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Role of RNA and protein in memory storage: A review
Abstract
The literature on the possible role of RNA and protein in long-term memory is reviewed in terms of the following five criteria: (1) The molecule should have a defined role in normal brain function. (2) There should be a quantitative and/or qualitative change in the molecule as a function of learning. (3) The molecule should lead to some type of relatively permanent change in the functioning of the brain. (4) Altered synthesis or utilization of the molecule should lead to predictable consequences for memory. (5) The localization of the response should be consistent in terms of the utilization of that portion of the brain in the particular task.
... Katz and Halstead (1950) suggested that biochemical memory traces in the nervous system might consist of nuclear proteins, modified as a consequence of experience. Other authors suggested cytoplasmic macromolecules as memory traces (see Uphouse et al., 1974, for a review of these early proposals). ...
The most widely accepted view of memory in the brain holds that synapses are the storage sites of memory, and that memories are formed through associative modification of synapses. This view has been challenged on conceptual and empirical grounds. As an alternative, it has been proposed that molecules within the cell body are the storage sites of memory, and that memories are formed through biochemical operations on these molecules. This paper proposes a synthesis of these two views, grounded in a computational theory of memory. Synapses are conceived as storage sites for the parameters of an approximate posterior probability distribution over latent causes. Intracellular molecules are conceived as storage sites for the parameters of a generative model. The theory stipulates how these two components work together as part of an integrated algorithm for learning and inference.
... It was even suggested at the time that the molecular processes that underlie learning and encode new memories may be ''continuous across the phyla (as genetic codes are) and therefore would be reasonably similar for a protozoan and a mammal" (Gershman et al., 2021;Gelber, 1962). These ideas were largely abandoned in the 1970s (Gaito, 1976;Glassman, 1969;Ungar, 1973;Uphouse et al., 1974) but have been rekindled in recent years (Gallistel, 2017;Langille and Gallistel, 2020;Mattick and Mehler, 2008;Abraham et al., 2019;Gallistel, 2018;Gallistel and Balsam, 2014;Queenan et al., 2017). The recent revival is rooted in the sobering realization that current theories of synaptic plasticity and network activity cannot explain learning, memory, and cognition (Gallistel and King, 2009;Gallistel and Matzel, 2013) and that several lines of evidence bring into question the theory that synaptic strengthening/weakening is the primary form of long-term information storage in the brain (Gallistel, 2017;Cai et al., 2012;Daou and Margoliash, 2020;Jirenhed et al., 2017;Johansson et al., 2014;Johansson et al., 2015;Pearce et al., 2017;Poo et al., 2016;Ryan et al., 2015;Santin and Schulz, 2019;Zhao et al., 2019). ...
Life is confronted with computation problems in a variety of domains including animal behavior, single-cell behavior, and embryonic development. Yet we currently do not know of a naturally existing biological system that is capable of universal computation, i.e., Turing-equivalent in scope. Generic finite-dimensional dynamical systems (which encompass most models of neural networks, intracellular signaling cascades, and gene regulatory networks) fall short of universal computation, but are assumed to be capable of explaining cognition and development. I present a class of models that bridge two concepts from distant fields: combinatory logic (or, equivalently, lambda calculus) and RNA molecular biology. A set of basic RNA editing rules can make it possible to compute any computable function with identical algorithmic complexity to that of Turing machines. The models do not assume extraordinarily complex molecular machinery or any processes that radically differ from what we already know to occur in cells. Distinct independent enzymes can mediate each of the rules and RNA molecules solve the problem of parenthesis matching through their secondary structure. In the most plausible of these models all of the editing rules can be implemented with merely cleavage and ligation operations at fixed positions relative to predefined motifs. This demonstrates that universal computation is well within the reach of molecular biology. It is therefore reasonable to assume that life has evolved – or possibly began with – a universal computer that yet remains to be discovered. The variety of seemingly unrelated computational problems across many scales can potentially be solved using the same RNA-based computation system. Experimental validation of this theory may immensely impact our understanding of memory, cognition, development, disease, evolution, and the early stages of life.
... It was even suggested at the time that the molecular processes that underlie learning and encode new memories may be ''continuous across the phyla (as genetic codes are) and therefore would be reasonably similar for a protozoan and a mammal" (Gershman et al., 2021;Gelber, 1962). These ideas were largely abandoned in the 1970s (Gaito, 1976;Glassman, 1969;Ungar, 1973;Uphouse et al., 1974) but have been rekindled in recent years (Gallistel, 2017;Langille and Gallistel, 2020;Mattick and Mehler, 2008;Abraham et al., 2019;Gallistel, 2018;Gallistel and Balsam, 2014;Queenan et al., 2017). The recent revival is rooted in the sobering realization that current theories of synaptic plasticity and network activity cannot explain learning, memory, and cognition (Gallistel and King, 2009;Gallistel and Matzel, 2013) and that several lines of evidence bring into question the theory that synaptic strengthening/weakening is the primary form of long-term information storage in the brain (Gallistel, 2017;Cai et al., 2012;Daou and Margoliash, 2020;Jirenhed et al., 2017;Johansson et al., 2014;Johansson et al., 2015;Pearce et al., 2017;Poo et al., 2016;Ryan et al., 2015;Santin and Schulz, 2019;Zhao et al., 2019). ...
Life is confronted with computation problems in a variety of domains including animal behavior, single-cell behavior, and embryonic development. Yet we currently have no biologically plausible model capable of universal computation, i.e., Turing-equivalent in scope. Network models (which include neural networks, intracellular signaling cascades, and gene regulatory networks) fall short of universal computation, but are assumed to be capable of explaining cognition and development. I present a class of models that bridge two concepts form distant fields: combinatory logic (or lambda calculus) and RNA molecular biology. A set of simple RNA editing rules can make it possible to compute any computable function with identical algorithmic complexity to that of Turing machines. The models do not assume extraordinarily complex molecular machinery or any processes that radically differ from what we already know to occur in cells. Distinct independent enzymes can mediate each of the rules and RNA molecules solve the problem of parenthesis matching through their secondary structure. This demonstrates that universal computation is well within the reach of molecular biology. It is therefore reasonable to assume that life has evolved - or possibly began with - a universal computer that yet remains to be discovered. The variety of seemingly unrelated computational problems across many scales can potentially be solved using the same RNA-based computation system. Experimental validation of this theory may greatly impact our understanding of memory, cognition, development, cancer, evolution, and the early stages of life.
