Significant Achievements in Space Bioscience 1958-1964

8/7/2019 Significant Achievements in Space Bioscience 1958-1964

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Space Biosc ience1958-1964C F S T I PRICE(S) $

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.1 N A SA SP-92

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Space Biosc ience1958-1964

1 9 6 6cientifit and Technicd I f rmation DivisionNATIONAL AERONAUTICS AND SPACE ADMINISTRATIONWashington, D.C.


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Foreword

H I S VOLUME IS ON E OF A SERIES which summarize theT rogress made during the period 1958 through 1964in discipline areas covered by the Space Science andApplications Program of the United States. In thisway, the contribution made by the National Aero-nautics and Space Administration is highlighted againstthe background of overall progress in each discipline.Succeeding issues will document .the results from lateryears.

T h e initial issue of this series appears in 10 volumes(NASA Special Publications 91 to 100) which describethe achievements in the following areas: Astronomy,Bioscience, Comrnunicacions and Navigation, Geodesy,Ionospheres and Radio Physics, Meteorology, Particlesand Fields, Planetary Atmospheres, Planetology, andSolar Physics.

Although we d o not here attempt to name those whohave contributed to our program during these first 6years, both in the experimental and theoretical researchand in the analysis, compilation, and reporting ofresults, nevertheless we wish to acknowledge all thecontributions to a very fruitful program in which thiscountry may take justifiable pride.

~ i -__- mi_._..-__n w m u 1 ; . I Y E W E L LAssociate Administrator f o r

Space Science and Ap plication s, N A S A


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PrefaceH I S SUMMARY OF CERTAIN ASPECTS of the space biology program ofT he National Aeronautics and Space Administration brings together

some results of NASA research and NASA-sponsored research undergrants and contracts from 1958 through 1964. Closely related researcheven though not sponsored by NASA is also included.

T h e space biology program has had a late start in comparison withthe space physics program, and only a token program existed before 1962.Much of the present research involves preparation of space-flight experi-ments an d obtainment of adequate baseline information. Perhaps halfthe research results reported are derived from the NASA program.Additional information is included from many other sources, especiallythe U.S. Air Force with its long history of work in aviation and aerospacemedicine.

Relatively few biological space-flight experiments have been under-taken. These have been to test life-support systems and to demonstrate,before manned space flight, an animals capability to survive. Fewcritical biological experiments have been placed in orbit by NASA, buta biosatellite program will soon make a detailed study of the fundamentalbiological effects of weightlessness, biorhythms, and radiation.

T h e search for extraterrestrial life has been limited to ground-basedresearch and planning for planetary and lunar landings. Life-detectionexperiments have been developed and tested, and an important andexciting program is being planned to detect and study extraterrestriallife, if it exists.Interest in space biology has been slow in developing, and there hasbeen some caution and controversy in the scientific community. How-ever, increased interest is starting to push forward the frontier of thisfiew aiid i x p x i ~ tciextifc fie!& 2nd f ~ m r e i.i!lonk appears to beoptimistic.

This summary was written and compiled by the members of the Bio-science Programs Division of the Office of Space Science and Applications.T h e report was edited and chapters 1, 3, 6, and 7 were written by Dale W.,Jenkins, Chief, Environmental Biology; chapter 2, by Gregg Mami-kunian, Staff Scientist, Exobiology; chapters 4 and 8, by Richard E.Bellevillc, Chief, Behavioral Biology; and chapter 5 , by George J. Jacobs,Chief, Physical Biology.

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Contents

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B A C K G R O U N DEXOBIOLOGY _ _ - _ _ - _ _ - _ - _ _ - _ _ _ _ _ _ - - - - - - - _ _ _ _ _ _ _ _ - - _ENVIRONMENTAL BIOLOGYBEHAVIORAL BIOLOGY _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _MOLECULAR BIOLOGY A N D BIOINSTRUMENTATION--FLIGHT PROGRAMS _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _MANNED SPACE FLIGHT _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _SIGNIFICANCE O F TH E ACHIEVEMENTS _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

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chapter 1Background

HE BIOLOGICAL P K O G K A M of the National Aeronautics and SpaceT dministration had a late start. A small life sciences group, organ-ized in 1958, was concerned with life support and use of primates forsystem and vehicle testing for the Mercury program. Thre e small sub-orbital flights of biological materials were flown in space.

T h e Bioscience Program Office of the Office of Space Science and Appli-cations was organized in 1962. T h e goals of the Bioscience Program are:(1) to determine if extraterrestrial life exists anywhere in the solar systemand to study its origin, nature, and level of development, if i t is present;(2 ) to determine the effects of space and planetary environments onEarth organisms, including man; (3 ) to conduct biological research todevelop life support and protective measures for extended manned spaceflight; and (4 ) to develop fundamental theories in biology relative toorigin, development, and relationship to environment. Research anddevelopment has been carried out to design life-detection experimentsand instruments for future flights to Mars and to develop experimentsto study the effects of the space environment on living organisms. A bio-satellite program, started in 1963, has the first of six flights scheduledfor 1966.Space exploration has demanded a rigorous development, especially i nthe biosciences area. Investigation of the solar system for exotic lifeforms, the environmental extremes to which Earth organisms (includingman) are being exposed, the possibilities for modification of planetaryenvironments by biological techniques yet to be developed, and theproblems of communication in biosystems are areas which have requiredrefinement of the theoretical framework oi biology before progress couldbe made rapidly enough to keep pace with technological advances intransportation.Of all the sciences, biology alone has not yet benefited from compari-sons with the universe beyond Earth. I t is reasonable to suppose thatbreakthroughs might be made in biology on the basis of comparisons


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+S P A C E B I O S C I E N C Ewith life from other worlds. Organisms elsewhere niay have foundalternatives to processes we think of as basic characteristics of life.

In contrast, physical science has advanced sufficieritly to provide a greatbody of laws which may be expressed in mathematical terms, and bywhich phenomena may be predicted with complete accuracy. A well-known characteristic of biological phenomena is variability. T h eDarwinian concept of evolution is perhaps the only pervading generali-zation in biology. This concept has been supported by evidence of ahereditary mechanism in the discovery of genes and gene mutations.

Space bioscience represents the convergence of many disciplines witha single orientation, whose direction is determined by the problems ofi~ ia nn cd pace travel which have, in tu rn , created a host of bioengineer-iiig problenis concerned with supporting inan in space.

Foremost among these questions is the possibility of the existence ofextraterrestrial life. T h e field which is concerned with the search forextraterrestrial life has come to be called exobiology. In addit ion tothe challenge of great technological problems which must be solved,exobiology is so closely related to the central scientific questions inbiological science that it is considered by some to be the most significantpursuit in all of science.

One of the mi jo r opportunities already presented by the advances inpropulsion systems is the abil ity to escape from the influence of the Earth,which has made possible the study of organism-environment relation-ships, particularly the role that environmental stimuli play i n the estab-lishment and maintenance of normal organization in living systems.

Transcending even these formidable objectives of space bioscience isan objective shared by all life sciences, the discovery of natures schemefor coding the messages contained in biological molecules. Extra-terrestrial biology seeks to find not only evidence of life now present, butthe vestigial chemicals of its previous existence. T h e ways and meanshave already been made available to study molecules on whose long,recorded messages is written the autobiography of evolution-the historyof living organisms extending back to the beginnings of life. On thissame basis, it is now within the realm of science to foresee the means ofpredicting the development of life from primordial, nonliving chemicalsystems. Closely allied to the search for extraterrestrial life is researchwhich seeks to identify the materials and the conditions which are theprerequisites of life.

Space bioscience research is now extending human knowledge of funda-mental biological phenomena, both in space an d on Earth, just as [liephysical sciences explore other aspects of the universe. T h e accomplish-men t of bioscience objectives is totally dependent upon advances in thetechnology of space flight. A highly developed launch-vehicle capability2


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. BACKGROLUVDis essential to accomplish the long-duration missions required in thesearch for extraterrestrial life.

Life on other planets in the solar system (with emphasis on Mars) willbe investigated.by full exploitation of space technology which will allowboth remote (orbiter) and direct (lander) observations of the planetaryatmosphere., surface, and subsurface. Certain characteristics of terrestriallife, such as growth and reproduction, provide a basis for relativelysimple experiments which may be used on early missions to detect theexistence of life on Mars. Later missions will provide extensive auto-matic laboratory capabilities for analyzing many samples taken fromvarious depths an d locations. Because of the hypothetical nature ofcurrent experiment designs, i t is likely that visual observations of theplanet will be required. Many technical problems are involved in stor-ing and transmitting the large amounts of data over planetary distances.Such visual observations might very well be crucial in interpreting resultsfrom other experiments. Critical to all exploration of the Moon andplanets are the requirements to: (1) prevent contamination of theenvironment with Earth organisms and preserve th e existing conditionsof the planet for biological exploration; (2) provide strict quarantine foranything returned to Earth from the Moon an d planets.

