Table 2. Size (standard length, millimeters) of adult male platyfish with different genotypes.pepa pep I pip'

Birth Y-Ir Y-Ir Y-lr Y-Br Y-Br Y-Brdate

Mean Range N Mean Range N Mean Range N

Pedigree 28282/27/71 28 1 32 30-34 4

Pedigree 29186/25/71 24.5 23-26 2 31 28-33.5 69/30/71 22.1 19-25 5 27.3 24.5-30 2

Pedigree 30302/15/72 25 1 29.3 28.5-31 33/17/72 22.7 21-23.5 8 27.4 27-29 4

Pedigree 29649/20/71 30.5 29.5-31 3 36.6 35-38 310/20/71 25.5 22.5-27 3 31.8 28-34.5 51/26/72 a 25.8 24-28 6 28.3 24.5-32 61/26/72 b 24.1 22-26 6 29 27.5-30.5 6

X-Y-Ir X-Y-Br

Pedigree 297411/1/71 24.7 23-26.5 13 29.9 27.5-32 15

mozygous for PI (large size), differingcompletely in breeding structure andappearance.The P locus of X. imaculatus is of

general significance not only for pitui-tary gland function in teleosts, but forall vertebrates including man. Thissystem may become an importantmodel for studying the genetic controlof endocrine structure and function.

KLAUS D. KALLMANOsborn Laboratories of MarineSciences, New York Aquarium,New York Zoological Society,Brooklyn 11224

MARTIN P. SCHREIBMANBrooklyn College of City Universityof New York. Brooklyn 11210

VALERIE BoRKosKIOsborn Laboratories of MarineSciences, New York Aquarium

Pl may specify two proteins differingin their efficiency in initiating morpho-genic changes. The P locus probablyexerts its effect directly on the pitui-tary gland, although the role of thehypothalamus should also be evaluated.The P gene also has an indirect ef-

fect on the adult size of male platyfish.Since growth rate decreases with in-creasing androgen production (9), earlymaturing males are significantly smallerthan late maturing ones. Size differ-ences between the two classes of maleswere absolute within seven broods,whereas in one (9/30/71) the largestirIr male surpassed the smallest IrBrmale by 0.5 mm (Table 2). The ex-ceptional BrBr male (2964-11) thatmatured at 18 weeks (Table 1), isalso the smallest homozygous Br maleobtained so far. Small and large fishgrow at the same rate, but the formerstop at an earlier age when they be-come sexually mature. Since these ob-servations did not differ throughoutthe year, natural daylight, which wasnot controlled, cannot account for thedifferences in growth and age of sexualmaturity.The P factors have manifested them-

selves both in the offspring of intra-and interstrain crosses. This poly-morphism is a natural component ofwild populations and is apparentlywidespread. We have found it in platy-fish stocks collected from four river sys-tems (10). The sex chromosome con-stitution of the males, XY or YY, hasnothing to do with adult size and ageof sexual maturation. pe and pl can beboth X- and Y-linked. An X chromo-some with pl has recently been identi-fied from the Belize population (10).

680

Similar variations in size and ageof sexual maturation have been re-ported for a number of other speciesof Xiphophorus (11), but little isknown about the genetics of these dif-ferences. The polymorphism at thislocus may have been important for theevolution of the genus, since variousbody parts show allometric growth andlarge males, for example, those of X.pygmaeus, not only assume a differenthabitus, but also develop structuresnot present in the smaller morphs(12). The allele for early gonadotropdifferentiation has become fixed in X.pygmaeus pygmaeus inhabiting the RioAxtla, and has led to a uniform popu-lation of small males. However, X.pygmaeus nigrensis in the Rio Choyis polymorphic at the P locus resultingin two kinds of males (13). Elmina-tion of pc from the Rio Choy wouldleave two populations: X. pygmaeuspygmaeus homozygous for pe (smallsize) and X. pygmaeus nigrensis ho-

References and Notes

1. K. D. Kallman, Zoologica New York 55, 1(1970).

2. Xiphophorus maculatus is polymorphic forsex chromosomes. Three kinds of females(XX, WX, WY) and two kinds of males (XY,YY) may occur within the same naturalpopulationi. YY males are not the result ofspecial laboratory crosses as are the YYmales of the rice fish, Oryzias latipes [T.Yamamoto, Genetics 50, 45 (1964)].

