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RNA metabolism in the rat brain during learning following intravenous and intraventricular injections of 3H-cytidine
Abstract
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.
It has been known for some time from neuroendocrine and behavioral studies that the brain is both a master controller of endocrine function and a target for these endocrine secretions. There exist complex feedback interactions between endocrine secretions and the brain which not only control the secretion of hypothalamic and pituitary hormones but also influence neural activity underlying behavior. It is only quite recently that we have come to appreciate the role of the entire limbic brain, and not just the hypothalamus, in these endocrine-brain interactions.
The state of sleep-wakefulness and the central metabolism of monoamines and proteins have been studied 6 hr after i.p. injection of DL-5-hydroxytryptophan (5 mg/kg) into cats pretreated (48 hr) with p-chlorophenylalanine (400 mg/kg). Insomnia and a marked diminution of the endogenous levels of 5-HT and 5-hydroxyindole-acetic acid without remarkable variation of NA were induced in the telencephalon of cats treated with p-chlorophenylalanine only. Injection of 5-hydroxytryptophan restored a normal sleep pattern and at the same time the metabolism of 5-HT increased significantly.
High resolution light microscopic autoradiography was used, together with regional surveys and combined aeridine orange staining, to define in rat hippocampus cellular and subcellular sites of concentration and retention of 3H dexamethasone and to compare the topographic pattern of labeling with that of 3H corticosterone. Nuclear uptake of 3H dexamethasone in the hippocampus is demonstrated for the first time in vivo. With 3H dexamethasone, strongest nuclear radioactive labeling was observed in certain glial cells throughout the hippocampus, followed by strong nuclear labeling in most neurons in area CA1 and in the adjacent dorsolateral subiculum and weak nuclear labeling in granule cells of the dentate gyrus. Neurons in areas CA2, CA3, CA4, and in the dorsomedial subiculum and indusium griseum showed little or no nuclear labeling after 3H dexamethasone. With 3H corticosterone, strongest nuclear labeling was observed in neurons in area CA2 and in the dorsomedial subiculum and indusium griseum, followed by area CA1, then CA3 and CA4; the dentate gyrus contained scattered strongly labeled cells among cells with intermediate nuclear labeling. At the subcellular level, evidence for both nuclear and cytoplasmic accumulation of label was found. The results indicate that dexamethasone and corticosterone have both nuclear and cytoplasmic binding sites and that particular patterns of target cell distribution exist, characteristic for each agent. This suggests a differential regulation of cellular functions for the two compounds. Corticosterone nuclear binding appears to be more extensive and encompasses regions with dexamethasone binding. Whether in certain of these common regions corticosterone binds to the same receptor as dexamethasone, which seems possible, or to different receptors, remains to be clarified.
An intercalated reinforcement technique is described. The method equates number of trials, response topography, sensory exposure,
and frequency of reinforcement between reversal Ss overtrained on a discrimination and then trained on reversals and nonreversal
Ss similarly overtrained but continued on the same discrimination. In extending previous successful work in the rat, monkeys
were trained on spatial discrimination and reversal tasks in the WGTA at 50%, 75%, and 100% reinforcement in order to test
the efficacy of learning by this procedure, and to compare the method with learning obtained by a more classical procedure.
Learning rates were the same by both procedures. This technique, therefore, appears useful for investigating neurochemical
(or other) correlates of learning unconfounded by performance or sensory effects.
Previously reported changes in the amount of 3H-L-fucose incorporated into glycoproteins of brain and liver after subcutaneous injections of precursor when mice were exposed to the training apparatus did not occur when the precursor was injected either intracranially or intravenously.The increased incorporation of subcutaneously injected 3H-L-fucose was attenuated by sham intracranial and by sham intravenous injections. Thus, the failure to detect incorporation changes when the precursor is administered through these routes may be partially due to stress caused by intracranial or intravenous injection.
The incorporation of 3H-uridine into RNA and selected nucleotides of mouse brain during avoidance training was assayed using intracranial injections of precursor. The net incorporation of uridine into total brain RNA, as assayed by three methods, was not detectably affected by training. Training induced a decrease in the amount of radioactivity recovered from the brain as uridine monophosphate, while concomitantly increasing the amount of radioactivity in substances that chromatographed as uridine diphosphate sugars. The results suggest that changes in the cerebral metabolism of uridine observed during this form of avoidance training may reflect changes in the cellular uptake of uridine, or the metabolism of carbohydrate compounds, rather than increases in the rate of cerebral RNA synthesis.
