DAVID A. BOOTH 2. Rockefeller University. Current evidence for the chemical trace is reviewed. The known capacities of. RNA mechanisms are discussed in ...
SEPTEMBER 1967
VOL. 68, No. 3
Psychological Bulletin VERTEBRATE BRAIN RIBONUCLEIC ACIDS AND MEMORY RETENTION1 DAVID A. BOOTH
2
Rockefeller University Current evidence for the chemical trace is reviewed. The known capacities of RNA mechanisms are discussed in relation to the time course of memory consolidation and ways in which information could be transferred into and out of a trace involving RNA. RNA analyses suggest characteristic synthesis correlated specifically with learning. Brain preparations from trained animals may transfer information, sometimes via peptides and possibly other materials, including RNA. These experiments, taken with indications from disruptive manipulations that temporary and permanent retention involve multiphasic and dissociable mechanisms, suggest that RNA synthesis may normally mediate consolidation but is not necessary to information-holding mechanisms of several hours' half-life, although protein synthesis dependent on RNA probably is necessary. Interdisciplinary work should be accelerated to clarify the implicated biochemical and neuroanatomical mechanisms and to substantiate specific correlations preliminarily made with consolidation.
THE CHEMICAL MEMORY TRACE It has been widely held that the long-term retention of environmental information available for subsequent use, which is known to occur in vertebrates in a variety of behavioral paradigms, must be mediated by permanent chemical changes in the central nervous system (Barondes, 1965; Deutsch, 1962; Glickman, 1961; Katz & Halstead, 1950; Moore & Mahler, 1965; Schmitt, 1964). These changes might range from irreversible steric rearrangements within or between molecules to multiple biosyntheses and degradations as in growth. Assuming that the psychological achievements of men and other animals are in fact dependent on known physical mechanisms, information being held in perseverating electrical activity of some sort appears to be
the only conceivable alternative, and the experimental evidence is against it. The possibility of memory storage in steady potential gradients seems to be excluded by evidence of survival of recall after massive cortical implantation of insulating sheets (Sperry & Miner, 1955) or conducting wires (Sperry, Miner, & Myers, 1955) in regions known to be important for retention of the visual discriminations used. Besides, any such standing potentials are likely to be the epiphenomena of chemical gradients or the summation of the effects of electrical activity in many cells. On the other hand, the existence of a permanent trace as varying potentials is taken to be excluded by the fact that it can survive a number of manipulations which would be expected to eliminate all signs of patterned 1 Earlier versions were written for research semi- electrophysiological activity for brief periods. nars at Yale and Rockefeller Universities with the encouragement of Neal Miller to whom the author is Prime examples are: marked reduction of much indebted for ideas and the incentive to experi- brain temperature for an hour or so (Sudak mentation in this area. Supported by United States & Essman, 1964); blocking the flow of Public Health Service Grants MH 02949 and 00647. oxygen-enriched blood for 8 minutes (Niel2 Now at the Laboratory of Experimental Psychology, Sussex University, England. Reprint re- son, Zimmerman, & Colliver, 1963); lowering quests c/o Professor N. E. Miller, Rockefeller Uni- air pressure by 60% for about 20 minutes versity, New York, N. Y. 10021. (Thompson & Preyer, 1956); immersion in 149
