Day 3: Movement of pigment granule in fish scales

by Charlotte Omoto

Introduction

Many animals including fish, amphibians, cephalopods like octopus, and arthropods like shrimp can change their coloration. Depending upon the circumstances, the change in coloration may serve to avoid predation or to attract mate. The chromatophores, cells containing pigment granules, can lighten by aggregating the pigment and darken by dispersing the pigment granules. In the zebrafish scales that we will use, there are different types chromatophores. There are melanocytes with black pigment, which have melanin, xanthophores with carotenoids, and perhaps erythrophores with red pigment. Although you may observe any of these, the melanocytes may be the easiest to observe.

Chromatophores are cells derived from the neural crest where our nerve cells originate so are sometimes called "paraneurons". The regulation of the pigment granule movement involves chemicals released by nerve terminals, neural transmitters, and endocrine hormones circulating in the blood. These chemicals bind to receptors on the chromatophore cell surface and cause a cascade of events inside the cells. The neural transmitter we will use is called norepinephrine. Hormones that we will use are melatonin and melanocyte stimulating hormone (MSH). MSH is a peptide hormone produced in the pituitary. Melatonin is produced in the pineal gland by modifications of the amino acid tryptophan, and is related in structure to another hormone, serotonin. In many vertebrates, melatonin release exhibits circadian regulation and is related to light levels. We will also use a chemical related to caffeine called isobutylmethylxanthine (IBMX).

In addition to pigment granule movement, zebrafish is a model vertebrate organism for studying development, among other things. For example Dr. Ho, here at Princeton, who donated the fish we are using, uses them to study embryonic development. http://www.molbio.princeton.edu/faculty/ho.html. For those of you interested in using zebrafish for other investigations, there is an electronic zebrafish guide at http://zfish.uoregon.edu/zf_info/zfbook/zfbk.html that gives more detail on caring for fish, and various techniques for using them.

The experiment

Prepare a microscope perfusion chamber

Suck up a fish scale up in a pasteur pipet and place a drop of fluid with the fish scale on a microscope slide. Place two thin coatings of vaseline on opposite sides of a coverslip (alternatively one can use double-stick tape). Gently place the coverslip on top of the fish scale so that the taped or vaseline edges makes contact with the edges of the glass slide, leaving the two sides open for introducing solution on one side and wicking the solution from the other.

Observe intact cells

Observe the cells on the scales. Depending on the type of optics you use, you may be able to see epithelial cells overlying the pigment cells. Melanocytes, the most visible are the ones with the big black blobs which are formed by the dispersed melanin granules. Check the appearance of these cells using different settings on the microscope. Choose the setting that you feel provides the best image of the cells, by changing the magnification, the condenser iris diaphragm and/or phase, if you have a phase-contrast microscope. Since you will need to decide whether the pigment granules are aggregated or dispersed, it may help to draw a diagram of their appearance before any treatment. Be sure that you can recognize aggregated and dispersed pigment granules before proceeding with an experiment.

Observe pigment granule aggregation and dispersion on intact cells

Now you are prepared to observe the effects of test solutions. Pick one of the solutions and place a drop on one side of the coverslip, while wicking the solution from the other side of the coverslip with a piece of filter paper. Use at least 2 to 3 drops of solution to make sure that you have exchanged the previous solution with your test solution. Observe the pigment cells over time, drawing the changed appearance of your selected cells. Make sure that the chamber does not dry up. This is best done by having excess solution by the coverslip. It is also advisable to turn down the intensity of the microscope lamp when you are not observing to prevent heating. When no more changes seem to be occurring, you can test the effect of the other solution. First rinse out the solution in the chamber with a couple of drops of TPS then introduce the other solution. Observe as before. If you have a poorly responding scale or the solution dries out, try a fresh scale from your petri dish.

Pre-Lab Preparation

Getting fish scales

Although we used zebrafish scales, most any (other than albino) fish scales will work, though they may have different responses to chemicals and different response rates, and may require a different saline solution to remain viable. For example, it is reported that goldfish xanthophores require 1/2 to 1 hour to complete aggregation and dispersion whereas squirrelfish erythrophores require only a few seconds! We pull the fish scales from anesthesized fish, and the fish are pretty much not much worse for wear, and can be returned to the fish tank. I keep these as ``pets'' in my lab between the lab exercises. Note, this procedure is different than the reference I give below where they leave the fish in the anesthetic until it dies. I found this not necessary by following the procedure below.

To anesthesize fish, prepare three 500 ml beakers with

1. anesthesizing solution\tab
   • 4.5 ml MS222 stock solution (0.4g/100ml water stored in dark bottle) from Sigma
   • 2 ml 1.5M Tris pH 8.8 (to neutralize pH)
   • 100 ml tank water
2. wash solution of 200 ml tank water
3. recovery solution of 500 ml tank water

• put one fish in anesthesizing solution 1-2 min
• you can use a bent spoon to scoop up the fish
• place fish dorsal slide up in a slit of foam under dissecting scope
• use sharp forceps to pull out scales from dark regions
• take 5 - 30 scales / fish depending on size of fish
• collect scales in TPS in small petri dish
• rinse fish in the second beaker (so that anesthetic is not placed back in the tank)
• let fish recover in the recovery tank before returning to main tank

Make buffers and solutions and have at room temperature

TPS (teleost physiological saline)		per. liter
NaCl	169. mM	9.87 g
KCl	5.36 mM	0.4 g
CaCl2	1.8 mM	18 ml 0.1M
MgCl2	1.3 mM	0.26 g
Tris	5.0 mM
Tris HCl		0.7 g
Tris base		0.067 g
glucose	5.6 mM	1.0 g

If made ahead of time, put in 100 ml aliquots and sterilize otherwise the glucose will allow things to grow in it.

Concentrations of test chemicals

chemical	fold X - stock solution recipe	final conc. in TPS
IBMX	100x - 0.11g/ml in DMSO in freezer	5 X 10-3 M
norepinephrine	100X - 0.017g/10 ml in DMSO in freezer	10-4 M
melatonin	1000x - 23mg in 10ml absolute ethanol	10-5 M
MSH	100 X - 1mg in 6.2ml of TPS	10-6 M

Reference

Nyquist, Sally E. & Toner, Kathryn Brady 1997. Pigment Granule Transport in Chromatophores. Chapter 8 volume 18 Tested Studies for Laboratory Teaching.

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