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The Effect of Short Term Experiences on the Incorporation of Uridine into RNA and Polysomes of Mouse Brain
The Effect of Short Term Experiences on the Incorporation of Uridine into RNA and Polysomes of Mouse Brain E D W A R D GL AS S MAN
AND
JOHN E. WILSON
Department of Biochemistry and the Neurobiology Curricuhm, University of North Carolina, Chapel Hill, North Carolina (U.S.A.)
There is a considerable amount of literature indicating that training experiences can have marked effects on the macromolecules of the nervous system. Unfortunately, a number of difficulties mar unequivocal interpretation of these studies. First, the behavioral task is often too long or is too complicated to allow clear-cut conclusions concerning the critical behavioral components responsible for the chemical response. Second, there is often no comparable examination of chemical changes in tissues other than the nervous system to be sure the response is specific to it. Third, often either the entire brain is homogenized or only a very small part is examined, and an extensive inventory of all the major portions of the brain and even of the cells involved in the response is not available. A lack of consideration of these problems has led many investigators to conclude that their findings are relevant to memory research when, in fact, there is often a lack of critical data to establish such relevance. These considerations have made it difficult, if not impossible, to draw any conclusions concerning the possible cause-and-effect relationships between the experience or the behavior and the chemical change. Nor are there any data to indicate the role that the chemicals are playing in the responding cells. This work is an attempt to approach this problem. The mouse was chosen because it has been used extensively in behavioral, biochemical, and genetical studies. To minimize genetic variation, only 6 to 8 week old males of strain C57B1/6J, supplied by the Jackson Laboratories, were used. Conditioned avoidance training was carried out in the jump box described previously (Zemp et al., 1966; Schlesinger and Wimer, 1967). It consists of a box divided into two sections with a common electric grid floor. One mouse is placed in each section. A light and a buzzer are attached to the outside of the box so that each mouse receives equal stimulation. The sections are identical except that one side has a shelf onto which the mouse in that section can jump. The light and buzzer are presented for 3 sec before the electric shock is applied. Initially, both mice jump in response to the shock and the animal that has the shelf uses it as a haven. The shock is terminated as soon as it does so. The mouse is then removed from the shelf and placed on the grid floor, and another trial then commences. The training lasts for 15 min, and between 30 and 35 trials are carried out during this period. The mouse that has the shelf will usually start to avoid the shock in response to light and buzzer by the fifth trial References p . 249
246
E. G L A S S M A N , J. E. W I L S O N
and is performing to a criterion of 9 out of 10 by 5 min. It should be noted that when the trained mouse avoids the shock, the animal on the other side also does not receive one. The untrained mouse also receives equivalent handling at random during the training. Thus with respect to lights, buzzers, shocks, handling, and injections, the untrained mouse is yoked to the trained mouse. The biochemical analysis utilizes a double isotope-labeling method. One mouse of a pair was injected intracranially with [14C]uridine,the other with [SHIuridine. Thirty minutes later, one of the mice was trained for 15 min in the jump box while the other served as the yoke animal. After the training period, the brains of both mice were homogenized simultaneously in the same homogenizer, after which the homogenate was fractionated into nuclei, ribosomes, and a supernatant fraction (Zemp et al., 1966). UMP was isolated from the supernatant fraction and RNA was extracted from each of the subcellular components : the amount of 14Cand 3H in each was determined (Zemp et al., 1966). The purpose of using two labels in this way is to avoid the problem of differential losses of RNA that occur during the complicated manipulations we had to employ to isolate the RNA. The ratio of 3H to 14C in the UMP is a useful indication of the relative efficiency of the injection and of the relative amount of uridine that entered brain cells, and is used to correct the observed ratio of 3H to 14C in the RNA. In 25 of such blind double-labeling experiments, all trained mice incorporated more radioactivity into RNA than did the untrained mice (Zemp et al., 1966). The average increase in RNA isolated from nuclei was 38 % and the range was 6.5 to 119%. The average increase in RNA isolated from the ribosomal pellet was 64% and the range was 7 to 180%. This difference between the incorporation into brain RNA of the trained and untrained mice cannot be unequivocally ascribed to increased synthesis of RNA in the trained animal. It could, for example, be due to decreased destruction of RNA, to increases in permeability of the cells or