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The Action of Various Pharmaco- 
 logical and other Chemical 
 Agents on the Chromatophores 
 of the Brook Trout Salvelinus 
 Fontinalis Mitchill 
 
 JOHN N. LOWE 
 
 From the Department of Zoology, University of 
 Wisconsin 
 
 Reprinted from The Journal of Experimental 
 Zoology, Vol. 23, No. 1, May, 1917 
 
 UNIVERSITY OF WISCONSIN 
 PH. D. THESIS \m^r 
 
I7<? 
 
 J? in 
 
 
Reprinted from The Journal of Experimental Zoology, Vol. 23, No. 1 
 
 May, 1917 
 
 ^o 
 
 THE ACTION OF VARIOUS PHARMACOLOGICAL AND 
 
 OTHER CHEMICAL AGENTS ON THE CHROMA- 
 
 TOPHORES OF THE BROOK TROUT SAL- 
 
 VELINUS FONTINALIS MITCHILL 
 
 JOHN N. LOWE 
 From the Department of Zoology, University of Wisconsin 
 
 THREE TEXT FIGURES AND ONE PLATE 
 
 CONTENTS 
 
 Material and methods 148 
 
 Reactions to gases 150 
 
 1. Oxygen 150 
 
 2. Carbon dioxide 150 
 
 Effect of distilled water 151 
 
 Reactions to salts 152 
 
 1. Effects of potassium salts 153 
 
 2. Effects of sodium salts 154 
 
 3. Discussion 156 
 
 Reactions to alcohols 163 
 
 1. Methyl alcohol 164 
 
 2. Ethyl alcohol .164 
 
 3. Propyl alcohol 167 
 
 Reactions to alkaloids 169 
 
 1. Strychnine 170 
 
 2. Picrotoxin 172 
 
 3. Morphine 173 
 
 4. Caffeine 174 
 
 5. Curara 176 
 
 6. Nicotine 178 
 
 7. Atropine. . •. 179 
 
 8. Cocaine ISO 
 
 9. Veratrine 181 
 
 10. Quinine 182 
 
 Summary 1S3 
 
 Bibliography 1S7 
 
 The reactions of melanophores (pigment cells) to pharmaco- 
 logically active agents have been but little investigated. In the 
 
 • 147 
 
148 JOHN N. LOWE 
 
 majority of the physiological researches upon the melanophores, 
 the experiments have only included the study of such physical 
 agents as light, heat, etc. The problem here undertaken was to 
 determine the reactions of the melanophores of young trout em- 
 bryos in response to changes in their chemical environment. The 
 trout embryos that were used in these experiments were too 
 young to react to a change in the light conditions, and through- 
 out the work gave no evidence of any psychic influence of the 
 pigment cells. 
 
 MATERIAL AND METHODS 
 
 Young brook trout embryos, from two days to two weeks after 
 hatching, were used. The melanophore of such young individuals 
 are dark, much branched cells with deep black or brown pigment 
 granules. These are the only kind of pigment cells present at 
 this time. The xanthophores, the yellow or reddish pigment 
 cells, appear after or a little before the yolk is absorbed. All the 
 experiments were performed before the xanthophores appeared. 
 After the yolk is absorbed the fish begin to react to the back 
 ground. When placed in a dark dish, they become dark; when 
 placed in a white dish, light in color. Microscopical examina- 
 tion shows that the pigment cells (melanophores) are expanded 
 in the dark colored individuals and contracted in the light ones. 
 The very young, two-day or two-week old embryos do not 
 respond to changes of the back ground. 
 
 This constant condition is taken as a known factor. The 
 contraction of the pigment cells was used as the criterion for 
 determining stimulation, and their expansion (relaxation) as a 
 mark of depression. The expansion of the pigment cells is char- 
 acterized by the peripheral migration of the pigment granules 
 within the processes of cell, and in contraction the movement is 
 centripetal. My reason for considering contraction as stimula- 
 tion and expansion as a depression is that certain reagents, alka- 
 loids for example, if used in high concentrations produce no ob- 
 servable change in the pigment cells which under normal con- 
 ditions are expanded. Small or 'therapeutic' doses produced a 
 
CHEMICAL AGENTS ON CHROMATOPHORES 149 
 
 contraction. Large doses produced an expansion of all the cells 
 which had contracted in the weak solution. Inasmuch as it has 
 been shown by various investigators that large doses of pharma- 
 cologically active agents produce a depression, and small doses 
 incite a stimulation in other tissues, it is inferred that the 
 condition is essentially the same with the melanophores. 
 
 All the chemicals used in these experiments were of Merck's, 
 Kahlbaum's and Baker's manufacture. The solutions were 
 made up with oxygenated distilled water. Chemically pure 
 oxygen was bubbled through the water before it was used. This 
 precaution was taken because the distilled water was very low in 
 oxygen content and in it the pigment cells contracted. When 
 oxygen was added no such contraction occurred. The details 
 of the way in which the solutions were prepared are given under 
 the respective heads. 
 
 The experiments with the salts and the alkaloids were carried 
 on in Syracuse watch glasses, which were kept covered to prevent 
 excessive evaporation. They were uncovered only when actual 
 observations were made. The amount of the solution used was 
 about 10 cc. Experiments were performed in stender dishes 
 of 50 cc. capacity as a check on the Syracuse watch glasses. 
 There was no difference in the results. The experiments with 
 volatile substances were carried on in wide-mouthed, glass 
 stoppered bottles, with a capacity of 50 cc. All precautions 
 were taken to prevent evaporation. 
 
 Most of the experiments were carried on at room temperatures 
 which varied between 69° and 72° F., although some were per- 
 formed at the fish hatchery where the temperatures were from 
 46° to 50° F. 
 
 The experiments with the alkaloids and alcohols were started 
 in solutions of 0.0001 per cent. The concentrations were in- 
 creased in multiples of ten. 
 
 The experiments were repeated ten to fifteen times for each 
 solution tested. In many cases the experiments were repeated 
 double the number, in order to eliminate all possible individual 
 Variation and errors. 
 
150 JOHN N. LOWE 
 
 I wish, here, to express my chief indebtedness to Prof. 
 M. F. Guyer, for his kindly criticism and suggestions during the 
 progress of the work. To Prof. A. S. Loevenhart, I wish to 
 acknowledge my appreciation of many courtesies extended. For 
 the privilege and use of the fish hatchery and trout embryos, I 
 desire to express my appreciation of the favor to Dean E. A. 
 Birge and Superintendent, James Nevin of the Wisconsin Fish 
 Commission. 
 
 Reactions to gases 
 
 1 . Oxygen. The oxygen used in these experiments was chemi- 
 cally pure. The pigment cells remained expanded in an atmos- 
 phere of oxygen, and the fish lived indefinitely. 
 
 The hydrogen used in these experiments was obtained by the 
 action of chemically pure hydrochloric acid on Merck's highest 
 purity zinc. The gas was passed through two towers of KOH 
 and then through two towers of distilled water, of which one 
 had red litmus, and the other blue litmus. The trout were in 
 the fifth tower. 
 
 The pigment cells contracted completely in four to six nun- • 
 utes when the embryos were exposed to hydrogen. If oxygen 
 was substituted before the fish died the pigment cells expanded. 
 If the oxygen was again replaced by hydrogen the pigment cells 
 contracted. The results of these experiments show (1) that the 
 absence of oxygen caused a contraction of the melanophores ; 
 (2) that the oxygen is necessary for the maintenance of the 
 expanded pigment cells. 
 
 2. Carbon dioxide. The carbon dioxide was generated through 
 the interaction of chemically pure hydrochloric acid on marble. 
 The gas was purified by being passed through a tower of sodium 
 bicarbonate and then through a tower of acidified lead acetate, 
 and lastly through two towers of distilled water. 
 
 The fish were exposed to water through which the carbon di- 
 oxide was bubbling in a steady slow stream. The carbon dioxide 
 produced a complete contraction of the pigment in two and one- 
 half minutes. The time of contraction was the same for all the 
 
CHEMICAL AGENTS ON CHROMATOPHORES 151 
 
 experiments performed. If an intense stream of oxygen was 
 bubbled at the same time with the carbon dioxide, the pigment 
 cells remained expanded. The proportion of the two gases which 
 maintained the expansion of the melanophores was not de- 
 termined. Briefly summarized the results prove that carbon 
 dioxide produces a contraction of the pigment cells of trout 
 embryos. The presence of oxygen antagonized the action of the 
 carbon dioxide. 
 
 Effects of distilled water 
 
 The first experiments that were performed were to determine 
 the effect of distilled water on the pigment cells of trout embryos. 
 The normally expanded pigment cells contracted in ten to twelve 
 minutes and the fish died usually in about twenty minutes — 
 differing somewhat with the individual lots of fish. After an in- 
 terval of ten to thirty minutes, following the initial contrac- 
 tion, the pigment cells began to expand. This secondary ex- 
 pansion of the melanophores in no way equaled the normal ex- 
 panded condition. The processes of the cells were short and 
 blunt. This expanded condition lasted for a short period; then 
 the walls of the melanophores began to break down and the cell 
 contents, viz., the pigment granules migrated into the inter- 
 spaces of the epidermal layer. Often the pigment cells disinte- 
 grated without a previous expansion. Spaeth ('13) obtained 
 essentially the same results with isolated scales of Fundulus in 
 which the chromatophores (1) expanded, (2) contracted, (3) 
 expanded a second time with a final degeneration. He did not 
 try oxygenated distilled water. If 2 cc. of boiled tap water were 
 added to 8 cc. of distilled water the results were the same. 
 Then boiled tap water was tried and the pigment cells con- 
 tracted in fourteen and twenty-two minutes. In distilled and 
 boiled tap water through which oxygen had been bubbled the 
 melanophores remained expanded and the fish lived indefinitely. 
 The conclusion was obvious. It was oxygen want and not the 
 absence of salts in the distilled water that caused the contrac- 
 tion of Jhe pigment cells and the death of the fish. 
 
152 JOHN N. LOWE 
 
 Reactions to salts 
 
 The problem of salt action is one of the most interesting 
 within the scope of physiology and has wide applications. The 
 relation of various salts to heart beat is a long debated question. 
 
 Howell ('98), p. 49, is of the opinion "that the inorganic salts 
 of the blood and liquids of the heart tissues especially of the 
 calcium compounds, stand in a peculiar and fundamental rela- 
 tion to' the initiation of the inner stimulus of the heart con- 
 tractions." Loeb ('00 a, '00 b) believes that the sodium cations 
 acting on the striped muscle to be the stimulating agents being 
 counteracted by the ions of potassium and calcium. The posi- 
 tion of Loeb is supported by Lingle ('00). Benedict ('05 and 
 '08) is of the opinion that the anion probably plays an important 
 role in the action of salt solutions upon heart beat. 
 
