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The incorporation of uridine into brain RNA during short experiences
Since neurobiological approaches to the elucidation of the mechanisms of learning and memory usually require the use of animal behaviors (conditioned reflexes, operant conditioning, etc.) as an essential tool for experimentation, studies in this field have been confronted with great difficulties, especially in quantitative analysis. When the historical breakthrough in the field of genetics revealed the role of DNA as the bearer of genetic information, neurobiologists also began to search for similar mechanisms of information processing involving base sequences of RNA and protein as memory-bearing substances in nerve cells. Others tried to analyze metabolic changes, especially of proteins and amino acids, in the brain of animals after learning.
This chapter discusses the effects of chronic ethanol ingestion on the brain of rodents. In brain tissue, besides their role in cellular metabolism, proteins and ribonucleic acids (RNA) are thought to have distinctive functions. There is strong evidence that mnemonic processes are clearly impaired as a function of alcohol ingestion, though the precise nature of this effect still requires further elucidation. Animal studies have also demonstrated impairment in brain functions following chronic ethanol ingestion. In a study described in the chapter, adverse effects of chronic ethanolism were observed in the in vivo labeling of transfer RNA, mitochondrial RNA, and ribosomal RNA. Inhibition of mitochondrial RNA as a result of ethanol is of great interest as most of the intramitochondrial RNA is not nuclear in origin and, therefore, the effects observed on this organelle are independent of the effects demonstrated on the nuclear fraction. Long-term ethanol ingestion was shown to have a differential effect on the labeling of rRNA in two different populations of ribosomes. Labeling of free rRNA was found to be decreased under conditions where labeling of bound rRNA was increased.
The content of free lysine in the brains of mice increased significantly during an appetitive training in which the mice were trained to touch a bar in order to get sweetened milk. The free lysine level reached a maximum at 20-30 min of training, and returned to control levels at 60 min. The specific activity of free lysine was significantly lower in the brains of trained mice than in controls at 20 and 30 min after either subcutaneous or intracerebral administration of the isotopically labeled compound. Subcutaneously injected radioactive lysine disappeared more rapidly from the blood of trained mice than from the blood of control mice during the interval from 20 to 60 min after injection. The specific activities of brain nuclear proteins from trained mice were significantly greater than those of controls after 20 min or more of training. These protein differences were more marked when expressed as relative specific activities that were corrected for changes of specific activity of free lysine that occurred during training.
RNA synthesis in the phenolic fractions of nuclei and cytoplasm from higher structures of the rat brain under normal conditions, during training in defensive movements in a maze, and in an active control (irregular presentation of stimuli evoking defensive movements) was investigated by electrophoresis in polyacrylamide gel. Behavioral stimulation and irregular presentation of the same stimuli to the animals as during training, but not leading to the formation of defensive skills, activates synthesis of 18S, 28S, and higher forms of RNA in the phenolic extracts of the nuclei. An increase in the incorporation of labeled precursor in the 18S region was found for cytoplasmic RNA.
The effect of different learning schedules massed or distributed practice conditions (3 trials a day for 3 days), on water-filled multiple T-maze learning ability of 8-week-old SPF Wistar-Imamichi rats was investigated. Although the mean number of errors decreased day by day in both groups, the number of errors in a given day and the total number of errors in 3 days did not differ significantly between the two groups. A tendency toward a decrease in the number of errors was observed as the trials prodeeded in the group with distributed practice but not in the group with massed practice. The result suggests that a certain time period for rest after each trial is necessary to acquire the memory.
In order to specify the role of brain electrical activity in learning, two experiments were performed. In Experiment 1, effects of whole-body hypothermia were studied on electrocorticogram and spontaneous spike activity: those two disappeared at about 7°C (body temperature) and their evolution was very similar all along hypothermia. In Experiment 2, partial learning or retention in a Skinner box was followed by hypothermia to 3–4°C. Testing was conducted 24 hr later in extinction. Cooling resulted in deficit in partial learning group but not in retention group. In the two groups, the animals' level of activity and food consumption were not modified. These results support the view that hypothermia acts upon consolidation processes and the possible mechanisms are discussed.
The acquisition of an optical discrimination reaction in rats is not significantly influenced by intraventricular injection of the nucleotide monophosphates uridine monophosphate (UMP), cytidine monophosphate (CMP), adenosine monophosphate (AMP), guanosine monophosphate (GMP), or thymidine monophosphate (TMP). The extinction, however, is affected differently. A significant delay is found after 100 μg UMP or CMP; GMP was only effective after 200 μg, AMP was without effect, and TMP enhanced the extinction. The extinction of a different conditioned avoidance reaction was also delayed by UMP or CMP.