T h e biological exploration of Mars is a scientific undertaking of thegreatest significance. Its realization will be a major milestone in thehistory of human achievement. T h e characterization of life, if present,an d study of the evolutionary processes involved and their relationshipto the evolution of terrestrial life would have a great scientific andphilosophical impact. What is at stake is nothing less than knowledgeof ou r place in nature.

Extended Earth orbital flights with subhuman specimens will be usedto determine the effects on Earth organisms of prolonged weightlessness,radiation, and removal from the influence of the Earths rotation. Suchflights of biosatellites an d other suitable spacecraft are expected to:(1) establish biological specifications for extending the durat ion ofmanned space flight; (2) provide a flexible means of testing unforeseencontingencies, thus providing an effective biological backup for mannedmissions; (3 ) yield experimentai data more rapidly by virtue of thegreater number and expendability of subjects; (4) anticipate possible de-layed effects appearing in later life or in subsequent generations, throughuse of animal subjects with more rapid development and aging; (5) de-velop and test new physiological instrumentation techniques, surgicalpreparations, prophylactic techniques, and therapeutic procedures whichare not possible o n human subjects; and (6) provide a broad backgroundof experience and data which will permit more accurate interpretationsof observed effects of space flight on living organisms, including man.

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chapter 2Exobiology

HE POSSIBILITY OF DISCOVERING AN INDEPLNDI-NT LIFI FORM on a planetT ther than Earth presents an unequaled challenge in the history ofscientific search. Therefore, the detection of life within the solar systemis a major objective of space research in the foreseeable future.

T h e scientific data presently available concerning the possible existenceof a Mart ian life form and the chemical constitution of the surface ofMars are disappointingly few. In fact, it is impossible to make a state-ment about any of the many surface features, other than the polar caps,with any degree of certainty. T h e observational results have beenaccounted for by many conflicting hypotheses which can only be resolvedby the accumulation of new evidence.

T h e arguments supporting the existence of Martian life (ref. 1) arebased on the tollowing observations:

(1) T h e various colors, including green, exhibited by the dark areas(2 ) T h e seasonal changes in the visual albedo and polarization of the(3) T h e ability of the dark areas to regenerate after an extensive dust-(4) T h e presence of absorption bands at 3.3p-3.7p, attributed toConflicting interpretations of the above obsci vations have been ad-

vanced. T h e argument based on the colors is inconclusive, and severalworkers have suggested that the color is a contrast effect with the bright-reddish continents. T h e meager quantitative data have been discussedby d p i k (ref. 2) who has reduced Kozyrevs photometric observations otthe very dark area of Syrtis Major to intrinsic reflectivities by allowingfor the estimated atmospheric attenuation and reflectivity. Kuiper (ref.3) similarly demonstrated the absence of the near-infrared refiection

dark areasstormorganic moiecuies

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S P A C E B I O S C I E N C E

maximum, which is characteristic of most green plants, indicating thatchlorophyll was not responsible for the color.

However,serious limitations are imposed on the second i f the severity of theMartian climate is considered. Fiicas (ref. 4)has photometrically meas-ured the seasonal changes in the fine structure of the dark areas of Marsand concludes that-

(1) The dark areas of Mars show periodic variation of intensity follow-in g the cycle of the darkening element

( 2 ) The average intensity of the dark area, not including the action ofthe darkening waves, increases from the poles toward the equator

(3) T h e action of each of the darkening waves decreases from the polestoward the equator. Thi s decrease is balanced in the equatorialzone by the combined action o f the twn darkening waves alter-nately originating at the two poles. T h e mechanism of thedarkness-gencrating elenicnt seems to bc- constant for all latitudesdur ing the Martian year.

The variation in intensity has been explained rcccntly by nonlifeniechanisnis for Depressio Hellespontica (an area showing one of thegreatest seasonal changes) (ref. 2). Similar nonlile nicchanisnis may beapplicable to the other dark regions, antl, thus, the darkening can beused only as circumstantial evidence in support of a Martian lifc form.

I f inorganic interpretations of the seasonal albetlo variation areaccepted, thcn an inorganic interpretation must a l s o be atlvancctl for thepolarimtion variation.

(1 ) A change in surface texture, caused b y wrying al)sorption o f atnios-phcric constituents, causing both the all,etlo antl po la riat ion tochange in the manner observed

(2) A change in surface texture, in which the surlace material becomesrougher, which also explains the ol)scm.etl polari/ation da ta(ref. 5)The third argument against the regenerative Ieaturc o f the dark areas

being a lire process has bee11 advanced b y Kuiller (ref. 6). I t is h e t l onatmosphcric circulation causing dust, prcsuiii; i l) ly l a v a , t o be blown onthe tlark areas oi Mars du rin g the late siininier, ;iiituiiin, an(1 winter, andthcn reinovd during the spring. M,lamik1ini:cn a n d \loorc 1i;ivc rcccntlyadvanced the siiiiilar explanation that t x ~ l ~ o i i : i ( ~ o u ~hondrites orasteroidal matter may induce the o l ) s c ~ v c . t l~ ~ l i ~ ~ i i ~ ~ i i i c ~ i i o i il t h e y art; I l ) l i l i t l i i l i t 011 the planets si1rI;ice. T h e ~)iil\.ei.i/etl 1101itli.i tic iliatcriiilw i l l exhibit a high degree of opacity due t o I o c ; t I i / : i t i , ) i i a i i t l , h(mc.e, :Ichange in polariiation characteristics ; i n d ;I rlec i~c;isc~n I)oIari/atio11

T h e second an d third arguments remain the most cogent.

Two possibilities can he suggested:


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E X O B I O L O G Yfollowing mixing of the chondritic material with indigenous surfaceminerals.

T h e fourth observational argument, the Sinton bands (ref. 7), has beenshown to be at least doubtfu l. Rea, Belsky, and Calvin (ref. 8) recordedinfrared reflection spectra for a large number of inorganic and organicsamples, including minerals an d biological specimens, for the purpose ofinterpreting the 3p-to-4~pectrum of Mars. These authors state th at aprevious suggestion that the Martian bands be attributed solely tocarbohydrates is not a required conclusion. At the same time they failto present a satisfactory alternate explanation, and the problem remainsunsolved. More recently, Rea et al. (ref. 9) noted the similarity betweenthe 3.58, and 3.69, minima in the Mart ian infrared spectra and those ofD,O-HDO-H,O mixtures and, particularly, of HDO.

With all this marked disagreement in interpreting the observationaldata concerning Mars, i t becomes clearly evident tha t an experimentalapproach to the detection of life on Mars should provide the maximumpositive information possible. Some life-detection experiments devel-oped with N A S A support have been summarized by Quimby (ref. 10).

T h e schema of the biological exploration of a planet is to conduct aseries of complementary experiments proceeding from general to specific.The general experiments will examine gross characteristics of the planetsenvironment an d surface for determining the probability of an activebiota (life). Data from the general experiments will be significant in-

(1) Defining the nature of specific experiments in which life detectionis the major objective; and(2) Providing a high degree of confidence in undertaking specific exper-

iments, since indications from the gross characterization of theplanet in question will influence the choice and design of thespecific experiments.

T h e biological exploration of planets is then to be defined as the searchlor those parameters relevant to the origin, development, sustenance, anddegradation of life in a planetary environment. Thi s definition will giverise to a critical question for each progressively specific and complex

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experiment to determine-(1) lheexistence of iife on the p ia~ i r i(2) T h e degree of similarity or dissimilarity (structure and function)

with respect to terrestrial life(3) T h e origin of this planetary lifeT h e immediate objective of the biological explorations of the planet

is to define the state of the planetary surface, which may exhibit thefollowing properties:

(1 ) A prebiota (defined as the absence of life)7


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SP ACE BIO SCIEN CE(2) An active biota (defined as the presence of life)(3) An extinct biota (defined as evidence of former life)T h e identification an d the detailed characterization of each of the

above stages of planetary development constitute the subject matter ofthe biological exploration of the planets and, specifically, Mars.

THE EXPERIMENTAL INVESTIGATION OF CHEMICAL EVOLUTIONAttempts have been made to simulate and approximate models of

primitive Earth conditions for abiogenic synthesis, and successful synthe-sis of essential biochemical constituents necessary for mainta ining li f thas been partly accomplished.

Urey (ref. 11) has clearly pointed out the possible role of a reducingatmosphere in the synthesis of prebiological organic molecules. Miller(ref. 12) synthesi7ed a variety of amino acids in a reducing atmosphere bymeans of an electrical discharge. A variety of organic compounds havebeen synthesized by the action of various energy sources upon reducingatmospheres, and several investigators have extended the Urey-Miller-type reactions to synthesize nucleic acid components (ref. 13) , adenosinetriphosphate (ref. 14), and a host of biologically essential organiccompounds.

It is likely that in the synthesis of organic moieties, simple and specificmolecules were first produced when the planets had a reducing atmos-phere. Further complexity or degradation of the organic compoundsproduced varied, depending on the geochemical changes of the planetssurface, the atmospheric constituents, the degree of interaction betweensurface and atmosphere, and the rate of the organic synthesis. Oparin(ref. 15) presented the most detailed mechanisms for the spontaneousgeneration of the first living organism arising in a sea of organic com-pounds synthesi/ed in a reducing atmosphere on Earth.