 Mathews ('04 a, '04 b, '05, '06) maintains that in the action 
 of salt solutions on motor nerves, colloids, and sea urchin eggs, 
 the ionic potential of the salt, which is the reciprocal of the 
 solution tension, is an important factor in ionic action. R. S. 
 Lillie ('11, '12 a, '12 b) working with the larvae of Arenicola 
 and eggs of starfish, and McClendon ('10) on sea urchin eggs put 
 forth the hypothesis that ionic action is due to the modification of 
 the permeability of the plasma membrane. Loeb ('00 b) holds 
 that ionic action is due to the formation of ion protein com- 
 pounds, that is that the ions of the salt combine directly in some 
 way with the protein molecules of the living protoplasm. True 
 and Kahlenberg ('96) working with plants (Lupinus albus) be- 
 lieve that the anion is unimportant in the toxic action of the 
 salt. 
 
 Spaeth ('13) working on the chromatophores in isolated scales 
 of Fundulus heteroclitus concludes that the anion in potassium 
 salts is of no importance in causing the initial contraction of the 
 chromatophores, but that in the secondary expansion of the 
 chromatophores the action of potassium is modified by the 
 anions. On the other hand, the duration of the sodium expan- 
 sion varies with the nature of the anion. 
 
CHEMICAL AGENTS ON CHROMATOPHORES 153 
 
 The above opinions tend to show that the part played by ions 
 in stimulation is by no means a settled question . In an attempt 
 to gain further insight into the subject brook trout embryos were 
 subjected to solutions of pure potassium and sodium salts. The 
 results have been so promising that the work is being extended to 
 numerous other salts. 
 
 The salts used were of the purest of Merck's, Kahlbaum's and 
 Baker's manufacture. The solutions were made up in a 0.2 
 molecular concentration with oxygenated distilled water. The 
 solutions of the iodides which readily undergo decomposition 
 were never older than thirty-six hours when used. 
 
 The experiments were carried on in Syracuse watch glasses in 
 about 10 cc. of the solution. At times small dishes of 25 to 50 
 cc. capacity were used. 
 
 1. Effects of potassium salts. When the trout embryos are 
 immersed in a 0.2 M. KI solution a rapid contraction of the nor- 
 mally expanded chromatophores results within two or three 
 minutes. They then appear as minute dots with no peripheral 
 processes. In placing a similar lot into a 0.2 M K 2 S0 4 equiva- 
 lent solution the change does not occur as rapidly, being com- 
 pleted in fifteen to twenty minutes. This at once suggested that 
 there is a specific difference in the rate of contraction for potas- 
 sium, varying with the anion. The experiments were extended 
 to include the following neutral salts of potassium, viz., K 2 S0 4 , 
 KC1, KBr, KNOs and KI. Practically the first experiment 
 showed that there was a distinct difference in the rate of con- 
 traction varying with the anion. The rate and intensity of the 
 contraction was most rapid in the order given (figs. 1, 2, 3, 4 
 and 5). 
 
 I> N0 3 > Br> Cl> S0 4 > 
 
 In KI the contraction was complete before it had even begun in 
 K( 1 or K0SO4. The experiments were repeated many times and 
 as a check several of my colleagues were asked to come in and 
 arrange the sets showing the greatest change. In all cases their 
 arrangement was in the -above order. This clearly indicates that 
 if contraction in the melanophore is specifically induced by the 
 
154 JOHN N. LOWE 
 
 cation of potassium, it is unqualifyingly modified by its anion or 
 the residual part of the undissociated molecules. 
 
 Another interesting feature observed was that after a longer 
 or a shorter interval after the first contraction there followed a 
 peripheral expansion of the pigment cells (figs. 6, 7, 8, 9 and 10), 
 that is, the pigment cells put out processes which became 
 longer and longer as time went on but which never reached the 
 original size they had before treatment with the potassium salt 
 solutions. This expansion set in earlier in KI where the contrac- 
 tion took place first, evidently the secondary expansion or pa- 
 ralysis is reciprocal of the first contraction. The expansion is 
 in the order of the first contraction (figs. 6, 7, 8, 9 and 10). 
 
 I> N0 3 > Br> Cl> S0 4 
 
 This peripheral migration of the pigment is in the nature of 
 a paralysis. The paralytic state (depression) is soon followed by 
 death of the pigment cell. The walls of the pigment cell dis- 
 integrate and the pigment granules flow into the interspaces of 
 the body tissues. Death of the cells takes place often before 
 the expansion is complete, and then premature disintegration of 
 the pigment cells occurs. The condition or extent of the de- 
 generation is dependent upon the 'physiological state' of the 
 melanophores and the individual fish. 
 
 The maintenance of the irritability of the melanophores fol- 
 lowed the same order, correlated with this was the longevity of 
 the fish. The fish lived the longest in K 2 S0 4 and KC1. They 
 died very rapidly in KI. 
 
 The reactions varied with the concentration of the solutions, 
 for in solutions of 0.1 M or less the changes were slightly slower. 
 Molecular solutions gave no results but killed the fish imme- 
 diately. 
 
 2. Effects of sodium salts. Here as in the potassium salts the 
 embryos used had their melanophores expanded. It was ob- 
 served that the neutral salts of sodium produced a contraction 
 of the melanophores very slowly. In some instances the contrac- 
 tion did not take place in 92 to 116 hours, especially in the solu- 
 tions of Na 2 S0 4 and NaCl. The contraction in Nal was complete 
 
CHEMICAL AGENTS ON CHROMATOPHORES 155 
 
 in five to forty-five minutes. It was confirmed by repeated ob- 
 servation, that these contractions, slow as they may be for 
 certain solutions (Na 2 S0 4 and NaCl), were in the following 
 order : 
 
 I> N0 3 > Br> Cl> S0 4 
 
 A number of experiments were tried to determine if the sodium 
 salts produced an expansion of the melanophores after the po- 
 tassium salt contraction. The embryos were exposed to KC1 
 from fifteen to twenty minutes when they were removed and 
 rinsed in water to free them of the excess of KC1. They were 
 now placed into the five neutral salts of sodium. The rate and 
 degree of expansion was in the following order: 
 
 S0 4 > Cl> Br> N0 3 > I 
 
 The expansion was most rapid and complete in Na 2 S0 4 and NaCl. 
 In Nal there was no expansion. 
 
 The experiments were repeated with embryos that were not 
 rinsed with water. The result was. the same as in those that 
 were washed in water. If the melanophores are contracted with 
 KI instead of KC1 the results are the same. 
 
 S0 4 > Cl> Br> N0 3 > I 
 
 It is interesting to note here that no expansion of the mel- 
 anophores occurred in the Nal solution. Is this because the 
 sodium cations are inhibited in permeating the cell membrane 
 due to the presence of the dissociated iodine anions or some other 
 factor? Are the cells permeable only to the iodine anions and 
 not to the cations of sodium? Hamburger and von Lier ('02) 
 claim that the blood corpuscles are permeable only for anions 
 and are not permeable to the cations. If the expansion of the 
 melanophore is specific for the sodium cation, it is overcome by 
 the antagonistic action of the iodine anion, which produces a 
 contraction. Nevertheless we must consider another factor, 
 that is, the action exerted by the residual undissociated molecule 
 which is present at all times in the solution. The expansion in- 
 
156 JOHN N. LOWE 
 
 duced by the sodium salts after a potassium salt contraction is 
 followed by a contraction of the melanophores in the visual 
 order. The position or order of the contraction was the same 
 as for the expansion of the melanophores; but with one excep- 
 tion where the NaN0 3 changed places with the NaBr. 
 
 S0 4 > Cl> N0 3 > B r > I 
 
 The extent to which the life of the fish and the irritability of the 
 melanophores are preserved is possibly the function of the 
 cation which is modified by the anion or the residual undisso- 
 ciated molecule. 
 
 3. Discussion. All these results seem to lend themselves to 
 the interpretation that salt solution having a common cation 
 are modified by their anions or the residual undissociated mole- 
 cule. This is clearly shown by the rate and degree of the con- 
 traction of the melanophores by the potassium salts, where the 
 contraction may be specific for the cation of potassium. Speath 
 ('13) p. 547 says in speaking of the action of potassium salts: 
 "The time of this contraction (K) is the same for the five salts 
 within the limits of the variation of the individual scales. Since 
 there is this common cation K+ in all five salts it seems prob- 
 able that the initial effect (contraction) is specific for the K+ 
 ions." My own results in the case of pigment cells of trout 
 embryos are contrary to this conclusion. If contraction is spe- 
 cific for the positive cation of potassium (K + ), it should be the 
 same in rate and degree in all the salts of potassium. Since the 
 rate and degree of the contraction are not the same for the five 
 potassium salts (figs. 1, 2, 3, 4, and 5) it must depend on some 
 other or some modifying factor which is responsible for this 
 difference. 
 
 A dissolved electrolyte conducts a current in proportion to the 
 extent that it is dissociated or ionized. Its maximum conduc- 
 tion will be at complete ionization which occurs at infinite dilu- 
 tion. Therefore the degree of the dissociation or the coefficient 
 of dissociations can be obtained from the conductivity of solu- 
 tion. The conductivity of an electrolyte divided by its num- 
 
CHEMICAL AGENTS ON CHROMATOPHORES 
 
 157 
 
 ber of gram equivalents in cms. is the molecular conductivity 
 of the substance written as A- However, the conductivity is at 
 its maximum at infinitely dilute solutions, therefore the value 
 A °= is taken as a measure of the total number of ions that are 
 produced by the dissociation of one gram equivalent of the 
 substance. Therefore d the degree of dissociation is directly 
 proportional to the conductivity; thus we have the simple form- 
 A 
 
 ula 
 
 The equivalent conductivity at infinite dilution 
 
 for KC1 is calculated to be 130.10. The equivalent conductivity 
 of a two-tenth molecular KC1 is 107.96 A 0.2 m. The degree of 
 
 A 0.2 M 107.96 
 
 dissociation at 18°C. is the ratio 
 
 or 82.98 
 
 A co ' ^ 130.10 
 per cent. The values obtained in this way may be regarded 
 only as approximate. The values are given in the following 
 table. 
 
 SALT 
 
 i KiSO< 
 
 KC1 
 
 KBr 
 
 KNO, 
 
 KI 
 
 Equivalent conductivity 
 infinite dilution A « 
 
 at 
 
 I 132.8 
 
 130.10 
 
 132.30 
 
 126.50 
 
 131 10 
 
 Equivalent conductivity 
 0.2 M dilution A 0.2 M 
 
 at 
 
 1 87.76 
 
 107.96 
 
 110.40 
 
 98.74 
 
 111.2 
 
 Per cent or degree of dissocia- 
 
 1 
 
 
 
 
 
 tion « = A 0.2 M 
 
 A = 
 
 
 66.03 
 
 82.98 
 
 83.44 
 
 7S.05 
 
 84.82 
 
 A study of the table leads one to believe that the rate and the 
 degree of the contraction are in some way correlated with the 
 degree of dissociation of the salts. The lowest rate and degree of 
 contraction was found in K 2 S0 4 , where the degree of dissociation 
 is 66.03 per cent. The most rapid and complete contraction 
 occurred in KI where the dissociation is 84.82 per cent. 
 