... As mentioned before, the rationale behind these experiments is that learning may induce the synthesis of macromolecules, which would modify synaptic connections in some unknown way, to establish the memory for the learning. These experiments have been recently summarized in detail by other reviewers (17,18). ...
A brief review is given of experiments which are concerned with the hypothesis that brain RNA and protein synthesis are directly involved in the establishment of long-term memory. It is concluded that these experiments neither support or refute this hypothesis. A convincing demonstration is lacking of interanimal memory transfer by injection of macromolecular extracts. The majority of experiments which attempt to correlate increased macromolecular synthesis with learning use radioactive precursor methods and these studies do not exclude possible changes in precursor specific activity as the cause of the increased labeling. Although some studies find directly observable changes in brain macromolecules in response to training, their relationship to memory formation is unclear. It is possible that these changes represent only an enhanced production of constitutive macromolecules in response to an increase in cerebral metabolism during training, rather than molecular changes that are directly involved with modifying synaptic connectivity. Inhibitors of cerebral protein synthesis block memory formation, but these drugs are not pharmacologically specific and this complicates the interpretation of these studies.
... Findings of early studies using intracerebral infusions of protein and RNA synthesis inhibitors (1)(2)(3)(4) suggested that experience-dependent alterations in gene expression within neurons may be required for the formation of long term memory (LTM). An early step in such inducible neuronal gene expression is the activation of constitutively expressed regulatory transcription factors, such as the cAMP response element binding (CREB) protein, through phosphorylation reactions mediated by second messenger-activated kinases (5)(6)(7)(8)(9)(10)(11). ...
Extensive evidence suggests that long term memory (LTM) formation is dependent on the activation of neuronal second messenger systems and requires protein synthesis. The cAMP response element binding protein (CREB) is a constitutively expressed regulatory transcription factor that couples changes in second messenger levels to changes in cellular transcription. Several recent studies suggest that CREB and related transcription factors regulate gene expression necessary for neuronal plasticity and LTM. However, the role of CREB, within defined mammalian brain structures, in mediating the cellular events underlying LTM formation has not been investigated. We examined whether CREB-mediated transcription within the dorsal hippocampus is critical to LTM consolidation of water maze spatial training, which is known to depend on dorsal hippocampal function. Pretraining infusions of antisense oligodeoxynucleotides (ODN) directed against CREB mRNA were used to disrupt hippocampal CREB protein levels in adult rats. Control groups received pretraining infusions of ODN of the same base composition but in a randomized order (scrambled ODN) or buffer. Task acquisition and memory up to 4 h (i.e., short term memory) were similar in CREB antisense ODN and control groups. In contrast, CREB antisense ODN-infused rats exhibited significantly impaired memory 48 h later (i.e., LTM). Moreover, administration of antisense ODN 1 day after training did not affect subsequent retention performance. These findings provide the first evidence that CREB-mediated transcription is integral to hippocampal-dependent memory consolidation processes.
... Adding to doubts over the specificity of protein synthesis inhibitors on translation is the fact that they are all significantly toxic at levels needed to cause amnesia. Yet, the antibiotics puromycin, anisomycin, ementine, cycloheximide and acetoxycycloheximide continue to be widely used to inhibit translation in vivo (Davis & Squire, 1984;Stork & Welzl, 1999;Uphouse, MacInnes, & Schlesinger, 1974). The following section summarizes some of the issues surrounding mechanisms of translation inhibition and toxicity. ...
A major component of consolidation theory holds that protein synthesis is required to produce the synaptic modification needed for long-term memory storage. Protein synthesis inhibitors have played a pivotal role in the development of this theory. However, these commonly used drugs have unintended effects that have prompted some to reevaluate the role of protein synthesis in memory consolidation. Here we review the role of protein synthesis in memory formation as proposed by consolidation theory calling special attention to the controversy involving the non-specific effects of a group of protein synthesis inhibitors commonly used to study memory formation in vivo. We argue that molecular and genetic approaches that were subsequently applied to the problem of memory formation confirm the results of less selective pharmacological studies. Thus, to a certain extent, the debate over the role of protein synthesis in memory based on interpretational difficulties inherent to the use of protein synthesis inhibitors may be somewhat moot. We conclude by presenting avenues of research we believe will best provide answers to both long-standing and more recent questions facing field of learning and memory.
Tested approach choices in genetically and environmentally manipulated quail chicks with pairs of stimuli identical in size and luminance but different in color, flicker, or both color and flicker. Data indicated comparable flicker and vastly different color preferences in Ss that were bidirectionally selected for color choices. In the choices between composite stimuli, flicker effects dominated over color effects in genetic controls, and color effects over flicker effects in selected Ss. Imprinting to colors modified color preferences, but imprinting to white or colored flicker did not change, or only marginally changed, flicker preferences. Flicker in testing stimuli, however, influenced the phenotypic expression of acquired color preferences. Implications concerning the nature of constitutional biases and constitution–environment interactions in early perception and perceptual learning are discussed. (12 ref)
The most widely accepted view of memory in the brain holds that synapses are the storage sites of memory, and that memories are formed through associative modification of synapses. This view has been challenged on conceptual and empirical grounds. As an alternative, it has been proposed that molecules within the cell body are the storage sites of memory, and that memories are formed through biochemical operations on these molecules. This paper proposes a synthesis of these two views, grounded in a computational model of memory. Synapses are conceived as storage sites for the parameters of an approximate posterior probability distribution over latent causes. Intracellular molecules are conceived as storage sites for the parameters of a generative model. The model stipulates how these two components work together as part of an integrated algorithm for learning and inference.
This chapter provides an overview of macromolecules and behavior. Much of the work on macromolecules has been associated with studies of learning and memory. The chapter presents two principal approaches to the relationships between molecules and behavior—correlative and the interventive. In the correlative approach, the aim is to detect a particular molecular change that occurs during the behavior and show that the change and the behavior cannot be separated. The interventive approach requires that interference with the particular molecular system should predictably affect the behavior. Inhibition of the process should impair or prevent the behavior, whereas facilitation may increase it. Most researchers have assumed that, at the very least, combinations of these two approaches are necessary to establish specific associations between molecular changes and behavior.
The sleep-wake cycle and multiple-unit activity following the administration of various doses of protein synthesis inhibitors was studied in unrestrained cats. Special care was taken to analyze the effects of those drugs on phasic and tonic rapid eye movement (REM) sleep periods. It was observed that protein synthesis inhibitors decreased specifically total REM sleep time, at the expense of wakefulness, without altering slow-wave sleep time. It was further noted that the reduction in REM sleep was the result of a decrease in the frequency of REM periods, rather than in the duration of each individual period. In addition, protein synthesis inhibitors decreased phasic REM sleep and notably prolonged tonic REM periods. Moreover the phasic bursts of multiple-unit activity normally seen during REM sleep in control recordings were practically absent during REM sleep after the administration of protein synthesis inhibitors. It is suggested that protein molecules may participate in the mechanisms which trigger REM sleep.