It is generally accepted that, under favorable conditions, life can ariseby spontaneous generation. A primary requirement for this ini tiation isthat there be abundant organic compounds concentrated in one or morespecific zones. These simple organic molecules would undergo modifica-tion to develop a greater structural complexity and specificity, finallygiving rise to a living organism. Therefore, because of the ease withwhich organic compounds can be synthesized under reducing conditions,planetary surfaces may contain an abu nd an t sourcc of similar organicmatter. However, difficulties arise in postulating steps for furtherorganization or modification of the above synthesizcd organic niatteiinto a living state. Most of the original organic matter produced in theprimary reducing atmospheres of the various planets may have beenqui te similar. However, major variations between planets, in chemical

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E X O B I O L O G Yevolution beyond the prebiotic stage, must have been the rule ratherthan the exception.

T h e primary interest in this area of research has been the realkation ofthe possible existence of organic molecules on planetary surfaces and,particularly, Mars. Pertinent synthesis may be either biological orabiological. Research conducted in the simulation of cosmochemicalsynthesis has used most of the available solar spectrum. Simulationexperiments devised to study the effects of these energies on the assumedearly atmosphere of the Earth have yielded products that play a domi-nant role in molecular and biochemical organization of the cell.

Calvin (ref. 16) irradiated water and carbon dioxide in a cyclotron,obtaining formaldehyde and formic acid. Miller (ref. 17) found thatwhen methane, ammonia, water, and hydrogen were subjected to a high-frequency electrical discharge, several amino acids were produced alongwith a variety of other organic compounds.

Corroborating experiments established that the synthesis of aminoacids occurred readily. The apparent mechanism for the production ofamino acids is as follows: aldehydes and hydrogen cyanide are synthe-sized in the gas phase by the electrical discharge. These substance1react together and also together with ammonia in the water phase of thesystem to give hydroxy and amino nitri les, which are then hydrolyzed tohydroxy an d amino acids. Among the major constituents were asparticacid, glutamic acid, glycine, a-alanine, and ,&alanine.

T h e Miller-Urey reaction mixture has been extended and severalmodifications introduced. Or6 (ref. 18) introduced hydrogen cyanideinto the system as the primary gas component. Adenine was obtainedwhen Or6 heated a concentrated solution of hydrogen cyanide in aqueousammonia for several days at temperatures up to 100 C. Adenine is anessential component of nucleic acids and of several important coenzymes.Guanine a nd urea were the two other products identified in the hydrogencyanide reaction. Or6 further obtained guanine and uracil as productsof nonenzymatic reactions by using certain purine intermediates asstarting materials.

?or?narr?perum:, (ref. 19) also obtained adenine upon irradiation ofmethane, ammonia, hydrogen, and water, using a high-energy electronbeam as the source of energy of irradiation. These results indicate thatadenine is very readily synthesized under abiotic conditions. Adenine,among the biologically important purines and pyrimidines, has thegreatest resonance energy, thus making its synthesis more likely andimparting greater radiation stability to the molecule.

T h e formation of adenine and guanine, the purines in RNA an dDNA, by a relatively simple abiological process lends further support

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S P A C E B I O S C I E N C Eto the hypothesis that essential biochemical constituents of life may haveoriginated on Ear th by a gradual chemical evolution and selection. Inthis respect, the examination of planetary surfaces-specifically Mars-presents practical implications for current research on the problem ofchemical evolution.

Lthen Ponnamperuma et al. (ref. 14) exposed adenine and ribose toultraviolet light in the presence 01 phosphate, adenosine was produced.\\hen the adenine and ribose were similarly exposed in the presence ofthe ethyl ester of polyphosphoric acid, adenosine diphosphate (ADP) andadenosine triphosphate (A TP) were produced. T h e abiological forma-tion of ATP was a major stride along the path of chemical evolution,since AT P is the principal free energy source of living organisms.

Oparin (ref. 15) postulated that a-amino acids could have been formednonbiologically from hydrocarbons, ammonia, and hydrogen cyanide ata time when the Earths atmosphere contained these substances in highconcentrations. Oparins hypothesis has received strong experimentalsupport, as evidenced by the work of Miller (ref. 12). Bernal (ref. 20)has emphasized the role played by ultraviolet light in the formation oforganic compounds a t a certain stage of the Earths evolution.

Genei ally it has been believed that the first proteins or foreproteinwere nonbiologically formed by the polycondensation of preformed freeamino acids (ref. 21). Akabori (ref. 2 2) proposed a hypothesis for theorigin 01 the foreprotein and speculated that it must have been producedthrough reactions consisting of the following three steps.

T h e first step is the formation of aminoacetonitrile from tornialdehyde,ammonia, an d hydrogen cyanide.

CH2O+NH,+HCK + 2N--CHZ--CN+HzOThe second is the polymerization of aminoacetonitrile on a solid sur-

face, probably absorbed on clay, followed by the hydrolysis of the polymeito polyglycine and ammonia.

x HZN-CHZ-CN + -NH-CHZ-C -) \NH

+X HZO1-N H-CH,-CO-), +xN H :2) and mesons are

Thebe flights gave no evidence of radiation damage.

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SPACE BIOSCIENCEshort flight durations of these vehicles did not provide substantial infor-mation. T h e USAF Discoverer satellite program has given impetus tocosmic-ray research and provided for longer staytimes.

I t has been difficult to separate radiation effects from other space-flightfactors; therefore, some of the alterations observed are still subject todebate. Vibration, acceleration, and weightlessness appear to be thethree most important additional parameters. Measurements of radiationdosagc have been made by chemical and photographic dosimetry, ionchambers, and biological dosimetry. All evidence to date indicates thatradiation exposure levcls arc not hazardous to man at present orbitalaltitudes u p to 200 nautical miles. Most biological materials flown so farhave been for the express purpose of investigating space-radiation levelsand effects. T h e biological materials have ranged f rom tissue culturesto entire organisms and from phage and bacterial cells to man. T h estudies have required much of the space an d weight resources allottedbiology by the U.S.S.R. antl the United States. They have been accom-panied by ground-based controls.

T h e Vostok series provided the following data:(1) A small, but statistically significant, increase was observed in the

percentage of chromosome aberrations i n the rootlet cells ofair-dried wheat and pea seeds after germination. In this caseonly, the increase did not depend on Hight duration.(2 j Lysogenic bacteria exhibited an increase of genetic alterations andincreased phage production. Length of Hight was associatedwith increased bacteriophage production by the lysogenic bac-teria. There was an increase of recessive lethals coupled withnonconvergence of chromosomes (sex linked) in the f ru it fly. Astimulation of cell division in wheat and pea seeds w;is otmrved.Cultures of human cells exposed to space-flight factors did notdiffer significantly from terrestrial controls with respect to suchindicators as proliferation rate, percentage of mortality antlmorphological, antigenic, antl cultural properties. Repeatedflights of the identical HeLa cells revealed that there was alonger latcnt period for restoration of growth capacity than incells carried in to space once or not flown at all.

(3) T h e most definite radiation cffccts observcd were only revcaletl ingenetic tests. N o harmful influence on those characteristicsaffecting the viability of thc organism has been discovered.

T h e Air Force Discoverer series launched froni the west coast had afew successful flights incorporating organisms. \Vith severe environ-mental stress and long recovery times, data on radiation exposure wereequivocal u p to Discovcrcr X V I I antl X V I l I when cultures of human28


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E N V I R O N M E N T A L B I O L O G Ytissue were flown, recovered, and assessed fo r radiation exposure effects.Comparison with ground-based controls revealed no measurabledifferences.

Radiation dosimetry from the Mercury series established that minimalexposures were encountered at those orbital altitudes. A typical exampleis the MA-8 flight of W. M. Schirra, Jr., during which the body surfacedosage was less than 30 millirads.

NASA has supported fundamental radiation studies at the Oak RidgeNational Laboratory and the Lawrence Radiation Laboratory. Empha-sis has been placed on the biological effects of high-energy proton radia-tion and particulate radiation from accelerators.

At the NASA Ames Research Center extensive fundamental studiesare being carried out on the effects of radiation, especially in the nervoussystem. It has been demonstrated that deposits accumulate in the brainfollowing exposure to large doses of ionizing particle radiation as wellas after X-irradiation. These deposits, referred to as a chemical lesion,result from a n accumulation of glycogen. T h e formation of thesedeposits during exposure to large doses of X-irradiation was no t increasedin environments of 99.5 percent oxygen and increased atmosphericpressure.

S I M U L A T I O N OF P L A N E T A R Y ( M A R T I A N ) E N V I R O N M E N T SAttempts have been made to simulate to some degree the various

parameters of the Martian environment, such as atmospheric composi-tion, pressure, radiation flux, temperatures, and the day-night as well asseasonal cycles. Certain factors for Mars cannot yet be simulated, suchas soil composition, gravitational field, magnetic field, and electrical field.

