Chapter III: Part 3

Another major hypothesis was that there were two or more stages in the information storage process. The final form the information took in the brain was called a brain engram, or memory trace. However, prior to the formation of the engram, a transitory process denoted as "reverberational memory" was postulated to exist for a relatively short time (minutes to hours) (refs. [ref.106] and [ref.107]). This hypothesis was used by Pauling to explain why an elderly chairman of a board could brilliantly summarize a complex 8-hour meeting and yet, after its conclusion and his return to his office, not even remember having attended the meeting. Thus, this individual’s reverberational memory functioned well, but advanced years had seriously impaired his brain’s ability to form a permanent engram. Similar, although less dramatic, observations in other situations are not uncommon. A wide variety of experiments have been conducted to study this aspect of memory and to relate it to the process whereby the information is transformed to a more stable form (refs. [ref.110]-[ref.112]).

More recently, the concept of a specific biochemical activity during the process 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. [ref.113] and [ref.114]), Bennett et al. ([ref.115]), Rosenzweig et al. (refs. [ref.116] and [ref.117]) support the view that alteration of the levels of acetylcholinesterase at cortical synapses play an important role in information storage. These studies will be discussed in a later section. However, these authors do not claim that the changes observed are unambiguously related to the storage of memory. It may well be that the alterations observed are in some way related to this process but are still secondary to some other, more basic, process.

An alternative hypothesis is that the information resides in its ultimate form in some more central structure of the neurone than the synapse. (It has even been postulated that the basic information is stored in nonneuronocortical material.) Perhaps Halstead was the first to postulate the involvement of nucleoprotein in this process ([ref.107]). From the biochemist’s point of view, this is an extremely attractive hypothesis. Both proteins and nucleic acids possess sufficient possible permutations of structure to permit storage of a lifetime’s accumulation of information in an organ the size of the brain. From the previously known ability of the nucleic acids to code genetic information, they are the prime suspects. However, from the known regulatory ability of nucleic acids in specific protein synthesis, it is possible that the final repository is protein.

Recent Biochemical Studies

Among the foremost investigators of the chemistry and biochemistry of the central nervous system is Holger Hyden at the University of Göteborg, Sweden. He and others (refs. [ref.118]-[ref.120]) have for many years performed elegant microanalytical studies of single nerve cells. The evidence which Hyden has obtained is consistent with the hypothesis that the initial electrical reverberations in the brain induce a change in the molecular structure of the ribonucleic acid (RNA) of the neurones which, in turn, leads to a subsequent deposition of specific proteins. It is well known from other investigations that a major role of RNA in any type of cell is to specify and mediate synthesis of the protein enzymes of the cells. Thus, in this hypothesis, it is only necessary to postulate the modification of brain RNA by the activities associated with reverberational memory. Particularly pertinent to this hypothesis are observations that—

(1) Large nerve cells have a very high rate of metabolism of RNA and
proteins, and, of the somatic cells, are the largest producers of
RNA.
(2) Vestibular stimulation by passive means leads to an increase in
the RNA content of the Deiters nerve cells of rabbits ([ref.121]).
The protein content of these cells is also increased.
(3) Changes in the RNA composition of neurones and glia of the
brainstem occur during a learning situation. Animals were trained
over a period of 4 to 5 days to climb a steeply inclined wire to
obtain food. The big nerve cells and the glia of their lateral
vestibular apparatus were analyzed, since the Deiters neurones
present in this structure are directly connected to the middle
ear. The amount of RNA was found to be increased in the nerve
cells; and, more significantly, the adenine-to-uracil ratio of
both the nuclear RNA of nerve cells and glia cells became
significantly increased ([ref.119]). A variety of control
experiments were conducted. Although there was an increase in RNA
content of these cells in animals exposed to passive stimulation,
there was no change in the ratio of adenine to uracil. Nerve cells
from the reticular formation, another portion of the brain, had
only an increased content of RNA with no base-ratio change.
Animals subjected to a stress experiment involving the vestibular
nucleus showed only an increase in content of RNA. Littermates
living in cages on the same diet as learning animals showed no
change in content of RNA. Thus, it would appear that the change in
the base ratio of the RNA synthesized is not due to increased
neurone function per se, but is more directly related to the
learning process. The fact that this was nuclear RNA implies that
it was immediately related to chromosomal DNA.
(4) Neuronal RNA with changed cytosine-guanine ratios synthesized
during a short period of induced protein synthesis could be
blocked by actinomycin D. It was concluded, therefore, that the
RNA was immediately DNA dependent and directly related to the
genetic apparatus.

Rats which were normally right handed were forced to modify their handedness in order to obtain food. The RNA of nerve cells in that part of the cortex, whose destruction destroys the ability to transfer handedness, was analyzed. A significant increase in RNA of nerve cells of the fifth to sixth cortical layers on the right side of the brain was observed. The corresponding nerve cells on the opposite side of the same brain served as controls. There was an increase in RNA and a significant increase in the purine bases relative to the pyrimidine bases in the learning side of the cortex. When the animals were not forced to learn a new procedure, only an increase of RNA was observed, with no change in base ratio.

Frank Morrell, head of the Neurology Department at Stanford Medical School, has also been active in this field during the past 6 years. He has found that if a primary epileptic lesion is induced on one side of the cortex, a secondary mirror lesion eventually develops in the contralateral homologous cortex. This secondary lesion, which showed self-sustaining epileptiform discharge, could be isolated, whereupon the epileptiform discharge disappeared. This was interpreted as learned behavior of the secondary lesion. From changes in the staining properties of the secondary lesion, Morrell concluded that changes in RNA had occurred in the cell. Changes in the composition of the RNA could not be shown by these techniques.