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carbon dioxide gas for 25 seconds (Paolino, Quartermain, & Miller, 1966; Taber & Banuazizi, 1966); and a single electric shock through the head, either with convulsive tonic extension (Heriot & Colman, 1962) or without (McGaugh & Alpern, 1966). It is not yet certain, however, that all of these procedures meet both the criteria of effectiveness in disrupting retention of information in alternating potentials. First, electrical silence during the manipulation must be established by cortical and depth recordings. Second, test performance must vary with interval between training and disruptive manipulation—poor at the shortest intervals, during which electrical activity is presumed to be necessary to the trace, while normal at some greater interval. Even those manipulations which satisfy these criteria are open to careful reexamination to check whether, for example, some interaction of the manipulation with some immediately preceding factor in training does not cause poor performance at the time of test. (The existence of the temporal gradient just mentioned excludes important direct effects on subsequent performance.) Despite all the work on the effects of a single electroconvulsive shock, aimed at establishing retrograde amnesia apart from aversive or conditioned interference effects (Chorover & Schiller, 1965; Hudspeth, McGaugh, & Thomson, 1964; Quartermain, Paolino, & Miller, 1965), it is still possible to suggest that some or perhaps all of the apparent amnesia is an unspecific motility or reactivity shift which is contingent on contiguity of footshock reinforcement and ECS (Chorover & Schiller, 1966). The metabolic changes caused by this pattern of stimulation—in brain amines (Mclver & Nielson, 1966; Nielson, 1966; Routtenberg & Kay, 1965), for example, or even in RNA metabolism (Essman, 1966)—might last hours or days. This type of experiment should therefore include control groups tested after much longer intervals than 1 day after ECS (Zinkin & Miller, 1967). The existence of the chemical trace will be taken for granted in the following discussion. The data which provoked this review came from several recent reports pointing to a specific role of ribonucleic acid (RNA) in
retention mechanisms (Albert, 1966a; Hyden & Egyhazi, 1964; Hyden & Lange, 1965). Thus a summary of the general function of RNA will first be presented, indicating how it might or might not be relevant to memory. This focus on RNA should not be taken to imply that special importance for memory of some other class of substances is not equally plausible and would not be of equal interest. CAPACITY OF BRAIN RNA FOR CONSOLIDATING CNS CHANGES Prolonged retention and much-delayed recall are of course in a trivial sense bound to be dependent on RNA, since functioning RNA and its synthesis are presumably essential for long-term maintenance of the vertebrate CNS (Briggs & Kitto, 1962; Dingman & Sporn, 1964) although the invertebrate stretch receptor neuron can retain electrical activity after 12-24 hours' treatment with the RNA synthesis inhibitor actinomycin D (Edstro'm & Grampp, 1965), and after 7-12 hours' suppression of RNA-dependent protein synthesis by puromycin (Toschi & Giacobini, 1965). Also, it is obvious that RNA changes cannot, on the other hand, be entirely sufficient for retention, let alone for learning or recall. Even if some RNA were capable of specifying correctly its relationship to a functioning organism other than the animal whose memory it originally carried, many homologies between the donor and recipient organisms would be additional necessary conditions for usable coding of environmental information, even though, in such case, the memory function of the RNA could survive the death or disruption of the donor animal. It should be clear, therefore, that the possible role of RNA in memory that is of interest would be that of chemical mediation necessary to the potential usefulness of an organism's past experience and necessary to no other aspect of performance. Practically all constituents of the CNS except for some structural components, such as parts of myelin, have a marked rate of breakdown (Khan & Wilson, 1965). Hence, unless some specially protected material is postulated, the material basis of the memory trace might be expected to require continuous