Caution is required in interpreting all simulation experiments. HOWEarth organisms respond to simulated Martian environments probablyhas nothing to do with life on Mars, but these experiments may showwhether or not anything in the environment of Mars makes life as weknow it impossible. We must expect that on Mars, life will have evolvedand have adapted over long periods of time under conditions which arequite different from conditions on Earth. T h e simulation experimentsalso provide some information about the possibility of contaminatingthe planet Mars, or any planet, with organisms from Earth. In addition,they give us some clues about the possibilities of adaptation and evolu-tion of life under these conditions.

From an evolutionary point of view, if life has developed on Mars, weexpect it to have evolved at least to a microbial stage. On Earth, micro-organisms are the most ubiquitous and numerous forms of life. Thisfact should be considered in studying extraterrestrial bodies.

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Micro-organisms have been selected as the best test organisms, andbacteria and fungi have been used because they are durable and easy togrow. Also, because of their rapid growth, many generations can bestudied in a relatively short period of time. The organisms includechemoautotrophic bacteria, which are able to synthesize their cell con-stituents f rom carbon dioxide by energy derived from inorganic reac-tions; anaerobic bacteria, which grow only in the absence of molecularoxygen; photoautotrophic plants such as algae, lichens, and more com-plex seed plants; and small terrestrial animals.

Organisms have been collected from tundra, desert, hot springs, alpine,and saline habitats to obtain species with specialized capabilities to con-serve water, balance osmotic discrepancies, store gases, accommodate totemperature extremes, and otherwise meet stresses. An attempt is madein these simulation experiments to extend these processes across thepossible overlapping microenvironments which Earth and Mars mayshare.

Scientists have developed various special environmental simulators,including Mars jars an d Marsariums. These have made possiblecontrolled temperatures, atmospheres, pressures, water activities, andsoil conditions for duplicating assumed Martian surface. A complexsimulator, developed b y Young et al. (ref. 52), reproduces the formationof a permafrost layer with some water tied u p in the form of ice beneaththe soil surface. This simulator serves as a model to study the wave ofdarkening, thus supporting the hypothesis that thc pole-to-equatorwave of darkening is correlated with the availability of subsurface water.T h e simulator is a heavily insulated 2-cu-ft capacity chamber with aninternal pressure of 0.1 atm. T h e chamber contains a soil mixture oflimonite and sand and an atmosphere of carbon dioxide and nitrogen.With the use of a liquid nitrogen heat exchanger at one end and anexternal battery of infrared lamps at the other end , the temperature simu-lates that of Mars from pole to equator. Thermocouples throughoutthe soil monitor the temperatures in the chamber.

Zhukova and Kondratyev (ref. 69) designed a structure tneasuringl00X 150x180 cm. Micro-organisms were placed at the surface of acopper bar made in a special groove separated by glass cloth. Copperwas selected as one of thc best heat-conduction materials pe rmitting arapid change of temperature. T h e lower end of the bar was immersedinto a mixture of dry ice and ethyl alcohol, which made it possible tocreate a temperature of - 6OO C. Heating was performed by an incandes-cent spiral.

AS the knowledge concerning the Martian environment becomes morerefined, scientists can more accurately simulate this environment under30


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E N V I R O N M E N T A L B I O L O G Ycontrolled conditions in the laboratory. Determination of the effects ofthe Mar tian environment on Earth organisms will permit better theori-zation on the forms of life we might find on Mars and will permit us toestimate the potential survival of Earth contaminants on Mars.

However, until the environmental conditions of Mars are definedmore accurately, the experiments must be changed continually to fitnewly determined conditions. Therefore, existing simulation data aremade less valid for comparison. T h e data resulting from the simulationexperiments for Mars have been compiled in table 11, and the experi-ments are summarized below.

T h e earliest simulation studies were carried out by the .4ir Force, andthe studies during the past 6 years have been supported by NASA.Recently, these studies have received less support or have been termi-nated in favor of critical studies on the effects of biologically importantenvironmental extreme factors on Earth organisms. These critical studiespermit establishing the extreme environmental factor parameters inwhich Earth life can grow or survive. These data will have valuableapplication to the consideration of life on any planet, to the design oflife-detection instruments, to the sterilization of space vehicles, and tothe problem of contamination of planets.

Some exploratory experimental studies are in progress to study thecapabilities of organisms to grow under the assumed conditions onJupiter. These include studies at high pressure with liquid ammonia,methane, and other reducing compounds.

Early experiments simulating Martian conditions using soil bacteriawere carried ou t by Davis an d Fulton (ref. 70) at the Air Force School ofAviation Medicine, San Antonio, Tex. Mixed populations of soilbacteria were pa t i n Mars jars with the following conditions: 65-mmHg pressure, 1 percent water or less, nitrogen atmosphere, rsandstone-lava soil, and a temperature day-night cycle of f 2 5 O to - 2 5 O C. T h emoisture was controlled by desiccating the soil and adding a givenamount of water. Experiments, conducted u p to 10 months, demon-strated that obligate aerobes died quickly. The anaerobes and spore-formers survived. Although a small increase in the total number oforganisms indicated growth, the increases in the number of bacteria mayhave been due to breaking up clumps of dirt .

Roberts and Irvine (ref. 71) reported that, in a simulated Martianenvironment, colony counts of a sporeforming bacterium, Ba ci l lus cereus,increased when 8 percent moisture was added. Moisture was consideredmore important than temperature or atmospheric gases inasmuch as asimulated Martian microenvironment containing 8 percent moisturepermitted germination an d growth of endospores of Cl os t r i d i um sporo-

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E N V I R O N M E N T A L B I O L O G Igenes . Increases in colony counts of Baci l lus ceieus appeared to beinfluenced by temperature cycling (ref. 72).

Studies of the effects of simulated Mart ian cnvironinents on spore-forming anaerobic bacteria were carried out by Hawrylewicz et al. (ref.49). Th ey showed that the encapsulated facultative anaerobe, Klebsiellap n e u m o n i a e , survived under simulated Martian atmosphere for 6 to 8months, but were less virulent than the freshly isolated organisms.Spores of the anaerobe C l o st r id i u m b o t u l i n u m survived 10 months in thesimulator. Hagen et al. (ref. 53) found that the addition of moisture todry-simulated Martian soil did not improve the survival of Baci l lussub t i l i s or Pseu dom ona s aeruginosa. Baci l lus cei eus spores survived,with added organic medium plus moisture, but n o germination of thespores resulted.

Hawrylewicz et al. (ref. 49) put rocks from Antarctica bearing variouslichens in simulated Martian conditions in a large desiccator. Th eyfound that the algal portion of a lichen, Trebozix ia c i ic i , showed onlyslight resistance to the Martian environment. Th ey also pointed ou t theeffect moisture had on the physical condition of lichens. The under-surface of a lichen has great water-absorbing capability, and the slightestamount of moisture on a rock surface is absorbed by the lichen which canturn green in 15 minutes.

Scher et al. (ref. 51) exposed desert soils to simulated environmentalconditions and diu rnal cycles of Mars. T h e atmosphere consisted of 95percent nitrogen and 5 percent carbon dioxide (no oxygen) and wasdried, using calcium sulfate as a desiccant. T h e total atmosphericpressure was 0.1 atm. T h e temperature ranged from - 6 O O to + ZOO C in24-hour cycles. One hour was spent at the maximum and a t the mini-mu m temperatures. T h e chambers were irradiated with ultraviolet,2537 A, with a dose of 10" ergs/cm2, which is comparable to a daily dosefound on Mars, an d easily exceeds the mean lethal dose for unprotectedbacteria. Soil aliquots were removed weekly and incubated at 30' C.T h e scoring was done both aerobically and anaerobically. Sporeformingobligate and facultative anaerobes, including Closti id iu m, Bac il lus , andPlanosarc ina , and nonsporeforming facultative anaerobes, includingP s e u d o m o n a s and R h o d o p s e u d o m o n a s , were found. T h e experimentalchambers were frozen and thawed cyclically up to 6 months. Organismsthat were able to survive the first freeze-thaw cycle were able to survivethe entire experiment. T h e ultraviolet irradiation did not kill sub-surface organisms, and a thin layer of soil served as an ultraviolet shield.A l l of the samples showed survivors.Young et al. (ref. 52) assumed that water is present on Mars, at least inmicroenvironments, and that nutr ients would be available. The primary

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S PA C E B I O S C I E N C E Iobjective of their experiments was to determine the likelihood of con-taminating Mars with Earth organisms should a space probe from Earthencounter an optimum microenvironment in terms of watFr and nutri-ents. T h e experiments used bacteria in liquid nutrient media. T heenvironment consisted of a carbon dioxide-nitrogen atmosphere, andthe temperature cycling was - 7 0 O to +25O C, with a maximum timeabove freezing of 4% hours. Aerobacter aerogenes and P s e u d o m o n a s SP.grew in nutrient medium under Martian treezing and thawing cycles.Atmospheric pressure was not a significant factor in the growth ofbacteria under these conditions.