At the University of California at Berkeley, Drs. Rosenzweig, Bennett, and Krech have conducted extensive studies related to this topic. These investigators have directed their efforts toward demonstrating alterations in the cerebral cortex of animals exposed to continuing learning situations or continuously deprived of sensory stimulation. In a recent publication ([ref.116]), which also summarizes a considerable amount of previous work, they report studies which demonstrate the following:

(1) Rats given enriched experience develop, in comparison with their
restricted littermates, greater weight and thickness of cortical
tissue and an associated proportional increase in total
acetylcholinesterase activity of the cortex.
(2) The gain in weight of cortical tissue is relatively larger than
the increase in enzymatic activity. Acetylcholinesterase activity
increases in other portions of the brain even though tissue weight
decreases.
(3) The changes appear in a variety of lines of rats, although
differing in amount between strains.
(4) The changes are observed in both the young and adult animals.

The previous studies were comparisons between experience-enriched animals and animals maintained in isolation. Animals which were housed in colonies, but given no special treatment, showed intermediate effects in those situations studied.

The Berkeley group emphasized that the finding of changes in the brain subsequent to experience does not prove that the changes have anything to do with memory storage, but do establish the fact that the brain can respond to environmental pressure. However, the results are compatible with the hypothesis that long-term memory storage involves the formation of new somatic connections among neurones. Calculations of the amount of additional material required to permit this to exist are compatible with the increases observed.

A number of investigators have studied the effects of antimetabolites and drugs on the learning process. Since their specific metabolic effects are known in other tissues, the rationale is that if these materials do interfere with memory, then specific types of metabolic activities may be implicated in the deposition of the engram.

One of the initial studies of this type was conducted by Dingman and Sporn ([ref.122]), presently at the National Institute of Mental Health. They showed that 8-azaguanine, a purine antagonist, injected intra-cisternally was incorporated into the RNA of the brains of rats. Associated with this incorporation was an impairment of the maze-learning ability of the animals. These findings have been confirmed.

Flexner and his associates injected puromycin, an inhibitor of protein synthesis, into the brains of mice, which were then trained to perform in a maze. Losses of short-term or long-term memory were obtained, depending upon the site of the injection. The results indicate that the hippocampal region is the site of recent memory.

The hippocampal region is of interest in connection with memory processes for a number of other reasons. Adey et al. ([ref.123]) and his group observed a transient fall in electrical impedance in this region when cats learned to perform in a T-maze in response to a visual cue. It was supposed that the electrodes were situated within glial cells of the dendritic zone of the hippocampal pyramidal cell layer. Extinction of the learned habit abolished the briefly evoked impedance changes, which subsequently reappeared with retraining.

A number of other studies more or less indirectly implicate RNA in the learning processes. For instance, in retinal cells of rabbits raised in darkness, there was virtually no ribonucleoprotein as compared with normal amounts in the cells of animals raised in light ([ref.124]). Further, maintenance of normal electrical activity of isolated perfused cat brains is highly dependent upon the presence of the ribonucleic acid precursors, uridine and cytidine, in the perfusate ([ref.125]), and severe derangements occur if any of a variety of pyrimidine antagonists are added ([ref.126]). Brief electrical stimulation of cat cortical tissue causes an increase in nucleic acid cytidine and adenine, thus indicating a synthesis of altered polynucleotides. Finally, injections of RNA in animals have shown interesting effects. When given at a dose of 116 mg/kg daily for 1 month, rats showed an enhanced response and greater resistance to extinction in a shock-motivated behavioral response. It has been shown by another group that injections of RNA enhance the ability of young animals to learn various tasks.

Planaria have been used in a variety of studies which seem to bear on the problem of memory. Quite recent evidence by Bennett, Calvin, and their associates has cast somewhat of a pall over the studies; nevertheless, the work may have some validity. Interest in the use of flatworms, particularly planaria, for study of memory began with a demonstration by McConnell that these simple animals could undergo conditioning ([ref.127]). Subsequently, it was found that some conditioning was retained when the animal was transected and allowed to regenerate. The retention of training was found in both new animals, although the very simple brain, really only two ganglia, was in the head section ([ref.128]).

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

(1) The two halves of a trained planaria were allowed to regenerate in
a solution containing RNA-destroying enzymes. Whereas the head
ends retained some training, no retention was observed in 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 become trained to a
criterion. It would appear that the digestive system of planaria
is so simple that the material responsible for the transfer of the
information was not broken down.
(3) When RNA, obtained from trained planaria, is injected into the
digestive tract of untrained animals, there is a transfer of
information.

NEUROPHYSIOLOGY²

² Excerpt from [ref.130].

Neurophysiological studies concern the functions of the nervous system—in particular the central nervous system (CNS)—under normal, simulated, and actual flight conditions. Of paramount importance is the maintenance of equilibrium and orientation in three-dimensional space. The ability of man and his close relatives among the vertebrates to maintain these functions depends on an integrated sensory input from the vestibular organ; the eyes; the interoceptors of the muscles, tendons, joints, and viscera; and the exteroceptors of the skin.

Certain parameters of the environmental and space-flight conditions drastically affect man’s ability to maintain equilibrium and spatial orientation. Centrifugal forces modify or reverse the directional vector of gravity. Linear acceleration may increase enormously, as may angular stimulation. The sensory organs listed above are unreliable under such conditions. The very organ which is designed specifically to furnish information on spatial orientation may malfunction in man while he is in flight. Thus, with respect to sensory orientation, these labyrinthine organs are by no means precision instruments.

