John Eric Wilson

Effect of sensory stimulation on the uptake and incorporation of radioactive lysine into protein of mouse brain and liver

Alterations in incorporation of tritiated lysine into protein of mouse brain and liver were observed following brief exposure to a variety of sensory stimuli. During a 15-min session, subjects were trained to perform a one-way active avoidance response or else were exposed to one of the stimulus components of the situation, including shocks, buzzers, lights, handling, and the apparatus alone. Twenty min after these behavioral treatments, tritiated lysine was injected subcutaneously, and its incorporation into total protein during a 10-min pulse was measured. Quiet mice, undisturbed until injection of the precursor, constituted the baseline group for biochemical comparisons. Most behavioral treatments increased the total amount of radioactivity in brain and liver. The treatments increased the incorporation of radioactivity into protein of both organs even more, thereby producing elevations of relative radioactivity (RR) of protein, a measure of the amount of radioactivity incorporated into protein relative to that in the acid-soluble pools. The RR increases following most of the behavioral experiences were approximately equal; however, exposure to lights or to the apparatus were less effective than the other treatments in eliciting these metabolic changes. The responses were greatly diminished in mice previously exposed to the treatments. Thus, the effectiveness of a stimulus in producing these metabolic alterations may depend upon its apparent magnitude and its novelty. The total radioactivity increases were larger in brain than in liver, while the RR increases were smaller in brain than in liver. Brain RR increases were of equal magnitude when the precursor was injected 5, 20, or 35 min after behavioral treatment, whereas the liver RR responses declined markedly over this period. Despite these differences, strong positive correlations between brain and liver across the various behavioral treatments existed. The RR changes occurred about equally in the cerebellum-brain stem, basal ganglia, hippocampus-septum, and ventral cortex, while the thalamus-hypothalamus and dorsal cortex showed smaller differences.

THE INCORPORATION OF RADIOACTIVE AMINO ACIDS INTO BRAIN SUBCELLULAR PROTEINS DURING TRAINING

—Total proteins, free amino acids, tritiated water and subcellular proteins of mouse brain were examined for changes in radioactivity during operant conditioning after subcutaneous administration of labelled amino acids. The conditioning was based on appetitive learning, using sweetened milk as a reward.

Brain phosphoproteins: the effect of short experiences on the phosphorylation of nuclear proteins of rat brain

In this paper we describe the effect of a short training experience on the appearance of radioactive phosphate in various chemicals in rat brain. There was little or no effect of training on radioactive phosphate in AMP, CMP, UMP, or GMP. The radioactivity was increased in nuclear nonhistone acid-extractable proteins, and possibly decreased in histones. Disc electrophoresis revealed that nuclear nonhistone acid-extractable protein is heterogeneous, containing at least 14 species of protein, only 3 of which are phosphorylated. Amino acid analysis of nuclear nonhistone acid-extractable protein showed that the difference in amount of radioactivity in brain nuclear nonhistone acid-extractable protein from the trained and untrained rats was due to an increase in the amount of radioactivity in phosphoserine, not phosphothreonine, although both contained radioactivity. Finally, an increase in the molar ratio of phosphoserine relative to serine in brain nuclear nonhistone acid-extractable protein from trained rats as compared to untrained ones was demonstrated.

Phosphorylation of Synaptic Membranes

In vivo and in vitro phosphorylation of synaptic membrane proteins were compared. In vivo phosphorylation was carried out by injecting rats intraventricularly with 1 mCi of 32Pi‐orthophosphate. After a 40‐min isotope incorporation period, rats were decapitated and synaptic membranes isolated. In vitro phosphorylation was accomplished by incubating unlabeled synaptic membranes, isolated from noninjected rats, with 5 μM‐[γ‐32P]ATP. In vivo and in vitro32P‐labeled synaptic membranes were then fractionated electrophoretically on an SDS‐polyacrylamide (7.5%) slab gel. The Coomassie blue protein patterns for in vivo and in vitro32P‐labeled synaptic membranes were identical. In contrast, autoradiographs of these gels showed striking differences in the pattern of phosphate incorporation. Cyclic‐AMP (10 μM) maximally stimulated) the in vitro phosphate labeling of two protein bands (80,000 and 55,000 apparent molecular weight) which were not substantially labeled by the in vivo procedure.

