Localization of pacemaking activity in early embryonic heart monitored using voltage-sensitive dye

Abstract

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 embryo¹. 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^2,3. Recently, potential sensitive dye-related absorption signals have provided a new method for monitoring spontaneous action potential activity in the early embryonic heart^4–7. 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)^8–10. 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^4,5, development of pacemaker potential^6,7 and cardiac rhythm generation⁷. With this method, originally introduced to record neuronal activity in invertebrate ganglia^11,12, 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–rhodanine dye (NK2761) as a potentiometric probe. This dye is an analogue of Dye XVII⁹ or Dye XXIII¹⁰.