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.

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. MATERIAL A N D METHODS Biochemical studiesYoung adult, albino mice weighing about 30 g were used. We are indebted to Dr. LEON GOLDMAN of the Lederle Laboratories Division of the American Cyanamid Company for our supply of puromycin dihydrochloride pentahydrate. L-Valine uniformly labelled with 14C with a specific radioactivity of 1.0 mc per mg was obtained from the New England Nuclear Corporation.Subcutaneous injection of puromycin and treatment of tissues. Our first experiments were directed towards establishing the maximum amount of puromycin which could be tolerated in a single, subcutaneous injection and then towards following, as a function of time after injection, the degree of suppression of incorporation of ['*C]valine into protein.Just before use, puromycin dihydrochloride was dissolved in 0.15 ml of water and the solution brought to pH 6 with 1 N-NaOH. This solution was injected subcutaneously over the middle of the back of the animal. At various times after administration of the puromycin, 0.10 ml of [14C]valine (4 p c per 30 g mouse) was injected subcutaneously over the back of the mouse. Forty minutes later the animal was killed by asphyxiation. Previous experience had shown that the rates of incorporation of labelled essential amino acids into the proteins of liver and cerebral cortex are approximately constant during this time interval (ROBERTS, FLEXNER and FLEXNER, 1959). In control experiments, the same amount of [Wlvaline was injected in the same way and the animal was killed 40 min later.In the earliest experiments, samples of cerebral cortex, liver, spleen, kidney (including cortex and medulla) and abdominal muscle were taken for analysis. In later experiments we confined our interests to several regions of the brain. In these last experiments, the brain was removed from the skull and samples were taken of the cerebellar cortex, the two thalami, corpora striata, hippocampi, and the cerebral cortex, the latter divided roughly into rostra1 and caudal halves. Blunt dissection of the brain to separate these regions required about 3 min. With few exceptions, samples of fresh tissue * This work was supported by grants B-514, M-3571 and RG-6970 from the U.S. Public Health Service, National Institutes ...

It is apparent that antibiotics are useful in differentiating different stages in the formation of memory. Puromycin gave the first indication that very early memory can be established and survive, for a short period at least, in spite of inhibition of protein synthesis (12). Injection of actinomycin D indicates that RNA synthesis is not essential during this early stage (13). The duration of this early period seems to vary with the inhibiting agent; with puromycin memory was notably degraded in less than an hour, but with actinomycin D or with acetoxycycloheximide it persisted for several hours or more. The fixation or consolidation of memory involves whatever processes give permanence to memory. These processes are disrupted when electroconvulsive shock is administered shortly after a learning experience, presumably because of the interference with organized patterns of neuronal electrical activity. Memory acquired in the presence of antibiotics appears to proceed to a stage beyond that based purely on electrical activity because the memory persists beyond the period usually reported as sensitive to electroconvulsive shock. Further work should show whether this stage is truly insensitive to electroconvulsive shock. Memory acquired in the presence of puromycin does not seem to achieve any durable consolidation. In contrast, memory acquired in the presence of or immediately before injection of acetoxycycloheximide does appear to initiate the later stages of consolidation, as permanent memory. reappears some days after the initial stages have become ineffective in controlling performance. Finally, puromycin has provided evidence of the enlarged area of the neocortex which participates as memory matures. Puromycin also indicates the time required for this maturation process. Since antibiotics have also been useful in studying learning and memory in goldfish (14), this approach seems to have general applicability in defining various stages in the process of memory formation. The initial purpose of these investigations was to determine the molecular basis of the "memory trace" This goal still remains distant, although there are some indications that protein synthesizing systems are involved. This objective, though of enormous interest, is to be regarded as only a necessary first step. Whether new proteins or some other molecules cause the changes in synapses thought to underlie memory, this knowledge of itself will contribute only a beginning to our understanding of the events which account for the functioning of the brain. A determination of the composition of computer components would provide very little information towards unraveling their function. As the experiments proceeded, however, information of a more general nature was being obtained. The identification of different stages of consolidation show how injections of antibiotics can supplement electroconvulsive shock as a way of disrupting the establishment of memory and how it can supplement ablation in destroying memory already laid down in a permanent mode....

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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, ROBERTS 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.