Silverman et al. (ref. 47) studied bacteria antl a fungus under extreme-bu t not Martian-conditions. Spores of five test organisms ( B . s u b t i hvar. niger , B . m e g a t e r i u m , B . s t ea?o t i i e rmoph i lus , C los t r id ium sporo-genes , and Asperg i l lu s n iger ) and soils were exposcd while under ultra-high vacuum to temperatures of from - 1 9 0 O to +17O0 C for 4 to 5 days.Up to 25 C there was no loss in viability; at higher temperatures, differ-ences in resistivity were observctl. At 8 8 O C, only B. sub t i / i s and A . n ig ersurvived in appreciable numbers; at 1 0 7 O C, only A . n i ge r spores sur-vived; none were recoverable after exposure to 120 C. R . subt i l i s sur-vived a t atmospheric pressure and 9 0 O C for 5 days, bu t none of the otherspores were viable after 2 days. Four groups of soil organisms (mesophilic,aerol)ic.,and anaerobic bacteria, molds, an d actinomycetes) were similarlytested in the vacuum chamber. From one sample only actinomycetessurvived 120 C, while one other soil sample yielded viable bacteria afterexposure to 170 C. Several organisms resisted 120O C in ultrahighvacuum for 4 to 5 days. IVhen irradiated with gamma rays from a cobalt60 source, differences were observed between vacuum-dried sporesirradiated while under vacuum antl those exposed to air imniecliatelybefore irradiation. A reduction of from one-third to one-ninth of theviability of spores irradiated in vacuum occurrccl with vacuuni-treatedspores irradiated in a ir.

Siege1 et al. (ref. 73), in approximate simulations of Martian environ-ments, studied tolerances of certain seed plants, such as cucumbers, corn,and winter rye, to low temperatures and lowered oxygen tensions.1.owered oxygen tensions enhanced the resistance o f seedlings, particu-1;irly cucumber ant1 rye to freeling. and lowered the niininiuni teniper;i-ture required for germination. Germination of seeds in the absence ofliquid water has also been studied. In this case, seeds of xerophytes havebeen suspended in air at 55-mni Hg pressure above water. T h e a ir wiibthus saturated. Germination was slow bu t did occur.

Siege1 et al. (refs. 7 3 and 74) found that the growth rate of severalhigher plants was enhanced by certain gases usually thought to be toxic,3f


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E N V I R O N M E N T A L BIOLOGYsuch as N2Q. This finding is significant inasmuch as the presence ofnitrogen oxides in the Martian atmosphere has been cited as evidencefor the nonexistence of plants on that planet by Kiess et al. (ref. 75).Exploratory survival tests showed that various mature plants, as well asthe ,larvae, pupae, and adult specimens of a coleopteran insect, wereundamaged when exposed to at least 40 hours of an atmosphere contain-ing 96.5 percent NzO, 0.7 percent O,, and 2.8 percent N,.

Lichens are of interest because of their ability to survive and thriveunder extreme environmental conditions on Earth. Biological activityof slow-growing lichens was detected by metabolic gas exchange, C 0 2detection being especially convenient. Siege1 points out that this methodis sensitive and nondestructive, to be preferred to staining techniques,which a t present are limited because they are only semiquantitative, sub-jective, and destructive of the lichen.A Russian study of simulated planetary environments has been per-formed with good simulation but for periods of only 2 to 6 hours. Com-ments on simulat ion experiments made by Zhukova and Kondratyev (ref.69) are presented as follows:

On the basis of modern conceptions on Martian conditions i t is difficult to imaginethat higher forms of an imals or plants exist on the planet. .4Martian change of sea-sons similar to that of our planet empoivers n s to think that there is a circulation ofan organic substance on Mars. which cannot exist without participation of microbicforms of life. Microorganisms are the most proba1,le inhabi tan ts of Mars although thepossibility is not excluded that their physiological features will be very specific. T ha tis why the solution of the problem concerning the character of life on Mars is of excep-tional interest. But still the answer to this question can be verified only b) simulatingMartian conditions, taking into account the information obtained from astrophysicists.

Exper iments aimed at creating artificial Martian climatic conditions have beenstarted qu it e recently; thei r number is not large cince they cannot be combined withthe results of numerous experiments investigating the elfect of extreme factors onmicroorganisms. T h e result of the effect of such physicochemical parameters of theniedium ;IS pressure, sharp temperature changes, the absence of oxygen antl insolation,tlepends on the ir combina tion and simu ltaneity . These examples convincingly showthat while simulating Martian conditions one should strive to the most comprehensivecomplex of simultaneously acting factors. T h e creation of individual climatic param-etei-s acting successively leads to absolutely different, often opposite results. I t shouldbe mentioned also that refusal to imitate insolation antl the performance of expeii-ments with specimens of soil which itself has protective effect oil cells ot microorgan-isms, Init n ot with pure cul ture of lracteria, are usual shortcomings in the bu l k ofstudies on this problem.

I t appears that organisms from Earth might survive in large numberswhen introduced to Martian environment. Whether these organismswill be capable of growth and explosive contamination of the planet in abiological sense or not is highly questionable. The likelihood of a norganism from Earth finding ideal conditions for growth on Mars seems

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extremely low. However, the likelihood of an organism from Earthserving as a contaminant for any life-detection device flown to Mars forthe purpose oi searching out carbon-based life is considerably higher.T h e chance that life has originated and evolved on Mars is a completelyseparate question an d much more difficult to answer.

It would be interesting to attempt to determine possible evolutionarytrends which might occur on a planet by means of selection of organisnisin a simulated planetary environment. Rapid genetic selection combinedwith radiation and chemicals to speed up mutation rate under theseconditions should reveal possible evolutionary trends under the planetaryenvironmental conditions. Th is could be attempted af ter the planetaryenvironments are more accurately defined.

E X TR E M E A N D L I M I T IN G E N V I R ON M E N T A L PA R AM E TE R S OF LIFET h e question of the existence of extraterrestrial life is one of the most

important and interesting biological questions facing mankind and hasbeen the subject of much controversial discussion and conjecture. Manyof the quantitative, and even qualitative, environniental constituents ofthe planets also are still subjects of controversy and speculation. Bestguesses about a relatively unknown planetary environment, combinedwith lack of information al)out the capa1)ilities ol Earth life to growi n extreme environments, (lo not provide the lxrsis for niaking informedscientific estimates.Life on Earth is usually considered to be relatively limited in its abilityto grow, reproduce, or survive in extreme environmental conditions.While many conimon plants and animals (including man) are quitesensitive to, or incapable of, surviving severe chemical and physicalchanges or extremes of environment, a large number of micro-organisnisare highly adapted and flourish in environments usually consideredlethal. Certain chemoautotrophic bacteria require high concentrationsof :tmntoni;i, niethane, or other chemicals to grow. .\n; ierol~ic acteriagrow only in the :ii)serice oL oxygen.

Besides adapting to the extremes of environntcnts on Earth, life is alsocapable of growing and reproducing under extreme environmental con-ditions not normally encountered; e.g., from a lew rad oE radiation innormal habitats to 106 or more rad froin artificial sources, froin 0.5 gaussof Earth niagnetisni to I ( i f 000 g a u s s i n manni;itle niagnetic fields, andfrom I-g force of gravity to 1 I O OO g. T h e extreme ranges of physicaland chemical environmental factors for growth, reimxluction, an d sur-vival forEarth micro-organisms are phenomenally large.Life is ubiquitous on Earth and is found in almost every possibleenvironment, including the most severe habitats, from the I)ottotn of the36


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E N V I R O N M E N T A L B I O L O G Yocean to the highest mountain tops and from cold Arctic habitats to hotsprings, as well as in volcanic craters, deep wells, salt flats, and mountainsnowfields. Earth life has become adapted to, and has invaded, nearlyevery habitat, no matter how severe. T he physiological and morpho-logical adaptations ot life are exceedingly diverse and complex.Surprisingly, the extreme parameters or ranges of the physical andchemical environmental factors permitting growth, reproduction, andother physiological processes of Ea rth organisms have not been criticallycompiled. A partial compilation of certain selected environmentalfactors has been made by Vallentyne (ref. 76). A compilation of availablepublished data on certain environmental extremes, particularly fromrecent NASA-supported research (compiled by Dale \V . Jenkins, in press),is presented in tables I11 to V I. These data can serve as a starting pointlor more intensive literature review by specialists, critical evaluation,standardi/ation of end points, and especially to point out areas where

This critical compilation involves a review of a very broad and com-plex range of subjects involved in many different disciplines with widelyscattered literature. Since the effects of many of the specific environ-mental factors are harmful, it is difficult to select a point on a scale fromno effect to death and use some criteria to say that normal or even mini-mal growth and reproduction are occurring. T h e effects of environ-mental factors are dependent on (1) the specific factor, times, (2) theConcentration or energy, times, (3) the time of exposure or application ofthe factor. Many reports, especially older ones, d o not give all of thenecessary data to permit proper evaluation. -4 complicating factor is thatthe effect of each factor depends on the other factors before, during, andafter its application. T he condition of the organism itself is a greatvariable. Proper evaluation requires the critical review by a variety ofbiological specialists, physicists, and chemists.