The use of classical histological methods and the observation of equilibrium disturbances resulting from operative interference with the internal ear have in the past been the two principal sources of knowledge concerning the structure and function of the labyrinth, but the answers given to various questions vary considerably in their value. The development of electrophysiological techniques and the refinement in recent years of the ultrastructural analysis by means of the electron microscope may allow more precise experimental studies of the correlation of function and structure.

Before considering vestibular impulses in their bulbar and descending spinal pathways, a recent study concerning the generation of impulses in the labyrinth must be mentioned. Von Bekesy’s finding ([ref.131]) of the direct current potentials in the cochlea aroused speculation about the existence of similar labyrinthine potentials. Such dc potentials were also detected in the semicircular canal of the guinea pig by Trincker ([ref.132]), who measured the potential changes in the endolymph, surface of the cupula, or side of the crista during cupular deflection. It seems likely, however, that the effects do not represent the physicochemical changes in the cupula but the electrical potentials in the nerve and nerve endings of the crista. Attempts at differentiating these effects have failed so far. Great expectations are brought by the advances of microchemistry, microphysiology, and physical chemistry with regard to the excitatory processes, the generation of the nerve impulse. Quite apart from a need to understand vestibular nerve discharges and patterns more adequately in such terms, the analysis of the vestibular system has in the past revealed general biological principles which were not readily discernible through the examination of other tissues ([ref.133]).

The neural connections of the vestibular organ consist of numerous chains of neurons, reciprocally linked in many ways and having their synapses in various anatomical nuclei. All the chains work in intimate collaboration, and the final pattern of reflex responses is attributable largely to the highly complex integrating activity of the center. The labyrinthine function is automatic, carried out in a reflex fashion: in other words, mostly below the level of consciousness. The brain centers through which the labyrinth elicits the various appropriate muscular reactions of the head, body, limbs, and eyes—the righting, the postural, and the ocular reflexes—represent an intricate mechanism. Before we can hope for a satisfactory understanding of their functional organization, we will have to know their anatomy in more detail. Thus, we are confronted with a fruitful field for the exploration of basic mechanisms of neuronal activity. Major advances dining the last years have provided us with new information about the neuroanatomy of the vestibular system (refs. [ref.134]-[ref.137]).

Vestibular impulses entering the brainstem ascend and descend the neuroaxis and cross the midline. It was previously believed that the vestibular apparatus had only subcortical projections. Recently, however, it has been established by means of electrophysiological methods that the organ is represented by a projection area in the cerebral cortex of some animals (refs. [ref.138]-[ref.141]). The use of brief electrical stimulation of the vestibular nerve in order to elicit a cortical response has been of great value for the mapping of these areas.

Among a great variety of sensory receptors, the vestibular ones are capable of evoking the most widespread somatovisceral effects throughout the body. Moreover, vestibular effects seem to be imperious and less dependent upon the state of readiness of the nervous system. As a consequence of the extensive distribution of vestibular effects, there are many opportunities for central integration. Proprioceptive and vestibular systems are both known to be active in posture and locomotion; streams of impulses arising from the receptors in each of these systems must converge to influence the activity of the final common path. The state of the motor centers of the spinal cord, as affected by vestibular stimulation, has been tested by dorsal root and other sensory input interventions. These experiments have provided us with insight into the mechanisms concerned with the vestibular control of spinal reflexes (refs. [ref.142]-[ref.146]).

It has long been known that the vestibular apparatus is essential for the development of motion sickness. Commonplace subjective experience of nausea relates to visceral changes mediated through autonomic efferent pathways and may ultimately involve rhythmic somatic nerve discharges to skeletal muscles responsible for retching and vomiting. However, very little is known about the central nervous mechanisms responsible for elaboration of the whole syndrome. Since the maintenance of vestibular bombardment for some length of time seems essential for the development of motion sickness, one would presume this to be an instance of slow temporal summation. Experimental findings demonstrate a powerful effect of temporal summation upon somatic motor outflow during vestibular stimulation ([ref.147]), and not upon parasympathetic outflow.

The practical implication of these studies is closely related to physiological effects of weightlessness. Based on experimental evidence from short weightless periods obtained in aircraft, it was concluded that "when the exposure becomes longer, there may develop minor physiologic disturbances which, if cumulative or irritating, may cause or enhance psychiatric symptoms" ([ref.148]). Although the zero-g condition, per se, does not cause spatial disorientation if visual cues are provided, the astronauts reported a temporary loss of orientation during the orbital flight while they were engaged in activities which diverted their attention. However, no disturbing sensory inputs were observed during the weightless period. Violent head maneuvers within the limited mobility of the helmet were performed in every direction without illusions or vertigo. The subjective sensations of "tumbling forward" after sustainer engine cutoff reported by the Mercury astronauts, and Titov’s motion sickness attacks, which were particularly dismaying during head movements, were well within the entire range of psychosomatic experiences already obtained during aerodynamic trajectories ([ref.149]). Interestingly enough it now appears that the otolithic output in mammals including man is the differential of linear acceleration, and therefore unaffected by zero g.

Of interest in this connection are the problems which may be encountered during and following long-term exposure to weightlessness. Although there is no evidence of adverse effects on operative behavior, the possibility of biological disturbances on a cellular or subcellular level, which may cause a deterioration of the somatic basis, has been repeatedly stressed. Whether effects of this sort will occur or whether the organism will be able to adapt is still an open question. Since motion sensitivity based on vestibular stimulation differs widely among individuals, the selection of astronauts may solve the problem of zero-g vestibular disturbance. Reports from the MA-8 (Sigma 7) and Vostok III and IV flights seem to support this assumption. Moreover, experiments are being made in the slow rotation room at the Naval School of Aviation Medicine to study the Coriolis effects which arise when "artificial gravity" is produced by angular acceleration. Since man can adapt to wave motion on shipboard within a few days, a similar process may be expected to occur in the case of long-term weightlessness ([ref.150]).

chapter 5

_Molecular Biology and Bioinstrumentation_

To support biological investigations in space and to accumulate baseline data needed for manned space flight, NASA has conducted a program in laboratory research and theory. A multidisciplinary approach has included such fields as ecology, physiology, organic and biological chemistry, engineering, electronics, and optics. Emphasis in this program has been placed on qualitative and theoretical rather than purely descriptive research, and the investigation of fundamental biological phenomena at all levels, from the molecular to the total life form.

