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Distribution of 3H-uridine-5 into brain RNA species of rats exposed to various training tasks—An electrophoretic analysis
Abstract
Operant schedules were used to isolate component parts of a training task and specific activities were determine for nuclear and cytoplasmic RNA species separated by polyacrylamide disc gel electrophoresis. Rats exposed to a stimulus or schedule change incorporated more radioactivity into nuclear rRNA and mRNA and cytoplasmic rRNA and tRNA than littermates exposed to no change from baseline training. Rats developing a change in response probability to a stimulus in the environment incorporated more radioactivity into cytoplasmic mRNA.
The substances that are synthesized in the nerve cell body either remain in the perikaryon, or are transported toward the periphery along the dendrites or axon. For the most part, the axonal and dendritic processes are metaboli cally dependent upon the synthetic capacity of the perikaryon. They require regular provisions of macromolecules and organelles, the great percentage of which is supplied by this active movement from the sites of synthesis.
This chapter discusses the effects of social deprivation on RNA and membrane-protein synthesis in the rat brain tissue. Animals deprived of contact with others of their species from an early age develop various anomalies of behavior. Social deprivation implies the absence of external stimuli, which apparently play the main role in the natural development of the brain and are a necessary condition for its normal metabolism. The mechanisms of transmission of information on nuclear structures remain unclear as well. The influence of the exogenic calcium on the RNA-synthesizing activity of the brain cell nuclei both in normally developing animals plays a major role. The exogenous calcium affects nuclei by altering the level of RNA synthesis in different ways.
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.
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.
— The incorporation in vivo of [3H]uridine into the RNA isolated from the free polyribosomes of rat cerebral cortex was studied. Sedimentation in sucrose gradients showed that initially (at times less than 60 min after injection of precursor) the label was associated with a heterodisperse species, while at longer times there was an increased coincidence of label with stable rRNA. Further fractionation was accomplished by means of differential extraction with phenol and analysis on polyacrylamide-agarose gels. Most of the rapidly labelled RNA was concentrated in a fraction obtained at pH 8-3 and 40°C. The base composition of this fraction differed greatly from that of rRNA, preribosomal RNA and DNA. Analysis by electrophoresis on polyacrylamide-agarose gels showed it to be composed of several distinct species in addition to residual 18 and 28S rRNA. Most of the latter was concentrated in a fraction extracted at pH 60 at 0°C.
—Nuclear RNA from neurones, astrocytes and other glial cells was pulse-labelled in vivo with [3H]uridine and analysed by sucrose density-gradient centrifugation after various periods of incorporation. Thirty min after the injection of the isotope, rapidly-labelled RNA appeared in all three cell types, a heterogeneous fraction sedimenting above 30S, the others at 25 and 12S. The transformation rate of the two latter components was equally rapid in all three types of nuclei studied. These components are assumed to be of messenger nature. The heavy fractions underwent transformations which in other cells have been described to lead to rRNA formation. The temporal pattern as well as the sequence of changes were similar in nuclei from neurones and astrocytes, the only difference being that a 35S intermediate was found in the former and a 32S in the latter. In non-astrocytic glial nuclei, synthesis and transformation of the 45S component were delayed as compared to the other cell types and the processing of this component may involve both a 32S and a 35S intermediate. Moreover, the radioactivity incorporated in all the nuclear RNA species was always lower in these cells.
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.
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.
MUCH circumstantial evidence implicates protein and RNA synthesis in memory consolidation and increasing interest in work directed toward elucidating the macromolecular chemical processes underlying learning1,2. A derepression model has been advocated by Bonner3, who also suggested a method by which the model might be tested. Newly induced RNA molecules would be gene products, present in the brains of learning animals but not naive animals, and they should be able to be pulse labelled and detected by competitive hybridization experiments.
MICE injected with radioactive uridine in a double isotope labelling
technique and then trained for 15 min to avoid a shock by jumping to a
shelf incorporated about 50% more radioactivity into brain
RNA1 and polysomes2 than untrained mice yoked to
them. Quiet mice, yoked mice, and mice that were subjected to thirty
electric shocks at random intervals for 15 min all incorporated less
radioactive uridine into brain RNA and polysomes than did the trained
mice1,2. This indicates that the lights, buzzers, shocks,
handling and activity have little effect.