T o determine the potential of Ear th organisms to survive or growunder other planetary environmental conditions, a number of experi-ments have been carried out attempting to simulate planetary environ-ments, especially of Mars, as reviewed previously. While the results areo f real interest, they do not provide much basic inkormation. Further, asthe Martian environment is more accurately defined, the experimentalconditions are changed. In addition, some experimenters have alteredcertain factors, such as water content, to allow for potential microhabitatsor for areas which might contain more water at certain times.

ritical experimentation is urgently needed.

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chapter 4Behavioral Biology

EFFECTS OF T H E SPACE E N V I R O N M E N T ON BEHAVIORASA WAS ESTABLISHED IN 1958, shortly after the Russian launching ofN the second Earth satellite Sputnik 11, the first vehicle to carry life

into orbit around the Earth. Th is accomplishment was preceded by thepioneering work of Henry et al. (ref. 77), in which animals were exposedbriefly to low-gravity states in Aerobee rockets. A motion-picture cameraphotographed the behavior of two white mice in rotating drums duringthis series of flights, which marked the first time that simple psychologicaltests were made on animals in the weightless condition. While thisbehavioral experiment was relatively simple, it provided the basic con-cepts for recent studies which involved rotation of animals during theweightless state. Subsequent flights such as Project MIA (Mouse-in-Able)reflected a preoccupation with physiologic measures (refs. 78 and 79),although the flights of Baker and Able included preflight and postflightperformance studies (ref. 80). Ables behavior was recorded in detail onin-flight film, but none of the behavior was programed or under experi-mental control.

T h e first flights in which behavior or performance was explicitly pro-gramed were those of Sam and Miss Sam in flights of the Little Joe rocketwith the Mercliry capsu!ei launched from Wallops Tsland in 1959 and1960 (ref. 81). T h e first major space achievement in the behavioralsciences was the successful in-flight measurement of the behavior of thechimpanzee Ham in early 1961, in which the pretrained animal per-formed throughout the flight. T h e second achievement along these lineswas in 1962 when the chimpanzee Enos made several orbits aro und Earthand performed continuously on a complex behavioral task. T h e taskswhich the animals performed during these flights have been describedin detail by Belleville et al. (ref. 82), and the results of the in-flight per-

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S P A C E B IOSC IE NC Eformance have been presented by Henry and Mosely (ret. 83). Theseearly flights provided much of the technological framework on whichcurrent biological experiments on organisms during flights of extendedduration are based. Due largely to the efor ts of GrunLke (rets. 84 and85) , the apparatus needed to sustain animals dur ing space flight, such a5iero-g watering and feeding devices, are now commonplace (ref. 86).Advanced systems of programing stimulus presentations and recordingresponses, developed for Project Mercury, may now be seen in manybasic research laboratories throughout the country.

Several other noteworthy advances have been niatle a 5 an outgrowthof the Mercury animal flights. Immediately betore the orbital flightMA-5, in which the chimpaniee Enos was employed, it was unexpectedlyfound that this 5-year-old animal was hypertensive. Subsequent centri-fuge studies showed that its vascular responses exceeded those of a controlgroup. Consideration ot the animal's preflight experience led to specula-tion concerning the origin of thi? hypertension. An explanation of thehigh-blood-pressure responses detected in Enos has been pursued byMeehan et al. (ref. 87). Persistent hypertension has been produced inother laboratory chirnpan~ees estrained in the same manner as thoseparticipating in space flight and exposed to tlenianciing perlormancetasks, a demonstration which has important iniplications for prolongedmanned space night and for cardiovascular medicine in general.

Studies more directly concerned with behavior and performance havebeen extended from those of Project Mercury. 'I'hese extensions havebeen in the following directions: ( 1 ) the establishment and maintenanceof complex behavioral repertoires under conditions of I u I I environmentalcontrol, (2) the refinement of behavioral techniques lor assessing sensoryand motor processes, anti (3) the mainten;ince o f sustained performance. ,under conditions of long-term isolation and confinement an d preliminaryextension of such experimental analysis to man.

Numerous studies with primate subjects, including. several at AmesResearch Center, have been devoted to developing methods for maintain-ing optimum perfornianc.e in cnvironnients with liniitetl sources ofstimulation. Monkeys, l x i l ) o o n s , a n d chinip;iniees, for example, havebeen isolated for periods of longer than 2 years with no tlecrenient inperformance on complicated behavioral tasks (ret'. 88). T h e behavioraltechniques used in these studies are closely related t o those ctiil)loyed onhuman subjects under NASA sponsorship at the LJniversity o f Maryland(rcf. 89). T h e essence of these techniclues is i n the 1)roI)cr prograniingof environnieiital stimuli (ref.90). It is not sufficient to provide the sub-ject with his physiological requirements for sttrvival, but hc must begiven the psychological nlotivation for using thesc I)rovisions. This44


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B E H . 1 V I O R A L BIOLOGYstatement, of course, is an oversimplification of the problem, but it servesto illustrate the essence of these experimental programs.

Gravity has long been known as one of the major factors influencingvarious life processes and the orientation of both plants and animals.One of the most challenging problems of space research has been to definethis influence more precisely. Related to the effect of gravity on livingprocesses is the problem of the effects of weightlessness. Of particularinterest to psychologists are the possible modifications an altered gravita-tional environment might produce in behavioral patterns basic to theanimals maintenance and survival, such as eating, sensory and discrimi-native processes, development and maturation, and learning capacity(ref. 91).

One prominent method of studying gravitational effects is to simulatean increase in gravity by centrifugation. Smith et al. (ref. 92) and Wingctet al. (ref. 93) have investigated the effects of long-term acceleration onbirds, primarily chickens, while Wunder (refs. 94 and 95) and hiscoworkers (refs. 96-99) have used frui t flies, mice, rats, hamsters, andturtles. T h e general findings are that, when animals are subjected to aprolonged period of acceleration of moderate intensity, they exhibittlecrcased growth, delayed maturation, and an increase in the size ofcertain muscles and organs, dependent on the species. Wit h regard tothe decreased growth effect, the data of these investigators show someexceptions. When the gravitational increase is kept below a certainlimit, growth was greater than that of controls in the frui t fly, turtle,mouse, and chicken. T h e limit below which enhancement of growthwas observed varied with thc species studied.

T h e data on food intake do not present a consistent picture. Wunder(ref. 94) found that food intake i n accelerated micc was markedly rcducedfrom that o f nonacceleratetl control animals. Smith, however, foundthat in chickens, food intake increased up to 36 perccnt over controlsand has derived an exponential relation between food intake andacceleration. After six generations of selective breeding, Smith has pro-duced a strain of chickens better adapted to prolongcd exposure to high g.

A very relevant finding of their research with birds was that exposureto chronic acceleration in some way appears to interfere with habituationto rotatory stimulation. Chickens who were being subjected to chronicacceleration were given repeated rotatory stimulation tests to estimatetheir labyrinthine sensitivity. This study revealed that centrifuged ani-mals showed a marked reduction in labyrinthine sensitivity. Thi s resultappeared to persist after the accelcration was terminated. In animalswho developed gait or postural difficulties as a result of acceleration,there was no evidence of a postnystagmus in response to the rotatory

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stimulation test, which the investigators point out may be evidence of alesion in the labyrinth o r its neural pathways.

Smith has implicated social factors as interfering with accelerationeffects. His subjects were typically accelerated four or six to a cage.When groups were mixed midway through the experiment, they exhibiteda higher mortality rate an d incidence of acceleration symptoms than didgroups whose constituency remained unchanged.

At the U.S. Naval School of Aerospace Medicine, numerous studieshave been conducted on the effects of slow rotation on the behavior antlphysiology of humans and animals (ref. 100). Rotation initially producesdecrements in performance, but adaptation to a rotating environmentensues quite rapidly (refs. 101-103). Perceptual distortion, nystagmus,nausea, antl other signs of tlisconifort are coninion responses to slowrotation. These symptoms are generally reduced with continuedexposure (adaptation). Interestingly, however, adaptation is delayedwhen the subjects are exposed to a fixed reference outside their rotatingenvironment.

At NASA-Ames, rodents have been used in experiments by Meissmanan d Seldeen to delimit the stimulus eBects of rotation. In these experi-ments the subjects must discriminate between different speeds of rotationin order to obtain food reinforcement. The results thus far provideevidence that these animals are capable of cliscrimiiiating between thedifferent speeds at which they are being rotated. T h e range of speedsstudicd was 0-25 rpm, with tests of discrimination being made a t inter-vals of less than 5 rpni. Experiments such as these will lead to thedevelopment of techniques for measuring rotational sensitivity in manyspecies, including man.

The optimum configuration of ni;unned spacecraft will depend, inpart, upon biomedical considerations. .4 voluminous litcraturc nowexists on the possible hazards to man of prolonged exposure to zero-gc.onditions. Should prolonged weightlessness provc t o be a wrious tletri-ment to health, consideration must be given to design concepts whichprovide artificial gravity.