 Potassium nitrate is out of place. It has a greater stimulating 
 action than its degree of dissociation would indicate. It should 
 fall between potassium sulphate and potassium chloride. The 
 possible explanation for this break in the series may be that the 
 
 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 23, NO. 1 
 
158 
 
 JOHN N. LOWE 
 
 TABLE 2 
 
 s ALT 
 
 I NajSO< 
 
 NaCl 
 
 NaBr 
 
 NaNOj 
 
 Nal 
 
 Equivalent conductivity at 
 infinite dilution A <* 
 
 > 111.5 
 
 108.99 
 
 112.0 
 
 105.99 
 
 109.9 
 
 Equivalent conductivity at 
 0.2 M dilution A 0.2 M 
 
 1 71.4 
 
 87.73 
 
 91.2 
 
 82.28 
 
 90.2 
 
 Per cent or degree of dissocia- 
 
 1 
 
 
 
 
 
 tion a = A 0.2 M 
 
 !■ 64.03 
 
 80.49 
 
 81.43 
 
 78.11 
 
 82.08 
 
 A * 
 
 J 
 
 
 
 
 
 nitrate anion exerts an independent action or it may form nitrites 
 which are more active. 
 
 In table 2 are shown the equivalent conductivities and degree 
 of dissociation of the sodium salts. 
 
 The values were calculated in the same manner as those for 
 the potassium salts. Here, as in the potassium salts, the reac- 
 tion of the melanophores was correlated with the degree of 
 dissociation. 
 
 There are two reactions of the melanophores which are char- 
 acteristic of the potassium salts: (1) a primary contraction, 
 (2) an expansion which is the sign of death or degeneration of 
 the cell. The cell wall breaks down and the pigment granules 
 escape into the surrounding tissues. The degree of the cytolysis 
 is directly proportional to the degree of dissociation of the salt. 
 In sodium salts we have two specific reactions: (1) the expansion 
 and maintenance of the expansion for a certain period of time, 
 (2) a slow contraction. The two reactions of sodium salts occur 
 in an inverse order to those of the potassium salts, where con- 
 traction is followed by a cytolytic expansion. The contraction 
 in sodium salts is not followed by a cytolytic expansion, but the 
 disintegration takes place directly from the contracted pigment 
 cell. This contraction in sodium salts is directly comparable 
 to the cytolytic expansion observed in potassium salts, for both 
 of these stages indicates the death of the pigment cell. 
 
 A. P. Mathews ('06) suggested that it is the ionic potential 
 of the ions, and not the difference of voltage between the plate of 
 
CHEMICAL AGENTS ON CHROMATOPHORES 
 
 159 
 
 a metal and any solution of its salts, but rather the difference 
 in pressure between a single ion and a single atom of the metal 
 that determines the chemical action of the ions. Since solution 
 tension is a measure of the difference in potential between the 
 solution which contains a known amount of the ions of the metal 
 and the metal itself, it is also the difference between the tendency 
 of an atom of the plate to become an ion. When applied to 
 living protoplasm the metal plate is replaced by the protoplasm. 
 The value varies with the amount of electrolytic dissociation 
 and the kind of plate present. 
 
 The solution tensions in volts of elements in normal ionic 
 solutions. 
 
 CI 1.694 
 
 K 2.92 Br 1.270 
 
 Na 2.54 1 0.797 
 
 N0 3 2.229 
 
 The ionic potential is the reciprocal of the solution tension. 
 Ionic potential is the tendency of any ion in any concentration 
 of solution to change into an atom of its metal. 
 
 The ionic potentials of the ions of metals in volts are: 
 
 CI 1.694 (?) 
 
 K 2.92 (?) Br 1.270 (?) 
 
 Na 2.54 (?) 1 0.797 (?) 
 
 N0 3 2.229 (?) 
 
 Mathews ('06) shows that the dissolving power of the salts 
 of sodium and potassiun for edestine, a globulin of the hemp 
 seed is in some way correlated with the ionic potential. 
 
 s„. T 
 
 IONIC POTENTIAL 
 
 NUMBER OF CUBIC 
 
 CENTIMETERS RE- 
 
 QUIREDTO DISSOLVE 
 
 ONE «:ram of 
 
 EDESTIN 
 
 KI 
 
 -2.123 
 
 -1.65 
 
 -1.226 
 
 -1.743 
 
 -1.270 
 
 -0.846 
 
 
 5.7 
 
 KBr 
 
 10 
 
 KC1 ... 
 
 15 1 
 
 Nal 
 
 5 7 
 
 NaBr 
 
 9 3 
 
 NaCl 
 
 12 8 
 
 
 
160 JOHN N. LOWE 
 
 The more negative the value for the ionic potential the greater 
 the solvent power of the salt for edestin. The negative value in 
 potassium is much greater than that in the sodium. In the table 
 we observe that it takes like amounts of the iodides and less of 
 the other sodium solutions to dissolve the edestin. However, we 
 should expect it to take less of the potassium salts than it does 
 of the sodium. I find this to be true for the pigment cells of 
 trout, where the potassium salts cause the contraction of the 
 pigment cells more rapidly than do the salts of sodium. Un- 
 fortunately the solutions tensions for sodium and potassium are 
 more or less indefinite which makes the results obtained for the 
 salts of these metals incomparable. The ionic potential is not 
 determined directly, but calculated only, thus making the ex- 
 planation more difficult. 
 
 The results obtained in experiments on the action of salts on 
 the pigment cells of trout are explicable on three assumptions; 
 
 (1) that it is the antagonistic action between anion and cation, 
 
 (2) that it is the independent action of the cation, (3) that the 
 reaction is modified by the residual undissociated molecule. 
 
 The antagonistic action between anions and cations has been 
 postulated by Mathews ('06), Benedict ('05, '08), and W. Koch 
 ('09). The increased action of different salts having the same 
 cation have been observed in different tissues. Loeb ('99) pro- 
 duced a better rhythmical contraction in striped muscle with 
 Nal than he did with NaCl. Zoethout ('04) confirmed this ob- 
 servation, and extended it to KI which increased the muscle 
 tone more than KC1. Benedict ('08) concluded that "the direct 
 production of rhythmic activity by means of a salt's action upon 
 heart muscle is due to the anion of the salt, while the chief 
 function of the cation is apparently to maintain such a tone of the 
 heart muscle that it will respond to the stimulus furnished by 
 the anion." Mathews ('02) has shown that the presence of 
 iodine, bromine anions stimulated the motor nerve more power- 
 fully than the chlorine anion. Speath ('13) observed that the 
 cytolytic expansion of the melanophores in potassium solutions, 
 varied with the anions, but he did not note a difference in the 
 rate of the primary contraction of the melanophores in the 
 
CHEMICAL AGENTS ON CHROMATOPHORES 161 
 
 different potassium salts. In sodium salts the expansion of 
 contracted melanophores varied with the anion, and the con- 
 traction following this expansion was correlated with the anions. 
 In neutral salts of potassium there are two constant results 
 produced on the pigment cells of trout; (1) a contraction of the 
 pigment cells, (2) a cytolytic expansion. The times for each 
 varied with the anion. If the antagonism existed between the 
 potassium cations K + , and the negative anions S0 4 -, Br-, 
 CI - , No 3 - , I - , it was the least effective in KI and most potent 
 in K 2 S0 4 . The order of contraction and expansion was 
 
 I> N0 3 > Br> Cl> SO4 
 In sodium salts there were two characteristic reactions, (1) an 
 expansion, (2) a contraction. The rate and degree of the ex- 
 pansion of the melanophores was greatest in Na 2 S0 4 and least 
 in Nal. The rate of contraction was rapid in Nal and least in 
 Na 2 S0 4 . The order of the expansion was 
 
 S0 4 > Cl> Br> N0 3 > I 
 
 The contraction rate of the pigment cells was inverse to the 
 above. 
 
 I> Br> N0 3 > Cl> SO 
 
 The cationic action was modified by the nature of the anion. 
 This anionic order was observed by Paul and Kronig ('96) on 
 the disinfecting power of mercuric salts of chloride bromide and 
 cyanide. Mathews ('06) has shown for the eggs of Fundulus 
 heteroclitus that the fatal dose varied with the anion. Loeb and 
 Cattell ('15) have shown that the hearts of Fundulus embryos, 
 previously poisoned by KC1, and recovered by sodium salts 
 was an anion effect inasmuch as it increased with the anion, 
 apparently in agreement with Hardy's rule (ion effect = ex- 
 ponential function of the valency) for the acetate was much 
 more efficient than the chloride. 
 
 2. That it is the cation of potassium or of sodium that causes 
 the reaction of the pigment cells of trout embryos. 
 
 Loeb ('10, '12) and Loeb and Wasteneys ('11a and 'lib) 
 maintain that there is an antagonism between the sodium cation 
 
162 JOHN N. LOWE 
 
 Na + and the potassium cation K + and not between the po- 
 tassium cation and the chlorine anion K + CI — . This is sup- 
 ported in part by the foregoing experiments on the pigment 
 cells of trout embryos. The pigment cells are expanded in 
 sodium salts after a potassium salt contraction. But this is not 
 true of all the salts of sodium. If the pigment cells are con- 
 tracted in KC1 or KI and are now placed in Nal there is no ex- 
 pansion. Apparently there is an antagonism between the 
 dissociated anions of (CI — and I — ) and the sodium cation 
 (Na +) for from the conditions of the experiment we should get 
 an expansion. It is probable that Loeb underestimated the 
 antagonism between the positive ions of K + and Na + and 
 their negative ions CI — . The longevity of the fish is better 
 protected in sodium salts than in potassium salts. But again 
 some of the sodium salts are more protective (Na 2 S0 4 or NaCl) 
 than others (Nal). That the potassium and sodium cations do 
 exert some such modifying action is undeniable, but to say that 
 it is independent of its anion is not warranted by the facts at 
 our command. 
 
 3) That it is the residual undissociated molecules in the 
 solution that modify the action of the salt. 
 
 In 0.2 M, KI the degree of dissociation is much greater than 
 in an equivalent 0.2 M, solution of K 2 S0 4 . Correspondingly KI 
 initiates more intense responsiveness of the pigment cells than 
 does K 2 S0 4 . The rate and degree of the reactions of the pigment 
 cells decline as the number of the undissociated molecules in- 
 creases. In the potassium salts the primary contraction and the 
 expansion vary with undissociated molecule, thus, 
 
 *0 4 > Cl> Br> NO, I 
 
 The degree of dissociation for 0.2 M solutions are 
 
 66.03 82.98 83.44 78.05 84.82 
 
 The fact that the nitrate is out of place was mentioned before. 
 As already stated, this may be due to the independent activity 
 of the nitrate, which may break down to form a nitrite. 
 