3. V. L. de Vlaming, J. Fish Biol. 4, 131 (1972).4. M. Gordon, The Care and Breeding of

Laboratory Animilals, E. J. Farris, Ed. (Wiley.New York, 1950), p. 345.

5. K. D. Kallman, Zoologica New York 50, 151(1965).

6. C. Grobstein, Univ. Calif. Publ. Zool. 47, 1(1940); J. Exp. Zool. 109, 215 (1948).

7. G. Pickford and J. W. Atz, The Physiologyof the Pituitar!' Gland of Fishes (New YorkZoological Society, New York, 1957).

8. M. P. Schreibman, Zoologica New York 49,217 (1964).

9. H. Cohen, ibid. 31, 121 (1946).It). K. D. Kallman and M. P. Schreibman,

uinpublished results.11. K. D. Kallman, Zoologica New York 56, 77

(1971); and R. Borowsky, Heredity 28,297 (1972); G. Peters, Z. Zool. Syst. Evolu-tions Forsch. 2, 185 (1964); C. D. Zander, Z.Vererbungsl. 96, 128 (1965).

12. D. E. Rosen, Bull. Fla. State Mus. Biol. Ser.5, 57 (1960).

13. K. D. Kallman, unpublished results.14. Supported in part by NIH grant 5 ROI

CA06665.1 January 1973; revised 5 April 1973 0

Perfluorocarbons Having a Short Dwell Time in the Liver

Abstract. Perfluorinated organic liquids are useful as high capacity oxygenand carbon dioxide solvents. After intravenous infusion mnost of these perfluori-nated emulsions are deposited in the liver and spleen in a mnatter of days, wherethey remain for the lifetime of the animal. Hence, while they may be usefulas isolated organ perfusion inedia their value as artificial blood is limited. Afamiiily of perfluorocarbons has now been discovered, which, although depositedin the liver after circulation in the blood, leave the liver to be excreted via thelungs and skin in a Inatter of days without apparent harmn to the animal.

Since 1966, when organic liquidbreathing was first reported, there havebeen over 100 publications concerningthe use of inert fluorochemicals (1)

in physiological research. These includereports of liquid breathing (2), oforgan perfusion (3), of infusion inwhole animals (4), and as radiographic

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Court File No. 27-CV-10-28862

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a.ents (5). A symposium held in 1969g,ave the state of the art as of that time(6).Most of these perfluorinated organic

liquids are synthesized for industrialuse because of their value as electrically-onconductive, chemically nonreactive,and heat stable liquid heat exchangers,leak detectors, and hydraulic fluids.Their high solubility for gases, a nui-sance in commerce, is a highly desiredproperty in biology. Including the iso-mers present there are hundreds ofcompounds of this general type, overa hundred of which we have studied.

Generally speaking, these perfluori-nated compounds (PFC) are excellent-olvents for a large variety of gases(7), are nonmetabolizable, and arealmost completely insoluble in water.As a class they are very poor solventsfor all but a few organic substances,and for these reasons can only be used,or intravascular gas transport in theform of oil-in-water emulsions.

Efforts are being made in variouslaboratories to develop artificial blood,based oni the use of oil-in-water PFCrniulsions, where the emulsion is made

with an emulsifying agent and mechani-cal energy. Such emulsions have re-niarkably low toxicity-we have 10 per-cent (by volunle) fluorocarbon emul-sions where the median lethal dose inIiice after a single intravenous injectionis 200 nil/kg, with the blood volumeof the niouse being probably about 60ml/kg. One of the main problems, ifnot the main probleni, with the use offluorocarbon eiiiulsions has been thatthey are deposited in the liver andspleen, where they reniain for the lifeof the animal. We niow report the dis-covery of a nuniber of closely relatedfluorocarbons that leave the liver andspleen of the niouse in a niatter ofdays.