No data exist on the minimum gravity requirenien ts necessary tosustain basic biological functions for extciitletl periods. .-I imit of 0.2 ghas been given as the lower level at which nian ca n w a l k unaided (ref.10.1). It has also been recommentled that angular velocity be maintainedat the lowest possible level in order to minimize the occurrence ofvcstil)ular disturbances. These recomiiicndii tions arc b:isetl on hunian-factor requirements, rathei- than upon biological considerations, whichmay significantly modify these values. I n recent studies, a techniquehas been devised which promises to Iirovide reliable criteria for biological46


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B E H A V I O R A L B I O L O G Yacceptability, since it is based on fundamental biological and behavioralprinciples.

As animals progress u p the evolutionary scale, their survival dependsless and less upon stereotyped physiological reactions which occur inreflex fashion, in response to environmental stimulation. In higherorganisms, survival depends more upon the capacity of organisms tomodify their behavior. At the highest levels of functional efficiency, theultimate form of adaptation is seen-the manipulation of the environ-ment by the organism. Developments in behavioral science now permitus to utilize the adaptive behavior of animals to investigate many prob-lems of biological interest. Recent studies on the self-selection of gravitylevels represent a further attempt to exploit the adaptive capacities ofanimals, in order to provide information relevant to problems of spaceexploration.

One such project allows animals to select their own gravity environ-ment in an apparatus designed to create g-forces through centrifugalaction by rotation at 60 rpm (ref. 10.3). T h e surface of this centrifuge isparabolic, so that the resultant of the centrifugal g and the Earth's gravityis always normal to the surface. \\'hen the animal moves away fromthe center, increasing the radius of rotation, it is exposed to increasinggravity. Motion toward the center reduces the gravity level. By thismeans, an animal is free to select its own gravity environment.

When the animal moves toward or away from the center, he is movingfrom one tangential velocity to another. He is therefore acted upon bya third force-due to Coriolis acceleration. T h e effects of Coriolis forcesare a major problem difficult to eliminate in studies such as these, butthey must be taken into account in the design of spacecraft which produceartificial gravity by rotation. Motion of the head in any direction notparallel to the centrifugal force vector would result in birarre stimulationof the semicircular canals and consequent motion sickness. This effectis likely to become even more pronounced if the sensitivity of theseorgans is increased by prolonged exposure to reduced gravity. Methodssuch as these are currently being clcvelopetl for cwitlucting :I refinedpsychophysical analysis of gravitv, including studies b y Lange andRroderson on the perception of angular, linear, and Coriolis acceleration.

T h e results of animal studies such as these will be of great value inarriving at a decisive judgment concerning the need for artificial gravityin a manned orbiting space station, or other vehicles designed for long-term occupancy.T o aid in the interpretation of in-flight data , other studies are under-way to determine the functions of the vestibular system, as a principalbrain center related to orientation in space and to the physiology of

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posture antl movement, as well as with the infiuences of acceleration,rotation, an d weightlessness. Experiments are presently being conductedon monkeys and cats in order to trace tlicsc co~iiplexneurological con-nections and to determine their functional organiiation.

BIOLOGICAL INFORMATION SYSTEMST h e nature of memory has been the subject of consitlerable speculation

in the past. It has long been felt intuitively that retention of informationin the central nervous system involves citlicr a n alteration of preexistingmaterial or structure, or, alternatively, synthesis o f illaterials not presentpreviously. The cellular site of operational alteration was unknown but,again intuitively, was felt to be closely associated with the synapses. T h eproblems faced by early investigators were great; but nevertheless muchinformation relevant to the question 0 1 biological inloriiiation storagewas obtained. With the relatively recent advent o l more refined toolsand methodologies, there has been rapid progress.

A significant amount of the work which has Iiccii conducted in thearea of biological information and communication systems is easilyclassificd as basic research (refs. 106-1 03). Ihis discussion will belimited to those aspects closely related t o the fields o f iiiolecu1;ir biologyand experiincntal psychology, which seem to have universal ;ipl)licationto all known animal life forms. Studies involving the basic principlesof acquisition, processing, storage, antl retrieval of information in livingsystems are emphasized.l iarly Work

Early speculations on the operational nature of niemory have beenbased upon relatively little experiiiiental evidence. Charles Darwinobserved that domestic rabbits had sniallcr br:iiii\ t l i m their wildcounterparts, and attributed this to lack o f exercise of their intellect,senses, and voluntary movements. Unfortunately, su l~sec~ucnttudies o fthe brains of men with greatly diflering intellectual capability did notsubstantiate the hypothesis. Idiots soiiietinics had larger brains thangeniuses. Later, an idea proposed b y Ranion y Cajal came into favor.Since brain cells did not increase in nunil)cr alter birth, he proposedthat niemory involved the establishinent o f new antl more extendedintercortical connections. Unfortunately, mcthotls were not availablcto test this hypothesis adequately and i t has relnainetl until quiterecently in the realm of conjecture.

Another major hypothesis was that there were tlvo or more stages inthe information storage process. The final form the information tookin the brain was called a brain engram, 01 memory trace. However,48


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B E H A V I O R A L BIOLOGYprior to the formation of the engram, a transitory process denoted asreverberational memory was postulated to exist for a relatively shorttime (minutes to hours) (refs. 106 and 107). This hypothesis was usedby Pauling to explain why an elderly chairman of a board could bril-liantly summarize a complex 8-hour meeting and yet, after its conclusionand his return to his office, not even remember having attended themeeting. Thus, this individuals reverberational memory functionedwell, but advanced years had seriously impaired his brains ability toform a permanent engram. Similar, although less dramatic, observationsin other situations are not uncommon. .4 wide variety of experimentshave been conducted to study this aspect of memory and to relate it tothe process whereby the information is transformed to a more stableform (refs. 110-1 12).More recently, the concept of a specific biochemical activity durin g theprocess of long-term storage of information has gained considerable favor.Initially, neither the site nor the nature of the change was well defined.Quite recent studies by Krech et al. (refs. 113 and 114), Bennett et al . (ref.115), Rosenzweig et al. (refs. 116 and 117) support the view that alterationof the levels of acetylcholinesterase at cortical synapses play an importantrole in information storage. These studies will be discussed in a latersection. However, these authors do not claim that the changes observedare unambiguously related to the storage of memory. It may well be thatthe alterations observed are in some way related to this process but arestill secondary to some other , more basic, process.

An alternative hypothesis is that the information resides in its ultimateform in some more central structure of the neurone than the synapse.( l t has even been postulated that the basic information is stored innonneuronocortical material.) Perhaps Halstead was the first to postu-late the involvement of nucleoprotein in this process (ref. 107). From thebiochemists point of view, this is an extremely attractive hypothesis.Both proteins and nucleic acids possess sufficient possible permutationsof structure to permit storage of a lifetimes accumulation of informationin a n organ the size of the brain. From the previously known ability ofthe nucleic acids to code genetic information, they are the prime suspects.However, from the known regulatory ability of nucleic acids in specificprotein synthesis, it is possible that the final repository is protein.Recent Biochemical Studies

Among the foremost investigators of the chemistry and biochemistryof the central nervous system is Holger Hyden at the University ofGoteborg, Sweden. He and others (refs. 118-120) have fo r many yearsperformed elegant microanalytical studies of single nerve cells. T h e evi-

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SPACE BIOSCIENCEdence which Hyden has obtained is consistent with the hypothesis thatthe initial electrical reverberations in the brain, induce a change in themolecular s tructure of the ribonucleic acid (RNA) of the neurones which,in turn, leads to a subsequent deposition of specific proteins. It is wellknown from other investigations that a major role of RNA ih any typeof cell is to specify and mediate synthesis of the protein enzymes of thecells. Thus, in this hypothesis, it is only necessary to postulate theinotlification of brain RNA by the activities associated with reverbera-tional memory. Particularly pertinent to this hypothesis are observationsthat-

( 1 ) Large nerve cells have a very high rate o f metabolism of RNA andproteins, and, of the somatic cells, are the largest producers ofRNA.

(2) Vestibular stimulation by passive means leads to an increase in theRNA content of the Deiters nerve cells of rabbits (ref. 121). T h eprotein content of these cells is also increased.