MOLECULAR BIOLOGY

Research in molecular biology has included chemical, physical, biological, and theoretical investigations of prebiological conditions on Earth and, possibly, on other planets; studies of cellular inclusions; genetic material (DNA and RNA) and coding; as well as energy transfer in biological systems.

The understanding of prebiological conditions on Earth, and possible conditions on other planets, depends upon the nature of the complex chemical species which might be encountered. Scientists have shown that biologically important compounds, such as amino acids, can be generated by applying an electrical discharge, ultraviolet radiation, or heat to a gaseous mixture. Biologically interesting compounds can be removed from such a system by condensation or absorption; however, in the limited time and space available in such experiments, many compounds are not produced in sufficient quantity to be measured.

The National Biomedical Research Foundation (NBRF) and the National Bureau of Standards (NBS) are conducting an investigation on equilibria in multielement systems. The distribution of molecular species at equilibrium is independent of the way equilibrium was reached and is dependent only on pressure, temperature, and elemental composition. Many of the conditions which might have arisen naturally can be approximated by thermodynamic equilibrium. Compounds which can be formed at equilibrium need no special mechanism to explain their presence. However, special mechanisms have to be sought for those compounds which could not be so produced and which would have been required for the structure and nutrition of the first living organisms.

In the absence of precise knowledge of the composition of the primitive planetary atmospheres, equilibrium concentrations with a wide range of temperatures, pressures, and elemental compositions are being investigated by NBRF and NBS. These investigators have postulated that the maximum atmospheric pressure may have approached 100 atm if the primitive Earth was sufficiently hot and if an appreciable portion of the water on Earth’s surface today was present on primitive Earth. (If the present oceans were to evaporate, the surface pressure would be approximately 300 atm.) Low pressures of 10⁻⁶ atm and temperatures between 500° and 1000° K are being used.

A large range of N, O, C, and H compositions are being investigated for interesting and plausible combinations of factors. In these calculations an IBM 7090 computer is being used to obtain data on a very large number of combinations of chemicals. Other chemical species will be added as the research continues. Some results of this study give an insight into the variety of biologically significant chemicals which might have existed during Earth’s primitive prebiological condition or may now exist on the surfaces and in the atmospheres of other planets (refs. [ref.151]-[ref.153]). The general method described by White et al. ([ref.152]), minimizing the free energy of the system, was used. The solution was approached by an iterative process, starting with an initial guess of concentrations of the compounds. At each step, _M_+1 linear equations are solved where _M_ is the number of elements in the system.

In addition to listing of the concentrations of all compounds included in each problem, the results of three-element problems have been expressed on a triangular composition diagram for convenience. A coarse grid of 60 points is used to survey all elemental compositions, with finer grids being used in regions of particular interest. The calculated concentrations of the compounds at each composition are stored, and finally a series of triangular diagrams is printed out, each showing the concentrations of as many as four compounds at the grid points.

Figure 2 shows the results obtained in the C, H, and O systems. Organic compounds in concentrations greater than 10⁻²⁰ mole fraction are found everywhere except where free O₂, is present. Solid carbon theoretically becomes stable along the lower dashed line at 500° K. However, reactions producing it are very slow. The supersaturated region beyond the line of potential carbon formation was also investigated. A threshold was found where polynuclear aromatic compounds are sufficiently concentrated to form a liquid phase. These conditions may have been involved in the primordial formation of asphaltic petroleum.

Jukes and associates ([ref.154]) at the University of California at Berkeley have been investigating the code for amino acids in protein synthesis, the key for translating the sequence of bases in DNA into the sequence of amino acids in proteins. The amino acid code was solely a matter of theory until Nirenberg and Matthaei ([ref.155]) at the National Institutes of Health carried out a crucial experiment. This experiment bridged the last remaining gap separating theoretical genetics and test-tube biochemistry. It now became experimentally possible to search for codes for all 20 amino acids concerned in the synthesis of proteins.

The amino acid bases of DNA are: A, adenine; C, cytosine; G, guanine; T, thymine; and U, uracil, which replaces thymine in RNA. There are only 16 ways of arranging A, C, G, and T in pairs. For this and other reasons it is thought that a triplet of three consecutive bases is needed to code for each amino acid. The sequences of bases in a strand of DNA are known to be unrestricted with respect to the order in which they occur; apparently any one of the four bases can be next to any of the other four, although, of course, each base must be paired with the corresponding complementary base in the adjacent strand. Since the same freedom is true of the amino acid sequences in the polypeptide chains of proteins, any one of the 20 amino acids can occur next to any other. Moreover, the sequences in DNA are subject to mutational changes in which one base replaces another, or bases are added to or deleted from the DNA. Such rearrangements plus the possibility of lengthening of DNA molecules are numerous enough to account for all the genetics of living forms since the first appearance of life on Earth.

