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R A Silver

Publications and source records attributed to R A Silver.

At least 19 recordsLinked to original sources
PubMed1998

Locus of frequency-dependent depression identified with multiple-probability fluctuation analysis at rat climbing fibre-Purkinje cell synapses.

1. EPSCs were recorded under whole-cell voltage clamp at room temperature from Purkinje cells in slices of cerebellum from 12- to 14-day-old rats. EPSCs from individual climbing fibre (CF) inputs were identified on the basis of their large size, paired-pulse depression and all-or-none appearance in response to a graded stimulus. 2. Synaptic transmission was investigated over a wide range of experimentally imposed release probabilities by analysing fluctuations in the peak of the EPSC. Release probability was manipulated by altering the extracellular [Ca2+] and [Mg2+]. Quantal parameters were estimated from plots of coefficient of variation (CV) or variance against mean conductance by fitting a multinomial model that incorporated both spatial variation in quantal size and non-uniform release probability. This 'multiple-probability fluctuation' (MPF) analysis gave an estimate of 510 +/- 50 for the number of functional release sites (N) and a quantal size (q) of 0.5 +/- 0.03 nS (n = 6). 3. Control experiments, and simulations examining the effects of non-uniform release probability, indicate that MPF analysis provides a reliable estimate of quantal parameters. Direct measurement of quantal amplitudes in the presence of 5 mM Sr2+, which gave asynchronous release, yielded distributions with a mean quantal size of 0.55 +/- 0.01 nS and a CV of 0.37 +/- 0.01 (n = 4). Similar estimates of q were obtained in 2 mM Ca2+ when release probability was lowered with the calcium channel blocker Cd2+. The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1 microM) reduced both the evoked current and the quantal size (estimated with MPF analysis) to a similar degree, but did not affect the estimate of N. 4. We used MPF analysis to identify those quantal parameters that change during frequency-dependent depression at climbing fibre-Purkinje cell synaptic connections. At low stimulation frequencies, the mean release probability (pr) was unusually high (0.90 +/- 0.03 at 0.033 Hz, n = 5), but as the frequency of stimulation was increased, pr fell dramatically (0.02 +/- 0.01 at 10 Hz, n = 4) with no apparent change in either q or N. This indicates that the observed 50-fold depression in EPSC amplitude is presynaptic in origin. 5. Presynaptic frequency-dependent depression was investigated with double-pulse and multiple-pulse protocols. EPSC recovery, following simultaneous release at practically all sites, was slow, being well fitted by the sum of two exponential functions (time constants of 0.35 +/- 0.09 and 3.2 +/- 0.4 s, n = 5). EPSC recovery following sustained stimulation was even slower. We propose that presynaptic depression at CF synapses reflects a slow recovery of release probability following release of each quantum of transmitter. 6. The large number of functional release sites, relatively large quantal size, and unusual dynamics of transmitter release at the CF synapse appear specialized to ensure highly reliable olivocerebellar transmission at low frequencies but to limit transmission at higher frequencies.

Animals↗
PubMed1996

Non-NMDA glutamate receptor occupancy and open probability at a rat cerebellar synapse with single and multiple release sites.