An injection of UMP, CMP, AMP or GMP increased the number of positive reactions during the first days of extinction.
It is concluded that an increased supply of pyrimidine-nucleotides is of significance for the consolidation of an acquired type of behaviour presumably through RNA-synthesis and its role in the protein synthesis in the nerve cells.
Glucose metabolism in the visual system of rat was studied after prolonged exposure to photic flash. Animals were unilaterally enucleated and local rates of glucose utilization were quantitively measured in optic centers on both sides of brain. We also measured the averaged visual evoked response and behavioral reactivity to photic flash. Prolonged stimulation resulted in a decrease in behavioral reactivity, a phenomenon known as habituation. After behavioral habituation had been observed there was an increase in the amplitude of VER and an increase in glucose utilization. Visual structures on both sides of brain responded in this way, but the relationship was not the same for all components of the system. This is probably determined by the number and types of retinofugal projections to the structures. We suggest that more cells are recruited into activity as the photic flash continues and that they eventually discharge in synchrony with the stimulus.
Amino acid incorporation into perikaryal and synaptosomal subcellular fractions of cortex and hippocampus was studied in rats trained to use the non-preferred paw in the retrieval of food.After 4 days of training, incorporation ofl-[3H]leucine into all subcellular fractions of hippocampus, except the perikaryal mitochondria, was significantly higher in trained than in control rats; the opposite was true in the perikaryal mitochondria. No differences between trained and control animals were observed in cortex after 4 days, or in hippocampus after 30 days of training. Experiments involving double labelling and electrophoresis indicate that the hippocampal changes at 4 days involve all the proteins of the various fractions, and are not restricted to one or a few protein species. Amino acid incorporationin vitro into isolated perikaryal mitochondria, and synaptosomal mitochondria and membranes, was the same in 4-day-trained and control rats.The data suggest that there is an increased metabolic demand in the hippocampi or rats trained for 4 days to transfer handedness.
An increase of radioactive phosphate incorporation into nonhistone acid-extractable protein occurs in the brain nuclei of naive mice or rats during 5 min of active one-way avoidance training. The increase is not triggered by motor activity, handling, or the experimental conditioning stimuli. An increase does occur, however, in the brains of previously trained animals when they perform the avoidance, and it occurs in rats trained on previous days when they are simply handled or placed in the training apparatus. This “reminder effect” does not occur in rats that have not been trained, but have repeatedly received only unavoidable footshock in the apparatus.
Stimuli such as water deprivation or salt loading which induce the secretion and synthesis of the neurohypophysial hormones also induce ‘activation’ of the glial cells (pituicytes) surrounding the neurosecretory axonal fibers and terminals in the neural lobe. It has been show that this is expressed in part by the ability of neural lobes from stimulated rats to shown an enhancedin vitro incorporation of [3H]uridine into total RNA extracted by alkaline hydrolysis. In order to characterize the newly synthesized RNAs, quantitative and reproducible methods were developed for the selective extraction and fractionation on glycerol gradients of microgram amounts of pulse-labeled RNA from neural tissues. These methods were applied in comparative studies on the RNA labeling patterns of cerebral cortex and of neural lobes pulsedin vivo andin vitro respectively with [3H]uridine and [32P]orthophosphate. In the former case, analysis of the RNA labeling profiles at various time periods, indicate that in the brain as in many eukaryotic cells, there is 45S precursor-28S and 18S product relationship; a 36–38S RNA appears to be a major component in the transformation of the 45S molecules to 32S RNA. Similar results were obtained in studies in which neural lobes from control and stimulated rats were pulsedin vitro with the above isotopic compounds. Furthermore, the dehydration stimulus resulted in both a generalized and a selective increase in the labelling of the various RNA species. This stimulated incorporation of [3H]uridine or [32P]orthophosphate into RNA was not uniform across the gradient and distinct differences in the pattern of labeling could be discerned. The greatest differences (i.e increase over control) were observed in the high molecular weight species and were apparent depending upon the RNA extraction procedure and the labeling period.
Goldfish were taught a new swimming skill by attaching a float to their ventral surface. The effect of training on the in vitro aminoacyl acceptor activity of tRNA from whole brain was studied. The results were as follows:(1) The total functional amount of tRNA remained constant 0–8 h after training.(2) Consistent and significant increases in leucyl-tRNA activity were observed in trained fish 2–8 h post-training.(3) This change was not caused by the (a) stress or intense physical exertion attendant to training, (b) minor surgical procedures involved, or (c) variation in the enzyme preparations employed.(4) No differences were found in the activities of several other tRNA species tested.Both our behavioral and biochemical findings correspond well with those reported by Shashoua18,19. The implications of these findings with respect to learning are discussed.
