The previous studies were comparisons between experience-enriched animals and animals maintained in isolation. Animals which were housed in colonies, but given no special treatment, showed intermediate effects in those situations studied.

The Berkeley group emphasized that the finding of changes in the brain subsequent to experience does not prove that the changes have anything to do with memory storage, but do establish the fact that the brain can respond to environmental pressure. However, the results are compatible with the hypothesis that long-term memory storage involves the formation of new somatic connections among neurones. Calculations of the amount of additional material required to permit this to exist are compatible with the increases observed.

A number of investigators have studied the effects of antimetabolites and drugs on the learning process. Since their specific metabolic effects are known in other tissues, the rationale is that if these materials do interfere with memory, then specific types of metabolic activities may be implicated in the deposition of the engram.

One of the initial studies of this type was conducted by Dingman and Sporn ([ref.122]), presently at the National Institute of Mental Health. They showed that 8-azaguanine, a purine antagonist, injected intra-cisternally was incorporated into the RNA of the brains of rats. Associated with this incorporation was an impairment of the maze-learning ability of the animals. These findings have been confirmed.

Flexner and his associates injected puromycin, an inhibitor of protein synthesis, into the brains of mice, which were then trained to perform in a maze. Losses of short-term or long-term memory were obtained, depending upon the site of the injection. The results indicate that the hippocampal region is the site of recent memory.

The hippocampal region is of interest in connection with memory processes for a number of other reasons. Adey et al. ([ref.123]) and his group observed a transient fall in electrical impedance in this region when cats learned to perform in a T-maze in response to a visual cue. It was supposed that the electrodes were situated within glial cells of the dendritic zone of the hippocampal pyramidal cell layer. Extinction of the learned habit abolished the briefly evoked impedance changes, which subsequently reappeared with retraining.

A number of other studies more or less indirectly implicate RNA in the learning processes. For instance, in retinal cells of rabbits raised in darkness, there was virtually no ribonucleoprotein as compared with normal amounts in the cells of animals raised in light ([ref.124]). Further, maintenance of normal electrical activity of isolated perfused cat brains is highly dependent upon the presence of the ribonucleic acid precursors, uridine and cytidine, in the perfusate ([ref.125]), and severe derangements occur if any of a variety of pyrimidine antagonists are added ([ref.126]). Brief electrical stimulation of cat cortical tissue causes an increase in nucleic acid cytidine and adenine, thus indicating a synthesis of altered polynucleotides. Finally, injections of RNA in animals have shown interesting effects. When given at a dose of 116 mg/kg daily for 1 month, rats showed an enhanced response and greater resistance to extinction in a shock-motivated behavioral response. It has been shown by another group that injections of RNA enhance the ability of young animals to learn various tasks.

Planaria have been used in a variety of studies which seem to bear on the problem of memory. Quite recent evidence by Bennett, Calvin, and their associates has cast somewhat of a pall over the studies; nevertheless, the work may have some validity. Interest in the use of flatworms, particularly planaria, for study of memory began with a demonstration by McConnell that these simple animals could undergo conditioning ([ref.127]). Subsequently, it was found that some conditioning was retained when the animal was transected and allowed to regenerate. The retention of training was found in both new animals, although the very simple brain, really only two ganglia, was in the head section ([ref.128]).

Apparently, some diffusely distributed component of the animal was responsible for retention of learning. Evidence has accumulated to indicate that this material is RNA. Among this evidence is the following:

  1. The two halves of a trained planaria were allowed to regenerate in a solution containing RNA-destroying enzymes. Whereas the head ends retained some training, no retention was observed in the animals derived from the tail end ([ref.129]).
  2. When pieces of trained planaria were fed to untrained animals, the untrained cannibal required a shorter time to become trained to a criterion. It would appear that the digestive system of planaria is so simple that the material responsible for the transfer of the information was not broken down.
  3. When RNA, obtained from trained planaria, is injected into the digestive tract of untrained animals, there is a transfer of information.