be explained by active propagation delay. This suggests that calcium spikes causing these signals can be evoked separately in different regions of the Purkinje cell dendritic field by long-lasting potentials which may reach local threshold at different times. 5. Calcium signals resulting from slow plateau after-potentials and the calcium spikes produced by them were also detected in all locations in the dendritic field. The relative distribution of amplitudes from these plateau signals was different from the distribution of evoked signals during current injection. 6. Climbing fibre synaptic activation produced calcium signals which were distributed over the dendritic arborization, but larger at the main dendritic tree where most of the synaptic contacts are located. 7. Calcium signals were also detected from the dendrites of other neurone types in the in vitro slice preparation. Thus, it is likely that these kind of measurements can be used to analyse the electroresponsiveness of many kinds of neurones in the mammalian brain.

Action Potentials↗
PubMed1987

Optical recording of calcium and voltage transients following impulses in cell bodies and processes of identified leech neurons in culture.

Optical methods were used to examine the spread of electrical potentials and the distribution and time course of calcium transients in individual identified nerve cells isolated from the leech. A photodiode array detected voltage transients by measuring absorbance changes of cells stained with the voltage-sensitive dye RH155 added to the bath. Calcium transients were recorded by measuring absorbance changes of the dye arsenazo III, which had been injected into the cells. In addition, Lucifer yellow was injected to outline the some and processes. Calcium changes resulting from individual action potentials were recorded from N, P, and Retzius cells without averaging. Signals from T cells and anterior pagoda (AP) cells were weaker but could be detected with averaging. These results are in accord with previous studies on calcium contributions to action potentials in these cells. For all cells, larger or wider action potentials gave bigger signals. Calcium changes from each of a train of action potentials were of equal amplitude, showing no sign of facilitation. Calcium transients from Retzius cells that had formed chemical synapses with P cells had properties similar to those of isolated cells. We were also able to detect responses from prolonged subthreshold depolarizations to -40 mV from a hyperpolarized membrane potential (-65 mV). These signals rose throughout the duration of the pulse (1-2 sec). With the photodiode array we mapped the distribution of the calcium signals. The amplitudes from each pixel are proportional to the amount of calcium entering that element in response to the stimulating pulse, if the simplifying assumption is made that the calcium buffering of the cytoplasm is uniform throughout the cell. The largest signals were detected over the axon stump left from the cell isolation procedure. Large signals were also detected from the soma. Weak signals were detected from the processes of some cells. From many Retzius cells, no signals at all were detected from the newly formed processes. Using the photodiode array, we also recorded voltage transients from the cells. Signals were recorded from all over the arborization of the neuron, with no obvious variation in time course, showing that the entire cell, including fine slender processes and broad growth cones, was essentially isopotential. Combining these observations with the measured distribution of calcium transients in the same cell suggests that the density of calcium channels in most cells is less in the outgrowing processes than in the soma or axon stump.

Action Potentials↗
PubMed1986

A TTX-resistant propagating calcium action potential.

The cross-commissural (CC) cell in the supraesophageal ganglion of the giant barnacle, Balanus nubilus, was stimulated intrasomatically and antidromically in normal saline and 3 X 10(-7) M tetrodotoxin (TTX) saline. The action potential in normal saline contained both sodium and calcium components, each independently capable of propagation. Evidence that the action potential in TTX saline was calcium dependent included: the amplitude of the spike in TTX saline increased monotonically with increasing calcium; it was blocked by the calcium channel blockers La, Ni, Cd and Co; and equimolar substitution of Ba or Sr for Ca in TTX saline supported regenerative activity. Evidence that the calcium component could propagate alone included: the distance over which the calcium action potential traveled exceeded the space constant of the axon; the biphasic nature of the extracellularly recorded action potential, which propagated to the axon in the nerve root, indicated inward regenerative current was occurring on the axon; electrotonically spread potentials were clearly distinguishable from active regenerative potentials. In addition, optical experiments using the calcium indicator dye arsenazo III (28) showed that the relative magnitude of the calcium signal was not diminished along the axon in TTX saline compared with normal saline. The critical external calcium concentration necessary to support the propagating action potential in TTX saline was estimated to be between 1.25 and 5 mM. To our knowledge, this is the first direct observation of a neuron with sodium and calcium channels of apparently normal kinetics where the calcium component alone can propagate in the absence of an outward current blocker. Our results suggest that there is a greater density of calcium channels on the CC axon than on the axons of other neurons.

Animals↗
PubMed1986

Regional properties of calcium entry in barnacle neurons determined with Arsenazo III and a photodiode array.