1. Excitatory postsynaptic currents (EPSCs) were recorded under whole-cell voltage clamp from granule cells in slices of rat cerebellum. EPSCs from individual mossy fibre inputs were identified by their all-or-none appearance in response to a graded stimulus. Excitatory synaptic transmission was investigated at room temperature (approximately 24 degrees C) and at near-physiological temperature (approximately 34 degrees C) by analysing current fluctuations in the peak and decay of the non-N-methyl-D-aspartate (non-NMDA) component of EPSCs. 2. In a subset of synapses the mean EPSC amplitude remained unchanged as the probability of transmitter release was substantially lowered by raising the extracellular [Mg2+] and lowering [Ca2+]. These synapses were considered to have only one functional release site. Single-site synapses had small EPSCs (139 +/- 16 pS, n = 5, at 24 degrees C) with a large coefficient of variation (c.v. = 0.23 +/- 0.02, n = 5) and an amplitude distribution that was well fitted by a Gaussian distribution in four out of five cases. The EPSC latency had a unimodal distribution and its standard deviation had a temperature dependence with a temperature coefficient (Q10; range, 24-35 degrees C) of 2.4 +/- 0.4 (n = 4). 3. Peak-scaled non-stationary fluctuation analysis of single-site EPSCs indicated that the mean conductance of the underlying non-NMDA channels was 12 +/- 2 pS (n = 4) at 35 degrees C. Upper and lower limits for mean channel open probability (Po), calculated from fluctuations in the EPSC peak amplitude, were 0.51 and 0.38, respectively. These estimates, together with the open probability of the channel when bound by transmitter, suggest that only about 50% of the non-NMDA channels were occupied following the release of a quantum of transmitter. 4. At some multi-site synapses EPSCs had a low c.v. (0.4 +/- 0.01, n = 5) at 34 degrees C and non-stationary fluctuation analysis gave a parabolic variance-mean current relationship. This suggests that practically all of the non-NMDA receptors were occupied by glutamate at the peak of EPSC. The channel open probability (Po = 0.84 +/- 0.03, n = 5) at these 'saturated' multi-site synapses will therefore equal the open probability of the channel when bound by transmitter (Po,max). 5. Non-stationary fluctuation analysis of EPSCs from 'saturating' multi-site synapses indicated that 170 +/- 40 postsynaptic non-NMDA channels were exposed to transmitter at the peak of the EPSC. The mean conductance of the synaptic channels was 10 +/- 2 pS (n = 5) at 34 degrees C. 6. At synapses with multiple release sites the EPSC decay time became faster when release probability was lowered (by reducing the external [Ca2+]/[Mg2+] ratio), indicating that the transmitter concentration profile depended on release probability. No such speeding of the EPSC decay was observed at single-site synapses. 7. Our results suggest that release of a packet of transmitter from a single release site does not saturate postsynaptic non-NMDA receptors at cerebellar mossy fibre-granule cell synapses. However, at multi-site synapses transmitter released from neighbouring sites can overlap, changing the transmitter concentration profile in the synaptic cleft. We conclude that the level of postsynaptic receptor occupancy can depend on the probability of transmitter release at individual multi-site synapses.

Animals↗
PubMed1996

Deactivation and desensitization of non-NMDA receptors in patches and the time course of EPSCs in rat cerebellar granule cells.

1. Spontaneous and evoked non-NMDA receptor-mediated EPSCs were recorded from cerebellar granule cells in slices at approximately 24 and approximately 34 degrees C. The EPSC decay was fitted with the sum of two exponential functions. 2. The time courses of non-NMDA receptor deactivation and desensitization were determined with fast concentration jumps of glutamate onto patches from cultured granule cells. Deactivation (decay time constant tau = 0.6 ms at 24 degrees C) was substantially faster than desensitization (tau = 4 ms). Both processes were fitted by single exponential functions. 3. The decay of the fast component of the spontaneous EPSC (tau EPSCfast = 0.9 ms at 23 degrees C) was marginally slower than deactivation but too fast to be determined by desensitization. Our results suggest that the decay of this component is set by both the rate of decline of transmitter concentration and channel deactivation. 4. A simple diffusion model predicts that the time course of transmitter in the cleft declines slowly during the later stages of its action. The slow phase of transmitter removal could account for the time course of the slow component of the spontaneous EPSC (tau EPSCslow = 8 ms at 23 degrees C).

Animals↗
PubMed1993

Glutamate uptake from the synaptic cleft does not shape the decay of the non-NMDA component of the synaptic current.

To study the role of glutamate uptake at central glutamatergic synapses, we used the uptake blocker L-transpyrrolidine-2,4-dicarboxylate (PDC). The effects of PDC on the glutamate uptake current in salamander retinal glia indicated that PDC competes with glutamate for transport on the uptake carrier and that 300 microM PDC should significantly reduce the uptake of glutamate during the synaptic current. In isolated rat hippocampal neurons, 300 microM PDC did not affect non-N-methyl-D-aspartate (NMDA) receptor currents, but reduced NMDA receptor currents by 30%. In hippocampal and cerebellar slices, whereas 300 microM PDC reduced the NMDA component of excitatory synaptic currents by 50%, it reduced the non-NMDA component only slightly with no change in its decay time constant. Thus, the decay rate of the non-NMDA component is not set by the rate of glutamate uptake from the synaptic cleft into the presynaptic terminal.

Animals↗
PubMed1993

Estimated conductance of glutamate receptor channels activated during EPSCs at the cerebellar mossy fiber-granule cell synapse.