Most of our knowledge is based on experiments with synthetic RNA carried out with extracts of _E. coli_. The majority of the work has been at Nirenberg’s laboratory at the National Institutes of Health and at Ochoa’s laboratory at New York University ([ref.155]). Various combinations of A, C, G, and U were used in preparing the synthetic RNA molecules that are used in experiments to explore the code. These molecules are made by incubating a mixture of ribonucleoside diphosphates with a specific enzyme, polynucleotide phosphorylase. An important property of this enzyme is that it condenses the nucleoside diphosphates into polynucleotide strands containing random sequences depending on the proportion of each base. For example, if the enzyme were furnished with a mixture of 5 parts of A and 1 part of C, it would make strands containing, on the average, 25 sequences of AAA, 5 of AAC, 5 of ACA, 5 of CAA, and 1 each of ACC, CAC, and CCA. The proportion of triplets within the strands of a polynucleotide is reflected in the proportion of amino acids in polypeptides that are obtained in the cell-free system. Most of the present knowledge of the amino acid code is based on this concept. All the proposed codes have been discovered by this experimental approach where synthetic RNA molecules are used as "artificial" messenger RNA.

Representative of another class of activities in molecular biology is the examination of passive ion flux across axon membranes. This work is being done by Goldman at the National Naval Medical Center. The question of stimulus transmission by nerve tissue is far from simple, and the ion concentrations associated with nerve membranes is a significant part of the answer. Because the space environment may very well produce alterations in these ion potentials, an investigation of their natures and significance becomes extremely important. A working theory is now being developed as a result of this study.

Vital cell processes, chemical transformations, and mechanisms that provide energy for cell maintenance and activity have been studied by Kiesow (refs. [ref.157] and [ref.158]) at the Naval Medical Research Institute. The common objective of all phases of this project is the elucidation of reaction steps in which energy and matter are transformed in living systems. Compared with _photo_synthetic organisms, _chemo_synthetic bacteria offer distinct advantages for the study of energy assimilation. These studies have led to the following experimental findings.

With the energy from oxidation of nitrite, NO₂— to nitrate, NO₃— as an _inorganic_ source, and with added _organic_ chemical energy from the hydrolysis of adenosinetriphosphate (ATP) to adenosinediphosphate (ADP) and inorganic phosphate, chemosynthetic bacteria are capable of reducing diphosphopyridinenucleotide (DPN⁺) to DPNH, in a coupled oxidoreduction-dephosphorylation. Thus, in the crucial step of chemosynthesis, _ATP is consumed, not produced_. However, in simultaneously proceeding cell respiration, the energy donor, DPNH, is oxidized and generates more ATP than is required for DPN⁺ reduction. This "breeder cycle" for DPNH—with different ratios of cell respiration and biosynthesis—results in a net production of either DPNH, or ATP, or both. Production of DPNH in the cycle leads immediately to the assimilation of C¹⁴ from HC¹⁴O₃—. These observations explain the bacteria’s energy source without the classical hypotheses of either direct phosphorylation or direct CO₂ reduction by inorganic chemical or electromagnetic energy. The cycle transforms the free energy of nitrite oxidation into the free energy of the organic compounds. Cell respiration and elementary biosynthesis proceed through structure-bound enzyme systems in the same fraction of subcellular particles. Three components, two cytochromes and one flavoprotein, have been identified. A thermodynamic analysis of the DPNH "breeder cycle" appears to be attainable by measurements of redox potentials and calorimetric determinations of heats of reaction.

Studies are also being conducted by Pollard and associates at Pennsylvania State University in an attempt to formulate a theoretical basis for the description of the processes of synthesis, growth, division, and differentiation of the living cell. Such a theory would be basic to an understanding of very primitive life forms or prebiological material which might be found elsewhere in the universe. For these purposes, studies are being undertaken in macromolecular reproduction which differ from the studies involving cellular genetic material. Theories concerning the problem of replication of cellular structures and information storage in two-dimensional systems are being developed. Theories are also being developed about the mechanisms which control and regulate receptor and enzymatic activities within the cell.

One study involved the rate of mutation in cells and disposed of the suggestion that the process of mutation consists of a "tunneling" of proton from one base to another in DNA. Such a suggestion can no longer be advanced as a major explanation of mutations.

Work is also being conducted on the centrifugation of cells of _E. coli_. It has been shown that cells exposed to as little as 100 g have a modification in their function. This has been looked at from the point of view of thymine uptake, which would be concerned with the formation of DNA, and also from the point of view of the induction of an enzyme, which would correspond to the transcription of the DNA. Preliminary experiments in the latter case indicate considerable centrifugation effect. The thymine uptake is affected, but not nearly as much as formerly thought. Further work is in progress in this area.

Important work has been completed on the cells of _E. coli_ grown on maltose, which can be induced to produce betagalactosidase by the addition of thiomethyl galactoside. If cells are irradiated shortly after induction, the transcription of the DNA ceases and the enzyme produced by the messenger RNA is observed to reach a maximum. This enables the calculation of the half-life of unstable messenger RNA. The half-life for this decay is readily measurable, and values are given over a temperature range of 17° C (5.2 minimum) to 45° C (0.56 minimum). These agree very well with half-lives measured by others by inducing for short times and measuring the course of enzyme formation. The rate of transcription is involved in the kinetics of cessation of enzyme induction, and the rate of transcription can be measured. Arrhenius plots for this rate and the rate of decay are given, and the activation energies measured are about 16 000 cal/mole. The cessation of transcription is linked to the degradation, possibly of only one strand, of DNA.

Pollard has suggested that one important action of ionizing radiation is concerned with the transcription of the genetic message into RNA. Clayton and Adler ([ref.159]) showed that induced catalase synthesis in _Rhodopseudomonas spheroides_ is inhibited by low doses of X-rays, giving experimental support to the idea. Pollard and Vogler ([ref.160]), using cells in which the process of induction involved permease, showed that there is some sensitivity to gamma radiation. Novelli et al. ([ref.161]) found a reduced sensitivity as compared with colony formation, but it is still a considerable sensitivity.