Calcium changes were simultaneously measured at many positions on individual neurons from the supraesophageal ganglion of the barnacle by detecting absorbance changes of the indicator dye Arsenazo III with a 10 X 10 photodiode array. These changes were correlated with positions on the stimulated cell determined from Lucifer yellow injections. Absorbance signals were found at all locations on the cells, demonstrating that calcium channels were distributed on the somata, axons, and neuropil processes. By comparing the amplitude of the signal with the membrane area in each region, we could measure the calcium entry per impulse per unit of surface in each part of the cell. Assuming that the properties of the calcium channels are the same in all regions, we determined that calcium channels were distributed uniformly along the commissural axon of one cell and were found at higher density in the neuropil. Because significant calcium changes are only detected when cells are depolarized above about -20 mV, the presence of absorbance signals indicates membrane depolarization above this level. We used this fact to show that calcium spikes propagate along the axon and into the neuropil of one cell, along the axon of another, and not at all in a third. Differences in time course of calcium transients were observed in different regions of cells. The recovery time course was faster at the edge of the cell body than in the center and faster in the neuropil than in the axon or soma. During trains of action potentials or during wide action potentials in tetraethylammonium (TEA) saline, the calcium signal reached a plateau in the neuropil while continuing to rise in the axon and soma.

Action Potentials↗
PubMed1985

Regional distribution of calcium influx into bursting neurons detected with arsenazo III.

Absorbance changes of the metallochromic indicator arsenazo III were used in conjunction with an array of 100 photodiodes to measure changes in intracellular calcium concentration at many positions simultaneously in identified neurons of the crab stomatogastric ganglion. When stimulated with intrasomatically injected current, several of these neurons showed calcium changes all over the cell, indicating that calcium channels were distributed widely in the neuropil and on the soma. When the membrane potential was allowed to oscillate without stimulation, absorbance oscillations were detected all over the neuropil but not in the soma. A comparison between the membrane potential recorded in the soma and the calcium signal in the neuropil shows that calcium entry followed the slow voltage oscillation with the peak calcium signal detected 50-150 msec after the end of the voltage plateau.

Animals↗
PubMed1984

Optical measurements of potential changes in axons and processes of neurons of a barnacle ganglion.

Optical techniques using voltage-sensitive dyes were used to record electrical events simultaneously from many positions on certain neurons of the barnacle supraesophageal ganglion. By signal-averaging, recordings with good signal-to-noise ratios and time resolution were obtained from fine dendritic processes as well as the axon and cell body. Controls established that the optical signals faithfully matched recordings made with intracellular electrodes for short times (5 to 10 msec), but deviations were observed at longer times. Pharmacological effects and photodynamic damage due to the dye were insignificant. The optical records were correlated with positions on the stimulated cell determined from Lucifer Yellow injections. This comparison demonstrated that signal-averaged records with large signal-to-noise ratios were obtained from those parts of the visual field which contained elements of the stimulated cell and hence could be attributed to specific locations on the cell. The quality of the optical signals were adequate to: (a) determine variations in the shape of action potentials in different parts of the cell; (b) demonstrate electrotonic spread of hyperpolarizing pulses; (c) determine the direction and velocity of action potential propagation, and (d) in some circumstances, using reasonable assumptions, estimate variations in the amplitude of action potentials or electrotonic pulses in different regions of the cell.

Action Potentials↗
PubMed1984

Regional variations in excitability of barnacle neurons.

Optical recording techniques using voltage-sensitive dyes were used to examine the initiation and propagation of action potentials within neurons of the supraesophageal ganglion of the giant barnacle, Balanus nubilus. When a neuron was stimulated with current injection into the soma, the site of spike initiation, determined as the location with the earliest time-to-peak, was usually in the axon, 100 to 200 micron from the soma. The soma spike was broader and often later, suggesting that the cell body was less excitable than the axon. The action potential was narrowest at the site of initiation and then widened before propagating uniformly down the axon. In most cases, somatically stimulated action potentials and electrotonic pulses propagated into the dendrites with little delay or change of shape, indicating that the electrotonic length of these processes was great. Several different kinds of experiments indicated that some dendrites of these cells are excitable. These included the observations that (a) spikes could be made to initiate earlier in the dendrites than in the axonal region to which they were connected, and (b) action potentials sometimes decremented less than subthreshold pulses along dendritic processes. However, in other cases a decline in amplitude and a widening of the action potential demonstrated passive propagation into the dendrites, suggesting that not all dendrites are equally excitable.

Action Potentials↗
PubMed1981

Simultaneous optical measurements of electrical activity from multiple sites on processes of cultured neurons.

Optical methods using changes in fluorescence and absorption of voltage-sensitive dyes were developed to record electrical activity from processes of nerve cells grown in monolayer culture. For transmission measurements, a merocyanine dye was discovered but was more sensitive than others previously tested on cultured neurons. Action potentials from the somata of these cells were detected without averaging, with a signal-to-noise ratio of 20:1. With this dye, electrical responses were simultaneously recorded from many points along the arborization of neuroblastoma cells by using a 10 X 10 array of photodiodes positioned in the microscope image plane. Frequently different processes had different shapes of electrical responses, suggesting regional specializations. Fluorescence measurements with an oxonol dye proved to be more sensitive than transmission measurements, particularly when recording from small processes. By changing the position of the cell relative to a laser microbeam while recording electrically from the cell body, it was possible to monitor the membrane potential in the cell body and in the process simultaneously. From the delay in response in the process, a lower limit for the mean conduction velocity of 0.2-0.6 m/sec was found for 2- to 6- micrometers processes. The mean space constants of processes were estimated by comparing the amplitudes of passive voltage responses in the cell body and growth cone. A lower limit of 400-950 micrometers was obtained for 4- to 7-micrometers processes.

Animals↗