The activities of RNA-dependent DNA polymerase and DNA-dependent DNA polymerase were measured in hippocampus of fast and slow learning Wistar rats. The RNA-dependent DNA polymerase activity in the hippocampus of fast learning rats exceeds two-fold that in the slow learning ones, while the rates of the DNA-dependent DNA polymerase activities are similar. A significant increase in RNA-dependent DNA polymerase only was found in the hippocampus of rats 20 min after training for the conditioned food response before the trace consolidation registered 40 min after the training session. The data obtained are consistent with the suggestion that reverse transcription plays an important role in memory consolidation.
Cycloheximide (CHX:1, 10 or 20 microg) was injected via indwelling cannulas into various regions of the rat brain and its effects on passive avoidance training were studied. Rats with 10 or 20 microg of CHX injected into the amygdala immediately after the training footshock exhibited amnesia for the learning experience when tested after 24 h. In contrast, animals injected with 20 microg of CHX at a site either in the internal capsule only 2 mm above the amygdaloid injection site or in the frontal cortex showed no retention deficit when tested after 24 h. A quantitative examination of protein synthesis in brain halves 30 min after unilateral injection of 20 microg of CHX into the amygdala demonstrated that total protein synthesis was inhibited by less than 10%. Autoradiographic studies revealed that this inhibition resulted from a profound, highly localized inhibition of protein synthesis in areas immediately adjacent to the cannula. A comparison of the regional patterns of protein synthesis inhibition caused by injection of CHX into either the amygdala or internal capsule suggested that CHX might produce amnesia by virtue of its localized effect on the amygdala. Control experiments revealed that injection of 20 microg CHX into the amygdala had no effect on short-term retention, or short-term performance. Injection of 20 microg of CHX into the amygdala 12 h after the footshock had no effect on long-term retention. The observed impairment of retention was shown to be dose-dependent as injection of 1 microg of CHX into the amygdala was without effect. In addition, it was demonstrated that the CHX-induced amnesia did not result from induction of local seizure activity. These data show that localized injections of small amounts of CHX into the amygdala can produce deficient memory of a training experience even though total brain protein synthesis is only slightly inhibited.
The neurochemical basis of memory has been approached experimentally in four different ways: the bioassay; the interventive approach; the interventive-correlative approach; and the correlative approach. These approaches are fundamentally similar to those used for the study of the chemistry of any behavior. Each of these approaches has serious limitations, and significant progress has only been made by combinations of more than one. I shall discuss each of these approaches in turn.
The present investigation sought to determine the effects of Anisomycin (A), Chloramphenicol (ChA), Vincristine (V), and Penicilline G on the sleep-wake cycle of rats. It was found that both high and low doses of anisomycin decreased rapid eye movement (REM) sleep, while only high doses of ChA and V produced such a decrease. Slow wave sleep (SWS) was unaffected by these drugs. Penicilline G, on the other hand, had no effect on the sleep-wake cycle. It was further shown that the reduction of REM sleep was the result of a decrease in the number of REM periods rather than in the duration of each individual period. These results suggest that protein synthesis may participate in the mechanisms that trigger REM sleep.
In this study adult Fischer, inbred rats experienced (1) training to avoid footshock (2) unavoidable footshock or (3) no training or footshock. Each animal was sacrificed at one of several time points (1-60 hours) following experience. Brain chromatin was extracted and used as template for RNA synthesis in vitro. Both groups which received the novel experience demonstrated greater template activity than the unshocked, untrained groups. This effect was brief. The two groups which received the experience did not differ from each other. These results suggest that a brief, novel experience can temporarily alter the transcriptional activity of brain chromatin.
Whole brain chromatin from enriched, isolated, or standard colony rats was measured for its capacity to support RNA synthesis in vitro. Chromatin from rats environmentally enriched from 28 to 60 days of age supported less RNA synthesis than did chromatin from the corresponding isolated and standard colony animals. However, when the differential rearing was maintained from 28 to 88 days of age, no rearing-related differences in chromatin-supported RNA synthesis were observed. These results are discussed in terms of environmental modulation of brain development.
— We have measured the effect of small variations in extracellular potassium concentrations ([K+]) upon the incorporation of radioactively labelled amino acid into the protein of the isolated guinea-pig hippocampal slice. The slice is super-perfused with glucose fortified buffer and maintains an ATP concentration of 33–36 nmol/mg protein and incorporates lysine into protein at a rate of 0.82 pmol/(ig protein/h. Within the range of extracellular K+ from 1.3 to 8.1 mil the change in the rate of lysine incorporation into protein is proportional to the logarithm of the extracellular K+ concentration. Incorporation increases by about 100% over this range. Measurements of the specific activity of the presumed intracellular amino acid pool indicate that the effect of changes in extracellular [K+] is to alter the rate of protein synthesis rather than alter the availability of radioactively labelled precursor. Altering extracellular [K+] does not affect tissue levels of ATP or creatine phosphate, indicating that the effect on amino acid incorporation does not result from an effect upon energy metabolism. It is suggested that this effect of extracellular [K.+] may be a means by which changes in cerebral electrical activity lead to changes in the rate of protein synthesis in brain.
Electroconvulsive shock or frontal cortex stimulation administered to rats at 5 but not at 180 minutes after an initial administration of morphine sulfate disrupted the development of one-trial tolerance to the analgesic effects of morphine sulfate. It is suggested that development of tolerance may be mediated by cellular mechanisms and memory processes similar to those thought to underlie conventional learning.
The question of learning from materials presented during sleep has been answered positively by Soviet studies and negatively by Western studies. However, procedural differences among studies have been confounded with the absence of an established criterion for sleep. The present paper reviews 11 studies in sleep learning for the potential practical value of sleep-assisted instruction (SAI). A strategy of optimizing compatibility between learning and sleep variables to support SAI is proposed within the context of both wake and sleep research on attention, perception, and memory. Age, sex, health, wake learning capacity, and suggestibility are important moderating variables in SAI. The individual's motivation and set, meaningful-relevant learning material, activation of low-voltage EEG sleep patterns and coordinated wake learning with extended training are tentatively deduced as necessary conditions in applied SAI. (111/2 p ref) (PsycINFO Database Record (c) 2012 APA, all rights reserved)
Myelencephalic grass frogs were trained to elevate the right forelimb to avoid a shock plate at several times in relation to administration of 3H-leucine and sacrifice. Total radioactivity (excluding 3H2O) and radioactivity in the soluble pool were significantly greater in the trained animals. The differences between trained and yoked animals decreased with increasing time, up to 30 min after training. The opposite trend was apparent in TCA insoluble material; differences between trained and yoked animals increased with increasing time after training. Similar experiments with 3H-inulin provide evidence that the effects were not due to generalized permeability increases or circulatory alterations.