We have analyzed the variance associated with the decay of the non-NMDA receptor component of synaptic currents, recorded from mossy fiber-granule cell synapses in cerebellar slices, to obtain a conductance estimate for the synaptic channel. Current fluctuations arising from the random channel gating properties were separated from those arising from the fluctuations in the population of channels by subtracting the mean excitatory postsynaptic current (EPSC) waveform scaled to the EPSC peak amplitude. A weighted mean single-channel conductance of approximately 20 pS was determined from the relationship between the mean current and the variance around the mean during the decay of evoked and spontaneous synaptic currents. This result suggests that high conductance non-NMDA channels, such as the 10-30 pS glutamate receptor channel previously characterized in granule cells, carry the majority of the fast component of the EPSC at this synapse. In addition, our data are consistent with the activation of surprisingly few (approximately 10) non-NMDA channels by a single packet of transmitter.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1992

Rapid-time-course miniature and evoked excitatory currents at cerebellar synapses in situ.

Neurotransmission from mossy fibre terminals onto cerebellar granule cells is almost certainly mediated by L-glutamate. By taking advantage of the small soma size, limited number of processes and short dendrite length of granule cells, we have obtained high-resolution recordings of spontaneous miniature excitatory postsynaptic currents (m.e.p.s.cs) and evoked currents in thin cerebellar slices. Miniature currents have a similar time-course and pharmacology to evoked currents and consist of an exceptionally fast non-NMDA (N-methyl-D-aspartate) component (measured rise-time, 200 microseconds; estimated pre-filtered rise-time less than 100 microseconds; decay time constant, tau = 1.0 ms), followed by 50 pS NMDA channel openings that are directly resolvable. We could find no evidence for the recent proposal that miniature currents in granule cells are mediated solely by NMDA channels with a novel time course. The non-NMDA receptor component of m.e.p.s.cs has a skewed amplitude distribution, which suggests potential complications for quantal analysis. The difference in time course between the m.e.p.s.cs reported here and other synaptic currents in the brain could reflect differences in synaptic function or electrotonic filtering; the relative contribution of these possibilities has yet to be established.

Animals↗
PubMed1992

Intracellular ion imaging using fluorescent dyes: artefacts and limits to resolution.

Development of highly efficient fluorescent ratio indicators has made imaging of ion concentrations within individual cells possible (Grynkiewicz et al. 1985; Tsien and Poenie 1986). Ion imaging is a complex technique and is therefore prone to artefacts. In this paper we investigate the limits of the technique and its potential pitfalls. The spatial resolution of an imaging system is determined for different cell geometries. We describe a technique to increase the time resolution of existing systems by using a single excitation wavelength to measure changes in ion concentration. We demonstrate examples of potential artefacts arising from hardware limitations, image processing and fundamental optics. Methods for recognition and minimization of these problems are discussed.

Calcium↗
PubMed1991

Mitogens induce calcium transients in both dividing and terminally differentiating keratinocytes.

During terminal differentiation, keratinocytes lose the ability to divide. One indicator of responsiveness to certain growth factors is a transient rise in the intracellular concentration of free calcium ions ([Ca2+]i). The aim of our experiments was to discover whether or not terminally differentiating keratinocytes have lost the ability to exhibit an increase in [Ca2+]i in response to factors that stimulate [3H]thymidine incorporation and increase [Ca2+]i in undifferentiated keratinocytes. [Ca2+]i was measured with the calcium indicator dye FURA-2 and by a ratio imaging method. Expression of involucrin, a precursor of the keratinocyte cornified envelope, was used as a marker of terminal differentiation. Measurements were made on stratified colonies of cells grown in standard medium (containing 1.8 mM calcium ions) and on cell monolayers in low calcium medium (0.1 mM). Treatment of serum-starved monolayers with substance P, bombesin or complete growth medium containing 10% fetal calf serum resulted in increased [3H]thymidine incorporation. A switch from low calcium to standard medium also stimulated [3H]thymidine incorporation whether or not the cells had been serum-starved. In each experiment some cells showed an increase in [Ca2+]i while others did not. However, the heterogeneity in the [Ca2+]i response did not reflect the terminal differentiation status of individual cells: both involucrin-positive and -negative cells were found in the responding and nonresponding populations. Involucrin-positive and -negative areas of stratified cultures also underwent a transient increase in [Ca2+]i in response to serum-containing medium. Our data therefore indicate that both proliferating (involucrin-negative) and post-mitotic, terminally differentiating (involucrin-positive) keratinocytes can respond to mitogenic stimuli by an increase in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood↗
PubMed1991

Expression of T-type calcium current precedes neurite extension in neuroblastoma cells.