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renewal. This biosynthesis would require enzymes, which are proteins or proteincontaining systems themselves subject to degradation, and the sole known function of RNA of all types is organizing the synthesis of proteins from amino acids in arrangements specified by the genes (Watson, 1963). It has sometimes been assumed that therefore some RNA, or even gene changes, must be involved in the chemical changes consolidating the memory trace if it is to be maintained for as long as the lifetime of the organism (Gaito & Zavala, 1964; Morrell, 1961). It is thought that the deoxyribonucleic acid (DNA) constituting the genes is stable in mature nondividing cells, and no DNA synthesis can be detected in most neurons (Messier & LeBlond, 1960; Pevzner, 1966), making these cells speculatively preferred candidates for the permanent trace over glial cells which might be apt to lose an acquired genetic expression during mitosis and cell growth. Hippocampal granule cells are an exception, since they are formed over many weeks postnatally in the rat by multiplication, migration, and differentiation of periventricular ependymal cells (Altman & Das, 1966a). No function of hippocampal granule cells in memory has been suggested, and undiscovered neuronal populations which divide in postnatal brain are presumably much smaller and are negligible. However, systematic mutation of chromosomal or other DNA—for example, by methylation (Hechter & Halkerston, 1964) —or some other specialization of brain-DNA function which is quantitatively minor cannot be excluded at present. Glial DNA synthesis, and therefore presumably proliferation, varies with gross differences in environmental stimulation (Altman & Das, 1964), further emphasizing that it may be unwise to consider neuronal function in isolation from possible neuronal-neuroglial interactions (Galambos, 1961; Hyden, 1960a). Intercellular interaction is, however, one example (Barondes, 1965; Monod & Jacob, 1961) of a cellular change whose biosynthetic maintenance may in some circumstances not require any qualitatively new activity in the cell, even if the change had to be initiated by some special mechanism. Yet another way in which nucleic acids would
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require no special role in memory would be if retention were mediated by some quantitatively minor component of the CNS which does not break down at an appreciable rate. For example, melanin is an extremely stable pigment known to occur in certain mammalian neurons (Crosby, Humphrey, & Lauer, 1962), although its distribution is reason to expect it to be totally unrelated to memory. It may be possible for membrane properties to be radically and permanently altered by the local formation of such molecules or of more easily detectable macromolecular complexes of such conformation as to be virtually inaccessible to degradative enzymes. This alteration might happen when a polyacidic compound formed a salt with a polybase or when an antigen reacted with an antibody to give a protein globule which happened to have a hydrophobic exterior (Booth, 1965; Meisler & McCluer, 1966; Van Sickle & McCluer, 1966). In whatever way it might be modified, local membrane structure could itself carry a large amount of information (Lehninger, 1965), and stable local specialization has been demonstrated for the soma membrane of Mauthner neurons (Diamond, 1963). Parts of the cell surface of amphibia eggs are sufficiently stable and differentiated to specify a good deal of developmental differentiation, possibly by means of their phospholipid constituents, although the effects may be dependent on an interaction between the crucial region of the cell cortex and the nuclear genes (Curtis, 1963). Despite all these alternatives, some results to be discussed below may be the first indications that not only protein synthesis but even the synthesis of nuclear RNA may be especially important for memory, even if not necessary a priori, and gene activation has increasingly been invoked as a possible memory mechanism (Bonner, 1966; Hyden, 1966; Meierson & Kruglikov, 1966; Young, 1965). Current theory of the general biochemical mechanisms of genetic expression will therefore be outlined, paying some attention to the maximum speed with which they are thought to operate, with a view to dealing with later questions about the speed of consolidation of the trace by what may be some specialization of nucleic acid function in the brain.