 In sodium salts the dissociation percentages are slightly less 
 than in potassium salts. The rate of contraction is much slower. 
 
CHEMICAL AGENTS ON CHROMATOPHORES 163 
 
 The fish live longer in sodium solutions, and the pigment cells 
 retained their irritability longer. The pigment cells expand if 
 transferred after they are contracted in potassium salts. Is 
 this reaction specific for the sodium ion or is it due to the in- 
 creased number of undissociated molecules in sodium solution? 
 It cannot be positively concluded whether this difference in 
 residual molecules is enough to account for the difference in the 
 rate of contraction of the melanophores in sodium and potas- 
 sium salt solutions. The ascribing of the principles of salt ac- 
 tion to the anion or cation without the consideration of the 
 residual undissociated molecule is just as out of proportion in 
 the field of physiology as to say that the undissociated alkaloids 
 and other substances have no action. 
 
 Reactions to alcohols 
 
 Whether or not alcohols have a stimulating action is a much 
 debated question. The Schmiedeberg school of pharmacolo- 
 gists maintains that alcohols produce no primary stimulation of 
 the central nervous system. According to this view the giving 
 alcohol to a mammal, if followed by an increased muscular ac- 
 tivity, is said to be due to the depression of the cerebral centers, 
 thus removing the restraint from the motor areas. Binz and 
 his followers hold to the view that alcohol first stimulates and 
 then depresses the nerve cells. 
 
 The literature on the pharmacological action of alcohol on the 
 heart and other tissues is very extensive, but to my knowledge 
 there are no records of any attempts to determine its action on 
 the melanophores. Whatever action the alcohols exert on the 
 melanophores will not settle the question whether alcohols 
 stimulate or depress the nervous system, as the melanophores in 
 the very nature of their origin and structure must be looked upon 
 as specialized mesenchymal cells. While it is not at all improb- 
 able that the general facts observed with melanophores may be 
 
 1 After these experiments were completed Spaeth 1916 published results, 
 where he subjected isolated scales of Fundulus to vapors of alcohol, ether and 
 chloroform and always obtained a contraction of the melanophores, and larger 
 amounts of these vapors inhibited the contraction. 
 
164 JOHN N. LOWE 
 
 true also of other tissues, I refrain from applying the results to 
 such an interpretation. The literature is used in a compara- 
 tive way, but not in the sense that the results obtained with 
 melanophores are directly comparable. 
 
 Ten per cent stock solutions of methyl (Sp. G. O. 796), ethyl 
 (Sp. G. O. 796-800) propyl (Sp. G. O. 8066) alcohols of Merck's 
 manufacture were made up with oxygenated distilled water. The 
 dilutions were made from these stock solutions with oxygenated 
 distilled water. The experiments were carried on in glass stop- 
 pered bottles of 75 cc. capacity. All work was done at room 
 temperature of 18° to 20°C. 
 
 /. Methyl alcohol. Overton ('01) showed that methyl alco- 
 hol has a less powerful narcotic action on tadpoles than ethyl 
 alcohol. Vernon ('11) confirmed that the same was true in the 
 depressing action of methyl alcohol on the heart muscle of a 
 turtle's heart. 
 
 Young brook trout embryos of the same age and condition were 
 subjected to the action of the respective alcohols of the various 
 concentrations. The contraction of the pigment cells was taken 
 as the criterion of stimulation, the relaxation (expansion) as 
 that of a depression. 
 
 Ethyl alcohol in solutions of 1.6 to 2.5 per cent produced a 
 complete contraction of the pigment cells. Methyl alcohol of an 
 equal concentration did not cause a contraction. In a 3.5 per 
 cent solution* there was a slight retraction of the pigment cells, 
 but the contraction was not complete. A 4.5 per cent solution 
 produced a complete contraction of the melanophores. Solu- 
 tions of 5 per cent to 5.5 per cent produced a slight contraction 
 of pigment cells This partial contraction was followed by an 
 immediate expansion. If embryos in which the melanophores 
 were just contracted in a 0.005 per cent strychnine solution were 
 subjected to 5 per cent to 5.5 per cent methyl alcohol the pig- 
 ment cells expanded. In 7 per cent to 10 per cent solutions of 
 methyl alcohol there was no visible change in the expanded 
 melanophores. 
 
 Thus it may be concluded that (1) methyl alcohol in high con- 
 centration acts as a depressing agent, (2) in medium concentra- 
 
CHEMICAL AGENTS ON CHROMATOPHORES 165 
 
 tion it has a stimulating action, and (3) in very weak solution it 
 has no effect on the melanophores of trout embryos. Methyl 
 alcohol has a less pronounced stimulation action than ethyl 
 alcohol on pigment cells of trout It was necessary to double 
 to concentration so as to bring about reactions in any way com- 
 parable to those produced by ethyl alcohol. The action of 
 methyl alcohol was less striking and the stages of stimulation 
 and relaxation were slower in appearing than in ethyl alcohol. 
 
 2. Ethyl alcohol. — When trout embryos were exposed to weak 
 solutions (0.01 per cent to 0.8 per cent) of ethyl alcohol, no change 
 took place in the pigment cells. The embryos did not show any 
 signs of depression and appeared perfectly normal. In solutions 
 of 1 per cent to 1.5 per cent the embryos became more restless 
 and the pigment cells exhibited a partial contraction. In con- 
 centrations of 1.6 per cent to 2.5 per cent of ethyl alcohol the 
 fish became more active, the pigment cells showed a complete 
 contraction; while in solutions of 3.0 per cent to 4.5 per cent 
 they showed a transitory contraction, followed by an expansion. 
 This result could be very easily overlooked. In 6 per cent to 
 10 per cent solutions the trout embryos died rapidly in from fif- 
 teen to twenty-five minutes, and there was no contraction of the 
 pigment cells. If embryos that had their pigment cells con- 
 tracted in the 2 per cent solution were transferred to a 7 per cent 
 the pigment cells expanded rapidly. 
 
 If the embryos in which the pigment cells were contracted were 
 transferred to a 4.5 per cent to 6 per cent solution an imme- 
 diate expansion resulted This expansion was due to the de- 
 pression caused by the high concentration of the alcohol, which 
 was far beyond the maximum threshold of stimulation. When 
 the fish which had their melanophores contracted in a 2 per cent 
 solution were placed in a 0.5 per cent solution they expanded. 
 Here the dilution ofHhe alcohol was below the threshold stimu- 
 lus. If embryos that were exposed to 1\ per cent solution for 
 an interval of four to six minutes, were placed in water or very 
 weak alcohol, there was observed a contraction of the pigment 
 cells which was of a very short duration. This result was no 
 doubt due to the washing out or the dilution of the alcohol within 
 
166 JOHN N. LOWE 
 
 the tissues, to the threshold stimulus and as the process of 
 dilution continued the point was reached where the concentration 
 fell below the threshold and a relaxation (expansion) of the 
 melanophores occurred. After a complete recovery of the em- 
 bryos from the effects of the alcohol the pigment cells reacted 
 normally to other stimuli. 
 
 These results show clearly that very weak solutions of ethyl 
 alcohol do not have any effect on the pigment cells of the trout 
 embryos. This is in harmony with the work of Kobert ('82) 
 on the frog's muscle, Lee and Salant ('02) on the gastrocnemius 
 muscle of the frog, and Carlson ('06) for the heart muscle and 
 heart ganglion of Limulus, all of whom observed that weak or 
 very weak solutions of ethyl alcohol had no stimulatory action. 
 Ethyl alcohol in contractions of 1.3 per cent to 2.5 per cent water 
 shows a decided stimulatory action on the pigment cells of brook 
 trout embryos. This is in accord with results of others on the 
 primary stimulation of ethyl alcohol. Pickering ('95) has shown 
 that alcohol excites the embryonic heart muscle of the chick. 
 Scheffer ('00) has observed that in the frog's gastrocnemius when 
 it was treated with alcohol the capacity for work was increased. 
 If the muscle was curanized the stimulating effect of alcohol 
 was nil. 0. Loeb ('05) has noted that in solutions of 0.13 to 
 0.3 per cent that the action of the isolated mammalian (cat) 
 heart was augmented. Wood and Hoyt ('05) have shown that 
 small amounts of ethyl alcohol increased the force of the heart 
 beat in the frog, snake, tortoise, and turtle. Lee and Salant 
 ('02) have demonstrated that in medium concentrations of ethyl 
 alcohol there was an increased rate of contraction and relaxa- 
 tion in frog's muscle (gastrocnemius). Carlson ('06) has ob- 
 served that for the heart muscle and heart ganglion of Limulus, 
 alcohol stimulated. Vernon ('10) has shown that alcohol has an 
 excitatory effect on the isolated heart of the turtle (Emys). The 
 ('02) observed a marked increase in the number of contractions 
 of the bell of the Medusa Gonionema in ethyl alcohol of 0.5 
 to 0.25 per cent. 
 
 In a strong concentration of 4.5 per cent there was a marked 
 depression or an expansion of the pigment cells. In this con- 
 
CHEMICAL AGENTS" ON CHROMATOPHORES 167 
 
 centration there was no primary stimulating period observed. 
 If it is to be found, it may be so short as to be very easily over- 
 looked. Alcohol in large amounts decreased the rate of contrac- 
 tion in the gastrocnemius frog's muscle, Lee and Salant ('02). 
 Romanes ('77) found that strong solutions of ethyl alcohol pro- 
 duced increased and spasmodic contraction of the medusa bells 
 of Sarsia (sp.) and Tiaropsis (sp.). These were followed by a 
 depression. 
 
 Lee ('02) observed that in solutions of ethyl alcohol of a greater 
 concentration than 2 per cent the contractions of the bell of the 
 medusa, Gonionema, were much reduced in volume and in 
 number. Dogiel ('77) has shown a depression in the heart 
 rhythm of Corethra plumicornis. Vernon ('10) observed that 
 large doses of ethyl alcohol depressed the rate and volume of 
 the contraction of a turtle's heart (Emys). 
 
 3. Propijl alcohol. Weak solutions of propyl alcohol 0.01 per 
 cent to 0.04 per cent did not effect the melanophores. In a 0.06 
 per cent there was a noticeable contraction of the pigment cells. 
 Solutions of 0.08 per cent to 0.125 per cent produced a rapid and 
 complete contraction In 0.7 per cent to 1.3 per cent the contrac- 
 tion was only temporary, and was followed by an immediate 
 relaxation of the pigment cells. A 1.5 per cent to 2 per cent 
 produced no visible change in the expanded melanophores, and 
 when embryos with contracted melanophores were exposed to 
 the solution the melanophores expanded. In these concentra- 
 tions there was observed a marked disintegration (cytolysis) 
 of the cells. Higher concentrations (2.5 per cent to 4 per cent) 
 killed the embryos without inducing any change in the expanded 
 pigment cells. Contracted cells exposed to these solutions ex- 
 panded instantaneously and after this response gave no reactions 
 to other stimuli. 
 