Hybridization to unique DNA by RNA from brain or liver of rats given varying degrees of experience was investigated. RNA from brain of environmentally enriched rats hybridized to more unique DNA than did brain RNA from nonenriched controls. No significant differences were observed with liver RNA. This provides preliminary evidence for an increased transcription of the unique sequences of DNA in the enriched animals. The technique used has great promise for the investigation of experientially-induced alterations in gene expression.
The hypothesis that two qualitatively different stages of cerebral protein synthesis (PS) are required for the formation of long-term memory (LTM) for an active-avoidance task was investigated in rats. Cytoplasmic PS was inhibited with anisomycin (ANI-5.0 mg subcutaneously). When ANI was injected at 15 min pre- and 30 min posttraining, so that cerebral PS was inhibited by 90% for 2 hours starting just before training. LTM formation was prevented. When ANI was given after training, it was not effective. Mitochondrial PS was inhibited with chloramphenicol (CAP-1.5 mg intracisternally). Inhibition occurred 40 min after the injection. CAP interfered with LTM formation only when injected between 15 and 55 min after training. From these data it was concluded that two stages of PS are required for the formation of LTM. The first one takes place in the cytoplasm, starts with the commencement of training and is independent of newly synthesized mRNA. The second stage takes place in mitochondria starting approximately 25 min after training and is dependent upon newly formed mRNA.
The possibility that the amnesia induced by protein synthesis inhibition is state dependent was investigated. Chicks injected with the protein synthesis inhibitor anisomycin and then trained in a single-trial passive avoidance learning task showed no recall for the task when tested 6 h later in the absence of the drug. If, however, the same chicks were subsequently retested 30 min after a second administration of the drug they demonstrated clear recall for the task. Control groups showed that this effect was not the result of the administration of anisomycin per se but was due to state-dependent recall. Quantitative morphological characterisation of synapses in a region of the chick forebrain (the intermediate part of the medial hyperstriatum ventrale (IMHV) previously shown to be involved in passive avoidance learning was performed. The characteristic increase in the length of the postsynaptic density in the left IMHV was only evident in chicks killed after the behavioural test in which they had demonstrated recall. No synaptic changes were observed in chicks in which state-dependent recall had been demonstrated in a previous test but which were killed after a test in which they appeared amnestic. These results suggest that a memory trace may be established even in the absence of protein synthesis but that this trace may not normally be accessible. It is also suggested that the synaptic changes observed following learning may be dependent on some aspect of the recall phenomenon.
The effect of the protein synthesis inhibitor anisomycin on the structural changes associated with passive avoidance learning in the chick was investigated. Chicks were trained when they were 24 h old by allowing them to peck at a shiny bead coated with either water or the aversive-tasting substance methylanthranilate (MeA). Chicks which peck the MeA-coated bead will on subsequent testing avoid pecking a similar, but water-coated bead. Behavioural testing was carried out 12 h after training and immediately afterwards the chicks were killed and their brains prepared for electron microscopy. A specific region of the forebrain, the intermediate and medial part of the hyperstriatum ventrale (IMHV) was investigated. When the IMHV of the MeA trained chicks was compared with that of water-trained controls structural changes of the synapse were detected. These changes involved a significant increase in the mean length of the postsynaptic density (LPSD) of symmetrical synapses in the left IMHV. Chicks injected with 0.8 mg of anisomycin 30 min before training with a MeA-coated bead showed aversion for the shiny bead when tested 12 h later. Electron microscopic analysis of the IMHV from these amnestic chicks showed no evidence for the change in LPSD demonstrated in the water-injected controls. These results are discussed in relation to the nature of the memory trace induced by training on a passive avoidance task.
Rats received intraventricular injections of whole brain ribonucleic acid (RNA) via a cannula over two consecutive days of training. Two tests were conducted 14 and 30 days later. Compared to saline controls, there was a significant treatment by test period interaction for errors. However, there was no reliable difference between groups in terms of errors or time. It is suggested that RNA injections do not reliably decrease the amount of time to complete a complex maze, but do produce an initial enhancement of performance in terms of errors.
Within the last two decades it has become evident that the brain exhibits a wide range of chemical and physiological responses to its sensory environment. Comparison of the brains of animals reared in relatively complex as opposed to deprived sensory environments has yielded significant insight into the nature of these effects, which are reviewed in this paper. Greater complexity of the sensory environment results in increased total cholinesterase and acetylcholinesterase enzyme activity, while other neurotransmitter related substances, the catecholamines, show more variable responses. RNA concentration is slightly greater as is DNA transcriptional activity, while protein precursor uptake shows a variety of regional and temporal patterns. In general, responses for most substances tend to show regional and temporal specificity with the largest effects most often in the occipital cortex. Electrophysiological measures have revealed shorter visual cortex evoked potential latencies and greater amounts of sleep in the complexity-reared subjects. The wide range of environmentally responsive parameters is consistent with an adaptive functioning of brain chemistry and physiology and with recent models in the physical sciences which view the universe as composed of dynamic webs of relationships rather than isolated independent units.
1.
Intrahippocampal injection of 5 μg serotonin into rats immediately after defensive conditioning leads to the development of retrograde amnesia. Injection of the same dose of serotonin inhibits incorporation of [3H]leucine into hippocampal proteins. An increase in the dose of serotonin injected to 10 μg has no effect on consolidation of conditioned reflexes once formed or on protein synthesis in the hippocampus.
2.
Addition of serotonin to the incubation medium containing surviving hippocampal slices leads to a decrease in the incorporation of [14C]leucine and an increase in the incorporation of [3H]fucose into total proteins of the hippocampal slices. The most profound inhibition of incorporation of [14C]leucine into hippocampal proteins is observed by the action of maximal and minimal serotonin concentrations.
3.
These results suggest that the participation of serotonin in the consolidation process is connected with its regulatory role in protein metabolism in the hippocampus.