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.
References in this bibliography have been selected from the Subject-Strain Bibliography of Inbred Strains of Mice, maintained at The Jackson Laboratory, which attempts to include all published papers dealing with specific inbred strains of mice, named genes in mice, or named transplantable tumors. We have selected all references which appear to be of behavioral interest, including reports of the effects of neurological mutations, but have omitted genetic studies conducted with these mutants. Studies using "white", "Swiss", or undesignated mice are not included. This bibliography covers literature published from 1922 through late 1973. The authors would like to be informed of omissions, and to receive reprints of omitted papers. The bibliography is divided into three sections. The first section includes all references in which a behavioral measure appears to be the variable of primary interest. This section is divided into 16 behavioral categories: activity, aggression, audiogenic seizures, communication, emotionality, feeding, learning, maternal, memory, psychomotor, regulation, reproduction, biorhythms, sensation, social, and miscellaneous. References are assigned to a category on the basis of their apparent emphasis. The second section includes all references in which the effects of a treatment on behavior appear to be the variables of primary interest. This section is divided into nine treatment categories: age, alcohol, central nervous system, mutations, neonatal and teratogenic, population size, pharmacological agents, genetic selection, and miscellaneous. Where multiple treatments were used, references are assigned to the category of the most important treatment. The third section contains reviews and theoretical references. Each item in the bibliography is assigned to a category in one of the three sections and given a reference number. At the end of each category is a list of the reference numbers from other categories which contain information pertinent to that category. References are arranged alphabetically within each category.
Ribonucleic acids (RNA) and proteins are known to have unique functions (1–4) in the central nervous system in addition to their general role in cellular metabolism. For example, alterations in RNA of neural tissue have been demonstrated following learning (5) or subsequent to hormonal treatment (6,7).
We are now at the stage of neurophysiology where learning and memory can be subjects of studies at a strictly molecular level, on the basis of the well-established finding that these higher nervous activities are sustained by, and formed in, the physicochemical events of specified neural mechanisms in the brain. As for the neurophysiological process of memory, much evidence has shown that short-term memory and long-term memory probably result from different molecular events in the brain, i.e., the former from reversible chemical modification of the synapses concerned, and the latter from reorganization of the synapses following synthesis of protein and its axonal transport, which causes the enduring consolidation of memories. How does the experience of individual organisms trigger the protein synthesis in the brain required for long-term memory? What is the role of protein molecules thus formed? What is the mechanism for the regulation of gene expression in the reorganization of neuronal circuits? Many such difficult problems need to be solved. Recently, cholinergic and glutamatergic neuron networks have attracted much attention because there is a strong possibility that they play a critical role in memory. The clinical implication of these findings in human memory deficit, as exemplified in senile dementia, further emphasizes the importance of neurobiological elucidation of the molecular mechanism for learning and memory.
— The methylated albumin-kieselguhr column first employed by SUEOKA and YAMANE (1962) to fractionate tRNA was modified and scaled down by a factor of 100 to permit the separation of 1 y% of goldfish brain tRNA. Double labelling, co-chromatography of single aminoacylated tRNA species demonstrated that the column can characterize isoaccepting tRNAs without serious loss of resolution. The procedure is simple and reproducible and can be useful for the rapid scanning of tRNAs from limited amounts of biological material. Further scaling down of the procedure appears to be feasible.
The incorporation of [3H]uridine-5-monophosphate (UMP) and [3H]orotic acid into neurones and glial cells of the rat hippocampus was studied microautoradiographically after intraventricular injection of these substances. In addition, RNA labelling was compared in neurones and glial cells during a learning experiment.(1)Incorporation of UMP and orotic acid into the neurones was higher than into the glial cells. While the amount of incorporated precursors into the neurones varied with time, the amount of label in the glial cells remained constant over an extended time interval.(2)The ratio of incorporation into the neurones to that into the glial cells was considerably higher with UMP as precursor than with orotic acid.(3)During a learning experiment, the neurones exhibited an increased incorporation of the injected UMP, whereas no significant changes in incorporation into the glial cells was observed.
Immediately after a brightness discrimination 3H-leucine was administered to rats intraperitoneally. One hour after injection the brains were prepared for microautoradiographical examination. In conditioned animals, as compared to the controls, the incorporation of leucine into neurons was increased in all structures of the hippocampal formation, in the visual cortex and cingulate cortex, whereas no increase in incorporation was found in other cortical structures as well as in thalamic and hypothalamic nuclei investigated.
1) An apparatus for electrophoresis of nuclei and for observation of nuclear migration is described.