 It is obvious from these results that the stimulation of the 
 pigment cells by propyl alcohol begins in solutions of lower con- 
 centrations than it does in ethyl and methyl alcohol. It will be 
 seen that my results for methyl, ethyl, and propyl alcohols are 
 in perfect agreement with the results on the toxicity of the 
 above alcohols of other investigators. 
 
168 
 
 JOHN N. LOWE 
 
 Joffroy and Serveaux ('95) studied the toxicity of alcohols 
 on mammals by intravenous injections. Bear ('98) introduced 
 the alcohol directly into the stomach of the mammals. Picaud 
 ('97) placed fish and amphibians in the solutions of the alcohols 
 and in this way determined the toxicity of the alcohols. Brad- 
 bury ('99) and Cololian ('01) used fish, Overton ('01) on tadpoles 
 employed the same method in their investigations. Wirgin ('04) 
 determined the concentrations at which the various alcohols in- 
 hibited the growth of Micrococcus pyogenes aureus. He also 
 investigated the laking power of the alcohols on the red corpuscles 
 of the rabbit. Vernon ('11) studied the depression of an iso- 
 lated tortoise heart by the alcohols. In table 3 the toxicity 
 of ethyl alcohol is taken as unity and the values given are the 
 comparative toxicities of the other alcohols. The values are 
 only approximate. 
 
 
 
 
 
 
 TABLE 3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 ' X F. 
 
 i « F- 
 
 
 
 
 
 
 
 
 
 
 
 
 
 ' ALCOHOL 
 
 *"s 
 
 s 
 
 
 I 
 
 a 
 
 Z" -* 
 
 O ° 
 
 < 
 _ 5 
 
 gd 
 
 
 2Sri o» 
 
 H g a: o 
 < a " H * 
 
 5 £*> «i 
 
 2 d e P 
 
 
 I 3 
 
 <* 
 
 < 
 
 < ~ 
 
 P 
 
 > ** 
 
 o 
 
 3 ^ 
 
 ?° 
 
 a o 
 > 
 
 - E o w 
 
 «l.B fc S 
 
 Methyl 
 
 46 
 
 o s 
 
 67 
 
 1 
 
 i 1 
 
 73 
 
 n 73 
 
 84 
 
 7? 
 
 0.45 
 
 0.55 
 
 Ethyl 
 
 i n 
 
 i n 
 
 1 
 
 1 
 
 1 
 
 1 
 
 1 
 
 1 
 
 1 
 
 1 
 
 1 
 
 
 3.5 
 
 2.0 
 
 2.0 
 
 1.0 
 
 3.6 
 
 2.0 
 
 1.5 
 
 2.1 
 
 2.1 
 
 2.0 
 
 3 
 
 
 
 The stimulation level is lowest in methyl alcohol (4.5 per 
 cent); next is ethyl alcohol (1.6 per cent to 2.5 per cent); and 
 lastly propyl alcohol (0.08 per cent to 0.125 per cent). This is 
 in harmony with the results of other investigators on the toxi- 
 city of alcohols where it was found that methyl was less potent 
 in bringing about narcosis, and the potency increased for the 
 other alcohols directly with the molecular weight. It was shown 
 by Baer ('98) that the toxicity of the alcohols varied directly as 
 their boiling points. Meyer ('99) and Overton ('01) discovered 
 that the narcotic action of the alcohols varied with their solvent 
 power for fats or lipoids. It may be suggested that in addition 
 to the above physical factors involved in the action of the alco- 
 hols, that the dielectric constant of the alcohols probably plays 
 
CHEMICAL AGENTS ON CHRUMATOPHORES 
 
 169 
 
 an important part in their action. It was observed that the 
 greater the dielectric constant of the alcohols used the lower the 
 stimulating or depressing power, and conversely the lower the 
 dielectric constant the more striking were the reactions. What- 
 ever may be the relation of these physical factors of the alco- 
 hols in stimulation or depression, their chemical structure must 
 not be overlooked; for as the length and complexity of the chain 
 in monohydric alcohols increases so does the strength of their 
 action. 
 
 ALCOHOLS 
 
 MOLECULAR 
 WEIGHT 
 
 CONSTANT 
 AT 20°C 
 
 BOILING POINT, °C. 
 
 STIMULATING 
 
 POWER — ETHYL 
 
 ALCOHOL TAKEN 
 
 AS 1 
 
 Methyl 
 
 Ethyl 
 
 32.03 
 46 05 
 60.06 
 
 31.2 
 25. S 
 
 22.0 
 
 65.7 
 78.4 
 97.4 
 
 0.45 
 
 1.0 
 
 
 2.0 
 
 
 
 Reactions to alkaloids 
 
 The study of the action of drugs on the pigment cells of trout 
 was undertaken with a threefold purpose, viz., to compare the 
 action of drugs on the pigment cells with that of other tissues; 
 second, to determine if possible the controlling mechanism of the 
 pigment cells, and third, to see if the drugs had a specific action 
 on the pigment cells. 
 
 The literature on the pharmacology of the pigment cells of fish 
 is not very extensive. The earliest historical record of experi- 
 ments on the action of drugs on the pigment cells is that of Redi 
 (1664), who observed that eels which died in a tobacco decoction 
 were light in color. Pouchet (76) observed that Gobius niger 
 changed in color when placed in strychnine. Morphine, qui- 
 nine, and santonin had no effect. Lode ('90) concluded that 
 curare destroyed the nerve endings of the pigments cells of 
 trout (Salmo fario). von Frisch ('11) found that chloral hy- 
 drate contracted the pigment cells of the minnow and crucion. 
 He also concluded that the action of cocaine was through the 
 central nervous system. 
 
 The pigment cell may be stimulated or depressed by the drug 
 acting: 1) on the pigment cell in such a way as to increase or 
 
170 JOHN N. LOWE 
 
 decrease its irritability; 2) on the nerve endings leading from the 
 ganglia controlling the pigment cells; 3) on the central nervous 
 system. I have no direct evidence to offer which will enable us 
 to determine which of these or combination of these three fac- 
 tors are operative in the action of the drugs on the pigment 
 cell, for I was unable to separate the nervous and pigment cell 
 tissues for experimental purposes. It is obvious that large 
 doses have no selective action. At certain optimal concentra- 
 tions all the drugs show a selective action on the pigment cells 
 or their controlling mechanism. This selective action of drugs 
 on the mechanisms of the pigment cells will further our knowl- 
 edge as to their function. 
 
 Fig. 1 A normal brook trout embryo showing the general alignment of (he 
 body. 
 
 In interpreting my results I have given special emphasis to 
 their relation in a comparative way to the observations of other 
 observers on various vertebrate and invertebrate tissues. This 
 comparative method makes the results easier of interpretation 
 and is less liable to lead to an erroneous conclusion. 
 
 The drugs used were all of Merck's manufacture. They were 
 dissolved in oxygenated distilled water. The stock solutions 
 were made up from 0.25 per cent to 0.5 per cent. Dilutions were 
 made from these solutions. The experiments were carried on in 
 Syracuse watch glasses in 10 cc. of the solution. These re- 
 sults were checked by experiments in small stender dishes of 
 50 cc. capacity. The conclusions are based on experiments re- 
 peated ten times in 1913 and again in 1914 another series of ten 
 was tried. Five to ten animals were used at one time in each 
 dilution. The trout embryos used were from four days to two 
 weeks after hatching. In no case were the individuals of the 
 different ages mixed. 
 
 2 Figures 1, 2, and 3 were drawn by Miss H. J. Wakeman. 
 
CHEMICAL AGENTS ON CHROMATOPHORES 171 
 
 Other experiments are in progress to determine the action of 
 drugs on pigment cells isolated from the nervous system. 
 
 1. Strychnine. In 0.5 per cent oxygenated solution death re- 
 sulted without primary stimulation of the pigment cells. In 
 0.05 per cent strychnine solution the results were the same. 
 Solutions of 0.005 per cent strychnine caused a contraction of the 
 pigment cells rapidly, the contraction was complete in five min- 
 utes. There was a remarkable thing observed in this concen- 
 tration of strychnine. The irritability of the fish was increased 
 to a high degree. The fish went into typical strychnine spasms. 
 The head was thrown backward and the tail curved upward and 
 forward, describing a half circle (as shown in text fig. 2). A 
 
 Fig. 2 Showing a brook trout embryo in a typical opisthotonos response in 
 0.005 per cent strychnine. 
 
 passing shadow over the disk brought on a new spasm. Shadows 
 in rapid succession increased the concavity backward. If the dish 
 was tapped very lightly the same responses occurred. This 
 period of heightened excitability lasted from ■ eight to twelve 
 minutes. During this interval the pigment cells remained con- 
 tracted (fig. 13). As this convulsive period disappeared, the pig- 
 ment cells expanded (fig. 14). This expansion showed that the 
 depression and paralysis of the pigment cell controlling mecha- 
 nism had occurred. In 0.0005 per cent the pigment cells were 
 contracted in ten minutes. No convulsions were observed in this 
 concentration. In weak solutions of 0.00005 per cent to 0.000025 
 per cent no contractions of the melanophores was produced. 
 
172 JOHN N. LOWE 
 
 Pouchet ('76) observed that the pigment cells of Gobius niger 
 contracted in strychnine solutions. Romanes ('77) noted that in 
 the medusa Sarsia (sp.) the swimming motions were much 
 accelerated by strychnine, also that convulsions occurred in this 
 and three other forms — Cyanaea capillata, Tiaropsis indicans, 
 and Tiaropsis diademta. Hedborn ('99) has shown that 
 strong doses of strychnine augment the beat of the isolated mam- 
 malian heart (cat). Dogiel ('77) demonstrated a slight increase 
 in the rate of the heart beat of Corethra larvae. Pickering ('93) 
 observed that weak solutions of strychnine had a primary stimu- 
 lating action on the heart muscle of an embryonic chick. Carlson 
 ('06) has found that strychnine in very weak concentrations had 
 a distinct stimulatory action on the heart ganglion of the Limu- 
 lus heart. Stronger solutions produced augmentation followed 
 by paralysis. He was unable to note any primary stimulation 
 on heart muscle. Laurens ('15) observed that if a drop or two 
 of a 1 per cent solution of strychnine was injected into the 
 body cavity of Ambly stoma larvae the pigment cells contracted. 
 