Cycloheximide (CXM) was found to be a potent inhibitor of protein synthesis in the nervous ganglia of praying mantis (Stagmatoptera biocellata) over a wide concentration range (from 1.75 μg/animal to 6.6 μg/animal) and for a substantial period after injection (more than 80% inhibition for a period of 2–3 hours after injection). When mantises were trained not to attack a mobile star, memory appreared to be disrupted by an injection of CXM administered shortly after training, but after two hours it becomes irressponsive to this agent. This finding is consistent with previous results [5] that reported a connection between formation of a memory trace and the apprearance of certain peptides of low molecular weight when mamtises were trained not to attack a mobile star. The concepts of “short term memory” and “long term memory” are reexamined in light of this and other works with praying mantis [9].
Using the conditioned feeding reflex model, a polymorphism for the rate of formation of this response was identified in a population of laboratory animals. Selection for high and low rate of the formation of this reflex resulted in significant differences in this character between two strains by the second generation. These differences were maintained in subsequent generations. Heterogeneity for the rate of the formation of conditioned response in the population is shown to be genetically determined. The RNA-dependent DNA-polymerase activity in the hippocampus of fast-learning rats exceeds twofold that in slow-learning rats, while the rates of the DNA-dependent DNA-polymerase activities are similar. A significant increase in RNA-dependent DNA-polymerase only was found in the hippocampus of rats 20 min after training for the conditioned food response before the trace consolidation registered 40 min after the training session.
Bitemporal intracerebral injections of puromycin in mice suppress indefinitely expression of memory of avoidance-discrimination learning. Ultrastructural studies of the entorhinal cortex of puromycin-treated mice revealed the following: (a) Abnormalities were not observed in presynaptic terminals and synaptic clefts; many postsynaptic dendrites or somas contained swollen mitochondria. (b) Dispersion of polyribosomes into single units or condensation of ribosomes into irregular aggregates with loss of "distinctiveness" was noted in a few neurons 7–27 hr after puromycin treatment. (c) Cytoplasmic aggregates of granular or amorphous material were frequently noted within otherwise normal neuronal perikarya. (d) Mitochondria in many neuronal perikarya and dendrites were swollen. Mitochondria in axons, presynaptic terminals, and glial cells were unaltered. The relationships between these lesions and the effect of puromycin on protein synthesis and memory are examined. It is suggested that the disaggregation of polysomes is too limited to explain the effect of puromycin on memory. Special emphasis is given to the swelling of mitochondria. The possible mechanisms and the significance of this lesion are discussed.
A role for macromolecules in the storage of behavioral information has long been championed.(1–5) This postulated role remains at present highly inferential, even though many investigators have attempted to confirm or to disprove it. Experiments have thus far produced too few constraints for those who would theorize on the biological basis of learning and memory. While we are clearly at a very speculative stage, the ranks of experimenters are growing, and with them the firm hope that an assault on this least-known aspect of living systems is underway.
In the last decade, studies of brain metabolism during simple behavioral experiences of short duration have provided evidence of measurable phenomena, particularly as regards changes in RNA metabolism. This area of investigation bears a logical resemblance to the study of electrical responses of brain during behavior, and can therefore be christened as chemoencephalography. The animal is subjected to a behavioral experience, sacrificed and the metabolic pattern of the brain at that point in time is analyzed. The intent is to discover metabolic events which may be specific to the experience and localized regionally in brain. These metabolic events may not necessarily be correlated in any simple way with the electrophysiology also occurring at the same time and may thus reflect neural processes different from those detectable by electrical signals.
One of the oldest and most intriguing problems in neurobiology is the elucidation of brain function. It has been apparent for a long time that the brain is essentially an electric organ which is capable of accrual, retention, and retrieval of information. In the past quarter of a century neurophysiologists made extensive studies of evoked electroencephalograms (EEG’s) from the brain under a variety of conditions, but none of these observations revealed any clues as to the mechanism underlying brain function. Ablation experiments did show that the information storage was not uniquely localized(1) and that, excluding evoked potentials due to arousal, no correlations were noted in EEG’s with conditioning or learning.
Because of its participation in so many important cellular regulatory processes, it is likely that cerebral protein synthesis is required for the cellular alterations which characterize “long-term” memory. a) The proteins whose synthesis might play a role in facilitating the formation of new functional synaptic relationships in the brain could fall into a variety of classes. They could be enzymes which regulate the synthesis of neurotransmitters, receptor proteins, structural proteins, or proteins which direct some specific type of intercellular recognition. Since little is known about all such proteins in the brain, and since the possible candidates which could be critical for the formation of new functional synapses are so great even within each of these classes, a “shot-gun” approach has been taken as a first step in implicating cerebral protein synthesis in the process of formation of long-term memory. This approach is based on the use of inhibitors of cerebral protein synthesis, particularly the cycloheximide class, with which we will be primarily concerned. Although it has not as yet helped to identify the existence of specific proteins or classes of proteins whose synthesis is critical for long-term memory storage, it has been useful in supporting the hypothesis that cerebral protein synthesis is indeed required for this process, and has provided some indication of the time relative to training when this process occurs. Other data which support this hypothesis are reviewed by Agranoff in Chapter 26.
A specific protein of the nervous system, S-100, appears to exist in multiple forms when subjected to acrylamide gel electrophoresis in the presence of either Ca 2+ or 8 M urea, but only in a single form in their absence. Calcium ion causes a limited conformational change in S-100 leading to a more unfolded structure in a region probably containing its single tryptophan, several of its tyrosine and phenylalanine, and two of its three cysteine residues. These effects are not seen with Mg 2+. Monovalent cations antagonize the effect of Ca 2+, K + being more effective than Na + . At physiological levels of K + and Na + the effects of Ca 2+ occur also in its physiological range.
It has often been assumed that memory depends upon the total action of the brain rather than upon some specialized intracerebral neuron mechanism. There is recent evidence, however, in support of the view that the recording of experience is localizable in the same sense that sensory functions and speech functions are localizable. Obviously, none of these subdivisions is separable from the work of the brain as a whole.
The following study shows that the capacity to record the daily current of conscious experience may be lost when there is bilateral destruction of a man's hippocampus and hippocampal gyrus. Functional paralysis of this recording mechanism does not, however, interfere with the patient's intellectual performance in other psychological tests not dependent on recent memory. Skills, language, and all those things which have already been learned are not lost.
