Neural control of tuneable skin iridescence in squid

Abstract

Fast dynamic control of skin coloration is rare in the animal kingdom, whether it be pigmentary or structural. Iridescent structural coloration results when nanoscale structures disrupt incident light and selectively reflect specific colours. Unlike animals with fixed iridescent coloration (e.g. butterflies), squid iridophores (i.e. aggregations of iridescent cells in the skin) produce dynamically tuneable structural coloration, as exogenous application of acetylcholine (ACh) changes the colour and brightness output. Previous efforts to stimulate iridophores neurally or to identify the source of endogenous ACh were unsuccessful, leaving researchers to question the activation mechanism. We developed a novel neurophysiological preparation in the squid Doryteuthis pealeii and demonstrated that electrical stimulation of neurons in the skin shifts the spectral peak of the reflected light to shorter wavelengths (greater than 145 nm) and increases the peak reflectance (greater than 245%) of innervated iridophores. We show ACh is released within the iridophore layer and that extensive nerve branching is seen within the iridophore. The dynamic colour shift is significantly faster (17 s) than the peak reflectance increase (32 s), revealing two distinct mechanisms. Responses from a structurally altered preparation indicate that the reflectin protein condensation mechanism explains peak reflectance change, while an undiscovered mechanism causes the fast colour shift.

Figure 1.

Neurally activated iridescence in squid iridophores. (a) Doryteuthis pealeii have conspicuous pigmentary chromatophores and underlying structurally coloured iridophores. Box shows the tissue location for fin preparations. (b) ‘Intact fin’ preparation yields a green iridophore phenotype when a nearby nerve branch is stimulated (5 V, 300 µs pulses at 10 Hz for 15 s) through a proximal suction pipette. (c) Fin iridophores at rest have low peak reflectance. (d) After electrical stimulation of the fin nerve, a field of colour-shifted iridophores is clearly visible (see also electronic supplementary material, movie S1). (e) Dorsal mantle iridophores, often larger than fin iridophores, at rest are barely visible. (f) After electrical stimulation of dorsal nerve branches (15 V, 300 µs pulses at 10 Hz for 15 s), dorsal mantle iridophores also respond with a large shift in peak colour and peak reflectance similar to those in the fin. (g) Mean reflectance spectra of fin iridophores after stimulation. (h) Fin iridophores that shifted the most in peak reflected wavelength also did so faster (they shifted at higher nm s^–1; colours inside circles represent peak of the iridophore spectra at the farthest wavelength shift). (i,j) Timing of the peak wavelength shift and peak reflectance increase, respectively. The step in the red trace indicates the stimulation timing. Grey, standard deviation. n = 4 animals and 13 iridophores with one to three repeat measures from each iridophore. Scale bars: (b) 1 cm, (c–f) 1 mm.

Figure 2.

Neurally tuneable structural coloration. (a–c) Examples of single iridophore coloration before and after stimulation to a single nerve. Mean maximum response spectra shown on the right (two or three measurements). (d) A preparation as in figure 1b, with subsequent removal of the layers below and above the iridophore layer, such that only the nerves, iridophores and connective tissue remain. In this preparation, neural stimulation (before and after 15 V, 300 µs pulses at 10 Hz for 15 s shown) activates iridophores, but the responses now show a red phenotype (n = 3 animals and 5 iridophores, with three repeat measures from each iridophore; see also electronic supplementary material, movie S2). (e) Representative example of a single red iridophore response to electrical stimulation with mean maximum response spectra (three measurements). Note that automatic brightness correction was applied between acquisitions for images in (a–c) and (e). (f) Plot of maximal wavelength versus maximal normalized reflectance. The points are at approximately 0.3 s intervals. The green (left) curve shows the mean iridophore response from intact skin (same data as figure 1g–j), while the red (right) curve shows the mean iridophore response from the open skin preparation shown in figure 2d. Dots display the wavelength of maximum peak reflectance at each sampled time point. Grey, standard deviation. Scale bars: (a–c,e) 200 µm, (d) 1 mm.

Figure 3.

Structural coloration and peak reflectance have separate mechanisms. (a) Neurally stimulated squid skin (15 V, 300 µs pulses at 10 Hz for 15 s), with three dissected openings (epidermis and chromatophores removed). Only the iridophores beneath the altered tissue show a red phenotype. (b) Application of 200 mM ACh to the same preparation does not recover the green phenotype. (c) Neurally stimulating tissue where the fin muscle and ventral skin (both below the iridophore layer) had been removed results in a red phenotype (see also electronic supplementary material, movie S3). (d) Application of 200 mM ACh to the type of preparation shown in (c) did not recover the green phenotype. (In the picture shown, the ventral surface is facing the viewer.) In (a–c), arrowheads indicate activated iridophores below or above removed layers. (e) Normalization of the peak reflectance change and colour shift for the red phenotype (red circles) and green phenotype (red–green circles) shows that the peak reflectance and colour shift changes are proportional for most of the red phenotype response, and that the decay phase also follows the same constant. However, the peak reflectance versus colour shift responses in the green phenotype are not proportional, and the rise and decay phases show different dynamics. Dots display the wavelength of maximum peak reflectance at each sampled time point. (a–d) Scale bars, 1 mm.

Figure 4.

Iridophores are associated with neural processes. (a) Nerves in red (filled with Lucifer yellow, tagged with Dylight 633, excited with 633 nm) can be easily traced among the distinctive chromatophores (Θ) and iridophores (Δ) (imaged as autofluorescence using 405 and 514 nm laser lines) that they innervate. Confocal maximum-intensity projection image from squid fin skin, cleared with TDE, whole-mounted and scanned (50 µm of tissue; 40 × 15 tile Z-scan). (b) Lucifer yellow (red) travelled along axons, which displayed forked branches. In blue, scattered iridocytes, which are unresponsive to ACh, can be seen (imaged as autofluorescence using 405 nm). (c) Tracing (green lines) of several small axons (red signal) that diverged from the main nerve and extended minute processes within an iridophore (seen as an area of increased blue fluorescence). Only branches that could be identified reliably were traced in this tissue volume, which is shown as a sum of all the optical sections. The white box is shown at a higher magnification in (d) where axons depart from the large nerve bundle (top right part of the image). (e) Thin (0.5–1 µm) neuronal processes (in red) deep within the iridophore are labelled with acetylated alpha-tubulin antibody. Fine iridophore structure (in blue) is seen owing to tissue autofluorescence. Maximum-intensity projection image (16 µm thick). Scale bars: (a) 500 µm, (b) 150 µm, (c) 200 µm, (d) 17 µm, (e) 25 µm.