1. N1E-115 mouse neuroblastoma cells morphologically differentiate by extending neurites in a period of seven days after addition of 2% DMSO to the culture medium. We used the whole-cell patch clamp technique to measure calcium currents in these cells under conditions where voltage clamp of the whole membrane was assured. 2. Current densities of both T and L type calcium currents were identical in cells included to differentiate with dibutyryl cyclic AMP and cells induced to differentiate with dimethylsulphoxide (DMSO). Cells differentiated with DMSO were used for all subsequent experiments. 3. All morphologically differentiated cells showed a T type calcium current. In contrast, a minority of morphologically undifferentiated cells did not show a T current. 4. Once expressed, both T and L currents did not change either in current density or in behaviour over a period of five days. 5. These data demonstrate that expression of a T current always precedes neurite extension, and suggest a role for calcium currents in triggering morphological differentiation.

Animals↗
PubMed1989

Elevated cytosolic calcium in the growth cone inhibits neurite elongation in neuroblastoma cells: correlation of behavioral states with cytosolic calcium concentration.

Schubert (1984) and Kater et al. (1988) have suggested that motility and growth at the neuronal growth cone is activated by an increase of cytosolic free calcium concentration ([Ca2+]i) above the levels found in quiescent growth cones. In order to test this model, we have used a digital imaging fluorescence microscope together with injection of the fluorescent indicator dye Fura-2 to measure [Ca2+]i in growth cones of a mammalian sympathetic neuron, the N1E-115 neuroblastoma cell. The behavior of individual growth cones, together with spontaneously varying levels of [Ca2+]i within the growth cone, were monitored for periods of up to several hours. [Ca2+]i in motile, advancing growth cones was low and equal to [Ca2+]i in quiescent growth cones. Higher values of [Ca2+]i were found in motile growth cones that were not advancing, suggesting that a small elevation of [Ca2+]i inhibits neurite extension. A further rise of [Ca2+]i above the level found in motile, nonadvancing growth cones appeared to inhibit motility and cause retraction of the growth cone back towards the cell body. Spatial gradients of [Ca2+]i within the growth cone were small and, where statistically significant, [Ca2+]i was lower by 5-10 nM in motile regions. Our results are incompatible with the model that a rise of [Ca2+]i is responsible for activating quiescent growth cones; however, our results suggest that in active growth cones [Ca2+]i can regulate morphology and behavior.

Animals↗
PubMed1985

Phase I-II trial of mitoxantrone in acute leukemia: an interim report.

We evaluated the effect of mitoxantrone (Novantrone; dihydroxyanthracenedione) in the treatment of refractory acute leukemia and acute leukemia in relapse. In this study, 70 patients are currently evaluable. Of the 25 patients who received mitoxantrone 10 mg/m2 X 5, two of 10 with ANLL in relapse, one of five with ALL in relapse achieved complete remission, and one of seven with blastic phase CML responded. At a dose of 12 mg/m2 X 5, nine of 22 patients with ANLL in relapse, one of five patients with blastic phase CML and none of the nine patients with ALL responded. At this dose all remissions occurred after one course of treatment. None of the patients with ANLL or ALL refractory to primary therapy achieved a remission. Toxicities encountered with both dose levels were comparable. However, second courses at 12 mg/m2 X 5 led to severe stomatitis and prolonged cytopenia. We conclude that mitoxantrone is effective therapy for ANLL in relapse and that 12 mg/m2 per day X 5 is the optimal dose schedule. A randomized trial comparing daunorubicin with mitoxantrone in combination with cytarabine in untreated patients with ANLL should answer whether mitoxantrone is less toxic and whether it should replace daunorubicin in standard induction therapy in ANLL.

Acute Disease↗
PubMed1980

Cults.

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Humans↗
PubMed1979

The use of the Vidal-Adrey external fixation system. Part 2: The treatment of infected and previously infected pseudarthrosis.

A total of 23 infected or previously infected non-unions were treated by the Vidal-Adrey modification of the Hoffmann apparatus. The external fixators were applied after decortication and bone grafting either immediately in cases with no evidence of infection or later in cases of draining, infected wounds. The average healing time for these non-unions, that is time when no external support was required, was 12 months after the initial application of the Vidal-Adrey external fixators. The results were considered good or acceptable in 21 of 23 cases. Union occurred in 21, although union did not necessarily mean a good result overall when all other factors (shortening, etc.) were considered. This fixation device, when correctly applied and in conjuction with adequate debridement and bone grafting is an excellent method for the treatment of severe non-unions where infection is or was a problem.

Adult↗