Josefa B. Flexner

Memory in Mice as Affected by Intracerebral Puromycin

The antibiotic, puromycin, caused loss of memory of avoidance discrimination learning in mice when injected intracerebrally. Bilateral injections of puromycin involving the hippocampi and adjacent temporal cortices caused loss of short-term memory; consistent loss of longer-term memory required injections involving, in addition, most of the remaining cortices. Spread of the effective memory trace from the temporal-hippocampal areas to wide areas of the cortices appears to require 3 to 6 days, depending upon the individual animal. Recent reversal learning was lost while longer-term initial learning was retained after bilateral injections into the hippocampal-temporal areas.

INHIBITION OF PROTEIN SYNTHESIS IN BRAIN AND LEARNING AND MEMORY FOLLOWING PUROMYCIN

ON the assumption that learning and memory may have a biochemical basis, the suggestion has become increasingly frequent during recent years that they may depend in some way on macromolecules such as nucleic acid or protein. The discovery by YARMOLINSKY and DE LA HABA (1960) that puromycin produces profound inhibition of protein synthesis in a cell-free system and the later demonstration that it efficiently suppresses protein synthesis in vivo (GORSKI, AIZAWA and MUELLER, 1961) led us to investigate its effect on the central nervous system. This report deals primarily with the substantial suppression of protein synthesis which has been produced in the brains of mice with puromycin, and with the performance of these mice in tests of their ability to learn and to retain memory of the learning experience.

LOSS OF MEMORY AS RELATED TO INHIBITION OF CEREBRAL PROTEIN SYNTHESIS

IT was previously reported (FLEXNER, FLEXNER and STELLAR, 1963) that the effective locus of the memory trace of simple maze learning in mice appears to spread from the hippocampi and temporal cortices to remaining areas of the neocortex in from 3-6 days after the learning experience. Memory before this period of transition has been called short-term or recent memory; after this period, longer-term memory. Recent memory can be consistently destroyed by bilateral temporal injections of puromycin which have been found to inhibit protein synthesis in the hippocampus and temporal cortex by at least 80 per cent for 8-10 hr. (FLEXNER, FLEXNER, OBERTS and DE LA HABA, 1964). Bilateral temporal injections are, however, without effect on longer-term memory, the loss of which depends upon the use of combined bilateral temporal plus ventricular plus frontal injections (FLEXNER et al., 1963). The present report is concerned with the inhibitory effects of these combined bilateral injections on cerebral protein synthesis. In addition, we have measured the rate of cerebral protein synthesis in the presence of several substances which are related to puromycin but which have no effect on memory.

Memory and cerebral protein synthesis in mice as affected by graded amounts of puromycin

Abstract The effects of intracerebral injections of graded amounts of puromycin on memory and on degree and duration of inhibition of cerebral protein synthesis have been studied in the mouse. As the concentration of the antibiotic was decreased it became progressively less effective in its behavioral and biochemical effects so that memory was retained in an increasing proportion of animals as the effect on protein synthesis diminished.

BIOCHEMICAL AND PHYSIOLOGICAL DIFFERENTIATION DURING MORPHOGENESIS‐XXIII. FURTHER OBSERVATIONS RELATING TO THE SYNTHESIS OF AMINO ACIDS AND PROTEINS BY THE CEREBRAL CORTEX AND LIVER OF THE MOUSE*

THE observations presented here in part supplement previous ones concerning the newborn mouse (FLEXNER, FLEXNER and ROBERTS, 1958). In major part, however, they relate to the adult animal. Both cerebral cortex and liver have been studied to obtain estimates of the following quantities: (a) the rate at which blood supplies glutamic acid to these tissues; (b) the rate at which the carbon of glucose is incorporated into several non-essential amino acids of the tissues; (c) the rate at which certain essential amino acids are supplied to the tissues by the blood; and (d) the rate at which these amino acids in the tissues are incorporated into protein. It has been our interest to study the effect of growth and maturation on these quantities. The findings of GAITONDE and RICHTER (1956) with methionine and of LAJTHA, FURST, GERSTEIN and WAELSCH (1957) and LAJTHA, FURST and WAELSCH (1957) with lysine leave no doubt that amino acids are incorporated at a substantial rate into the proteins of the adult as well as the newborn brain.