Effects of calcium, strontium, and barium ions on phosphorylation of hippocampal proteins in vitro.

Abstract: Calcium ion alone or in the presence of added calmodulin stimulated in vitro transfer of 32P from [γ32P]ATP into several proteins of mitochondrial and synaptosomal particulate fractions from rat brain. Strontium ion was capable of substituting for calcium ion in this stimulation, but barium ion lacked this capacity. These results bring into question the hypothesis that calciumdependent protein phosphorylation of synaptic proteins is intrinsic to neurotransmitter release during neurotransmission, but they do not rule out that possibility.

Environmental stimulation and fucose incorporation into brain and liver glycorproteins

Abstract Changes were observed in the amount of radioactive fucose incorporated into glycoproteins of brain and liver when mice were exposed to different environments. Mice were injected subcutaneously with 3H-L-fucose, placed in a small chamber with an electrifiable grid floor for 15 min, and killed 1 min later. Exposure to the apparatus without shocks increased the levels of incorporation in both and liver compared to mice placed in individual cages after the injection. Increasing amounts of footshock reduced the level of incorporation. Five to 40 sec of footshock resulted in incorporation levels inversely proportional to the number of shocks.

Phosphorylation of proteins of synaptosome enriched fractions of brain during short term training experience : the effects of various behavioral treatments

The relationship between the phosphorylation of synaptosomal proteins and the behavior of mice subjected to foot-shock avoidance conditioning has been studied. It appears that the increased phosphorylation of these proteins in the trained mouse is specific to the training, since mice that were merely exposed to the training stimuli or performed the avoidance after they had been previously trained did not show the response. However, mice that extinguished the learned behavior did show such increases. These changes may be relevant to the establishment of neural pathways that enable storage of memory of a novel experience.

Brain uridine monophosphate: reduced incorporation of uridine during avoidance learning

Abstract Avoidance training produced no detectable changes in the radioactivity incorporated from [5-3H]uridine into total RNA of mouse brain, but decreased the radioactivity in brain uridine monophosphate. The chemical response was largest in the subcortical forebrain, and did not occur in yoked control mice. This phenomenon has important implications for the interpretation of previous experiments, which employed the amount of radioactivity in uridine monophosphate as the pool correction factor in studying the effects of avoidance training upon the incorporation of uridine into brain RNA.

Phosphorylation of proteins from the brains of mice subjected to short term behavioral experiences

Publisher Summary This chapter describes the phosphorylation of proteins obtained from enriched synaptosomal fractions from the brains of mice subjected to short training experiences. An outline of the experiment is presented by a diagram in the chapter. One of a pair of 6-8-week-old male mice was injected intracranially with approximately 20 μCi [ 32 P] orthophosphate in 20 μl water, while the other was injected with 20 μCi of carrier-free [ 33 P] orthophosphate from ICN, Irvine, Calif., U.S.A. Twenty-nine minutes after injection one mouse was placed in a jump-box training apparatus and trained. The untrained mouse stayed in the home cage during the entire 45 min and referred as Quiet. At the end of the training, both mice were decapitated after minimal ether anesthetization. The brains were removed and the olfactory bulbs and cerebellum excised. Proteins of synaptosome-enriched fractions of mouse brain exhibited an increase in the incorporation of radioactive phosphate during 15 min of avoidance conditioning. The increase was initiated between 5 and 10 min of the training and could be detected 30 min beyond the training period.

Brain function and macromolecules. Viii. Uridine incorporation into brain polysomes of hypophysectomized rats and ovariectomized mice during avoidance conditioning.

Summary 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.