This inability to record new experience is not found in cases of strictly unilateral
The functions of the temporal lobe, and its preponderant role in the integrative aspects of social behavior, are well known (Bucy and Klüver,13 1939) and have been the subject of recent reviews (Adey,2 1959; Baldwin and Bailey,8 1958). To some extent, physiological dissection of the picture of "psychic blindness" characterizing the complete bilateral temporal lobectomy has been possible with more limited resections, with aspects of hypersexuality, loss of memory, taming, and exaggerated rage responses, each reported as the dominant aspect of a variety of limited resections in man and animals (Adey,1 1958; Bard and Mountcastle,10 1948; Green, Clemente, and de Groot,24 1958; Schreiner and Kling,45 1953; Scoville and Milner,46 1957).
Historically, the limbic lobe, set as a ring of cortex at the medial margin of the hemisphere, has been described as a somatic integrating center concerned with the integration of somatic influxes
A DNA-RNA successive competition experiment in which DNA is hybridized first with brain RNA from rats subjected to forced motor activity and then with brain RNA from shock avoidance trained rats suggests that unique RNA species are synthesized during this latter task.
A DNA-RNA successive competition experiment in which DNA is hybridized first with RNA from nonbehaving rats and then with RNA from shock avoidance trained rats suggests that unique RNA species are synthesized during this task.
Sprague-Dawley albino rats were given maze exploration and escape training in a straight-runway maze. Ss in Control Group 1 were given only maze exploration, and those in Control Group 2 had no maze experiences. Half the Ss from each of the groups were sacrificed immediately, and the remainder were sacrificed 2 h after completing training. The brains were analyzed for protein, RNA, and total nitrogen. A significant increase in brain protein was found for the 2-h sacrifice escape condition. Moreover, estimates of NPN (nonprotein nitrogen) were significantly lower in this experimental condition.
Ninety-six goldfish, divided into six groups, were trained in a classical conditioning experiment in a single hour-long session. Three of the groups were injected intracranially over the brain with puromycin 0, 24, and 48 h after training; the three control groups were injected with saline. Tested 3 days after training, the puromycin groups all showed a marked retention deficit compared to the controls. In another experiment, two groups of 16 fish were given 4 days of training and were then injected immediately with puromycin or saline. There were indications of interference in this experiment also. These experiments are at variance with recent hypotheses that puromycin interferes with the consolidation stage of memory.
Memory storage is one of the principal functions of higher nervous systems. Yet, until recently it has eluded intensive biological investigation. The reason for this is quite apparent. The storage of memory in the nervous system is believed to be mediated by an alteration in the functional relationship among large numbers of neurons. Lack of knowledge about general processes of cellular regulation and the control of intercellular relationships would preclude serious study of mechanisms for the formation of new functional interneuronal relationships. Recent advances in understanding of biological regulatory processes have, however, made possible the initiation of attempts to understand the mechanisms by which memory is stored in the brain. An important finding of this research is that general biological regulatory mechanisms are frequently mediated by the synthesis of new protein. In the course of such studies, a number of inhibitors of protein or ribonucleic acid synthesis were discovered. Studies with these inhibitors helped establish the critical role of protein synthesis in cellular regulation. Because it could be readily imagined that new functional interneuronal relationships could also be established by protein synthesis, these inhibitors have been employed for studies of the possible role of cerebral protein synthesis in memory storage. There is now considerable evidence that inhibitors of cerebral protein synthesis interfere with memory storage. Because all such experiments involve the introduction of a complex reagent with multiple effects and the measurement of alterations in a complex process, the interpretation of the results are difficult, particularly for the casual reader in this field. This chapter focuses on the important variables that influence the types of results that have been found.
When rats trained on a continuous avoidance schedule with an R-S interval of 30 sec were changed to a shorter R-S interval of 15 sec, the response rate increased immediately and then decreased to a new stable level as the new temporal discrimination was acquired. In contrast animals injected into the cerebral ventricles with 8-azaguanine immediately before the R-S interval was altered showed a delayed change in response rate and this delay was correlated with the concentration of the dose injected. This and other supporting evidence are consistent with the hypothesis that inhibition of protein synthesis in the hippocampus resulted in the impairment of learning and in cellular changes observed histologically two weeks thereafter.
It is apparent that antibiotics are useful in differentiating different stages in the formation of memory. Puromycin gave the first indication that very early memory can be established and survive, for a short period at least, in spite of inhibition of protein synthesis. Injection of actinomycin D indicates that RNA synthesis is not essential during this early stage. The duration of this early period seems to vary with the inhibiting agent; with puromycin memory was notably degraded in less than an hour, but with actinomycin D or with acetoxycycloheximide it persisted for several hours or more.
The fixation or consolidation of memory involves whatever processes give permanence to memory. These processes are disrupted when electroconvulsive shock is administered shortly after a learning experience, presumably because of the interference with organized patterns of neuronal electrical activity. Memory acquired in the presence of antibiotics appears to proceed to a stage beyond that based purely on electrical activity because the memory persists beyond the period usually reported as sensitive to electroconvulsive shock. Further work should show whether this stage is truly insensitive to electroconvulsive shock. Memory acquired in the presence of puromycin does not seem to achieve any durable consolidation. In contrast, memory acquired in the presence of or immediately before injection of acetoxycycloheximide does appear to initiate the later stages of consolidation, as permanent memory. reappears some days after the initial stages have become ineffective in controlling performance.
Finally, puromycin has provided evidence of the enlarged area of the neocortex which participates as memory matures. Puromycin also indicates the time required for this maturation process.
Since antibiotics have also been useful in studying learning and memory in goldfish, this approach seems to have general applicability in defining various stages in the process of memory formation.
The initial purpose of these investigations was to determine the molecular basis of the "memory trace." This goal still remains distant, although there are some indications that protein synthesizing systems are involved. This objective, though of enormous interest, is to be regarded as only a necessary first step. Whether new proteins or some other molecules cause the changes in synapses thought to underlie memory, this knowledge of itself will contribute only a beginning to our understanding of the events which account for the functioning of the brain. A determination of the composition of computer components would provide very little information towards unraveling their function.
As the experiments proceeded, however, information of a more general nature was being obtained. The identification of different stages of consolidation show how injections of antibiotics can supplement electroconvulsive shock as a way of disrupting the establishment of memory and how it can supplement ablation in destroying memory already laid down in a permanent mode. Applied to larger animals the localization of various regions sensitive or insensitive to the action of the drugs should become more definitive. We hope that such experiments will contribute increasingly to the general problem of brain function.
Changes in brain through experience, demanded by learning theories, are found in experiments with rats.