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Many genes in one cell at any time are held inactive, some at least by complex formation with repressers arising from other genes (Allfrey, Faulkner, & Mirsky, 1964). In bacteria, an inactive gene can begin transcription within a minute or two (Jacob & Monod, 1961b), perhaps by the cell's loss of a required substrate (derepression) or by arrival of a new substance capable of interaction with the enzyme specified by that gene (induction). In higher organisms, some hormones apparently act directly on the genes or on associated repressers: Testosterone appears to increase gene activity in the prostate (Williams-Ashman, Liao, Hancock, Jurkowitz, & Silverman, 1964); ecdysone, a steroid controlling insect development, can diffuse through a larva into the cells and cause a readily visible puffing at localized bands on the salivary gland chromosomes, all in as little as 30 minutes (Karlson, 1962) and with gene activation preceding other effects (Clever, 1966). Transplantation of nuclei between amphibian cells has demonstrated effects of cytoplasm on the expression of nuclear genes and their conservation through inactivation and reactivation (Gurdon, 1966). It is widely thought that each gene is a potential template for synthesis of a "messenger" RNA (m-RNA) molecule whose structure in turn determines the amino acid sequence in a given protein such as an enzyme (Jacob & Monod, 1961a). Assuming that transcription of gene DNA occurs by a mechanism analogous to DNA replication in cell division, each of the four major cyclic bases (adenine, guanine, cytidine, and thymine) characteristic of the DNA chain is matched by a DNA-dependent RNA polymerase enzyme to its complementary base (uracil, cytidine, guanine, and adenine, respectively) in a ribonucleotide (composed of the base, ribose, and phosphate) and these monomers are linked in the sequence thus specified by the genetic DNA to form the polymeric m-RNA. It has been calculated that a few seconds are sufficient for the synthesis of an m-RNA molecule of hundreds of thousands of nucleotides (Ruckenstein & Simon, 1966). In the case of RNA synthesis in embryo ganglia in vitro stimulated by a nerve-growth factor, it is possible to detect
the greater rate of incorporation of a radioisotopically labeled uracil derivative into RNA within 15 minutes (Toschi, Gandini, & Angeletti, 1964), and, within 30 minutes, apparently ribosomal membrane precursors can be seen by electron microscope (Grain, Benitez, & Vatter, 1964). Brain tissue has very high RNA polymerase activity, even higher than the liver, which makes large quantities of protein, and cerebral cortex was the most active tissue of any assayed (Bondy & Waelsch, 1965), presumably giving it a high capacity for m-RNA synthesis on demand. This capacity may well be modulated not only by growth factors, but also by hormones, as is known for RNA synthesis in other tissues (Bransome & Chargaff, 1964; Korner, 196S; Weber, Singhal, Stamm, & Srivstava, 196S). In a matter of minutes (Lathan & Darnell, 196S), the m-RNA can leave the cell nucleus. When functioning in protein synthesis, it is generally associated with ribosomes, cytoplasmic particles composed of RNA and protein sometimes found attached to endoplasmic membranes, a network which characterizes cells very active in protein synthesis, such as neurons or some liver cells (Palay & Palade, 1955). Ribosomal RNA is of two types, one containing about 1,500 nucleotides, the other about twice as many per molecule, both species being transcribed from nuclear DNA. The life cycle of ribosomes and their association with m-RNA is under intensive study at present, although not in brain tisssue. Definite incorporation of radioactive base precursor into ribosomal RNA in cerebral cortex has been found within 3 minutes in adult rats but not in 4-day-old rats (Adams, 1966). The evidence shows that during protein synthesis several ribosomes at a time pass along the m-RNA chain, forming a polysome. Examples of polysomes were first obtained from bacteria (Risebrough, Tissieres, & Watson, 1962) but have now been seen in the mammalian CNS (Campbell, Mahler, Moore, & Tewari, 1966; Ekholm & Hyden, 1965). For interconnection to form protein, a relatively low-molecular-weight (about 100 nucleotides) soluble type of RNA (s-RNA or t-RNA) transports amino acids into
VERTEBRATE
