Adrian Dunn

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.

Brain ganglioside preparation: phosphotungstic acid precipitation as an alternative to dialysis

IN THE procedure commonly used for the isolation of brain gangliosides (Suzu~r, 1965) the tissue is homogenized in chloroform-methanol and the gangliosides are separated from the chloroform-methanol extract by partition into an upper aqueous layer. Dialysis is then used to separate the gangliosides from the small molecules extracted. This dialysis procedure has the disadvantages that it is time-consuming (3 days), that losses are incurred in the transfer procedures, that there is the possibility of loss of samples due to leakage of the dialysis sacs, and that materials in the dialysis membrane may interfer with assays used for gangliosides. Furthermore, KANFER & SPIELVOGEL (1973) have recently reported that gangliosides may pass through the dialysis membrane if their concentration inside the sac is too low. To circumvent these problems the use of phosphotungstic acid to precipitate gangliosides from aqueous solutions has been investigated in this study.

Brain gangliosides: Increased incorporation of (1-3H)glucosamine during training☆

The incorporation of (1-3H)glucosamine into biochemical fractions of brain was studied in mice trained in a conditioned avoidance jump-up task, in mice yoked to the trained animals, and in undisturbed mice in their home cages. The (3H)glucosamine was injected subcutaneously 15 min before 15 min of training, and the mice killed after a total pulse time of one hour. There was a consistent and significant increase of about 21 percent of the incorporation of the 3H into the brain ganglioside fraction when trained mice were compared with quiet mice. This increase was not observed in any of the other chemical fractions studied, including the total chloroform-methanol-soluble compounds, the non-ganglioside lipids, and the chloroform-methanol-insoluble compounds (radioactivity principally in glycoprotein). Yoked mice showed an intermediate level of incorporation, exhibiting only a 12 percent increase in the ganglioside fraction. When the individual ganglioside species were analyzed by thin-layer chromatography the changed incorporation was not specific for any ganglioside species.

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.

The dependence of brain ATP content on cerebral electroshock current.

A question of continuing interest is the nature of the biochemical lesion responsible for the amnesia caused by cerebral electroshock. Very many studies have been concerned with this problem, but apparently the effect of electroshock is so gross that almost all brain chemicals that have been studied are affected (see ref. 6). It is likely that many of the changes result from the convulsions induced by the electroshock. Since the occurrence of bodily convulsions is not necessary for the amnesia3,7,9,11,12, examination of the biochemical effects of subconvulsive electroshock may simplify the problem. The convulsions associated with electroshock may be prevented either by the use of anesthetics or by decreasing the electroshock current. The effect of subconvulsive electroshock on the incorporation of radioactive amino acids into brain protein has been examined using both these techniques. Cotman et aL ~ showed that diethyl ether, which inhibited the convulsions but not the brain seizure, did not prevent the inhibition of amino acid incorporation caused by the electroshock. Significant inhibitions of the incorporation were only observed when brain seizures occurred. Dunn 4 showed that the inhibition of amino acid incorporation was linearly correlated with the current used in the electroshock; the presence or absence of convulsions did not especially disturb this correlation. A similar relationship between electroshock current and retrograde amnesia has also been observed 8,7, 9,12. Thus a convenient way of studying the interrelationships of the effects of electroshock is to relate the behavioral, electrophysiological and biochemical data to the electroshock current. The depression of amino acid incorporation into brain protein may be caused by a depression of cerebral ATP levels or the cation imbalance caused by the electroshock4, s. Severe disturbances of brain ATP are induced by the electroconvulsive shock s, but, since these may be prevented by administration of oxygen 1, the disturbances may be provoked by the anoxia associated with the convulsions, rather than the electroshock itself. Nevertheless, anesthetization of mice with secobarbitone prevented the convulsions but not the depression of ATP associated with electroconvul-

Use of cetyl trimethylammonium bromide for estimation of the in vivo incorporation of radioactive precursors into RNA

Abstract RNA can be precipitated from cell homogenates or ribosomes with cetyl trimethylammonium bromide more specifically than with trichloroacetic or perchloric acids. In this way radioactivity not present in RNA is not occluded in the precipitate as occurs with TCA or PCA. The precipitate can be collected on glass-fiber filters from which the RNA can be quantitatively eluted with Soluene 100 for the accurate determination of 3 H and/or 14 C radioactivity. These techniques have been used to estimate in vivo RNA synthesis in mouse brain.

Stimulation increases incorporation of [3H]lysine into rat brain and liver proteins

Exposure of rats to electric footshocks or merely to the footshock apparatus increased the incorporation of [3H]lysine into brain and liver protein. The effect was present in both fore-and hindbrain. The footshock treatment was the more effective stimulus, producing larger and more significant changes. These results resemble those previously observed in C57Bl/6J mice, and thus suggest that altered protein or amino acid metabolism is a general response to stress in rodents.