 All the above experiments on other tissues show that strych- 
 nine has a primary stimulating action and especially on the 
 motor ganglia. From the evidence of Ballowitz ('93) who dem- 
 onstrated that the pigment cells of fish have a connection with 
 the nervous system, and from the fact that strychnine stimu- 
 lates the nervous system, we are warranted in concluding that 
 strychnine acts directly on the nervous mechanism controlling 
 the melanophores of trout embryos, rather than on the melano- 
 phores themselves. The seat of strychnine poisoning is in the 
 spinal cord, therefore, the melanophores of trout embryos are 
 in all probability controlled in part by the spinal nervous 
 system. 
 
 2. Picrotoxin. Picrotoxin is used as a fish poison. It pro- 
 duces a medullary stimulation and ultimately results in death. 
 When trout embryos are exposed to a 0.25 per cent solution of 
 picrotoxin the pigment cells contract rapidly. The contraction 
 is complete in two to five minutes. The contraction remains 
 for forty-eight to sixty-four hours, if the fish are kept in this 
 solution. The fish live in 0.25 per cent solution for one hundred 
 
CHEMICAL AGENTS ON CHROMATOPHORES 173 
 
 and twenty-six hours. There is no convulsive period. In a 
 weak solution of 0.025 per cent of pictoroxin the contraction is 
 slightly less rapid, and lasts indefinitely (fig. 11). 
 
 When the tail is cut away the pigment cells in the tail portion 
 expand (fig. 12). They remain expanded for six hours and then 
 degeneration sets in. The melanophores in the anterior or head 
 end remain contracted. The contraction continues for eight to 
 twelve hours and then disintegration of the pigment cells occurs. 
 This justifies the conclusion that the reactions of the pigment 
 cells of trout embryos are in some way controlled by the higher 
 nerve centers. 
 
 If the pigment cells that are contracted in picrotoxin are ex- 
 panded in 0.2 M. NaCl and are now placed in picrotoxin the 
 contraction is much slower than the first time. The sodium 
 chloride seems to counteract the action of the picrotoxin. 
 
 8. Morphine. In embryos exposed to 0.5 per cent solution of 
 morphine hydrochloride the pigment cells remain expanded. 
 In a 0.12 per cent most of the pigment cells were expanded but 
 there were a few isolated areas that, showed a contraction. After 
 an exposure of three hours these isolated areas ' of contracted 
 pigment cells had increased. In a 0.06 per cent solution of 
 morphine the result was the same. In a 0.012 per cent solution 
 no change occurred, all the pigment cells remained expanded. 
 There was no contraction of the pigment cells in a 0.005 per 
 cent solution. Pigment cells contracted by picrotoxin, potas- 
 sium iodide or strychnine were expanded by morphine. Ac- 
 cording to Pouchet ('76), morphine did not cause any change 
 in the pigment cells of Gobius niger. Romanes ('77) has found 
 that in Aurelia aurita morphine had a highly depressing action. 
 Pickering ('93) found that morphine afcetate depressed the 
 action of the heart muscle of embryo chicks. Cushny ('10) says 
 that the action of morphine on the central nervous system is a 
 mixture of stimulation and depression which are not equally 
 marked throughout the system ; also, ' ' there is a selective action 
 on the medulla oblongata in which certain centers are entirely 
 paralyzed before neighboring ones undergo any distinct modifi- 
 cation." Waller ('96) found that morphine applied directly to 
 the nerve had but little effect on its irritability. 
 
 THE JOURNAL OP EXPERIMENTAL ZOOLOGY. VOL. 23, NO 1 
 
174 JOHN N. LOWE 
 
 The explanation for the localized areas of contracted pigment 
 cells may depend upon the selective action of morphine upon the 
 nervous system. 
 
 The foregoing experiments support the conclusion that the 
 pigment cells are controlled by the medulla or the spinal cord. 
 It is probable that the localized areas of expanded and contracted 
 pigment cells are in direct response to the mixture of stimulations 
 and depressions caused by the action of morphine on the me- 
 dulla. Or if the pigment cells are controlled by the reflex irri- 
 tability of the spinal cord which may be depressed for a period 
 and then may be followed by an increased irritability. On the 
 latter hypothesis all the pigment cells should contract during 
 the heightened irritability or expand during the diminished 
 irritability; but since this is not the case it is probable that all 
 the regions of 'the spinal cord are not involved at the same time. 
 
 4. Caffeine. In embryos exposed to a 0.2 per cent to 0.25 per 
 cent solution of caffeine citrate no change occurred in the pig- 
 ment cells. The animals died in a much distorted condition. 
 The pigment cells disintegrated in two hours. A 0.05 per cent 
 solution of caffeine citrate caused the pigment cells to con- 
 tract in 4.25 minutes. There was a peculiar twitching of the 
 muscles which lasted twelve minutes. A depression occurred in 
 fourteen minutes. The pigment cells expanded very rapidly. 
 In 0.025 per cent caffeine citrate solution contraction of the pig- 
 ment cells took place in 5.25 minutes. The depression or paraly- 
 sis was elicited in thirty minutes in some, while in others it took 
 forty-five to sixty minutes. A solution of 0.005 per cent caffeine 
 citrate caused no contraction of the pigment cells in two and 
 one-half hours. 
 
 The convulsions observed were quite similar to those that 
 occurred in strychnine. In caffeine the responses to shadows 
 were absent. If the dish was jarred the reactions were weaker 
 and lasted a short interval. These reactions occurred in solu- 
 tions ten times as strong as in strychnine. The response was not 
 opisthotonus, but the head was drawn toward one side and the 
 tail toward the other. There was no difference in the sides to 
 which the curvature occurred (as shown in text fig. 3). The 
 
CHEMICAL AGENTS ON CHROMATOTHORES 175 
 
 animal was in the form of the letter S. The convulsive period 
 lasted a short time and gave from one to six spasmodic reactions. 
 The pigment cells remained contracted during this period. As 
 the convulsive tremors gave way to a complete paralysis the 
 pigment cells expanded. The convulsive period and the con- 
 traction of the pigment were simultaneous. Weak solutions of 
 0.0005 per cent had no effect on the trout embryos or their pig- 
 ment cells. 
 
 If the embryos are removed from a 0.05 per cent caffeine citrate 
 solution during the period of convulsions, and if the poison is 
 washed out rapidly there is a complete recovery. The pigment 
 cells expand normally. If removed during paralysis after con- 
 
 Fig. 3 Showing a brook trout embryo in a typical caffeine convulsion 
 
 vulsions the fish may recover very slowly or not at all. In 
 weaker solutions of 0.01 per cent to 0.025 per cent there are no 
 convulsions, but only a contraction of the pigment cells; there 
 is a complete recovery when they are placed in water. 
 
 Carlson ('06) has shown that caffeine caused a primary aug- 
 mentation in the heart muscle and primary stimulation of the 
 heart ganglion of Limulus. Hedborn ('99) observed that caf- 
 feine stimulated the isolated mammalian heart (cat). Pickering 
 ('93) observed an increase in the number of heart beats in the 
 embryo chick's heart, and concluded from his work that it is 
 not necessary to introduce a nervous hypothesis to explain the 
 action of caffeine. Romanes (77) has found in Sarsia (sp.) 
 exposed to a sea water saturated with caffeine there was a great 
 increase of the contraction and at the same time a diminution 
 
176 JOHN N. LOWE 
 
 of the potency of the beat. Soon the pulsations became of a 
 fluttering nature and spontaneous movements ceased. 
 
 In the pigment cells of trout there is a stimulation which is 
 at its height during the convulsive period. This is soon followed 
 by a paralysis and an expansion of the pigment cells results. 
 There is a direct resemblance in the results obtained with caffeine 
 and strychnine, in that the reflex irritability is remarkably in- 
 creased. The pigment cells contract in both instances during the 
 convulsive period. There is a similarity in the results on the 
 pigment cells of trout and the work of other investigators on 
 other tissues. Caffeine may act directly on the pigment cells as 
 it does on muscle (Pickering, '93, Carlson, '06), or it may 
 stimulate the reflex centers in the medulla and spinal cord, 
 which give off the fibers which control the pigment cells. 
 
 ■5. Curara. In very strong solutions of curara of 2 per cent 
 to 1 per cent, a few pigment cells contracted. When trout em- 
 bryos were exposed to a 0.5 per cent solution they moved about 
 rapidly for eight to ten minutes. In one case one showed a 
 complete contraction of the melanophores in three minutes 
 while the other nine fish in the same lot showed no change. 
 The one that showed this contraction had its pigment cells com- 
 pletely expanded in thirteen minutes. In 0.25 per cent solution 
 there occurred a partial contraction of the pigment cells. The 
 pigment cells along the lateral line were not contracted. The 
 fish died in an hour and were covered with a colorless jelly- 
 like slime. In the following dilutions of curara, viz., 0.05 per 
 cent, 0.025 per cent and 0.001 per cent a partial contraction of 
 the pigment cells occurred in two minutes and thirty seconds. 
 In a 0.0025 per cent curara solution the change took place in 
 fourteen to forty minutes. In all the experiments the contrac- 
 tion of the pigment cells was not evenly distributed but occurred 
 in spots (fig. 15). The tail portion showed many contracted 
 pigment cells, but in the head region there were the largest num- 
 ber of contracted melanophores. Along the lateral line the pig- 
 ment cells remained expanded. After fourteen or fifteen min- 
 utes all the contracted pigment cells were expanded. This 
 mixture of responses was constant for all the experiments. 
 
CHEMICAL AGENTS ON CHROMATOPHORES 177 
 
 Pouchet ('76) observed that curara did not modify the reac- 
 tion of the pigment cells of turbot, viz., the pigment cells re- 
 mained in an expanded condition. Lode ('90) has found that 
 subcutaneous injection of a mixture of curara and glycerine 
 caused a dark coloration in the adult trout (Salmo fario). He 
 ligated the aorta and found that in the anterior end with the 
 intact circulation expansion of the pigment cells occurred, while 
 in the posterior end with the interrupted circulation, the pig- 
 ment cells remained contracted. These experiments are not 
 conclusive because the removal of the circulation interfered 
 with normal metabolism of the cells. Moreover, the pigment 
 cells are very sensitive to the changes in their oxygen supply. 
 He observed that if the spinal cord of a curarized trout was 
 stimulated, no contraction of the pigment cells occurred. If the 
 pigment cells were stimulated directly, the pigment cells con- 
 tracted. He concluded that the curara destroyed the nerve 
 endings but did not affect the pigment cells. Laurens ('15) 
 found that if Amblystoma larvae were placed in a 0.2 per cent 
 solution of curara their movements were abolished and the 
 pigment cells remained expanded under all conditions. He con- 
 cluded that this failure on the part of the pigment cells to react 
 was probably due to the direct effect of the solution on the ani- 
 mal; or asphyxiation of the larvae by the curara, and the con- 
 sequent increased amount of C0 2 in the blood may have caused 
 the melanophores to remain expanded. If a small amount of 1 
 per cent solution of curara was injected into the body cavity the 
 larvae were rendered unmotile but the melanophores reacted to 
 light (expanded) and to darkness (contracted) as usual. He 
 concluded here that this experiment did not prove that curara 
 had no effect on the melanophores, for it has been shown that 
 melanophores will contract and expand after all nervous con- 
 nections have been destroyed. 
 
