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Introduction
Background adaptation is the process of changing color to blend with the environment or to signal other animals. Lower vertebrates use melanophores to accomplish this process. Melanophores are specialized skin cells containing thousands of melanosomes. These are small membrane-enclosed organelles containing black or brown pigment melanin (Jayawickreme & Lerner n. d.). Melanosomes can aggregate at the cell center, making the cells look pale. Alternatively, they can disperse throughout the cell, making it dark. The transport of melanosomes is regulated by neuronal or hormonal external stimuli (Hedberg 2009). In the following experiments, the effects of different hormones were tested on the melanophore of the zebrafish. These experiments were aimed at illustrating the principles of cell surface receptors to signal transduction mechanisms.
Methods
Scales containing melanophores from the zebrafish were obtained from a salt solution. A 30µl of the salt solution was placed in a well on a glass slide and a scale was placed in the well, lying flat. At the beginning of the experiment, melanophores were observed using a light microscope. 7 to 10 melanophores on each scale were used to determine the state of dispersal or aggregation. Dispersal or aggregation was then qualitatively scored as follows: Dispersal 1(D1) – Small amount of dispersal, Dispersal 2 (D2)-Large amount of dispersal, Aggregation 1 (A1)-Small amount of aggregation and Aggregation 2 (A2)-Large amount of aggregation.
In experiment 1, the saline solution was removed and replaced with 40µl of 10-11M MCH. Dispersal or aggregation of pigment granules was observed for more than two minutes and recorded. The MCH was then removed and replaced with a higher concentration of the same solution. This process was repeated in increasing concentrations as follows: 10-11M, 10-10M, 10-9M and 10-8M. In experiment II, adrenaline and isoprenaline were used. In the first trial, 10-6M of adrenaline was used and in the second trial, increasing concentrations were added as follows: 10-8M, 3 x 10-8M, 10-7M, 3 x 10-7M and 10-6M. Isoprenaline was then added in increasing concentrations as follows: 10-7M, 10-6M, 10-5M and 10-4M in the first trial. In trial 2, the effect of isoprenaline was observed in the presence of MCH and Fish Ringer (FR) on different scales. Using the dose that caused aggregation in experiment one (10-6M), maximum aggregation was allowed to occur. Thereafter, increasing isoprenaline doses were added on one scale. On another scale, FR was added instead of isoprenaline.
In experiment 3, 10-6M of phentolamine and propanol were applied alone for ten minutes. Without washing out the phentolamine, a cocktail of adrenaline and phentolamine was added. The same procedure was repeated with propanol. In trial 1, 10-6 M of phentolamine was used with increasing concentrations of adrenaline as follows: 10-8M, 3 X 10-8M, 10-7M, 3 X 10-7M and 10-6M. Propanol was also used with the same doses of adrenaline. In experiment 4, 10-5M Acetycholine was used in trial 1. In trial 2, increasing concentrations of acetylcholine were applied as follows: 10-7M, 3 x 10-7M, 10-6M, 3 x 10-6M and 10-5M.
In practical 6 (experiment 1), two scales were incubated with 10-9M MCH until there was no further aggregation of melanophores. They were then washed in FR for ten seconds and 10-5M of forskolin (2µL 10-3M forskolin + 198 µL FR) added to one scale, while FR was added to the other scale. These were observed for 5 minutes and comparisons were made. In experiment 2, instead of forskolin, 10-3M 3-isobutyl-1-methylxanthine (IBMX) (3µL 10-1M IBMX + 297µl of FR) was applied. In experiment 3, 10-4M 8-bromo cAMP (2µL 10-2M 8-bromo cAMP + 198µL FR) was used instead of forskolin. In experiment 4, 10-4M Ca ionophore (2µL 10-2M Ca ionophore + 198 µl FR) was applied and observed for 5 minutes. In experiment 5, one scale was incubated with 10-3M neomycin (20 µL 10-2M neomycin+ 180 µL FR) for 10 minutes and another with adrenaline. The scale with neomycin was added increasing concentrations of adrenaline as follows: 10-8M, 3 X 10-8, 10-7, 3 X 10-7M and 10-6M.
In experiment 6, a scale was incubated with Arginine Vasotocin (AVT) concentrations as follows: 10-8M, 3 x 10-8M, 10-7M, 3 x 10-7M and 10-6M. In trial 2, two scales were incubated with 10-9M MCH then the doses of AVT were applied on one scale and FR to the other. In experiment 7, 10-4M of 8-Bromo cGMP (2 µL 10-2m 8-Bromo-cGMP+198 µL FR) was applied to a scale and observed for five minutes. In trial 2, two scales were incubated with 10-9M MCH then 10-4M of 8-Bromo cGMP was added to one scale and FR to the other. In experiment 8, 10-4M of Sodium Nitroprusside (SNP) (10µL OOF 10-2M + 990µl FR) was added to a scale and observed for five minutes. In trial 2, two scales were incubated with 10-9M MCH. They were then washed for 10 seconds with FR. 10-4M SNP was added to one scale and FR to the other scale. These were observed for five minutes.