their nuclei to uridine, to a decrease in the synthesis of endogenous uridine, or to any one of a number of other alternatives. Thus we refer only to the change in incorporation and do not specify a mechanism. By injecting radioactive uridine intraperitoneally as well as intracranially, it was possible to show that there were no differences between trained and untrained animals in the incorporation of radioactive uridine into liver and kidney RNA or polysomes, even though these same animals showed pronounced differences in the brain (Zemp et al., 1966; Adair et al., 1968a). It was concluded, therefore, that although tissues other than the liver and kidney might be responding, the effect may be specific to brain. The effect also seems to be limited to specific areas in the brain. Gross dissection revealed that the increased incorporation of uridine into RNA took place entirely in the diencephalon and associated structures (Zemp et al., 1967); there was a small but significant decrease in the cortex. Autoradiography confirmed this (Kahan et al., 1970). Although there was similar labeling in comparable neurons in all mice,’the trained mouse showed more incorporation into many neurons in the limbic system, whereas the untrained yoke mouse incorporated more [3H]uridine into some neurons
URIDINE INCORPORATION INTO B R A I N
RNA
A N D BEHAVIOR
247
in the neocortex. These results indicate how important it is to know the full extent of the involvement of the brain in chemical responses to behavioral stimuli in order to gain proper perspective on the phenomenon being observed. Yoked and quiet mice had similar amounts of incorporation of radioactive uridine into brain RNA and polysomes (Zemp et al., 1966; Adair et al., 1968b). It was concluded that non-specific stimuli, such as the lights, buzzers, shocks, and handling that the yoke mouse received are not sufficient to have any effect by themselves. To test this idea further, the incorporation of radioactive uridine into RNA taken from the brains of quiet mice was compared with that in mice subjected to 30 electric shocks given at random over the 15-min period. The data clearly show that the radioactivity in the RNA and polysomes was similar in both mice, thus giving further credence to the idea that mere stimuli and activity were not responsible for the effect we observed (Zemp et al., 1966; Adair et al., 1968b). There are, however, many differences between the trained mouse and the yoke mouse in addition to learning. For example, the trained mouse has a change in cue and his attention is now directed at a stimulus that the untrained mouse probably views as benign. Also, the stresses on the trained mouse are different, as are his responses to them. Finally, the trained mouse jumps more often than the yoke mouse and there is a difference in the quality of the jump, in that the trained mouse quickly learns to organize his locomotion to reach the shelf, while the untrained mouse jumps with random purpose. To examine some of these alternatives, a mouse was trained for two successive days with 15-min sessions in the jump box. On the third day, this mouse was injected with radioactive uridine, using the double-labeling method for polysomes, and was then required t o perform the jump box task. The incorporation of radioactive uridine into brain polysomes was similar to that of a yoked mouse (Adair et al., 1968). It was therefore concluded that the organized locomotion, the changed cue and attention, and those stress factors that operate after the animal has learned and is performing the task are not responsible for the chemical changes we observed. It is, of course, possible that the prior trained mouse had habituated to the training apparatus and the handling, and thus experienced reduced stress. This cannot be ruled out, but the animals do show fear reactions when placed into the training apparatus. Furthermore, mice previously exposed to the training situation as yoke mice (15-min sessions on two successivedays) learned the task perfectly well on the third day and showed the increased incorporation into brain polysomes. Other behaviors related to the jump box were also tested and the results are clear. When the animal learned to jump to the shelf in response to a conditioned stimulus, the increased incorporation of radioactive uridine into brain polysomes was observed (Adair et al., 1968b). Thus, the process of changing cue (Le., learning) seems to be relevant to the chemical change we observed (Adair et al., 1968b). Enough information is still not available to reach an unequivocal conclusion concerning cause-and-effect relationships or the biological significance of these results. Although the training experience causes both a change in behavior (learning) and in chemicals in the brain, there are no data that indicate whether either of these changes References p. 249
248
E. GLASSMAN, J. E. W IL SON