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juxtaposition within the ribosome in a sequence which is specified by the m-RNA nucleotide sequence. Each of the 20 fundamental amino acids is thought to have its specific t-RNA, and each amino acyl-t-RNA combination has a selective affinity for a certain few of 64 logically possible trios of base sequences in the m-RNA template (Marshall, Caskey, & Nirenberg, 1967). Thus the sequence of base-trio "codons" in the gene DNA is transcribed via the complementary m-RNA "anticodons" into the amino acid sequence which in turn determines the functional properties of a protein molecule. The maximum rate of ribosome function is very high, and in about 1 second several moderately sized molecules may well be synthesized off one m-RNA molecule (Eigen & DeMaeyer, 1965). In bacteria, m-RNA is very short-lived and its rate of synthesis controls the rate of protein synthesis; but some mammalian m-RNA is relatively stable (Marks, Burka, & Schlessinger, 1962; Revel & Hiatt, 1964), and other factors can be important in regulating ribosome function. Pep tides such as growth hormone (Korner, 1961) and adrenocorticotrophic hormone (Farese & Reddy, 1963) accelerate protein synthesis at the ribosome. ACTH also activates a transfer enzyme (Scribna & Reddy, 1963) and nuclear RNA polymerase (Bransome & Chargaff, 1964). Furthermore, the abundance of a protein is by no means entirely dependent on the rate of synthesis. Generally brain protein is rapidly broken down, and it is conceivable that these degralative processes are selectively inhibited by ions, substrates, or other molecules; the hormonal control of liver enzyme levels certainly involves such mechanisms as well as influences on protein synthesis (Schimke, 1964). Neurons are distinguished by an extremely high rate of protein synthesis, comparable with immature cells and greater than even liver cells (Hyden, 1960b). It is conceivable that some as yet physically undetected brain function is implicated over and above mere maintenance of extremely long axonal processes or of specialized neurosecretion. Among the candidates could be a retention-mediating process such as the continual differentiation of somatodendritic mem-
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branes under the influence of locally changing patterns of electrical or metabolic activity. There is also a protein synthesizing system in brain mitochondria (Campbell et al., 1966; Klee & Sokoloff, 1965) which may be partially independent of nuclear DNA and ribosomes (Humm & Humm, 1966), and which may be organized by mitochondrial DNA, RNA polymerase, and RNA. Since a major function of mitochondria is the generation of energy-rich substrates, it is conceivable that the functioning of this system is rapidly and reversibly altered by changing cellular energy consumption, or it might even undergo irreversible or self-maintaining changes. This process could be especially important in synaptic function, as mitochondria are very abundant in nerve endings (Gray & Whittaker, 1962). The nucleic acid functions which the brain is already known to have in common with other vertebrate tissues and with microorganisms, together with the speed with which these mechanisms may well be able to operate, suggest that synthetic or degradative changes could take place rapidly enough for chemical trace consolidation within as brief a time as a few minutes, even if gene activation were involved, unless some presynaptic change was required. Within the brain, in that case, up to 10 days are needed for migration of material from the nucleus (Barondes, 1964), presumably intracellularly down the axon (Ochs, 1966). Recently there have been several remarkably similar theories of memory fixation by gene activation (Hyden & Eghazi, 1964; Hyden & Lange, 1965; John, 1967; Young, 1965). Information might be incorporated in changes in transcription rates of functionally interdependent genes such as those which occur in differentiation and regeneration under the influence of extracellular interactions. These theories suppose that some aspect of postsynaptic excitation or of presynaptic vesicular modification might trigger synthesis or migration of a derepressor of a local type or of an activity-specific type which initiates further synthesis of homologous molecules. Such further synthesis facilitates, in the one case, general sensitivity in the same locale of the neuronal membrane or, in the other
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case, transmission of the same activity pattern. From the neurology of octopus memory, it has been hypothesized that the essential change is marked activation of small inhibitory neurons which then close one of alternative pathways by release of long-acting inhibitory transmitter and simultaneous initiation of an increased rate of its synthesis (Young, 196S). Negative feedback and selective differentiation of response has cybernetic advantages as a learning mechanism, and postulation of merely a frequency threshold as a trigger for consolidation requires no special system of molecular recognition and re-recognition of some parameter of electrical activity in both read-in and read-out. It should be noted that RNA synthesis is not necessarily implied even if change in RNA function is part of consolidation. In addition to the possibility of modulation of translation speed of stable m-RNA, interchangeable active and inactive forms of t-RNA have been demonstrated (Gartland & Sueoka, 1966; Lindahl, Adams, & Fresco, 1966). Even if RNA synthesis is involved, it need not be DNA-dependent: In a ciliate organism at least, a stable m-RNA from a paramecium can acquire the virus-like