Results
In experiment one, higher concentrations of MCH led to large amounts of Aggregation.
MCH dose-response effect
In experiment 2, a high concentration of adrenaline led to small amounts of aggregations (trial 1) while a gradual increase in concentrations led to large amounts of concentration. (trial 2).
Adrenaline Trial 1
Adrenaline Trial 2
Application of higher concentrations of Isoprenaline led to an increase in dispersal.
Isoprenaline Trial 1
Isoprenaline Trial 2
The MCH added did not have any effect on the action of isoprenaline.
Phentolamine: alpha-adrenergic receptor antagonist
Phentolamine caused large amounts of dispersal while the cocktail caused aggregation.
Propranolol: beta-adrenergic receptor antagonist
Propanolol caused aggregation which was increased by addition of adrenaline
Antagonist Trial 1: Phentolamine (10-6M) + increasing doses of adrenaline
Increased doses led to further dispersal
Antagonist Trial 2: Propranolol (10-6M) + increasing doses of adrenaline
Increased doses of adrenaline led to aggregation
Acetylcholine Trial 1
Acetylcholine led to aggregation in small amounts
Experiment 1: Forskolin
The Forskolin and the FR showed small amounts of dispersal
Experiment 2: 3-isobutyl-1-methylxanthine (IBMX)
The IBMX showed large amounts of dispersal while the FR showed no change
Experiment 3: 8-bromo cAMP
The 8-bromo cAMP showed dispersal while FR showed no change.
Experiment 4: Calcium ionophore
Calcium ionophore resulted in small amounts of aggregation
Experiment 5: Neomycin
The neomycin in the presence of adrenaline led to large amounts of aggregation
In this one we use Adr instead of the Fish Ringer.
Experiment 6: Arginine vasotocin
AVT in the presence of MCH led to small amounts of dispersal
Experiment 7: 8-bromo cGMP
8-bromo cGMP led to large amounts of aggregation but when they were already aggregated, it led to large amounts of dispersal
Experiment 8: SNP
SNP led to aggregation but when aggregation was already present, it led to large amounts of dispersal.
Conclusion
Hormones stimulate receptors that either lower or raise intracellular second messenger levels of cAMP. cAMP levels determine the color of cells because it controls the molecular motors responsible for positioning pigment within the cell (Jayawickreme & Lerner). In this experiment, the hormones used are melanin-concentrating hormone (MCH), adrenaline, and isoprenaline. Melanosomes aggregates in response to MCH in a dose-dependent manner (Logan, Burn & Jackson 2006) as shown in the results. Adrenaline acts on both alpha and beta-adrenergic receptors which is why it resulted in aggregation. Isoprenaline activates beta-adrenergic receptors that are linked to the cAMP pathway and resulted in dispersal. Results of the phentolamine and adrenaline cocktail also indicated that adrenaline binds to alpha-adrenergic receptors (Thaler & Haimo 1992) because the adrenaline was not inhibited from binding leading to aggregation. Acetylcholine has only a small effect on the melanophores and this was achieved at the highest dosage.
Results of forskolin, IBMX AND 8-bromo cAMP experiments support data from other sources. These three increase the intracellular level of cAMP leading to dispersal as shown in the results. The presence of forskolin and MCH together produced very little dispersal indicating that they act in opposition in the cAMP pathway (Logan et al. 2009). Neomycin and calcium ionophore had similar results. These two increase the level of intracellular Ca2+ (another messenger) and this may be the reason why it led to aggregation. Results of these experiments supported other experiments that show that many lower vertebrates like zebrafish display a rapid physiological color change in response to hormones (Logan et al., p. 1 2006). Data obtained from the experiments is also similar to that obtained in previous physiology classes. There are a few errors that may have occurred in one of the experiments. In practical 6, experiment 1, the control (FR) had the same result as the forskolin. This, and the lack of support from other sources of data, makes the result of this particular experiment inconclusive.
Reference List
Hedberg, D 2009, Melanosome transfer, photoreception and toxicity assays in Melanophores, Web.
Jayawickreme, CK, & Lerner, MR n.d., Melanophore recombinant receptor systems. Web.
Logan D, Burn FS, & Jackson, J 2006, ‘Regulation of pigmentation in Zebrafish Melanophores’, Pigment Cell Research, vol. 19, no. 3, pp. 206-219.
Thaler, C D, & Haimo, L T 1992, ‘Control of organelle transport in melanophores: Regulation of CA2+ and cAMP levels’, Cell Motility and the Cytoskeleton, vol. 22, no. 3, pp. 175-184.
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