The process of induction of an enzyme indicates that the transcription of the genetic message is repressed by something which can be acted on by a small molecule, the inducer, to remove repression and permit the formation of messenger RNA, which then acts to make the enzyme. The messenger RNA undergoes decay through a process which is still not clear. Very elegant measurements by Kepes ([ref.162]) show that for the messenger RNA for betagalactosidase, the half-life is 1.02 min at 37° C and 2.05 min at 25° C. The time of onset of enzyme formation after induction was found to be about 3 minutes.

If the process of transcription is indeed sensitive to ionizing radiation, then the irradiation of cells which have just been induced should show formation of the enzyme to the extent of formation of new messenger RNA within a few minutes, plus the formation of the enzyme while the messenger RNA is decaying. This pattern was found by Clayton and Adler. The experiments conducted by Pollard and associates amplify and extend their work and also agree with the work of Kepes ([ref.162]).

BIOINSTRUMENTATION

Fernandez-Moran (refs. [ref.163]-[ref.165]), at the University of Chicago, has devised a new multielectrode electrostatic lens which he has incorporated into an electron microscope. This necessitated the development of a novel high-voltage power source and voltage regulator of extreme stability and accuracy. Some promising work has now been done on superconducting lenses. In a series of experiments with a simple electron microscope without pole pieces, using high-field superconducting niobium-zirconium solenoid lenses in an open air core, liquid helium Dewar, electron microscopic images of test specimens have been recorded while operating at 32 200 gauss in a persistent current mode, with regulated accelerating potentials of 4 to 8 kilovolts. These preliminary experiments have demonstrated the exceptional stability of the images (both short term and long term) over a period of 4 to 8 hours and the relatively high quality of the images.

Progress has been made on the viscosimeter for high intrinsic viscosities. This is now working, and the viscosity of DNA preparations has been measured. It is hoped to use the viscosimeter to study the variation in DNA viscosity as a function of the cell cycle.

An instrument is under development by Wald at the University of Pittsburgh to automatically analyze cytogenetic material and, thus, extend cytogenetic methodology both for research and as a biological monitoring procedure, using automatic electronic scanning and computer analysis of chromosomes. Chromosomal aberrations can thus be monitored under unusual and abnormal conditions such as weightlessness and radiation, since chromosomes are very sensitive to stress situations. In this device a sample will be prepared and automatically inserted under a microscope lens. The device will then scan, identify, and photograph on 35-mm film a predetermined number of mitotic cells and process the film. The data will be recorded under the direct control of a digital computer. The computer will perform a detailed quantitative analysis of the pictorial data.

Significant effort has been expended in the development of instrumentation for measuring and recording electrophysiological information. One such instrument, developed by the Franklin Institute, Philadelphia, Pa., is a temperature-sensing microprobe. This microprobe is an implantable and remote broadcasting instrument. These developments are associated, in part, with training programs so that competent individuals may be trained not only in electronics but also in the biological uses of the devices they construct.

A project of interest, conducted at the Stanford Research Institute, is the investigation of the uses of an extremely sensitive method for measuring magnetic susceptibility having the possibility of detecting macroscopic quantum effects in macromolecules of biological interest. Good progress has been made in the first 15 months of a project devoted to the development and initial use of equipment specifically designed for this purpose. A new superconducting circuit, together with superconducting magnetic shields, has been constructed. This apparatus can measure the magnetic susceptibility of small organic samples at temperatures between 1° and 300° K in fields up to 40 000 gauss. It can detect flux changes of 10⁷ gauss-cm², which is equivalent to detecting a change in specific susceptibility of 1 in 10⁹ in a 100-mg sample under an applied field of 10 000 gauss.

Several hundred preliminary measurements were made on samples of coronene. The most reliable of these were in agreement with published values of the magnetic susceptibility of coronene. Experience during these measurements led to changes which have resulted in an apparatus well suited to the measurements on macromolecules. An improved version of the superconducting circuit now available shows promise of a further improvement in sensitivity by a factor of more than a thousand ([ref.166]).

Living organisms possess many unique processes and systems which are complex and poorly understood. The new theoretical approaches, combined with laboratory studies, are expected to result in advances which will expand both our scientific and technological horizons.

chapter 6

_Flight Programs_

BALLOONS

Biological and medical experiments carried out on balloon flights, both manned and unmanned, antedate the establishment of NASA. Aside from the early use of balloons in flights that could be called simply flight-survival studies, balloons have made important contributions to our present knowledge of the effects of cosmic radiation and to various aspects of space travel.

The achievements of the Strato-Lab and Man High series by the U.S. Navy and Air Force include a wealth of information on balloon travel and on the survival of man at altitudes close to and above 100 000 feet. Generally, balloon launches of animals, which reached a maximum in 1953 when 23 balloons were released, have established the feasibility of a program of extended manned balloon flights to high altitudes.

Atmospheric life studies outside the area of cosmic radiation effects have been comparatively few. Results from two manned flights, Strato-Lab I and II, indicate that the flights did produce pronounced changes in white blood cell count; however, the data suggest that these changes were due to psychological rather than physical stress. Exposure to altitudes above 90 000 feet for a total of 62 hours did not produce any general behavioral change in two Java monkeys, according to other balloon flights. Many of these flights were effective in testing equipment, telemetering devices, and in pointing the way for other flights.