CYCLOHEXIMIDE (`Actidione'), an antifungal antibiotic, isolated from Streptomyces griseus, was reported by Kerridge1 to inhibit protein synthesis in Saccharomyces carlsbergenesis. These observations have been substantiated by other workers2,4. Recent investigations indicate that the changes produced by cycloheximide in intermediate metabolism, such as amino-acid, organic acid, adenosine triphosphate (ATP), and other nucleotide production, is not responsible for the inhibition of protein synthesis3,4. The data presented here will show that incorporation of leucine labelled with carbon-14 into protein in a cell-free system from Saccharomyces pastorianus is inhibited by low concentrations of cycloheximide.
Administration of cycloheximide (actidione) to male mice of the C3H/HeJ strain inhibited the in vivo incorporation of L-[U-14C]leucine into liver proteins. This inhibition was approximately 20 % with 0.2 mg of cycloheximide/100 g of body weight and 62-65 % with 0.4 mg/100 mg of body weight. A maximum inhibition of 94-96% was reached with 8-10 mg of cycloheximide/100 g of body weight. The inhibitory effect of cycloheximide was observed almost immediately after injection, reached a maximum after 1-2 hr, and was still pronounced after 24 hr. Experiments with a cell-free amino acid incorporating system containing normal mouse liver supernatant demonstrated that liver ribosomal preparations isolated from cycloheximide-treated animals or homogenates performed as well as those isolated in the absence of this antibiotic. In contrast, a decreased capacity of normal liver ribosomal prepara-tions to incorporate L-[U-14C]leucine was noted when the liver supernatant utilized in the incorporation assay was prepared from a cycloheximide-treated animal. The breakdown of liver polysomes following administration of actinomycin or ethionine was inhibited by injection of cycloheximide. Similarly, the reassembly of polysomes produced by reversing the effect of ethionine by methionine and adenosine triphosphate was inhibited in the presence of cycloheximide.
This study is another in a series of investigations attempting to define the changes in the brain as a consequence of environmental manipulation. Cortical depth and neuron size measurements were taken on brains of rats from enriched (ECT) and impoverished (IC) environments.
Celloidin embedded, 10 μ, transverse sections of formalin fixed rat brains were stained wiith thionin. Ten representative sections from each brain were taken, utilizing subcortical landmarks to insure uniform sampling. Cortical depths were measured on microslide projected drawings beginning at the midline and proceeding laterally. The number of readings per hemisphere ranged from 41 to 46. The greatest increases in depth were found in the sections from the posterior commissure landmark. At this position the increases in four regions, proceeding medially to laterally, were as follows: A, 5.2% (p < 0.001); B, 6.3% (p < 0.001); C, 3.2% (p < 0.01); D, 1.6% (NS).
Perikarya and nuclear size measurements were taken with a planimeter on microfilm projected outlines. The cortex was divided into upper, middle and lower levels, and within each third, cell size increases were noted, with the outer third showing the greatest increases. The ECT perikarya were greater than the IC by 17.7% (p < 0.001) in the upper level, by 7.2% (p < 0.05) in the middle level, and by 11.6% (p < 0.01) in the lower level.
The first part of this paper deals with rat brain anatomy as affected by variations of starting age and of duration of exposure to enriched (complex) or impoverished (restricted) environmental conditions. Cerebral cortical depth measurements and weights of brain samples are compared. The second part of this report is concerned with an attempt to establish the extent to which the enriched condition or the improverished condition is responsible for the histological brain changes; this is done by comparison with brains from animals in the standard colony environment. The results indicate that both larger and more extensive cortical depth effects are found in rats exposed for 30 days rather than 80 days to their respective environments. The effects of enrichment are more prominent in the dorsal cortical segments, whereas the effects of impoverishment are more evident in the lateral cortical segments. More precise localization of cortical changes is obtained by measuring the depths at many segments than by taking the weights of relatively large blocks of tissue.
We have previously reported anatomical and chemical changes in the cerebral cortex of rats living in an enriched, stimulating environment. The present study includes additional histological measures such as more extensive depth measures, differential cell counts, and cell size measurements.
Celloidin-embedded, thionin-stained sections of the visual cortex from environmentally enriched rats and their impoverished littermates were measured. Two methods for calculating cortical depths were used: one, with an ocular micrometer taking eight measures lateral to the elevation in the corpus callosum on each hemisphere; two, on enlarged photographs taking 51 measurements 2 mm apart on each hemisphere. With the ocular micrometer method the cortical depth of the enriched brains was 6.4% greater than the depth of the impoverished cortex. On the enlarged photographs the more medial depths in the enriched rats showed greater increases than the lateral depths.
Overlapping photomicrographs were made of a medial cortical area 1.00 mm by 0.75 mm on each transverse section and were combined into one composite picture for each animal. Differential cell counts from the composite pictures were determined independently by two technicians. Results indicate a 14% (p<0.01) increase in glia among the enriched rats. No significant differences between enriched and impoverished groups were found in the perikarya and nuclear circumferences as measured with a planimeter from camera lucida drawings.
Cycloheximide, injected subcutaneously after training in a passive avoidance task resulted in impaired performances on the retest given 7 days later. A gradient was observed for this effect, injections given immediately after training producing most impairment and injections given 2 h after training producing none.
The utilization of systemically injected leucine-H3 by nerve cells was investigated autoradiographically in exercised and unexercised rats. Handled rats reared in an enriched environment were run daily for a long period in a motor-driven exercise wheel to adapt them to forced exercise. On the day of administration of the radiochemical the “exercised” animals were forced to run for 1 hr prior and 1 hr after the injection, whereas the “unexercised” animals rested before and after injection. Microdensitometric evaluation of autoradiographic grain density over single nerve cells in several brain regions indicated that there were no significant differences in the utilization of the radiochemical by proteins of the brain in the two groups of animals, though there was a slight trend of increased incorporation of leucine-H3 in the exercised group of animals. The results suggest that the presumed functional activation of the brain by this sensori-motor task need not lead to enhanced protein metabolism if the stress produced by forced exercise is reduced or eliminated by pre-adaptation to the task.
Since stimulation of RNA synthesis in animals usually involves a hormonal intermediate it was felt that hormones might be involved in the observed increases in incorporation of uridine into polysomes and RNA of brain during avoidance training previously reported from this laboratory. Both hypophysectomized rats and ovariectomized mice learned the avoidance response normally and exhibited increases in incorporation of radioactive precursors into brain polysomes when compared with yoked or quiet animals. Intact female mice did not perform as well as ovariectomized mice and showed random variations in phosphate incorporation into brain polysomes. It is concluded that the adrenal, the pituitary, the testis and the ovary are not necessary for avoidance conditioning or for the increased incorporation of radioactive precursors into RNA that accompanies it.