Eniulsioiis are niade with 5 percentPluronic F68 in the water phase, with10 percent by volume (about 19 per-cent by weight) PFC liquid and withthe mixture being subjected to a briefsonication or to a pressure of about550 kg/cm2 in a Gaulin homogenizer.For any given surfactant concentration,each PFC has a characteristic rate atwhich it breaks down to particles whencontinuously sonicated aiid a charac-teristic point at which the particles canbe made no smaller, as judged by opti-cal measurenients (Fig. 1). The emul-sion was centrifuged to remove largeparticles, and each emulsion to be testedwas injected intravenously into the tailveins of 200 albino Swiss mnice at a17 AUGUST 1973

Time (hours)

Fig. 1. Emulsion absorbance plottedagainst ultrasonication time. Emulsionswere circulated through a Branson auto-tLuned sonicator cell at 10°C, samplestrearns were flowed through spectro-photometer cuLvettes having appropriatelight paths, and the optical density wasrecorded. The optical density was cal-cLtlated to accouint for the length of thecell path.

rate of 2 nil/miiii. The niice were killedin groups at intervals thereafter, andtheir livers and spleens were analyzedfor PFC by specific gravity, by directconibLstion to fluoride ion with theLise of sodiLni biphenyl (8), and oftenby hexane extraction and gas chroma-tography. The fluoride ion concentra-tion was measured with an OrionlanthanuLm fluoride electrode. The PFCliquids were stoichionietrically con-verted to fluoride ion by the biphenylniethod.

50-

40

o20-

0

*' 10-

t~~~1 - qpitD

- PP9

1 2 3 4 5 7 8 9 10 11 12Time (weeks)

Fig. 2. Per-fluorochemical content of themouse liver plotted against time. Theamount of PFC expressed as the percent-alge of initial dose remaining in the liveiof Swiss albino mice injected with 10percent PFC emulsion in 5 percent Plu-ronic F68 and killed at var-ious intervalsafter injection is plotted. Groups of sixmice were killed at I hour, 1 and 5 days,and 2, 3, 4, 8, 12, and 20 weeks. Liversand spleens were analyzed in dLuplicate forPFC by the sodium biphenyl procedure.Each point repr-esents the average, andthe vertical bars represent the standarddeviation for six mice. Approximately 8percent of the initial dose appeared in thespleen, and a similar rate of decreasein PFC was observed for PP5 and PP9.

Perfluorodecalin and perfluorometh-yldecalin left the liver in a matter ofdays while P1lD remained for months(Fig. 2). After 5 months the percentageof PFC of the injected dose remainingfor PIID, PP5, and PP9 was 34, 2.3,and 1.3. We think that at least partof this residual PFC was due to im-purities in the liquid. Other PFC tested,including FC75, FC47, PID, E4, E3,Fomblin L, and Fomblin Z remainedfor months, probably for the lifetimeof the animal. We still have dogs withapparently as much PFC in their liversas was there 4 years ago when thePFC was first administered.

Examination by gas chromatographyand a 63Ni electron capture detector ofthe breath of mice having PP5 or PP9in their livers revealed the presence ofenough of these substances to more orless account for their disappearance atthe rate shown in Fig. 2. The breathof mice just injected with FC47, wherethe concentration in the blood was high(about 5 percent by volume), or fromniice that had been injected monthsbefore, was analyzed by gas chroma-tography and no detectable PFC wasfound. The smallest aniount of FC47detectable with this instrument is about200 pg.

In addition, we have found that per-fluorodiniethyldecalin perfluoro-(1,3-di-methyl) cyclohexane, perfluoromethyl-cyclohexane, and a mixture of per-fluorodecalin and perfluoro-( 1-3-di-methyl) cyclohexane leave the liverrapidly. In this unique family of per-fluorinated decalins and cyclohexanes,those having the higher vapor pressuresleave the most rapidly. It is easily dem-onstrated that these substances are pres-ent in the breath of mice and cats inlarge concentrations. Further, by plac-ing a cup on the aninials' skin, samplingthe gas space, and analyzing by gaschromatography we can show that itis diffusing through the skin.