2) Intact nuclei isolated from rat cerebral cortex migrate in an electric field. At the stationary level, the nuclei migrate to the positive electrode at all pH values of the medium above pH 4.0. In 10 mM phosphate buffer, the peak velocity is observed at approximately pH 6.8. The nuclei present the same behaviour independent of the buffer used: Tris, sodium or potassium phosphate, or sodium bicarbonate. Analysis of the population of velocity values obtained indicates that there are at least two distinct groups within the nuclear population. Red blood cells isolated from the same animals were used for reference, as well as for calibration of the electrophoretic chamber.
3) The electrophoretic mobility of the nuclei is found to be a function of the molarity of the buffer used. It decreases as the molarity of the buffer increases.
4) Nuclei were treated with cations at different concentrations. Treatment with divalent cations at a concentration of 4 mM results in a decrease of the electrophoretic mobility.
5) Intact nuclei obtained from cerebral grey and white matter of human origin also migrate in an electric field. Tumor nuclei, also from human cerebral material, migrate faster than those from normal grey matter.
6) The effect of an applied electric field on the nuclear morphology is also discussed.
7) The results are discussed in relation to what is presently known concerning the charge properties of intact cells and cellular organelles.
The influence of UMP on the incorporation of labeled guanosine into the rat hippocampus during an optical discrimination test was examined by using the microautoradiographic method and comparing the findings for trained animals with those for the corresponding active controls.Under learning conditions, an increased incorporation of guanosine was observed. This increase in incorporation was significantly enhanced by simultaneous application of UMP.These results support the hypothesis that the retention-extending effect of UMP is due to an increase in RNA synthesis.
The incorporation of [3H]lysine into cerebral cortex acid-insoluble material in rats subjected to forced exercise and in control rats has been followed. Decreases in incorporation rate in the exercised rats were observed as compared to the controls.
Studies of the precursor pool parameters have been carried out in order to assess the possibility that the observed changes in incorporation were due to changes in the pool metabolism rather than in the protein metabolism. In particular a time course of the pool activity has been followed, and the pool size and specific activity at the end of the experimental period determined. No changes in pool metabolism that could explain the changes in observed incorporations were found. The only changes that were found in the reverse direction to that which could be required to account for the incorporation figures.
It is suggested that the observed differences in incorporation represent a difference in the rate of protein synthesis in the cerebral cortex. The question of whether this difference is due to motor activity or to stress is discussed.
— Amino acid incorporation in vivo was investigated in the cortex and hippocampus of rats raised in enriched and deprived environments for various periods of time following weaning. At early times after weaning (7 days), the incorporation of l-[3H]leucine into all sub-cellular fractions of both cortex and hippocampus was higher in enriched than in deprived rats. At 16 days, incorporation into synaptosomal sub-cellular fractions was higher in enriched than in deprived hippocampus, and lower in enriched than in deprived cortex; incorporation into perikaryal fractions of both brain regions was the same in the two groups of animals. Incorporation into subcortical nuclear protein fractions was higher in enriched rats at this time. At 35 days, the only difference between enriched and deprived rats was a lower incorporation into cortical synaptosomal sub-fractions in the former. Experiments involving double labelling and electrophoresis indicate that there is no stimulation or inhibition of the synthesis of any particular protein in hippocampal nuclear and synaptosomal sub-fractions of enriched rats. Synaptosomal proteins of cortex have a greater half-life in enriched than in deprived rats; proteins of perikaryal fractions of cortex, and of all fractions of hippocampus, are turning over at the same rate in enriched and deprived animals.
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.
Effects of physical dependence upon ethanol on the polyribosomal properties and the reconstitution of the rough endoplasmic reticulum (RER) of the brain has been examined. The purified free polyribosomes (polysomes), membrane-bound polysomes, and a fraction of RER membrane that has been stripped of polysomes were isolated from rat brain. RNA yields, amino acid incorporation activities, and electron micrographs established the purity of stripped membranes, but no differences were detected following the ethanol treatment. For the polyribosomal fractions, the stability of the mRNA/ribosomal complex was decreased after ethanol dependence as was the in vitro translation of endogenous mRNA. In the reconstitution reaction, the incubation of membranes from ethanol-treated animals with either source of purified bound polysomes resulted in higher yields of protein than when control membranes were used. The above results suggest that ethanol dependence affects the properties of both the RNA and membrane components of the RER of brain.