 Carlson ('06) has shown that in weak solutions of curara there 
 was a primary stimulation of the heart ganglion of Limulus. It 
 had a little effect on the heart muscle. Young ('81) observed 
 that in Mya (sp.) and Solen (sp.) there was a distinct accelera- 
 tion in the number of heart beats, and sometimes a diminution, 
 
178 JOHN N. LOWE 
 
 and even a complete arrest. Plateau ('80) found that curara 
 did not modify the frequency of the amplitude of the Decapod 
 heart. Larger doses diminished the amplitude. Dogiel (77) 
 showed there was a primary stimulation by curara of the heart 
 of Corethra plumicornis. Boehm and Tillie ('04) have observed 
 a primary stimulation of the isolated mammalian heart (dog). 
 
 Since Curara stimulates the central nervous system Cushny 
 ('10) and other ganglia Carlson ('06), it is possible that it acts 
 as a stimulant on the medulla and spinal cord which transmit 
 the impulse to the chromatophores, and a contraction results. 
 Later as the curara destroys the nerve end plates the stimulus 
 does not reach the pigment cell from the center. The pigment 
 cells retain their independent irritability for a long time. The 
 mixture of contracted and expanded melanophores is probably 
 due to the unequal action of the curara on the peripheral nervous 
 mechanism of the melanophores. 
 
 6. Nicotine. When trout embryos were exposed to 0.5 per cent 
 nicotine solution, their muscles twitched for a moment and then 
 all activity ceased. The heart beat continued for twenty-eight 
 minutes. There was no change in the pigment cells. The pig- 
 ment cells disintegrated soon after death. There was a very 
 marked maceration of all the tissues. The whole fish was cov- 
 ered by a colorless slime. In a 0.125 per cent nicotine solution 
 there was a slight primary contraction of the melanophores 
 which was followed almost simultaneously by an expansion. 
 The eyes bulged out of the head which caused the fish to ap- 
 pear grotesque. A 0.005 per cent solution of nicotine caused a 
 complete contraction of the pigment cells in two and one-half 
 minutes. The paralytic expansion occurred eight minutes after 
 the contraction. In 0.0025 per cent nicotine the contraction 
 time was the same as in the preceding experiment. The period 
 of paralysis was delayed which occurred in eleven minutes. A 
 nicotine solution of 0.0005 per cent produced a complete con- 
 traction in eleven minutes. The paralysis or depression of the 
 pigment cells appeared in thirty-five minutes. In a 0.0001 per 
 cent nicotine solution there occurred only a very slight change 
 in the form of the pigment cells. In diluted 0.00005 per cent 
 
CHEMICAL AGENTS ON CHROMATOPHORES 179 
 
 no change was elicited. In all the cases where paralytic expan- 
 sion occurred, it appeared first on the ventral side. The reason 
 for this is not understood. 
 
 Redi (1634) according to van Rynberk ('05) observed that 
 eels which died in a tobacco solution became lighter in color. 
 Cushny ('10) says, that in nicotine the spinal cord is thrown 
 into a condition of exaggerated irritability and that the medulla 
 seems to be involved to a greater degree than the spinal cord. 
 The stimulation does not involve the higher brain centers. Carl- 
 eon ('06) observed that nicotine in weak solutions stimulated the 
 heart ganglion of the Limulus heart. This primary stimulation 
 was followed by a depression. There was no primary stimula- 
 tion of the heart muscle. Gee ('13) has found that in a solution 
 of 0.00066 per cent of nicotine leeches were vigorously stimu- 
 lated, which was followed by a depression of movements. Ro- 
 manes ('77) found that violent spasms were incited in the 
 medusae Sareia (sp.) and Tiaropsis when exposed to nicotine. 
 He also observed various distortions. Langley and Dickson 
 ('90) concluded that nicotine acts directly on the nerve cells and 
 not on the muscle. 
 
 It is known that nicotine first stimulates and later paralyzes 
 the ganglionic cells of the sympathetic system, whether applied 
 directly to them or injected into the circulation. It is quite 
 probable that nicotine affects the sympathetic system of the 
 pigment cells, for there is first a contraction of the cells which is 
 later followed by an expansion. 
 
 7. Atropine. Strong solutions (0.5 per cent) produced no change 
 in the pigment cells. The fish lived four hours in this concen- 
 tration. In 0.025 per cent solution of atropine sulphate, there 
 was no change in the pigment cells. All possible concentrations 
 were tried, but none of them produced a contraction of the 
 pigment cells. 
 
 Pigment .cells were contracted in 0.005 per cent strychnine 
 solution and then were transferred to solutions of 0.05 per cent 
 to 0.0025 per cent of atropine, where all the pigment cells ex- 
 panded rapidly. The expansion was complete from two to 
 four minutes. Pigment cells contracted in potassium salts were 
 expanded just as those that were contracted by strychnine. 
 
180 JOHN N. LOWE 
 
 All the experiments show conclusively that atropine does not 
 have any direct stimulating action on the pigment cells of trout 
 embryos. Cushny ('10) says, that atropine acts on the higher 
 centers of the brain and less on the lower divisions, viz., the 
 medulla and the spinal cord, which is just the reverse of strych- 
 nine. This acts on the lower centers and not on the central 
 system. The results obtained justify the conclusion that the 
 pigment cells are controlled by the lower reflex centers. 
 
 Romanes {"17) in his experiments on the medusae Sarsia (sp.) 
 and Tiaropsis found that atropine caused convulsive swimming 
 movements by a marked depression. Pickering ('93) showed that 
 0.012 gm. of atropine to 1 cc. of normal saline solution reduced 
 the normal heart beat of the embryonic heart of the chick. 
 Carlson ('06) found that atropine stimulated the heart ganglion 
 and not the muscle of the Limulus heart. Cushny ('10) says most 
 secretions are depressed by the administration of atropine. 
 This is not due to the inactivation of the secretory cells, but to 
 the failure of the nervous impulses. The action of atropine on 
 other tissues, from all evidence, shows us that it does not act 
 directly on the vascular and secretory elements, but on their 
 nerve terminations. It is therefore possible that atropine acts 
 on the pigment cells through their nerve fibers, paralyzing them, 
 but does not act directly on the pigment cells. 
 
 8. Cocaine. One-half per cent solutions killed the trout em- 
 bryos rapidly. There was a momentary stimulation of the pig- 
 ment cells which was followed almost simultaneously by an 
 expansion of the pigment cells. In a 0.125 per cent solution of 
 cocaine the behavior of the pigment cells was the same as in the 
 0.5 per cent solution. In 0.025 per cent to 0.05 per cent cocaine 
 solution the pigment cells were contracted in four minutes. The 
 contraction was followed by an expansion. Solutions of 0.005 
 per cent of cocaine produced a complete contraction in five 
 minutes. The expansion followed in twelve minutes. Very 
 weak, 0.00033 per cent solutions, had no effect on the melano- 
 phores. 
 
 These results show that cocaine has a primary stimulating 
 action on the pigment cells of trout embryos. This primary 
 
CHEMICAL AGENTS ON CHROMATOPHORES 181 
 
 stimulation is followed by an expansion of the pigment cells. 
 The action of cocaine on the nervous system is in a series, 
 namely, the cerebrum is first affected, then the cerebellum and 
 medulla, and lastly the spinal cord. It also acts on the sen- 
 sory fibers and their terminations. 
 
 Von Frisch ('11) observed that the local application of cocaine 
 caused the contraction of the melanophore in the minnow and 
 Carssius (sp.). An injection of a 5 per cent solution into the body 
 cavity caused a contraction of the pigment cells after the sym- 
 pathetic nerves were severed. He concluded that the action 
 of cocaine was through the central nervous system. Carlson 
 ('06) showed that weak solutions of cocaine had a primary stim- 
 ulating action on the heart ganglion of Limulus, but had no 
 effect on the heart muscle. Hedborn ('99) observed a slight 
 primary stimulating action on the isolated heart of the cat. 
 
 It is probable that cocaine acts on the reflex center which 
 controls the pigment cells. It may act on the nerve endings of 
 the pigment cells. It is obvious that it will require a great deal 
 more of work to determine the relation of cocaine to the pigment 
 cells before any generalization can be made. 
 
 9. Veratrine. Solutions of veratrine of 0.5 per cent concen- 
 tration caused a rapid contraction of the pigment cells. The 
 contraction was complete in two minutes. Paralysis set in at 
 six minutes, and pigment cells were completely expanded in 
 two more minutes. In a 0.25 per cent solution the stages were 
 the same. In a 0.005 per cent veratrine solution the pigment 
 cells were completely contracted in nine minutes. The first 
 signs of paralysis appeared in eighteen minutes. Veratrine 
 was a very active agent in causing the contraction of the pig- 
 ment cells. Dilutions of 0.0005 per cent to 0.00005 per cent 
 caused a contraction of the pigment cells in eighteen to twenty 
 minutes. The paralytic expansion occurred in thirty minutes. 
 A solution of 0.00001 per cent veratrine caused no change in 
 the pigment cells. 
 
 Veratrine acts on the medullary center and the spinal cord, 
 where a marked increase of irritability is elicited. After large 
 doses there is a paralysis of the centers. It acts on the periph- 
 
182 JOHN N. LOWE 
 
 eral ganglia and nerve endings. It is highly probable that the 
 action of veratrine on the pigment cells is through the lower 
 centers of the nervous system rather than local. 
 
 Carlson ('06) found that weak solutions of veratrine had a 
 primary stimulating action on the heart ganglion of Limulus. In 
 strong solutions the period of stimulation was followed by a de- 
 pression in two minutes. The ganglion free heart did not re- 
 spond to the poison. Plateau ('78) observed a primary stimu- 
 lation in the heart of Carcinus moenas and Homarus (sp.) which 
 was followed by a depression. Romanes ('77) found- that in the 
 medusa Sarsia (sp.) the first .effect of veratrine was an increase in 
 the number and potency of the contractions. This period of 
 increased responsiveness was followed by a gradual depression 
 into complete quiescence. 
 
 Summarizing the action of veratrine on the pigment cells, it 
 may be stated, that it first stimulates the contraction of the 
 pigment cells. This period of stimulation is followed later by a 
 paralysis of the mechanism controlling the pigment cells. The 
 pigment cells are expanded during this period of depression. 
 This is in harmony with the observations of other workers on 
 various tissues, where there is observed a primary stimulation 
 followed by a depression. 
 