Analysis of the liver of a mousewhich received (per kilogram of bodyweight) 100 nil of 10 percent FC47(0.2 ml of FC47) 8 months earlierrevealed the preseiice of 5.1 percentof FC47 (by volume). The liver wasallowed to dry for 4 days over silicagel. Analysis of the gas above thesilica gel showed no trace of FC47.The liver was removed, powdered in a

mortar, and reanalyzed for FC47. Itcontained 8 percent PFC (by volume)per gram of dried liver. FC47 has avapor pressture of only about 2 torr.But even PID with a vapor pressureof 13 torr remaiined in the liver in-

681

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definitely. The vapor pressure of per-fluorodecalin is 14 torr and that ofperfluoromethyldecalin is 5 torr.Our results show that there are

classes of PFC which form some kindof chemical bond with the liver sub-stance. Such PFC contain atoms otherthan carbon and fluorine. All of themcontain either a C-O-C or a C-N-Clinkage. One possibility for such acoupling is that the unshared electronpair on the oxygen or nitrogen atommay passively form bonds with thesubstance of the liver. If the bindingwas to protein then it is surprising thatit was not bound as much to, say,muscle. Ullrich's finding (9) that per-fluorohexane is bound to, or at leastinteracts with, cytochrome P450 sug-gests that certain PFC may form com-plexes with certain iron-containing pro-teins. The straight-chain fluorocarbonsstudied so far, on the other hand, rapid-ly leave the liver of the intact animal.

Another possibility is that an activemetabolic process is involved not onlyin the binding, but in the release fromthe liver. It may be, for example, thatthe perfluorocyclocarbons are activelyexcreted by the liver because they re-semble steroid fragments.

This new family of compounds con-taining only carbon and fluorine andhaving cyclic structures may make pos-sible PFC emulsions capable of beingsafely used in intact animals.

LELAND C. CLARK, JR.FERNANDO BECATTINI

SAMUEL KAPLAN, VIRGINIA OBROCKDAVID COHEN, CHARLES BECKER

Children's Hospital ResearchFoundation, Cincinnati, Ohio 45229

References and Notes

1. The nomenclature for these compounds isnot universally accepted. Perfluorochemical isused here to mean organic compounds thathave been fluorinated until no hydrogen re-mains. Fluorocarbon refers to compoundshaving only fluorine and carbon in the mole-cule. PP5 is cis- and tris-perfluorodecalin andsome impurities. PP9 is a mixture of isomersof perfluoromethyldecalin, some PP5, andsome impurities. PP5 and PP9 are tradenames of I.S.C. Chemicals, Ltd. PIID is aperfluorodiisopropoxybutane synthesized bythe Allied Chemical Company. After thismanuscript was read in proof we foundthat our perfluorodimethyldecalin (product10964, PCR, Fla.) has the same infrared spec-trum as the monomethyl compound.

2. L. C. Clark, Jr., and F. Gollan, Science 152,1755 (1966); F. Gollan, J. McDermott, A. E.Johnson, R. Namon, Fed. Proc. 29, 1725(1970); J. H. Modell, J. Newby, B. C. Ruiz,ibid., p. 1731; M. M. Patel, P. Szanto, B.Yates, D. M. Long, ibid., p. 1740; D. J. Sass,E. L. Ritman, P. E. Caskey, N. Bancherol,E. H. Wood, J. Appl. Physiol. 32, 451 (1972).

3. F. Gollan and L. C. Clark, Jr., Physiologist9, 191 (1966); H. A. Sloviter and T. Kami-moto, Nature 216, 458 (1967); L. Triner,M. Verosky, D. V. Habif, G. G. Nahas,Fed. Proc. 29, 1778 (1970); H. Brown andW. G. Hardison, Surgerv 71, 388 (1972).