Chronic ethanol ingestion or cycloheximide treatment results in alterations in the properties and synthesis of protein and RNA of polyribosomes in the whole brain. To analyze the effects on a homogeneous neural cell population, Cox astrocytoma (glioma) cells were grown in tissue culture media with 100 mM ethanol or 0.017 mg/ml of cycloheximide. When ethanol had been present for ten days, the cell densities remained unchanged but had markedly reduced RNA and protein contents. Furthermore, the ethanol treatment reduced the whole-cell pulse-labeling of RNA with (5–3H) orotic acid and protein with (14C) leucine in the postmitochondrial supernatant. These results suggest that chronic ethanol treatment reduced the whole cell synthesis of RNA and protein or increased their degradation. Analysis of the polyribosomes on sucrose density gradients showed that dense polyribosomal chains were decreased after the ethanol treatment, supporting the concept that the polyribosomes were degraded with an alteration in the metabolism of mRNA. The cell-free incorporation of (14C) leucine into hot TCA precipitable protein by the purified polyribosomes in the presence of ATP, GTP, a heterologous source of soluble factors, and endogenous mRNA was also reduced following the ethanol treatment, further indicating that the previous chronic exposure to ethanol had inhibited the translation of mRNA. When the control cells were grown in the presence of cycloheximide for one hour prior to harvesting, the cell densities remained unchanged, but again, as with the ethanol treatment, the polyribosomal protein and RNA yields decreased. In contrast to ethanol, however, cycloheximide treatment caused increases both in the whole-cell incorporation of labeled RNA and protein precursors into the supernatant fraction and in the cell-free incorporation of (14C) leucine into protein. These results suggest that, like the ethanol effects, cycloheximide reduces the total polyribosomes, but unlike the ethanol effects, the remaining polyribosomes have stable mRNA and rapidly incorporate radioactive amino acids, even more than untreated controls. The one-hour cycloheximide treatment also caused an increase in the ratio of dense polyribosomes to monosomes plus 40s and 60s ribosomal subunits of control cells. In addition, it increased the incorporation of the labeled precursor into protein in the polyribosomal region of the sucrose gradients of both control and ethanol treated cells, suggesting that cycloheximide inhibited the termination step of protein synthesis. When cycloheximide was present for 24 hours prior to harvesting, the ethanol treated cells, in contrast to the controls, still had increased cell-free incorporation of amino acid into protein, indicating that the stimulatory effects of cycloheximide are prolonged to 24 hours when ethanol is present. Thus, while the ethanol treatment in general inhibits polyribosomal biogenesis in the cells, it alters the complex of stimulating and then inhibiting effects of cycloheximide by preserving cycloheximide's stimulating effects on the amino acid incorporation activities of the polyribosomes.
Decreased protein synthesis in a cell free system of brain has been reported for male adult rats following chronic ethanol ingestion. To assess the developmental and maturational changes occurring in the neonatal brain, the effects of pre- and postnatal maternal ingestion of ethanol were determined. For these studies young female rats were given a 10% w/v ethanol/water solution for varying periods after impregnation and seven days post pregnancy. Data showed that maternal ethanol ingestion produced a large deficit in the in vitro incorporation of (14C) leucine into the hot TCA extractable residue of ribosomes of neonatal brain. Maximum inhibition was obtained when ethanol was given postnatally. To determine the molecular sites of ethanol's action, ribosomes and pH 5 enzymes from the adult and neonatal brain were examined. Data showed that the highest activity was obtained with control neonatal brain enzymes and ribosomal fractions.
Using microautoradiography ex vivo we tested the effect of forced running on a roller drum for 3 h on the nuclear incorporation of [5-3H uridine] in mouse brain. Specific neuron types with increased nuclear labelling included primary motor cortex layer 5 nerve cells with nuclei greater than 12 μm (+38%) and large neuron nuclei in putamen (+58%). Mice running for 45 min do not show any change in the labelling of nerve cell nuclei compared with mice moving freely in the cage. The [3H]uridine uptake in other cell types, e.g. other neurons in cortical layer 5, neurons in sensory cortex and in the other cell layers in motor cortex, were not different from control mice.
We conclude that RNA synthesis is normally low in adult mouse brain, but that physical exercise stimulates RNA synthesis in specific populations of large neurons in the motor system.
After injection of leucine-H(3), members from eight triplets of rats were assigned to one of three treatment groups: avoidance learning, the same handling and shocks without opportunity to learn, or passive control. Autoradiographic grains over all nuclei were counted "blind." In the hippocampus, the number of grains for the learning rats was reliably greater than that for either control. The differences approached statistical significance in the entorhinal cortex and the septal area but not in other brain areas or in the liver. Although the processes responsible for the increased incorporation are not yet defined, these changes probably are the result of learning rather than of stress.