Stratoscope I and II, originally undertaken by the Office of Naval Research (ONR), are projects involving various astronomical observations with the aid of a balloon-borne telescope and television and camera systems. NASA cooperated with ONR on Stratoscope II (36-inch telescope compared with Stratoscope I’s 12-inch telescope) which has already resulted in significant discoveries about the nature of the planets and stars. Water vapor has been identified in the atmosphere of cool red stars and an analysis of the Martian spectra showed a greater abundance of carbon dioxide than had previously been believed. Since the balloon-borne telescope was carried beyond Earth’s obscuring atmosphere, the Stratoscope projects have yielded valuable photographs of the Sun, stars, and various planets.

ROCKETS AND SATELLITES

Historically, biological experiments aboard rockets and satellites have been limited to a "piggyback" and "noninterference" basis on military rockets. For the past few years, however, as the effort toward manned space flight leading to lunar and Martian landings increased, more attention was devoted to experiments designed to show the effects of the space environment on living systems. As in the balloon flight programs, the U.S. Army, Navy, and Air Force played an important role, reaching what might be considered a high point with the successful launch and recovery of a ballistic rocket experiment with monkeys Able and Baker. Aerobee rockets as well as Thor IRBM’s carried biological payloads consisting of mice and monkeys on six launches, contributing to our knowledge of the effects of weightlessness and radiation on higher animals.

Van der Wal and Young ([ref.78]) used Thor-Able combinations to serve as boosters for lifting a 20-pound biocapsule to a peak altitude of 1400 miles and over a distance of about 5300 miles from Cape Canaveral to the west coast of Africa. Weightlessness was attained for a period of almost 40 minutes. During reentry into the atmosphere, a peak deceleration of about 60 g was reached. Each of the three capsules flown carried one mouse (Mouse-in-Able); two of the mice were instrumented for heart-rate telemetry. Although all three mice were lost, the two experiments with Laska and Benji yielded physiological results.

The experimenters designed effective instrumentation for registering the electrical activity of the mouse’s heart through a single commutated telemetry channel. Records were obtained for both animals during various portions of the flight. The results indicate that both animals were alive when the nose cones hit the water.

Two South American squirrel monkeys (Gordo and Baker) and a rhesus monkey (Able) were launched into space from Cape Canaveral in 1958 and 1959 by U.S. Army Jupiter missiles. The vehicles reached speeds of approximately 10 000 mph and altitudes of 300 miles on flights which lasted about 15 min.

Time courses of cardiac and respiratory rates ([ref.80]) of the two squirrel monkeys showed that the noise of the engine at liftoff immediately produced an increase in their heart rates. Respiration also increased temporarily, but slowed later with increasing acceleration. Heart rates fluctuated considerably during launch acceleration, which reached about 15 g at cutoff.

The period of free flight and weightlessness was characterized by pronounced fluctuations of heart activity in the postacceleration phase. Thereafter, the heart rate of Baker remained relatively constant, whereas the cardiac activity of Gordo fluctuated markedly and decreased slowly almost to the end of his flight. Slight changes, which were transient and not pathological in nature, were also noted in the electrocardiogram. Gordo’s respiration was very shallow during maximum launch acceleration, when Baker’s reached its highest value, only to be approximated again during reentry when forces of about 35 g were encountered.

Able’s cardiac and respiratory rates indicated that, after an initial startle reaction, the heart rate dropped transiently and then increased steeply, reaching a maximum of 259 during the 10-second interval at peak acceleration. Respiration increased only slightly throughout the launching phase. There was a period of tachycardia during postacceleration weightlessness, after which the heart rate declined steadily and was disturbed only by several startling missile events. At the end of the subgravity phase, Able’s cardiac rate was slightly below normal.

Although the periods of high g force and free flight were short, the extremes were considerable, and the changes from one state to the next were rapid. In spite of this, the cardiovascular, hemodynamic, and electrocardiographic phenomena were remarkably well maintained. Apparently the animals were not in serious plight at any time. That psychological factors entered into the observed phenomena is clearly evident from the increase in cardiac rate associated with the noise of the engine prior to liftoff and also from the cinematographic record of facial expressions. Nevertheless, the integrated responses indicated that the animals’ physiological states remained sufficiently normal to insure a safe flight.

LITTLE JOE FLIGHTS

The first step in an attempt at animal verification of the adequacy of the Mercury flight program was the development of two tests by NASA in collaboration with the U.S. Air Force School of Aviation Medicine in which there would be a biomedical evaluation of the accelerations experienced during the abort of a Mercury flight at and shortly after liftoff. These flights were launched at the NASA Wallops Station with a Little Joe solid-fuel launch vehicle.

Two Little Joe launches were made with activation of the escape rockets during the boost phase to secure maximum acceleration; only a brief period of weightlessness was attained. The first launch was on December 4, 1959, and the other on January 21, 1960. A 36 by 18-inch sealed, 125-pound, cylindrical capsule containing the subject, an 8-pound _Macaca mulatta_, the necessary life-support system, and associated instrumentation was flown in a "boilerplate" model of the Mercury spacecraft. The rhesus monkeys were named "Sam" and "Miss Sam."

The flight profile included maximum accelerations of about 10 to 12 g and periods of about 3 minutes at 0±0.02 g. The peak altitude obtained in the last ballistic flight was about 280 000 feet. The experimental capsule was pressurized at 1 atmosphere with 100 percent oxygen at the start of the experiment and fell to just below a half atmosphere of oxygen due to breathing during flight. The capsule temperature was kept between 10° and 20° C in both flights.

The measurements taken from the rhesus monkeys were the electrocardiogram, respiration, body temperature, eye movements, and bar pressing, but only partial results were obtained in the first flight. Oxygen tension, total pressure, capsule temperature, and relative humidity were recorded. Both animals were recovered alive and did not show pathologic alterations in their physiologic and psychological reactions.