is the cause of the other, whether they might even be completely unrelated responses to two different input stimuli. This problem is common to all research on the nervous system where an experience has more than one behavioral, biological, and chemical response, Whether this chemical response has anything to do with the learning process per se or with a response incidental to this process, such as emotional responses, cannot be answered at this time. Many of the 14C and 3H-labeled RNA mixtures from yoke and trained animals were sedimented in sucrose gradients to see if the increased radioactivity was located in a single species of RNA that might have a unique function (Zemp e l al., 1966). In all cases, the increased radioactivity associated with the RNA of the trained mouse was located throughout the gradient and was quite heterogenous with respect to sedimentation rate. The patterns of radioactivity were of the same general shape for RNA from brain, liver, and kidney from trained mice and untrained mice. Thus the increased incorporation in brain RNA resembled that found when RNA synthesis is stimulated in liver by hydrocortisone or in uterus by estrogen. It was concluded that the increased radioactivity was not confined to a single species of RNA, but was distributed among many species, and that a general increase was observed in the synthesis of rapidly labeled RNA due to a metabolic stimulation of the cells involved. That there is also increased incorporation into polysomes of the brain during the training experience (Adair et al., 1968 a and b) further substantiates this idea. Thus no evidence was found for an RNA with a function that does not involve protein synthesis; indeed, the RNA found was similar to RNA extracted from other tissues. Further work is needed to establish whether this RNA is involved in the amount of proteins already present. Because hormones are among the few substances in the body that have been shown to affect RNA synthesis, it was of interest to ascertain the effect of various hormonal influences on this chemical response to experiential stimulation. In these experiments we were concerned mainly with whether the experience stimulated a chain of events which involved secretion of hormones from the adrenals or the pituitary which then led to the increased incorporation into RNA or polysomes in the brain. We have been able to show that mice adrenalectomized seven days prior to the training were able to learn the jump-box task and did show the increased incorporation into polysomes (Adair et al., 1968b). Unfortunately, technical reasons prevented us from testing hypophysectomized mice in the same way. However, we have been carrying out studies on the effects of short term avoidance conditioning in the rat (Coleman, 1969). The training is similar to that used in the mice and the apparatus consisted of a runway with an escape platform at one end. The rats were hypophysectomized 7 days prior to use and were in fairly good health at the time of training. The double labeling method using [I4C]- and [3H] uridine was used. Hypophysectomized rats resembled normal rats in that both groups showed increased incorporation of radioactive uridine into brain RNA as compared to quiet or yoked rats. We conclude that the pituitary gland and the adrenal gland play no acute role in the increased incorporation into RNA and polysomes during the 15-min experience. That is, the activity of these glands is not a necessary step prior to the-change in RNA during the
URIDINE INCORPORATION INTO B R A I N
RNA
A N D BEHAVIOR
249
time of training. We have yet to explore whether secretions from these glands that are not used up in the seven days after the gland was removed might be necessary for the maintenance of proper brain function in relation to this chemical response. REFERENCES ADAIR,LINDAB., WILSON,J. E. AND GLASSMAN, E. (1968a) Proc. Natl. Acad. Sci. U S . , 61, 917-922 ADAIR,LINDAB., WILSON, J. E., ZEMP,J. W. AND GLASSMAN, E. (1968b) Proc. Narl. Acad. Sci. U S . , 606-613. COLEMAN, M. S., Ph. D. 17resis, The Department of Biochemistry, University of North Carolina, Chapel Hill, North Carolina, U.S.A., 1969 KAHAN,B., KRIGMAN, M. R., WILSON,J. E. AND GLASSMAN, E., (1970) Proc. Natl. Acad. Sci. U.S., in press. SCHLESINGER, K. AND WIMER,R.(1967) J. Comp. Physiol. Psychol., 63, 139-141. ZEMP,J. W., WILSON,J. E., SCHLESINGER, K., BOGGAN,W. 0.AND GLASSMAN, E. (1966) Proc. Natl. Acad, Sci. U S . , 55, 1423-1431. ZEMP,J. W., WILSON,J. E. AND GLASSMAN, E. (1967) Proc. Natl. Acad. Sci. US.,58, 1120-1125.
DISCUSSION SACHAR: Since there are differences between trained and yoked animals in adrenal response, have you treated animals with ACTH or corticosteroids to see if this provokes changes in uptake of radioactive uridine? GLASSMAN: Adrenalectomy, in a large series of animals, had no effect on the ability of these animals to learn the task or on the ability to show the RNA effect. Hypophysectomy in about 6 8 pairs of rats in a related paradigm, also failed to affect learning and uridine incorporation, that is: if the animals learn, they show the RNA effect. I don’t know how to reconcile this with Gispen’s results, but as I was pointing out in the discussion of his paper, the peptide may be restoring the ability of the animal to learn and this in turn may affect the RNA effect. The peptide might affect the polysomes indirectly, in that the chemical response only occurs in animals that learn. I do not think there is any basic disagreement between Gispen’s and my results.