Injection of actinomycin D into the hippocampal area of rats impaired their performance of a previously learned position discrimination task in a T-maze. Three experiments were conducted to test whether the impairment was due to retroactive interference with some aspect of memory or due to proactive interference with the performance at the time of retention test. When the animals were tested within a few days after actinomycin injection, their performance of the task was slightly impaired regardless of when the task had been learned originally; when they were tested more than four days after injection, the performance was severely impaired, again regardless of the time of original learning. In contrast to the findings in the goldfish and in the mouse, the actinomycin effect in the rat appears to be proactive only, unrelated to memory.
Bowman and Strobel [5] reported increased incorporation of tritiated cytidine into hippocampal RNA of rats learning 60 min of repeated spatial reversals for water reinforcement in an automated Y-maze. The present studies replicated this effect 3 times using intravenous (IV) injections of tritiated cytidine, but failed to observe such an effect in 3 other replications using intracranial (IC) injections of tritiated cytidine. Various control conditions appear to rule out injection trauma as a reason for the lauter failure. The observation of equal precursor distribution across brain regions following IV injections and of unequal distribution following IC injections, coupled with the autoradiographic observations of Altman and Chorover [1] of steep hypothalamic distribution gradients of IC injected nucleoside in the cat, indicate that regional metabolic measurements following IC injections of precursors do not equally assess the contributions of all brain cells and can lead to serious quantitative errors. This point needs reemphasis in view of the widespread use of IC injections in psychoneurochemical research. Concerning the possibility of stress or corticosterone effect on the observed hippocampal RNA effect, there were no differences found in plasma corticosterone concentrations between the learning and control rats at sacrifice.
Cycloheximide-treated mice and saline controls were given one-trial training in a black and white, step-through, passive avoidance box. The following parameters were shown to affect the degree of amnesia in cycloheximide-treated animals: (a) shock intensity; (b) shock duration; (c) original latency to enter the shock compartment; and, (d) retention interval (periods used were 24 hr and 1, 2, and 3 weeks). When those parameters are measured and controlled, highly consistent amnestic effects can be obtained. In the present experiment, certain groups showed over 80% amnesia while the controls showed less than 2% amnesia. Amnesia could best be obtained in passive avoidance when animals were trained to just under the asymptote of the acquisition curve. Measures of inhibition of protein synthesis by cycloheximide showed differences among 5 strains, but there were no significant individual differences within a strain.
The utilization of systemically injected leucine-H3 by nerve cells was investigated autoradiographically in rats reared in a restricted and enriched environment. Microdensitometric evaluation of autoradiographic grain density over single nerve cells in several brain regions indicated a significant increase in the utilization of the radiochemical by proteins of the brain in the restricted group of animals. This finding supports the hypothesis that the presumed activation of neuronal circuits by chronic behavioral engagement, represented by the enriched environment, need not lead to enhanced protein metabolism; instead, it is the stress produced by handling and the injection procedure in the previously unhandled, restricted animals that leads under these conditions to increased utilization of the labeled precursor of proteins.
The utilization of systemically injected leucine-H3 by the brain was compared in visually-deprived rats and rats receiving prolonged training in a maze on a series of visual pattern-discrimination tasks. Microdensitometric evaluation of autoradiographic grain density over single nerve cells in a variety of visual and nonvisual brain regions indicated that there was no significant difference in the utilization of the radiochemical by proteins of the brain in the two groups of animals; though there was a trend of increased uptake in several visual and nonvisual structures in the visually-deprived animals. This result supports our previous conclusion that the presumed functional activation of the brain by behavioral engagement need not lead to increased protein metabolism, as measured by our technique. The slight increase in the utilization of leucine-H3 in the visually-deprived animals can be attributed to the greater stress produced by the injection procedure in these unhandled animals.
Fish given a single electroconvulsive shock or intracranial administration of puromycin, acetoxycycloheximide, or KCl immediately following shock avoidance training show amnesia on retraining days later. When the treatments are given 24 hr after training no amnesia develops. But amnesia can be obtained 24 hr after training with KCl, puromycin and acetoxycycloheximide, if the fish are replaced in the intertrial environment for a brief period just prior to injection. Implications of these results to the consolidation hypothesis of memory formation are discussed. Previously unreported results on the amnesic effects of intracranial KCl in goldfish are also described.
The release of [14C]amino acid-containing polypeptides from rabbit reticulocyte ribosomes by puromycin has been studied and compared to the release occurring in a hemoglobin synthesizing cell-free system. Unlike the more naturally occurring release of a completed peptide chain, the puromycin-produced release of polypeptide from the ribosome does not require an ATP-generating system or the supernatant fraction, and occurs maximally in 1 or 2 min. Thus, completion of a chain is apparently a requisite for the naturally occurring release, but not for puromycin release. The polypeptide released by puromycin is not hemoglobin but resembles the completed molecule in having N-terminal valine. [14C]Puromycin, synthesized by methylating the tyrosyl analog with [14C]diazomethane is bound by the soluble polypeptide released from the ribosome. Saturation of the binding site on the puromycin-released polypeptide occurs at a puromycin concentration equivalent to that producing maximal inhibition of [14C]amino acid incorporation in a cell-free system and maximal release of polypeptide from the ribosomes. One residue of puromycin is bound to the released polypeptide for every N-terminal valine or one residue per polypeptide chain. It is hypothesized that puromycin may displace the soluble RNA binding the polypeptide chain to the ribosome in its action in releasing the chain from the ribosome.
Instead of exposing rats to environmental complexity (EC) for 24 hr a day as in previous experiments, we exposed some groups to EC for either 2, 2, or hr a day. In five experiments, brain values of the various EC groups were compared with those of littermates kept continuously in the isolated condition (IC) for the duration of the experiment. A few hours a day of EC were found to produce significant changes in activities of acetylcholinesterase and cholinesterase and in weights of brain samples; these changes were quite similar to those produced by 24 hr a day. In Experiments I and II, hr and hr groups were in standard colony (SC) conditions when they were not in EC. Each of these experiments also included a 24-hr EC group, and the experiments lasted for eight weeks. Experiment III included a 2-hr EC group and an SC group; the main differences from IC were found to be due to EC and not to SC. In Experiments IV and V, the enriched-experience groups spent 2 hr a day in EC and 22 hr in IC, and the experiments lasted only one month. Two hr a day removal from IC to EC over a 30-day period produced significant cerebral effects.