32 male Holtzman rats were pretrained to asymptote on a Y maze spatial discrimination for water reinforcement. Tritiated cytidine was then injected intravenously as an RNA precursor. Experimental Ss were immediately subjected to an average of 5.6 successive spatial reversals to criterion, whereas the controls were continued on the original discrimination, both for 60 min., before decapitation. RNA synthesis proved to be 25% higher in the experimental hippocampus, and pools of tritiated cytidine or its metabolites were 15 and 17% higher in the experimental hippocampus and pyriform cortex. Since sensory and motor factors were equated between groups, these chemo-EEG effects were probably related either to frustration of nonreward or, considering the hippocampal literature, perhaps more likely to learning or memory processes. (32 ref.) (PsycINFO Database Record (c) 2012 APA, all rights reserved)
7 INBRED STRAINS OF MICE AND 4 F1 HYBRID GROUPS WERE TESTED FOR ACQUISITION AND EXTINCTION OF AN AVOIDANCE RESPONSE IN AN APPARATUS DESIGNED TO PRODUCE RAPID TRAINING OF LABORATORY MICE. THE 7 INBRED STRAINS SHOWED DIFFERENCES IN RATE OF ACQUISITION AND EXTINCTION; CORRELATIONS OF MEASURES OF ACQUISITION AND EXTINCTION WERE SHOWN TO DEPEND ON GENOTYPE. THE 4 F1 HYBRID GROUPS SHOWED PERFORMANCE SUPERIOR TO THE AVERAGE PERFORMANCE OF THE PARENTAL STRAINS; THIS RESULT IS INTERPRETED AS AN INSTANCE OF BEHAVIORAL HETEROSIS.
Specific stimulation of the ganglion cells of the frog retina leads to a sharp increase in the synthetic processes in the nucleus and the cytoplasm. It is expressed in an increased amount of protein and RNA (determined cyto-chemically) which is in full agreement with electron microscopic findings showing the following changes in the structure of these cells. 1. (1) Appearance of specially structured zones in the caryoplasm-chromocentres. 2. (2) Increase in the perinuclear space. 3. (3) Considerable increase in the total number of RNP granules in the cytoplasm. 4. (4) Change in the character of the arrangement of RNP granules in the cytoplasm (their appearance in zones of nuclear pores, at the external nuclear membranes and on the membranes of the endoplasmic reticulum). 5. (5) Change in the mitochrondria which increase in length. 6. (6) Change in the ratio of components of Golgi's apparatus (disappearance of flat cylinders and predominance of the vacuolar component).
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.
By comparison with a photoreceptor cell that has been exposed to light for at least several hours, a cell from an animal grown in darkness is deficient in both rough and smooth endoplasmic reticulum. The free ribosomes of cells under either condition appear to be single or randomly clumped. Illumination of a previously light deprived cell stimulates the formation of free polyribosomes having a characteristic, probably helical, organizaation. They are found at first in association with the rough endoplasmic reticulum, and after 30 minutes, fill the cell. The polyribosomes are mostly gone after two hours of illumination. Illumination also stimulates an increase in the amount of endoplasmic reticulum, which reaches its maximum density by 12 hours of illumination. The discussion is concerned with the possible significance of these light induced ultrastructural changes to the protein metabolism of the photo-receptor cell.
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.
were unable to state whether the increases represented a generalized change throughout the brain or a relatively large increase in a smaller area of the brain. In an attempt to answer this question, we have dissected the brains of trained and untrained mice into six areas and have analyzed each area for incorporation of radioactivity into RNA. The results indicate that the increases represent a localized rather than a general change. We had hoped that the results might also help to clarify which aspect of the training situation (i.e., learning, stress, jumping, attention, etc.) was the cause of the increased labeling. However, the localization found does not enable us to make such a distinction and we must await further studies now in progress using autoradiography that may permit more precise localization. Materials and Methods.-Male C57B1/6J mice, six to eight weeks old, from the Jackson Laboratories were used as the experimental animals. For each experiment a double labeling method involving two mice was used.1 One of the mice received 10 ,uc of uridine-5-H3 (Nuclear-Chicago, sp. act. 2 c/mmole, conc. 1 mc/ml) and the other received 2 jAc of uridine-2-C'4 (Nuclear-Chicago, sp. act. 1040 mc/mmole, conc. 200 Ac/ml). Injections were made intracranially into the frontal lobe to a depth of 3 mm on both sides of the midline. Thirty minutes after the injections, one of the mice, chosen randomly, was trained in the jump box and the other served as an untrained yoked control. The training apparatus and procedure have been described previously.1 Briefly, we conditioned the mouse to avoid an electric shock by jumping to a shelf when a light and a buzzer were presented for three seconds prior to the onset of the shock. The training lasted 15 minutes, during which time an average of 30 trials was completed. The untrained, yoked control animal received the light, buzzer, and electric shock to the same extent as the trained animal but could not avoid shock. At the end of training (45 min after the injections), the two mice were sacrificed and their brains were dissected into the following six parts, as described in Figure 1: olfactory bulbs and tracts (0), ventral hippocampus and temporal areas (H), frontal areas (F), parietal and occipital areas (P), cerebellum (C), and diencephalon (D) with anatomically associated areas. Corresponding areas from the trained and untrained mice were combined and homogenized. Nuclei and the RP pellet were obtained by differential centrifugation, the RNA was isolated, and the radioactivity was determined by procedures previously described.' Total RNA was determined in a perchloric acid precipitate from each fraction following extraction of lipids. The lipid-free residue was hydrolyzed with 1 N NaOH and the amount of RNA determined by the orcinol method.2
In agreement with the data of Beach et al. (these Proceedings, 62, 692 (1969)) on the effects of avoidance training on protein synthesis, we report autoradiographic evidence for the increased incorporation of radioactive uridine into limbic system structures of the brains of trained mice. There is also a decreased incorporation into the outer layers of the neocortex. The observed responses in labeling are restricted to the nuclei of neurons. The significance of these changes in brain function is not known but they may be related to those processes of the memory consolidation phenomenon affected by inhibitors of protein and RNA synthesis.
