Detection of intracellular Ca²⁺ transients in sympathetic neurones using arsenazo III

Abstract

Changes in cytosolic calcium ion concentration ([Ca²⁺]_i) have been implicated in a wide variety of cellular stimulus–transduction roles^1–3. In nerve cells, it is believed that electrical activity raises [Ca²⁺]_i, by allowing influx of Ca²⁺ through voltage-dependent channels in the surface membrane^4,5. Elevation of neuronal [Ca²⁺]_i may also occur due to release of Ca²⁺ from intracellular storage sites^6–8. Transient increases in [Ca²⁺]_i are thought to trigger neurotransmitter release⁹, and to modulate axonal transport¹⁰, energy metabolism¹¹ and growth cone movement¹². Intracellular Ca²⁺ also appears to regulate membrane potassium channels and thereby to regulate electrical excitability¹³. Although [Ca²⁺]_i transients have been measured in a few giant invertebrate neurones¹⁴, detection of such transients in a vertebrate neurone has not been previously reported. We have measured [Ca²⁺]_i in bullfrog sympathetic neurones by photometry of a microinjected calcium indicator dye, arsenazo III (refs 14–16), and report here that action potentials and voltage-clamped depolarizations cause long-lasting increases in [Ca²⁺]_i. Also, exposure to the drug theophylline can cause spontaneous periodic increases in [Ca²⁺]_i. Comparisons of [Ca²⁺]_i signals with simultaneous intracellular recordings of membrane potential suggest that the kinetics of the post-tetanic hyperpolarization (PTH) following a series of action potentials or the spontaneous hyperpolarizations induced by theophylline directly reflect the kinetics of the [Ca²⁺]_i, transient.