682

4. L. C. Clark, Jr., Edmund-Hall Lecture, Uni-versity of Louisville Sigma Xi, 19 May 1967(W. Welch, Louisville Times 20 May 1967);R. P. Geyer, R. G. Monroe, K. Taylor,Organ Perfusion and Preservation (Appleton-Century-Crofts, New York, 1968), pp. 85-96;H. A. Sloviter, M. Petkovic, S. Ogoshi,H. Yamada, J. Appl. Physiol. 27, 666 (1969);L. C. Clark, Jr., S. Kaplan, F. Becattini,J. Thorac. Cardiovasc. Surg. 60, 757 (1970);L. C. Clark, Jr., F. Becattini, S. Kaplan,Triangle 11, 115 (1972).

5. D. M. Long, M.-S. Liu, P. S. Szanto, D. P.Alrenga, M. M. Patel, M. V. Rios, L. M.Nyhaus, Radiologist 105, 323 (1972).

6. L. C. Clark, Jr., Fed. Proc. 29, 1696 (1970).7. T. M. Reed, III, Fluorine Chemistry (Aca-

demic Press, New York, 1964), vol. 5, pp.133-231.

8. Blood samples and tissue homogenates werepipetted directly into an excess of sodium

the locus coeruleus, although these ratsmotor and exploratory activity.

Studies with the Falck-Hillarp histo-chemical technique (1) reveal the pres-ence of a network of norepinephrine-containing nerve terminals in themammalian cerebral cortex. Studies withlesions (2) and stimulation (3) showthat these terminals are derived fromcell bodies situated in the nucleus locuscoeruleus in the floor of the fourth ven-tricle. Electrical self-stimulation can beobtained through electrode tips in closeproximity to this nucleus (4), and thisnoradrenergic system may be one oftwo catecholamine-containing systemsthat will support this behavior (5, 6).On the basis of theoretical considera-tions, it has been proposed (5, 6a)that the norepinephrine-containing neu-

biphenyl reagent, where the fluorine on. th,compound was converted to fluoride ion; aftedilution with buffer the F- activity was measured with an Orion electrode. The method ibased in part upon that of P. P. Wheeleand M. I. Fauth [Anal. Chem. 38, 1971(1966)].

9. V. Ullrich and H. Diehl, Eur. J. Biochet20, 509 (1971).

10. Supported in part by grants HL12419 ancHD05221 from the National Institutes olHealth and a grant from the SouthwesterrOhio Chapter of the American Heart As-sociation. Dr. D. Steible and Mrs. DSchwartz assisted with some of the analytical work. We have received gifts from all o;the manufacturers of perfluorochemicals, bVwe are especially indebted to the Allie&Chemical Company.

16 April 1973; revised 11 May 1973

showed normal weight gain and normai

rons arising from the cell bodies of thelocus coeruleus function as a "reinforce.ment" system in the sense that thisterm is used in theories of learning.Our experiments were designed tc

test the theory that the norepinephrinecontaining neurons innervating thecerebral cortex form an essential com-ponent of the mechanisms of learning.Male hooded Lister rats '(initial weight,200 ± 10 g) were anesthetized withpentobarbitone, immobilized in a Koptstereotaxic apparatus, and had burnholes drilled bilaterally in the skullover the cerebellum. In one group (BH)of six rats,-no further procedures werecarried out before the wound was re-sutured. In three further groups ofrats, bilateral electrolytic lesions weremade by passing a charge of 15 to 20millicoulombs through the bare tip ofa varnished steel electrode to an analcathode. In one group (CB) of six rats,the electrode was located at symmetri-cally placed points in the cerebellar

Fig. 1. Histological preparations of theregion of the locus coeruleus in the floorof the fourth ventricle. (A) The locuscoeruleus on each side has been ablated,although cells of the mesencephalic tractof the trigeminal nerve (mesV), situatedlaterally to the locus coeruleus, have beenspared. This rat showed no increase inrunning speed in the course of behavioraltesting. (B) The locus coeruleus (LC) isintact on both sides in a rat in group BH.

SCIENCE. VOL. 181

Impaired Learning and Decreased Cortical Norepinephrineafter Bilateral Locus Coeruleus Lesions

Abstract. Bilateral lesions of the nucleus locus coeruleus in rats deplete thecerebral cortex of norepinephrine and significantly diminish the rate of increas&of running for food reward in a simple L-shaped runway. As assessed in thissituation, learning was absent in those rats with the most complete ablations oj

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