The effects of proprioceptive and exteroceptive stimulation and of vestibular stimulation on cerebellar Purkinje cell RNA content and composition were studied in male albino rats. After stimulation involving extero-and proprioceptors a significant increase of RNA was found in the vermian part of the lobulus centralis, the pyramis, the copula pyramidis and the parafloculus. No such increase was found in the lateral parts of the lobulus centralis, lateral parts of the culmen, the simplex, crus I or in the nodulus. After vestibular stimulation an increase of RNA content occurred in the lobulus centralis and the nodulus but not in the pyramis, the copula pyramidis or in the paraflocculus. A significant decrease of the purine/pyrimidine quotient was found in the vermian part of the lobulus centralis and in the nodulus of rats receiving vestibular stimulation. A comparison between the neuroanatomical and electrophysiological findings in this field has been presented. The conclusions are drawn that Purkinje cells increase their RNA content upon stimulation and that this condition may be used for studies of functional localization in the cerebellum.
Acquisition of new swimming skills by goldfish during learning experiments is accompanied by changes in the base ratio of RNA. Several hypotheses are put forward to explain these changes.
Abstract— —Studies were undertaken to correlate the changes in the synthesis of brain nuclear RNA during olfactory stimulation in saltwater catfish (Galeichthys felis). Catfish allowed to swim for 1 hr in sea water containing morpholine (10−4 M) showed an increase in brain nuclear RNA and a change in base ratios in contrast to controls in plain sea water. These changes in brain nuclear RNA were reversed within 24 hr to the levels of unstimulated controls when morpholine stimulated fish were transferred to fresh sea water.
In a split-brain preparation in an isolated catfish head, one naris was washed with morpholine in sea water (10−6 M), while the other naris was washed with plain sea water. The stimulated half of the brain, compared to the unstimulated half, showed the same changes in nuclear RNA as those noted in free swimming catfish. Brain cytoplasmic fractions did not exhibit any changes in RNA following olfactory stimulation. Amyl acetate, shrimp extract, and extracts from red fish skin as odorants also elicited changes in brain nuclear RNA. With each odorant there was an increase in amount of RNA and also a change in base ratio, where the base ratio changes were different for each odorant tested. With camphor as an odorant, there was an increase in brain nuclear RNA, while with menthol as an odorant, there was a decrease in brain nuclear RNA. In both instances the base ratio of the RNA did not change in contrast to the controls. These studies suggest that olfactory stimulants affect a change in content and character of the RNA in brain nuclei, whereas irritants to the olfactory epithelium change the content of brain nuclear RNA but do not alter the base ratio.
It was shown that puromycin administered to mice 1 or more days after maze-learning blocks expression of memory; the blockage can be removed by intracerebral injections of saline. We present evidence that intracerebral injections of saline are relatively ineffective in restoring memory when puromycin is administered either before or immediately after training; in these two situations puromycin appears to interfere with consolidation of memory.
—Investigations of rat brain RNA were carried out by phenol extraction of the whole brain and chromatographic fractionation into ribosomal RNA and transfer RNA.
(1) The amounts of both RNA species increase in the course of the animal's development reaching a maximum at about the tenth week of life. The ratio of both species remains constant throughout the growth to the twentieth week. After the rats had been trained how to reach their forage by balancing on a stressed rope, the rRNA content was found to be significantly higher, whereas the tRNA content was unchanged.
