We have developed a vibrating calcium-specific electrode to measure minute extracellular calcium gradients and thus infer the patterns of calcium currents that cross the surface of various cells and tissues. Low-resistance calcium electrodes (routinely approximately 500 M omega) are vibrated by means of orthogonally stacked piezoelectrical pushers, driven by a damped square wave at an optimal frequency of 0.5 Hz. Phase- sensitive detection of the electrode signal is performed with either analogue or digital electronics. The resulting data are superimposed on a video image of the preparation that is being measured. Depending on the background calcium concentration, this new device can readily and reliably measure steady extracellular differences of calcium concentration which are as small as 0.01% with spatial and temporal resolutions of a few microns and a few seconds, respectively. The digital version can attain a noise level of less than 1 microV. In exploratory studies, we have used this device to map and measure the patterns of calcium currents that cross the surface of growing fucoid eggs and tobacco pollen, moving amebae and Dictyostelium slugs, recently fertilized ascidian eggs, as well as nurse cells of Sarcophaga follicles. This approach should be easily extendable to other specific ion currents.

An ion-selective vibrating-microelectrode system, which was originally used to measure extracellular Ca(2+) gradients generated by Ca(2+) currents, was used to study K(+), H(+) and Ca(2+) transport in intact maize (Zea mays L.) roots and individual maize suspension cells. Comparisons were made between the vibrating ion-selective microelectrode, and a technique using stationary ion-selective microelectrodes to measure ionic gradients in the unstirred layer at the surface of plant roots. The vibrating-microelectrode system was shown to be a major improvement over stationary ion-selective microelectrodes, in terms of sensitivity and temporal resolution. With the vibrating ion microelectrode, it was easy to monitor K(+) influxes into maize roots in a background K(+) concentration of 10 mM or more, while stationary K(+) electrodes were limited to measurements in a background K(+) concentration of 0.3 mM or less. Also, with this system it was possible to conduct a detailed study of root Ca(2+) transport, which was previously not possible because of the small fluxes involved. For example, we were able to investigate the effect of the excision of maize roots on Ca(2+) influx. When an intact maize root was excised from the seedling at a position 3 cm from the site of measurement of Ca(2+) transport, a rapid fourfold stimulation of Ca(2+) influx was observed followed by dramatic oscillations in Ca(2+) flux, oscillating between Ca(2+) influx and efflux. These results clearly demonstrate that wound or perturbation responses of plant organs involve transient alterations in Ca(2+) transport, which had previously been inferred by demonstrations of touch-induced changes in cytoplasmic calcium. The sensitivity of this system allows for the measurement of ion fluxes in individual plant cells. Using vibrating K(+) and H(+)electrodes, it was possible to measure H(+)efflux and both K(+) influx and efflux in individual maize suspension cells under different conditions. The availability of this technique will greatly improve our ability to study ion transport at the cellular level, in intact plant tissues and organs, and in specialized cells, such as root hairs or guard cells.

In embryos of the equally cleaving marine gastropod Patella vulgata, the mesodermal stem cell is determined during the interval between the fifth and sixth cleavage by means of cellular interactions between one of the four vegetally located macromeres with the overlying animal micromeres. Shortly before and during this interaction phase an extracellular matrix (ECM) is present between the interacting cells. In this study the glycosylation-perturbing ionophore monensin was used to investigate the possible morphogenetic significance of the ECM. Incubation of 32-cell-stage Patella embryos in 10 M monensin results in radialized embryos in which none of the four macromeres interacts with the overlying animal micromeres. None of the macromeres is determined, therefore, to form mesoderm in such embryos. Trochophore larvae reared from these embryos retain their radial symmetry, as is indicated by the presence of four shell glands and four blastopore- or stomodeum-like invaginations in these larvae. The monensin-treated embryos probably secrete abnormal ECM that does not provide the proper conditions for the blastomeres to stretch and interact with the micromeres. Changes in intracellular ionic concentrations may also be involved.

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Early embryos of Patella vulgata have been injected with Lucifer Yellow. No restriction of dye spread was found. We show that later in the development, the larval trochophore stage present evidence of compartments of cell communication. These dye compartments coincide with different presumptive regions.