The incorporation of cytidine into total RNA in six regions of the brain was assayed in mice during three behavioral treatments. Following the injection of 3H-cytidine, reversal mice were trained to reverse repeatedly their side preference in a Y-maze for water reinforcement during a 60 min session. Nonreversal mice were reinforced for choosing the previously correct alley during 60 min. Quiet mice were kept isolated after the injection. Only small increases in the incorporation of precursor into RNA were observed. Incorporation of 3H-cytidine was not altered in the hippocampi of reversal mice. These findings suggest that previously reported elevations of the incorporation of cytidine into hippocampal RNA during spatial reversal training may not be general across species or may depend upon special experimental conditions.
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 ribosomes of the CA1 and CA3 pyramidal cells of hipocampus were investigated by morphometric methods after the acquisition of a shock-motivated brightness discrimination in rats. A significant increase in the total number of ribosomes was observed in CA1 cells of trained animals and in CA3 cells of both active controls and trained rats. A significant increase in membrane-bound ribosomes was obtained in CA1 and CA3 cells after training only. The results confirm the suggestion of an increased protein synthesis in hippocampal neurons during and after the acquisition of a brightness discrimination, as we have concluded from out previous investigations on the incorporation of labeled amino acids under identical experimental conditions. The results lead to the assumption that the protein synthesis in some neuronal cells may probably differ not only quantitatively, but also qualitatively in trained and untrained animals.
Operant schedules were used to isolate components of a training task and the distribution of radioactivity into various brain areas was studied using high resolution autoradiography. Rats exposed to a visual stimulus change showed more labelling in hippocampal area CA1 than littermates exposed to no stimulus change. Rats exposed to a non-cued contingency change showed higher labelling in hippocampal areas CA2 and CA4 than littermates exposed to a cued contingency change. The results suggest that hippocampal changes in RNA synthesis may be related more to the aversive conditions of the training task than to specific learning changes.
Operant schedules were used to isolate component parts of a training task and rates of incorporation of 3H-uridine into the brain and brain RNA were determined. Rats that developed a discrimination in responding to a visual stimulus absorbed more radioactivity into the brain and incorporated a higher percentage of this radioactivity into total and cytoplasmic RNA than littermates exposed to the visual stimulus only. Of the component parts of the training task, the discrimination accounted for the greatest increase in absorption of radioactivity and incorporation of it into RNA. The schedule change had the second largest effect and the stimulus change the least.
The effect of injection by intracranial and systemic routes on the localization of [3H]uridine incorporated into RNA of mouse brain was quantitated autoradiographically. The pattern of labeled RNA 45 min after intraventricular or intracisternal injection was highly variable throughout the brain, and activity was localized in structures adjacent to the ventricles or subarachnoid space. The amount incorporated was inversely related to distance from these cerebrospinal fluid compartments with little incorporation observed at distances greater than 200 microns. In contrast to intracranial administration, intrapleural and subcutaneous routes yielded a more homogeneous distribution of incorporated uridine throughout the brain. Regional differences in the autoradiographic pattern following systemic administration were correlated with regional differences in cell density. The results clearly indicate that injection route is a potent factor affecting the localization of agents used to study brain.
High resolution light microscopic autoradiography was used, together with regional surveys and combined acridine orange staining, to define in rat hippocampus cellular and subcellular sites of concentration and retention of 3H dexamethasone and to compare the topographic pattern of labeling with that of 3H corticosterone. Nuclear uptake of 3H dexamethasone in the hippocampus is demonstrated for the first time in vivo. With 3H dexamethasone, strongest nuclear radioactive labeling was observed in certain glial cells throughout the hippocampus, followed by strong nuclear labeling in most neurons in area CA1 and in the adjacent dorsolateral subiculum and weak nuclear labeling in granule cells of the dentate gyrus. Neurons in areas CA2, CA3, CA4, and in the dorsomedial subiculum and indusium griseum showed little or no nuclear labeling after 3H dexamethasone. With 3H corticosterone, strongest nuclear labeling was observed in neurons in area CA2 and in the dorsomedial subiculum and indusium griseum, followed by area CA1, then CA3 and CA4; the dentate gyrus contained scattered strongly labeled cells among cells with intermediate nuclear labeling. At the subcellular level, evidence for both nuclear and cytoplasmic accumulation of label was found. The results indicate that dexamethasone and corticosterone have both nuclear and cytoplasmic binding sites and that particular patterns of target cell distribution exist, characteristic for each agent. This suggests a differential regulation of cellular functions for the two compounds. Corticosterone nuclear binding appears to be more extensive and encompasses regions with dexamethasone binding. Whether in certain of these common regions corticosterone binds to the same receptor as dexamethasone, which seems possible, or to different receptors, remains to be clarified.
