Calcium transient activity in cultured murine neural crest cells is regulated at the IP(3) receptor.

In a previous study we have shown that cultured neural crest cells exhibit spontaneous calcium transients and that these events are required for neurogenesis. In this study, we examine the mechanism that generates these calcium transients. Extracellular Ca(2+) modulates calcium transient activity. Lanthanum (La(3+)), a general calcium channel antagonist and zero extracellular Ca(2+), reduces the percentage of cells exhibiting calcium transients (26.2 and 40. 5%, respectively) and decreases calcium spiking frequency (4.5 to 1. 0 and 2.5 to 1.0 spikes/30 min, respectively). Intracellular calcium stores also contribute to the generation of calcium transients. Depleting the calcium stores of the endoplasmic reticulum (ER) reduces the percentage of active cells (15.7%) and calcium spiking frequency (2.8 to 1.5 spikes/30 min). Ryanodine (100 microM), which blocks calcium release regulated by the ryanodine receptor (RyR), had no effect on calcium transient activity. Blocking inositol 1,4, 5-triphosphate receptor (IP(3)R)-dependent calcium release, with elevated extracellular Mg(2+) (20 mM), abolished calcium transient activity. Mg(2+) did not block caffeine-sensitive calcium release (RyR-dependent) or voltage dependent calcium channels. Mg(2+) also suppressed thimerosal-induced calcium oscillations (IP(3)R-dependent). Small increases in the intracellular calcium concentration ([Ca(2+)](i)), increases the percentage of active cells and the calcium spiking frequency, while larger increases in [Ca(2+)](i) block the transients. Reducing intracellular IP(3) levels reduces the percentage of active cells and the calcium spiking frequency. We conclude that the mechanism for generating spontaneous calcium transients in cultured neural crest cells fits the model for IP(3)R-dependent calcium excitability of the ER.

Spontaneous calcium transients are required for neuronal differentiation of murine neural crest.

We have shown that cultured mouse neural crest (NC) cells exhibit transient increases in intracellular calcium. Up to 50% of the cultured NC-derived cells exhibited calcium transients during the period of neuronal differentiation. As neurogenic activity declined, so did the percentage of active NC-derived cells and their calcium spiking frequency. The decrease in calcium transient activity correlated with a decreased sensitivity to thimerosal, which sensitizes inositol 1,4,5-triphosphate receptors. Thimerosal increased the frequency of oscillations in active NC-derived cells and induced them in a subpopulation of quiescent cells. As neurogenesis ended, NC-derived cells became nonresponsive to thimerosal. Using the expression of time-dependent neuronal traits, we determined that neurons exhibited spontaneous calcium transients as early as a neuronal phenotype could be detected and continued through the acquisition of caffeine sensitivity, soon after which calcium transient activity stopped. A subpopulation of nonneuronal NC-derived cells exhibited calcium transient activity within the same time frame as neurogenesis in culture. Exposing NC-derived cells to 20 mM Mg(2+) blocked calcium transient activity and reduced neuronal number without affecting the survival of differentiated neurons. Using lineage-tracing analysis, we found that 50% of active NC-derived cells gave rise to clones containing neurons, while inactive cells did not. We hypothesize that calcium transient activity establishes a neuronal competence for undifferentiated NC cells.

Neurons differentiating from murine neural crest in culture exhibit sensory or sympathetic-like calcium currents.

The trunk neural crest gives rise to peripheral sensory and sympathetic neurons. In culture, neural crest cells can be induced to differentiate into either neuronal phenotype. Few studies have examined the differentiation of physiological properties in cultures of neural crest cells. Using whole-cell recordings, our study examined the effects of growth factors on high-voltage-activated calcium current profiles exhibited by neurons differentiating in culture. We compared these profiles with those exhibited by sensory and sympathetic neurons. Neural crest cells in culture gave rise to neurons with calcium current profiles identical to either sensory or sympathetic neurons, depending on the growth conditions. On average, the calcium current profile for sensory neurons was 23% (L), 51% (N), and 12% (P), while sympathetic neurons had a similar L-type current (20%), higher N-type (76%), and lower P-type (4%). Neural crest cells cultured with human leukemia inhibitory factor plus somite cells produced neurons with a sympathetic-like calcium current profile (L: 17%, N: 75%, and P: 4%). However, murine leukemia inhibitory factor (L: 25%, N: 52%, and P: 13%) and ciliary neurotrophic factor (L: 18%, N: 49%, and P: 9%) plus somite cells produced neurons with sensory-like calcium current profiles. These growth conditions did not modify the calcium current profiles of neurons cultured from embryonic and neonatal ganglia. Similarly, murine leukemia inhibitory factor produced a greater percentage of neurons (57%) with sensitivity to capsaicin (sensory phenotype) than human leukemia inhibitory factor (3%). Physiological traits can be a useful tool for the determination of neuronal phenotype in culture where other traits may be less stable.

