Puromycin and cycloheximide: different effects on hippocampal electrical activity

Abstract. Mice were injected in the temporal region of the brain with cycloheximide or puromycin; both agents markedly inhibit protein synthesis in the brain. Recordings of electrical activity were made in the hippocampal region 5 hours after injection of these drugs. The amplitude and frequency observed in records from mice injected with cycloheximide were indistinguishable from those injected with saline alone. Records from puromycin-injected mice were strikingly abnormal. This finding may contribute to the differences in behavioral effects of intracerebral injections of the two inhibitors of protein synthesis studied.     

Memory in the Japanese quail: effects of puromycin and acetoxycycloheximide

Intracerebral injections of puromycin produced memory deficits in naive quail trained to discriminate between red and green stimuli. Puromycin aminonucleoside, acetoxycycloheximide, and saline had no such effect. After a single reversal of the visual cues, naive quail treated with puromycin performed better than control birds. Also, puromycin had no effect on performance when injected into previously trained animals. High doses both of puromycin and acetoxycycloheximide inhibited ribonucleic acid and protein synthesis to a similar extent, while low doses of puromycin inhibited only protein synthesis. Since only puromycin inhibited memory, the basis for its effect appears more likely to be mediated by the action of peptidyl-puromycin rather than by the quantitative inhibition of macromolecular synthesis or by some nonspecific toxic action.     

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.     

Puromycin effect on memory may be due to occult seizures

Intracerebral injections of puromycin, which have been shown to impair memory 3 hours after training, increase the susceptibility of mice to seizures after administration of normally subconvulsive doses of pentylenetetrazol. Cycloheximide, which antagonizes the puromycin-induced amnesia 3 hours after training, also antagonizes the puromycin effect on susceptibility to seizure. The anticonvulsant diphenylhydantoin antagonizes the puromycin effect on memory. The puromycin effect on memory may be due to occult seizures.     

Memory in mice analyzed with antibiotics. Antibiotics are useful to study stages of memory and to indicate molecular events which sustain memory

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. Applied to larger animals the localization of various regions sensitive or insensitive to the action of the drugs should become more definitive. We hope that such experiments will contribute increasingly to the general problem of brain function.     

Amnesia or reversal of forgetting by anticholinesterase, depending simply on time of injection

The effect of intracerebral injections of the anticholinesterase drug diisopropyl fluorophosphate in rats was to produce good recall of an otherwise almost forgotten habit learned 28 days before. The same injections produced temporary amnesia for the same habit, otherwise well remembered, learned 14 days before. The injections had no ef fect on the memory of the same habit when it was only partly learned 14 days before. The results support the hy pothesis that the physiological basis of memory lies in an increase, and for getting in a decrease, in synaptic con ductance.     

Memory traces: experimental separation by cycloheximide and electroconvulsive shock

Mice given cycloheximide or saline were trained with a single trial. Electroconvulsive shock was administered to both groups at various times after training. Cycloheximide led to memory that decayed with time. Cycloheximide plus electroconvulsive shock produced complete amnesia at times when neither treatment alone produced amnesia. Only two types of processes appear to support memory storage in our study.     

Memory: modification of anisomycin-induced amnesia by stimulants and depressants

Mice were trained in a passive (foot shock)avoidance task. When administered after training, the stimulants caffeine or nicotine blocked amnesia for the task that had been produced by injections of the protein synthesis inhibitor anisomycin given prior to training. With foot shock at a higher intensity, anisomycin did not produce amnesia by itself, but the administration of the depressants chloral hydrate or sodium phenobarbital after training did cause amnesia. Stimulants and depressants did not have an appreciable influence on the overall degree of protein synthesis inhibition produced by anisomycin. The results support the hypothesis that arousal after training is an important factor in the conversion of short-term to long-term memory.     

Memory-blocking agents: effects on olfactory discrimination in homing salmon

Homing salmon were injected intracranially with puromycin, actinomycin D, or cycloheximide. From 4 to 7 hours after such treatment these agents markedly inhibited olfactory bulbar discrimination between home water and other natural waters, including spawning sites for other groups of salmon. At longer intervals after treatment there was a partial restoration of olfactory memory-based discrimination. The dosages of the inhibitors used could be shown to have depressed incorporation of H(3)-leucine into protein by 78 percent or of H(3)-uridine into RNA by 41 percent in the salmon brains 4 hours after intracranial injection. These findings suggest that acute blockage of RNA synthesis or protein synthesis can interfere with long-term olfactory memory in anadromous salmon, at least as this function can be analyzed by electrophysiological methods. This implies that long-term olfactory memory depends upon continued metabolism of RNA and continued protein synthesis.     

Puromycin: effect on memory of mice when injected with various cations

Puromycin dihydrochloride neutralized with bases of potassium, lithium, calcium, or magnesium fails to block expression of memory of maze learning in mice, unlike puromycin neutralized with NaOH. This failure may be due to cationic binding at anionic membrane sites with a resultant exclusion of sufficient peptidyl-puromycin to make it ineffective in blocking memory.     

