Significant Achievements in Space Bioscience 1958-1964 Part 7

A significant amount of the work which has been conducted in the area of biological information and communication systems is easily cla.s.sified as "basic research" (refs. [ref.106]-[ref.109]). This discussion will be limited to those aspects closely related to the fields of molecular biology and experimental psychology, which seem to have universal application to all known animal life forms. Studies involving the basic principles of acquisition, processing, storage, and retrieval of information in living systems are emphasized.

Early Work

Early speculations on the operational nature of memory have been based upon relatively little experimental evidence. Charles Darwin observed that domestic rabbits had smaller brains than their wild counterparts, and attributed this to lack of exercise of their intellect, senses, and voluntary movements. Unfortunately, subsequent studies of the brains of men with greatly differing intellectual capability did not substantiate the hypothesis. Idiots sometimes had larger brains than geniuses. Later, an idea proposed by Ramon y Cajal came into favor. Since brain cells did not increase in number after birth, he proposed that memory involved the establishment of new and more extended intercortical connections.

Unfortunately, methods were not available to test this hypothesis adequately and it has remained until quite recently in the realm of conjecture.

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 acc.u.mulation 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 Goteborg, Sweden. He and others (refs. [ref.118]-[ref.120]) have for many years performed elegant microa.n.a.lytical 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 a.s.sociated 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 pa.s.sive 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 a.n.a.lyzed, 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 pa.s.sive 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 a.n.a.lyzed. 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 h.o.m.ologous 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 a.s.sociated 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 Sp.o.r.n ([ref.122]), presently at the National Inst.i.tute of Mental Health.

They showed that 8-azaguanine, a purine antagonist, injected intra-cisternally was incorporated into the RNA of the brains of rats.

a.s.sociated with this incorporation was an impairment of the maze-learning ability of the animals. These findings have been confirmed.

Flexner and his a.s.sociates 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 a.s.sociates 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 acc.u.mulated 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.

NEUROPHYSIOLOGY

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 s.p.a.ce.

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 s.p.a.ce-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 cla.s.sical histological methods and the observation of equilibrium disturbances resulting from operative interference with the internal ear have in the past been the two princ.i.p.al 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 a.n.a.lysis 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 ca.n.a.l 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 a.n.a.lysis 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 c.u.mulative 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 t.i.tov'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 a.s.sumption. 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