New applications of laser microbeam irradiation to cell and developmental biology include a new instrument with a tunable wavelength (217- to 800-nanometer) laser microbeam and a wide range of energies and exposure durations (down to 25 × 10
-12
second). Laser microbeams can be used for microirradiation of selected nucleolar genetic regions and for laser microdissection of mitotic and cytoplasmic organelles. They are also used to disrupt the developing neurosensory appendages of the cricket and the imaginal discs of
Drosophila
.
Laser microirradiation of neonatal rat (1 to 2-day-old) ventricular cells in tissue culture results in overt changes in contractility. The intracellular study of their ongoing electrical activity prior to, during, and after laser microirradiation demonstrates that definite membrane alteration occurs concomitantly with induced contractile responses. Although all ventricular cells are depolarized by laser microirradiation, the ultimate response elicited seems to differ according to the type of myocardial cell impaled. Typical fibrillation potentials were induced mainly in pacemaker cells.
Intracellular analysis of neonatal rat (1-2 day old) ventricular cells in culture shows that contracting myocardial cells exhibit an array of different patterns of spontaneous electrical activity. Resting membrane potentials varied between -40 mV and -98 mV. Our results indicate that some cultured cells show resting membrane potentials, overshoot, and total spike amplitude values comparable to those normally found in neonatal and adult rat heart. A low ratio of pacemaker (40%) to nonpacemaker cells (60%) and low incidence of hyperpolarizing after-potentials (35%) were found. La3+ application (0.1-4.0 mM) induced progressive cell depolarization, concomitant diminution in discharge frequency, and marked alteration of action potential configuration. A parallel decline in frequency and strength of rhythmic contractions was observed. Abolition of contractility occurred only in association with depolarization and complete disappearance of action potentials. Recovery of electrical and contractile activity followed medium replacement. Our results indicate that La3+ does not act as a specific excitation-contraction (E-C) uncoupler in the cultured cells but has multiple effects upon their normal electrical characteristics.
Rat myocardial cells in vitro were irradiated in individual mitochondria with an argon ion laser microbeam. The contractile respone termed fibrillation in single and multicellular groups of both ventricle and auricle cells were compared. Specific correlations were made between fibrillation duration, the number of cells in the group, and the number of times the cells had fibrillated. Correlations were also made for the number of laser shots needed to induce fibrillation and the number of cells in the group. Another set of correlations were made between the pre-irradiation beat frequency and the beat frequency following recovery. Several differences and similarities of the above parameters were detected between auricle and ventricle cells. A comparison of the morphology and ultrastructure of auricle and ventricle cells also revealed significant differences.
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