Polyacrylamide gel electrophoresis (PAGE) provides a versatile, gentle, high resolution method for fractionation and physical-chemical
characterization of molecules on the basis of size, conformation, and net charge. The polymerization reaction can be rigorously
controlled to provide uniform gels of reproducible, measurable pore size over a wide range. This makes it possible to obtain
reproducible relative mobility (Rf) values as physical-chemical constants. Application and extension of Ogston's (random fiber) model for a gel allows for
calculation of molecular volume, surface area, or radius, free mobility, and valence from RJ measurements at several gel concentrations, to calculate gel concentration for optimal resolution, and to predict behavior
of macromolecules on gel gradients by computerized methods. Extension of classical moving boundary theory has been used to
generate multiphasic buffer systems (providing selective stacking, unstacking, restacking, and preparative steady-state-stacking)
with known operating characteristics for any pH at 0° and 25°C. A general strategy for isolation of macromolecules and for macromolecular mapping has been developed. Preparative
scale PAGE is operational for milligram loads and feasible for gram quantities.
All subjects were killed by immersion in liquid nitrogen, followed by the extraction and purification of brain RNA. DNA was extracted from the brains of two non-injected control subjects, with which a series of membranes was prepared with 5 ?g DNA/membrane.Single hybrid saturation curves were obtained using control RNA to determine the amount of RNA necessary to saturate the DNA membranes. During subsequent double hybridization, this quantity of unlabelled competitor RNA from control subjects was annealed with DNA, followed by the addition of labelled RNA from experimental subjects.The double hybridization data indicate an absence of obvious differences between control and experimental RNA, suggesting that no unique RNA species are synthesized during visual stimulation.
A method for separating high molecular weight RNA or DNA by electrophoresis on polyacrylamide gels has been described.Ten viral nucleic acid species and four Escherichia coli RNA species have been used to calibrate the system and demonstrate a general relationship between the logarithm of the molecular weight and the relative electrophoretic mobility.The elution of viral RNA from gel slices and the demonstration of infectivity after electrophoresis are described.
—Tritium-labelled RNA precursors were injected at 30 min intervals into the fourth ventricle of rats or rabbits. After 4 h the nuclei from neurones, astrocytes, and other glial cells were isolated and RNA extracted. Investigations were performed in order to establish optimum conditions for RNA extraction from this particular material.
The sedimentation patterns obtained in sucrose gradients were similar to those of nuclear RNA from other mammalian tissues and showed the presence of RNA species with high specific activities in the region of the gradient between 10S and 16S and above 28S. All three types of nuclei contained a 45S and a 38S RNA. Moreover, a 32S component could be identified in astrocytic nuclei, a 35S fraction in neuronal nuclei, and both a 32S and 35S RNA in nuclei from glial cells. The nuclei from the various cell types also differ with respect to the rate of incorporation of the label into the nuclear RNA, being four times higher in astrocytic and neuronal nuclei than in those derived from the other glial cells.
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.
—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.
A method for estimation of purified RNA using cupric ion catalyzed orcinol has been studied and improved. If such an assay is carried out under the optimal conditions described here it would appear to be the most sensitive, accurate, and rapid method for routine RNA estimation currently available. The improved conditions include a combination of several factors such as replacement of the usual FeCl3 by a copper salt, increasing the concentration of orcinol in the reaction mixture, and increasing the heating time from the commonly used 20 minutes to 35 minutes.
RESULTS of two previous studies1,2 using successive competition DNA-RNA hybridization procedures suggested the synthesis of ``unique'' species of RNA during a one way active shock avoidance task. Here we describe two experiments in which brain RNA from shock avoidance trained rats competed with brain RNA from shocked yoke controls. The results of both experiments suggest the presence of ``unique'' RNA species during learning.
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.
RNA from animal cells
- J Darneu
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RNA from animal cells
- Darnell