property of programming RNA synthesis (Gibson & Sonneborn, 1964). Memory might then depend on "plasmagenes" (Wright, 1945) in mature neurons, or, if circumstantial evidence of glial-neuronal RNA transfer is substantiated (Hyden & Pigon, 1960), mutant glial RNA might conceivably act as a virus in adjacent neurons. At least one other tissue system has specialized to provide a means of forming and permanently maintaining a re-recognizing reaction to an unlimited variety of environmental stimuli of a certain sort: the lymphoid-immune response to a chemical antigen by synthesis of a sterically matching antibody protein. The analogy with psychic memory should not, perhaps, be regarded as strong, since the only type of information retained concerns the shape of molecules interacting directly with the system, but the necessary adaptation of general nucleic acid function is a fertile source of speculation and evidence from which to form theories of the memory trace. It has been suggested (Hood,
Gray, & Dreyer, 1966) that antibody synthesis depends in part on genes containing ambiguous triplets whose protein expression is specified for a given cell line by the antigen to which the parent cell has reacted. These genes might be related virally (by insertion into the chromosome) or enzymically (at the protein synthesis stage) to normally specified protein to form the total immunoglobin antibody. Obviously, possibilities for nucleic acid function are so varied that neither the necessity nor the impossibility of specific functions of RNA in memory can be prejudged, nor can the ways in which it might be involved. We are entirely dependent on current and much future investigation both of basic nucleic acid biochemistry, especially in brain tissue, and of the special problems of biochemistry of memory. RETENTION MECHANISMS AND THEIR "READ-IN" AND "READ-OUT" IN RELATION TO RNA We know that vast amounts of information are carried through the CNS in sequences of neuronal action potentials, and that their interactions as postsynaptic graded potentials on somatodendritic membranes are key processes in the transformation and translocation of electrical activity necessary to functional integration, although other less well understood physiological processes may also be important. Yet a major difficulty in even speculative construction of theoretical mechanisms by which the physicochemical patterns of electrical activity or their immediate metabolic consequences are converted into a more stable chemical trace, and vice versa in retrieval, is the absence of an adequate outline of how the environmental information to be retained is coded in neural activity. It is unknown whether the necessary and normally sufficient electrical activity is very localized or widespread, or how dependent this is on the task, or whether it is characterized by its sheer novelty compared with all previous activity which has affected the CNS or by some special qualities of impulse frequence (John, 1962), multineuronal field potentials (Adey, Dunlop, & Hendrix, 1960), or conjunction of input sources (Landauer,
VERTEBRATE BRAIN RNA AND MEMORY RETENTION
1964). Results indicating a spread of taskspecific potentials through the brain (John & Killam, 1960) and the development of hippocampal conductivity changes characteristic of successful attention to the task (Adey, Dunlop, & Hendrix, 1960) provide data which should prove important in eventual descriptions of brain mechanisms in learning. The problems of describing the translation of psychological perception, integration, and action into the neurophysiological mechanisms that are necessary to and perhaps characteristic of such achievements (Fields & Abbott, 1963; Morrell, 1961; Uttal, 196S) are greater than sometimes seems to be assumed. The importance of psychological categories and the "external world" in functional description of the CNS should not be underestimated by neurophysiologists studying single neurons and neuronal systems (J. Lettvin, cited by Kreiling, 1966; McCulloch, 1952) any more than biochemists and psychologists with relevant interests can ignore the neuroanatomical and electrophysiological knowledge and the problems involved. Even if the vague assumption is made that the adequate CNS input-output relationships are somehow represented analogously in electrical activity during acquisition and during retrieval, a number of chemical theories of memory have been totally inadequate for the dual problem of preserving the information content of the trace while translating both from electrical activity into the chemical changes it causes in consolidation and then back from the chemical trace to facilitation of the required patterns of electrical activity. The postulated read-in transformations have been arbitrarily related to the read-out transformations, and hence the theories have not suggested an explanation of the chemical basis of memory but have merely listed chemical changes which may be uniquely necessary, usually including RNA synthesis (Gaito, 1963; Hyd