(3 ) Changes in the RNA composition of neurones and glia of thebrainstem occur during a learning situation. Animals weretrained over a period of 4 to 5 days to climb a steeply inclinedwire to obtain food. T h e big nerve cells and the glia of theirlateral vestibular apparatus were analyzed, since the Deitersneurones present in this structure are directly connected to themiddlc ear. T he amount of RNA was found to be increased inthe nerve cells; and, more significantly, the adenine-to-uracilratio of both the nuclear RNA of nerve cells and glia cellsbecame significantly increased (ref. 119). ,-Iariety of controlexperiments were conducted. Although there was an increasein RNA content of these cells in animals exposed to passivestimulation, there was no change in th e ratio of adenine touracil. Nerve cells from the reticular formation, another por-tion of the brain, had only an increased content ol KNA with nobase-ratio change. Animals subjected to a stress experimentinvolving the vestibular nucleus showed only an increase incontent of RNA. Littermates living in cages on the same dietas learning animals showed no change in content of RN A.Thus , it would appear that the change in the base ratio of theK N A synthesi/etl is not due t o increased neiirone function perse, but is more directly related to the learning process. T h e factthat this was nuclear RN,4 implies that i t JW S immediatelyrelated to chromosomal DNA.(4)Neuronal RNA with changed cytosine-giianiile ratios syiithesiicdduring a short period of induced protein synthesis could be

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BEH .4 VI O RAL B I O L O G Yblocked by actinomycin D. I t was concluded, therefore, that theR N A was immediately DNA dependent and directly related tothe genetic apparatus.

Rats which were normally right handed were forced to modify theirhandedness in order to obtain food. T h e RNA of nerve cells in thatpa rt of the cor.tex, whose destruction destroys the ability to transferhandedness, was analyzed. A significant increase i n RNA of nerve cellsof the fifth to sixth cortical layers on the right side of the brain wasobserved. T h e corresponding nerve cells on the opposite side of thesame brain served as controls. There was a n increase in R N A and asignificant increase in the purine bases relative to the pyrimidine basesin the learning side of the cortex. When the animals were not forced tolearn a new procedure, only an increase of R N A was observed, with nochange in base ratio.

Frank Morrell, head of the Neurology Department a t Stanford Medi-cal School, has also been active in this field during the past 6 years. Hehas found that if a primary epileptic lesion is induced on one side of thecortex, a secondary mirror lesion eventually develops in the contralateralhomologous cortex. This secondary lesion, which showed self-sustainingepileptiform discharge, could be isolated, whereupon the epileptiformdischarge disappeared. This was interpreted as learned behavior of thesecondary lesion. From changes in the staining properties of thesecondary lesion, Morrell concluded that changes in RNA had occurredin the cell. Changes in the composition of the R N A could not be shownby these techniques.

At the University of California at Berkeley, Drs. Kosenzweig, Bennett,and Krech have conducted extensive studies related to this topic. Theseinvestigators have directed their efforts toward demonstrating alterationsin the cerebral cortex of animals exposed to continuing learning situa-tions or continuously deprived of sensory stimulation. In a recent publi-cation (ref. 116), which also summarizes a considerable amount ofprevious work, they report studies which demonstrate the following:

(1) Rats given enriched experience develop, in comparison with theirrestricted littermates, greater weight and thickness of corticaltissue and an associated proportional increase in totalacetylcholinesterase activity of the cortex.(2) Th e gain in weight of cortical tissue is relatively larger than theincrease in enzymatic activity. Acetylcholinesterase activityincreases in other portions of the brain even though tissue weightdecreases.

(3) T h e changes appear in a variety of lines of rats, although differingin amount between strains.

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S P A C E B I O S C l E N C E

(4) T h e changes are observed in both the young antl adult animals.The previous studies were comparisons between experience-enriched

animals and animals maintained in isolation. Animals which werehoused in colonies, but given no special treatment, showed intermediateeffects in those situations studied.

T h e Berkeley group emphasized that the finding of changes in thebrain subsequent to experience does not prove that the changes haveanything to do with memory storage, but do establish the fact that thebrain can respond to environmental pressure. However, the results arecompatible with the hypothesis that long-term memory storage involvesthe formation of new somatic connections aniong neurones. Calcula-tions of the amount of additional material required to permit this to existare compatible with the increases observed.

A number of investigators have studied the effects of antimetabolitesand drugs on the learning process. Since their specific metabolic effectsare known in other tissues, the rationale is that if these materials d ointerfere with memory, then specific types of metabolic activities may beimplicated in the deposition of the engram.

One of the initial studies of this type was conducted by Dingman an dSporn (ref. 122), presently at the National Institute of Mental Health.They showed that 8-azaguanine, a purine antagonist, injected intra-cisternally MW incoq)or;itetI into the R N A o f the I)rains ol rats. Associ-ated with this incorporation was an impairment o f the maze-learningability of the animals. l'hese findings have been confirmed.

Flexner antl his associates injected puromycin, a n inhibitor of proteinsynthesis, in to the brains of mice, which were then trained to perform ina maze. Losses of short-term or long-term nieinory were obta ined ,depending upon the site of the injection. T h e results indicate tha t thehippncampal region is the site of recent memory.

T h e hippocampal region is of interest in connection with memoryprocesses for a number of other reasons. Adey et al. (rcf. 123) and hisgroup observed a transient fall in electrical impetlmce in this regionwhen cats lcarned to perforni in a T-maze in response to a visual cue. Itwas supposed that the electrodes were situated within glial cells of thedcntlritic zone of the hippocampal pyramidal cell layer. Extinction oEthe learned habit almlished the briefly evoked impedance changes,which subsequently reappeared with retraining.

A number of other studies more or lcss indirectly implicate RNA inthe learning processes. For instance, in retinal cells o f rabbits raised indarkness, tlic1.e was virtually no ribonucleoprotein as compared withnormal amounts in the cells of animals raised in light (ref. 124). Further,maintenance of normal electrical activity of isolated 1xrfusc.d cat brains52


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B E H A V I O R A L B I O L O G Yis highly dependent upon the presence of the ribonucleic acid precursors,uridine and cytidine, in the perfusate (ref. 125), and severe derangementsoccur if any of a variety of pyrimidine antagonists are added (ref. 126).Brief electrical stimulation of cat cortical tissue causes an increase innucleic acid cytidine and adenine, thus indicating a synthesis of alteredpolynucleotides. Finally, injections of RNA in animals have showninteresting effects. When given at a dose of 116 mg/kg daily for 1 month,rats showed an enhanced response and greater resistance to extinction ina shock-motivated behavioral response. It has been shown by anothergroup that injections of RN A enhance the ability of young animals tolearn various tasks.

Planaria have been used in a variety of studies which seem to bear onthe problem of memory. Quite recent evidence by Bennett, Calvin, andtheir associates has cast somewhat of a pall over the studies; nevertheless,the work may have some validity. Interest in the use of flatworms, par-ticularly planaria, for study of memory began with a demonstration byMcConnell that these simple animals could undergo conditioning (ref.127). Subsequently, it was found that some conditioning was retainedwhen the animal was transected and allowed to regenerate. T h e reten-tion of training was found in both new animals, although the very simplebrain, really only two ganglia, was in the head section (ref. 128).

Apparently, some diffusely distributed component of the animal wasresponsible for retention of learning. Evidence has accumulated toindicate that this material is RNA. Among this evidence is the following:

(1) T h e two halves of a trained planaria were allowed to regeneratein a solution containing RNA-destroying enLymes. Whereasthe head ends retained some training, no retention was observedin the animals derived from the tail end (ref. 129).(2) When pieces of trained planaria were fed to untrained animals,the untrained cannibal required a shorter time to becometrained to a criterion. I t would appear that the digestive systemof planaria is so simple that the material responsible for thetransfer of the information was not broken down.

(3) When RNA, obtained from trained planaria, is injected into thedigestive tract of untrained d i i i i i i zk , t k r c is 2 trmsfer nfinformation.

NEUROPHYSIOLOGYNeurophysiological studies concern the functions of the nervous system-in part icular the central nervous system (CNS)-under normal, simu-

lated, and actual flight conditions. Of paramount importance is the~-

2 Eweipt from ret. 130.5 3


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maintenance of equilibrium and orientation in three-dimensional space.The ability of man and his close relatives among the vertebrates to main-tain these functions depends on an integrated sensory input from thevestibular organ; the eyes; the interoccptors of the muscles, tendons,joints, and viscera; and the exteroceptors of the skin.

Certain parameters of the environmental and space-Hight conditionsdrastically affect mans ability to maintain equi1il)riuni and spatialorientation. Centrifugal forccs modify or reverse the directional vectorof gravity. Linear acceleration may increase enormously,as may angularstimulation. T h e sensory organs listed above are unreliablc under suchconditions. T h e very organ which is designed specifically to furnishinformation on spatial orientation may malfunction in man while he isin Bight. Thus , with respect to sensory orientation, these labyrinthineorgans are by no means prccision instruments.T h e use of classical histological methods and the observation ofequilibrium disturbances resulting from operative interfcrencc with theinternal ear have in the past been the two principal sources of knowledgeconcerning the structure and function of the labyrinth, but the answersgiven to various questions vary considerably in their value. T h e tlevelop-ment of electrophysiological techniques and the refincment in recentyears of the ultrastructural analysis by means of the electron microscopemay allow morc precise experimental studies o f thc corrclation of func-tion and structure.Before considering vestibular impulses in their bulbar and descendingspinal pathways, a recent study concerning the generation of iinpulsesin the labyrinth must be mentioned. Von Bekesys finding (ref. 131) ofthe direct current potentials in the cochlea aroused speculation aboutthe existenc

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Significant Achievements in Space Bioscience 1958-1964