(2) The portion of ribosomes bound in polysome complexes decreases with increasing age of rats. Conditioning of the animals brings about again an increase in polysome content. It is supposed that this reflects an enhanced synthesis of specific proteins in young developing rats and in the course of conditioning.
(3) In young rats a second valine specific tRNA could be found as a minor component in addition to the major valyl-tRNA. This additional component disappears as the animals advance to an age of 3 weeks and it could not be detected in the brains of rats after training experiments. In tRNAs specific for the amino acids leucine, lysine and phenylalanine no kind of deviation could be stated.
When 2 micrograms of antinomycin D was injected intracranially into goldfish immediately after a training session, the formation
of long-term memory of a shock-avoidance was blocked. The results are discussed in relation to similar findings with acetoxycycloheximide
and puromycin in the goldfish and with apparently conflicting results in the mouse.
and their glia were analyzed from that part of the brain stem which was functionally involved in the establishment of this complex motor and sensory performance. The nuclear and cytoplasmic RNA of the big Deiters' neurons of the lateral vestibular nucleus were analyzed with respect to amount per cell and base ratio composition. During learning, an increased synthesis of neuronal RNA was found, and the nuclear RNA showed a changed base composition with an increased adenine-touracil ratio. Similar, but not identical, glial RNA changes were found during learning.' Several control experiments were performed involving a stress experiment, vestibular stimulation, and also RNA analyses in a part of the brain outside of the vestibular nuclei. Only in the Deiters' neurons and glia were significant RNA base ratio changes found during learning, indicating a nuclear synthesis of small fraction(s) of RNA with highly specific base ratios. In the present study, single cortical neurons were analyzed during transfer of handedness in rats. The advantage in this experiment is that the control material is obtained from the same brain. Experimental Setup and Material.-White rats of the Sprague-Dawley strain weighing about 150 gm were used. The experimental setup consisted of a large wooden box with a glass cylinder (diameter 1 cm) placed 5 cm from the floor, into the opening of which the rat had to reach in order to grab small pieces of food. On the first day of the experiment the pieces of the food pellets (4 gm per day) were placed close to the opening, offering no difficulties for the animals to reach them with the preferred paw. Each rat was permitted to show by 25 reachings which hand it preferred. In this initial short test, 23 out of 25 reaches were demanded as a criterion for handedness. The conditions to be fulfilled in this respect have been studied earlier by Peterson3 and Wentworth.4 For our experiments, right-handed rats were forced to use the left hand to retrieve the daily ration of 4 gm of food per day (at 10 A.M. and 3 P.M., 25 min each time) from deep down in the glass cylinder. In order to force the hungry animals to transfer to the left hand, a wooden wall was arranged close to and parallel to the left side of the glass cylinder which was most effective in prohibiting the use of the right hand. In Figure 1 is plotted the number of reaches during the morning period of 25 min for 5 rats and for 5 days. On an average each rat performed 400-500 reaches during the 4-day period of the experiment. We also tested and confirmed the findings reported in the literature that once a shift in handedness had occurred by as few as 200 forced reaches, the animal proceeded with the "new" hand even when tested 9 months after the transfer.4 By using a stereotactic technique, Peterson and Devine5 found indications for a critical area of the rat cortex controlling handedness which involved the cells in layers 5 and 6. These authors point out, however, that the critical area varies individually and probably encompasses a greater area of the cortex than the 0.5-1 mm3 indicated by the results. We chose, however, neurons and glia of layers 5 and 6 from an area 2.7-lmm lateral and 1.6-mm rostral from the bregma.5 These nerve cell bodies together with the first part of the dentrites contain a small amount of RNA, averaging 22 ,uug. Therefore, about 10 nerve cells were used for each quantitative analysis.
The polypeptide chain which constitutes the first ten amino acids of the ACTH molecule inhibits extinction of a shuttle-box avoidance response. If the phenylalanine molecule in the 7th position of this peptide is replaced by its dextrorotatory form, extinction is facilitated.
Polysomes have been isolated from rat brain and characterized by their appearance in the electron microscope and by their sedimentation in sucrose density gradients. Rats were isolated for 3 days in the dark and were then returned to the light for 15 minutes. The polysomes in brain, but not in liver, decreased in rats deprived of light and increased in those stimulated with light. These findings together with an increased capacity for protein synthesis in the brain in vitro and in vivo suggest that an increase in the activity of messenger RNA in the brain may result from environmental changes.
Incorporation of radioactive precursors into polysomes and RNA of mammalian brain during short term behavioral experiences
- Coleman
Local cerebral circulation at rest and during altered cerebral activity induced by anesthesia or visual stimulation
- Sokoloff