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First published online April 23, 2004
Journal of Experimental Biology 207, 1759-1769 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00955

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The role of muscarinic receptors and intracellular Ca²⁺ in the spectral reflectivity changes of squid iridophores

Lydia M. Mäthger^1,2,*, Toby F. T. Collins^1,2, and Pedro A. Lima^1,

¹ The Marine Biological Association of the United Kingdom, The Laboratory, Citadel Hill, Plymouth PL1 2PB, UK
² Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK

^* Author for correspondence at present address: Vision, Touch and Hearing Research Centre, University of Queensland, Brisbane, Queensland 4072, Australia (e-mail: l.mathger{at}uq.edu.au)

Accepted 24 February 2004

In this paper we describe changes in spectral reflectivity of the light reflectors (iridophores) of the squid Alloteuthis subulata. The spectral changes that can be seen in living squid, can also be brought about by superfusing whole skin preparations with acetylcholine (ACh) (20 µmol l^-1) and muscarine (30 µmol l^-1) but not nicotine (up to 50 mmol l^-1), suggesting that cholinergic muscarinic receptors are involved. Changing the osmolarity of the external solution had no effect on spectral reflectivity. To study the iridophores at the cellular level, iridophores were isolated enzymatically. Lucifer Yellow filled the iridophores uniformly, showing cellular individuality. Isolated iridophore cells were loaded with Fura-2 AM and cytoplasmic Ca²⁺ was recorded ratiometrically. Intracellular Ca²⁺ (resting concentration at 66.16 nmol l^-1) increased transiently after addition of ACh (50 µmol l^-1), muscarine (25 µmol l^-1), but not nicotine (up to 5 mmol l^-1). Ca²⁺ also increased when superfused with potassium chloride (10 mmol l^-1) and caffeine (2.5 mmol l^-1). Hypo- and hyperosmotic solutions had no effects on the cytoplasmic Ca²⁺. By presenting direct evidence that iridophores are polarised cellular structures containing Ca²⁺ stores and that they are activated via cholinergic muscarinic receptors, we demonstrate that Ca²⁺ is involved in the reflectivity changes of the iridophores of A. subulata. Specimens were prepared for transmission electron microscopy. It was found that the orientations of the plates with respect to the skin surface are in good agreement with the expected orientations based on the prediction that the iridophores act as multilayer reflectors.

Key words: squid, Alloteuthis subulata, invertebrate, multilayer reflector, reflectivity, iridophore, calcium, acetylcholine, muscarine receptor

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