Abstract. Puromycin was injected bitemporally in mice one day after training in a Y-maze. Eight days later various psychotropic drugs were injected intraperitoneally or subcutaneously at maximum tolerable doses. Ten days after the drug injection the mice were tested for their memory of the maze-learning. Memory was lost in control animals injected with saline but restored in most of the animals injected with imipramine, tranylcypromine, or D-amphetamine. Some indication of restoration was observed after injection of reserpine or Ldopa. These results suggest that the blockage caused by puromycin is due to adsorption of peptidyl-puromycin to adrenergic sites and that these sites may be involved in the memory trace.For the first three days after training, memory of Y-maze learning in mice is blocked by bitemporal injections of puromycin that primarily involve the hippocampal areas. The change in behavior is not attributable to lack of ability or motivation to perform, since the mice usually retrain readily. Also, mice trained initially to one arm of the Y-maze, retrained three weeks later to the opposite arm, and injected bitemporally with puromycin 1 day later forget their most recent training and revert with a high level of performance to the first behavior pattern.1 In these circumstances the loss of memory caused by puromycin cannot be attributed to its inhibition of protein synthesis; no such effect is observed when acetoxycycloheximide or a mixture of puromycin and acetoxycycloheximide is substituted for the puromycin even though these injections produce a more drastic and longer-lasting inhibition of protein synthesis. The blockage caused by puromycin can be removed by intracerebral injections of several inorganic solutions; thus, it is apparent that puromycin does not destroy the memory pattern but suppresses its expression.3' 4 Radioactive puromycin has been found to remain in the brain for prolonged periods (held as peptidyl-puromycin5) and is presently assumed to be the blocking agent.The evidence that norepinephrine influences affective states, possibly by acting as a modulator otI cholinergic systems, has been summarized.6 In addition, drugs reducing the level of norepiniephrine (reserpine and alpha-methyl-paratyrosine) can cause a temporary failure to perform a well-learned conditioned avoidance response.7' 8 The suggestion has been made that the biogenic amines do in fact play an important role in the processes responsible for memory.9 310

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In an earlier report' evidence was presented that puromycin given subcutaneously to mice is without effect on memory of simple maze performance although it appeared to inhibit cerebral protein synthesis by about 80 per cent for a period lasting from the second to the eighth hour after the injection. Subsequentlyit was found that both recent and longer-term memory of maze performance can be destroyed by intracerebral injections of puromycin in mice.2 Destruction of recent memory appeared to be primarily related to the effects of intracerebral puromycin on the hippocampi and temporal cortices, whereas the additional exposure of the remainder of the cortex to the antibiotic appeared necessary to produce loss of longer-term memory. The principal known effect of puromycin is to inhibit protein synthesis. If this effect is related to loss of memory, it would be expected that intracerebral injections would produce a more profound or more enduring inhibition of protein synthesis in the brain than occurs with subcutaneous injections. We have determined the degree and duration of inhibition of protein synthesis using bilateral temporal injections2 which uniformly cause loss of recent memory and compared them to the inhibition following subcutaneously injected puromycin. In addition, we have observed the behavioral and chemical effects of another inhibitor of protein synthesis, chloramphenicol.Materials and Methods.-We are indebted to Dr. Leon Goldman of the Lederle Laboratories Division of the American Cynamid Corporation for our supply of puromycin. The l-valine-C'4 was uniformly labeled and had a specific radioactivity of about 50 uc/0.03 mg. Young adult albino mice weighing about 30 gm were used.At various times after bilateral temporal or subcutaneous injections of puromycin or bilateral temporal injections of chloramphenicol, 1.3 / C of valine-C14 was injected subcutaneously. Forty minutes later' the animal was killed by asphyxiation. To establish normal rates of incorporation of valine into protein, the same amount of valine-C'4 was injected in the same way and for the same period of time in untreated animals. As the quantity of radiovaline was not varied in proportion to the size of the animal, the observed quantity of radioactivity in pool and protein fractions was numerically adjusted to compensate for the variable dilution.Six samples of brain weighing 18-30 mg were routinely taken; these consisted of the two hippocampi, thalami, corpora striata, and of frontal, parietal, and temporal cortices. Procedures previously used for preparation of samples' have been simplified with, we believe, improvements in accuracy. Tissue was dissolved in 1 ml 0.1 N NaOH, and aliquots were taken for determination of protein in duplicate.3 Protein was then precipitated with 3 vol of 12 per cent TCA and the supernatant fluid saved for measurement of pool radiovaline. After thorough washing 1165