 10. Quinine. Quinine in a 0.5 per cent maintained for a long 
 time the pigment cells in an expanded condition. Dilutions were 
 made from 0.25 per cent to 0.0005 per cent of quinine hydrochlo- 
 ride solution, and in all of these dilutions no change occurred in 
 the pigment cells. Solutions of 0.000033 per cent to 0.0000165 
 per cent gave the same result. 
 
 The pigment cells were first contracted in picrotoxin and were 
 then placed in the quinine solutions of 0.025 per cent to 0.005 
 per cent and in every case a rapid expansion of the pigment cells 
 occurred. The rapidity of the expansion was greater in the 
 quinine than it was in the ordinary process of washing out of 
 the picrotoxin. 
 
 Quinine differs from most drugs in that its action is very 
 widespread, and it is often called a general protoplasmic poison. 
 Binz ('68) observed that quinine inhibited the beat of the cilia 
 
CHEMICAL AGENTS ON CHROMATOPHORES 183 
 
 in protozoans. Also, that it stopped the movements of the 
 leucocytes. Santesson ('93) found that quinine depressed the 
 rhythm of an isolated frog's heart. Hedborn ('99) observed 
 that quinine depressed an isolated mammalian heart (cat). 
 O. and R. Hertwig ('87) observed that sperm treated with qui- 
 nine had their movement paralyzed. Eggs when treated with 
 quinine after the sperm entered, the conjugation of the pro- 
 nuclei was delayed. Carlson ('06) has found that quinine did 
 not stimulate the ganglion or the muscle of the Limulus heart. 
 It is obvious from the experiments that quinine exhibits no 
 primary stimulating action on the pigment cells of trout em- 
 bryos. Any accurate interpretation of the depressing action of 
 quinine is not possible, since the drug acts in the same way on 
 the nervous tissues and the pigment cells as well. 
 
 SUMMARY 
 
 1. The experiments were performed on the melanophores 
 (pigment cells) of the brook trout embryos, Salvelinus fontinalis 
 Mitchill. Such young trout have only one kind of pigment 
 cells, the melanophores. The young two-day or two-week old 
 trout do not yet react to back ground. The first sign of reac- 
 tion to back ground appears only after the yolk is absorbed. 
 
 2. In the presence of oxygen the pigment cells remain ex- 
 panded and the fish live indefinitely. When hydrogen (oxygen 
 want) is substituted for the oxygen the pigment cells contract 
 and the embryos die. Oxygen is necessary for the maintenance 
 of the expansion of the melanophores and life of the trout 
 embryos. 
 
 3. Carbon dioxide excess caused a contraction of the melano- 
 phores. If oxygen was bubbled with the carbon dioxide, the 
 presence of the oxygen had an antagonistic action. 
 
 4. Distilled water caused a rapid contraction. A mixture of 
 distilled and boiled tap water gave the same result. In boiled 
 tap water the pigment cells contracted. Oxygenated distilled 
 water and boiled tap water maintained the pigment cells in a 
 normal expanded condition. It was the absence of oxygen and 
 not of the salts that caused the contraction. 
 
184 JOHN N. LOWE 
 
 5. In the potassium salts, K 2 S0 4 , KC1, KBr, KN0 3 , and KI 
 there occurred a rapid contraction of the expanded melano- 
 phores. The rate and degree of the contraction was the order 
 given 
 
 I> NO,> Br> Cl> S0 4 
 
 This primary contraction was followed by a cytolytic de- 
 generation (expansion) . The time required for the appearance 
 of this degeneration was greatest in 
 
 I> N0 3 > Br> Cl> S0 4 
 
 If the contraction or degeneration of the melanophores is 
 specific for the potassium cation, it is unqualifiedly modified by 
 its anion, or the residual part of the undissociated molecules. 
 
 6. The neutral salts of sodium, Na 2 S0 4 , NaCl, NaBr, NaN0 3 , 
 and Xal, caused a slow contraction of the melanophores. The 
 contraction was most rapid in Nal and slowest in Na 2 S0 4 and 
 other salts were intermediate as 
 
 I> N0 3 > Br> Cl> S0 4 
 
 Degeneration appeared first in Nal and last, in Na 2 S0 4 and 
 varied in this order 
 
 I> N0 3 > Br> Cl> S0 4 
 
 The irritability of the chromatophores and life of the fish was 
 maintained longest in Na 2 S0 4 and NaCl from (118 to 132 hours) 
 in Nal from one to two and one-half hours. 
 
 7. The pigment cells that were contracted in potassium salts, 
 when placed in sodium salt they expanded. The order of ex- 
 pansion was 
 
 S0 4 > Cl> Br> N0 3 > I 
 
 There was no expansion in Nal. 
 
 8. The results obtained in the experiments on the action of 
 the salts on the pigment cells of trout are probable to be ex- 
 plained on one or more of three assumptions: (1) That it is due 
 to the antagonistic action between anion and cation; (2) that it 
 is the independent action of the cation; (3) that reaction of the 
 melanophores is likely modified by the undissociated molecule. 
 
CHEMICAL AGENTS ON CHROMATOPHORES 185 
 
 9. The narcosis or depression of the pigment cells of trout by 
 the homologous alcohols corresponds very closely to their nar- 
 cotic action as determined by Overton and numerous other 
 investigators. 
 
 10. Very dilute solutions of methyl, ethyl, and propyl alcohols 
 exert no action on the pigment cells of trout. 
 
 11. The pigment cells of trout embryos respond to alcoholic 
 stimuli. Their mode of reaction is comparable to .the reaction 
 of other tissues to alcohols inasmuch as they are stimulated by 
 small doses and depressed by large doses. 
 
 12. Strychnine in moderate doses causes a primary con- 
 traction of the expanded melanophores. Large doses cause a 
 depression without a primary stimulation (contraction). The 
 action of the strychnine is on the nervous system rather than 
 on the pigment cells directly. 
 
 13. The action of picrotoxin causes a rapid contraction of 
 the pigment cells. The mechanism controlling the pigment 
 cells is in the higher centers, because if the spinal cord is severed 
 the pigment cells expanded. 
 
 14. Morphine induces a contraction of the melanophores in 
 isolated areas. This is probably due to the selective action of 
 morphine upon the nervous system. Large doses produce no 
 change in the expanded melanophores. Morphine expands the 
 pigment cells that were contracted in picrotoxin, KC1, and 
 strychnine. 
 
 15. Curara causes a mixture of responses, that is, there are 
 areas of expanded and contracted melanophores. This is likely 
 due to the unequal action of the curara.on the peripheral nervous 
 mechanism of the melanophores. 
 
 16. Medium solutions of nicotine cause a contraction of the 
 pigment cells. Strong nicotine solutions have no effect on the 
 pigment cells. The action of nicotine is directly on the nervous 
 controlling mechanism of the pigment cells. 
 
 17. Atropine in all concentrations has no stimulating action 
 on the pigment cells of trout. Atropine paralyzes the fine nerve 
 connections of the pigment cells. 
 
 . 
 
186 JOHN N. LOWE 
 
 18. Cocaine has a primary stimulating action on the pigment 
 cells of trout. This action is probably on the nerve endings of 
 the pigment cells that connect them with the reflex center. 
 
 19. Veratrine causes a primary contraction of the pigment 
 cells which is followed by a rapid depression (expansion). The 
 action of veratrine is through the reflex center of the spinal 
 cord and medulla rather than local. 
 
 20. Quinine exhibits no primary stimulating action on the 
 pigment cells. The drug has no selective action on tissues, 
 therefore it is a general 'protoplasmic poison.' 
 
CHEMICAL AGENTS ON CHROMATOPHORES 187 
 
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 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL- 23, NO 1 
 
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CHEMICAL AGENTS ON CHROMATOPHORES 191 
 
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PLATE 1 
 
 EXPLANATION OP FIGURES 
 
 In figures 1 to 10 are shown five sets of brook trout embryos which were ex- 
 posed to the action to 0.2 M solutions of Kl, KN0 3 , KBr, KC1, and K 2 S0 4 , 1 to 
 5 for an interval of fifteen minutes, and figures 6 to 10 for a period of three 
 hours. 
 
 1 The melanophores were completely contracted in Kl. 
 
 2 The contraction was not as pronounced in KN0 3 . 
 
 .3 In KBr the melanophores had longer processes than in the two preceding 
 solutions. 
 
 4 In KC1 the processes were more distinct and showed the finer arboriza- 
 tions. 
 
 5 An exposure of fifteen minutes to K2SO4 produced no observable change 
 in tlie melanophores. 
 
 6. After an exposure of three hours to Kl the melanophores showed ;i distinct 
 secondary expansion. 
 
 7 In KNO3 an exposure of three hours produced a less extensive secondary 
 expansion than Kl. 
 
 8 In KBr the processes were very much shorter than in- Kl and KN0 3 . 
 
 9 After three hours in KC1 the melanophores were still spherical, but there 
 was a suggestion toward a peripheral migration of the pigment as indicated by 
 the swollen condition of the cells. 
 
 10 After three 'hours in K2SO4 there was no expansion of the melanophores. 
 
 11 All melanophores contracted, photograph taken after twenty-four hours 
 of exposure to Picrotoxin. 
 
 12 The melanophores expanded after severing the tail in an individual which 
 was exposed to Picrotoxin for twenty-four hours. Photograph taken five min- 
 utes after cutting. 
 
 13 All melanophores contracted during the period of Strychnine con- 
 vulsions. 
 
 14 Showing the expansion of the melanophores after the strychnine convul- 
 sion had subsided. 
 
 15 The contracted and expanded melanophores as they occurred in 0.0025 
 per cent curara 
 
CHEMICAL AGENTS ON C 
 
 JOHN N. LO 
 
 HROMATOPHORES 
 
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THE JOURNAL OF EXPERIMENTAL ZOOLOGY 
 
 VOLUME 23, NUMBER 1, MAY, 1917 
 
 CONTENTS 
 
 Harley N. Gould. Studies on sex in the hermaphrodite mullusc Crepidula plana. I. 
 History of the sexual cycle. Eighty-five figures 1 
 
 Henry Laurens and J. W. Williams. Photomechanical changes in the retina of normal 
 and transplanted eyes of Amblystoma larvae. Three text figures and one plate. ... 71 
 
 Franklin Pearce Reagan. The role of the auditory sensory epithelium in the forma- 
 tion of the stapedial plate. Ten figures 85 
 
 Edwin Cableton MacDowell. Bristle inheritance in Drosophila. II. Selection. 
 Ten figures 109 
 
 John N. Lowe. The action of various pharmacological and other chemical agents on 
 the chromatophores of the brook trout Salvelinus fontinalis Mitchill. Three text 
 figures and one plate 147 
 
 Henry Laurens. The reactions of the melanophores of Amblystoma tigrinum larvae to 
 light and darkness. Six figures 195 
 
 Carey Pratt McCord and Floyd P. Allen. Evidences associating pineal gland function 
 with alterations in pigmentation. Seven figures 207 
 
 THE WAVERLY PRESS 
 
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