Lactic dehydrogenases of the developing cerebral cortex and liver of the mouse and guinea pig

Abstract Using cellulose ion exchangers and starch gel electrophoresis, four components of LDH have been found in the cerebral cortex of mouse and guinea pig. The several components tested could also be distinguished by their ratios of rates of reduction of DPN and two of its analogs. These components apparently each contribute to a different degree to the increase in LDH activity of the cortex which occurs during development. Maturation of the liver is accompanied by a reduction in the number of components of LDH in both mouse and guinea pig. With the liver of the mouse, peaks of LDH activity in the chromatograms which corresponded in their position with those from cerebral cortex also gave the same ratios of reduction of DPN and its analogs.

Dose-response relationships in attenuation of puromycin-induced amnesia by neurohypophyseal peptides

Intracerebral injections of puromycin one day after training of mice in a Y-maze cause amnesia when the animals are tested 7 days later. This amnesia was shown to be attenuated by various neurohypophyseal hormones, analogs and fragments, administered subcutaneously immediately after training. Dose-response relationships have been obtained for the attenuation of puromycin-induced amnesia in mice by selected neurohypophyseal peptides. All of the compounds tested reduce the amnesia in a dose-related way, suggesting that these peptides may interact with specific receptors to induce their central effect. Among the peptides studied the two most potent--i.e., those that cause substantial retention of memory at the lowest doses--are the neurohypophyseal hormone arginine vasopressin and Z-prolyl-leucyl-glycinamide (Z-MIF).

Intracerebral Saline: Effect on Memory of Trained Mice Treated with Puromycin

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.

Distribution, survival and biological effects in mice of a behaviorally active, enzymatically stable peptide: Pharmacokinetics of cyclo(Leu-Gly) and puromycin-induced amnesia

Cyclo(Leu-Gly), the enzymatically resistant diketopiperazine formally derived from the C-terminal dipeptide sequence of oxytocin, exhibits activity in several behavioral systems. The distribution of cyclo(Leu-14C(U)Gly) in brain, and the time course of the disappearance of this labeled peptide from brain and plasma after subcutaneous injection into mice have been studied. The intact peptide was distributed equally in the five cerebral areas studied, for up to 96 hours after injection. Two exponential components were determined for peptide disappearance rates in plasma and brain; peptide half-lives in plasma up to 10 hr and from 24--96 hr after injection were, respectively, 0.8 and 33 hr; in brain, 1.0 and 42 hr. The peptide was found to accumulate in brain intracellular space to some degree. The time course of distribution of labeled cyclo(Leu-Gly) in subcellular fractions of mouse brain was also examined, and the concentration of peptide in the synaptosomal fraction was significantly correlated with the degree of protection against puromycin-induced amnesia of a maze-learning test. The results obtained not only confirm that cyclo(Leu-Gly) penetrates brain tissue intact and remains intact after peripheral administration in order to exert its behavioral effects, but, moreover, suggest an intriguing dynamic relationship between peptide concentration in the synaptosomal fraction and behavioral activity.

ADH and related peptides: Effect of pre- or posttraining treatment on puromycin amnesia ☆

The peptide Z-Pro-Leu-Gly-NH2 attenuated puromycin-induced amnesia in mice when administered 5 days prior to training, while arginine vasopressin, lysine vasopressin and cyclo(Leu-Gly), were effective when given 24 hr before training. The activity of all peptides to inhibit puromycin-induced amnesia decreased as the interval after training and before peptide administration increased, suggesting that the peptides influence memory processes rather than generalized arousal mechanisms.