The effect of sound level, temperature and dehydration on the brainstem auditory evoked potential in anuran amphibians.

Brainstem auditory evoked potentials (BAEPs) were used to examine the effects of sound level, temperature, and dehydration on the auditory pathway of three species of anuran amphibians: Rana pipiens, Bufo americanus and B. terrestris. BAEP latency, amplitude and a measure of threshold were determined for all stimulus and test conditions. Threshold values obtained with this technique were similar to other neural measures of threshold in anurans, and were stable for repeated measures within 12 h and over three days. Transient changes in temperature caused non-linear changes in BAEP threshold and latency. Above 20 degrees C small threshold shifts were elicited, while below 20 degrees C we observed rapid deterioration of threshold. Animals acclimated to a cold temperature (14 degrees C) were acoustically less sensitive than warm (21 degrees C) animals, even when both groups were tested at colder temperatures. Because peripheral components of the BAEP were most affected by both transient and acclimation (longer term) cooling and warming, the sensory epithelium appears to be the most temperature-sensitive component of the auditory pathway. Dehydrated frogs showed no auditory dysfunction until a critical level of dehydration was reached. More dehydration-resistant species (B. terrestris and B. americanus) were less susceptible to BAEP degradation near their critical dehydration level.

An aging model for surface acoustic wave devices.

The authors present a study of the phase-aging kinetics of a 591.2 MHz quartz-crystal surface acoustic wave (SAW) filter intended for application in an undersea telecommunication system. At aging temperatures from 50 to 140 degrees C, a previously established SAW-device aging model describes the time dependence of the phase aging. The results of an investigation of the temperature dependence of the coefficients in this aging model allows the authors to extend the model, capturing both the time and the temperature dependence of the degradation. They then identify and assess the sources of variation, or error, affecting the data and model, estimate the distributions of the errors, and incorporate these error distributions in the extended aging model. This leads to a composite aging model that describes the time and temperature dependence of the complete phase-aging distribution. The authors use this composite model to predict end-of-life phase-aging distributions, demonstrating that the devices exhibit the high level of stability required by the application.

Ventriculo-cisternal perfusion of twelve amino acids in the rabbit.

The clearances of twelve amino acids from the ventricles during ventriculo-cisternal perfusion in the rabbit have been measured; uptake by the brain was also measured and this permitted the separate computation of loss to brain and loss to blood during the perfusion. Clearance under carrier-free conditions was greater than when a concentration of 5mM unlabeled amino acid was present in the perfusion fluid. Brain uptake was also usually reduced by the presence of unlabeled amino acid due presumably to suppression of accumulation by brain cells. Reduction of transport across the blood-brain barrier would tend to increase brain uptake, and there was some evidence for a balance between the two opposing tendencies. Inhibition of clearance of a given labeled amino acid could be brought about by unlabeled amino acids of different molecular species. In general, the amino acids fell into three categories: neutral, acidic, and basic, and there was some overlap between them; of the neutral amino acids the A- and L-classification of Christensen was valid, although once again there was some overlap. If, during ventriculo-cisternal perfusion of a labeled amino acid, the activity of this labeled amino acid in the blood was raised well above that in the inflowing perfusion fluid, the labeled amino acid continued to be cleared from the perfusion fluid, suggesting uphill transport. On this basis it was suggested that the normally low concentrations of amino acids in the cerebrospinal fluid (CSF), by comparison with those in plasma, were due to an active transport from the CSF to the blood. Substrate-facilitated transport, whereby the penetration of labeled amino acid into the perfusion fluid from blood could be accelerated by adding unlabeled amino acid to the perfusion fluid, or vice versa, was demonstrated.