AND
JOHN E. WILSON
Department of Biochemistry and the Neurobiology Curricuhm, University of North Carolina, Chapel Hill, North Carolina (U.S.A.)
There is a considerable amount of literature indicating that training experiences can have marked effects on the macromolecules of the nervous system. Unfortunately, a number of difficulties mar unequivocal interpretation of these studies. First, the behavioral task is often too long or is too complicated to allow clear-cut conclusions concerning the critical behavioral components responsible for the chemical response. Second, there is often no comparable examination of chemical changes in tissues other than the nervous system to be sure the response is specific to it. Third, often either the entire brain is homogenized or only a very small part is examined, and an extensive inventory of all the major portions of the brain and even of the cells involved in the response is not available. A lack of consideration of these problems has led many investigators to conclude that their findings are relevant to memory research when, in fact, there is often a lack of critical data to establish such relevance. These considerations have made it difficult, if not impossible, to draw any conclusions concerning the possible cause-and-effect relationships between the experience or the behavior and the chemical change. Nor are there any data to indicate the role that the chemicals are playing in the responding cells. This work is an attempt to approach this problem. The mouse was chosen because it has been used extensively in behavioral, biochemical, and genetical studies. To minimize genetic variation, only 6 to 8 week old males of strain C57B1/6J, supplied by the Jackson Laboratories, were used. Conditioned avoidance training was carried out in the jump box described previously (Zemp et al., 1966; Schlesinger and Wimer, 1967). It consists of a box divided into two sections with a common electric grid floor. One mouse is placed in each section. A light and a buzzer are attached to the outside of the box so that each mouse receives equal stimulation. The sections are identical except that one side has a shelf onto which the mouse in that section can jump. The light and buzzer are presented for 3 sec before the electric shock is applied. Initially, both mice jump in response to the shock and the animal that has the shelf uses it as a haven. The shock is terminated as soon as it does so. The mouse is then removed from the shelf and placed on the grid floor, and another trial then commences. The training lasts for 15 min, and between 30 and 35 trials are carried out during this period. The mouse that has the shelf will usually start to avoid the shock in response to light and buzzer by the fifth trial References p . 249
246
E. G L A S S M A N , J. E. W I L S O N
and is performing to a criterion of 9 out of 10 by 5 min. It should be noted that when the trained mouse avoids the shock, the animal on the other side also does not receive one. The untrained mouse also receives equivalent handling at random during the training. Thus with respect to lights, buzzers, shocks, handling, and injections, the untrained mouse is yoked to the trained mouse. The biochemical analysis utilizes a double isotope-labeling method. One mouse of a pair was injected intracranially with [14C]uridine,the other with [SHIuridine. Thirty minutes later, one of the mice was trained for 15 min in the jump box while the other served as the yoke animal. After the training period, the brains of both mice were homogenized simultaneously in the same homogenizer, after which the homogenate was fractionated into nuclei, ribosomes, and a supernatant fraction (Zemp et al., 1966). UMP was isolated from the supernatant fraction and RNA was extracted from each of the subcellular components : the amount of 14Cand 3H in each was determined (Zemp et al., 1966). The purpose of using two labels in this way is to avoid the problem of differential losses of RNA that occur during the complicated manipulations we had to employ to isolate the RNA. The ratio of 3H to 14C in the UMP is a useful indication of the relative efficiency of the injection and of the relative amount of uridine that entered brain cells, and is used to correct the observed ratio of 3H to 14C in the RNA. In 25 of such blind double-labeling experiments, all trained mice incorporated more radioactivity into RNA than did the untrained mice (Zemp et al., 1966). The average increase in RNA isolated from nuclei was 38 % and the range was 6.5 to 119%. The average increase in RNA isolated from the ribosomal pellet was 64% and the range was 7 to 180%. This difference between the incorporation into brain RNA of the trained and untrained mice cannot be unequivocally ascribed to increased synthesis of RNA in the trained animal. It could, for example, be due to decreased destruction of RNA, to increases in permeability of the cells or their nuclei to uridine, to a decrease in the synthesis of endogenous uridine, or to any one of a number of other alternatives. Thus we refer only to the change in incorporation and do not specify