Puromycin: action on neuronal mitochondria

Puromycin, in dosages that inhibit cerebral protein synthesis and expression of memory in mice, produces swelling of neuronal mitochondria. Acetoxycycloheximide, which inhibits cerebral protein synthesis to the same extent as puromycin, fails to produce swelling of neuronal mitochondria. Puromycin and heximide mixtures produce severe inhibition of protein synthesis, but result in a minimal swelling of neuronal mitochondria and in a decrease of peptidylpuromycin complexes to a level of 30 percent of that following the injection of puromycin alone. It is concluded that swelling of neuronal mitochondria in the presence of puromycin is not due to inhibition of cerebral protein synthesis per se, but is related to a specific action of puromycin on ribosomal protein synthesis. The findings are consistent with the hypothesis that peptidyl-puromycin complexes are responsible for mitochondrial swelling.     

Actinomycin D blocks formation of memory of shock-avoidance in goldfish

When 2 micrograms of antinomycin D was injected intracranially into goldfish immediately after a training session, the formation of long-term memory of a shock-avoidance was blocked. The results are discussed in relation to similar findings with acetoxycycloheximide and puromycin in the goldfish and with apparently conflicting results in the mouse.     

Reticular stimulation and chlorpromazine: an animal model for schizophrenic overarousal

It has been postulated that certain schizophrenic patients are in a state of continual central excitation and that improvement in these patients after treatment with chlorpromazine is a result of the action of the drug in reducing this excitation. A model was developed to test this postulated state of central excitation. Rats were electrically stimulated in the mesencephalic reticular formation while performing a simple attention task. Stimulation or treatment with chlorpromazine impaired the performance of the animals; however, the two treatments together resulted in performance indistinguishable from that seen after injections of saline alone.     

Retention deficit correlated with a deficit in the corticoid response to stress

Previous research has indicated that intracerebral injection of potassium chloride, and the resultant electrical silence of the brain, lead to the attenuation of a previously conditioned emotional response; this response may reflect conditioned fear. The data reported here indicate that the normal mobilization of corticosteroids, perhaps a second index of emotionality, is also attenuated by such injections.     

PROLONGING EFFECTIVE PENICILLIN ACTION

(1) Single injections of penicillin in saline administered intramuscularly with ice applications at the site of the injection produced and maintained bacteriostatic levels for six to twelve hours in nine out of ten patients, nine of whom were diagnosed as having gonorrhea and one urethritis with extracellular diplococci. (2) There was one failure, Subject No. 10, who received only 30,000 units. (3) Eight of the patients with gonorrhea and the one patient with urethritis were cured (bacteriologically and clinically) by this single intramuscular injection of penicillin.     

Puromycin and retention in the goldfish

A first experiment compared the behavior of goldfish injected with puromycin immediately after each of a weekly series of brief discriminative training sessions in the shuttlebox to that of appropriate controls. Discrimination was not prevented, nor was escape from shock impaired, but probability of response to the conditioned stimuli, both positive and negative, was reduced substantially. These results suggest that puromycin interferes with the consolidation of conditioned fear. The null outcome of a second experiment, in which all training was given in a single long session instead of a series of short sessions, suggests (contrary to recent indications) that consolidation begins in the training session. The conditioned-fear hypothesis is supported by the results of a third experiment in which the animals were shocked upon entering a goalbox to which they had previously learned to swim for food; animals injected with puromycin, immediately after the shock, entered the goalbox more readily 1 week later than did appropriate controls.     

Puromycin effect on successive phases of memory storage

Mice injected bitemporally with puromycin 5 hours before training learned to escape or to avoid shock by choosing the correct limb of a Y-maze. When retested 15 minutes after training they had normal retention. In the ensuing 2(3/4) hours the animals injected with puromycin, unlike the controls, showed a progressive decrease of savings to less than 7 percent.     

The effect of B-pyracin and the Lactobacillus casei factor upon hemoglobin regeneration following hemorrhage

In severe experimental hemorrhagic anemia in hens, the injection of beta-pyracin and Lactobacillus casei factor, alone and in combination, was found to hasten the regeneration of hemoglobin. When these factors were administered together, however, the rise in hemoglobin was much more rapid and the level reached was higher than in the lots administered the factors alone. The hemoglobin levels in the control lots rose above the "pre-bleeding" values and then began an almost immediate decline to approximately the original level. In the lots receiving beta-pyracin and Lactobacillus casei factor, alone or in combination, the hemoglobin levels also rose above the "pre-bleeding" values but were maintained for some time after injections were discontinued before declining to the original values.     

Influence of anesthesia on experimental western equine encephalomyelitis

Anesthesia, by ether, is effective in the treatment of western equine encephalomyelitis in mice. Of mice treated with deep ether anesthesia soon after the intracerebral injection of western equine virus, only 58 per cent developed the disease as compared with 92.4 per cent of control animals. When anesthesia was delayed the approximate length of the incubation period, 60 per cent of the animals developed the disease as compared with 92.4 per cent of the controls. In addition, ether anesthesia delays the development of central nervous system symptoms not only when administered soon after the injection of the virus but also when administered after the disease has progressed far enough to cause objective signs of encephalitis.     

Photic responses in hyperkinesis of childhood

Intravenous injections of drugs that stimulate the central nervous system decreased photic driving responses and photoinyoclonic responses in hyperkinetic children. Neither injections of saline in these children nor injections of stimulant drugs in normal subjects produced such diminution. The neurophysiological implications are discussed.