Direct intracellular measurement of electrical events in the early embryonic heart is impossible because the cells are too small and frail to be impaled with microelectrodes; it is also not possible to apply conventional electrophysiological techniques to the early embryonic heart. For these reasons, complete understanding of the ontogeny of electrical activity and related physiological functions of the heart during early development has been hampered. Optical signals from voltage-sensitive dyes have provided a new powerful tool for monitoring changes in transmembrane voltage in a wide variety of living preparations. With this technique it is possible to make optical recordings from the cells that are inaccessible to microelectrodes. An additional advantage of the optical method for recording membrane potential activity is that electrical activity can be monitored simultaneously from many sites in a preparation. Thus, applying a multiple-site optical recording method with a 100- or 144-element photodiode array and voltage-sensitive dyes, we have been able to monitor, for the first time, spontaneous electrical activity in prefused cardiac primordia in the early chick embryos at the six- and the early seven-somite stages of development. We were able to determine that the time of initiation of the contraction is the middle period of the nine-somite stage. In the rat embryonic heart, the onset of spontaneous electrical activity and contraction occurs at the three-somite stage. In this review, a new view of the ontogenetic sequence of spontaneous electrical activity and related physiological functions such as ionic properties, pacemaker function, conduction, and characteristics of excitation-contraction coupling in the early embryonic heart are discussed.

Early in cardiogenesis, heart primordia are brought together at the midline and fuse with each other progressively caudally-- this results in the formation of the primitive tubular heart which begins beating spontaneously at the middle period of the 9-somite developmental stage in the chick embryo. However, in these very early stages of development, the myocardial cells are small and technically difficult to impale with microelectrodes; thus electrophysiological studies on the very early embryonic heart are rare. Recently, potential sensitive dye-related absorption signals have provided a new method for monitoring spontaneous action potential activity in the early embryonic heart. This technique is based on the observation that changes in potential across membrane(s) stained with certain voltage-sensitive dyes are accompanied by changes in their optical properties (absorption, fluorescence, and/or birefringence). Using absorption signals, we have already demonstrated in embryonic pre-beating chick heart in the 7-8-somite stages, the occurrence of action potential activity, development of pacemaker potential and cardiac rhythm generation. With this method, originally introduced to record neuronal activity in invertebrate ganglia, many cells or portions of the preparation can be monitored simultaneously. Accordingly we have expanded the optical recording apparatus to monitor simultaneously spontaneous action potentials from five portions of an early embryonic heart, and report here experiments carried out on the embryonic hearts of chicks (white Leghorn) at the 7-11-somite developmental stages, corresponding to 25-35 h of incubation. The hearts attached to the embryo were stained with a merocyanine-rhodamine dye (NK2761) as a potentiometric probe. This dye is analogue of Dye XVII or Dye XXIII.

In an effort to increase the utility of optical methods for measuring membrane potential in excitable cells, an additional 369 dyes were tested on giant axons from the squid. Several promising dyes with relatively large absorption and fluorescence signals are described. In addition, a simple modification of the apparatus led to a sixfold increase in the size of dye-related birefringence signals. In preparations with a suitable geometry, these signals are as large as absorption signals but photodynamic damage and bleaching are eliminated when wavelengths longer than the absorption band are used.

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