a mechanism. By injecting radioactive uridine intraperitoneally as well as intracranially, it was possible to show that there were no differences between trained and untrained animals in the incorporation of radioactive uridine into liver and kidney RNA or polysomes, even though these same animals showed pronounced differences in the brain (Zemp et al., 1966; Adair et al., 1968a). It was concluded, therefore, that although tissues other than the liver and kidney might be responding, the effect may be specific to brain. The effect also seems to be limited to specific areas in the brain. Gross dissection revealed that the increased incorporation of uridine into RNA took place entirely in the diencephalon and associated structures (Zemp et al., 1967); there was a small but significant decrease in the cortex. Autoradiography confirmed this (Kahan et al., 1970). Although there was similar labeling in comparable neurons in all mice,’the trained mouse showed more incorporation into many neurons in the limbic system, whereas the untrained yoke mouse incorporated more [3H]uridine into some neurons
URIDINE INCORPORATION INTO B R A I N
RNA
A N D BEHAVIOR
247
in the neocortex. These results indicate how important it is to know the full extent of the involvement of the brain in chemical responses to behavioral stimuli in order to gain proper perspective on the phenomenon being observed. Yoked and quiet mice had similar amounts of incorporation of radioactive uridine into brain RNA and polysomes (Zemp et al., 1966; Adair et al., 1968b). It was concluded that non-specific stimuli, such as the lights, buzzers, shocks, and handling that the yoke mouse received are not sufficient to have any effect by themselves. To test this idea further, the incorporation of radioactive uridine into RNA taken from the brains of quiet mice was compared with that in mice subjected to 30 electric shocks given at random over the 15-min period. The data clearly show that the radioactivity in the RNA and polysomes was similar in both mice, thus giving further credence to the idea that mere stimuli and activity were not responsible for the effect we observed (Zemp et al., 1966; Adair et al., 1968b). There are, however, many differences between the trained mouse and the yoke mouse in addition to learning. For example, the trained mouse has a change in cue and his attention is now directed at a stimulus that the untrained mouse probably views as benign. Also, the stresses on the trained mouse are different, as are his responses to them. Finally, the trained mouse jumps more often than the yoke mouse and there is a difference in the quality of the jump, in that the trained mouse quickly learns to organize his locomotion to reach the shelf, while the untrained mouse jumps with random purpose. To examine some of these alternatives, a mouse was trained for two successive days with 15-min sessions in the jump box. On the third day, this mouse was injected with radioactive uridine, using the double-labeling method for polysomes, and was then required t o perform the jump box task. The incorporation of radioactive uridine into brain polysomes was similar to that of a yoked mouse (Adair et al., 1968). It was therefore concluded that the organized locomotion, the changed cue and attention, and those stress factors that operate after the animal has learned and is performing the task are not responsible for the chemical changes we observed. It is, of course, possible that the prior trained mouse had habituated to the training apparatus and the handling, and thus experienced reduced stress. This cannot be ruled out, but the animals do show fear reactions when placed into the training apparatus. Furthermore, mice previously exposed to the training situation as yoke mice (15-min sessions on two successivedays) learned the task perfectly well on the third day and showed the increased incorporation into brain polysomes. Other behaviors related to the jump box were also tested and the results are clear. When the animal learned to jump to the shelf in response to a conditioned stimulus, the increased incorporation of radioactive uridine into brain polysomes was observed (Adair et al., 1968b). Thus, the process of changing cue (Le., learning) seems to be relevant to the chemical change we observed (Adair et al., 1968b). Enough information is still not available to reach an unequivocal conclusion concerning cause-and-effect relationships or the biological significance of these results. Although the training experience causes both a change in behavior (learning) and in chemicals in the brain, there are no data that indicate whether either of these changes References p. 249
248
E. GLASSMAN, J. E. W IL SON