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.
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.
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 effects of two different training procedures on the incorporation of [5-3H]- orotic acid into total brain and liver RNA of the newtsTriturus cristatus andPleurodeles waltl are reported.Triturus cristatus trained to spatial discrimination reinforced by food or trained to avoid darkness by aversive electrical shocks incorporated more [5-3H]orotic acid into their brain RNA after training than did yoked newts or untreated newts.Pleurodeles waltl trained to avoid darkness incorporated more than the untreated group after training ceased while the yoked animals incorporated less than the untreated group into their brain RNA. No changes occurred in the incorporation into the liver RNA of any of the experimental newts.
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)
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.
The measurement of the adrenocortical hormones, Cortisol and corticosterone, m blood plasma has previously best been done
by procedures employing varying degrees of purification of the hormone, followed by photometric or fluorimetric quantitation.
These methods have been relatively difficult or laborious, particularly in the case of the procedures that most specifically
measure the hormoneof intent. These limitations are considerably overcome by new assays for Cortisol and corticosterone based
on the principle of competitive protein-binding of the steroids in the presence of radioactively labeled steroid of the same
molecular species. These methods are described and data on the specificity of such a method for corticosterone are presented
and compared with one of the better fluorimetric assays.
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
Base composition measurements were used as a criterion of RNA changes in goldfish brain. RNA synthesized during the acquisition of new swimming skills was found to have a uridine/cytidine ratio 20-80 per cent higher than that of RNA formed under nonlearning conditions. A variety of behavioral situations were used to demonstrate that these RNA changes were not due to such factors as intense physical exertion, passive responses to stress, or the intense electrical activity of the brain and convulsive behavior produced by KCl injections. The RNA changes were produced by two behavioral situations; (1) during the process of acquiring new swimming skills, and (2) as a result of attempts to master an impossible task. The results suggest that the modified RNA synthesis taking place during the acquisition of new behavioral patterns is probably not specific as to the particular information content being stored, but may be required for the consolidation step of new information storage.
MILD electroshock is used as an aversive stimulus in many kinds of
learning experiments with rats and mice. Some workers have correlated
changes in brain RNA concentration with learning1-3 so it
seemed of interest to isolate, as well as possible, the elements in a
learning situation and to determine their contribution to the observed
changes in RNA.
Protein synthesis was studied in the pyramidal nerve cells of the CA3 region in the hippocampus of rats uring a behavioral test involving transfer of handedness. A new electrophoretic technique was used for separation of 10(-7) to 10(-9) gram of protein and radiometric determination of the various protein fractions. After interventricular administration of (3)H-leucine, protein synthesis of two fast-moving fractions was significantly higher bilaterally in the hippocampus of the trained rats. There was also a trend to lateralization of the highest protein synthesis to the learning side.
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— In the cat, intraventricularly injected [14c]leucine does not appear to penetrate into the cerebral tissue, whereas intravenously injected [14c]leucine readily penetrates the blood-brain barrier. The latter route of administration of [14c]leucine produces rather uniform distribution of radioactivity in cortical and subcortical regions as well as diencephalic, lower brain stem, and cerebellar regions. Data consistent with compartmentation of the glutamate-glutamine system were observed in all regions except the cerebellum and head of the caudate nucleus. In the latter two areas, the ratios of the specific activity of glutamine to glutamic acid was less than 1, whereas in all other areas it was greater than 1. The turnover rate of the brain protein was fastest in the cerebellum and neocortex and slowest in the caudate nucleus and in the pons and medulla.