MERCURY ANIMAL TEST FLIGHTS

In the Mercury animal test program a Redstone missile carried the chimpanzee Ham on a ballistic flight to a height of 155 miles to provide animal verification of the success with which the Mercury system could be applied to manned flight. The male chimpanzee was trained to perform a two-phased reaction task during the 16 minutes of flight. The chimpanzee Enos was put into orbit for 3 hours and 20 minutes. Results of the two flights gave the following information:

(1) Pulse and respiration rates during both the ballistic (MR-2) and
the orbital (MA-5) flights remained within normal limits
throughout the weightless state. Effectiveness of heart action, as
evaluated from the electrocardiograms and pressure records, was
also unaffected by the flights.
(2) Blood pressures, both arterial and venous, were not significantly
changed from preflight values during 3 hours of the weightless
state.
(3) The performance of a series of tasks involving continuous and
discrete avoidance, fixed ratio responses for food reward, delayed
response for a fluid reward, and solution of a simple oddity
problem was unaffected by the weightless state.
(4) Animals trained in the laboratory to perform during simulated
acceleration, noise, and vibration of launch and reentry were able
to maintain performance throughout an actual flight.

From the results of the MR-2 and MA-5 flights, the following conclusions were drawn:

(1) The numerous objectives of the Mercury animal test program were
met. The MR-2 and MA-5 tests preceded the first ballistic and
orbital manned flights, respectively, and provided valuable
training in countdown procedures and range monitoring and recovery
techniques. The bioinstrumentation was effectively tested and the
adequacy of the environmental control system was demonstrated.
(2) A 7-minute (MR-2) and a 3-hour (MA-5) exposure to the weightless
state were experienced by the subjects in an experimental design
which left visual and tactile references unimpaired. There was no
significant change in the physiological state or performance of
the animals as measured during a series of tasks of graded
motivation and difficulty.
(3) Questions were answered concerning the physical and mental demands
that the astronauts would encounter during space flight, and it
was shown that these demands would not be excessive.
(4) It was also demonstrated that the young chimpanzee can be trained
to be a highly reliable subject for space-flight studies.

The suborbital ballistic flight of Ham on January 31, 1961, was the prelude to Alan R. Shepard’s suborbital space flight, while the orbital flight of Enos on November 29, 1961, preceded the orbital flight of John H. Glenn.

The fact that we now categorize these events as belonging to the rather distant past, although they occurred only about 4 years ago, serves to emphasize the pace of development in the exploration of space. While the chimpanzee program may pale in the light of subsequent successes, its scientific and technological contribution should not be overlooked.

The significance of this project can be fully appreciated, and its contribution judged, only by considering the lack of knowledge existing at the time of its conception. In addition to its essential training function, this project verified the feasibility of manned space flight through operational tests of the Mercury life-support system. It demonstrated that complex behavioral processes and basic physiological functions remained essentially unperturbed during brief exposures to space flight. The Mercury chimpanzee program marked the first time that physiological and behavioral assessment techniques were combined for evaluating the functional efficiency of the total organism in space.

Perhaps the ultimate contribution of this program was in providing the framework of knowledge upon which future scientific experiments on biological organisms, exposed to flights of extended durations, must be based. Biosatellite experiments designed to seek more subtle and elusive effects of prolonged space flight on biological functioning will require even more refined and difficult techniques, but will depend heavily on the groundwork laid in these early steps of Project Mercury.

A summary of the more important animal suborbital and orbital flights during the period 1957 to 1964 is presented in table VII.

In another NASA-supported flight, _NERV_ 1, various experiments were carried in a suborbital flight of 20 minutes. _Neurospora_ molds showed a surprisingly high level of mutation, but the control molds also had high rates.

The Discoverer XVII and XVIII flights, to which the Air Force contributed, resulted in many interesting findings relative to the responses of living systems to space flight. On the Discoverer XVII flight, samples of human gamma globulin and rabbit antiserum specific for human gamma globulin showed an increase in reactivity, and samples of synovial and conjunctival cells showed no changes in their cytological characteristics.

Discoverer XVIII was launched during a massive solar flare which lasted for the first 13 hours of the 48-orbit, 3-day flight. _Neurospora conidia_, nerve tissue, algae, human bone marrow, eyelid tissue, gamma globulin, and cancer cells were put in orbit. The results indicated that biological specimens may be able to withstand radiation from solar flares with a minimum of shielding and that aluminum shielding may be better than lead.

In 1949, the U.S.S.R. began a systematic, uninterrupted research program in biological space experimentation. They have studied the effects of physical stress, immune reactions, psychobiology and behavior, genetics, and responses to environmental factors such as spacecraft dynamics and ambient radiation. The organisms and biological materials included tobacco mosaic and influenza viruses; T2 and T4 bacteriophage; _Bacillus aerogenes_; lysogenic bacteria; _Clostridium butyricum_; _Escherichia coli_; actinomycetes; yeasts; _Chlorella pyrenoidosa_; seeds of fir, pine, onion, corn, lettuce, wheat, cabbage, carrot, buckwheat, cucumber, beet, _Euonymus_, fennel, mustard, pea, broad bean, tomato, and nutmeg; _Tradescantia paludosa_; _Ascaris_ eggs; snail spawn; _Drosophila melanogaster_; loach roe; frog eggs and sperm; guinea pigs; mice; rats; hamsters; rabbits; dogs; monkeys; human and rabbit skin; HeLa tissue cultures and other tissues (refs. [ref.167] and [ref.168]).

Table VII.—_Orbital and Suborbital Animal Flights for 1957-64_