is the cause of the other, whether they might even be completely unrelated responses to two different input stimuli. This problem is common to all research on the nervous system where an experience has more than one behavioral, biological, and chemical response, Whether this chemical response has anything to do with the learning process per se or with a response incidental to this process, such as emotional responses, cannot be answered at this time. Many of the 14C and 3H-labeled RNA mixtures from yoke and trained animals were sedimented in sucrose gradients to see if the increased radioactivity was located in a single species of RNA that might have a unique function (Zemp e l al., 1966). In all cases, the increased radioactivity associated with the RNA of the trained mouse was located throughout the gradient and was quite heterogenous with respect to sedimentation rate. The patterns of radioactivity were of the same general shape for RNA from brain, liver, and kidney from trained mice and untrained mice. Thus the increased incorporation in brain RNA resembled that found when RNA synthesis is stimulated in liver by hydrocortisone or in uterus by estrogen. It was concluded that the increased radioactivity was not confined to a single species of RNA, but was distributed among many species, and that a general increase was observed in the synthesis of rapidly labeled RNA due to a metabolic stimulation of the cells involved. That there is also increased incorporation into polysomes of the brain during the training experience (Adair et al., 1968 a and b) further substantiates this idea. Thus no evidence was found for an RNA with a function that does not involve protein synthesis; indeed, the RNA found was similar to RNA extracted from other tissues. Further work is needed to establish whether this RNA is involved in the amount of proteins already present. Because hormones are among the few substances in the body that have been shown to affect RNA synthesis, it was of interest to ascertain the effect of various hormonal influences on this chemical response to experiential stimulation. In these experiments we were concerned mainly with whether the experience stimulated a chain of events which involved secretion of hormones from the adrenals or the pituitary which then led to the increased incorporation into RNA or polysomes in the brain. We have been able to show that mice adrenalectomized seven days prior to the training were able to learn the jump-box task and did show the increased incorporation into polysomes (Adair et al., 1968b). Unfortunately, technical reasons prevented us from testing hypophysectomized mice in the same way. However, we have been carrying out studies on the effects of short term avoidance conditioning in the rat (Coleman, 1969). The training is similar to that used in the mice and the apparatus consisted of a runway with an escape platform at one end. The rats were hypophysectomized 7 days prior to use and were in fairly good health at the time of training. The double labeling method using [I4C]- and [3H] uridine was used. Hypophysectomized rats resembled normal rats in that both groups showed increased incorporation of radioactive uridine into brain RNA as compared to quiet or yoked rats. We conclude that the pituitary gland and the adrenal gland play no acute role in the increased incorporation into RNA and polysomes during the 15-min experience. That is, the activity of these glands is not a necessary step prior to the-change in RNA during the
URIDINE INCORPORATION INTO B R A I N
RNA
A N D BEHAVIOR
249
time of training. We have yet to explore whether secretions from these glands that are not used up in the seven days after the gland was removed might be necessary for the maintenance of proper brain function in relation to this chemical response. REFERENCES ADAIR,LINDAB., WILSON,J. E. AND GLASSMAN, E. (1968a) Proc. Natl. Acad. Sci. U S . , 61, 917-922 ADAIR,LINDAB., WILSON, J. E., ZEMP,J. W. AND GLASSMAN, E. (1968b) Proc. Narl. Acad. Sci. U S . , 606-613. COLEMAN, M. S., Ph. D. 17resis, The Department of Biochemistry, University of North Carolina, Chapel Hill, North Carolina, U.S.A., 1969 KAHAN,B., KRIGMAN, M. R., WILSON,J. E. AND GLASSMAN, E., (1970) Proc. Natl. Acad. Sci. U.S., in press. SCHLESINGER, K. AND WIMER,R.(1967) J. Comp. Physiol. Psychol., 63, 139-141. ZEMP,J. W., WILSON,J. E., SCHLESINGER, K., BOGGAN,W. 0.AND GLASSMAN, E. (1966) Proc. Natl. Acad, Sci. U S . , 55, 1423-1431. ZEMP,J. W., WILSON,J. E. AND GLASSMAN, E. (1967) Proc. Natl. Acad. Sci. US.,58, 1120-1125.
DISCUSSION SACHAR: Since there are differences between trained and yoked animals in adrenal response, have you treated animals with ACTH or corticosteroids to see if this provokes changes in uptake of radioactive uridine? GLASSMAN: Adrenalectomy, in a large series of animals, had no effect on the ability of these animals to learn the task or on the ability to show the RNA effect. Hypophysectomy in about 6 8 pairs of rats in a related paradigm, also failed to affect learning and uridine incorporation, that is: if the animals learn, they show the RNA effect. I don’t know how to reconcile this with Gispen’s results, but as I was pointing out in the discussion of his paper, the peptide may be restoring the ability of the animal to learn and this in turn may affect the RNA effect. The peptide might affect the polysomes indirectly, in that the chemical response only occurs in animals that learn. I do not think there is any basic disagreement between Gispen’s and my results.